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Coates BS, Walden KKO, Lata D, Vellichirammal NN, Mitchell RF, Andersson MN, McKay R, Lorenzen MD, Grubbs N, Wang YH, Han J, Xuan JL, Willadsen P, Wang H, French BW, Bansal R, Sedky S, Souza D, Bunn D, Meinke LJ, Miller NJ, Siegfried BD, Sappington TW, Robertson HM. A draft Diabrotica virgifera virgifera genome: insights into control and host plant adaption by a major maize pest insect. BMC Genomics 2023; 24:19. [PMID: 36639634 PMCID: PMC9840275 DOI: 10.1186/s12864-022-08990-y] [Citation(s) in RCA: 11] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/21/2022] [Accepted: 11/04/2022] [Indexed: 01/15/2023] Open
Abstract
BACKGROUND Adaptations by arthropod pests to host plant defenses of crops determine their impacts on agricultural production. The larval host range of western corn rootworm, Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae), is restricted to maize and a few grasses. Resistance of D. v. virgifera to crop rotation practices and multiple insecticides contributes to its status as the most damaging pest of cultivated maize in North America and Europe. The extent to which adaptations by this pest contributes to host plant specialization remains unknown. RESULTS A 2.42 Gb draft D. v. virgifera genome, Dvir_v2.0, was assembled from short shotgun reads and scaffolded using long-insert mate-pair, transcriptome and linked read data. K-mer analysis predicted a repeat content of ≥ 61.5%. Ortholog assignments for Dvir_2.0 RefSeq models predict a greater number of species-specific gene duplications, including expansions in ATP binding cassette transporter and chemosensory gene families, than in other Coleoptera. A majority of annotated D. v. virgifera cytochrome P450s belong to CYP4, 6, and 9 clades. A total of 5,404 transcripts were differentially-expressed between D. v. virgifera larvae fed maize roots compared to alternative host (Miscanthus), a marginal host (Panicum virgatum), a poor host (Sorghum bicolor) and starvation treatments; Among differentially-expressed transcripts, 1,908 were shared across treatments and the least number were between Miscanthus compared to maize. Differentially-expressed transcripts were enriched for putative spliceosome, proteosome, and intracellular transport functions. General stress pathway functions were unique and enriched among up-regulated transcripts in marginal host, poor host, and starvation responses compared to responses on primary (maize) and alternate hosts. CONCLUSIONS Manual annotation of D. v. virgifera Dvir_2.0 RefSeq models predicted expansion of paralogs with gene families putatively involved in insecticide resistance and chemosensory perception. Our study also suggests that adaptations of D. v. virgifera larvae to feeding on an alternate host plant invoke fewer transcriptional changes compared to marginal or poor hosts. The shared up-regulation of stress response pathways between marginal host and poor host, and starvation treatments may reflect nutrient deprivation. This study provides insight into transcriptomic responses of larval feeding on different host plants and resources for genomic research on this economically significant pest of maize.
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Affiliation(s)
- Brad S. Coates
- grid.508983.fCorn Insects & Crop Genetics Research Unit, USDA-ARS, 2310 Pammel Dr, 532 Science II, Iowa State University, Ames, IA 50011 USA
| | - Kimberly K. O. Walden
- grid.35403.310000 0004 1936 9991Roy J. Carver Biotechnology Center, University of Illinois at Champaign-Urbana, Urbana, IL USA
| | - Dimpal Lata
- grid.62813.3e0000 0004 1936 7806Department of Biology, Illinois Institute of Technology, Chicago, IL USA
| | | | - Robert F. Mitchell
- grid.267474.40000 0001 0674 4543University of Wisconsin Oshkosh, Oshkosh, WI USA
| | - Martin N. Andersson
- grid.4514.40000 0001 0930 2361Department of Biology, Lund University, Lund, Sweden
| | - Rachel McKay
- grid.267474.40000 0001 0674 4543University of Wisconsin Oshkosh, Oshkosh, WI USA
| | - Marcé D. Lorenzen
- grid.40803.3f0000 0001 2173 6074Department of Entomology and Plant Pathology, North Carolina State University, Raleigh, NC USA
| | - Nathaniel Grubbs
- grid.40803.3f0000 0001 2173 6074Department of Entomology and Plant Pathology, North Carolina State University, Raleigh, NC USA
| | - Yu-Hui Wang
- grid.40803.3f0000 0001 2173 6074Department of Entomology and Plant Pathology, North Carolina State University, Raleigh, NC USA
| | - Jinlong Han
- grid.40803.3f0000 0001 2173 6074Department of Entomology and Plant Pathology, North Carolina State University, Raleigh, NC USA
| | - Jing Li Xuan
- grid.40803.3f0000 0001 2173 6074Department of Entomology and Plant Pathology, North Carolina State University, Raleigh, NC USA
| | - Peter Willadsen
- grid.40803.3f0000 0001 2173 6074Department of Entomology and Plant Pathology, North Carolina State University, Raleigh, NC USA
| | - Huichun Wang
- grid.24434.350000 0004 1937 0060Department of Entomology, University of Nebraska, Lincoln, NE USA
| | - B. Wade French
- grid.508981.dIntegrated Crop Systems Research Unit, USDA-ARS, Brookings, SD USA
| | - Raman Bansal
- grid.512850.bUSDA-ARS, San Joaquin Valley Agricultural Sciences Center, Parlier, CA USA
| | - Sammy Sedky
- grid.512850.bUSDA-ARS, San Joaquin Valley Agricultural Sciences Center, Parlier, CA USA
| | - Dariane Souza
- grid.15276.370000 0004 1936 8091Department of Entomology, University of Florida, Gainesville, FL USA
| | - Dakota Bunn
- grid.62813.3e0000 0004 1936 7806Department of Biology, Illinois Institute of Technology, Chicago, IL USA
| | - Lance J. Meinke
- grid.24434.350000 0004 1937 0060Department of Entomology, University of Nebraska, Lincoln, NE USA
| | - Nicholas J. Miller
- grid.62813.3e0000 0004 1936 7806Department of Biology, Illinois Institute of Technology, Chicago, IL USA
| | - Blair D. Siegfried
- grid.15276.370000 0004 1936 8091Department of Entomology, University of Florida, Gainesville, FL USA
| | - Thomas W. Sappington
- grid.508983.fCorn Insects & Crop Genetics Research Unit, USDA-ARS, 2310 Pammel Dr, 532 Science II, Iowa State University, Ames, IA 50011 USA
| | - Hugh M. Robertson
- grid.35403.310000 0004 1936 9991Department of Entomology, University of Illinois at Champaign-Urbana, Urbana, IL USA
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Kuznetsov AV, Javadov S, Grimm M, Margreiter R, Ausserlechner MJ, Hagenbuchner J. Crosstalk between Mitochondria and Cytoskeleton in Cardiac Cells. Cells 2020; 9:cells9010222. [PMID: 31963121 PMCID: PMC7017221 DOI: 10.3390/cells9010222] [Citation(s) in RCA: 53] [Impact Index Per Article: 10.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/03/2019] [Revised: 01/10/2020] [Accepted: 01/13/2020] [Indexed: 12/28/2022] Open
Abstract
Elucidation of the mitochondrial regulatory mechanisms for the understanding of muscle bioenergetics and the role of mitochondria is a fundamental problem in cellular physiology and pathophysiology. The cytoskeleton (microtubules, intermediate filaments, microfilaments) plays a central role in the maintenance of mitochondrial shape, location, and motility. In addition, numerous interactions between cytoskeletal proteins and mitochondria can actively participate in the regulation of mitochondrial respiration and oxidative phosphorylation. In cardiac and skeletal muscles, mitochondrial positions are tightly fixed, providing their regular arrangement and numerous interactions with other cellular structures such as sarcoplasmic reticulum and cytoskeleton. This can involve association of cytoskeletal proteins with voltage-dependent anion channel (VDAC), thereby, governing the permeability of the outer mitochondrial membrane (OMM) to metabolites, and regulating cell energy metabolism. Cardiomyocytes and myocardial fibers demonstrate regular arrangement of tubulin beta-II isoform entirely co-localized with mitochondria, in contrast to other isoforms of tubulin. This observation suggests the participation of tubulin beta-II in the regulation of OMM permeability through interaction with VDAC. The OMM permeability is also regulated by the specific isoform of cytolinker protein plectin. This review summarizes and discusses previous studies on the role of cytoskeletal proteins in the regulation of energy metabolism and mitochondrial function, adenosine triphosphate (ATP) production, and energy transfer.
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Affiliation(s)
- Andrey V. Kuznetsov
- Cardiac Surgery Research Laboratory, Department of Cardiac Surgery, Innsbruck Medical University, 6020 Innsbruck, Austria;
- Department of Paediatrics I, Medical University of Innsbruck, 6020 Innsbruck, Austria;
- Correspondence: (A.V.K.); (J.H.); Tel.: +43-512-504-27815 (A.V.K.); +43-512-504-81578 (J.H.)
| | - Sabzali Javadov
- Department of Physiology, School of Medicine, University of Puerto Rico, San Juan, PR 00936-5067, USA;
| | - Michael Grimm
- Cardiac Surgery Research Laboratory, Department of Cardiac Surgery, Innsbruck Medical University, 6020 Innsbruck, Austria;
| | - Raimund Margreiter
- Department of Visceral, Transplant and Thoracic Surgery, Medical University of Innsbruck, 6020 Innsbruck, Austria;
| | | | - Judith Hagenbuchner
- Department of Paediatrics II, Medical University of Innsbruck, 6020 Innsbruck, Austria
- Correspondence: (A.V.K.); (J.H.); Tel.: +43-512-504-27815 (A.V.K.); +43-512-504-81578 (J.H.)
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Tian X, Wang Y, Fan X, Shi Y, Zhang W, Hou Q, Liu R, Zhou G. Expression of Pork Plectin during Postmortem Aging. JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY 2019; 67:11718-11727. [PMID: 31518118 DOI: 10.1021/acs.jafc.9b03040] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/10/2023]
Abstract
The current study investigated the distribution and degradation of pork plectin during postmortem aging. Longissimus thoracis (LT) muscles from 12 pig carcasses were vacuum-packaged and aged at 4 °C for 0 h, 6 h, 12 h, 1 day, 3 days, 7 days, and 13 days. Immunofluorescence analysis showed that pork plectin was distributed in a honeycomb-like pattern in the cross section and a regularly striated pattern in the longitudinal section. However, plectin was found preferentially expressed in fibers that were stained with high anti-fast-MyHC. Double immunostaining revealed the colocalization of plectin and desmin in the cytoplasm and beneath the sarcolemma. Western blot analysis showed that the amount of intact plectin was rapidly reduced during the early postmortem aging (P < 0.05) and almost disappeared at day 3. The degraded 240 kDa plectin accumulated fast and was further cleaved after 3 days of aging (P < 0.05). The plectin degradation could be significantly blocked by calpain inhibitor MDL-28170 rather than caspase-3 inhibitor Ac-DEVD-CHO (P < 0.05). Double immunostaining of μ-calpain and plectin showed a large amount of overlap at 0 h and 3 days of postmortem. Accordingly, these findings showed that plectin was preferentially expressed in fast muscle fiber and regularly distributed along with desmin at the strategic cellular sites. Plectin suffered a prominent and prompt degradation during postmortem aging, which might be attributed to μ-calpain.
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Affiliation(s)
- Xiaona Tian
- Key Laboratory of Meat Processing and Quality Control, MOE, Key Laboratory of Meat Processing, MOA, Jiangsu Synergetic Innovation Center of Meat Processing and Quality Control, College of Food Science and Technology , Nanjing Agricultural University , Nanjing 210095 , P. R. China
| | - Yingying Wang
- Key Laboratory of Meat Processing and Quality Control, MOE, Key Laboratory of Meat Processing, MOA, Jiangsu Synergetic Innovation Center of Meat Processing and Quality Control, College of Food Science and Technology , Nanjing Agricultural University , Nanjing 210095 , P. R. China
| | - Xiaoquan Fan
- Key Laboratory of Meat Processing and Quality Control, MOE, Key Laboratory of Meat Processing, MOA, Jiangsu Synergetic Innovation Center of Meat Processing and Quality Control, College of Food Science and Technology , Nanjing Agricultural University , Nanjing 210095 , P. R. China
| | - Yingwu Shi
- Key Laboratory of Meat Processing and Quality Control, MOE, Key Laboratory of Meat Processing, MOA, Jiangsu Synergetic Innovation Center of Meat Processing and Quality Control, College of Food Science and Technology , Nanjing Agricultural University , Nanjing 210095 , P. R. China
| | - Wangang Zhang
- Key Laboratory of Meat Processing and Quality Control, MOE, Key Laboratory of Meat Processing, MOA, Jiangsu Synergetic Innovation Center of Meat Processing and Quality Control, College of Food Science and Technology , Nanjing Agricultural University , Nanjing 210095 , P. R. China
| | - Qin Hou
- Key Laboratory of Meat Processing and Quality Control, MOE, Key Laboratory of Meat Processing, MOA, Jiangsu Synergetic Innovation Center of Meat Processing and Quality Control, College of Food Science and Technology , Nanjing Agricultural University , Nanjing 210095 , P. R. China
| | - Rui Liu
- College of Food Science and Engineering , Yangzhou University , Yangzhou 225127 , Jiangsu , China
| | - Guanghong Zhou
- Key Laboratory of Meat Processing and Quality Control, MOE, Key Laboratory of Meat Processing, MOA, Jiangsu Synergetic Innovation Center of Meat Processing and Quality Control, College of Food Science and Technology , Nanjing Agricultural University , Nanjing 210095 , P. R. China
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Pressler K, Mitterer F, Vorkapic D, Reidl J, Oberer M, Schild S. Characterization of Vibrio cholerae's Extracellular Nuclease Xds. Front Microbiol 2019; 10:2057. [PMID: 31551990 PMCID: PMC6746945 DOI: 10.3389/fmicb.2019.02057] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/30/2019] [Accepted: 08/20/2019] [Indexed: 12/22/2022] Open
Abstract
The Gram-negative bacterium Vibrio cholerae encodes two nucleases, Dns and Xds, which play a major role during the human pathogen's lifecycle. Dns and Xds control three-dimensional biofilm formation and bacterial detachment from biofilms via degradation of extracellular DNA and thus contribute to the environmental, inter-epidemic persistence of the pathogen. During intestinal colonization the enzymes help evade the innate immune response, and therefore promote survival by mediating escape from neutrophil extracellular traps. Xds has the additional function of degrading extracellular DNA down to nucleotides, which are an important nutrient source for V. cholerae. Thus, Xds is a key enzyme for survival fitness during distinct stages of the V. cholerae lifecycle and could be a potential therapeutic target. This study provides detailed information about the enzymatic properties of Xds using purified protein in combination with a real time nuclease activity assay. The data define an optimal buffer composition for Xds activity as 50 mM Tris/HCl pH 7, 100 mM NaCl, 10 mM MgCl2, and 20 mM CaCl2. Moreover, maximal activity was observed using substrate DNA with low GC content and ambient temperatures of 20-25°C. In silico analysis and homology modeling predicted an exonuclease domain in the C-terminal part of the protein. Biochemical analyses with truncated variants and point mutants of Xds confirm that the C-terminal region is sufficient for nuclease activity. We also find that residues D787 and H837 within the predicted exonuclease domain are key to formation of the catalytic center.
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Affiliation(s)
| | - Fabian Mitterer
- Institute of Molecular Biosciences, University of Graz, Graz, Austria
| | - Dina Vorkapic
- Institute of Molecular Biosciences, University of Graz, Graz, Austria
| | - Joachim Reidl
- Institute of Molecular Biosciences, University of Graz, Graz, Austria
- BioTechMed-Graz, Graz, Austria
| | - Monika Oberer
- Institute of Molecular Biosciences, University of Graz, Graz, Austria
- BioTechMed-Graz, Graz, Austria
| | - Stefan Schild
- Institute of Molecular Biosciences, University of Graz, Graz, Austria
- BioTechMed-Graz, Graz, Austria
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Sonam S, Srnak JA, Perry KJ, Henry JJ. Molecular markers for corneal epithelial cells in larval vs. adult Xenopus frogs. Exp Eye Res 2019; 184:107-125. [PMID: 30981716 DOI: 10.1016/j.exer.2019.04.010] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/11/2019] [Accepted: 04/08/2019] [Indexed: 12/14/2022]
Abstract
Corneal Epithelial Stem Cells (CESCs) and their proliferative progeny, the Transit Amplifying Cells (TACs), are responsible for maintaining the integrity and transparency of the cornea. These stem cells (SCs) are widely used in corneal transplants and ocular surface reconstruction. Molecular markers are essential to identify, isolate and enrich for these cells, yet no definitive CESC marker has been established. An extensive literature survey shows variability in the expression of putative CESC markers among vertebrates; being attributed to species-specific variations, or other differences in developmental stages of these animals, approaches used in these studies and marker specificity. Here, we expanded the search for CESC markers using the amphibian model Xenopus laevis. In previous studies we found that long-term label retaining cells (suggestive of CESCs and TACs) are present throughout the larval basal corneal epithelium. In adult frogs, these cells become concentrated in the peripheral cornea (limbal region). Here, we used immunofluorescence to characterize the expression of nine proteins in the corneas of both Xenopus larvae and adults (post-metamorphic). We found that localization of some markers change between larval and adult stages. Markers such as p63, Keratin 19, and β1-integrin are restricted to basal corneal epithelial cells of the larvae. After metamorphosis their expression is found in basal and intermediate layer cells of the adult frog corneal epithelium. Another protein, Pax6 was expressed in the larval corneas, but surprisingly it was not detected in the adult corneal epithelium. For the first time we report that Tcf7l2 can be used as a marker to differentiate cornea vs. skin in frogs. Tcf7l2 is present only in the frog skin, which differs from reports indicating that the protein is expressed in the human cornea. Furthermore, we identified the transition between the inner, and the outer surface of the adult frog eyelid as a key boundary in terms of marker expression. Although these markers are useful to identify different regions and cellular layers of the frog corneal epithelium, none is unique to CESCs or TACs. Our results confirm that there is no single conserved CESC marker in vertebrates. This molecular characterization of the Xenopus cornea facilitates its use as a vertebrate model to understand the functions of key proteins in corneal homeostasis and wound repair.
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Affiliation(s)
- Surabhi Sonam
- Department of Cell and Developmental Biology, University of Illinois, 601 S. Goodwin Avenue, Urbana, IL, 61801, USA
| | - Jennifer A Srnak
- Department of Cell and Developmental Biology, University of Illinois, 601 S. Goodwin Avenue, Urbana, IL, 61801, USA
| | - Kimberly J Perry
- Department of Cell and Developmental Biology, University of Illinois, 601 S. Goodwin Avenue, Urbana, IL, 61801, USA
| | - Jonathan J Henry
- Department of Cell and Developmental Biology, University of Illinois, 601 S. Goodwin Avenue, Urbana, IL, 61801, USA.
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Abstract
Many diseases are related to age, among these neurodegeneration is particularly important. Alzheimer's disease Parkinson's and Glaucoma have many common pathogenic events including oxidative damage, Mitochondrial dysfunction, endothelial alterations and changes in the visual field. These are well known in the case of glaucoma, less in the case of neurodegeneration of the brain. Many other molecular aspects are common, such as the role of endoplasmic reticulum autophagy and neuronal apoptosis while others have been neglected due to lack of space such as inflammatory cytokine or miRNA. Moreover, the loss of specific neuronal populations, the induction of similar mechanisms of cell injury and the deposition of protein aggregates in specific anatomical areas are very similar events between these diseases. Intracellular and/or extracellular accumulation of protein aggregates is a key feature of many neurodegenerative disorders. The existence of abnormal protein aggregates has been documented in the RGCs of glaucomatous patients such as the anomalous Tau protein or the β-amyloid accumulations. Intra-cell catabolic processes also appear to be common in both glaucoma and neurodegeneration. They also help us to understand how the basis between these diseases is common and how the visual aspects can be a serious problem for those who are affected.
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Affiliation(s)
- Sergio Claudio Saccà
- Department of Head/Neck Pathologies, St Martino Hospital, Ophthalmology Unit, Genoa, Italy.
| | - Carlo Alberto Cutolo
- Department of Neuroscience, Rehabilitation, Ophthalmology, Genetics and Maternal and Child Science, University of Genoa, Policlinico San Martino Hospital, Eye Clinic Genoa, Genoa, Italy
| | - Tommaso Rossi
- Department of Head/Neck Pathologies, St Martino Hospital, Ophthalmology Unit, Genoa, Italy
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Miao Z, Ali A, Hu L, Zhao F, Yin C, Chen C, Yang T, Qian A. Microtubule actin cross-linking factor 1, a novel potential target in cancer. Cancer Sci 2017; 108:1953-1958. [PMID: 28782898 PMCID: PMC5623738 DOI: 10.1111/cas.13344] [Citation(s) in RCA: 38] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/20/2017] [Revised: 07/28/2017] [Accepted: 08/02/2017] [Indexed: 01/09/2023] Open
Abstract
Cancer is a polygenic disease characterized by uncontrolled growth of normal body cells, deregulation of the cell cycle as well as resistance to apoptosis. The spectraplakin protein microtubule actin cross-linking factor 1 (MACF1) plays an essential function in various cellular processes, including cell proliferation, migration, signaling transduction and embryo development. MACF1 is also involved in processes such as metastatic invasion in which cytoskeleton organization is a critical element that contributes to tumor progression in various human cancers. Aberrant expression of MACF1 initiates the tumor cell proliferation, and migration and metastasis in numerous cancers, such as breast cancer, colon cancer, lung cancer and glioblastoma. In this review, we summarized the current knowledge of MACF1 and its critical role in different human cancers. This will be helpful for researchers to investigate the novel functional role of MACF1 in human cancers and as a potential target to enhance the efficacy of therapeutic treatment modalities.
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Affiliation(s)
- Zhiping Miao
- Laboratory for Bone Metabolism, Key Laboratory for Space Bioscience and Biotechnology, School of Life Sciences, Northwestern Polytechnical University, Xi'an, Shaanxi, China.,Shenzhen Research Institution of Northwestern Polytechnical University, Shenzhen, China
| | - Arshad Ali
- Laboratory for Bone Metabolism, Key Laboratory for Space Bioscience and Biotechnology, School of Life Sciences, Northwestern Polytechnical University, Xi'an, Shaanxi, China.,Shenzhen Research Institution of Northwestern Polytechnical University, Shenzhen, China
| | - Lifang Hu
- Laboratory for Bone Metabolism, Key Laboratory for Space Bioscience and Biotechnology, School of Life Sciences, Northwestern Polytechnical University, Xi'an, Shaanxi, China.,Shenzhen Research Institution of Northwestern Polytechnical University, Shenzhen, China
| | - Fan Zhao
- Laboratory for Bone Metabolism, Key Laboratory for Space Bioscience and Biotechnology, School of Life Sciences, Northwestern Polytechnical University, Xi'an, Shaanxi, China.,Shenzhen Research Institution of Northwestern Polytechnical University, Shenzhen, China
| | - Chong Yin
- Laboratory for Bone Metabolism, Key Laboratory for Space Bioscience and Biotechnology, School of Life Sciences, Northwestern Polytechnical University, Xi'an, Shaanxi, China.,Shenzhen Research Institution of Northwestern Polytechnical University, Shenzhen, China
| | - Chu Chen
- Hong-Hui Hospital, Xi'an Jiaotong University College of Medicine, Xi'an, Shaanxi, China
| | - Tuanmin Yang
- Hong-Hui Hospital, Xi'an Jiaotong University College of Medicine, Xi'an, Shaanxi, China
| | - Airong Qian
- Laboratory for Bone Metabolism, Key Laboratory for Space Bioscience and Biotechnology, School of Life Sciences, Northwestern Polytechnical University, Xi'an, Shaanxi, China.,Shenzhen Research Institution of Northwestern Polytechnical University, Shenzhen, China
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Ali A, Hu L, Zhao F, Qiu W, Wang P, Ma X, Zhang Y, Chen L, Qian A. BPAG1, a distinctive role in skin and neurological diseases. Semin Cell Dev Biol 2017. [PMID: 28627382 DOI: 10.1016/j.semcdb.2017.06.005] [Citation(s) in RCA: 18] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
Spectraplakins are multifunctional cytoskeletal linker proteins that act as important communicators, connecting cytoskeletal components with each other and to cellular junctions. Bullous pemphigoid antigen 1 (BPAG1)/dystonin is a member of spectraplakin family and expressed in various tissues. Alternative splicing of BPAG1 gene produces various isoforms with unique structure and domains. BPAG1 plays crucial roles in numerous biological processes, such as cytoskeleton organization, cell polarization, cell adhesion, and cell migration as well as signaling transduction. Genetic mutation of BPAG1 isoforms is the miscreant of epidermolysis bullosa and multifarious, destructive neurological diseases. In this review, we summarize the recent advances of BPAG1's role in various biological processes and in skin and neurological diseases.
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Affiliation(s)
- Arshad Ali
- Laboratory for Bone Metabolism, Key Laboratory for Space Bioscience and Biotechnology, School of Life Sciences, Northwestern Polytechnical University, Xi'an, Shaanxi 710072, PR China; Shenzhen Research Institution of Northwestern Polytechnical University, Shenzhen, 518057, PR China; Northwestern Polytechnical University-Hong Kong Baptist University Joint Research Centre for Translational Medicine on Musculoskeletal Health in Space, Xi'an, 710072, PR China
| | - Lifang Hu
- Laboratory for Bone Metabolism, Key Laboratory for Space Bioscience and Biotechnology, School of Life Sciences, Northwestern Polytechnical University, Xi'an, Shaanxi 710072, PR China; Shenzhen Research Institution of Northwestern Polytechnical University, Shenzhen, 518057, PR China; Northwestern Polytechnical University-Hong Kong Baptist University Joint Research Centre for Translational Medicine on Musculoskeletal Health in Space, Xi'an, 710072, PR China
| | - Fan Zhao
- Laboratory for Bone Metabolism, Key Laboratory for Space Bioscience and Biotechnology, School of Life Sciences, Northwestern Polytechnical University, Xi'an, Shaanxi 710072, PR China; Shenzhen Research Institution of Northwestern Polytechnical University, Shenzhen, 518057, PR China; Northwestern Polytechnical University-Hong Kong Baptist University Joint Research Centre for Translational Medicine on Musculoskeletal Health in Space, Xi'an, 710072, PR China
| | - Wuxia Qiu
- Laboratory for Bone Metabolism, Key Laboratory for Space Bioscience and Biotechnology, School of Life Sciences, Northwestern Polytechnical University, Xi'an, Shaanxi 710072, PR China; Shenzhen Research Institution of Northwestern Polytechnical University, Shenzhen, 518057, PR China; Northwestern Polytechnical University-Hong Kong Baptist University Joint Research Centre for Translational Medicine on Musculoskeletal Health in Space, Xi'an, 710072, PR China
| | - Pai Wang
- Laboratory for Bone Metabolism, Key Laboratory for Space Bioscience and Biotechnology, School of Life Sciences, Northwestern Polytechnical University, Xi'an, Shaanxi 710072, PR China; Shenzhen Research Institution of Northwestern Polytechnical University, Shenzhen, 518057, PR China; Northwestern Polytechnical University-Hong Kong Baptist University Joint Research Centre for Translational Medicine on Musculoskeletal Health in Space, Xi'an, 710072, PR China
| | - Xiaoli Ma
- Laboratory for Bone Metabolism, Key Laboratory for Space Bioscience and Biotechnology, School of Life Sciences, Northwestern Polytechnical University, Xi'an, Shaanxi 710072, PR China; Shenzhen Research Institution of Northwestern Polytechnical University, Shenzhen, 518057, PR China; Northwestern Polytechnical University-Hong Kong Baptist University Joint Research Centre for Translational Medicine on Musculoskeletal Health in Space, Xi'an, 710072, PR China
| | - Yan Zhang
- Laboratory for Bone Metabolism, Key Laboratory for Space Bioscience and Biotechnology, School of Life Sciences, Northwestern Polytechnical University, Xi'an, Shaanxi 710072, PR China; Shenzhen Research Institution of Northwestern Polytechnical University, Shenzhen, 518057, PR China; Northwestern Polytechnical University-Hong Kong Baptist University Joint Research Centre for Translational Medicine on Musculoskeletal Health in Space, Xi'an, 710072, PR China
| | - Lei Chen
- Laboratory for Bone Metabolism, Key Laboratory for Space Bioscience and Biotechnology, School of Life Sciences, Northwestern Polytechnical University, Xi'an, Shaanxi 710072, PR China; Shenzhen Research Institution of Northwestern Polytechnical University, Shenzhen, 518057, PR China; Northwestern Polytechnical University-Hong Kong Baptist University Joint Research Centre for Translational Medicine on Musculoskeletal Health in Space, Xi'an, 710072, PR China
| | - Airong Qian
- Laboratory for Bone Metabolism, Key Laboratory for Space Bioscience and Biotechnology, School of Life Sciences, Northwestern Polytechnical University, Xi'an, Shaanxi 710072, PR China; Shenzhen Research Institution of Northwestern Polytechnical University, Shenzhen, 518057, PR China; Northwestern Polytechnical University-Hong Kong Baptist University Joint Research Centre for Translational Medicine on Musculoskeletal Health in Space, Xi'an, 710072, PR China.
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Schweiggert J, Panigada D, Tan AN, Liakopoulos D. Kar9 controls the nucleocytoplasmic distribution of yeast EB1. Cell Cycle 2016; 15:2860-2866. [PMID: 27625073 DOI: 10.1080/15384101.2016.1231282] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/21/2022] Open
Abstract
The precise temporal and spatial concentration of microtubule-associated proteins (MAPs) within the cell is fundamental to ensure chromosome segregation and correct spindle positioning. MAPs form an intricate web of interactions among each other and compete for binding sites on microtubules. Therefore, when assessing cellular phenotypes upon MAP up- or downregulation, it is important to consider the protein interaction network between individual MAPs. Here, we show that changes in the amounts of the spindle positioning factor Kar9 specifically affect the distribution of yeast EB1 on spindle microtubules, without influencing other microtubule-associated interacting partners of Kar9, i.e. yeast XMAP215 and CLIP-170. Alterations in the distribution of yeast EB1 explain chromosome segregation defects upon knockout, overexpression or stabilization of Kar9 and provide an example for non-linear effects on MAP behavior after perturbation of their equilibrium.
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Affiliation(s)
- Jörg Schweiggert
- a Centre de Recherche en Biologie cellulaire de Montpellier (CRBM), CNRS UMR 523 , Montpellier , France.,b Biochemistry Centre Heidelberg (BZH) , Heidelberg , Germany.,c The Hartmut Hoffmann-Berling International Graduate School of Molecular and Cellular Biology, University of Heidelberg , Heidelberg , Germany
| | - Davide Panigada
- a Centre de Recherche en Biologie cellulaire de Montpellier (CRBM), CNRS UMR 523 , Montpellier , France
| | - Ann Na Tan
- b Biochemistry Centre Heidelberg (BZH) , Heidelberg , Germany
| | - Dimitris Liakopoulos
- a Centre de Recherche en Biologie cellulaire de Montpellier (CRBM), CNRS UMR 523 , Montpellier , France.,c The Hartmut Hoffmann-Berling International Graduate School of Molecular and Cellular Biology, University of Heidelberg , Heidelberg , Germany
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10
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Baas PW, Rao AN, Matamoros AJ, Leo L. Stability properties of neuronal microtubules. Cytoskeleton (Hoboken) 2016; 73:442-60. [PMID: 26887570 PMCID: PMC5541393 DOI: 10.1002/cm.21286] [Citation(s) in RCA: 217] [Impact Index Per Article: 24.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/10/2015] [Revised: 02/02/2016] [Accepted: 02/12/2016] [Indexed: 01/12/2023]
Abstract
Neurons are terminally differentiated cells that use their microtubule arrays not for cell division but rather as architectural elements required for the elaboration of elongated axons and dendrites. In addition to acting as compression-bearing struts that provide for the shape of the neuron, microtubules also act as directional railways for organelle transport. The stability properties of neuronal microtubules are commonly discussed in the biomedical literature as crucial to the development and maintenance of the nervous system, and have recently gained attention as central to the etiology of neurodegenerative diseases. Drugs that affect microtubule stability are currently under investigation as potential therapies for disease and injury of the nervous system. There is often a lack of consistency, however, in how the issue of microtubule stability is discussed in the literature, and this can affect the design and interpretation of experiments as well as potential therapeutic regimens. Neuronal microtubules are considered to be more stable than microtubules in dividing cells. On average, this is true, but in addition to an abundant stable microtubule fraction in neurons, there is also an abundant labile microtubule fraction. Both are functionally important. Individual microtubules consist of domains that differ in their stability properties, and these domains can also differ markedly in their composition as well as how they interact with various microtubule-related proteins in the neuron. Myriad proteins and pathways have been discussed as potential contributors to microtubule stability in neurons. © 2016 Wiley Periodicals, Inc.
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Affiliation(s)
- Peter W Baas
- Department of Neurobiology and Anatomy, Drexel University College of Medicine, Philadelphia, Pennsylvania, USA.
| | - Anand N Rao
- Department of Neurobiology and Anatomy, Drexel University College of Medicine, Philadelphia, Pennsylvania, USA
| | - Andrew J Matamoros
- Department of Neurobiology and Anatomy, Drexel University College of Medicine, Philadelphia, Pennsylvania, USA
| | - Lanfranco Leo
- Department of Neurobiology and Anatomy, Drexel University College of Medicine, Philadelphia, Pennsylvania, USA
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11
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Regulation of a Spindle Positioning Factor at Kinetochores by SUMO-Targeted Ubiquitin Ligases. Dev Cell 2016; 36:415-27. [PMID: 26906737 DOI: 10.1016/j.devcel.2016.01.011] [Citation(s) in RCA: 20] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/17/2015] [Revised: 12/04/2015] [Accepted: 01/14/2016] [Indexed: 12/17/2022]
Abstract
Correct function of the mitotic spindle requires balanced interplay of kinetochore and astral microtubules that mediate chromosome segregation and spindle positioning, respectively. Errors therein can cause severe defects ranging from aneuploidy to developmental disorders. Here, we describe a protein degradation pathway that functionally links astral microtubules to kinetochores via regulation of a microtubule-associated factor. We show that the yeast spindle positioning protein Kar9 localizes not only to astral but also to kinetochore microtubules, where it becomes targeted for proteasomal degradation by the SUMO-targeted ubiquitin ligases (STUbLs) Slx5-Slx8. Intriguingly, this process does not depend on preceding sumoylation of Kar9 but rather requires SUMO-dependent recruitment of STUbLs to kinetochores. Failure to degrade Kar9 leads to defects in both chromosome segregation and spindle positioning. We propose that kinetochores serve as platforms to recruit STUbLs in a SUMO-dependent manner in order to ensure correct spindle function by regulating levels of microtubule-associated proteins.
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12
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Cheng C, Nowak RB, Fowler VM. The lens actin filament cytoskeleton: Diverse structures for complex functions. Exp Eye Res 2016; 156:58-71. [PMID: 26971460 DOI: 10.1016/j.exer.2016.03.005] [Citation(s) in RCA: 56] [Impact Index Per Article: 6.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/07/2015] [Revised: 03/01/2016] [Accepted: 03/07/2016] [Indexed: 01/05/2023]
Abstract
The eye lens is a transparent and avascular organ in the front of the eye that is responsible for focusing light onto the retina in order to transmit a clear image. A monolayer of epithelial cells covers the anterior hemisphere of the lens, and the bulk of the lens is made up of elongated and differentiated fiber cells. Lens fiber cells are very long and thin cells that are supported by sophisticated cytoskeletal networks, including actin filaments at cell junctions and the spectrin-actin network of the membrane skeleton. In this review, we highlight the proteins that regulate diverse actin filament networks in the lens and discuss how these actin cytoskeletal structures assemble and function in epithelial and fiber cells. We then discuss methods that have been used to study actin in the lens and unanswered questions that can be addressed with novel techniques.
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Affiliation(s)
- Catherine Cheng
- Department of Cell and Molecular Biology, The Scripps Research Institute, La Jolla, CA 92037, USA
| | - Roberta B Nowak
- Department of Cell and Molecular Biology, The Scripps Research Institute, La Jolla, CA 92037, USA
| | - Velia M Fowler
- Department of Cell and Molecular Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.
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13
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14
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Gu Y, Wang G, Fang N. Simultaneous single-particle superlocalization and rotational tracking. ACS NANO 2013; 7:1658-1665. [PMID: 23363388 DOI: 10.1021/nn305640y] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/01/2023]
Abstract
Superlocalization of single molecules and nanoparticles has become an essential procedure to bring new insights into nanoscale structures and dynamics of biological systems. In the present study, superlocalization is combined with the newly introduced differential interference contrast (DIC) microscopy-based single-particle orientation and rotational tracking. The new technique overcomes the difficulty in localization of the antisymmetric DIC point spread function by using a dual-modality microscope configuration for simultaneous rotational tracking and localization of single gold nanorods with nanometer-scale precision. The new imaging setup has been applied to study the steric hindrance induced by relatively large cargos in the microtubule gliding assay and to track nanocargos in the crowded cellular environment. This technique has great potential in the study of biological processes where both localization and rotational information are required.
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Affiliation(s)
- Yan Gu
- Ames Laboratory, U.S. Department of Energy, and Department of Chemistry, Iowa State University, Ames, Iowa 50011, USA
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15
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Rahimi N, Rezazadeh K, Mahoney JE, Hartsough E, Meyer RD. Identification of IGPR-1 as a novel adhesion molecule involved in angiogenesis. Mol Biol Cell 2012; 23:1646-56. [PMID: 22419821 PMCID: PMC3338432 DOI: 10.1091/mbc.e11-11-0934] [Citation(s) in RCA: 47] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/16/2023] Open
Abstract
IGPR-1 is a novel adhesion molecule that regulates cell–cell interaction. IGPR-1 associates with several SH3-containing proteins, including SPIN90/WISH, and regulates capillary tube formation of primary endothelial cells. Angiogenesis—the growth of new blood vessels from preexisting vessels—is an important physiological process and is considered to play a key role in tumor growth and metastasis. We identified the immunoglobulin-containing and proline-rich receptor-1 (IGPR-1, also called TMIGD2) gene as a novel cell adhesion receptor that is expressed in various human organs and tissues, mainly in cells with epithelium and endothelium origins. IGPR-1 regulates cellular morphology, homophilic cell aggregation, and cell–cell interaction. IGPR-1 activity also modulates actin stress fiber formation and focal adhesion and reduces cell migration. Silencing of expression of IGPR-1 by small interfering RNA (siRNA) and by ectopic overexpression in endothelial cells showed that IGPR-1 regulates capillary tube formation in vitro, and B16F melanoma cells engineered to express IGPR-1 displayed extensive angiogenesis in the mouse Matrigel angiogenesis model. Moreover, IGPR-1, through its proline-rich cytoplasmic domain, associates with multiple Src homology 3 (SH3)–containing signaling proteins, including SH3 protein interacting with Nck (SPIN90/WISH), bullous pemphigoid antigen-1, and calcium channel β2. Silencing of expression of SPIN90/WISH by siRNA in endothelial cells showed that SPIN90/WISH is required for capillary tube formation. These features of IGPR-1 suggest that IGPR-1 is a novel receptor that plays an important role in cell–cell interaction, cell migration, and angiogenesis.
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Affiliation(s)
- Nader Rahimi
- Departments of Pathology and Ophthalmology, School of Medicine, Boston University, Boston, MA 02118, USA.
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16
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Desclaux M, Teigell M, Amar L, Vogel R, Gimenez y Ribotta M, Privat A, Mallet J. A novel and efficient gene transfer strategy reduces glial reactivity and improves neuronal survival and axonal growth in vitro. PLoS One 2009; 4:e6227. [PMID: 19597552 PMCID: PMC2705675 DOI: 10.1371/journal.pone.0006227] [Citation(s) in RCA: 38] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/01/2009] [Accepted: 06/10/2009] [Indexed: 12/21/2022] Open
Abstract
Background The lack of axonal regeneration in the central nervous system is attributed among other factors to the formation of a glial scar. This cellular structure is mainly composed of reactive astrocytes that overexpress two intermediate filament proteins, the glial fibrillary acidic protein (GFAP) and vimentin. Indeed, in vitro, astrocytes lacking GFAP or both GFAP and vimentin were shown to be the substrate for increased neuronal plasticity. Moreover, double knockout mice lacking both GFAP and vimentin presented lower levels of glial reactivity in vivo, significant axonal regrowth and improved functional recovery in comparison with wild-type mice after spinal cord hemisection. From these results, our objective was to develop a novel therapeutic strategy for axonal regeneration, based on the targeted suppression of astroglial reactivity and scarring by lentiviral-mediated RNA-interference (RNAi). Methods and Findings In this study, we constructed two lentiviral vectors, Lv-shGFAP and Lv-shVIM, which allow efficient and stable RNAi-mediated silencing of endogenous GFAP or vimentin in vitro. In cultured cortical and spinal reactive astrocytes, the use of these vectors resulted in a specific, stable and highly significant decrease in the corresponding protein levels. In a second model — scratched primary cultured astrocytes — Lv-shGFAP, alone or associated with Lv-shVIM, decreased astrocytic reactivity and glial scarring. Finally, in a heterotopic coculture model, cortical neurons displayed higher survival rates and increased neurite growth when cultured with astrocytes in which GFAP and vimentin had been invalidated by lentiviral-mediated RNAi. Conclusions Lentiviral-mediated knockdown of GFAP and vimentin in astrocytes show that GFAP is a key target for modulating reactive gliosis and monitoring neuron/glia interactions. Thus, manipulation of reactive astrocytes with the Lv-shGFAP vector constitutes a promising therapeutic strategy for increasing glial permissiveness and permitting axonal regeneration after central nervous system lesions.
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Affiliation(s)
- Mathieu Desclaux
- Biotechnology and Biotherapy, Centre de Recherche de l'Institut du Cerveau et de la Moelle Epiniere, Centre National de la Recherche Scientifique (CNRS) UMR 7225, Institut National de la Santé et de la Recherche Médicale (INSERM) UMRS 975, Université Pierre et Marie Curie (UPMC) - Hôpital de la Pitié Salpêtrière, Paris, France
| | | | - Lahouari Amar
- Biotechnology and Biotherapy, Centre de Recherche de l'Institut du Cerveau et de la Moelle Epiniere, Centre National de la Recherche Scientifique (CNRS) UMR 7225, Institut National de la Santé et de la Recherche Médicale (INSERM) UMRS 975, Université Pierre et Marie Curie (UPMC) - Hôpital de la Pitié Salpêtrière, Paris, France
| | - Roland Vogel
- Biotechnology and Biotherapy, Centre de Recherche de l'Institut du Cerveau et de la Moelle Epiniere, Centre National de la Recherche Scientifique (CNRS) UMR 7225, Institut National de la Santé et de la Recherche Médicale (INSERM) UMRS 975, Université Pierre et Marie Curie (UPMC) - Hôpital de la Pitié Salpêtrière, Paris, France
| | - Minerva Gimenez y Ribotta
- Consejo Superior de Investigationes Cientifícas (CSIC), Universidad Miguel Hernández (UMH), Instituto de Neurociencias de Alicante, Sant Joan D'Alacant Nacional, España
| | - Alain Privat
- Institut National de la Santé et de la Recherche Médicale (INSERM) U583, Physiopathologie et Thérapie des Déficits Sensoriels et Moteurs, Institut des Neurosciences de Montpellier (INM), Université Montpellier 2, Montpellier, France
- * E-mail:
| | - Jacques Mallet
- Biotechnology and Biotherapy, Centre de Recherche de l'Institut du Cerveau et de la Moelle Epiniere, Centre National de la Recherche Scientifique (CNRS) UMR 7225, Institut National de la Santé et de la Recherche Médicale (INSERM) UMRS 975, Université Pierre et Marie Curie (UPMC) - Hôpital de la Pitié Salpêtrière, Paris, France
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17
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Prasain N, Stevens T. The actin cytoskeleton in endothelial cell phenotypes. Microvasc Res 2009; 77:53-63. [PMID: 19028505 PMCID: PMC2700738 DOI: 10.1016/j.mvr.2008.09.012] [Citation(s) in RCA: 221] [Impact Index Per Article: 13.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/17/2008] [Accepted: 09/26/2008] [Indexed: 10/21/2022]
Abstract
Endothelium forms a semi-permeable barrier that separates blood from the underlying tissue. Barrier function is largely determined by cell-cell and cell-matrix adhesions that define the limits of cell borders. Yet, such cell-cell and cell-matrix tethering is critically reliant upon the nature of adherence within the cell itself. Indeed, the actin cytoskeleton fulfills this essential function, to provide a strong, dynamic intracellular scaffold that organizes integral membrane proteins with the cell's interior, and responds to environmental cues to orchestrate appropriate cell shape. The actin cytoskeleton is comprised of three distinct, but inter-related structures, including actin cross-linking of spectrin within the membrane skeleton, the cortical actin rim, and actomyosin-based stress fibers. This review addresses each of these actin-based structures, and discusses cellular signals that control the disposition of actin in different endothelial cell phenotypes.
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Affiliation(s)
- Nutan Prasain
- Department of Molecular and Cellular Pharmacology College of Medicine, University of South Alabama, Mobile, AL 36688, USA
- Center for Lung Biology, College of Medicine, University of South Alabama, Mobile, AL 36688, USA
| | - Troy Stevens
- Department of Molecular and Cellular Pharmacology College of Medicine, University of South Alabama, Mobile, AL 36688, USA
- Center for Lung Biology, College of Medicine, University of South Alabama, Mobile, AL 36688, USA
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18
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Wu X, Kodama A, Fuchs E. ACF7 regulates cytoskeletal-focal adhesion dynamics and migration and has ATPase activity. Cell 2008; 135:137-48. [PMID: 18854161 PMCID: PMC2703712 DOI: 10.1016/j.cell.2008.07.045] [Citation(s) in RCA: 221] [Impact Index Per Article: 13.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/20/2008] [Revised: 07/02/2008] [Accepted: 07/30/2008] [Indexed: 11/17/2022]
Abstract
Coordinated interactions between microtubule (MT) and actin cytoskeletons are involved in many polarized cellular processes. Spectraplakins are enormous (>500 kDa) proteins able to bind both MTs and actin filaments (F-actin) directly. To elucidate the physiological significance and functions of mammalian spectraplakin ACF7, we've conditionally targeted it in skin epidermis. Intriguingly, ACF7 deficiency compromises the targeting of microtubules along F-actin to focal adhesions (FAs), stabilizes FA-actin networks, and impairs epidermal migration. Exploring underlying mechanisms, we show that ACF7's binding domains for F-actin, MTs, and MT plus-end proteins are not sufficient to rescue the defects in FA-cytoskeletal dynamics and migration functions of ACF7 null keratinocytes. We've uncovered an intrinsic actin-regulated ATPase domain in ACF7 and demonstrate that it is both functional and essential for these roles. Our findings provide insight into the functions of this important cytoskeletal crosslinking protein in regulating dynamic interactions between MTs and F-actin to sustain directional cell movement.
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Affiliation(s)
- Xiaoyang Wu
- The Howard Hughes Medical Institute and Laboratory of Mammalian Cell Biology and Development, Rockefeller University, New York, NY 10065, USA
| | - Atsuko Kodama
- The Howard Hughes Medical Institute and Laboratory of Mammalian Cell Biology and Development, Rockefeller University, New York, NY 10065, USA
| | - Elaine Fuchs
- The Howard Hughes Medical Institute and Laboratory of Mammalian Cell Biology and Development, Rockefeller University, New York, NY 10065, USA
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19
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Sun N, Critchley DR, Paulin D, Li Z, Robson RM. Identification of a repeated domain within mammalian alpha-synemin that interacts directly with talin. Exp Cell Res 2008; 314:1839-49. [PMID: 18342854 DOI: 10.1016/j.yexcr.2008.01.034] [Citation(s) in RCA: 40] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/13/2007] [Revised: 01/25/2008] [Accepted: 01/31/2008] [Indexed: 12/21/2022]
Abstract
The type VI intermediate filament (IF) protein synemin is a unique member of the IF protein superfamily. Synemin associates with the major type III IF protein desmin forming heteropolymeric intermediate filaments (IFs) within developed mammalian striated muscle cells. These IFs encircle and link all adjacent myofibrils together at their Z-lines, as well as link the Z-lines of the peripheral layer of cellular myofibrils to the costameres located periodically along and subjacent to the sarcolemma. Costameres are multi-protein assemblies enriched in the cytoskeletal proteins vinculin, alpha-actinin, and talin. We report herein a direct interaction of human alpha-synemin with the cytoskeletal protein talin by protein-protein interaction assays. The 312 amino acid insert (SNTIII) present only within alpha-synemin binds to the rod domain of talin in vitro and co-localizes with talin at focal adhesion sites within mammalian muscle cells. Confocal microscopy studies showed that synemin co-localizes with talin within the costameres of human skeletal muscle cells. Analysis of the primary sequences of human alpha- and beta-synemins revealed that SNTIII is composed of seven tandem repeats, each containing a specific Ser/Thr-X-Arg-His/Gln (S/T-X-R-H/Q) motif. Our results suggest human alpha-synemin plays an essential role in linking the heteropolymeric IFs to adherens-type junctions, such as the costameres within mammalian striated muscle cells, via its interaction with talin, thereby helping provide mechanical integration for the muscle cell cytoskeleton.
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Affiliation(s)
- Ning Sun
- Muscle Biology Group, Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, IA 50011-3260, USA
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20
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Marceau N, Schutte B, Gilbert S, Loranger A, Henfling MER, Broers JLV, Mathew J, Ramaekers FCS. Dual roles of intermediate filaments in apoptosis. Exp Cell Res 2007; 313:2265-81. [PMID: 17498695 DOI: 10.1016/j.yexcr.2007.03.038] [Citation(s) in RCA: 55] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/19/2007] [Revised: 03/05/2007] [Accepted: 03/12/2007] [Indexed: 02/06/2023]
Abstract
New roles have emerged recently for intermediate filaments (IFs), namely in modulating cell adhesion and growth, and providing resistance to various forms of stress and to apoptosis. In this context, we first summarize findings on the IF association with the cell response to mechanical stress and growth stimulation, in light of growth-related signaling events that are relevant to death-receptor engagement. We then address the molecular mechanisms by which IFs can provide cell resistance to apoptosis initiated by death-receptor stimulation and to necrosis triggered by excessive oxidative stress. In the same way, we examine IF involvement, along with cytolinker participation, in sequential caspase-mediated protein cleavages that are part of the overall cell death execution, particularly those that generate new functional IF protein fragments and uncover neoantigen markers. Finally, we report on the usefulness of these markers as diagnostic tools for disease-related aspects of apoptosis in humans. Clearly, the data accumulated in recent years provide new and significant insights into the multiple functions of IFs, particularly their dual roles in cell response to apoptotic insults.
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Affiliation(s)
- Normand Marceau
- Centre de recherche en cancérologie de l'Université Laval and L'Hôtel-Dieu de Québec (CHUQ), Québec, Canada G1R 2J6
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21
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Hirsch J, Hansen KC, Sapru A, Frank JA, Chalkley RJ, Fang X, Trinidad JC, Baker P, Burlingame AL, Matthay MA. Impact of low and high tidal volumes on the rat alveolar epithelial type II cell proteome. Am J Respir Crit Care Med 2007; 175:1006-13. [PMID: 17363773 PMCID: PMC1899270 DOI: 10.1164/rccm.200605-621oc] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/05/2006] [Accepted: 03/13/2007] [Indexed: 11/16/2022] Open
Abstract
RATIONALE Mechanical ventilation with high tidal volumes leads to increased permeability, generation of inflammatory mediators, and damage to alveolar epithelial cells (ATII). OBJECTIVES To identify changes in the ATII proteome after two different ventilation strategies in rats. METHODS Rats (n = 6) were ventilated for 5 hours with high- and low tidal volumes (VTs) (high VT: 20 ml/kg; low VT: 6 ml/kg). Pooled nonventilated rats served as control animals. ATII cells were isolated and lysed, and proteins were tryptically cleaved into peptides. Cellular protein content was evaluated by peptide labeling of the ventilated groups with (18)O. Samples were fractionated by cation exchange chromatography and identified using electrospray tandem mass spectrometry. Proteins identified by 15 or more peptides were statistically compared using t tests corrected for the false discovery rate. MEASUREMENTS AND MAIN RESULTS High Vt resulted in a significant increase in airspace neutrophils without an increase in extravascular lung water. Compared with low-VT samples, high-VT samples showed a 32% decrease in the inositol 1,4,5-trisphosphate 3 receptor (p < 0.01), a 34% decrease in Na(+), K(+)-ATPase (p < 0.01), and a significantly decreased content in ATP synthase chains. Even low-VT samples displayed significant changes, including a 66% decrease in heat shock protein 90-beta (p < 0.01) and a 67% increase in mitochondrial pyruvate carboxylase (p < 0.01). Significant differences were found in membrane, acute phase, structural, and mitochondrial proteins. CONCLUSIONS After short-term exposure to high-VT ventilation, significant reductions in membrane receptors, ion channel proteins, enzymes of the mitochondrial energy system, and structural proteins in ATII cells were present. The data supports the two-hit concept that an unfavorable ventilatory strategy may make the lung more vulnerable to an additional insult.
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Affiliation(s)
- Jan Hirsch
- Cardiovascular Research Institute, University of California, San Francisco, CA 94143-0130, USA.
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22
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Abstract
Upon infection, virions or subviral nucleoprotein complexes are transported from the cell surface to the site of viral transcription and replication. During viral egress, particles containing viral proteins and nucleic acids again move from the site of their synthesis to that of virus assembly and further to the plasma membrane. Because free diffusion of molecules larger than 500 kDa is restricted in the cytoplasm, viruses as well as cellular organelles employ active, energy-consuming enzymes for directed transport. This is particularly evident in the case of neurotropic viruses that travel long distances in the axon during retrograde or anterograde transport. Viruses use two strategies for intracellular transport: Viral components either hijack the cytoplasmic membrane traffic or they interact directly with the cytoskeletal transport machinery. In this review we describe how viruses--particularly members of the Herpesviridae, Adenoviridae, Parvoviridae, Poxviridae, and Baculoviridae--make use of the microtubule and the actin cytoskeleton. Analysing the underlying principles of viral cytosolic transport will be helpful in the design of viral vectors to be used in research as well as human gene therapy, and in the identification of new antiviral target molecules.
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Affiliation(s)
- K Döhner
- Department of Virology, Hannover Medical School, Carl-Neuberg-Str 1, 30625 Hannover, Germany
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23
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Hudson TY, Fontao L, Godsel LM, Choi HJ, Huen AC, Borradori L, Weis WI, Green KJ. In vitro methods for investigating desmoplakin-intermediate filament interactions and their role in adhesive strength. Methods Cell Biol 2005; 78:757-86. [PMID: 15646638 DOI: 10.1016/s0091-679x(04)78026-7] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 03/12/2023]
Affiliation(s)
- Tracie Y Hudson
- Department of Pathology, Northwestern University Feinberg School of Medicine, Chicago, Illinois 60611, USA
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24
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Mollet G, Silbermann F, Delous M, Salomon R, Antignac C, Saunier S. Characterization of the nephrocystin/nephrocystin-4 complex and subcellular localization of nephrocystin-4 to primary cilia and centrosomes. Hum Mol Genet 2005; 14:645-56. [PMID: 15661758 DOI: 10.1093/hmg/ddi061] [Citation(s) in RCA: 117] [Impact Index Per Article: 5.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/07/2023] Open
Abstract
Nephrocystin and nephrocystin-4 are newly identified proteins involved in familial juvenile nephronophthisis, an autosomal recessive nephropathy characterized by cyst formation and renal fibrosis. Nephrocystin is an adaptor protein that is able to associate with signaling molecules involved in cell adhesion and actin cytoskeleton organization, such as p130Cas, Pyk2, tensin and filamins. Nephrocystin was recently shown to interact and to co-localize with the microtubule component beta-tubulin to the primary cilia in renal epithelial cells, an organelle known to play a key role in the pathogenesis of cystic kidney diseases. In this study, we demonstrated that nephrocystin-4 also localizes to the primary cilia in polarized epithelial tubular cells, particularly at the basal bodies, and associates with microtubule component alpha-tubulin, suggesting a common role for the nephrocystin proteins in ciliary function. However, the co-localization of nephrocystin-4 with the microtubules is not restricted to the primary cilia, as nephrocystin-4 was also detected at the centrosomes of dividing cells and close to the cortical actin cytoskeleton in polarized cells. We also detected p130Cas and Pyk2 in the nephrocystin-4-containing complex, confirming the role of the nephrocystin proteins in cell-cell and cell-matrix adhesion signaling events. Finally, we refined the structural and functional regions involved in the interaction between nephrocystin and nephrocystin-4. These data suggest that nephrocystin and nephrocystin-4 belong to a multifunctional complex localized in actin- and microtubule-based structures involved in cell-cell and cell-matrix adhesion signaling as well as in cell division.
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Affiliation(s)
- Géraldine Mollet
- INserm U574, Hôpital Necker-Enfants Malades, Université René Descartes, Paris, France
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Green KJ, Böhringer M, Gocken T, Jones JCR. Intermediate filament associated proteins. ADVANCES IN PROTEIN CHEMISTRY 2005; 70:143-202. [PMID: 15837516 DOI: 10.1016/s0065-3233(05)70006-1] [Citation(s) in RCA: 69] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/20/2022]
Abstract
Intermediate filament associated proteins (IFAPs) coordinate interactions between intermediate filaments (IFs) and other cytoskeletal elements and organelles, including membrane-associated junctions such as desmosomes and hemidesmosomes in epithelial cells, costameres in striated muscle, and intercalated discs in cardiac muscle. IFAPs thus serve as critical connecting links in the IF scaffolding that organizes the cytoplasm and confers mechanical stability to cells and tissues. However, in recent years it has become apparent that IFAPs are not limited to structural crosslinkers and bundlers but also include chaperones, enzymes, adapters, and receptors. IF networks can therefore be considered scaffolding upon which associated proteins are organized and regulated to control metabolic activities and maintain cell homeostasis.
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Affiliation(s)
- Kathleen J Green
- Departments of Pathology and Dermatology and R.H. Lurie Cancer Center, Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60611, USA
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Kalinin AE, Kalinin AE, Aho M, Uitto J, Aho S. Breaking the Connection: Caspase 6 Disconnects Intermediate Filament-Binding Domain of Periplakin from its Actin-Binding N-Terminal Region. J Invest Dermatol 2005; 124:46-55. [PMID: 15654952 DOI: 10.1111/j.0022-202x.2004.23507.x] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
Abstract
Periplakin is a member of the plakin family of cytolinkers that connect cytoskeletal networks to each other as well as to the cell junctional complexes. Here, we demonstrate a direct molecular interaction between actin and periplakin. Furthermore, the oligomerization state of periplakin was shown to determine specificity of its binding to intermediate filaments (IF) in vitro. Both the filament association and the cell membrane localization of periplakin were confirmed in the cells overexpressing human periplakin. Double labeling of the N- and C-terminally tagged periplakin revealed unexpected lack of co-localization of periplakin ends in a confluent culture, and separation of the periplakin ends was even more pronounced in apoptotic cells. Western analysis revealed that after induction of apoptosis, periplakin becomes cleaved close to its C-terminal tail. Only the distinct cleavage products, but not the full-length periplakin, were present in the cells detached from the solid support during the apoptotic process. We show that caspase 6 cleaves periplakin at an unconventional recognition site, amino acid sequence TVAD. Thus, the separation of periplakin ends disconnects the actin-binding head-rod domain from the IF-binding C-terminal domain. We show that specific cleavage products co-exist with the full-length periplakin in cells, suggesting physiological consequences due to their altered binding specificities.
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Affiliation(s)
- Andrey E Kalinin
- Laboratory of Skin Biology, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, Maryland, USA
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Abstract
Reactive gliosis is a prominent result of many types of insult to the central nervous system (CNS) and leads to the formation of glial scar that impedes the regeneration of axons. The intermediate filament protein vimentin is found in pathology of the CNS, mainly in the vicinity of injuries to the CNS. In the present study we investigated the role of vimentin in the formation of glial scars in vitro and in vivo by using immunohistochemistry, Western blot analysis, and in situ hybridization. In vitro experiments showed that the intensity of immunofluorescent labeling for vimentin and glial fibrillary acidic protein (GFAP) was consistently decreased in astrocytes after transfection with a retrovirus carrying antisense complementary DNA (cDNA) for vimentin. Transfection also inhibited the growth of astrocytes and decreased the expression of vimentin mRNA. In vivo studies demonstrated that transfection with the retrovirus carrying the antisense cDNA vimentin inhibited the upregulation of vimentin and GFAP in stab wounds in rat cerebrum. These results suggest that vimentin may play a key role in the formation of glial scars in the CNS.
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Affiliation(s)
- Jiangkai Lin
- Department of Neurosurgery, Southwest Hospital, College of Medicine, Third Military Medical University, Chongqing, The People's Republic of China.
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Aho S. Plakin proteins are coordinately cleaved during apoptosis but preferentially through the action of different caspases. Exp Dermatol 2004; 13:700-7. [PMID: 15500642 DOI: 10.1111/j.0906-6705.2004.00217.x] [Citation(s) in RCA: 35] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/11/2023]
Abstract
In epithelial cells, cell-cell and cell-matrix junctions, desmosomes and hemidesmosomes, provide anchorage sites for the keratin-intermediate filaments. The plakin proteins desmoplakin (DP), plectin, and periplakin represent intracellular constituents of these adhesion junctions. In staurosporine-treated apoptotic HaCaT cells, DP, plectin, and periplakin became cleaved coordinately with the elimination of keratins 10 and 14, while involucrin, actin, and keratin 18 displayed considerable stability. The caspase inhibitor zVAD-fmk prevented both the cell detachment and protein cleavage, indicating the function of caspases in these events. Closer examination in vitro revealed that while caspases 2 and 4 most efficiently cleaved DP, and plectin served as a target for caspases 3 and 7, periplakin as well as keratins were cleaved by caspase 6. The involvement of multiple caspases in the destruction of epithelial cell integrity ensures the efficient elimination of cytoskeleton, but also provides specificity for selectively targeting individual adhesion molecules.
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Affiliation(s)
- Sirpa Aho
- Department of Dermatology and Cutaneous Biology, Thomas Jefferson University, Philadelphia, PA 19107, USA.
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Mukhopadhyay R, Kumar S, Hoh JH. Molecular mechanisms for organizing the neuronal cytoskeleton. Bioessays 2004; 26:1017-25. [PMID: 15351972 DOI: 10.1002/bies.20088] [Citation(s) in RCA: 59] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/07/2022]
Abstract
Neurofilaments and microtubules are important components of the neuronal cytoskeleton. In axons or dendrites, these filaments are aligned in parallel arrays, and separated from one another by nonrandom distances. This distinctive organization has been attributed to cross bridges formed by NF side arms or microtubule-associated proteins. We recently proposed a polymer-brush-based mechanism for regulating interactions between neurofilaments and between microtubules. In this model, the side arms of neurofilaments and the projection domains of microtubule-associated proteins are highly unstructured and exert long-range repulsive forces that are largely entropic in origin; these forces then act to organize the cytoskeleton in axons and dendrites. Here, we review the biochemical, biophysical, genetic and cell biological data for the polymer-brush and cross-bridging models. We explore how the data traditionally used to support cross bridging may be reconciled with a polymer-brush mechanism and compare the implications of recent experimental insights into axonal transport and physiology for each model.
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30
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Aho S, Li K, Ryoo Y, McGee C, Ishida-Yamamoto A, Uitto J, Klement JF. Periplakin gene targeting reveals a constituent of the cornified cell envelope dispensable for normal mouse development. Mol Cell Biol 2004; 24:6410-8. [PMID: 15226441 PMCID: PMC434234 DOI: 10.1128/mcb.24.14.6410-6418.2004] [Citation(s) in RCA: 40] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
The members of the plakin family of proteins serve as epidermal cytolinkers and components of cell-cell and cell-matrix adhesion complexes, i.e., desmosomes and hemidesmosomes, respectively. Periplakin is a recently characterized member of this family. Human and mouse periplakin genomic loci are conserved, and the proteins are highly homologous, suggesting a role for periplakin in vertebrate physiology. In order to evaluate the functional role of periplakin, we generated periplakin null mice through targeted homologous recombination of mouse embryonic stem cells, followed by development of Ppl(-/-) mice. Mice homozygous for the targeted allele were born in the expected Mendelian frequency, developed normally, possessed grossly normal epidermis and hair, and were healthy and fertile. The epidermal barrier appeared to develop normally during fetal days E15.5 to E16.5, and the cornified envelope and desmosomes in the newborn mice were ultrastructurally normal. No compensatory increase in the expression of other epithelial proteins was detected in the neonatal mouse epidermis lacking periplakin. Consequently, the primary role of periplakin may not relate to the physiology of the cornified cell envelope in epidermal keratinocytes but may reside in the challenges, which normal laboratory mice do not encounter.
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Affiliation(s)
- Sirpa Aho
- Department of Dermatology and Cutaneous Biology, Jefferson Medical College, Thomas Jefferson University, Philadelphia, PA 19107, USA.
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Nemoto T, Kojima T, Oshima A, Bito H, Kasai H. Stabilization of Exocytosis by Dynamic F-actin Coating of Zymogen Granules in Pancreatic Acini. J Biol Chem 2004; 279:37544-50. [PMID: 15184362 DOI: 10.1074/jbc.m403976200] [Citation(s) in RCA: 105] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
Reorganization of F-actin in the apical region of mouse pancreatic acinar cells during Ca(2+)-dependent exocytosis of zymogen granules was investigated by two-photon excitation microscopy with intact acini. Granules were rapidly coated with F-actin in response to either agonist stimulation or photolysis of a caged-Ca(2+) compound. Such F-actin coating occurred exclusively at the surface of granules undergoing exocytosis and was prevented either by latrunculin-A, which inhibits actin polymerization, or by Clostridium botulinum exoenzyme C3, which inhibits the small GTPase Rho. Latrunculin-A or exoenzyme C3 also triggered the formation of vacuoles in acinar cells, a characteristic of acute pancreatitis. Stimulation of acini with high concentrations of cholecystokinin, which cause acute pancreatitis in mice, also impaired the F-actin coating of granules and induced vacuole formation. Latrunculin-A reduced the latency to exocytosis but did not affect the total number of exocytic events, suggesting that F-actin slows and further stabilizes exocytosis by facilitating F-actin coating. Rho-dependent F-actin coating of granule membranes thus stabilizes exocytic structures and is necessary for physiological progression of sequetial compound exocytosis in the exocrine pancreas and for prevention of acute pancreatitis.
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Affiliation(s)
- Tomomi Nemoto
- Department of Cell Physiology, National Institute for Physiological Sciences, and Graduate University of Advanced Studies, 5-1 Higashiyama, Myodaiji-cho, Okazaki, Aichi 444-8787, Japan
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Tasanen K, Tunggal L, Chometon G, Bruckner-Tuderman L, Aumailley M. Keratinocytes from patients lacking collagen XVII display a migratory phenotype. THE AMERICAN JOURNAL OF PATHOLOGY 2004; 164:2027-38. [PMID: 15161638 PMCID: PMC1615787 DOI: 10.1016/s0002-9440(10)63762-5] [Citation(s) in RCA: 100] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Accepted: 02/06/2004] [Indexed: 10/18/2022]
Abstract
Acquired or inherited junctional epidermolysis bullosa are skin diseases characterized by a separation between the epidermis and the dermis. In inherited nonlethal junctional epidermolysis bullosa, genetic analysis has identified mutations in the COL17A1 gene coding for the transmembrane collagen XVII whereas patients with acquired diseases have autoantibodies against this protein. This suggests that collagen XVII participates in the adhesion of basal keratinocytes to the extracellular matrix. To test this hypothesis, we studied the behavior of keratinocytes with null mutations in the COL17A1 gene. Initial adhesion of mutant cells to laminin 5 was comparable to controls and similarly dependent on alpha3beta1 integrins. The spreading of mutant cells was, however, enhanced, suggesting a propensity to migrate, which was confirmed by migration assays. In addition, laminin 5 deposited by collagen XVII-deficient keratinocytes was scattered and poorly organized, suggesting that correct integration of laminin 5 within the matrix requires collagen XVII. This assumption was supported by the co-distribution of the two proteins in the matrix of normal human keratinocytes and by protein-protein-binding assays showing that the C-terminus of collagen XVII binds to laminin 5. Together, the results unravel an unexpected role of collagen XVII in the regulation of keratinocyte migration.
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Affiliation(s)
- Kaisa Tasanen
- Department of Dermatology, University of Oulu, Oulu, Finland
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Schneider N, Weber I, Faix J, Prassler J, Müller-Taubenberger A, Köhler J, Burghardt E, Gerisch G, Marriott G. A Lim protein involved in the progression of cytokinesis and regulation of the mitotic spindle. ACTA ACUST UNITED AC 2004; 56:130-9. [PMID: 14506710 DOI: 10.1002/cm.10139] [Citation(s) in RCA: 50] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/10/2022]
Abstract
DdLimE regulates cell motility and cytokinesis in Dictyostelium. To specify its function, we generated knock-out mutants and analyzed mitosis by marking the mitotic apparatus with GFP-alpha-tubulin. Characteristic of DdLimE-null cells is a late reversal of cytokinesis caused by backward movement of the incipient daughter cells. This process of "retro-cytokinesis" is accompanied by a delay in disassembly of the mitotic spindle. The length of interphase microtubules is increased and their depolymerization at prophase is impaired. These data indicate that DdLimE links the cortical actin network, where it is located, to the microtubule system, whose dynamics it regulates.
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Affiliation(s)
- Natalie Schneider
- University of Wisconsin-Madison, Department of Physiology, Madison, WI 53706, USA
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Lee M, Lee S, Zadeh AD, Kolodziej PA. Distinct sites in E-cadherin regulate different steps in Drosophila tracheal tube fusion. Development 2004; 130:5989-99. [PMID: 14597571 DOI: 10.1242/dev.00806] [Citation(s) in RCA: 43] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/16/2023]
Abstract
We have investigated how E-cadherin controls the elaboration of adherens junction associated cytoskeletal structures crucial for assembling tubular networks. During Drosophila development, tracheal branches are joined at branch tips through lumens that traverse doughnut-shaped fusion cells. Fusion cells form E-cadherin contacts associated with a track that contains F-actin, microtubules, and Shot, a plakin that binds F-actin and microtubules. Live imaging reveals that fusion occurs as the fusion cell apical surfaces meet after invaginating along the track. Initial track assembly requires E-cadherin binding to beta-catenin. Surprisingly, E-cadherin also controls track maturation via a juxtamembrane site in the cytoplasmic domain. Fusion cells expressing an E-cadherin mutant in this site form incomplete tracks that contain F-actin and Shot, but lack microtubules. These results indicate that E-cadherin controls track initiation and maturation using distinct, evolutionarily conserved signals to F-actin and microtubules, and employs Shot to promote adherens junction-associated cytoskeletal assembly.
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Affiliation(s)
- Mihye Lee
- Department of Cell and Developmental Biology, Center for Molecular Neuroscience, Program in Developmental Biology, Vanderbilt University Medical Center, Nashville TN 37232-2175, USA
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35
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Young KG, Pool M, Kothary R. Bpag1 localization to actin filaments and to the nucleus is regulated by its N-terminus. J Cell Sci 2003; 116:4543-55. [PMID: 14576348 DOI: 10.1242/jcs.00764] [Citation(s) in RCA: 41] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022] Open
Abstract
Plakins are a family of giant cytoskeleton binding proteins. One member of this group is bullous pemphigoid antigen 1 (Bpag1)/dystonin, which has neuronal and muscle isoforms that consist of actin-binding and microtubule-binding domains at either end separated by a plakin domain and several spectrin repeats. The better-characterized epithelial isoform has only the plakin domain in common with the neuronal and muscle isoforms. Here, we have analyzed the localization of muscle/neuronal (Bpag1a/b) isoforms and the epithelial (Bpag1e) isoform within C2C12 myoblast cells. Although an antibody specific to Bpag1a/b isoform 2 detected protein co-aligning actin stress fibers, this same antibody and two Bpag1e antibodies predominantly detected protein in the nuclei. A Bpag1a/b isoform 2 N-terminal fusion protein containing the plakin domain also localized to actin stress fibers and to nuclei. Within the plakin domain, we characterized a functional nuclear localization signal, which was responsible for localization of the fusion protein to the nucleus. Bpag1a/b isoform 1 N-terminal fusion proteins differed in their interaction with the actin cytoskeleton and with their ability to localize to the nucleus, suggesting that Bpag1 isoforms with different N-termini have differing roles. These results show the importance of N-terminal domains in dictating the localization and function of Bpag1 isoforms. We provide the first indication that Bpag1 is not strictly a cytoplasmic/membrane protein but that it can also localize to the nucleus.
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Affiliation(s)
- Kevin G Young
- Ottawa Health Research Institute, 501 Smyth Road, Ottawa, Ontario, Canada K1H 8L6, and Center for Neuromuscular Disease, and Departments of Cellular and Molecular Medicine, and Medicine, University of Ottawa, Ottawa, Ontario, Canada K1H 8M5
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36
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García-Alvarez B, Bobkov A, Sonnenberg A, de Pereda JM. Structural and functional analysis of the actin binding domain of plectin suggests alternative mechanisms for binding to F-actin and integrin beta4. Structure 2003; 11:615-25. [PMID: 12791251 DOI: 10.1016/s0969-2126(03)00090-x] [Citation(s) in RCA: 81] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/28/2023]
Abstract
Plectin is a widely expressed cytoskeletal linker. Here we report the crystal structure of the actin binding domain of plectin and show that this region is sufficient for interaction with F-actin or the cytoplasmic region of integrin alpha6beta4. The structure is formed by two calponin homology domains arranged in a closed conformation. We show that binding to F-actin induces a conformational change in plectin that is inhibited by an engineered interdomain disulfide bridge. A two-step induced fit mechanism involving binding and subsequent domain rearrangement is proposed. In contrast, interaction with integrin alpha6beta4 occurs in a closed conformation. Competitive binding of plectin to F-actin and integrin alpha6beta4 may rely on the observed alternative binding mechanisms and involve both allosteric and steric factors.
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Affiliation(s)
- Begoña García-Alvarez
- Program on Cell Adhesion, The Burnham Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA
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37
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Fontao L, Favre B, Riou S, Geerts D, Jaunin F, Saurat JH, Green KJ, Sonnenberg A, Borradori L. Interaction of the bullous pemphigoid antigen 1 (BP230) and desmoplakin with intermediate filaments is mediated by distinct sequences within their COOH terminus. Mol Biol Cell 2003; 14:1978-92. [PMID: 12802069 PMCID: PMC165091 DOI: 10.1091/mbc.e02-08-0548] [Citation(s) in RCA: 82] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/30/2002] [Revised: 12/11/2002] [Accepted: 12/27/2002] [Indexed: 01/06/2023] Open
Abstract
The bullous pemphigoid antigen 1 (BP230) and desmoplakin (DP) are members of the plakin protein family of cytolinkers. Despite their homology, their COOH termini selectively bind distinct intermediate filaments (IFs). We studied sequences within their COOH termini required for their interaction with the epidermal keratins K5/K14, the simple epithelial keratins K8/K18, and type III IF vimentin by yeast three-hybrid, cell transfection, and overlay assays. The results indicate that BP230 interacts with K5/K14 but not with K8/K18 or vimentin via a region encompassing both the B and C subdomains and the COOH extremity, including a COOH-terminal eight-amino-acid stretch. In contrast, the C subdomain with the COOH-terminal extremity of DP interacts with K5/K14 and K8/K18, and its linker region is able to associate with K8/K18 and vimentin. Furthermore, the potential of DP to interact with IF proteins in yeast seems to be regulated by phosphorylation of Ser 2849 within its COOH terminus. Strikingly, BP230 and DP interacted with cytokeratins only when both type I and type II keratins were present. The head and tail domains of K5/K14 keratins were dispensable for their interaction with BP230 or DP. On the basis of our findings, we postulate that (1) the binding specificity of plakins for various IF proteins depends on their linker region between the highly homologous B and C subdomains and their COOH extremity and (2) the association of DP and BP230 with both epidermal and simple keratins is critically affected by the tertiary structure induced by heterodimerization and involves recognition sites located primarily in the rod domain of these keratins.
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Affiliation(s)
- Lionel Fontao
- Department of Dermatology, University Hospital, Geneva, Switzerland CH-1211
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Taylor W, Gokay KE, Capaccio C, Davis E, Glucksberg M, Dean DA. The effects of cyclic stretch on gene transfer in alveolar epithelial cells. Mol Ther 2003; 7:542-9. [PMID: 12727118 PMCID: PMC4394637 DOI: 10.1016/s1525-0016(03)00041-8] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/09/2023] Open
Abstract
Cyclic stretch has been shown to alter cell physiology, cytoskeletal structure, signal transduction, and gene expression in a variety of cell types. To determine the effects of stretch on the gene transfer process, we compared the transfection efficiencies of human A549 cells grown either statically or exposed to 10% cyclic stretch (Delta surface area) at 60 cycles/min (1 Hz) for 24 hours prior to, and/or after transfection with pEGFP-N1 and pCMV-lux-DTS using lipoplex or electroporation. Stretching the cells prior to transfection had no effect on gene transfer. By contrast, cyclic, but not continuous, stretch applied immediately after transfection for as little as 30 minutes resulted in a 10-fold increase in gene transfer and expression by either transfection technique. These stretch conditions did not result in rupture of the plasma membrane based on the fact that DNA was unable to enter stretched cells unless either an electric field was applied or the DNA was complexed with liposomes. Taken together with the timing of the stretch response and the known effects of stretch on transcription, these findings suggest that cyclic stretch may be altering the intracellular transport of plasmids to increase gene expression.
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Affiliation(s)
- Winna Taylor
- Division of Pulmonary and Critical Care Medicine, Northwestern University Medical School, Chicago, IL 60611, USA
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Liu M, Li XL, Hassel BA. Proteasomes modulate conjugation to the ubiquitin-like protein, ISG15. J Biol Chem 2003; 278:1594-602. [PMID: 12426315 DOI: 10.1074/jbc.m208123200] [Citation(s) in RCA: 63] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
ISG15 is a ubiquitin-like protein that is induced by interferon and microbial challenge. Ubiquitin-like proteins are covalently conjugated to cellular proteins and may intersect the ubiquitin-proteasome system via common substrates or reciprocal regulation. To investigate the relationship between ISG15 conjugation and proteasome function, we treated interferon-induced cells with proteasome inhibitors. Surprisingly, inhibition of proteasomal, but not lysosomal, proteases dramatically enhanced the level of ISG15 conjugates. The stimulation of ISG15 conjugates occurred rapidly in the absence of protein synthesis and was most dramatic in the cytoskeletal protein fraction. Inhibition of ISG15 conjugation by ATP depletion abrogated the proteasome inhibitor-dependent increase in ISG15 conjugates, suggesting that the effect was mediated by de novo conjugation, rather than protection from proteasomal degradation or inhibition of ISG15 deconjugating activity. The increase in ISG15 conjugates did not occur through a stabilization of the ISG15 E1 enzyme, UBE1L. Furthermore, simultaneous modification of proteins by both ISG15 and ubiquitin did not account for the proteasome inhibitor-dependent increase in ISG15 conjugates. These findings provide the first evidence for a link between ISG15 conjugation and proteasome function and support a model in which proteins destined for ISG15 conjugation are proteasome-regulated.
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Affiliation(s)
- Mingjuan Liu
- Program in Molecular and Cell Biology, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA
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40
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Huen AC, Park JK, Godsel LM, Chen X, Bannon LJ, Amargo EV, Hudson TY, Mongiu AK, Leigh IM, Kelsell DP, Gumbiner BM, Green KJ. Intermediate filament-membrane attachments function synergistically with actin-dependent contacts to regulate intercellular adhesive strength. J Cell Biol 2002; 159:1005-17. [PMID: 12499357 PMCID: PMC2173978 DOI: 10.1083/jcb.200206098] [Citation(s) in RCA: 131] [Impact Index Per Article: 5.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/30/2022] Open
Abstract
By tethering intermediate filaments (IFs) to sites of intercellular adhesion, desmosomes facilitate formation of a supercellular scaffold that imparts mechanical strength to a tissue. However, the role IF-membrane attachments play in strengthening adhesion has not been directly examined. To address this question, we generated Tet-On A431 cells inducibly expressing a desmoplakin (DP) mutant lacking the rod and IF-binding domains (DPNTP). DPNTP localized to the plasma membrane and led to dissociation of IFs from the junctional plaque, without altering total or cell surface distribution of adherens junction or desmosomal proteins. However, a specific decrease in the detergent-insoluble pool of desmoglein suggested a reduced association with the IF cytoskeleton. DPNTP-expressing cell aggregates in suspension or substrate-released cell sheets readily dissociated when subjected to mechanical stress whereas controls remained largely intact. Dissociation occurred without lactate dehydrogenase release, suggesting that loss of tissue integrity was due to reduced adhesion rather than increased cytolysis. JD-1 cells from a patient with a DP COOH-terminal truncation were also more weakly adherent compared with normal keratinocytes. When used in combination with DPNTP, latrunculin A, which disassembles actin filaments and disrupts adherens junctions, led to dissociation up to an order of magnitude greater than either treatment alone. These data provide direct in vitro evidence that IF-membrane attachments regulate adhesive strength and suggest furthermore that actin- and IF-based junctions act synergistically to strengthen adhesion.
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Affiliation(s)
- Arthur C Huen
- Department of Pathology, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA
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41
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Karashima T, Watt FM. Interaction of periplakin and envoplakin with intermediate filaments. J Cell Sci 2002; 115:5027-37. [PMID: 12432088 DOI: 10.1242/jcs.00191] [Citation(s) in RCA: 69] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Periplakin is a component of desmosomes and the epidermal cornified envelope. Its N-terminal domain interacts with the plasma membrane; it heterodimerises with envoplakin via its rod domain; and its C-terminus interacts with intermediate filaments. Periplakin has the shortest C-terminus of the plakin family, comprising only the linker domain found in all conventional plakins. By transient transfection of COS7 cells and primary human epidermal keratinocytes with deletion mutants of the periplakin C-terminus we mapped sequences required for intermediate filament interaction to two regions of the linker motif that are most highly conserved amongst the plakins. The results were confirmed by overlay assays of the binding of in vitro translated periplakin constructs to keratins and vimentin. We found that envoplakin and periplakin could still associate with each other when parts of their rod domains were deleted and, surprisingly, that removal of the entire rod domain did not completely inhibit their interaction. Co-transfection of constructs containing the C-termini of envoplakin and periplakin suggested that the periplakin C-terminus may stabilise the interaction of the envoplakin C-terminus with intermediate filaments. We conclude that the periplakin C-terminus plays an important role in linking periplakin and envoplakin to intermediate filaments.
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Affiliation(s)
- Tadashi Karashima
- Keratinocyte Laboratory, Cancer Research UK, 44 Lincoln's Inn Fields, London WC2A 3PX, UK
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42
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Li S, Finley J, Liu ZJ, Qiu SH, Chen H, Luan CH, Carson M, Tsao J, Johnson D, Lin G, Zhao J, Thomas W, Nagy LA, Sha B, DeLucas LJ, Wang BC, Luo M. Crystal structure of the cytoskeleton-associated protein glycine-rich (CAP-Gly) domain. J Biol Chem 2002; 277:48596-601. [PMID: 12221106 DOI: 10.1074/jbc.m208512200] [Citation(s) in RCA: 70] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
Cytoskeleton-associated proteins (CAPs) are involved in the organization of microtubules and transportation of vesicles and organelles along the cytoskeletal network. A conserved motif, CAP-Gly, has been identified in a number of CAPs, including CLIP-170 and dynactins. The crystal structure of the CAP-Gly domain of Caenorhabditis elegans F53F4.3 protein, solved by single wavelength sulfur-anomalous phasing, revealed a novel protein fold containing three beta-sheets. The most conserved sequence, GKNDG, is located in two consecutive sharp turns on the surface, forming the entrance to a groove. Residues in the groove are highly conserved as measured from the information content of the aligned sequences. The C-terminal tail of another molecule in the crystal is bound in this groove.
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Affiliation(s)
- Songlin Li
- Southeast Collaboratory for Structural Genomics, University of Georgia, Athens 30602, USA
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43
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Chan W, Kozma R, Yasui Y, Inagaki M, Leung T, Manser E, Lim L. Vimentin intermediate filament reorganization by Cdc42: involvement of PAK and p70 S6 kinase. Eur J Cell Biol 2002; 81:692-701. [PMID: 12553669 DOI: 10.1078/0171-9335-00281] [Citation(s) in RCA: 29] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
Rho family GTPases play a major role in actin cytoskeleton reorganization. Recent studies have shown that the activation of Rho family GTPases also induces collapse of the vimentin intermediate filament (IF) network in fibroblasts. Here, we report that Cdc42V12 induces the reorganization of vimentin IFs in Hela cells, and such reorganization is independent of actin and microtubule status. We analyzed the involvement of three serine/threonine kinase effectors, MRCK, PAK and p70 S6K in the Cdc42-induced vimentin reorganization. Surprisingly, the ROK-related MRCK is not involved in this IF reorganization. We detected phosphorylation of vimentin Ser72, a site phosphorylated by PAK, after Cdc42 activation. PAK inhibition partially blocked Cdc42-induced vimentin IF collapse suggesting the involvement of other effectors. We report that p70 S6 kinase (S6K)1 participates in this IF rearrangement since the inhibitor rapamycin or a dominant inhibitory S6K could reduce the Cdc42V12 or bradykinin-induced vimentin collapse. Further, inhibition of PAK and S6K in combination very effectively prevents Cdc42-induced vimentin IF collapse. Conversely, only in combination active PAK and S6K could induce a vimentin IF rearrangement that mimics the Cdc42 effect. Thus, Cdc42-induced vimentin reorganization involves PAK and, in a novel cytoskeletal role, p70 S6K.
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Affiliation(s)
- Wing Chan
- Glaxo-IMCB Group, Institute of Molecular and Cell Biology, Singapore, Singapore.
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44
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Kazerounian S, Uitto J, Aho S. Unique role for the periplakin tail in intermediate filament association: specific binding to keratin 8 and vimentin. Exp Dermatol 2002; 11:428-38. [PMID: 12366696 DOI: 10.1034/j.1600-0625.2002.110506.x] [Citation(s) in RCA: 62] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/23/2022]
Abstract
Plectin, desmoplakin, and the 230-kDa bullous pemphigoid antigen (BPAG1), members of the plakin family of proteins, are multifunctional cytolinkers, connecting the cytoskeletal structures to the cell adhesion complexes. Envoplakin and periplakin are components of the cornified envelope, but less is known about their role in tissues other than the stratified epithelium. Our tissue-wide survey utilizing RT-PCR revealed that periplakin, like plectin and desmoplakin, has a wide tissue distribution, but envoplakin expression is limited to certain tissues only, and BPAG1 is clearly specific for epidermal keratinocytes. Plectin, desmoplakin and BPAG1 are known to bind to the intermediate filaments through their C-terminal domains. The short C-terminal domain of periplakin is composed only of the linker domain, a region highly homologous between the plakin proteins. Here we demonstrate, through the use of yeast two-hybrid assay, a specific interaction of the periplakin linker domain with keratin 8 and vimentin. Co-expression of each plakin linker domain with keratin 8 revealed that periplakin and BPAG1 linkers co-localize with keratin signals in HaCaT cells, plectin and desmoplakin linkers were detected both in the nucleus and in cytoplasm together with the overexpressed keratin 8, while envoplakin linker localized independently into the nucleus. These results suggest that, in spite of its high homology and structural similarity with envoplakin, periplakin is functionally closer to the well-characterized plakin proteins plectin and desmoplakin, and thus may function tissue-wide as a scaffolding protein in intermediate filament assembly.
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Affiliation(s)
- Shideh Kazerounian
- Department of Dermatology and Cutaneous Biology, Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA
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45
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Leonova EV, Lomax MI. Expression of the mouse Macf2 gene during inner ear development. BRAIN RESEARCH. MOLECULAR BRAIN RESEARCH 2002; 105:67-78. [PMID: 12399109 DOI: 10.1016/s0169-328x(02)00394-7] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/23/2022]
Abstract
Plakins, a family of linker proteins that connect cytoskeletal elements to cellular junctions and the extracellular matrix, are primarily responsible for the mechanical properties of cells and tissues. They include desmoplakin, envoplakin, plectin, dystonin/BPAG1, and Kakapo. Mutations in plakins cause several skin, muscular and neurological disorders. Macrophins are a recently discovered subfamily of plakins with binding domains for actin, intermediate filaments and microtubules. Characteristic features of macrophins include variable actin binding domains, a central rod domain containing both plectin and spectrin repeats, and a C-terminus containing EF hands and GAS2/GAR22 domain. We have examined expression of mouse Macf2, encoding macrophin-2, in adult tissues and in the developing, neonatal, and mature inner ear by in situ hybridization. Northern blot analysis identified three large tissue-specific Macf2 transcripts: a 16-kb mRNA in skeletal muscle and heart, a 15-kb mRNA in brain, and a 9-kb mRNA in RNA from ovary plus uterus. In situ hybridization of the developing mouse inner ear indicated that Macf2 is expressed in the otocyst at day 12.5, in the sensory epithelium by embryonic day 16.5, and in both inner and outer hair cells by day 16.5. Macf2 is expressed in the bodies of both sensory and motor neurons in the central and peripheral nervous system, including the auditory pathway. The Macf2 protein could be involved in the regulation of cytoskeletal connections to cellular junctions and play an important structural role in organs, such as the inner ear, that are subjected to strong mechanical forces.
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MESH Headings
- Animals
- Animals, Newborn
- Cell Adhesion/genetics
- Central Nervous System/embryology
- Central Nervous System/growth & development
- Central Nervous System/metabolism
- Cytoskeleton/genetics
- Cytoskeleton/metabolism
- Ear, Inner/embryology
- Ear, Inner/growth & development
- Ear, Inner/metabolism
- Female
- Fetus
- Ganglia/embryology
- Ganglia/growth & development
- Ganglia/metabolism
- Gene Expression Regulation, Developmental/genetics
- Hair Cells, Auditory, Inner/embryology
- Hair Cells, Auditory, Inner/growth & development
- Hair Cells, Auditory, Inner/metabolism
- Hair Cells, Auditory, Outer/embryology
- Hair Cells, Auditory, Outer/growth & development
- Hair Cells, Auditory, Outer/metabolism
- Humans
- Intercellular Junctions/genetics
- Intercellular Junctions/metabolism
- Mice
- Mice, Inbred C57BL
- Microfilament Proteins
- Neurons/cytology
- Neurons/metabolism
- Pregnancy
- RNA, Messenger/metabolism
- Spectrin/genetics
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Affiliation(s)
- Elena V Leonova
- Department of Otolaryngology/Head-Neck Surgery, Kresge Hearing Research Institute, The University of Michigan, 1150 W Medical Center Dr 9301E MSRB III, Box 0648, Ann Arbor, MI 48109-0648, USA
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46
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Zhen YY, Libotte T, Munck M, Noegel AA, Korenbaum E. NUANCE, a giant protein connecting the nucleus and actin cytoskeleton. J Cell Sci 2002; 115:3207-22. [PMID: 12118075 DOI: 10.1242/jcs.115.15.3207] [Citation(s) in RCA: 221] [Impact Index Per Article: 9.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
NUANCE (NUcleus and ActiN Connecting Element) was identified as a novel protein with an α-actinin-like actin-binding domain. A human 21.8 kb cDNA of NUANCE spreads over 373 kb on chromosome 14q22.1-q22.3. The cDNA sequence predicts a 796 kDa protein with an N-terminal actin-binding domain, a central coiled-coil rod domain and a predicted C-terminal transmembrane domain. High levels of NUANCE mRNA were detected in the kidney, liver,stomach, placenta, spleen, lymphatic nodes and peripheral blood lymphocytes. At the subcellular level NUANCE is present predominantly at the outer nuclear membrane and in the nucleoplasm. Domain analysis shows that the actin-binding domain binds to Factin in vitro and colocalizes with the actin cytoskeleton in vivo as a GFP-fusion protein. The C-terminal transmembrane domain is responsible for the targeting the nuclear envelope. Thus, NUANCE is the firstα-actinin-related protein that has the potential to link the microfilament system with the nucleus.
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Affiliation(s)
- Yen-Yi Zhen
- Institute for Biochemistry, Medical Faculty, University of Cologne, 50931 Cologne, Germany
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47
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Marceau N, Loranger A, Gilbert S, Daigle N, Champetier S. Keratin-mediated resistance to stress and apoptosis in simple epithelial cells in relation to health and disease. Biochem Cell Biol 2002. [PMID: 11716296 DOI: 10.1139/o01-138] [Citation(s) in RCA: 58] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/12/2023] Open
Abstract
Epithelial cells such as hepatocytes exhibit highly polarized properties as a result of the asymmetric distribution of subsets of receptors at unique portions of the surface membrane. While the proper targeting of these surface receptors and maintenance of the resulting polarity depend on microtubules (MTs), the Golgi sorting compartment, and different actin-filament networks, the contribution of keratin intermediate filaments (IFs) has been unclear. Recent data show that the latter cytoskeletal network plays a predominant role in providing resistance to various forms of stress and to apoptosis targeted to the surface membrane. In this context, we first summarize our knowledge of the domain- or assembly-related features of IF proteins and the dynamic properties of IF networks that may explain how the same keratin pair K8/K18 can exert multiple resistance-related functions in simple epithelial cells. We then examine the contribution of linker protein(s) that integrate interactions of keratin IFs with MTs and the actin-cytoskeleton network, polarity-dependent surface receptors and cytoplasmic organelles. We next address likely molecular mechanisms by which K8/K18 can selectively provide resistance to a mechanical or toxic stress, or to Fas-mediated apoptosis. Finally, these issues on keratin structure-function are examined within a context of pathological anomalies emerging in tissue architecture as a result of natural or targeted mutations, or posttranslational modifications at specific amino acid residues. Clearly. the data accumulated in recent years provide new and significant insights on the role of K8/K18, particularly under conditions where polarized cells resist to stressful or apoptotic insults.
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Affiliation(s)
- N Marceau
- Centre de recherche en cancérologie et Departement de médecine, Université Laval, Quebec, QC, Canada.
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48
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Karki S, Ligon LA, DeSantis J, Tokito M, Holzbaur ELF. PLAC-24 is a cytoplasmic dynein-binding protein that is recruited to sites of cell-cell contact. Mol Biol Cell 2002; 13:1722-34. [PMID: 12006665 PMCID: PMC111139 DOI: 10.1091/mbc.02-02-0011] [Citation(s) in RCA: 17] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/11/2022] Open
Abstract
We screened for polypeptides that interact specifically with dynein and identified a novel 24-kDa protein (PLAC-24) that binds directly to dynein intermediate chain (DIC). PLAC-24 is not a dynactin subunit, and the binding of PLAC-24 to the dynein intermediate chain is independent of the association between dynein and dynactin. Immunocytochemistry using PLAC-24-specific polyclonal antibodies revealed a punctate perinuclear distribution of the polypeptide in fibroblasts and isolated epithelial cells. However, as epithelial cells in culture make contact with adjacent cells, PLAC-24 is specifically recruited to the cortex at sites of contact, where the protein colocalizes with components of the adherens junction. Disruption of the cellular cytoskeleton with latrunculin or nocodazole indicates that the localization of PLAC-24 to the cortex is dependent on intact actin filaments but not on microtubules. Overexpression of beta-catenin also leads to a loss of PLAC-24 from sites of cell-cell contact. On the basis of these data and the recent observation that cytoplasmic dynein is also localized to sites of cell-cell contact in epithelial cells, we propose that PLAC-24 is part of a multiprotein complex localized to sites of intercellular contact that may function to tether microtubule plus ends to the actin-rich cellular cortex.
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Affiliation(s)
- Sher Karki
- Department of Physiology, School of Medicine, University of Pennsylvania, Philadelphia 19104-6085, USA
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49
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Song Y, Maul RS, Gerbin CS, Chang DD. Inhibition of anchorage-independent growth of transformed NIH3T3 cells by epithelial protein lost in neoplasm (EPLIN) requires localization of EPLIN to actin cytoskeleton. Mol Biol Cell 2002; 13:1408-16. [PMID: 11950948 PMCID: PMC102278 DOI: 10.1091/mbc.01-08-0414] [Citation(s) in RCA: 40] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/16/2023] Open
Abstract
Epithelial protein lost in neoplasm (EPLIN) is a cytoskeleton-associated protein characterized by the presence of a single centrally located lin-11, isl-1, and mec-3 (LIM) domain. We have reported previously that EPLIN is down-regulated in transformed cells. In this study, we have investigated whether ectopic expression of EPLIN affects transformation. In untransformed NIH3T3 cells, retroviral-mediated transduction of EPLIN did not alter the cell morphology or growth. NIH3T3 cells expressing EPLIN, however, failed to form colonies when transformed by the activated Cdc42 or the chimeric nuclear oncogene EWS/Fli-1. This suppression of anchorage-independent growth was not universal because EPLIN failed to inhibit the colony formation of Ras-transformed cells. Interestingly, the localization of EPLIN to the actin cytoskeleton was maintained in the EWS/Fli-1- or Cdc42-transformed cells, but not in Ras-transformed cells where it was distributed heterogeneously in the cytoplasm. Using truncated EPLIN constructs, we demonstrated that the NH(2)-terminal region of EPLIN is necessary for both the localization of EPLIN to the actin cytoskeleton and suppression of anchorage-independent growth of EWS/Fli-1-transformed cells. The LIM domain or the COOH-terminal region of EPLIN could be deleted without affecting its cytoskeletal localization or ability to suppress anchorage-dependent growth. Our study indicates EPLIN may function in growth control by associating with and regulating the actin cytoskeleton.
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Affiliation(s)
- Yuhong Song
- Department of Medicine, Jonsson Comprehensive Cancer Center, Molecular Biology Institute, University of California at Los Angeles School of Medicine, Los Angeles, CA 90095, USA
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50
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Lee S, Kolodziej PA. The plakin Short Stop and the RhoA GTPase are required for E-cadherin-dependent apical surface remodeling during tracheal tube fusion. Development 2002; 129:1509-20. [PMID: 11880359 DOI: 10.1242/dev.129.6.1509] [Citation(s) in RCA: 72] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022]
Abstract
Cells in vascular and other tubular networks require apical polarity in order to contact each other properly and to form lumen. As tracheal branches join together in Drosophila melanogaster embryos, specialized cells at the junction form a new E-cadherin-based contact and assemble an associated track of F-actin and the plakin Short Stop (shot). In these fusion cells, the apical surface determinant Discs Lost (Dlt) is subsequently deposited and new lumen forms along the track. In shot mutant embryos, the fusion cells fail to remodel the initial E-cadherin contact, to make an associated F-actin structure and to form lumenal connections between tracheal branches. Shot binding to F-actin and microtubules is required to rescue these defects. This finding has led us to investigate whether other regulators of the F-actin cytoskeleton similarly affect apical cell surface remodeling and lumen formation. Expression of constitutively active RhoA in all tracheal cells mimics the shot phenotype and affects Shot localization in fusion cells. The dominant negative RhoA phenotype suggests that RhoA controls apical surface formation throughout the trachea. We therefore propose that in fusion cells, Shot may function downstream of RhoA to form E-cadherin-associated cytoskeletal structures that are necessary for apical determinant localization.
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Affiliation(s)
- Seungbok Lee
- Department of Cell Biology and Center for Molecular Neuroscience, C-2210 Medical Center North,Vanderbilt University Medical Center, Nashville, TN 37232-0295, USA
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