|
1
|
Guo JY, Xu K, Wang XH, Li XM, Ku YP, Zeng L, Wan B, Yang GY, Wang J, Chu BB, Pan JJ, Hao WB. Host factor DIAPH1 regulates pseudorabivirus replication by modulating the dynamics of cytoskeleton. Int J Biol Macromol 2025; 298:140112. [PMID: 39842589 DOI: 10.1016/j.ijbiomac.2025.140112] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/07/2024] [Revised: 01/05/2025] [Accepted: 01/18/2025] [Indexed: 01/24/2025]
Abstract
As obligate parasites, viruses exploit host cell organelles and molecular components to complete their life cycle. Among which, viruses firstly hijack the cytoskeleton of host cells to ensure their efficiently cell entry and replication. Although formin family members play a key role in both microfilament and microtubule cytoskeletal remodeling, few studies addressed the detailed function and mechanism of formins in the process of viral infection. Here, we showed that sus scrofa DIAPH1 was involved in the regulation of cytoskeletal dynamics during PRV replication. Firstly, we found that DIAPH1 showed significant changes in the expression level and intracellular localization during PRV infection of PK-15 cells. Next, inhibition of DIAPH1 by RNA interference or small molecular inhibitor SMIFH2 was found to diminish the outcome of PRV infection. Besides, DIAPH1 partially co-localized with actin and tubulin in PRV-infected cells. Cross-talk occurred between microfilaments and microfilaments, which also had an influence on the intracellular localization of DIAPH1. What's more, inhibition of DIAPH1 induced the reorganization of microfilament and the stability of microtubule. These results suggested that DIAPH1 regulated PRV infection by remodeling microfilament and microtubule cytoskeletal dynamics.
Collapse
Affiliation(s)
- Jie-Yuan Guo
- College of Veterinary Medicine, Henan Agricultural University, Zhengzhou 450046, China; Key Laboratory of Animal Biochemistry and Nutrition, Ministry of Agriculture and Rural Affairs of the People's Republic of China, Zhengzhou 450046, China; Key Laboratory of Veterinary Biotechnology of Henan Province, Zhengzhou 450046, China
| | - Kun Xu
- College of Veterinary Medicine, Henan Agricultural University, Zhengzhou 450046, China; Key Laboratory of Animal Biochemistry and Nutrition, Ministry of Agriculture and Rural Affairs of the People's Republic of China, Zhengzhou 450046, China; Key Laboratory of Veterinary Biotechnology of Henan Province, Zhengzhou 450046, China
| | - Xiao-Han Wang
- College of Veterinary Medicine, Henan Agricultural University, Zhengzhou 450046, China; Key Laboratory of Animal Biochemistry and Nutrition, Ministry of Agriculture and Rural Affairs of the People's Republic of China, Zhengzhou 450046, China; Key Laboratory of Veterinary Biotechnology of Henan Province, Zhengzhou 450046, China
| | - Xin-Man Li
- College of Veterinary Medicine, Henan Agricultural University, Zhengzhou 450046, China; Key Laboratory of Animal Biochemistry and Nutrition, Ministry of Agriculture and Rural Affairs of the People's Republic of China, Zhengzhou 450046, China; Key Laboratory of Veterinary Biotechnology of Henan Province, Zhengzhou 450046, China
| | - Yan-Pei Ku
- College of Veterinary Medicine, Henan Agricultural University, Zhengzhou 450046, China; Key Laboratory of Animal Biochemistry and Nutrition, Ministry of Agriculture and Rural Affairs of the People's Republic of China, Zhengzhou 450046, China; Key Laboratory of Veterinary Biotechnology of Henan Province, Zhengzhou 450046, China
| | - Lei Zeng
- College of Veterinary Medicine, Henan Agricultural University, Zhengzhou 450046, China; Key Laboratory of Animal Biochemistry and Nutrition, Ministry of Agriculture and Rural Affairs of the People's Republic of China, Zhengzhou 450046, China; Key Laboratory of Veterinary Biotechnology of Henan Province, Zhengzhou 450046, China
| | - Bo Wan
- College of Veterinary Medicine, Henan Agricultural University, Zhengzhou 450046, China; Ministry of Education Key Laboratory for Animal Pathogens and Biosafety, Zhengzhou 450046, China
| | - Guo-Yu Yang
- Key Laboratory of Animal Biochemistry and Nutrition, Ministry of Agriculture and Rural Affairs of the People's Republic of China, Zhengzhou 450046, China; Key Laboratory of Veterinary Biotechnology of Henan Province, Zhengzhou 450046, China; Henan University of Animal Husbandry and Economy, Zhengzhou 450047, China
| | - Jiang Wang
- College of Veterinary Medicine, Henan Agricultural University, Zhengzhou 450046, China; Key Laboratory of Animal Biochemistry and Nutrition, Ministry of Agriculture and Rural Affairs of the People's Republic of China, Zhengzhou 450046, China; Key Laboratory of Veterinary Biotechnology of Henan Province, Zhengzhou 450046, China; Ministry of Education Key Laboratory for Animal Pathogens and Biosafety, Zhengzhou 450046, China
| | - Bei-Bei Chu
- College of Veterinary Medicine, Henan Agricultural University, Zhengzhou 450046, China; Key Laboratory of Animal Biochemistry and Nutrition, Ministry of Agriculture and Rural Affairs of the People's Republic of China, Zhengzhou 450046, China; Key Laboratory of Veterinary Biotechnology of Henan Province, Zhengzhou 450046, China; Ministry of Education Key Laboratory for Animal Pathogens and Biosafety, Zhengzhou 450046, China
| | - Jia-Jia Pan
- College of Veterinary Medicine, Henan Agricultural University, Zhengzhou 450046, China; Key Laboratory of Animal Biochemistry and Nutrition, Ministry of Agriculture and Rural Affairs of the People's Republic of China, Zhengzhou 450046, China; Key Laboratory of Veterinary Biotechnology of Henan Province, Zhengzhou 450046, China; Ministry of Education Key Laboratory for Animal Pathogens and Biosafety, Zhengzhou 450046, China.
| | - Wen-Bo Hao
- College of Veterinary Medicine, Henan Agricultural University, Zhengzhou 450046, China.
| |
Collapse
|
|
2
|
He S, Zhou Z, Cheng MY, Hao X, Chiang T, Wang Y, Zhang J, Wang X, Ye X, Wang R, Steinberg GK, Zhao Y. Advances in moyamoya disease: pathogenesis, diagnosis, and therapeutic interventions. MedComm (Beijing) 2025; 6:e70054. [PMID: 39822761 PMCID: PMC11733107 DOI: 10.1002/mco2.70054] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/15/2024] [Accepted: 12/12/2024] [Indexed: 01/19/2025] Open
Abstract
Moyamoya disease (MMD) is a type of cerebrovascular disease characterized by occlusion of the distal end of the internal carotid artery and the formation of collateral blood vessels. Over the past 20 years, the landscape of research on MMD has significantly transformed. In this review, we provide insights into the pathogenesis, diagnosis, and therapeutic interventions in MMD. The development of high-throughput sequencing technology has expanded our understanding of genetic susceptibility, identifying MMD-related genes beyond RNF213, such as ACTA2, DIAPH1, HLA, and others. The genetic susceptibility of MMD to its pathological mechanism was summarized and discussed. Based on the second-hit theory, the influences of inflammation, immunity, and environmental factors on MMD were also appropriately summarized. Despite these advancements, revascularization surgery remains the primary treatment for MMD largely because of the lack of effective in vivo and in vitro models. In this study, 16 imaging diagnostic methods for MMD were summarized. Regarding therapeutic intervention, the influences of drugs, endovascular procedures, and revascularization surgeries on patients with MMD were discussed. Future research on the central MMD vascular abnormalities and peripheral circulating factors will provide a more comprehensive understanding of the pathogenic mechanisms of MMD.
Collapse
Affiliation(s)
- Shihao He
- Department of NeurosurgeryPeking Union Medical College Hospital, Peking Union Medical College and Chinese Academy of Medical SciencesBeijingChina
- Department of NeurosurgeryStanford University School of MedicineStanfordCaliforniaUSA
| | - Zhenyu Zhou
- Department of NeurosurgeryBeijing Tiantan Hospital, Capital Medical UniversityBeijingChina
| | - Michelle Y. Cheng
- Department of NeurosurgeryStanford University School of MedicineStanfordCaliforniaUSA
| | - Xiaokuan Hao
- Department of NeurosurgeryBeijing Tiantan Hospital, Capital Medical UniversityBeijingChina
| | - Terrance Chiang
- Department of NeurosurgeryStanford University School of MedicineStanfordCaliforniaUSA
| | - Yanru Wang
- Department of NeurosurgeryBeijing Tiantan Hospital, Capital Medical UniversityBeijingChina
| | - Junze Zhang
- Department of NeurosurgeryBeijing Tiantan Hospital, Capital Medical UniversityBeijingChina
- Department of PathologyStanford University School of MedicineStanfordCaliforniaUSA
| | - Xilong Wang
- Department of NeurosurgeryBeijing Tiantan Hospital, Capital Medical UniversityBeijingChina
| | - Xun Ye
- Department of NeurosurgeryBeijing Tiantan Hospital, Capital Medical UniversityBeijingChina
| | - Rong Wang
- Department of NeurosurgeryBeijing Tiantan Hospital, Capital Medical UniversityBeijingChina
| | - Gary K. Steinberg
- Department of NeurosurgeryStanford University School of MedicineStanfordCaliforniaUSA
| | - Yuanli Zhao
- Department of NeurosurgeryPeking Union Medical College Hospital, Peking Union Medical College and Chinese Academy of Medical SciencesBeijingChina
| |
Collapse
|
|
3
|
Kurasawa S, Ganaha A, Ayabe S, Yoshiki A, Kawama F, Kitayama S, Tabuchi K, Yamashita K, Ueyama T. Hearing loss occurs prior to thrombocytopenia in both mice and humans with DFNA1. FASEB J 2025; 39:e70309. [PMID: 39831886 DOI: 10.1096/fj.202402118r] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/06/2024] [Revised: 12/06/2024] [Accepted: 12/27/2024] [Indexed: 01/22/2025]
Abstract
DFNA1 (deafness, nonsyndromic autosomal dominant 1), initially identified as nonsyndromic sensorineural hearing loss, has been associated with an additional symptom: macrothrombocytopenia. However, the timing of the onset of hearing loss (HL) and thrombocytopenia has not been investigated, leaving it unclear which occurs earlier. Here, we generated a knock-in (KI) DFNA1 mouse model, diaphanous-related formin 1 (DIA1)KIΔv3/KIΔv3, in which Aequorea coerulescens green fluorescent protein (AcGFP)-tagged human DIA1(p.R1213X) was knocked into the ATG site of Dia1. Additionally, the exon 7 of Dia1 was deleted using genome editing to knock out (KO) Dia1-v3, a specific variant of Dia1. AcGFP-DIA1(p.R1213X) expression and endogenous DIA1 KO were confirmed in cochleae and platelets. Hearing function in DIA1KIΔv3/KIΔv3, but not DIA1KIΔv3/+ mice, evaluated by auditory brainstem response, was significantly worse at low frequencies compared to wild-type (WT) mice starting at 3 months of age (3M), with progressive deterioration. Using confocal microscopy and scanning electron microscopy, various stereociliary deformities were identified in the cochleae of DIA1KIΔv3/KIΔv3 mice. Platelet counts in DIA1KIΔv3/KIΔv3, but not DIA1KIΔv3/+ mice, were significantly lower than those in WT mice at 12M, but not at 6M. Furthermore, in a cohort of eight patients with DFNA1 harboring the p.R1213X mutation, HL preceded thrombocytopenia in three individuals. Thus, in both mice and humans, though HL and thrombocytopenia are progressive, HL manifests earlier than thrombocytopenia. Unlike myosin heavy chain 9 (MYH9)-related diseases, thrombocytopenia cannot be a predictive marker for HL in DFNA1. Nevertheless, monitoring platelet counts could provide insights into the progression of the hearing impairments in patients with DFNA1.
Collapse
Affiliation(s)
- Shunkou Kurasawa
- Laboratory of Molecular Pharmacology, Biosignal Research Center, Kobe University, Kobe, Japan
- Department of Otolaryngology-Head and Neck Surgery, University of Tsukuba, Tsukuba, Japan
| | - Akira Ganaha
- Department of Otolaryngology-Head and Neck Surgery, International University of Health and Welfare Narita Hospital, Narita, Japan
- Department of Otorhinolaryngology-Head and Neck Surgery, University of the Ryukyus, Okinawa, Japan
| | - Shinya Ayabe
- Experimental Animal Division, RIKEN BioResource Research Center, Tsukuba, Japan
| | - Atsushi Yoshiki
- Experimental Animal Division, RIKEN BioResource Research Center, Tsukuba, Japan
| | - Fumiya Kawama
- Laboratory of Molecular Pharmacology, Biosignal Research Center, Kobe University, Kobe, Japan
| | - Shota Kitayama
- Laboratory of Molecular Pharmacology, Biosignal Research Center, Kobe University, Kobe, Japan
| | - Keiji Tabuchi
- Department of Otolaryngology-Head and Neck Surgery, University of Tsukuba, Tsukuba, Japan
| | | | - Takehiko Ueyama
- Laboratory of Molecular Pharmacology, Biosignal Research Center, Kobe University, Kobe, Japan
| |
Collapse
|
|
4
|
Ni C, Zhou L, Yang S, Ran M, Luo J, Cheng K, Huang F, Tang X, Xie X, Qin D, Mei Q, Wang L, Xiao J, Wu J. Oxymatrine, a novel TLR2 agonist, promotes megakaryopoiesis and thrombopoiesis through the STING/NF-κB pathway. J Pharm Anal 2025; 15:101054. [PMID: 39906691 PMCID: PMC11791361 DOI: 10.1016/j.jpha.2024.101054] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/03/2024] [Revised: 07/17/2024] [Accepted: 07/22/2024] [Indexed: 02/06/2025] Open
Abstract
Radiation-induced thrombocytopenia (RIT) faces a perplexing challenge in the clinical treatment of cancer patients, and current therapeutic approaches are inadequate in the clinical settings. In this research, oxymatrine, a new molecule capable of healing RIT was screened out, and the underlying regulatory mechanism associated with magakaryocyte (MK) differentiation and thrombopoiesis was demonstrated. The capacity of oxymatrine to induce MK differentiation was verified in K-562 and Meg-01 cells in vitro. The ability to induce thrombopoiesis was subsequently demonstrated in Tg (cd41:enhanced green fluorescent protein (eGFP)) zebrafish and RIT model mice. In addition, we carried out network pharmacological prediction, drug affinity responsive target stability assay (DARTS) and cellular thermal shift assay (CETSA) analyses to explore the potential targets of oxymatrine. Moreover, the pathway underlying the effects of oxymatrine was determined by Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses, Western blot (WB), and immunofluorescence. Oxymatrine markedly promoted MK differentiation and maturation in vitro. Moreover, oxymatrine induced thrombopoiesis in Tg (cd41:eGFP) zebrafish and accelerated thrombopoiesis and platelet function recovery in RIT model mice. Mechanistically, oxymatrine directly binds to toll-like receptor 2 (TLR2) and further regulates the downstream pathway stimulator of interferon genes (STING)/nuclear factor-kappaB (NF-κB), which can be blocked by C29 and C-176, which are specific inhibitors of TLR2 and STING, respectively. Taken together, we demonstrated that oxymatrine, a novel TLR2 agonist, plays a critical role in accelerating MK differentiation and thrombopoiesis via the STING/NF-κB axis, suggesting that oxymatrine is a promising candidate for RIT therapy.
Collapse
Affiliation(s)
- Chengyang Ni
- Sichuan Key Medical Laboratory of New Drug Discovery and Druggability, Luzhou Key Laboratory of Activity Screening and Druggability Evaluation for Chinese Materia Medica, School of Pharmacy, Southwest Medical University, Luzhou, Sichuan, 646000, China
| | - Ling Zhou
- Sichuan Key Medical Laboratory of New Drug Discovery and Druggability, Luzhou Key Laboratory of Activity Screening and Druggability Evaluation for Chinese Materia Medica, School of Pharmacy, Southwest Medical University, Luzhou, Sichuan, 646000, China
| | - Shuo Yang
- Sichuan Key Medical Laboratory of New Drug Discovery and Druggability, Luzhou Key Laboratory of Activity Screening and Druggability Evaluation for Chinese Materia Medica, School of Pharmacy, Southwest Medical University, Luzhou, Sichuan, 646000, China
| | - Mei Ran
- Sichuan Key Medical Laboratory of New Drug Discovery and Druggability, Luzhou Key Laboratory of Activity Screening and Druggability Evaluation for Chinese Materia Medica, School of Pharmacy, Southwest Medical University, Luzhou, Sichuan, 646000, China
| | - Jiesi Luo
- School of Basic Medical Sciences, Southwest Medical University, Luzhou, Sichuan, 646000, China
- Education Ministry Key Laboratory of Medical Electrophysiology, Southwest Medical University, Luzhou, Sichuan, 646000, China
| | - Kui Cheng
- Guangdong Provincial Key Laboratory of New Drug Screening and NMPA Key Laboratory for Research and Evaluation of Drug Metabolism, School of Pharmaceutical Sciences, Southern Medical University, Guangzhou, 510515, China
| | - Feihong Huang
- Sichuan Key Medical Laboratory of New Drug Discovery and Druggability, Luzhou Key Laboratory of Activity Screening and Druggability Evaluation for Chinese Materia Medica, School of Pharmacy, Southwest Medical University, Luzhou, Sichuan, 646000, China
| | - Xiaoqin Tang
- Sichuan Key Medical Laboratory of New Drug Discovery and Druggability, Luzhou Key Laboratory of Activity Screening and Druggability Evaluation for Chinese Materia Medica, School of Pharmacy, Southwest Medical University, Luzhou, Sichuan, 646000, China
| | - Xiang Xie
- Public Center of Experimental Technology, Model Animal and Human Disease Research of Luzhou Key Laboratory, School of Basic Medical Sciences, Southwest Medical University, Luzhou, Sichuan, 646000, China
| | - Dalian Qin
- Sichuan Key Medical Laboratory of New Drug Discovery and Druggability, Luzhou Key Laboratory of Activity Screening and Druggability Evaluation for Chinese Materia Medica, School of Pharmacy, Southwest Medical University, Luzhou, Sichuan, 646000, China
| | - Qibing Mei
- Sichuan Key Medical Laboratory of New Drug Discovery and Druggability, Luzhou Key Laboratory of Activity Screening and Druggability Evaluation for Chinese Materia Medica, School of Pharmacy, Southwest Medical University, Luzhou, Sichuan, 646000, China
| | - Long Wang
- Sichuan Key Medical Laboratory of New Drug Discovery and Druggability, Luzhou Key Laboratory of Activity Screening and Druggability Evaluation for Chinese Materia Medica, School of Pharmacy, Southwest Medical University, Luzhou, Sichuan, 646000, China
| | - Juan Xiao
- Department of Cardiovascular Medicine, The Affiliated Hospital of Southwest Medical University, Luzhou, Sichuan, 646000, China
| | - Jianming Wu
- School of Basic Medical Sciences, Southwest Medical University, Luzhou, Sichuan, 646000, China
- Education Ministry Key Laboratory of Medical Electrophysiology, Southwest Medical University, Luzhou, Sichuan, 646000, China
| |
Collapse
|
|
5
|
de Castro Sampaio SS, Ramalho MCC, de Souza CS, de Almeida Rodrigues B, de Mendonça GRS, Lazarini M. RHO subfamily of small GTPases in the development and function of hematopoietic cells. J Cell Physiol 2025; 240:e31469. [PMID: 39434451 DOI: 10.1002/jcp.31469] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/25/2024] [Revised: 09/16/2024] [Accepted: 10/03/2024] [Indexed: 10/23/2024]
Abstract
RHOA, RHOB, and RHOC comprise a subfamily of RHO GTPase proteins famed for controlling cytoskeletal dynamics. RHO proteins operate downstream of multiple signals emerging from the microenvironment, leading to diverse cell responses, such as proliferation, adhesion, and migration. Therefore, RHO signaling has been centrally placed in the regulation of blood cells. Despite their high homology, unique roles of RHOA, RHOB, and RHOC have been described in hematopoietic cells. In this article, we overview the contribution of RHO proteins in the development and function of each blood cell lineage. Additionally, we highlight the aberrations of the RHO signaling pathways found in hematological malignancies, providing clues for the identification of new therapeutic targets.
Collapse
Affiliation(s)
| | | | - Caroline Santos de Souza
- Department of Clinical and Experimental Oncology, Federal University of São Paulo, São Paulo, Brazil
| | | | | | - Mariana Lazarini
- Department of Clinical and Experimental Oncology, Federal University of São Paulo, São Paulo, Brazil
| |
Collapse
|
|
6
|
Tang Y, Wang H, Zhang Z, Yao Y, Han Y, Wu D. DIAPH1 mutations predict a favorable outcome for de novo MDS. Cancer Lett 2024; 598:217125. [PMID: 39084456 DOI: 10.1016/j.canlet.2024.217125] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/03/2024] [Revised: 07/10/2024] [Accepted: 07/17/2024] [Indexed: 08/02/2024]
Abstract
DIAPH1, a member of the formins family and a Rho effector, was found to be involved in thrombocytopoiesis, and the process of MDS in mice with unknown pathogenesis. In this study, we reported a preliminary study about the heterogeneity in the clinical features and outcomes of DIAPH1 mutations in MDS. DIAPH1 frameshift mutations were identified in 20 out of 88 MDS patients, including 11 frameshift mutations locating at 140892588-141000567 (5q31.3), which causes structure changes at FH1 domain. DIAPH1 mutated cases were correlated with lower megakaryocyte dysplasia in lower-risk patients (IPSS-M score <0) at first diagnosis, and higher megakaryocyte counts pre-transplant. The megakaryopoiesis-related genes: GP1BA and SETBP1 mutation were positively and negatively associated with DIAPH1 mutation, respectively. DIAPH1 mutated cases showed superior overall survival of all patients and low-risk cohorts. In conclusion, we found DIAPH1 frameshift mutations are implicated in megakaryopoiesis of MDS and correlated with superior prognosis.
Collapse
Affiliation(s)
- Yaqiong Tang
- National Clinical Research Center for Hematologic Diseases, Jiangsu Institute of Hematology, The First Affiliated Hospital of Soochow University, Suzhou, China; Institute of Blood and Marrow Transplantation, Collaborative Innovation Center of Hematology, Soochow University, Suzhou, China; Key Laboratory of Thrombosis and Hemostasis of Ministry of Health, Suzhou, China
| | - Hong Wang
- National Clinical Research Center for Hematologic Diseases, Jiangsu Institute of Hematology, The First Affiliated Hospital of Soochow University, Suzhou, China; Institute of Blood and Marrow Transplantation, Collaborative Innovation Center of Hematology, Soochow University, Suzhou, China; Key Laboratory of Thrombosis and Hemostasis of Ministry of Health, Suzhou, China
| | - Ziyan Zhang
- National Clinical Research Center for Hematologic Diseases, Jiangsu Institute of Hematology, The First Affiliated Hospital of Soochow University, Suzhou, China; Institute of Blood and Marrow Transplantation, Collaborative Innovation Center of Hematology, Soochow University, Suzhou, China; Key Laboratory of Thrombosis and Hemostasis of Ministry of Health, Suzhou, China
| | - Yifang Yao
- Suzhou Hongci Hematology Hospital, Suzhou, China
| | - Yue Han
- National Clinical Research Center for Hematologic Diseases, Jiangsu Institute of Hematology, The First Affiliated Hospital of Soochow University, Suzhou, China; Institute of Blood and Marrow Transplantation, Collaborative Innovation Center of Hematology, Soochow University, Suzhou, China; Key Laboratory of Thrombosis and Hemostasis of Ministry of Health, Suzhou, China; State Key Laboratory of Radiation Medicine and Protection, Soochow University, Suzhou, China.
| | - Depei Wu
- National Clinical Research Center for Hematologic Diseases, Jiangsu Institute of Hematology, The First Affiliated Hospital of Soochow University, Suzhou, China; Institute of Blood and Marrow Transplantation, Collaborative Innovation Center of Hematology, Soochow University, Suzhou, China; Key Laboratory of Thrombosis and Hemostasis of Ministry of Health, Suzhou, China; State Key Laboratory of Radiation Medicine and Protection, Soochow University, Suzhou, China.
| |
Collapse
|
|
7
|
Wang M, Zhang B, Jin F, Li G, Cui C, Feng S. Exosomal MicroRNAs: Biomarkers of moyamoya disease and involvement in vascular cytoskeleton reconstruction. Heliyon 2024; 10:e32022. [PMID: 38868045 PMCID: PMC11168404 DOI: 10.1016/j.heliyon.2024.e32022] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/25/2024] [Revised: 05/23/2024] [Accepted: 05/27/2024] [Indexed: 06/14/2024] Open
Abstract
Moyamoya disease currently lacks a suitable method for early clinical screening.This study aimed to identify a simple and feasible clinical screening index by investigating microRNAs carried by peripheral blood exosomes. Experimental subjects participated in venous blood collection, and exosomes were isolated using Exquick-related technology. Sequencing was performed on the extracted exosomal ribonucleic acids (RNAs) to identify differential microRNAs. Verification of the results involved selecting relevant samples from the genetic database. The study successfully pinpointed a potential marker for early screening, hsa-miR-328-3p + hsa-miR-200c-3p carried by peripheral blood exosomes. Enrichment analysis of target genes revealed associations with intercellular junctions, impaired cytoskeletal regulation, and increased fibroblast proliferation, leading to bilateral internal carotid artery neointimal expansion and progressive stenosis. These findings establish the diagnostic value of hsa-miR-328-3p+hsa-miR-200c-3p in screening moyamoya disease, while also contributing to a deeper understanding of its underlying pathophysiology. Significant differences in microRNA expressions derived from peripheral blood exosomes were observed between moyamoya disease patients and control subjects. Consequently, the utilization of peripheral blood exosomes, specifically hsa-miR-328-3p + hsa-miR-200c-3p, holds potential for diagnostic screening purposes.
Collapse
Affiliation(s)
- Mengjie Wang
- Clinical College of Neurology, Neurosurgery and Neurorehabilitation, Tianjin Medical University, Tianjin, 300070, China
- Department of Neurosurgery, Affiliated Hospital of Jining Medical University, No.133, Lotus Road, Jining, Shandong, China
| | - Bin Zhang
- Department of Central Laboratory, Affiliated Hospital of Jining Medical University, No.133, Lotus Road, Jining, Shandong, China
| | - Feng Jin
- Qingdao Central Hospital, University of Health and Rehabilitation Sciences (Qingdao Central Hospital), 266042, Qingdao, Shandong, China
| | - Genhua Li
- Department of Neurosurgery, Affiliated Hospital of Jining Medical University, No.133, Lotus Road, Jining, Shandong, China
| | - Changmeng Cui
- Department of Neurosurgery, Affiliated Hospital of Jining Medical University, No.133, Lotus Road, Jining, Shandong, China
| | - Song Feng
- Department of Neurosurgery, Affiliated Hospital of Jining Medical University, No.133, Lotus Road, Jining, Shandong, China
- Qingdao Central Hospital, University of Health and Rehabilitation Sciences (Qingdao Central Hospital), 266042, Qingdao, Shandong, China
| |
Collapse
|
|
8
|
Li Z, Su M, Xie X, Wang P, Bi H, Li E, Ren K, Dong L, Lv Z, Ma X, Liu Y, Zhao B, Peng Y, Liu J, Liu L, Yang J, Ji P, Mei Y. mDia formins form hetero-oligomers and cooperatively maintain murine hematopoiesis. PLoS Genet 2023; 19:e1011084. [PMID: 38157491 PMCID: PMC10756686 DOI: 10.1371/journal.pgen.1011084] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/12/2023] [Accepted: 11/22/2023] [Indexed: 01/03/2024] Open
Abstract
mDia formin proteins regulate the dynamics and organization of the cytoskeleton through their linear actin nucleation and polymerization activities. We previously showed that mDia1 deficiency leads to aberrant innate immune activation and induces myelodysplasia in a mouse model, and mDia2 regulates enucleation and cytokinesis of erythroblasts and the engraftment of hematopoietic stem and progenitor cells (HSPCs). However, whether and how mDia formins interplay and regulate hematopoiesis under physiological and stress conditions remains unknown. Here, we found that both mDia1 and mDia2 are required for HSPC regeneration under stress, such as serial plating, aging, and reconstitution after myeloid ablation. We showed that mDia1 and mDia2 form hetero-oligomers through the interactions between mDia1 GBD-DID and mDia2 DAD domains. Double knockout of mDia1 and mDia2 in hematopoietic cells synergistically impaired the filamentous actin network and serum response factor-involved transcriptional signaling, which led to declined HSPCs, severe anemia, and significant mortality in neonates and newborn mice. Our data demonstrate the potential roles of mDia hetero-oligomerization and their non-rodent functions in the regulation of HSPCs activity and orchestration of hematopoiesis.
Collapse
Affiliation(s)
- Zhaofeng Li
- Hunan Provincial Key Laboratory of Animal Model and Molecular Medicine, Hunan University, Changsha, China
- School of Biomedical Sciences, Hunan University, Changsha, China
| | - Meng Su
- Hunan Provincial Key Laboratory of Animal Model and Molecular Medicine, Hunan University, Changsha, China
- School of Biomedical Sciences, Hunan University, Changsha, China
| | - Xinshu Xie
- Hunan Provincial Key Laboratory of Animal Model and Molecular Medicine, Hunan University, Changsha, China
- School of Biomedical Sciences, Hunan University, Changsha, China
| | - Pan Wang
- Department of Pathology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, United States of America
| | - Honghao Bi
- Department of Pathology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, United States of America
| | - Ermin Li
- Department of Pathology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, United States of America
| | - Kehan Ren
- Department of Pathology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, United States of America
| | - Lili Dong
- Hunan Provincial Key Laboratory of Animal Model and Molecular Medicine, Hunan University, Changsha, China
- School of Biomedical Sciences, Hunan University, Changsha, China
| | - Zhiyi Lv
- Hunan Provincial Key Laboratory of Animal Model and Molecular Medicine, Hunan University, Changsha, China
- School of Biomedical Sciences, Hunan University, Changsha, China
| | - Xuezhen Ma
- Hunan Provincial Key Laboratory of Animal Model and Molecular Medicine, Hunan University, Changsha, China
- School of Biomedical Sciences, Hunan University, Changsha, China
| | - Yijie Liu
- Department of Pathology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, United States of America
| | - Baobing Zhao
- Department of Pharmacology, School of Pharmaceutical Sciences, Cheeloo College of Medicine, Shandong University, Jinan, Shandong, China
| | - Yuanliang Peng
- Department of Hematology, the Second Xiangya Hospital; Molecular Biology Research Center, School of Life Sciences; Hunan Province Key Laboratory of Basic and Applied Hematology, Central South University; Changsha, China
| | - Jing Liu
- Department of Hematology, the Second Xiangya Hospital; Molecular Biology Research Center, School of Life Sciences; Hunan Province Key Laboratory of Basic and Applied Hematology, Central South University; Changsha, China
| | - Lu Liu
- Hunan Provincial Key Laboratory of Animal Model and Molecular Medicine, Hunan University, Changsha, China
- School of Biomedical Sciences, Hunan University, Changsha, China
| | - Jing Yang
- Department of Pathology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, United States of America
| | - Peng Ji
- Department of Pathology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, United States of America
| | - Yang Mei
- Hunan Provincial Key Laboratory of Animal Model and Molecular Medicine, Hunan University, Changsha, China
- School of Biomedical Sciences, Hunan University, Changsha, China
| |
Collapse
|
|
9
|
Kelliher S, Gamba S, Weiss L, Shen Z, Marchetti M, Schieppati F, Scaife C, Madden S, Bennett K, Fortune A, Maung S, Fay M, Ní Áinle F, Maguire P, Falanga A, Kevane B, Krishnan A. Platelet proteo-transcriptomic profiling validates mediators of thrombosis and proteostasis in patients with myeloproliferative neoplasms. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2023:2023.10.23.563619. [PMID: 37961700 PMCID: PMC10634751 DOI: 10.1101/2023.10.23.563619] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Indexed: 11/15/2023]
Abstract
Patients with chronic Myeloproliferative Neoplasms (MPN) including polycythemia vera (PV) and essential thrombocythemia (ET) exhibit unique clinical features, such as a tendency toward thrombosis and hemorrhage, and risk of disease progression to secondary bone marrow fibrosis and/or acute leukemia. Although an increase in blood cell lineage counts (quantitative features) contribute to these morbid sequelae, the significant qualitative abnormalities of myeloid cells that contribute to vascular risk are not well understood. Here, we address this critical knowledge gap via a comprehensive and untargeted profiling of the platelet proteome in a large (n= 140) cohort of patients (from two independent sites) with an established diagnosis of PV and ET (and complement prior work on the MPN platelet transcriptome from a third site). We discover distinct MPN platelet protein expression and confirm key molecular impairments associated with proteostasis and thrombosis mechanisms of potential relevance to MPN pathology. Specifically, we validate expression of high-priority candidate markers from the platelet transcriptome at the platelet proteome (e.g., calreticulin (CALR), Fc gamma receptor (FcγRIIA) and galectin-1 (LGALS1) pointing to their likely significance in the proinflammatory, prothrombotic and profibrotic phenotypes in patients with MPN. Together, our proteo-transcriptomic study identifies the peripherally-derived platelet molecular profile as a potential window into MPN pathophysiology and demonstrates the value of integrative multi-omic approaches in gaining a better understanding of the complex molecular dynamics of disease.
Collapse
Affiliation(s)
- Sarah Kelliher
- School of Medicine, University College Dublin, Dublin, Ireland
- Stanford University School of Medicine, Stanford University, Stanford, CA, USA
- Department of Haematology, Mater Misericordiae University Hospital, Dublin, Ireland
- UCD Conway SPHERE Research Group, University College Dublin, Dublin, Ireland
| | - Sara Gamba
- Department of Immunohematology and Transfusion Medicine, Hospital Papa Giovanni XXIII, Bergamo, Italy
| | - Luisa Weiss
- UCD Conway SPHERE Research Group, University College Dublin, Dublin, Ireland
- School of Biomolecular and Biomedical Science, University College Dublin, Dublin, Ireland
| | - Zhu Shen
- Stanford University School of Medicine, Stanford University, Stanford, CA, USA
| | - Marina Marchetti
- Department of Immunohematology and Transfusion Medicine, Hospital Papa Giovanni XXIII, Bergamo, Italy
| | - Francesca Schieppati
- Department of Immunohematology and Transfusion Medicine, Hospital Papa Giovanni XXIII, Bergamo, Italy
| | - Caitriona Scaife
- UCD Conway Institute for Biomolecular and Biomedical Research, University College Dublin, Dublin, Ireland
| | - Stephen Madden
- Data Science Centre, Royal College of Surgeons in Ireland, Dublin, Ireland
| | - Kathleen Bennett
- School of Population Health, RCSI University of Medicine and Health Sciences, Dublin, Ireland
| | - Anne Fortune
- School of Medicine, University College Dublin, Dublin, Ireland
- Department of Haematology, Mater Misericordiae University Hospital, Dublin, Ireland
| | - Su Maung
- School of Medicine, University College Dublin, Dublin, Ireland
- Department of Haematology, Mater Misericordiae University Hospital, Dublin, Ireland
| | - Michael Fay
- School of Medicine, University College Dublin, Dublin, Ireland
- Department of Haematology, Mater Misericordiae University Hospital, Dublin, Ireland
| | - Fionnuala Ní Áinle
- School of Medicine, University College Dublin, Dublin, Ireland
- Department of Haematology, Mater Misericordiae University Hospital, Dublin, Ireland
- UCD Conway SPHERE Research Group, University College Dublin, Dublin, Ireland
- School of Medicine, Royal College of Surgeons in Ireland
| | - Patricia Maguire
- UCD Conway SPHERE Research Group, University College Dublin, Dublin, Ireland
- School of Biomolecular and Biomedical Science, University College Dublin, Dublin, Ireland
- UCD Institute for Discovery, University College Dublin, Dublin, Ireland
| | - Anna Falanga
- Department of Immunohematology and Transfusion Medicine, Hospital Papa Giovanni XXIII, Bergamo, Italy
- University of Milano-Bicocca, Department of Medicine and Surgery, Monza, Italy
| | - Barry Kevane
- School of Medicine, University College Dublin, Dublin, Ireland
- Department of Haematology, Mater Misericordiae University Hospital, Dublin, Ireland
- UCD Conway SPHERE Research Group, University College Dublin, Dublin, Ireland
| | - Anandi Krishnan
- Stanford University School of Medicine, Stanford University, Stanford, CA, USA
- Rutgers University, Piscataway, NJ
- Stanford Cancer Institute, Stanford, CA, USA
| |
Collapse
|
|
10
|
He S, Hao X, Liu Z, Wang Y, Zhang J, Wang X, Di F, Wang R, Zhao Y. Association between DIAPH1 variant and posterior circulation involvement with Moyamoya disease. Sci Rep 2023; 13:10732. [PMID: 37400591 DOI: 10.1038/s41598-023-37665-1] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/11/2023] [Accepted: 06/25/2023] [Indexed: 07/05/2023] Open
Abstract
Moyamoya disease (MMD) is a chronic and progressive cerebrovascular stenosis or occlusive disease that occurs near Willis blood vessels. The aim of this study was to investigate the mutation of DIAPH1 in Asian population, and to compare the angiographic features of MMD patients with and without the mutation of the DIAPH1 gene. Blood samples of 50 patients with MMD were collected, and DIAPH1 gene mutation was detected. The angiographic involvement of the posterior cerebral artery was compared between the mutant group and the non-mutant group. The independent risk factors of posterior cerebral artery involvement were determined by multivariate logistic regression analysis. DIAPH1 gene mutation was detected in 9 (18%) of 50 patients, including 7 synonymous mutations and 2 missense mutations. However, the incidence of posterior cerebral artery involvement in mutation positive group was very higher than that in mutation negative group (77.8% versus 12%; p = 0.001). There is an association between DIAPH1 mutation and PCA involvement (odds ratio 29.483, 95% confidence interval 3.920-221.736; p = 0.001). DIAPH1 gene mutation is not a major genetic risk gene for Asian patients with moyamoya disease but may play an important role in the involvement of posterior cerebral artery.
Collapse
Affiliation(s)
- Shihao He
- Department of Neurosurgery, Beijing Tiantan Hospital, Capital Medical University, Beijing, 100070, China
- Department of Neurosurgery, Stanford University School of Medicine, 300 Pasteur Drive (R281), Stanford, CA, 94305-5327, USA
| | - Xiaokuan Hao
- Department of Neurosurgery, Beijing Tiantan Hospital, Capital Medical University, Beijing, 100070, China
| | - Ziqi Liu
- Department of Neurosurgery, Beijing Tiantan Hospital, Capital Medical University, Beijing, 100070, China
| | - Yanru Wang
- Department of Neurosurgery, Beijing Tiantan Hospital, Capital Medical University, Beijing, 100070, China
| | - Junze Zhang
- Department of Neurosurgery, Beijing Tiantan Hospital, Capital Medical University, Beijing, 100070, China
| | - Xilong Wang
- Department of Neurosurgery, Beijing Tiantan Hospital, Capital Medical University, Beijing, 100070, China
| | - Fei Di
- Department of Neurosurgery, The Affiliated Children's Hospital, Capital Institute of Pediatrics, Beijing, 100020, China
| | - Rong Wang
- Department of Neurosurgery, Beijing Tiantan Hospital, Capital Medical University, Beijing, 100070, China.
- China National Clinical Research Center for Neurological Diseases, Beijing, 100070, China.
- Center of Stroke, Beijing Institute for Brain Disorders, Beijing, 100069, China.
| | - Yuanli Zhao
- Department of Neurosurgery, Beijing Tiantan Hospital, Capital Medical University, Beijing, 100070, China.
- China National Clinical Research Center for Neurological Diseases, Beijing, 100070, China.
- Center of Stroke, Beijing Institute for Brain Disorders, Beijing, 100069, China.
- Beijing Institute of Brain Disorders, Collaborative Innovation Center for Brain Disorders, Capital Medical University, Beijing, 100069, China.
| |
Collapse
|
|
11
|
Yang S, Tang X, Wang L, Ni C, Wu Y, Zhou L, Zeng Y, Zhao C, Wu A, Wang Q, Xu X, Wang Y, Chen R, Zhang X, Zou L, Huang X, Wu J. Targeting TLR2/Rac1/cdc42/JNK Pathway to Reveal That Ruxolitinib Promotes Thrombocytopoiesis. Int J Mol Sci 2022; 23:16137. [PMID: 36555781 PMCID: PMC9787584 DOI: 10.3390/ijms232416137] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/18/2022] [Revised: 12/13/2022] [Accepted: 12/15/2022] [Indexed: 12/23/2022] Open
Abstract
BACKGROUND Thrombocytopenia has long been considered an important complication of chemotherapy and radiotherapy, which severely limits the effectiveness of cancer treatment and the overall survival of patients. However, clinical treatment options are extremely limited so far. Ruxolitinib is a potential candidate. METHODS The impact of ruxolitinib on the differentiation and maturation of K562 and Meg-01 cells megakaryocytes (MKs) was examined by flow cytometry, Giemsa and Phalloidin staining. A mouse model of radiation-injured thrombocytopenia (RIT) was employed to evaluate the action of ruxolitinib on thrombocytopoiesis. Network pharmacology, molecular docking, drug affinity responsive target stability assay (DARTS), RNA sequencing, protein blotting and immunofluorescence analysis were applied to explore the targets and mechanisms of action of ruxolitinib. RESULTS Ruxolitinib can stimulate MK differentiation and maturation in a dose-dependent manner and accelerates recovery of MKs and thrombocytopoiesis in RIT mice. Biological targeting analysis showed that ruxolitinib binds directly to Toll Like Receptor 2 (TLR2) to activate Rac1/cdc42/JNK, and this action was shown to be blocked by C29, a specific inhibitor of TLR2. CONCLUSIONS Ruxolitinib was first identified to facilitate MK differentiation and thrombocytopoiesis, which may alleviate RIT. The potential mechanism of ruxolitinib was to promote MK differentiation via activating the Rac1/cdc42/JNK pathway through binding to TLR2.
Collapse
Affiliation(s)
- Shuo Yang
- School of Pharmacy, Southwest Medical University, Luzhou 646000, China
| | - Xiaoqin Tang
- School of Pharmacy, Southwest Medical University, Luzhou 646000, China
| | - Long Wang
- School of Pharmacy, Southwest Medical University, Luzhou 646000, China
| | - Chengyang Ni
- School of Pharmacy, Southwest Medical University, Luzhou 646000, China
| | - Yuesong Wu
- School of Pharmacy, Southwest Medical University, Luzhou 646000, China
| | - Ling Zhou
- School of Pharmacy, Southwest Medical University, Luzhou 646000, China
| | - Yueying Zeng
- School of Pharmacy, Southwest Medical University, Luzhou 646000, China
| | - Chunling Zhao
- School of Basic Medical Sciences, Southwest Medical University, Luzhou 646000, China
| | - Anguo Wu
- School of Pharmacy, Southwest Medical University, Luzhou 646000, China
| | - Qiaozhi Wang
- School of Basic Medical Sciences, Southwest Medical University, Luzhou 646000, China
| | - Xiyan Xu
- School of Basic Medical Sciences, Southwest Medical University, Luzhou 646000, China
| | - Yiwei Wang
- School of Basic Medical Sciences, Southwest Medical University, Luzhou 646000, China
| | - Rong Chen
- School of Basic Medical Sciences, Southwest Medical University, Luzhou 646000, China
| | - Xiao Zhang
- School of Basic Medical Sciences, Southwest Medical University, Luzhou 646000, China
| | - Lile Zou
- School of Basic Medical Sciences, Southwest Medical University, Luzhou 646000, China
| | - Xinwu Huang
- School of Pharmacy, Southwest Medical University, Luzhou 646000, China
| | - Jianming Wu
- School of Basic Medical Sciences, Southwest Medical University, Luzhou 646000, China
- Education Ministry Key Laboratory of Medical Electrophysiology, Southwest Medical University, Luzhou 646000, China
| |
Collapse
|
|
12
|
Englert M, Aurbach K, Becker IC, Gerber A, Heib T, Wackerbarth LM, Kusch C, Mott K, Araujo GHM, Baig AA, Dütting S, Knaus UG, Stigloher C, Schulze H, Nieswandt B, Pleines I, Nagy Z. Impaired microtubule dynamics contribute to microthrombocytopenia in RhoB-deficient mice. Blood Adv 2022; 6:5184-5197. [PMID: 35819450 PMCID: PMC9631634 DOI: 10.1182/bloodadvances.2021006545] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/05/2021] [Accepted: 06/30/2022] [Indexed: 11/30/2022] Open
Abstract
Megakaryocytes are large cells in the bone marrow that give rise to blood platelets. Platelet biogenesis involves megakaryocyte maturation, the localization of the mature cells in close proximity to bone marrow sinusoids, and the formation of protrusions, which are elongated and shed within the circulation. Rho GTPases play important roles in platelet biogenesis and function. RhoA-deficient mice display macrothrombocytopenia and a striking mislocalization of megakaryocytes into bone marrow sinusoids and a specific defect in G-protein signaling in platelets. However, the role of the closely related protein RhoB in megakaryocytes or platelets remains unknown. In this study, we show that, in contrast to RhoA deficiency, genetic ablation of RhoB in mice results in microthrombocytopenia (decreased platelet count and size). RhoB-deficient platelets displayed mild functional defects predominantly upon induction of the collagen/glycoprotein VI pathway. Megakaryocyte maturation and localization within the bone marrow, as well as actin dynamics, were not affected in the absence of RhoB. However, in vitro-generated proplatelets revealed pronouncedly impaired microtubule organization. Furthermore, RhoB-deficient platelets and megakaryocytes displayed selective defects in microtubule dynamics/stability, correlating with reduced levels of acetylated α-tubulin. Our findings imply that the reduction of this tubulin posttranslational modification results in impaired microtubule dynamics, which might contribute to microthrombocytopenia in RhoB-deficient mice. Importantly, we demonstrate that RhoA and RhoB are localized differently and have selective, nonredundant functions in the megakaryocyte lineage.
Collapse
Affiliation(s)
- Maximilian Englert
- Institute of Experimental Biomedicine, University Hospital, University of Würzburg, Würzburg, Germany
- Rudolf Virchow Center for Integrative and Translational Bioimaging, University of Würzburg, Würzburg, Germany
| | - Katja Aurbach
- Institute of Experimental Biomedicine, University Hospital, University of Würzburg, Würzburg, Germany
- Rudolf Virchow Center for Integrative and Translational Bioimaging, University of Würzburg, Würzburg, Germany
| | - Isabelle C. Becker
- Institute of Experimental Biomedicine, University Hospital, University of Würzburg, Würzburg, Germany
- Rudolf Virchow Center for Integrative and Translational Bioimaging, University of Würzburg, Würzburg, Germany
| | - Annika Gerber
- Institute of Experimental Biomedicine, University Hospital, University of Würzburg, Würzburg, Germany
- Rudolf Virchow Center for Integrative and Translational Bioimaging, University of Würzburg, Würzburg, Germany
| | - Tobias Heib
- Institute of Experimental Biomedicine, University Hospital, University of Würzburg, Würzburg, Germany
- Rudolf Virchow Center for Integrative and Translational Bioimaging, University of Würzburg, Würzburg, Germany
| | - Lou M. Wackerbarth
- Institute of Experimental Biomedicine, University Hospital, University of Würzburg, Würzburg, Germany
- Rudolf Virchow Center for Integrative and Translational Bioimaging, University of Würzburg, Würzburg, Germany
| | - Charly Kusch
- Institute of Experimental Biomedicine, University Hospital, University of Würzburg, Würzburg, Germany
- Rudolf Virchow Center for Integrative and Translational Bioimaging, University of Würzburg, Würzburg, Germany
| | - Kristina Mott
- Institute of Experimental Biomedicine, University Hospital, University of Würzburg, Würzburg, Germany
| | - Gabriel H. M. Araujo
- Institute of Experimental Biomedicine, University Hospital, University of Würzburg, Würzburg, Germany
- Rudolf Virchow Center for Integrative and Translational Bioimaging, University of Würzburg, Würzburg, Germany
| | - Ayesha A. Baig
- Institute of Experimental Biomedicine, University Hospital, University of Würzburg, Würzburg, Germany
- Rudolf Virchow Center for Integrative and Translational Bioimaging, University of Würzburg, Würzburg, Germany
| | - Sebastian Dütting
- Institute of Experimental Biomedicine, University Hospital, University of Würzburg, Würzburg, Germany
- Rudolf Virchow Center for Integrative and Translational Bioimaging, University of Würzburg, Würzburg, Germany
| | - Ulla G. Knaus
- Conway Institute, School of Medicine, University College Dublin, Dublin, Ireland; and
| | | | - Harald Schulze
- Institute of Experimental Biomedicine, University Hospital, University of Würzburg, Würzburg, Germany
| | - Bernhard Nieswandt
- Institute of Experimental Biomedicine, University Hospital, University of Würzburg, Würzburg, Germany
- Rudolf Virchow Center for Integrative and Translational Bioimaging, University of Würzburg, Würzburg, Germany
| | - Irina Pleines
- Institute of Experimental Biomedicine, University Hospital, University of Würzburg, Würzburg, Germany
- Rudolf Virchow Center for Integrative and Translational Bioimaging, University of Würzburg, Würzburg, Germany
| | - Zoltan Nagy
- Institute of Experimental Biomedicine, University Hospital, University of Würzburg, Würzburg, Germany
- Rudolf Virchow Center for Integrative and Translational Bioimaging, University of Würzburg, Würzburg, Germany
| |
Collapse
|
|
13
|
Singh AA, Kharwar A, Dandekar MP. A Review on Preclinical Models of Ischemic Stroke: Insights Into the Pathomechanisms and New Treatment Strategies. Curr Neuropharmacol 2022; 20:1667-1686. [PMID: 34493185 PMCID: PMC9881062 DOI: 10.2174/1570159x19666210907092928] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/20/2021] [Revised: 07/21/2021] [Accepted: 08/26/2021] [Indexed: 11/22/2022] Open
Abstract
BACKGROUND Stroke is a serious neurovascular problem and the leading cause of disability and death worldwide. The disrupted demand to supply ratio of blood and glucose during cerebral ischemia develops hypoxic shock, and subsequently necrotic neuronal death in the affected regions. Multiple causal factors like age, sex, race, genetics, diet, and lifestyle play an important role in the occurrence as well as progression of post-stroke deleterious events. These biological and environmental factors may be contributed to vasculature variable architecture and abnormal neuronal activity. Since recombinant tissue plasminogen activator is the only clinically effective clot bursting drug, there is a huge unmet medical need for newer therapies for the treatment of stroke. Innumerous therapeutic interventions have shown promise in the experimental models of stroke but failed to translate it into clinical counterparts. METHODS Original publications regarding pathophysiology, preclinical experimental models, new targets and therapies targeting ischemic stroke have been reviewed since the 1970s. RESULTS We highlighted the critical underlying pathophysiological mechanisms of cerebral stroke and preclinical stroke models. We discuss the strengths and caveats of widely used ischemic stroke models, and commented on the potential translational problems. We also describe the new emerging treatment strategies, including stem cell therapy, neurotrophic factors and gut microbiome-based therapy for the management of post-stroke consequences. CONCLUSION There are still many inter-linked pathophysiological alterations with regards to stroke, animal models need not necessarily mimic the same conditions of stroke pathology and newer targets and therapies are the need of the hour in stroke research.
Collapse
Affiliation(s)
- Aditya A. Singh
- Department of Pharmacology and Toxicology, National Institute of Pharmaceutical Education and Research (NIPER), Hyderabad, Balanagar, TS 500037, India
| | - Akash Kharwar
- Department of Pharmacology and Toxicology, National Institute of Pharmaceutical Education and Research (NIPER), Hyderabad, Balanagar, TS 500037, India
| | - Manoj P. Dandekar
- Department of Pharmacology and Toxicology, National Institute of Pharmaceutical Education and Research (NIPER), Hyderabad, Balanagar, TS 500037, India,Address correspondence to this author at the Department of Pharmacology and Toxicology, National Institute of Pharmaceutical Education and Research (NIPER), Hyderabad, Balanagar, TS 500037, India; Tel: +91-40-23074750; E-mail:
| |
Collapse
|
|
14
|
Tilburg J, Becker IC, Italiano JE. Don't you forget about me(gakaryocytes). Blood 2022; 139:3245-3254. [PMID: 34582554 PMCID: PMC9164737 DOI: 10.1182/blood.2020009302] [Citation(s) in RCA: 26] [Impact Index Per Article: 8.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/23/2021] [Accepted: 09/08/2021] [Indexed: 11/20/2022] Open
Abstract
Platelets (small, anucleate cell fragments) derive from large precursor cells, megakaryocytes (MKs), that reside in the bone marrow. MKs emerge from hematopoietic stem cells in a complex differentiation process that involves cytoplasmic maturation, including the formation of the demarcation membrane system, and polyploidization. The main function of MKs is the generation of platelets, which predominantly occurs through the release of long, microtubule-rich proplatelets into vessel sinusoids. However, the idea of a 1-dimensional role of MKs as platelet precursors is currently being questioned because of advances in high-resolution microscopy and single-cell omics. On the one hand, recent findings suggest that proplatelet formation from bone marrow-derived MKs is not the only mechanism of platelet production, but that it may also occur through budding of the plasma membrane and in distant organs such as lung or liver. On the other hand, novel evidence suggests that MKs not only maintain physiological platelet levels but further contribute to bone marrow homeostasis through the release of extracellular vesicles or cytokines, such as transforming growth factor β1 or platelet factor 4. The notion of multitasking MKs was reinforced in recent studies by using single-cell RNA sequencing approaches on MKs derived from adult and fetal bone marrow and lungs, leading to the identification of different MK subsets that appeared to exhibit immunomodulatory or secretory roles. In the following article, novel insights into the mechanisms leading to proplatelet formation in vitro and in vivo will be reviewed and the hypothesis of MKs as immunoregulatory cells will be critically discussed.
Collapse
Affiliation(s)
- Julia Tilburg
- Vascular Biology Program, Boston Children's Hospital, Boston, MA
| | | | | |
Collapse
|
|
15
|
Kimmerlin Q, Strassel C, Eckly A, Lanza F. The tubulin code in platelet biogenesis. Semin Cell Dev Biol 2022; 137:63-73. [PMID: 35148939 DOI: 10.1016/j.semcdb.2022.01.010] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/18/2021] [Revised: 01/12/2022] [Accepted: 01/31/2022] [Indexed: 11/28/2022]
Abstract
Blood platelets are small non-nucleated cellular fragments that prevent and stop hemorrhages. They are produced in the bone marrow by megakaryocytes through megakaryopoiesis. This intricate process involves profound microtubule rearrangements culminating in the formation of a unique circular sub-membranous microtubule array, the marginal band, which supports the typical disc-shaped morphology of platelets. Mechanistically, these processes are thought to be controlled by a specific tubulin code. In this review, we summarize the current knowledge on the key isotypes, notably β1-, α4A- and α8-tubulin, and putative post-translational modifications, involved in platelet and marginal band formation. Additionally, we provide a provisional list of microtubule-associated proteins (MAPs) involved in these processes and a survey of tubulin variants identified in patients presenting defective platelet production. A comprehensive characterization of the platelet tubulin code and the identification of essential MAPs may be expected in the near future to shed new light on a very specialized microtubule assembly process with applications in platelet diseases and transfusion.
Collapse
Affiliation(s)
- Quentin Kimmerlin
- Université de Strasbourg, INSERM, EFS Grand-Est, BPPS UMR-S1255, FMTS, Strasbourg, France.
| | - Catherine Strassel
- Université de Strasbourg, INSERM, EFS Grand-Est, BPPS UMR-S1255, FMTS, Strasbourg, France.
| | - Anita Eckly
- Université de Strasbourg, INSERM, EFS Grand-Est, BPPS UMR-S1255, FMTS, Strasbourg, France.
| | - François Lanza
- Université de Strasbourg, INSERM, EFS Grand-Est, BPPS UMR-S1255, FMTS, Strasbourg, France.
| |
Collapse
|
|
16
|
Flat W, Borowski S, Paraschiakos T, Blechner C, Windhorst S. DIAPH1 facilitates paclitaxel-mediated cytotoxicity of ovarian cancer cells. Biochem Pharmacol 2021; 197:114898. [PMID: 34968485 DOI: 10.1016/j.bcp.2021.114898] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/07/2021] [Revised: 12/14/2021] [Accepted: 12/14/2021] [Indexed: 11/17/2022]
Abstract
The chemotherapeutic agent paclitaxel (PTX) selectively binds to and stabilizes microtubule (MTs). Also, the activated formin Diaphanous Related Formin 1 (DIAPH1) binds to MTs and increases its stability. In a recent study, we found that high DIAPH1 levels correlated with increased survival of ovarian cancer (Ovca) patients. A possible explanation for this finding is that Ovca cells with high DIAPH1 levels are more sensitive to PTX. To examine this assumption, in this study the effect of DIAPH1 depletion on PTX-mediated cytotoxicity of OVCAR8 and OAW42 cells was analyzed. Our data showed that down-regulation of DIAPH1 expression decreased PTX sensitivity in both cell lines by reducing apoptosis or necrosis. Analysis of MT stability by Western blotting revealed a decreased concentration of stable, detyrosinated MTs in PTX-treated DIAPH1 knock-down compared to control cells. Also, in fixed metaphase cells the level of stable, detyrosinated spindle MTs decreased in cells with reduced DIAPH1 expression. In vitro analysis with recombinant DIAPH1 protein showed that PTX and DIAPH1 exhibited additive effects on MT-polymerization, showing that also in a cell-free system DIAPH1 increased the effect of PTX on MT-stability. Together, our data strongly indicate that DIAPH1 increases the response of Ovca cells to PTX by enhancing PTX-mediated MT-stability.
Collapse
Affiliation(s)
- Wilhelm Flat
- Department of Biochemistry and Signal Transduction, University Medical Center Hamburg-Eppendorf, Martinistrasse 52, D-20246 Hamburg, Germany
| | - Sarah Borowski
- Department of Biochemistry and Signal Transduction, University Medical Center Hamburg-Eppendorf, Martinistrasse 52, D-20246 Hamburg, Germany
| | - Themistoklis Paraschiakos
- Department of Biochemistry and Signal Transduction, University Medical Center Hamburg-Eppendorf, Martinistrasse 52, D-20246 Hamburg, Germany
| | - Christine Blechner
- Department of Biochemistry and Signal Transduction, University Medical Center Hamburg-Eppendorf, Martinistrasse 52, D-20246 Hamburg, Germany
| | - Sabine Windhorst
- Department of Biochemistry and Signal Transduction, University Medical Center Hamburg-Eppendorf, Martinistrasse 52, D-20246 Hamburg, Germany.
| |
Collapse
|
|
17
|
Abstract
Almost 25 years have passed since a mutation of a formin gene, DIAPH1, was identified as being responsible for a human inherited disorder: a form of sensorineural hearing loss. Since then, our knowledge of the links between formins and disease has deepened considerably. Mutations of DIAPH1 and six other formin genes (DAAM2, DIAPH2, DIAPH3, FMN2, INF2 and FHOD3) have been identified as the genetic cause of a variety of inherited human disorders, including intellectual disability, renal disease, peripheral neuropathy, thrombocytopenia, primary ovarian insufficiency, hearing loss and cardiomyopathy. In addition, alterations in formin genes have been associated with a variety of pathological conditions, including developmental defects affecting the heart, nervous system and kidney, aging-related diseases, and cancer. This review summarizes the most recent discoveries about the involvement of formin alterations in monogenic disorders and other human pathological conditions, especially cancer, with which they have been associated. In vitro results and experiments in modified animal models are discussed. Finally, we outline the directions for future research in this field.
Collapse
Affiliation(s)
| | - Miguel A. Alonso
- Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas, Universidad Autónoma de Madrid, 28049 Madrid, Spain;
| |
Collapse
|
|
18
|
Ruiz HH, Nguyen A, Wang C, He L, Li H, Hallowell P, McNamara C, Schmidt AM. AGE/RAGE/DIAPH1 axis is associated with immunometabolic markers and risk of insulin resistance in subcutaneous but not omental adipose tissue in human obesity. Int J Obes (Lond) 2021; 45:2083-2094. [PMID: 34103691 PMCID: PMC8380543 DOI: 10.1038/s41366-021-00878-3] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/08/2020] [Revised: 05/19/2021] [Accepted: 05/27/2021] [Indexed: 02/05/2023]
Abstract
BACKGROUND/OBJECTIVES The incidence of obesity continues to increase worldwide and while the underlying pathogenesis remains largely unknown, nutrient excess, manifested by "Westernization" of the diet and reduced physical activity have been proposed as key contributing factors. Western-style diets, in addition to higher caloric load, are characterized by excess of advanced glycation end products (AGEs), which have been linked to the pathophysiology of obesity and related cardiometabolic disorders. AGEs can be "trapped" in adipose tissue, even in the absence of diabetes, in part due to higher expression of the receptor for AGEs (RAGE) and/or decreased detoxification by the endogenous glyoxalase (GLO) system, where they may promote insulin resistance. It is unknown whether the expression levels of genes linked to the RAGE axis, including AGER (the gene encoding RAGE), Diaphanous 1 (DIAPH1), the cytoplasmic domain binding partner of RAGE that contributes to RAGE signaling, and GLO1 are differentially regulated by the degree of obesity and/or how these relate to inflammatory and adipocyte markers and their metabolic consequences. SUBJECTS/METHODS We sought to answer this question by analyzing gene expression patterns of markers of the AGE/RAGE/DIAPH1 signaling axis in abdominal subcutaneous (SAT) and omental (OAT) adipose tissue from obese and morbidly obese subjects. RESULTS In SAT, but not OAT, expression of AGER was significantly correlated with that of DIAPH1 (n = 16; [Formula: see text], [0.260, 1.177]; q = 0.008) and GLO1 (n = 16; [Formula: see text], [0.364, 1.182]; q = 0.004). Furthermore, in SAT, but not OAT, regression analyses revealed that the expression pattern of genes in the AGE/RAGE/DIAPH1 axis is strongly and positively associated with that of inflammatory and adipogenic markers. Remarkably, particularly in SAT, not OAT, the expression of AGER positively and significantly correlated with HOMA-IR (n = 14; [Formula: see text], [0.338, 1.249]; q = 0.018). CONCLUSIONS These observations suggest associations of the AGE/RAGE/DIAPH1 axis in the immunometabolic pathophysiology of obesity and insulin resistance, driven, at least in part, through expression and activity of this axis in SAT.
Collapse
Affiliation(s)
- Henry H Ruiz
- Diabetes Research Program, Division of Endocrinology, Diabetes and Metabolism, Department of Medicine, New York University Grossman School of Medicine, New York, NY, USA.
| | - Anh Nguyen
- Cardiovascular Division, Department of Medicine and Cardiovascular Research Center, University of Virginia, Charlottesville, VA, USA
| | - Chan Wang
- Division of Biostatistics, Department of Population Health, New York University Grossman School of Medicine, New York, NY, USA
| | - Linchen He
- Division of Biostatistics, Department of Population Health, New York University Grossman School of Medicine, New York, NY, USA
| | - Huilin Li
- Division of Biostatistics, Department of Population Health, New York University Grossman School of Medicine, New York, NY, USA
| | - Peter Hallowell
- General Surgery Division, Department of Surgery, University of Virginia, Charlottesville, VA, USA
| | - Coleen McNamara
- Cardiovascular Division, Department of Medicine and Cardiovascular Research Center, University of Virginia, Charlottesville, VA, USA
| | - Ann Marie Schmidt
- Diabetes Research Program, Division of Endocrinology, Diabetes and Metabolism, Department of Medicine, New York University Grossman School of Medicine, New York, NY, USA
| |
Collapse
|
|
19
|
Mbiandjeu S, Balduini A, Malara A. Megakaryocyte Cytoskeletal Proteins in Platelet Biogenesis and Diseases. Thromb Haemost 2021; 122:666-678. [PMID: 34218430 DOI: 10.1055/s-0041-1731717] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/20/2022]
Abstract
Thrombopoiesis governs the formation of blood platelets in bone marrow by converting megakaryocytes into long, branched proplatelets on which individual platelets are assembled. The megakaryocyte cytoskeleton responds to multiple microenvironmental cues, including chemical and mechanical stimuli, sustaining the platelet shedding. During the megakaryocyte's life cycle, cytoskeletal networks organize cell shape and content, connect them physically and biochemically to the bone marrow vascular niche, and enable the release of platelets into the bloodstream. While the basic building blocks of the cytoskeleton have been studied extensively, new sets of cytoskeleton regulators have emerged as critical components of the dynamic protein network that supports platelet production. Understanding how the interaction of individual molecules of the cytoskeleton governs megakaryocyte behavior is essential to improve knowledge of platelet biogenesis and develop new therapeutic strategies for inherited thrombocytopenias caused by alterations in the cytoskeletal genes.
Collapse
Affiliation(s)
- Serge Mbiandjeu
- Department of Molecular Medicine, University of Pavia, Pavia, Italy
| | | | | |
Collapse
|
|
20
|
Mitochondrial dynamics and reactive oxygen species initiate thrombopoiesis from mature megakaryocytes. Blood Adv 2021; 5:1706-1718. [PMID: 33720340 DOI: 10.1182/bloodadvances.2020002847] [Citation(s) in RCA: 13] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/02/2020] [Accepted: 01/20/2021] [Indexed: 02/07/2023] Open
Abstract
Blood platelets are essential for controlling hemostasis. They are released by megakaryocytes (MKs) located in the bone marrow, upon extension of cytoplasmic protrusions into the lumen of bone marrow sinusoids. Their number increases in postpulmonary capillaries, suggesting a role for oxygen gradient in thrombopoiesis (ie, platelet biogenesis). In this study, we show that initiation of thrombopoiesis from human mature MKs was enhanced under hyperoxia or during pro-oxidant treatments, whereas antioxidants dampened it. Quenching mitochondrial reactive oxygen species (mtROS) with MitoTEMPO decreased thrombopoiesis, whereas genetically enhancing mtROS by deacetylation-null sirtuin-3 expression increased it. Blocking cytosolic ROS production by NOX inhibitors had no impact. Classification according to the cell roundness index delineated 3 stages of thrombopoiesis in mature MKs. Early-stage round MKs exhibited the highest index, which correlated with low mtROS levels, a mitochondrial tubular network, and the mitochondrial recruitment of the fission activator Drp1. Intermediate MKs at the onset of thrombopoiesis showed high mtROS levels and small, well-delineated mitochondria. Terminal MKs showed the lowest roundness index and long proplatelet extensions. Inhibiting Drp1-dependent mitochondrial fission of mature MKs by Mdivi-1 favored a tubular mitochondrial network and lowered both mtROS levels and intermediate MKs proportion, whereas enhancing Drp1 activity genetically had opposite effects. Reciprocally, quenching mtROS limited mitochondrial fission in round MKs. These data demonstrate a functional coupling between ROS and mitochondrial fission in MKs, which is crucial for the onset of thrombopoiesis. They provide new molecular cues that control initiation of platelet biogenesis and may help elucidate some unexplained thrombocytopenia.
Collapse
|
|
21
|
Actin/microtubule crosstalk during platelet biogenesis in mice is critically regulated by Twinfilin1 and Cofilin1. Blood Adv 2021; 4:2124-2134. [PMID: 32407474 DOI: 10.1182/bloodadvances.2019001303] [Citation(s) in RCA: 17] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/03/2019] [Accepted: 03/13/2020] [Indexed: 01/24/2023] Open
Abstract
Rearrangements of the microtubule (MT) and actin cytoskeleton are pivotal for platelet biogenesis. Hence, defects in actin- or MT-regulatory proteins are associated with platelet disorders in humans and mice. Previous studies in mice revealed that loss of the actin-depolymerizing factor homology (ADF-H) protein Cofilin1 (Cof1) in megakaryocytes (MKs) results in a moderate macrothrombocytopenia but normal MK numbers, whereas deficiency in another ADF-H protein, Twinfilin1 (Twf1), does not affect platelet production or function. However, recent studies in yeast have indicated a critical synergism between Twf1 and Cof1 in the regulation of actin dynamics. We therefore investigated platelet biogenesis and function in mice lacking both Twf1 and Cof1 in the MK lineage. In contrast to single deficiency in either protein, Twf1/Cof1 double deficiency (DKO) resulted in a severe macrothrombocytopenia and dramatically increased MK numbers in bone marrow and spleen. DKO MKs exhibited defective proplatelet formation in vitro and in vivo as well as impaired spreading and altered assembly of podosome-like structures on collagen and fibrinogen in vitro. These defects were associated with aberrant F-actin accumulation and, remarkably, the formation of hyperstable MT, which appears to be caused by dysregulation of the actin- and MT-binding proteins mDia1 and adenomatous polyposis coli. Surprisingly, the mild functional defects described for Cof1-deficient platelets were only slightly aggravated in DKO platelets suggesting that both proteins are largely dispensable for platelet function in the peripheral blood. In summary, these findings reveal critical redundant functions of Cof1 and Twf1 in ensuring balanced actin/microtubule crosstalk during thrombopoiesis in mice and possibly humans.
Collapse
|
|
22
|
Heib T, Hermanns HM, Manukjan G, Englert M, Kusch C, Becker IC, Gerber A, Wackerbarth LM, Burkard P, Dandekar T, Balkenhol J, Jahn D, Beck S, Meub M, Dütting S, Stigloher C, Sauer M, Cherpokova D, Schulze H, Brakebusch C, Nieswandt B, Nagy Z, Pleines I. RhoA/Cdc42 signaling drives cytoplasmic maturation but not endomitosis in megakaryocytes. Cell Rep 2021; 35:109102. [PMID: 33979620 DOI: 10.1016/j.celrep.2021.109102] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/27/2020] [Revised: 02/20/2021] [Accepted: 04/18/2021] [Indexed: 12/15/2022] Open
Abstract
Megakaryocytes (MKs), the precursors of blood platelets, are large, polyploid cells residing mainly in the bone marrow. We have previously shown that balanced signaling of the Rho GTPases RhoA and Cdc42 is critical for correct MK localization at bone marrow sinusoids in vivo. Using conditional RhoA/Cdc42 double-knockout (DKO) mice, we reveal here that RhoA/Cdc42 signaling is dispensable for the process of polyploidization in MKs but essential for cytoplasmic MK maturation. Proplatelet formation is virtually abrogated in the absence of RhoA/Cdc42 and leads to severe macrothrombocytopenia in DKO animals. The MK maturation defect is associated with downregulation of myosin light chain 2 (MLC2) and β1-tubulin, as well as an upregulation of LIM kinase 1 and cofilin-1 at both the mRNA and protein level and can be linked to impaired MKL1/SRF signaling. Our findings demonstrate that MK endomitosis and cytoplasmic maturation are separately regulated processes, and the latter is critically controlled by RhoA/Cdc42.
Collapse
Affiliation(s)
- Tobias Heib
- Institute of Experimental Biomedicine, University Hospital, University of Würzburg, 97080 Würzburg, Germany; Rudolf Virchow Center, University of Würzburg, 97080 Würzburg, Germany
| | - Heike M Hermanns
- Department of Internal Medicine II, Hepatology Research Laboratory, University Hospital Würzburg, 97080 Würzburg, Germany
| | - Georgi Manukjan
- Institute of Experimental Biomedicine, University Hospital, University of Würzburg, 97080 Würzburg, Germany; Rudolf Virchow Center, University of Würzburg, 97080 Würzburg, Germany
| | - Maximilian Englert
- Institute of Experimental Biomedicine, University Hospital, University of Würzburg, 97080 Würzburg, Germany; Rudolf Virchow Center, University of Würzburg, 97080 Würzburg, Germany
| | - Charly Kusch
- Institute of Experimental Biomedicine, University Hospital, University of Würzburg, 97080 Würzburg, Germany; Rudolf Virchow Center, University of Würzburg, 97080 Würzburg, Germany
| | - Isabelle Carlotta Becker
- Institute of Experimental Biomedicine, University Hospital, University of Würzburg, 97080 Würzburg, Germany; Rudolf Virchow Center, University of Würzburg, 97080 Würzburg, Germany
| | - Annika Gerber
- Institute of Experimental Biomedicine, University Hospital, University of Würzburg, 97080 Würzburg, Germany; Rudolf Virchow Center, University of Würzburg, 97080 Würzburg, Germany
| | - Lou Martha Wackerbarth
- Institute of Experimental Biomedicine, University Hospital, University of Würzburg, 97080 Würzburg, Germany; Rudolf Virchow Center, University of Würzburg, 97080 Würzburg, Germany
| | - Philipp Burkard
- Institute of Experimental Biomedicine, University Hospital, University of Würzburg, 97080 Würzburg, Germany; Rudolf Virchow Center, University of Würzburg, 97080 Würzburg, Germany
| | - Thomas Dandekar
- Department of Bioinformatics, Biocenter, University of Würzburg, 97074 Würzburg, Germany
| | - Johannes Balkenhol
- Department of Internal Medicine II, Hepatology Research Laboratory, University Hospital Würzburg, 97080 Würzburg, Germany; Department of Bioinformatics, Biocenter, University of Würzburg, 97074 Würzburg, Germany
| | - Daniel Jahn
- Department of Internal Medicine II, Hepatology Research Laboratory, University Hospital Würzburg, 97080 Würzburg, Germany
| | - Sarah Beck
- Institute of Experimental Biomedicine, University Hospital, University of Würzburg, 97080 Würzburg, Germany; Rudolf Virchow Center, University of Würzburg, 97080 Würzburg, Germany
| | - Mara Meub
- Department of Biotechnology and Biophysics, Biocenter, University of Würzburg, 97074 Würzburg, Germany
| | - Sebastian Dütting
- Institute of Experimental Biomedicine, University Hospital, University of Würzburg, 97080 Würzburg, Germany; Rudolf Virchow Center, University of Würzburg, 97080 Würzburg, Germany
| | - Christian Stigloher
- Imaging Core Facility, Biocenter, University of Würzburg, 97074 Würzburg, Germany
| | - Markus Sauer
- Department of Biotechnology and Biophysics, Biocenter, University of Würzburg, 97074 Würzburg, Germany
| | - Deya Cherpokova
- Institute of Experimental Biomedicine, University Hospital, University of Würzburg, 97080 Würzburg, Germany; Rudolf Virchow Center, University of Würzburg, 97080 Würzburg, Germany
| | - Harald Schulze
- Institute of Experimental Biomedicine, University Hospital, University of Würzburg, 97080 Würzburg, Germany; Rudolf Virchow Center, University of Würzburg, 97080 Würzburg, Germany
| | - Cord Brakebusch
- Biotech Research and Innovation Centre (BRIC), University of Copenhagen, 2200 Copenhagen, Denmark
| | - Bernhard Nieswandt
- Institute of Experimental Biomedicine, University Hospital, University of Würzburg, 97080 Würzburg, Germany; Rudolf Virchow Center, University of Würzburg, 97080 Würzburg, Germany.
| | - Zoltan Nagy
- Institute of Experimental Biomedicine, University Hospital, University of Würzburg, 97080 Würzburg, Germany; Rudolf Virchow Center, University of Würzburg, 97080 Würzburg, Germany
| | - Irina Pleines
- Institute of Experimental Biomedicine, University Hospital, University of Würzburg, 97080 Würzburg, Germany; Rudolf Virchow Center, University of Würzburg, 97080 Würzburg, Germany.
| |
Collapse
|
|
23
|
Nishimura Y, Shi S, Zhang F, Liu R, Takagi Y, Bershadsky AD, Viasnoff V, Sellers JR. The formin inhibitor SMIFH2 inhibits members of the myosin superfamily. J Cell Sci 2021; 134:237818. [PMID: 33589498 PMCID: PMC8121067 DOI: 10.1242/jcs.253708] [Citation(s) in RCA: 41] [Impact Index Per Article: 10.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/23/2020] [Accepted: 02/03/2021] [Indexed: 12/31/2022] Open
Abstract
The small molecular inhibitor of formin FH2 domains, SMIFH2, is widely used in cell biological studies. It inhibits formin-driven actin polymerization in vitro, but not polymerization of pure actin. It is active against several types of formin from different species. Here, we found that SMIFH2 inhibits retrograde flow of myosin 2 filaments and contraction of stress fibers. We further checked the effect of SMIFH2 on non-muscle myosin 2A and skeletal muscle myosin 2 in vitro, and found that SMIFH2 inhibits activity of myosin ATPase and the ability to translocate actin filaments in the gliding actin in vitro motility assay. Inhibition of non-muscle myosin 2A in vitro required a higher concentration of SMIFH2 compared with that needed to inhibit retrograde flow and stress fiber contraction in cells. We also found that SMIFH2 inhibits several other non-muscle myosin types, including bovine myosin 10, Drosophila myosin 7a and Drosophila myosin 5, more efficiently than it inhibits formins. These off-target inhibitions demand additional careful analysis in each case when solely SMIFH2 is used to probe formin functions. This article has an associated First Person interview with Yukako Nishimura, joint first author of the paper.
Collapse
Affiliation(s)
- Yukako Nishimura
- Mechanobiology Institute (MBI), National University of Singapore, Singapore 117411, Singapore
| | - Shidong Shi
- Mechanobiology Institute (MBI), National University of Singapore, Singapore 117411, Singapore
| | - Fang Zhang
- National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA
| | - Rong Liu
- National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA
| | - Yasuharu Takagi
- National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA
| | - Alexander D Bershadsky
- Mechanobiology Institute (MBI), National University of Singapore, Singapore 117411, Singapore.,Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot 7610001, Israel
| | - Virgile Viasnoff
- Mechanobiology Institute (MBI), National University of Singapore, Singapore 117411, Singapore.,CNRS UMI 3639 BMC, Singapore 117411, Singapore.,Department of Biological Sciences, National university of Singapore, Singapore 117558, Singapore
| | - James R Sellers
- National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA
| |
Collapse
|
|
24
|
Vainchenker W, Arkoun B, Basso-Valentina F, Lordier L, Debili N, Raslova H. Role of Rho-GTPases in megakaryopoiesis. Small GTPases 2021; 12:399-415. [PMID: 33570449 PMCID: PMC8583283 DOI: 10.1080/21541248.2021.1885134] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/27/2023] Open
Abstract
Megakaryocytes (MKs) are the bone marrow (BM) cells that generate blood platelets by a process that requires: i) polyploidization responsible for the increased MK size and ii) cytoplasmic organization leading to extension of long pseudopods, called proplatelets, through the endothelial barrier to allow platelet release into blood. Low level of localized RHOA activation prevents actomyosin accumulation at the cleavage furrow and participates in MK polyploidization. In the platelet production, RHOA and CDC42 play opposite, but complementary roles. RHOA inhibits both proplatelet formation and MK exit from BM, whereas CDC42 drives the development of the demarcation membranes and MK migration in BM. Moreover, the RhoA or Cdc42 MK specific knock-out in mice and the genetic alterations in their down-stream effectors in human induce a thrombocytopenia demonstrating their key roles in platelet production. A better knowledge of Rho-GTPase signalling is thus necessary to develop therapies for diseases associated with platelet production defects. Abbreviations: AKT: Protein Kinase BARHGEF2: Rho/Rac Guanine Nucleotide Exchange Factor 2ARP2/3: Actin related protein 2/3BM: Bone marrowCDC42: Cell division control protein 42 homologCFU-MK: Colony-forming-unit megakaryocyteCIP4: Cdc42-interacting protein 4mDIA: DiaphanousDIAPH1; Protein diaphanous homolog 1ECT2: Epithelial Cell Transforming Sequence 2FLNA: Filamin AGAP: GTPase-activating proteins or GTPase-accelerating proteinsGDI: GDP Dissociation InhibitorGEF: Guanine nucleotide exchange factorHDAC: Histone deacetylaseLIMK: LIM KinaseMAL: Megakaryoblastic leukaemiaMARCKS: Myristoylated alanine-rich C-kinase substrateMKL: Megakaryoblastic leukaemiaMLC: Myosin light chainMRTF: Myocardin Related Transcription FactorOTT: One-Twenty Two ProteinPACSIN2: Protein Kinase C And Casein Kinase Substrate In Neurons 2PAK: P21-Activated KinasePDK: Pyruvate Dehydrogenase kinasePI3K: Phosphoinositide 3-kinasePKC: Protein kinase CPTPRJ: Protein tyrosine phosphatase receptor type JRAC: Ras-related C3 botulinum toxin substrate 1RBM15: RNA Binding Motif Protein 15RHO: Ras homologousROCK: Rho-associated protein kinaseSCAR: Suppressor of cAMP receptorSRF: Serum response factorSRC: SarcTAZ: Transcriptional coactivator with PDZ motifTUBB1: Tubulin β1VEGF: Vascular endothelial growth factorWAS: Wiskott Aldrich syndromeWASP: Wiskott Aldrich syndrome proteinWAVE: WASP-family verprolin-homologous proteinWIP: WASP-interacting proteinYAP: Yes-associated protein
Collapse
Affiliation(s)
- William Vainchenker
- INSERM, UMR 1287, Gustave Roussy, Equipe Labellisée LNCC, Villejuif, France.,Université Paris Saclay, UMR 1287, Gustave Roussy, Villejuif, France.,Gustave Roussy, UMR 1287, Gustave Roussy, Villejuif, France.,GrEX, Sorbonne Paris Cité, Paris, France
| | - Brahim Arkoun
- INSERM, UMR 1287, Gustave Roussy, Equipe Labellisée LNCC, Villejuif, France.,Université Paris Saclay, UMR 1287, Gustave Roussy, Villejuif, France.,Gustave Roussy, UMR 1287, Gustave Roussy, Villejuif, France.,GrEX, Sorbonne Paris Cité, Paris, France
| | - Francesca Basso-Valentina
- INSERM, UMR 1287, Gustave Roussy, Equipe Labellisée LNCC, Villejuif, France.,Université Paris Saclay, UMR 1287, Gustave Roussy, Villejuif, France.,Gustave Roussy, UMR 1287, Gustave Roussy, Villejuif, France.,Université Sorbonne Paris Cité/Université Paris Dideront, Paris, France
| | - Larissa Lordier
- INSERM, UMR 1287, Gustave Roussy, Equipe Labellisée LNCC, Villejuif, France.,Université Paris Saclay, UMR 1287, Gustave Roussy, Villejuif, France.,Gustave Roussy, UMR 1287, Gustave Roussy, Villejuif, France
| | - Najet Debili
- INSERM, UMR 1287, Gustave Roussy, Equipe Labellisée LNCC, Villejuif, France.,Université Paris Saclay, UMR 1287, Gustave Roussy, Villejuif, France.,Gustave Roussy, UMR 1287, Gustave Roussy, Villejuif, France
| | - Hana Raslova
- INSERM, UMR 1287, Gustave Roussy, Equipe Labellisée LNCC, Villejuif, France.,Université Paris Saclay, UMR 1287, Gustave Roussy, Villejuif, France.,Gustave Roussy, UMR 1287, Gustave Roussy, Villejuif, France
| |
Collapse
|
|
25
|
Zuidscherwoude M, Haining EJ, Simms VA, Watson S, Grygielska B, Hardy AT, Bacon A, Watson SP, Thomas SG. Loss of mDia1 and Fhod1 impacts platelet formation but not platelet function. Platelets 2020; 32:1051-1062. [PMID: 32981398 PMCID: PMC8635707 DOI: 10.1080/09537104.2020.1822522] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/01/2022]
Abstract
An organized and dynamic cytoskeleton is required for platelet formation and function. Formins are a large family of actin regulatory proteins which are also able to regulate microtubule dynamics. There are four formin family members expressed in human and mouse megakaryocytes and platelets. We have previously shown that the actin polymerization activity of formin proteins is required for cytoskeletal dynamics and platelet spreading using a small molecule inhibitor. In the current study, we analyze transgenic mouse models deficient in two of these proteins, mDia1 and Fhod1, along with a model lacking both proteins. We demonstrate that double knockout mice display macrothrombocytopenia which is due to aberrant megakaryocyte function and a small decrease in platelet lifespan. Platelet function is unaffected by the loss of these proteins. This data indicates a critical role for formins in platelet and megakaryocyte function.
Collapse
Affiliation(s)
- Malou Zuidscherwoude
- Institute of Cardiovascular Sciences, College of Medical and Dental Sciences, University of Birmingham, Birmingham, UK
- Centre of Membrane Proteins and Receptors (COMPARE), University of Birmingham and University of Nottingham, Midlands, UK
| | - Elizabeth J. Haining
- Institute of Cardiovascular Sciences, College of Medical and Dental Sciences, University of Birmingham, Birmingham, UK
| | - Victoria A. Simms
- Institute of Cardiovascular Sciences, College of Medical and Dental Sciences, University of Birmingham, Birmingham, UK
| | - Stephanie Watson
- Institute of Cardiovascular Sciences, College of Medical and Dental Sciences, University of Birmingham, Birmingham, UK
| | - Beata Grygielska
- Institute of Cardiovascular Sciences, College of Medical and Dental Sciences, University of Birmingham, Birmingham, UK
| | - Alex T. Hardy
- Institute of Cardiovascular Sciences, College of Medical and Dental Sciences, University of Birmingham, Birmingham, UK
| | - Andrea Bacon
- Genome Editing Facility, Technology Hub, College of Medical and Dental Sciences, University of Birmingham, Birmingham, UK
| | - Stephen P. Watson
- Institute of Cardiovascular Sciences, College of Medical and Dental Sciences, University of Birmingham, Birmingham, UK
- Centre of Membrane Proteins and Receptors (COMPARE), University of Birmingham and University of Nottingham, Midlands, UK
| | - Steven G. Thomas
- Institute of Cardiovascular Sciences, College of Medical and Dental Sciences, University of Birmingham, Birmingham, UK
- Centre of Membrane Proteins and Receptors (COMPARE), University of Birmingham and University of Nottingham, Midlands, UK
| |
Collapse
|
|
26
|
Bächer C, Bender M, Gekle S. Flow-accelerated platelet biogenesis is due to an elasto-hydrodynamic instability. Proc Natl Acad Sci U S A 2020; 117:18969-18976. [PMID: 32719144 PMCID: PMC7431004 DOI: 10.1073/pnas.2002985117] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022] Open
Abstract
Blood platelets are formed by fragmentation of long membrane extensions from bone marrow megakaryocytes in the blood flow. Using lattice-Boltzmann/immersed boundary simulations we propose a biological Rayleigh-Plateau instability as the biophysical mechanism behind this fragmentation process. This instability is akin to the surface tension-induced breakup of a liquid jet but is driven by active cortical processes including actomyosin contractility and microtubule sliding. Our fully three-dimensional simulations highlight the crucial role of actomyosin contractility, which is required to trigger the instability, and illustrate how the wavelength of the instability determines the size of the final platelets. The elasto-hydrodynamic origin of the fragmentation explains the strong acceleration of platelet biogenesis in the presence of an external flow, which we observe in agreement with experiments. Our simulations then allow us to disentangle the influence of specific flow conditions: While a homogeneous flow with uniform velocity leads to the strongest acceleration, a shear flow with a linear velocity gradient can cause fusion events of two developing platelet-sized swellings during fragmentation. A fusion event may lead to the release of larger structures which are observable as preplatelets in experiments. Together, our findings strongly indicate a mainly physical origin of fragmentation and regulation of platelet size in flow-accelerated platelet biogenesis.
Collapse
Affiliation(s)
- Christian Bächer
- Biofluid Simulation and Modeling, Theoretische Physik VI, University of Bayreuth, 95447 Bayreuth, Germany;
| | - Markus Bender
- Institute of Experimental Biomedicine I, University Hospital and Rudolf Virchow Center, 97080 Würzburg, Germany
| | - Stephan Gekle
- Biofluid Simulation and Modeling, Theoretische Physik VI, University of Bayreuth, 95447 Bayreuth, Germany;
| |
Collapse
|
|
27
|
Karki NR, Ajebo G, Savage N, Kutlar A. DIAPH1 Mutation as a Novel Cause of Autosomal Dominant Macrothrombocytopenia and Hearing Loss. Acta Haematol 2020; 144:91-94. [PMID: 32594080 DOI: 10.1159/000506727] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/07/2020] [Accepted: 02/22/2020] [Indexed: 12/18/2022]
Abstract
Macrothrombocytopenia (MTP) is a group of rare disorders characterized by giant platelets, thrombocytopenia, and variable association with abnormal bleeding. Inherited MTP are frequently misdiagnosed as immune thrombocytopenia. Associated second-organ manifestation can help narrow down syndromic MTPs. We describe a case of autosomal dominant sensorineural hearing loss and MTP caused by a gain of function mutation in DIAPH1. This mutation causes altered megarkaryopoiesis and platelet cytoskeletal deregulation. Although hearing loss and MTP were likely progressive, clinically significant bleeding was not observed. DIAPH1-related MTP can be distinguished clinically from MYH9 mutation by the absence of cataracts and glomerular disease.
Collapse
Affiliation(s)
- Nabin Raj Karki
- Division of Hematology/Oncology, Georgia Cancer Center, Augusta University, Augusta, Georgia, USA,
| | - Germame Ajebo
- Division of Hematology/Oncology, Georgia Cancer Center, Augusta University, Augusta, Georgia, USA
| | - Natasha Savage
- Department of Pathology, Augusta University, Augusta, Georgia, USA
| | - Abdullah Kutlar
- Division of Hematology/Oncology, Georgia Cancer Center, Augusta University, Augusta, Georgia, USA
| |
Collapse
|
|
28
|
Green HLH, Zuidscherwoude M, Alenazy F, Smith CW, Bender M, Thomas SG. SMIFH2 inhibition of platelets demonstrates a critical role for formin proteins in platelet cytoskeletal dynamics. J Thromb Haemost 2020; 18:955-967. [PMID: 31930764 PMCID: PMC7186844 DOI: 10.1111/jth.14735] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/19/2019] [Accepted: 01/07/2020] [Indexed: 12/24/2022]
Abstract
BACKGROUND Reorganization of the actin cytoskeleton is required for proper functioning of platelets following activation in response to vascular damage. Formins are a family of proteins that regulate actin polymerization and cytoskeletal organization via a number of domains including the FH2 domain. However, the role of formins in platelet spreading has not been studied in detail. OBJECTIVES Several formin proteins are expressed in platelets so we used an inhibitor of FH2 domains (SMIFH2) to uncover the role of these proteins in platelet spreading and in maintenance of resting platelet shape. METHODS Washed human and mouse platelets were treated with various concentrations of SMIFH2 and the effects on platelet spreading, platelet size, platelet cytoskeletal dynamics, and organization were analyzed using fluorescence and electron microscopy. RESULTS Pretreatment with SMIFH2 completely blocks platelet spreading in both mouse and human platelets through effects on the organization and dynamics of actin and microtubules. However, platelet aggregation and secretion are unaffected. SMIFH2 also caused a decrease in resting platelet size and disrupted the balance of tubulin post-translational modification. CONCLUSIONS These data therefore demonstrated an important role for formin-mediated actin polymerization in platelet spreading and highlighted the importance of formins in cross-talk between the actin and tubulin cytoskeletons.
Collapse
Affiliation(s)
- Hannah L. H. Green
- Institute of Cardiovascular SciencesUniversity of BirminghamBirminghamUK
- Present address:
School of Cardiovascular Medicine & SciencesBHF Centre of Research ExcellenceKing's College LondonLondonUK
| | - Malou Zuidscherwoude
- Institute of Cardiovascular SciencesUniversity of BirminghamBirminghamUK
- Centre of Membrane Proteins and Receptors (COMPARE)University of Birmingham and University of NottinghamMidlandsUK
| | - Fawaz Alenazy
- Institute of Cardiovascular SciencesUniversity of BirminghamBirminghamUK
| | | | - Markus Bender
- Institute of Experimental Biomedicine – Chair IUniversity Hospital and Rudolf Virchow CenterWürzburgGermany
| | - Steven G. Thomas
- Institute of Cardiovascular SciencesUniversity of BirminghamBirminghamUK
- Centre of Membrane Proteins and Receptors (COMPARE)University of Birmingham and University of NottinghamMidlandsUK
| |
Collapse
|
|
29
|
Le Bagge S, Fotheringham AK, Leung SS, Forbes JM. Targeting the receptor for advanced glycation end products (RAGE) in type 1 diabetes. Med Res Rev 2020; 40:1200-1219. [PMID: 32112452 DOI: 10.1002/med.21654] [Citation(s) in RCA: 30] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/26/2019] [Revised: 11/09/2019] [Accepted: 11/12/2019] [Indexed: 12/18/2022]
Abstract
Type 1 diabetes (T1D) is one of the most common chronic diseases manifesting in early life, with the prevalence increasing worldwide at a rate of approximately 3% per annum. The prolonged hyperglycaemia characteristic of T1D upregulates the receptor for advanced glycation end products (RAGE) and accelerates the formation of RAGE ligands, including advanced glycation end products, high-mobility group protein B1, S100 calcium-binding proteins, and amyloid-beta. Interestingly, changes in the expression of RAGE and these ligands are evident in patients before the onset of T1D. RAGE signals via various proinflammatory cascades, resulting in the production of reactive oxygen species and cytokines. A large number of proinflammatory ligands that can signal via RAGE have been implicated in several chronic diseases, including T1D. Therefore, it is unsurprising that RAGE has become a potential therapeutic target for the treatment and prevention of disease. In this review, we will explore how RAGE might be targeted to prevent the development of T1D.
Collapse
Affiliation(s)
- Selena Le Bagge
- Glycation and Diabetes, Translational Research Institute (TRI), Mater Research Institute-The University of Queensland (MRI-UQ), Brisbane, Queensland, Australia.,School of Biomedical Sciences, The University of Queensland, Brisbane, Queensland, Australia
| | - Amelia K Fotheringham
- Glycation and Diabetes, Translational Research Institute (TRI), Mater Research Institute-The University of Queensland (MRI-UQ), Brisbane, Queensland, Australia.,School of Biomedical Sciences, The University of Queensland, Brisbane, Queensland, Australia
| | - Sherman S Leung
- Glycation and Diabetes, Translational Research Institute (TRI), Mater Research Institute-The University of Queensland (MRI-UQ), Brisbane, Queensland, Australia.,School of Biomedical Sciences, The University of Queensland, Brisbane, Queensland, Australia
| | - Josephine M Forbes
- Glycation and Diabetes, Translational Research Institute (TRI), Mater Research Institute-The University of Queensland (MRI-UQ), Brisbane, Queensland, Australia.,Baker IDI Heart and Diabetes Institute, Melbourne, Victoria, Australia.,Mater Clinical School, The University of Queensland, Brisbane, Queensland, Australia
| |
Collapse
|
|
30
|
Association of DIAPH1 gene polymorphisms with ischemic stroke. Aging (Albany NY) 2020; 12:416-435. [PMID: 31899686 PMCID: PMC6977662 DOI: 10.18632/aging.102631] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/23/2019] [Accepted: 12/23/2019] [Indexed: 12/29/2022]
Abstract
DIAPH1 is a formin protein involved in actin polymerization with important roles in vascular remodeling and thrombosis. To investigate potential associations of DIAPH1 single-nucleotide polymorphisms (SNPs) with hypertension and stroke, 2,012 patients with hypertension and 2,210 controls, 2,966 stroke cases [2,212 ischemic stroke (IS), 754 hemorrhagic stroke (HS)] and 2,590 controls were enrolled respectively in the case-control study. A total of 4,098 individual were included in the cohort study. DIAPH1 mRNA expression was compared between 66 IS [43 small artery occlusion (SAO) and 23 large-artery atherosclerosis (LAA)] and 58 controls. Odds ratio (OR), hazard ratio (HR) and 95% confidence interval (CI) were calculated by logistic and cox regression analysis. Rs7703688 T>C variation was significantly associated with an increased risk of IS [OR (95% CI) was 1.721 (1.486-1.993), P=4.139×10-12]. Association of rs7703688 with stroke risk was further validated in the cohort study [adjusted HRs (95% CIs) for additive and recessive models were 1.385 (1.001-1.918), P=0.049, and 2.882 (1.038-8.004), P=0.042, respectively)]. DIAPH1 mRNA expression was significantly downregulated in IS. In SAO stroke subtype, DIAPH1 expression has an increased trend among rs251019 genotypes (Ptrend=0.048). These novel findings suggest that DIAPH1 variation contributes to genetic susceptibility to stroke risk, especially the SAO subtype of IS.
Collapse
|
|
31
|
Lei XH, Yang YQ, Ma CY, Duan EK. Induction of differentiation of human stem cells ex vivo: Toward large-scale platelet production. World J Stem Cells 2019; 11:666-676. [PMID: 31616542 PMCID: PMC6789181 DOI: 10.4252/wjsc.v11.i9.666] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/14/2019] [Revised: 05/12/2019] [Accepted: 08/27/2019] [Indexed: 02/06/2023] Open
Abstract
Platelet transfusion is one of the most reliable strategies to cure patients suffering from thrombocytopenia or platelet dysfunction. With the increasing demand for transfusion, however, there is an undersupply of donors to provide the platelet source. Thus, scientists have sought to design methods for deriving clinical-scale platelets ex vivo. Although there has been considerable success ex vivo in the generation of transformative platelets produced by human stem cells (SCs), the platelet yields achieved using these strategies have not been adequate for clinical application. In this review, we provide an overview of the developmental process of megakaryocytes and the production of platelets in vivo and ex vivo, recapitulate the key advances in the production of SC-derived platelets using several SC sources, and discuss some strategies that apply three-dimensional bioreactor devices and biochemical factors synergistically to improve the generation of large-scale platelets for use in future biomedical and clinical settings.
Collapse
Affiliation(s)
- Xiao-Hua Lei
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China
| | - Yi-Qing Yang
- Faculty of Laboratory Medical Science, Hebei North University, Zhangjiakou 075000, Hebei Province, China
| | - Chi-Yuan Ma
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China
| | - En-Kui Duan
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China
| |
Collapse
|
|
32
|
Lei XH, Yang YQ, Ma CY, Duan EK. Induction of differentiation of human stem cellsex vivo: Toward large-scale platelet production. World J Stem Cells 2019. [DOI: dx.doi.org/10.4252/wjsc.v11.i9.666] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
|
|
33
|
Ghalloussi D, Dhenge A, Bergmeier W. New insights into cytoskeletal remodeling during platelet production. J Thromb Haemost 2019; 17:1430-1439. [PMID: 31220402 PMCID: PMC6760864 DOI: 10.1111/jth.14544] [Citation(s) in RCA: 26] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/23/2018] [Accepted: 06/12/2019] [Indexed: 12/16/2022]
Abstract
The past decade has brought unprecedented advances in our understanding of megakaryocyte (MK) biology and platelet production, processes that are strongly dependent on the cytoskeleton. Facilitated by technological innovations, such as new high-resolution imaging techniques (in vitro and in vivo) and lineage-specific gene knockout and reporter mouse strains, we are now able to visualize and characterize the molecular machinery required for MK development and proplatelet formation in live mice. Whole genome and RNA sequencing analysis of patients with rare platelet disorders, combined with targeted genetic interventions in mice, has led to the identification and characterization of numerous new genes important for MK development. Many of the genes important for proplatelet formation code for proteins that control cytoskeletal dynamics in cells, such as Rho GTPases and their downstream targets. In this review, we discuss how the final stages of MK development are controlled by the cellular cytoskeletons, and we compare changes in MK biology observed in patients and mice with mutations in cytoskeleton regulatory genes.
Collapse
Affiliation(s)
- Dorsaf Ghalloussi
- McAllister Heart Institute, University of North Carolina at Chapel Hill, Chapel Hill, NC
| | - Ankita Dhenge
- McAllister Heart Institute, University of North Carolina at Chapel Hill, Chapel Hill, NC
| | - Wolfgang Bergmeier
- McAllister Heart Institute, University of North Carolina at Chapel Hill, Chapel Hill, NC
- Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, NC
| |
Collapse
|
|
34
|
Kim BJ, Ueyama T, Miyoshi T, Lee S, Han JH, Park HR, Kim AR, Oh J, Kim MY, Kang YS, Oh DY, Yun J, Hwang SM, Kim NKD, Park WY, Kitajiri SI, Choi BY. Differential disruption of autoinhibition and defect in assembly of cytoskeleton during cell division decide the fate of human DIAPH1-related cytoskeletopathy. J Med Genet 2019; 56:818-827. [DOI: 10.1136/jmedgenet-2019-106282] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/12/2019] [Revised: 06/24/2019] [Accepted: 07/09/2019] [Indexed: 12/25/2022]
Abstract
BackgroundDiaphanous-related formin 1 (DIA1), which assembles the unbranched actin microfilament and microtubule cytoskeleton, is encoded by DIAPH1. Constitutive activation by the disruption of autoinhibitory interactions between the N-terminal diaphanous inhibitory domain (DID) and C-terminal diaphanous autoregulatory domain (DAD) dysregulates DIA1, resulting in both hearing loss and blood cell abnormalities.Methods and resultsHere, we report the first constitutively active mutant in the DID (p.A265S) of humans with only hearing loss and not blood cell abnormality through whole exome sequencing. The previously reported DAD mutants and our DID mutant (p.A265S) shared the finding of diminished autoinhibitory interaction, abnormally upregulated actin polymerisation activity and increased localisations at the plasma membrane. However, the obvious defect in the DIA1-driven assembly of cytoskeleton ‘during cell division’ was only from the DAD mutants, not from p.A265S, which did not show any blood cell abnormality. We also evaluated the five DID mutants in the hydrophobic pocket since four of these five additional mutants were predicted to critically disrupt interaction between the DID and DAD. These additional pathogenic DID mutants revealed varying degrees of defect in the DIA1-driven cytoskeleton assembly, including nearly normal phenotype during cell division as well as obvious impaired autoinhibition, again coinciding with our key observation in DIA1 mutant (p.A265S) in the DID.ConclusionHere, we report the first mutant in the DID of humans with only hearing loss. The differential cell biological phenotypes of DIA1 during cell division appear to be potential determinants of the clinical severity of DIAPH1-related cytoskeletopathy in humans.
Collapse
|
|
35
|
Mendelson A, Strat AN, Bao W, Rosston P, Fallon G, Ohrn S, Zhong H, Lobo C, An X, Yazdanbakhsh K. Mesenchymal stromal cells lower platelet activation and assist in platelet formation in vitro. JCI Insight 2019; 4:126982. [PMID: 31434805 DOI: 10.1172/jci.insight.126982] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/20/2018] [Accepted: 07/23/2019] [Indexed: 01/01/2023] Open
Abstract
The complex process of platelet formation originates with the hematopoietic stem cell, which differentiates through the myeloid lineage, matures, and releases proplatelets into the BM sinusoids. How formed platelets maintain a low basal activation state in the circulation remains unknown. We identify Lepr+ stromal cells lining the BM sinusoids as important contributors to sustaining low platelet activation. Ablation of murine Lepr+ cells led to a decreased number of platelets in the circulation with an increased activation state. We developed a potentially novel culture system for supporting platelet formation in vitro using a unique population of CD51+PDGFRα+ perivascular cells, derived from human umbilical cord tissue, which display numerous mesenchymal stem cell (MSC) properties. Megakaryocytes cocultured with MSCs had altered LAT and Rap1b gene expression, yielding platelets that are functional with low basal activation levels, a critical consideration for developing a transfusion product. Identification of a regulatory cell that maintains low baseline platelet activation during thrombopoiesis opens up new avenues for improving blood product production ex vivo.
Collapse
Affiliation(s)
| | | | | | | | | | | | | | | | - Xiuli An
- Laboratory of Membrane Biology, New York Blood Center (NYBC), New York, New York, USA
| | | |
Collapse
|
|
36
|
Schurr Y, Spindler M, Kurz H, Bender M. The cytoskeletal crosslinking protein MACF1 is dispensable for thrombus formation and hemostasis. Sci Rep 2019; 9:7726. [PMID: 31118482 PMCID: PMC6531446 DOI: 10.1038/s41598-019-44183-6] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/01/2019] [Accepted: 04/27/2019] [Indexed: 12/15/2022] Open
Abstract
Coordinated reorganization of cytoskeletal structures is critical for key aspects of platelet physiology. While several studies have addressed the role of microtubules and filamentous actin in platelet production and function, the significance of their crosstalk in these processes has been poorly investigated. The microtubule-actin cross-linking factor 1 (MACF1; synonym: Actin cross-linking factor 7, ACF7) is a member of the spectraplakin family, and one of the few proteins expressed in platelets, which possess actin and microtubule binding domains thereby facilitating actin-microtubule interaction and regulation. We used megakaryocyte- and platelet-specific Macf1 knockout (Macf1fl/fl, Pf4-Cre) mice to study the role of MACF1 in platelet production and function. MACF1 deficient mice displayed comparable platelet counts to control mice. Analysis of the platelet cytoskeletal ultrastructure revealed a normal marginal band and actin network. Platelet spreading on fibrinogen was slightly delayed but platelet activation and clot traction was unaffected. Ex vivo thrombus formation and mouse tail bleeding responses were similar between control and mutant mice. These results suggest that MACF1 is dispensable for thrombopoiesis, platelet activation, thrombus formation and the hemostatic function in mice.
Collapse
Affiliation(s)
- Yvonne Schurr
- Institute of Experimental Biomedicine - Chair I, University Hospital and Rudolf Virchow Center, Würzburg, Germany
| | - Markus Spindler
- Institute of Experimental Biomedicine - Chair I, University Hospital and Rudolf Virchow Center, Würzburg, Germany
| | - Hendrikje Kurz
- Institute of Experimental Biomedicine - Chair I, University Hospital and Rudolf Virchow Center, Würzburg, Germany
| | - Markus Bender
- Institute of Experimental Biomedicine - Chair I, University Hospital and Rudolf Virchow Center, Würzburg, Germany.
| |
Collapse
|
|
37
|
Meiring JCM, Bryce NS, Niño JLG, Gabriel A, Tay SS, Hardeman EC, Biro M, Gunning PW. Tropomyosin concentration but not formin nucleators mDia1 and mDia3 determines the level of tropomyosin incorporation into actin filaments. Sci Rep 2019; 9:6504. [PMID: 31019238 PMCID: PMC6482184 DOI: 10.1038/s41598-019-42977-2] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/11/2018] [Accepted: 04/11/2019] [Indexed: 12/31/2022] Open
Abstract
The majority of actin filaments in human cells exist as a co-polymer with tropomyosin, which determines the functionality of actin filaments in an isoform dependent manner. Tropomyosin isoforms are sorted to different actin filament populations and in yeast this process is determined by formins, however it remains unclear what process determines tropomyosin isoform sorting in mammalian cells. We have tested the roles of two major formin nucleators, mDia1 and mDia3, in the recruitment of specific tropomyosin isoforms in mammals. Despite observing poorer cell-cell attachments in mDia1 and mDia3 KD cells and an actin bundle organisation defect with mDia1 knock down; depletion of mDia1 and mDia3 individually and concurrently did not result in any significant impact on tropomyosin recruitment to actin filaments, as observed via immunofluorescence and measured via biochemical assays. Conversely, in the presence of excess Tpm3.1, the absolute amount of Tpm3.1-containing actin filaments is not fixed by actin filament nucleators but rather depends on the cell concentration of Tpm3.1. We conclude that mDia1 and mDia3 are not essential for tropomyosin recruitment and that tropomyosin incorporation into actin filaments is concentration dependent.
Collapse
Affiliation(s)
- Joyce C M Meiring
- Cellular and Genetic Medicine Unit, School of Medical Sciences, University of New South Wales, Sydney, NSW, 2052, Australia
| | - Nicole S Bryce
- Cellular and Genetic Medicine Unit, School of Medical Sciences, University of New South Wales, Sydney, NSW, 2052, Australia
| | - Jorge Luis Galeano Niño
- Single Molecule Science, School of Medical Sciences, University of New South Wales, Sydney, NSW, 2052, Australia
| | - Antje Gabriel
- Cellular and Genetic Medicine Unit, School of Medical Sciences, University of New South Wales, Sydney, NSW, 2052, Australia.,Pharmaceutical Biology, Center for Drug Research, Ludwig-Maximilians-Universität, Munich, Germany
| | - Szun S Tay
- Single Molecule Science, School of Medical Sciences, University of New South Wales, Sydney, NSW, 2052, Australia
| | - Edna C Hardeman
- Cellular and Genetic Medicine Unit, School of Medical Sciences, University of New South Wales, Sydney, NSW, 2052, Australia
| | - Maté Biro
- Single Molecule Science, School of Medical Sciences, University of New South Wales, Sydney, NSW, 2052, Australia
| | - Peter W Gunning
- Cellular and Genetic Medicine Unit, School of Medical Sciences, University of New South Wales, Sydney, NSW, 2052, Australia.
| |
Collapse
|
|
38
|
Miller EW, Blystone SD. The carboxy-terminus of the formin FMNL1ɣ bundles actin to potentiate adenocarcinoma migration. J Cell Biochem 2019; 120:14383-14404. [PMID: 30977161 DOI: 10.1002/jcb.28694] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/30/2018] [Revised: 02/25/2019] [Accepted: 02/28/2019] [Indexed: 12/31/2022]
Abstract
The formin family of proteins contributes to spatiotemporal control of actin cytoskeletal rearrangements during motile cell activities. The FMNL subfamily exhibits multiple mechanisms of linear actin filament formation and organization. Here we report novel actin-modifying functions of FMNL1 in breast adenocarcinoma migration models. FMNL1 is required for efficient cell migration and its three isoforms exhibit distinct localization. Suppression of FMNL1 protein expression results in a significant impairment of cell adhesion, migration, and invasion. Overexpression of FMNL1ɣ, but not FMNL1β or FMNL1α, enhances cell adhesion independent of the FH2 domain and FMNL1ɣ rescues migration in cells depleted of all three endogenous isoforms. While FMNL1ɣ inhibits actin assembly in vitro, it facilitates bundling of filamentous actin independent of the FH2 domain. The unique interactions of FMNL1ɣ with filamentous actin provide a new understanding of formin domain functions and its effect on motility of diverse cell types suggest a broader role than previously realized.
Collapse
Affiliation(s)
- Eric W Miller
- Department of Cell and Developmental Biology, SUNY Upstate Medical University, Syracuse, New York
| | - Scott D Blystone
- Department of Cell and Developmental Biology, SUNY Upstate Medical University, Syracuse, New York
| |
Collapse
|
|
39
|
Microtubule plus-end tracking Adenopolyposis Coli negatively regulates proplatelet formation. Sci Rep 2018; 8:15808. [PMID: 30361531 PMCID: PMC6202313 DOI: 10.1038/s41598-018-34118-y] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/13/2017] [Accepted: 10/03/2018] [Indexed: 12/17/2022] Open
Abstract
Platelets are produced upon profound reorganization of mature megakaryocytes (MK) leading to proplatelet elongation and release into the blood stream, a process termed thrombopoiesis. This highly dynamic process requires microtubules (MT) reorganization by mechanisms that are still incompletely understood. Adenomatous polyposis coli (APC) is a microtubule plus-end tracking protein involved in the regulation of MT in a number of cell systems and its inactivation has been reported to alter hematopoiesis. The aim of our study was to investigate the role of APC in megakaryopoiesis and the final steps of platelet formation. Down-regulation of APC in cultured human MK by RNA interference increased endomitosis and the proportion of cells able to extend proplatelets (68.8% (shAPC1) and 52.5% (shAPC2) vs 28.1% in the control). Similarly an increased ploidy and amplification of the proplatelet network were observed in MK differentiated from Lin- cells of mice with APC-deficiency in the MK lineage. In accordance, these mice exhibited increased platelet counts when compared to wild type mice (1,323 ± 111 vs 919 ± 52 platelets/µL; n = 12 p 0.0033**). Their platelets had a normal size, ultrastructure and number of microtubules coils and their main functions were also preserved. Loss of APC resulted in lower levels of acetylated tubulin and decreased activation of the Wnt signaling pathway. Thus, APC appears as an important regulator of proplatelet formation and overall thrombopoiesis.
Collapse
|
|
40
|
van Dijk J, Bompard G, Cau J, Kunishima S, Rabeharivelo G, Mateos-Langerak J, Cazevieille C, Cavelier P, Boizet-Bonhoure B, Delsert C, Morin N. Microtubule polyglutamylation and acetylation drive microtubule dynamics critical for platelet formation. BMC Biol 2018; 16:116. [PMID: 30336771 PMCID: PMC6194603 DOI: 10.1186/s12915-018-0584-6] [Citation(s) in RCA: 32] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/20/2018] [Accepted: 10/03/2018] [Indexed: 11/10/2022] Open
Abstract
Background Upon maturation in the bone marrow, polyploid megakaryocytes elongate very long and thin cytoplasmic branches called proplatelets. Proplatelets enter the sinusoids blood vessels in which platelets are ultimately released. Microtubule dynamics, bundling, sliding, and coiling, drive these dramatic morphological changes whose regulation remains poorly understood. Microtubule properties are defined by tubulin isotype composition and post-translational modification patterns. It remains unknown whether microtubule post-translational modifications occur in proplatelets and if so, whether they contribute to platelet formation. Results Here, we show that in proplatelets from mouse megakaryocytes, microtubules are both acetylated and polyglutamylated. To bypass the difficulties of working with differentiating megakaryocytes, we used a cell model that allowed us to test the functions of these modifications. First, we show that α2bβ3integrin signaling in D723H cells is sufficient to induce β1tubulin expression and recapitulate the specific microtubule behaviors observed during proplatelet elongation and platelet release. Using this model, we found that microtubule acetylation and polyglutamylation occur with different spatio-temporal patterns. We demonstrate that microtubule acetylation, polyglutamylation, and β1tubulin expression are mandatory for proplatelet-like elongation, swelling formation, and cytoplast severing. We discuss the functional importance of polyglutamylation of β1tubulin-containing microtubules for their efficient bundling and coiling during platelet formation. Conclusions We characterized and validated a powerful cell model to address microtubule behavior in mature megakaryocytes, which allowed us to demonstrate the functional importance of microtubule acetylation and polyglutamylation for platelet release. Furthermore, we bring evidence of a link between the expression of a specific tubulin isotype, the occurrence of microtubule post-translational modifications, and the acquisition of specific microtubule behaviors. Thus, our findings could widen the current view of the regulation of microtubule behavior in cells such as osteoclasts, spermatozoa, and neurons, which express distinct tubulin isotypes and display specific microtubule activities during differentiation. Electronic supplementary material The online version of this article (10.1186/s12915-018-0584-6) contains supplementary material, which is available to authorized users.
Collapse
Affiliation(s)
- Juliette van Dijk
- Universités de Montpellier, 34293, Montpellier, France.,CRBM, CNRS, UMR 5237, 1919 Route de Mende, 34293, Montpellier, France
| | - Guillaume Bompard
- Universités de Montpellier, 34293, Montpellier, France.,IGH, CNRS UMR9002, 141, rue de la Cardonille, 34396, Montpellier, France
| | - Julien Cau
- Universités de Montpellier, 34293, Montpellier, France.,IGH, CNRS UMR9002, 141, rue de la Cardonille, 34396, Montpellier, France.,Montpellier Rio Imaging, 34293, Montpellier, France
| | - Shinji Kunishima
- Department of Advanced Diagnosis, National Hospital Organization Nagoya Medical Center, 4-1-1 Sannomaru, Naka-ku, Nagoya, 4600001, Japan.,Present address: Department of Medical Technology, Gifu University of Medical Science, Seki, Gifu, 5013892, Japan
| | - Gabriel Rabeharivelo
- Universités de Montpellier, 34293, Montpellier, France.,CRBM, CNRS, UMR 5237, 1919 Route de Mende, 34293, Montpellier, France
| | - Julio Mateos-Langerak
- Universités de Montpellier, 34293, Montpellier, France.,IGH, CNRS UMR9002, 141, rue de la Cardonille, 34396, Montpellier, France.,Montpellier Rio Imaging, 34293, Montpellier, France
| | - Chantal Cazevieille
- Universités de Montpellier, 34293, Montpellier, France.,INM, INSERM UMR1051, 34293, Montpellier, France
| | - Patricia Cavelier
- Universités de Montpellier, 34293, Montpellier, France.,IGMM, CNRS, UMR 5535, 1919 Route de Mende, 34293, Montpellier, France
| | - Brigitte Boizet-Bonhoure
- Universités de Montpellier, 34293, Montpellier, France.,IGH, CNRS UMR9002, 141, rue de la Cardonille, 34396, Montpellier, France
| | - Claude Delsert
- Universités de Montpellier, 34293, Montpellier, France.,CRBM, CNRS, UMR 5237, 1919 Route de Mende, 34293, Montpellier, France.,3AS Station Expérimentale d'Aquaculture Ifremer, Chemin de Maguelone, 34250, Palavas-les-Flots, France
| | - Nathalie Morin
- Universités de Montpellier, 34293, Montpellier, France. .,CRBM, CNRS, UMR 5237, 1919 Route de Mende, 34293, Montpellier, France.
| |
Collapse
|
|
41
|
Abstract
PURPOSE OF REVIEW Platelets are small, anucleate cells that circulate within the blood and play essential roles in preserving vascular integrity. However, abnormalities in either platelet production or destruction can result in thrombocytopenia, clinically defined by a platelet count lower than 150 000/μL of whole blood. Thrombocytopenia is frequently associated with impaired hemostatic responses to vascular injury and can be life-threatening because of bleeding complications. Megakaryocytes are the precursor cells responsible for platelet production, a process commonly referred to as thrombopoiesis. This review specifically discusses how perturbation of molecular mechanisms governing megakaryocyte differentiation and development manifest in various forms of thrombocytopenia. RECENT FINDINGS This review highlights the identification of novel transcriptional regulators of megakaryocyte maturation and platelet production. We also provide an update into the essential role of cytoskeletal regulation in thrombopoiesis, and how both megakaryopoiesis and platelet production are altered by anticancer therapeutics. Lastly, we focus on recent investigative approaches to treat thrombocytopenia and discuss future prospects in the field of megakaryocyte research. SUMMARY In patients where thrombocytopenia is not due to heightened platelet destruction or clearance, defects in megakaryocyte development should be considered.
Collapse
Affiliation(s)
- Harvey G Roweth
- Division of Hematology, Brigham and Women's Hospital and Department of Medicine, Harvard Medical School, Harvard Institutes of Medicine, Boston, Massachusetts, USA
| | | | | |
Collapse
|
|
42
|
Contact inhibition controls cell survival and proliferation via YAP/TAZ-autophagy axis. Nat Commun 2018; 9:2961. [PMID: 30054475 PMCID: PMC6063886 DOI: 10.1038/s41467-018-05388-x] [Citation(s) in RCA: 199] [Impact Index Per Article: 28.4] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/06/2017] [Accepted: 07/02/2018] [Indexed: 12/12/2022] Open
Abstract
Contact inhibition enables noncancerous cells to cease proliferation and growth when they contact each other. This characteristic is lost when cells undergo malignant transformation, leading to uncontrolled proliferation and solid tumor formation. Here we report that autophagy is compromised in contact-inhibited cells in 2D or 3D-soft extracellular matrix cultures. In such cells, YAP/TAZ fail to co-transcriptionally regulate the expression of myosin-II genes, resulting in the loss of F-actin stress fibers, which impairs autophagosome formation. The decreased proliferation resulting from contact inhibition is partly autophagy-dependent, as is their increased sensitivity to hypoxia and glucose starvation. These findings define how mechanically repressed YAP/TAZ activity impacts autophagy to contribute to core phenotypes resulting from high cell confluence that are lost in various cancers. At high cell density or when plated on soft matrix, YAP/TAZ are redistributed from the nucleus to the cytosol, becoming transcriptionally inactive. Here the authors show that at high cell density, autophagosome formation is impaired due to reduced YAP/TAZ-dependent transcription of actomyosin genes
Collapse
|
|
43
|
Pleines I, Cherpokova D, Bender M. Rho GTPases and their downstream effectors in megakaryocyte biology. Platelets 2018; 30:9-16. [DOI: 10.1080/09537104.2018.1478071] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/27/2022]
Affiliation(s)
- Irina Pleines
- Institute of Experimental Biomedicine I, University Hospital and Rudolf Virchow Center, University of Würzburg, Würzburg, Germany
| | - Deya Cherpokova
- Program in Cellular and Molecular Medicine, Boston Children’s Hospital, and Department of Pediatrics, Harvard Medical School, Boston, MA, USA
| | - Markus Bender
- Institute of Experimental Biomedicine I, University Hospital and Rudolf Virchow Center, University of Würzburg, Würzburg, Germany
| |
Collapse
|
|
44
|
Zuidscherwoude M, Green HLH, Thomas SG. Formin proteins in megakaryocytes and platelets: regulation of actin and microtubule dynamics. Platelets 2018; 30:23-30. [PMID: 29913076 PMCID: PMC6406210 DOI: 10.1080/09537104.2018.1481937] [Citation(s) in RCA: 23] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022]
Abstract
The platelet and megakaryocyte cytoskeletons are essential for formation and function of these cells. A dynamic, properly organised tubulin and actin cytoskeleton is critical for the development of the megakaryocyte and the extension of proplatelets. Tubulin in particular plays a pivotal role in the extension of these proplatelets and the release of platelets from them. Tubulin is further required for the maintenance of platelet size, and actin is the driving force for shape change, spreading and platelet contraction during platelet activation. Whilst several key proteins which regulate these cytoskeletons have been described in detail, the formin family of proteins has received less attention. Formins are intriguing as, although they were initially believed to simply be a nucleator of actin polymerisation, increasing evidence shows they are important regulators of the crosstalk between the actin and microtubule cytoskeletons. In this review, we will introduce the formin proteins and consider the recent evidence that they play an important role in platelets and megakaryocytes in mediating both the actin and tubulin cytoskeletons.
Collapse
Affiliation(s)
- Malou Zuidscherwoude
- a Institute of Cardiovascular Sciences , University of Birmingham , Birmingham , UK.,b Centre of Membrane Proteins and Receptors (COMPARE) , University of Birmingham and University of Nottingham , Midlands , UK
| | - Hannah L H Green
- a Institute of Cardiovascular Sciences , University of Birmingham , Birmingham , UK
| | - Steven G Thomas
- a Institute of Cardiovascular Sciences , University of Birmingham , Birmingham , UK.,b Centre of Membrane Proteins and Receptors (COMPARE) , University of Birmingham and University of Nottingham , Midlands , UK
| |
Collapse
|
|
45
|
Hua K, Ferland RJ. Primary cilia proteins: ciliary and extraciliary sites and functions. Cell Mol Life Sci 2018; 75:1521-1540. [PMID: 29305615 PMCID: PMC5899021 DOI: 10.1007/s00018-017-2740-5] [Citation(s) in RCA: 60] [Impact Index Per Article: 8.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/08/2017] [Revised: 12/21/2017] [Accepted: 12/27/2017] [Indexed: 02/07/2023]
Abstract
Primary cilia are immotile organelles known for their roles in development and cell signaling. Defects in primary cilia result in a range of disorders named ciliopathies. Because this organelle can be found singularly on almost all cell types, its importance extends to most organ systems. As such, elucidating the importance of the primary cilium has attracted researchers from all biological disciplines. As the primary cilia field expands, caution is warranted in attributing biological defects solely to the function of this organelle, since many of these "ciliary" proteins are found at other sites in cells and likely have non-ciliary functions. Indeed, many, if not all, cilia proteins have locations and functions outside the primary cilium. Extraciliary functions are known to include cell cycle regulation, cytoskeletal regulation, and trafficking. Cilia proteins have been observed in the nucleus, at the Golgi apparatus, and even in immune synapses of T cells (interestingly, a non-ciliated cell). Given the abundance of extraciliary sites and functions, it can be difficult to definitively attribute an observed phenotype solely to defective cilia rather than to some defective extraciliary function or a combination of both. Thus, extraciliary sites and functions of cilia proteins need to be considered, as well as experimentally determined. Through such consideration, we will understand the true role of the primary cilium in disease as compared to other cellular processes' influences in mediating disease (or through a combination of both). Here, we review a compilation of known extraciliary sites and functions of "cilia" proteins as a means to demonstrate the potential non-ciliary roles for these proteins.
Collapse
Affiliation(s)
- Kiet Hua
- Department of Neuroscience and Experimental Therapeutics, Albany Medical College, 47 New Scotland Avenue, MC-136, Albany, NY, 12208, USA.
| | - Russell J Ferland
- Department of Neuroscience and Experimental Therapeutics, Albany Medical College, 47 New Scotland Avenue, MC-136, Albany, NY, 12208, USA.
- Department of Neurology, Albany Medical College, Albany, NY, 12208, USA.
| |
Collapse
|
|
46
|
Influence of Adalimumab on the Expression Profile of Genes Associated with the Histaminergic System in the Skin Fibroblasts In Vitro. BIOMED RESEARCH INTERNATIONAL 2018; 2018:1582173. [PMID: 29487864 PMCID: PMC5816894 DOI: 10.1155/2018/1582173] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 09/11/2017] [Revised: 11/26/2017] [Accepted: 12/10/2017] [Indexed: 12/15/2022]
Abstract
Objective The aim of this study was to evaluate the influence of adalimumab on expression profile of genes associated with the histaminergic system in Normal Human Dermal Fibroblast (NHDF) cells stimulated with 8.00 μg/ml of adalimumab and the identification of miRNAs regulating these genes' expression. Methods NHDFs were cultured with or without the presence of adalimumab for 2, 8, and 24 hours. The expression profile of genes and miRNA were determined with the use of microarray technology. Results Among 22283 ID mRNA, 65 are associated with the histaminergic system. It can be observed that 15 mRNAs differentiate NHDFs cultures with adalimumab form control. The analysis of miRNAs showed that, among 1105 ID miRNA, 20 miRNAs are differentiating in cells treated with adalimumab for 2 hours, 9 miRNA after 8 hours, and only 3 miRNAs after 24 hours. Conclusion It was also determined that miRNAs play certain role in the regulation of the expression of genes associated with the histaminergic system. The results of this study confirmed the possibility of using both genes associated with this system as well as miRNAs regulating their expression, as complementary molecular markers of sensitivity to the adalimumab treatment.
Collapse
|
|
47
|
Messaoudi K, Ali A, Ishaq R, Palazzo A, Sliwa D, Bluteau O, Souquère S, Muller D, Diop KM, Rameau P, Lapierre V, Marolleau JP, Matthias P, Godin I, Pierron G, Thomas SG, Watson SP, Droin N, Vainchenker W, Plo I, Raslova H, Debili N. Critical role of the HDAC6-cortactin axis in human megakaryocyte maturation leading to a proplatelet-formation defect. Nat Commun 2017; 8:1786. [PMID: 29176689 PMCID: PMC5702605 DOI: 10.1038/s41467-017-01690-2] [Citation(s) in RCA: 34] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/20/2016] [Accepted: 10/10/2017] [Indexed: 01/08/2023] Open
Abstract
Thrombocytopenia is a major side effect of a new class of anticancer agents that target histone deacetylase (HDAC). Their mechanism is poorly understood. Here, we show that HDAC6 inhibition and genetic knockdown lead to a strong decrease in human proplatelet formation (PPF). Unexpectedly, HDAC6 inhibition-induced tubulin hyperacetylation has no effect on PPF. The PPF decrease induced by HDAC6 inhibition is related to cortactin (CTTN) hyperacetylation associated with actin disorganization inducing important changes in the distribution of megakaryocyte (MK) organelles. CTTN silencing in human MKs phenocopies HDAC6 inactivation and knockdown leads to a strong PPF defect. This is rescued by forced expression of a deacetylated CTTN mimetic. Unexpectedly, unlike human-derived MKs, HDAC6 and CTTN are shown to be dispensable for mouse PPF in vitro and platelet production in vivo. Our results highlight an unexpected function of HDAC6–CTTN axis as a positive regulator of human but not mouse MK maturation. Histone deacetylase (HDAC) inhibitors, a class of cancer therapeutics, cause thrombocytopenia via an unknown mechanism. Here, the authors show that HDAC6 inhibition impairs proplatelet formation in human megakaryocytes, and show that this is linked to hyperacetylation of the actin-binding protein cortactin.
Collapse
Affiliation(s)
- Kahia Messaoudi
- Institut National de la Santé et de la Recherche Médicale, UMR 1170, Equipe labellisée par la Ligue Nationale contre le Cancer, 94805, Villejuif, France.,Paris-Saclay University, UMR 1170, 94805, Villejuif, France.,Gustave Roussy, 94805, Villejuif, France.,Paris7 Diderot University, 75013, Paris, France
| | - Ashfaq Ali
- Institut National de la Santé et de la Recherche Médicale, UMR 1170, Equipe labellisée par la Ligue Nationale contre le Cancer, 94805, Villejuif, France.,Paris-Saclay University, UMR 1170, 94805, Villejuif, France.,Gustave Roussy, 94805, Villejuif, France.,Paris7 Diderot University, 75013, Paris, France
| | - Rameez Ishaq
- Institut National de la Santé et de la Recherche Médicale, UMR 1170, Equipe labellisée par la Ligue Nationale contre le Cancer, 94805, Villejuif, France.,Paris-Saclay University, UMR 1170, 94805, Villejuif, France.,Gustave Roussy, 94805, Villejuif, France.,Paris7 Diderot University, 75013, Paris, France
| | - Alberta Palazzo
- Institut National de la Santé et de la Recherche Médicale, UMR 1170, Equipe labellisée par la Ligue Nationale contre le Cancer, 94805, Villejuif, France.,Paris-Saclay University, UMR 1170, 94805, Villejuif, France.,Gustave Roussy, 94805, Villejuif, France.,Paris7 Diderot University, 75013, Paris, France
| | - Dominika Sliwa
- Institut National de la Santé et de la Recherche Médicale, UMR 1170, Equipe labellisée par la Ligue Nationale contre le Cancer, 94805, Villejuif, France.,Paris-Saclay University, UMR 1170, 94805, Villejuif, France.,Gustave Roussy, 94805, Villejuif, France
| | - Olivier Bluteau
- Institut National de la Santé et de la Recherche Médicale, UMR 1170, Equipe labellisée par la Ligue Nationale contre le Cancer, 94805, Villejuif, France.,Paris-Saclay University, UMR 1170, 94805, Villejuif, France.,Gustave Roussy, 94805, Villejuif, France
| | - Sylvie Souquère
- CNRS-UMR-9196, Institut Gustave Roussy, 94805, Villejuif, France
| | - Delphine Muller
- Institut National de la Santé et de la Recherche Médicale, UMR 1170, Equipe labellisée par la Ligue Nationale contre le Cancer, 94805, Villejuif, France.,Paris-Saclay University, UMR 1170, 94805, Villejuif, France.,Gustave Roussy, 94805, Villejuif, France
| | - Khadija M Diop
- Genomic Platform, Institut Gustave Roussy, 94805, Villejuif, France
| | - Philippe Rameau
- Gustave Roussy, Integrated Biology Core Facility, 94805, Villejuif, France
| | | | - Jean-Pierre Marolleau
- Clinical Hematology and Cell Therapy Department, Amiens Hospital, UPJV University EA4666, 80054, Amiens, France
| | - Patrick Matthias
- Friedrich Miescher Institute for Biomedical Research, 4002, Basel, Switzerland
| | - Isabelle Godin
- Institut National de la Santé et de la Recherche Médicale, UMR 1170, Equipe labellisée par la Ligue Nationale contre le Cancer, 94805, Villejuif, France.,Paris-Saclay University, UMR 1170, 94805, Villejuif, France.,Gustave Roussy, 94805, Villejuif, France
| | - Gérard Pierron
- CNRS-UMR-9196, Institut Gustave Roussy, 94805, Villejuif, France
| | - Steven G Thomas
- Institute of Cardiovascular Sciences, The Medical School, University of Birmingham, Edgbaston, Birmingham, B15 2TT, UK.,Centre of Membrane Proteins and Receptors (COMPARE), Universities of Birmingham and Nottingham, Midlands, UK
| | - Stephen P Watson
- Institute of Cardiovascular Sciences, The Medical School, University of Birmingham, Edgbaston, Birmingham, B15 2TT, UK.,Centre of Membrane Proteins and Receptors (COMPARE), Universities of Birmingham and Nottingham, Midlands, UK
| | - Nathalie Droin
- Institut National de la Santé et de la Recherche Médicale, UMR 1170, Equipe labellisée par la Ligue Nationale contre le Cancer, 94805, Villejuif, France.,Paris-Saclay University, UMR 1170, 94805, Villejuif, France.,Gustave Roussy, 94805, Villejuif, France.,Genomic Platform, Institut Gustave Roussy, 94805, Villejuif, France
| | - William Vainchenker
- Institut National de la Santé et de la Recherche Médicale, UMR 1170, Equipe labellisée par la Ligue Nationale contre le Cancer, 94805, Villejuif, France.,Paris-Saclay University, UMR 1170, 94805, Villejuif, France.,Gustave Roussy, 94805, Villejuif, France
| | - Isabelle Plo
- Institut National de la Santé et de la Recherche Médicale, UMR 1170, Equipe labellisée par la Ligue Nationale contre le Cancer, 94805, Villejuif, France.,Paris-Saclay University, UMR 1170, 94805, Villejuif, France.,Gustave Roussy, 94805, Villejuif, France
| | - Hana Raslova
- Institut National de la Santé et de la Recherche Médicale, UMR 1170, Equipe labellisée par la Ligue Nationale contre le Cancer, 94805, Villejuif, France.,Paris-Saclay University, UMR 1170, 94805, Villejuif, France.,Gustave Roussy, 94805, Villejuif, France
| | - Najet Debili
- Institut National de la Santé et de la Recherche Médicale, UMR 1170, Equipe labellisée par la Ligue Nationale contre le Cancer, 94805, Villejuif, France. .,Paris-Saclay University, UMR 1170, 94805, Villejuif, France. .,Gustave Roussy, 94805, Villejuif, France.
| |
Collapse
|
|
48
|
Abstract
PURPOSE OF REVIEW Inherited thrombocytopenias are a heterogeneous group of diseases caused by mutations in many genes. They account for approximately only 50% of cases, suggesting that novel genes have yet to be identified for a comprehensive understanding of platelet biogenesis defects. This review provides an update of the last year of discoveries on inherited thrombocytopenias focusing on the molecular basis and potential pathogenic mechanisms affecting megakaryopoiesis and platelet production. RECENT FINDINGS Most of the novel discoveries are related to identification of mutations in novel inherited thrombocytopenia genes using a next-generation sequencing approach. They include MECOM, DIAPH1, TRPM7, SRC, FYB, and SLFN14, playing different roles in megakaryopoiesis and platelet production. Moreover, it is worth mentioning data on hypomorphic mutations of FLI1 and the association of single nucleotide polymorphisms, such as that identified in ACTN1, with thrombocytopenia. SUMMARY Thanks to the application of next-generation sequencing, the number of inherited thrombocytopenia genes is going to increase rapidly. Considering the wide genetic heterogeneity (more than 30 genes), these technologies can also be used for diagnostic purpose. Whatever is the aim, extreme caution should be taken in interpreting data, as inherited thrombocytopenias are mainly autosomal dominant diseases caused by variants of apparent unknown significance.
Collapse
|
|
49
|
Ganaha A, Kaname T, Shinjou A, Chinen Y, Yanagi K, Higa T, Kondo S, Suzuki M. Progressive macrothrombocytopenia and hearing loss in a large family with DIAPH1
related disease. Am J Med Genet A 2017; 173:2826-2830. [DOI: 10.1002/ajmg.a.38411] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/01/2017] [Revised: 07/27/2017] [Accepted: 07/29/2017] [Indexed: 01/17/2023]
Affiliation(s)
- Akira Ganaha
- Department of Otorhinolaryngology-Head and Neck Surgery; University of the Ryukyus; Okinawa Japan
| | - Tadashi Kaname
- Department of Genome Medicine; National Center for Child Health and Development; Tokyo Japan
| | - Ayano Shinjou
- Department of Otorhinolaryngology-Head and Neck Surgery; University of the Ryukyus; Okinawa Japan
| | - Yasutsugu Chinen
- Department of Pediatrics; Faculty of Medicine; University of the Ryukyus; Nishihara Japan
| | - Kumiko Yanagi
- Department of Genome Medicine; National Center for Child Health and Development; Tokyo Japan
| | - Teruyuki Higa
- Department of Otorhinolaryngology-Head and Neck Surgery; University of the Ryukyus; Okinawa Japan
| | - Shunsuke Kondo
- Department of Otorhinolaryngology-Head and Neck Surgery; University of the Ryukyus; Okinawa Japan
| | - Mikio Suzuki
- Department of Otorhinolaryngology-Head and Neck Surgery; University of the Ryukyus; Okinawa Japan
| |
Collapse
|
|
50
|
De Conto F, Fazzi A, Razin SV, Arcangeletti MC, Medici MC, Belletti S, Chezzi C, Calderaro A. Mammalian Diaphanous-related formin-1 restricts early phases of influenza A/NWS/33 virus (H1N1) infection in LLC-MK2 cells by affecting cytoskeleton dynamics. Mol Cell Biochem 2017; 437:185-201. [PMID: 28744815 DOI: 10.1007/s11010-017-3107-9] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/13/2017] [Accepted: 07/01/2017] [Indexed: 12/15/2022]
Abstract
Viruses depend on cellular machinery to efficiently replicate. The host cytoskeleton is one of the first cellular systems hijacked by viruses in order to ensure their intracellular transport and promote the development of infection. Our previous results demonstrated that stable microfilaments and microtubules interfered with human influenza A/NWS/33 virus (H1N1) infection in semi-permissive LLC-MK2 cells. Although formins play a key role in cytoskeletal remodelling, few studies addressed a possible role of these proteins in development of viral infection. Here, we have demonstrated that mammalian Diaphanous-related formin-1 (mDia1) is involved in the control of cytoskeleton dynamics during human influenza A virus infection. First, by employing cytoskeleton-perturbing drugs, we evidenced a cross-talk occurring between microtubules and microfilaments that also has implications on the intracellular localization of mDia1. In influenza A/NWS/33 virus-infected LLC-MK2 cells, mDia1 showed a highly dynamic intracellular localization and partially co-localized with actin and tubulin. A depletion of mDia1 by RNA-mediated RNA interference was found to improve the outcome of influenza A/NWS/33 virus infection and to increase the dynamics of microfilament and microtubule networks in LLC-MK2 cells. Consistent with these findings, observations made in epithelial respiratory cells from paediatric patients with acute respiratory disease assessed that the expression of mDia1 is stimulated by influenza A virus but not by respiratory syncytial virus. Taken together, the obtained results suggest that mDia1 restricts the initiation of influenza A/NWS/33 virus infection in LLC-MK2 cells by counteracting cytoskeletal dynamics.
Collapse
Affiliation(s)
- Flora De Conto
- Department of Medicine and Surgery, University of Parma, Parma, Italy.
| | - Alessandra Fazzi
- Department of Medicine and Surgery, University of Parma, Parma, Italy
| | - Sergey V Razin
- Institute of Gene Biology, Russian Academy of Sciences and Lomonosow Moscow State University, Moscow, Russia
| | | | | | - Silvana Belletti
- Department of Medicine and Surgery, University of Parma, Parma, Italy
| | - Carlo Chezzi
- Department of Medicine and Surgery, University of Parma, Parma, Italy
| | - Adriana Calderaro
- Department of Medicine and Surgery, University of Parma, Parma, Italy
| |
Collapse
|