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1
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Zou H, Wang Y, Luo G, Huang S. The biomechanical phenomena observed in the cell invasion pathway of porcine epidemic diarrhea virus: a review. Arch Virol 2025; 170:139. [PMID: 40418401 DOI: 10.1007/s00705-025-06326-1] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/05/2024] [Accepted: 01/08/2025] [Indexed: 05/27/2025]
Abstract
Porcine epidemic diarrhea virus (PEDV) is the primary pathogen responsible for highly contagious intestinal infections in pigs, which results in significant economic losses to the global animal husbandry industry. PEDV is an enveloped virus that enters cells via endocytosis, a process that is dependent on the binding of the viral surface S protein to a receptor on the host cell membrane. This results in a series of biomechanical alterations that drive the fusion of the viral and host cell membranes. These alterations stabilise the binding of the virus to the receptor and also affect the tension and the curvature of the plasma membrane and the formation of endocytic vesicles. A comprehensive understanding of the mechanism by which PEDV enters cells and the biomechanical changes that accompany this process is of paramount importance for the development of PEDV inhibitors, vaccines, and disease prevention and control strategies. Here, we review the general mechanism of PEDV entry, the biomechanical phenomena that occur during endocytosis, and the potential applications of biomechanics in antiviral therapy. It is anticipated that by gaining insight into these mechanisms, novel approaches to regulating viral entry pathways through mechanical interference, vaccine development, and antiviral drug design can be explored.
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Affiliation(s)
- Hong Zou
- College of Animal Science & Technology, Chongqing Three Gouges Vocational College, Chongqing, China
| | - Yi Wang
- College of Animal Science & Technology, Chongqing Three Gouges Vocational College, Chongqing, China
| | - Gan Luo
- Wanzhou Center for Animal Husbandry Industry Development of Chongqing, Chongqing, China.
| | - Shilei Huang
- College of Animal Science & Technology, Chongqing Three Gouges Vocational College, Chongqing, China.
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2
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Alonso A, Endres RG, Kirkegaard JB. Local Clustering and Global Spreading of Receptors for Optimal Spatial Gradient Sensing. PHYSICAL REVIEW LETTERS 2025; 134:158401. [PMID: 40315515 DOI: 10.1103/physrevlett.134.158401] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 09/20/2024] [Accepted: 03/11/2025] [Indexed: 05/04/2025]
Abstract
Spatial information from cell-surface receptors is crucial for processes that require signal processing and sensing of the environment. Here, we investigate the optimal placement of such receptors through a theoretical model that minimizes uncertainty in gradient estimation. Without requiring a priori knowledge of the physical limits of sensing or biochemical processes, we reproduce the emergence of clusters that closely resemble those observed in real cells. On perfect spherical surfaces, optimally placed receptors spread uniformly. When perturbations break their symmetry, receptors cluster in regions of high curvature, massively reducing estimation uncertainty. This agrees in many scenarios with mechanistic models that minimize elastic preference discrepancies between receptors and cell membranes. We further extend our model to motile receptors responding to cell-shape changes and external fluid flow, demonstrating the biological relevance of our model. Our findings provide a simple and utilitarian explanation for receptor clustering at high-curvature regions when high sensing accuracy is paramount.
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Affiliation(s)
- Albert Alonso
- Niels Bohr Institute, University of Copenhagen, Copenhagen, Denmark
| | - Robert G Endres
- Imperial College, Department of Life Sciences and Centre for Integrative Systems Biology and Bioinformatics, London, United Kingdom
| | - Julius B Kirkegaard
- Niels Bohr Institute, University of Copenhagen, Copenhagen, Denmark
- University of Copenhagen, Department of Computer Science, Copenhagen, Denmark
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3
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Lu CH, Lee CE, Nakamoto ML, Cui B. Cellular Signaling at the Nano-Bio Interface: Spotlighting Membrane Curvature. Annu Rev Phys Chem 2025; 76:251-277. [PMID: 40258240 PMCID: PMC12043246 DOI: 10.1146/annurev-physchem-090722-021151] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 04/23/2025]
Abstract
No longer viewed as a passive consequence of cellular activities, membrane curvature-the physical shape of the cell membrane-is now recognized as an active constituent of biological processes. Nanoscale topographies on extracellular matrices or substrate surfaces impart well-defined membrane curvatures on the plasma membrane. This review examines biological events occurring at the nano-bio interface, the physical interface between the cell membrane and surface nanotopography, which activates intracellular signaling by recruiting curvature-sensing proteins. We encompass a wide range of biological processes at the nano-bio interface, including cell adhesion, endocytosis, glycocalyx redistribution, regulation of mechanosensitive ion channels, cell migration, and differentiation. Despite the diversity of processes, we call attention to the critical role of membrane curvature in each process. We particularly highlight studies that elucidate molecular mechanisms involving curvature-sensing proteins with the hope of providing comprehensive insights into this rapidly advancing area of research.
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Affiliation(s)
- Chih-Hao Lu
- Department of Chemistry, Stanford University, Stanford, California, USA;
- Wu-Tsai Neuroscience Institute and Sarafan ChEM-H Institute, Stanford University, Stanford, California, USA
| | - Christina E Lee
- Wu-Tsai Neuroscience Institute and Sarafan ChEM-H Institute, Stanford University, Stanford, California, USA
- Biophysics Program, Stanford University School of Medicine, Stanford, California, USA
| | - Melissa L Nakamoto
- Department of Chemistry, Stanford University, Stanford, California, USA;
- Wu-Tsai Neuroscience Institute and Sarafan ChEM-H Institute, Stanford University, Stanford, California, USA
| | - Bianxiao Cui
- Department of Chemistry, Stanford University, Stanford, California, USA;
- Wu-Tsai Neuroscience Institute and Sarafan ChEM-H Institute, Stanford University, Stanford, California, USA
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4
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Bonazzi F, Weikl TR. Membrane-mediated interactions between arc-shaped particles strongly depend on membrane curvature. NANOSCALE 2025; 17:6841-6853. [PMID: 39964755 DOI: 10.1039/d4nr04674a] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 02/20/2025]
Abstract
Besides direct molecular interactions, proteins and nanoparticles embedded in or adsorbed to membranes experience indirect interactions that are mediated by the membranes. Membrane-mediated interactions between curvature-inducing proteins or nanoparticles can lead to assemblies of particles that generate highly curved spherical or tubular membrane shapes, but have mainly been quantified for planar or weakly curved membranes. In this article, we systematically investigate the membrane-mediated interactions of arc-shaped particles adsorbed to a variety of tubular and spherical membrane shapes with coarse-grained modelling and simulations. These arc-shaped particles induce membrane curvature by binding to the membrane with their inner, concave side akin to N-BAR domain proteins. We determine both the pairwise interaction free energy, which includes entropic contributions due to rotational entropy loss at close particle distances, and the pairwise interaction energy without entropic components from particle distributions observed in the simulations. For membrane shapes with small curvature, the membrane-mediated interaction free energies of particle pairs exceed the thermal energy kBT and can lead to particle ordering and aggregation. The interactions strongly decrease with increasing curvature of the membrane shape and are minimal for tubular shapes with membrane curvatures close to the particle curvature.
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Affiliation(s)
- Francesco Bonazzi
- Max Planck Institute of Colloids and Interfaces, Am Mühlenberg 1, 14476 Potsdam, Germany.
| | - Thomas R Weikl
- Max Planck Institute of Colloids and Interfaces, Am Mühlenberg 1, 14476 Potsdam, Germany.
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5
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Wang W, Zhao J, Li H, Huang D, Fu S, Li Z. Autophagy-related biomarkers identified in sepsis-induced ARDS through bioinformatics analysis. Sci Rep 2025; 15:7864. [PMID: 40050379 PMCID: PMC11885441 DOI: 10.1038/s41598-025-92409-7] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/08/2024] [Accepted: 02/27/2025] [Indexed: 03/09/2025] Open
Abstract
While dysregulated autophagy has been linked to acute respiratory distress syndrome (ARDS) development in sepsis, the exact regulatory mechanisms driving this process remain unclear. This study systematically investigated autophagy-related genes in sepsis-induced ARDS using integrative bioinformatics, including weighted gene coexpression network analysis (WGCNA), differential gene expression analysis (DEGs), receiver operating characteristic (ROC) curve analysis, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis, protein‒protein interaction (PPI) network analysis, and immune infiltration analysis. Hub genes were further validated by qPCR in Beas-2B cells receiving lipopolysaccharide (LPS) stimulation. We identified 18 autophagy-related DEGs with diagnostic potential for sepsis-induced ARDS. These DEGs were linked to endocytosis, protein kinase inhibition, and enigmatic Ficolin-1-rich granules. The downregulated hallmark signaling pathways involved apoptosis, complement, IL-2/STAT5, and KRAS signaling. Immune infiltration analysis revealed alterations in 7 immune cell subsets, including CD8 + T-cell exhaustion, natural killer cell reduction, and the type 1 helper T-cell response. When Beas-2B cells were treated with LPS, we discovered that 6 out of the 18 hub genes were significantly downregulated. Our findings provide novel insights into autophagy-mediated ARDS pathogenesis in sepsis. The hub genes represent promising candidates for clinical biomarker development and therapeutic targeting, which necessitates further validation.
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Affiliation(s)
- Wei Wang
- Department of Surgical Intensive Care Unit, First Affiliated Hospital, School of Medicine, Zhejiang University, 79 Qingchun Road, Hangzhou, 310003, China
| | - Jianfeng Zhao
- Department of Surgical Intensive Care Unit, First Affiliated Hospital, School of Medicine, Zhejiang University, 79 Qingchun Road, Hangzhou, 310003, China
| | - Hui Li
- Department of Surgical Intensive Care Unit, First Affiliated Hospital, School of Medicine, Zhejiang University, 79 Qingchun Road, Hangzhou, 310003, China
| | - Dabing Huang
- Department of Surgical Intensive Care Unit, First Affiliated Hospital, School of Medicine, Zhejiang University, 79 Qingchun Road, Hangzhou, 310003, China
| | - Shuiqiao Fu
- Department of Surgical Intensive Care Unit, First Affiliated Hospital, School of Medicine, Zhejiang University, 79 Qingchun Road, Hangzhou, 310003, China.
| | - Zhitao Li
- Department of Surgical Intensive Care Unit, First Affiliated Hospital, School of Medicine, Zhejiang University, 79 Qingchun Road, Hangzhou, 310003, China.
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6
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Karbanová J, Thamm K, Fargeas CA, Deniz IA, Lorico A, Corbeil D. Prominosomes - a particular class of extracellular vesicles containing prominin-1/CD133? J Nanobiotechnology 2025; 23:61. [PMID: 39881297 PMCID: PMC11776279 DOI: 10.1186/s12951-025-03102-w] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/17/2024] [Accepted: 01/09/2025] [Indexed: 01/31/2025] Open
Abstract
Extracellular membrane vesicles (EVs) offer promising values in various medical fields, e.g., as biomarkers in liquid biopsies or as native (or bioengineered) biological nanocarriers in tissue engineering, regenerative medicine and cancer therapy. Based on their cellular origin EVs can vary considerably in composition and diameter. Cell biological studies on mammalian prominin-1, a cholesterol-binding membrane glycoprotein, have helped to reveal new donor membranes as sources of EVs. For instance, small EVs can originate from microvilli and primary cilia, while large EVs might be produced by transient structures such as retracting cellular extremities of cancer cells during the mitotic rounding process, and the midbody at the end of cytokinesis. Here, we will highlight the various subcellular origins of prominin-1+ EVs, also called prominosomes, and the potential mechanism(s) regulating their formation. We will further discuss the molecular and cellular characteristics of prominin-1, notably those that have a direct effect on the release of prominin-1+ EVs, a process that might be directly implicated in donor cell reprogramming of stem and cancer stem cells. Prominin-1+ EVs also mediate intercellular communication during embryonic development and adult homeostasis in healthy individuals, while disseminating biological information during diseases.
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Affiliation(s)
- Jana Karbanová
- Biotechnology Center (BIOTEC) and Center for Molecular and Cellular Bioengineering, Technische Universität Dresden, Tatzberg 47-49, 01307, Dresden, Germany.
- Tissue Engineering Laboratories, Medizinische Fakultät der Technischen Universität Dresden, Fetscherstr. 74, 01307, Dresden, Germany.
- Tissue Engineering Laboratories, Biotechnology Center, Technische Universität Dresden, Tatzberg 47-49, 01307, Dresden, Germany.
| | - Kristina Thamm
- Biotechnology Center (BIOTEC) and Center for Molecular and Cellular Bioengineering, Technische Universität Dresden, Tatzberg 47-49, 01307, Dresden, Germany
- Tissue Engineering Laboratories, Medizinische Fakultät der Technischen Universität Dresden, Fetscherstr. 74, 01307, Dresden, Germany
- denovoMATRIX GmbH, Tatzberg 47, 01307, Dresden, Germany
| | - Christine A Fargeas
- Biotechnology Center (BIOTEC) and Center for Molecular and Cellular Bioengineering, Technische Universität Dresden, Tatzberg 47-49, 01307, Dresden, Germany
- Tissue Engineering Laboratories, Medizinische Fakultät der Technischen Universität Dresden, Fetscherstr. 74, 01307, Dresden, Germany
| | - Ilker A Deniz
- Biotechnology Center (BIOTEC) and Center for Molecular and Cellular Bioengineering, Technische Universität Dresden, Tatzberg 47-49, 01307, Dresden, Germany
- Tissue Engineering Laboratories, Medizinische Fakultät der Technischen Universität Dresden, Fetscherstr. 74, 01307, Dresden, Germany
| | - Aurelio Lorico
- College of Osteopathic Medicine, Touro University Nevada, 874 American Pacific Drive, Henderson, NV, 89014, USA
| | - Denis Corbeil
- Biotechnology Center (BIOTEC) and Center for Molecular and Cellular Bioengineering, Technische Universität Dresden, Tatzberg 47-49, 01307, Dresden, Germany.
- Tissue Engineering Laboratories, Medizinische Fakultät der Technischen Universität Dresden, Fetscherstr. 74, 01307, Dresden, Germany.
- Tissue Engineering Laboratories, Biotechnology Center, Technische Universität Dresden, Tatzberg 47-49, 01307, Dresden, Germany.
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7
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Song S, Li T, Stevens AO, Shorty T, He Y. Molecular Dynamics Reveal Key Steps in BAR-Related Membrane Remodeling. Pathogens 2024; 13:902. [PMID: 39452773 PMCID: PMC11510478 DOI: 10.3390/pathogens13100902] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/03/2024] [Revised: 10/08/2024] [Accepted: 10/13/2024] [Indexed: 10/26/2024] Open
Abstract
Endocytosis plays a complex role in pathogen-host interactions. It serves as a pathway for pathogens to enter the host cell and acts as a part of the immune defense mechanism. Endocytosis involves the formation of lipid membrane vesicles and the reshaping of the cell membrane, a task predominantly managed by proteins containing BAR (Bin1/Amphiphysin/yeast RVS167) domains. Insights into how BAR domains can remodel and reshape cell membranes provide crucial information on infections and can aid the development of treatment. Aiming at deciphering the roles of the BAR dimers in lipid membrane bending and remodeling, we conducted extensive all-atom molecular dynamics simulations and discovered that the presence of helix kinks divides the BAR monomer into two segments-the "arm segment" and the "core segment"-which exhibit distinct movement patterns. Contrary to the prior hypothesis of BAR domains working as a rigid scaffold, we found that it functions in an "Arms-Hands" mode. These findings enhance the understanding of endocytosis, potentially advancing research on pathogen-host interactions and aiding in the identification of new treatment strategies targeting BAR domains.
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Affiliation(s)
- Shenghan Song
- Department of Chemistry & Chemical Biology, The University of New Mexico, Albuquerque, NM 87131, USA
| | - Tongtong Li
- Department of Chemistry & Chemical Biology, The University of New Mexico, Albuquerque, NM 87131, USA
| | - Amy O. Stevens
- Department of Chemistry & Chemical Biology, The University of New Mexico, Albuquerque, NM 87131, USA
| | - Temair Shorty
- Department of Chemistry & Chemical Biology, The University of New Mexico, Albuquerque, NM 87131, USA
| | - Yi He
- Department of Chemistry & Chemical Biology, The University of New Mexico, Albuquerque, NM 87131, USA
- Translational Informatics Division, Department of Internal Medicine, The University of New Mexico, Albuquerque, NM 87131, USA
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8
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Gao J, Hou R, Hu W, Weikl TR, Hu J. Which Coverages of Arc-Shaped Proteins Are Required for Membrane Tubulation? J Phys Chem B 2024; 128:4735-4740. [PMID: 38706129 DOI: 10.1021/acs.jpcb.4c01019] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 05/07/2024]
Abstract
Arc-shaped BIN/Amphiphysin/Rvs (BAR) domain proteins generate curvature by binding to membranes and induce membrane tubulation at sufficiently large protein coverages. For the amphiphysin N-BAR domain, Le Roux et al., Nat. Commun. 2021, 12, 6550, measured a threshold coverage of 0.44 ± 0.097 for nanotubules emerging from the supported lipid bilayer. In this article, we systematically investigate membrane tubulation induced by arc-shaped protein-like particles with coarse-grained modeling and simulations and determine the threshold coverages at different particle-particle interaction strengths and membrane spontaneous curvatures. In our simulations, the binding of arc-shaped particles induces a membrane shape transition from spherical vesicles to tubules at a particle threshold coverage of about 0.5, which is rather robust to variations of the direct attractive particle interactions or spontaneous membrane curvature in the coarse-grained model. Our study suggests that threshold coverages of around or slightly below 0.5 are a general requirement for membrane tubulation by arc-shaped BAR domain proteins.
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Affiliation(s)
- Jie Gao
- Kuang Yaming Honors School, Nanjing University, Nanjing 210023, China
- Department of Polymer Science and Engineering, Key Laboratory of High Performance Polymer Material and Technology of Ministry of Education, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210023, China
| | - Ruihan Hou
- Kuang Yaming Honors School, Nanjing University, Nanjing 210023, China
- Department of Polymer Science and Engineering, Key Laboratory of High Performance Polymer Material and Technology of Ministry of Education, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210023, China
| | - Wenbing Hu
- Department of Polymer Science and Engineering, Key Laboratory of High Performance Polymer Material and Technology of Ministry of Education, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210023, China
| | - Thomas R Weikl
- Department of Bio-Molecular Systems, Max Planck Institute of Colloids and Interfaces, Science Park Golm, 14424 Potsdam, Germany
| | - Jinglei Hu
- Kuang Yaming Honors School, Nanjing University, Nanjing 210023, China
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9
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McCright J, Yarmovsky J, Maisel K. Para- and Transcellular Transport Kinetics of Nanoparticles across Lymphatic Endothelial Cells. Mol Pharm 2024; 21:1160-1169. [PMID: 37851841 PMCID: PMC10923144 DOI: 10.1021/acs.molpharmaceut.3c00720] [Citation(s) in RCA: 1] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/20/2023]
Abstract
Lymphatic vessels have received significant attention as drug delivery targets, as they shuttle materials from peripheral tissues to the lymph nodes, where adaptive immunity is formed. Delivery of immune modulatory materials to the lymph nodes via lymphatic vessels has been shown to enhance their efficacy and also improve the bioavailability of drugs when delivered to intestinal lymphatic vessels. In this study, we generated a three-compartment model of a lymphatic vessel with a set of kinematic differential equations to describe the transport of nanoparticles from the surrounding tissues into lymphatic vessels. We used previously published data and collected additional experimental parameters, including the transport efficiency of nanoparticles over time, and also examined how nanoparticle formulation affected the cellular transport mechanisms using small molecule inhibitors. These experimental data were incorporated into a system of kinematic differential equations, and nonlinear, least-squares curve fitting algorithms were employed to extrapolate transport coefficients within our model. The subsequent computational framework produced some of the first parameters to describe transport kinetics across lymphatic endothelial cells and allowed for the quantitative analysis of the driving mechanisms of transport into lymphatic vessels. Our model indicates that transcellular mechanisms, such as micro- and macropinocytosis, drive transport into lymphatics. This information is crucial to further design strategies that will modulate lymphatic transport for drug delivery, particularly in diseases like lymphedema, where normal lymphatic functions are impaired.
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Affiliation(s)
- Jacob McCright
- Department of Bioengineering, University of Maryland College Park, College Park, Maryland 20742, United States
| | - Jenny Yarmovsky
- Department of Bioengineering, University of Maryland College Park, College Park, Maryland 20742, United States
| | - Katharina Maisel
- Department of Bioengineering, University of Maryland College Park, College Park, Maryland 20742, United States
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10
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Li Q, Lu J, Liu J, Li J, Zhang G, Du G, Chen J. High-throughput droplet microfluidics screening and genome sequencing analysis for improved amylase-producing Aspergillus oryzae. BIOTECHNOLOGY FOR BIOFUELS AND BIOPRODUCTS 2023; 16:185. [PMID: 38031105 PMCID: PMC10685594 DOI: 10.1186/s13068-023-02437-6] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 09/01/2023] [Accepted: 11/21/2023] [Indexed: 12/01/2023]
Abstract
BACKGROUND The exceptional protein secretion capacity, intricate post-translational modification processes, and inherent safety features of A. oryzae make it a promising expression system. However, heterologous protein expression levels of existing A. oryzae species cannot meet the requirement for industrial-scale production. Therefore, establishing an efficient screening technology is significant for the development of the A. oryzae expression system. RESULTS In this work, a high-throughput screening method suitable for A. oryzae has been established by combining the microfluidic system and flow cytometry. Its screening efficiency can reach 350 droplets per minute. The diameter of the microdroplet was enlarged to 290 µm to adapt to the polar growth of A. oryzae hyphae. Through enrichment and screening from approximately 450,000 droplets within 2 weeks, a high-producing strain with α-amylase increased by 6.6 times was successfully obtained. Furthermore, 29 mutated genes were identified by genome resequencing of high-yield strains, with 15 genes subjected to editing and validation. Two genes may individually influence α-amylase expression in A. oryzae by affecting membrane-associated multicellular processes and regulating the transcription of related genes. CONCLUSIONS The developed high-throughput screening strategy provides a reference for other filamentous fungi and Streptomyces. Besides, the strains with different excellent characteristics obtained by efficient screening can also provide materials for the analysis of genetic and regulatory mechanisms in the A. oryzae expression system.
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Affiliation(s)
- Qinghua Li
- Science Center for Future Foods, Jiangnan University, 1800 Lihu Road, Wuxi, 214122, Jiangsu, China
- National Engineering Research Center for Cereal Fermentation and Food Biomanufacturing, Jiangnan University, 1800 Lihu Road, Wuxi, 214122, Jiangsu, China
| | - Jinchang Lu
- Science Center for Future Foods, Jiangnan University, 1800 Lihu Road, Wuxi, 214122, Jiangsu, China
- National Engineering Research Center for Cereal Fermentation and Food Biomanufacturing, Jiangnan University, 1800 Lihu Road, Wuxi, 214122, Jiangsu, China
| | - Jingya Liu
- Science Center for Future Foods, Jiangnan University, 1800 Lihu Road, Wuxi, 214122, Jiangsu, China
- National Engineering Research Center for Cereal Fermentation and Food Biomanufacturing, Jiangnan University, 1800 Lihu Road, Wuxi, 214122, Jiangsu, China
| | - Jianghua Li
- Science Center for Future Foods, Jiangnan University, 1800 Lihu Road, Wuxi, 214122, Jiangsu, China
- School of Biotechnology and Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, 1800 Lihu Road, Wuxi, 214122, Jiangsu, China
| | - Guoqiang Zhang
- Science Center for Future Foods, Jiangnan University, 1800 Lihu Road, Wuxi, 214122, Jiangsu, China.
- National Engineering Research Center for Cereal Fermentation and Food Biomanufacturing, Jiangnan University, 1800 Lihu Road, Wuxi, 214122, Jiangsu, China.
| | - Guocheng Du
- Science Center for Future Foods, Jiangnan University, 1800 Lihu Road, Wuxi, 214122, Jiangsu, China
- School of Biotechnology and Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, 1800 Lihu Road, Wuxi, 214122, Jiangsu, China
- The Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, 1800 Lihu Road, Wuxi, 214122, Jiangsu, China
| | - Jian Chen
- Science Center for Future Foods, Jiangnan University, 1800 Lihu Road, Wuxi, 214122, Jiangsu, China
- National Engineering Research Center for Cereal Fermentation and Food Biomanufacturing, Jiangnan University, 1800 Lihu Road, Wuxi, 214122, Jiangsu, China
- School of Biotechnology and Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, 1800 Lihu Road, Wuxi, 214122, Jiangsu, China
- The Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, 1800 Lihu Road, Wuxi, 214122, Jiangsu, China
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11
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Lu CH, Tsai CT, Jones Iv T, Chim V, Klausen LH, Zhang W, Li X, Jahed Z, Cui B. A NanoCurvS platform for quantitative and multiplex analysis of curvature-sensing proteins. Biomater Sci 2023; 11:5205-5217. [PMID: 37337788 PMCID: PMC10809791 DOI: 10.1039/d2bm01856j] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 06/21/2023]
Abstract
The cell membrane is characterized by a rich variety of topographical features such as local protrusions or invaginations. Curvature-sensing proteins, including the Bin/Amphiphysin/Rvs (BAR) or epsin N-terminal homology (ENTH) family proteins, sense the bending sharpness and the positive/negative sign of these topographical features to induce subsequent intracellular signaling. A number of assays have been developed to study curvature-sensing properties of proteins in vitro, but it is still challenging to probe low curvature regime with the diameter of curvature from hundreds of nanometers to micrometers. It is particularly difficult to generate negative membrane curvatures with well-defined curvature values in the low curvature regime. In this work, we develop a nanostructure-based curvature sensing (NanoCurvS) platform that enables quantitative and multiplex analysis of curvature-sensitive proteins in the low curvature regime, in both negative and positive directions. We use NanoCurvS to quantitatively measure the sensing range of a negative curvature-sensing protein IRSp53 (an I-BAR protein) and a positive curvature-sensing protein FBP17 (an F-BAR protein). We find that, in cell lysates, the I-BAR domain of IRSp53 is able to sense shallow negative curvatures with the diameter-of-curvature up to 1500 nm, a range much wider than previously expected. NanoCurvS is also used to probe the autoinhibition effect of IRSp53 and the phosphorylation effect of FBP17. Therefore, the NanoCurvS platform provides a robust, multiplex, and easy-to-use tool for quantitative analysis of both positive and negative curvature-sensing proteins.
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Affiliation(s)
- Chih-Hao Lu
- Department of Chemistry, Stanford University, Stanford, CA, USA.
| | - Ching-Ting Tsai
- Department of Chemistry, Stanford University, Stanford, CA, USA.
| | - Taylor Jones Iv
- Department of Chemistry, Stanford University, Stanford, CA, USA.
| | - Vincent Chim
- Department of Chemistry, Stanford University, Stanford, CA, USA.
| | - Lasse H Klausen
- Department of Chemistry, Stanford University, Stanford, CA, USA.
| | - Wei Zhang
- Department of Chemistry, Stanford University, Stanford, CA, USA.
| | - Xiao Li
- Department of Chemistry, Stanford University, Stanford, CA, USA.
| | - Zeinab Jahed
- Department of Chemistry, Stanford University, Stanford, CA, USA.
| | - Bianxiao Cui
- Department of Chemistry, Stanford University, Stanford, CA, USA.
- Wu-Tsai Neuroscience Institute and ChEM-H institute, Stanford University, Stanford, CA, USA
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12
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Lemaigre C, Ceuppens A, Valades-Cruz CA, Ledoux B, Vanbeneden B, Hassan M, Zetterberg FR, Nilsson UJ, Johannes L, Wunder C, Renard HF, Morsomme P. N-BAR and F-BAR proteins-endophilin-A3 and PSTPIP1-control clathrin-independent endocytosis of L1CAM. Traffic 2023; 24:190-212. [PMID: 36843549 DOI: 10.1111/tra.12883] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/15/2022] [Revised: 02/07/2023] [Accepted: 02/18/2023] [Indexed: 02/28/2023]
Abstract
Recent advances in the field demonstrate the high diversity and complexity of endocytic pathways. In the current study, we focus on the endocytosis of L1CAM. This glycoprotein plays a major role in the development of the nervous system, and is involved in cancer development and is associated with metastases and poor prognosis. Two L1CAM isoforms are subject to endocytosis: isoform 1, described as a clathrin-mediated cargo; isoform 2, whose endocytosis has never been studied. Deciphering the molecular machinery of isoform 2 internalisation should contribute to a better understanding of its pathophysiological role. First, we demonstrated in our cellular context that both isoforms of L1CAM are mainly a clathrin-independent cargo, which was not expected for isoform 1. Second, the mechanism of L1CAM endocytosis is specifically mediated by the N-BAR domain protein endophilin-A3. Third, we discovered PSTPIP1, an F-BAR domain protein, as a novel actor in this endocytic process. Finally, we identified galectins as endocytic partners and negative regulators of L1CAM endocytosis. In summary, the interplay of the BAR proteins endophilin-A3 and PSTPIP1, and galectins fine tune the clathrin-independent endocytosis of L1CAM.
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Affiliation(s)
- Camille Lemaigre
- UCLouvain, Louvain Institute of Biomolecular Science and Technology, Group of Molecular Physiology, Louvain-la-Neuve, Belgium
| | - Apolline Ceuppens
- UCLouvain, Louvain Institute of Biomolecular Science and Technology, Group of Molecular Physiology, Louvain-la-Neuve, Belgium
| | - Cesar Augusto Valades-Cruz
- Institut Curie, Université PSL, U1143 INSERM, UMR3666 CNRS, Cellular and Chemical Biology unit, Paris, France.,SERPICO Project Team, UMR144 CNRS Institut Curie, PSL Research University, Paris, France.,SERPICO Project Team, Inria Centre Rennes-Bretagne Atlantique, Campus Universitaire de Beaulieu, Rennes, France
| | - Benjamin Ledoux
- UCLouvain, Louvain Institute of Biomolecular Science and Technology, Group of Molecular Physiology, Louvain-la-Neuve, Belgium
| | - Bastien Vanbeneden
- UCLouvain, Louvain Institute of Biomolecular Science and Technology, Group of Molecular Physiology, Louvain-la-Neuve, Belgium
| | | | | | - Ulf J Nilsson
- Department of Chemistry, Lund University, Lund, Sweden
| | - Ludger Johannes
- Institut Curie, Université PSL, U1143 INSERM, UMR3666 CNRS, Cellular and Chemical Biology unit, Paris, France
| | - Christian Wunder
- Institut Curie, Université PSL, U1143 INSERM, UMR3666 CNRS, Cellular and Chemical Biology unit, Paris, France
| | - Henri-François Renard
- UNamur, NARILIS, Unité de recherche en biologie cellulaire animale (URBC), Namur, Belgium
| | - Pierre Morsomme
- UCLouvain, Louvain Institute of Biomolecular Science and Technology, Group of Molecular Physiology, Louvain-la-Neuve, Belgium
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13
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Kunduri G, Acharya U, Acharya JK. Lipid Polarization during Cytokinesis. Cells 2022; 11:3977. [PMID: 36552741 PMCID: PMC9776629 DOI: 10.3390/cells11243977] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/21/2022] [Revised: 12/01/2022] [Accepted: 12/02/2022] [Indexed: 12/13/2022] Open
Abstract
The plasma membrane of eukaryotic cells is composed of a large number of lipid species that are laterally segregated into functional domains as well as asymmetrically distributed between the outer and inner leaflets. Additionally, the spatial distribution and organization of these lipids dramatically change in response to various cellular states, such as cell division, differentiation, and apoptosis. Division of one cell into two daughter cells is one of the most fundamental requirements for the sustenance of growth in all living organisms. The successful completion of cytokinesis, the final stage of cell division, is critically dependent on the spatial distribution and organization of specific lipids. In this review, we discuss the properties of various lipid species associated with cytokinesis and the mechanisms involved in their polarization, including forward trafficking, endocytic recycling, local synthesis, and cortical flow models. The differences in lipid species requirements and distribution in mitotic vs. male meiotic cells will be discussed. We will concentrate on sphingolipids and phosphatidylinositols because their transbilayer organization and movement may be linked via the cytoskeleton and thus critically regulate various steps of cytokinesis.
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Affiliation(s)
- Govind Kunduri
- Cancer and Developmental Biology Laboratory, National Cancer Institute, Frederick, MD 21702, USA
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14
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Miao X, Wu J, Chen H, Lu G. Comprehensive Analysis of the Structure and Function of Peptide:N-Glycanase 1 and Relationship with Congenital Disorder of Deglycosylation. Nutrients 2022; 14:nu14091690. [PMID: 35565658 PMCID: PMC9102325 DOI: 10.3390/nu14091690] [Citation(s) in RCA: 7] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/25/2022] [Revised: 04/13/2022] [Accepted: 04/15/2022] [Indexed: 02/01/2023] Open
Abstract
The cytosolic PNGase (peptide:N-glycanase), also known as peptide-N4-(N-acetyl-β-glucosaminyl)-asparagine amidase, is a well-conserved deglycosylation enzyme (EC 3.5.1.52) which catalyzes the non-lysosomal hydrolysis of an N(4)-(acetyl-β-d-glucosaminyl) asparagine residue (Asn, N) into a N-acetyl-β-d-glucosaminyl-amine and a peptide containing an aspartate residue (Asp, D). This enzyme (NGLY1) plays an essential role in the clearance of misfolded or unassembled glycoproteins through a process named ER-associated degradation (ERAD). Accumulating evidence also points out that NGLY1 deficiency can cause an autosomal recessive (AR) human genetic disorder associated with abnormal development and congenital disorder of deglycosylation. In addition, the loss of NGLY1 can affect multiple cellular pathways, including but not limited to NFE2L1 pathway, Creb1/Atf1-AQP pathway, BMP pathway, AMPK pathway, and SLC12A2 ion transporter, which might be the underlying reasons for a constellation of clinical phenotypes of NGLY1 deficiency. The current comprehensive review uncovers the NGLY1’ssdetailed structure and its important roles for participation in ERAD, involvement in CDDG and potential treatment for NGLY1 deficiency.
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Affiliation(s)
- Xiangguang Miao
- Queen Mary School, Nanchang University, No. 1299 Xuefu Avenue, Honggutan New District, Nanchang 330036, China;
| | - Jin Wu
- Laboratory of Translational Medicine Research, Department of Pathology, Deyang People’s Hospital, No. 173 First Section of Taishanbei Road, Jingyang District, Deyang 618000, China;
- Deyang Key Laboratory of Tumor Molecular Research, No. 173 First Section of Taishanbei Road, Jingyang District, Deyang 618000, China
- Department of Molecular & Cellular Biology, Roswell Park Comprehensive Cancer Center, Elm and Carlton Streets, Buffalo, NY 14263, USA
| | - Hongping Chen
- Department of Histology and Embryology, Medical College of Nanchang University, Nanchang 330006, China
- Correspondence: (H.C.); (G.L.); Tel.: +86-188-0147-4087 (G.L.)
| | - Guanting Lu
- Laboratory of Translational Medicine Research, Department of Pathology, Deyang People’s Hospital, No. 173 First Section of Taishanbei Road, Jingyang District, Deyang 618000, China;
- Deyang Key Laboratory of Tumor Molecular Research, No. 173 First Section of Taishanbei Road, Jingyang District, Deyang 618000, China
- Correspondence: (H.C.); (G.L.); Tel.: +86-188-0147-4087 (G.L.)
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15
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Binkle L, Klein M, Borgmeyer U, Kuhl D, Hermey G. The adaptor protein PICK1 targets the sorting receptor SorLA. Mol Brain 2022; 15:18. [PMID: 35183222 PMCID: PMC8858569 DOI: 10.1186/s13041-022-00903-0] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/06/2021] [Accepted: 02/07/2022] [Indexed: 11/26/2022] Open
Abstract
SorLA is a member of the Vps10p-domain (Vps10p-D) receptor family of type-I transmembrane proteins conveying neuronal endosomal sorting. The extracellular/luminal moiety of SorLA has a unique mosaic domain composition and interacts with a large number of different and partially unrelated ligands, including the amyloid precursor protein as well as amyloid-β. Several studies support a strong association of SorLA with sporadic and familial forms of Alzheimer’s disease (AD). Although SorLA seems to be an important factor in AD, the large number of different ligands suggests a role as a neuronal multifunctional receptor with additional intracellular sorting capacities. Therefore, understanding the determinants of SorLA’s subcellular targeting might be pertinent for understanding neuronal endosomal sorting mechanisms in general. A number of cytosolic adaptor proteins have already been demonstrated to determine intracellular trafficking of SorLA. Most of these adaptors and several ligands of the extracellular/luminal moiety are shared with the Vps10p-D receptor Sortilin. Although SorLA and Sortilin show both a predominant intracellular and endosomal localization, they are targeted to different endosomal compartments. Thus, independent adaptor proteins may convey their differential endosomal targeting. Here, we hypothesized that Sortilin and SorLA interact with the cytosolic adaptors PSD95 and PICK1 which have been shown to bind the Vps10p-D receptor SorCS3. We observed only an interaction for SorLA and PICK1 in mammalian-two-hybrid, pull-down and cellular recruitment experiments. We demonstrate by mutational analysis that the C-terminal minimal PDZ domain binding motif VIA of SorLA mediates the interaction. Moreover, we show co-localization of SorLA and PICK1 at vesicular structures in primary neurons. Although the physiological role of the interaction between PICK1 and SorLA remains unsolved, our study suggests that PICK1 partakes in regulating SorLA’s intracellular itinerary.
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16
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Lysine acetylation regulates the interaction between proteins and membranes. Nat Commun 2021; 12:6466. [PMID: 34753925 PMCID: PMC8578602 DOI: 10.1038/s41467-021-26657-2] [Citation(s) in RCA: 37] [Impact Index Per Article: 9.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/15/2018] [Accepted: 09/30/2021] [Indexed: 11/23/2022] Open
Abstract
Lysine acetylation regulates the function of soluble proteins in vivo, yet it remains largely unexplored whether lysine acetylation regulates membrane protein function. Here, we use bioinformatics, biophysical analysis of recombinant proteins, live-cell fluorescent imaging and genetic manipulation of Drosophila to explore lysine acetylation in peripheral membrane proteins. Analysis of 50 peripheral membrane proteins harboring BAR, PX, C2, or EHD membrane-binding domains reveals that lysine acetylation predominates in membrane-interaction regions. Acetylation and acetylation-mimicking mutations in three test proteins, amphiphysin, EHD2, and synaptotagmin1, strongly reduce membrane binding affinity, attenuate membrane remodeling in vitro and alter subcellular localization. This effect is likely due to the loss of positive charge, which weakens interactions with negatively charged membranes. In Drosophila, acetylation-mimicking mutations of amphiphysin cause severe disruption of T-tubule organization and yield a flightless phenotype. Our data provide mechanistic insights into how lysine acetylation regulates membrane protein function, potentially impacting a plethora of membrane-related processes. Lysine acetylation regulates the function of soluble proteins in vivo, yet it remains largely unexplored whether lysine acetylation regulates the function of membrane proteins. Here, the authors map lysine acetylation predominantly in membrane-interaction regions in peripheral membrane proteins and show with three candidate proteins how lysine acetylation is a regulator of membrane protein function.
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17
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Zhu FY, Wang LL, Meng TG, Wang RL, Yang ZX, Cao Y, Zhu GY, Jin Z, Gao LL, Zeng WT, Wang ZB, Sun QY, Zhang D. Inhibiting bridge integrator 2 phosphorylation leads to improved oocyte quality, ovarian health and fertility in aging and after chemotherapy in mice. NATURE AGING 2021; 1:1010-1023. [PMID: 37118338 DOI: 10.1038/s43587-021-00133-4] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Received: 01/09/2021] [Accepted: 10/04/2021] [Indexed: 04/30/2023]
Abstract
Female ovaries degenerate about 20 years earlier than testes leading to reduced primordial follicle reserve and a reduction in oocyte quality. Here we found that bridge integrator 2 (BIN2) is enriched in mouse ovaries and oocytes and that global knockout of this protein improves both female fertility and oocyte quality. Quantitative ovarian proteomics and phosphoproteomics showed that Bin2 knockout led to a decrease in phosphorylated ribosomal protein S6 (p-RPS6), a component of the mammalian target of rapamycin pathway and greatly increased nicotinamide nucleotide transhydrogenase (NNT), the free-radical detoxifier. Mechanistically, we find that phosphorylation of BIN2 at Thr423 and Ser424 leads to its translocation from the membrane to the cytoplasm, subsequent phosphorylation of RPS6 and inhibition of Nnt translation. We synthesized a BIN2-penetrating peptide (BPP) designed to inhibit BIN2 phosphorylation and found that a 3-week BPP treatment improved primordial follicle reserve and oocyte quality in aging and after chemotherapy-induced premature ovarian failure without discernible side effects.
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Affiliation(s)
- Feng-Yu Zhu
- State Key Lab of Reproductive Medicine, Nanjing Medical University, Nanjing, China
| | - Li-Li Wang
- State Key Lab of Reproductive Medicine, Nanjing Medical University, Nanjing, China
| | - Tie-Gang Meng
- Fertility Preservation Lab and Guangdong-Hong Kong Metabolism & Reproduction Joint Laboratory, Reproductive Medicine Center, Guangdong Second Provincial General Hospital, Guangzhou, China
- State Key Lab of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China
| | - Ruo-Lei Wang
- State Key Lab of Reproductive Medicine, Nanjing Medical University, Nanjing, China
| | - Zhi-Xia Yang
- State Key Lab of Reproductive Medicine, Nanjing Medical University, Nanjing, China
| | - Ying Cao
- State Key Lab of Reproductive Medicine, Nanjing Medical University, Nanjing, China
| | - Gang-Yi Zhu
- State Key Lab of Reproductive Medicine, Nanjing Medical University, Nanjing, China
| | - Zhen Jin
- State Key Lab of Reproductive Medicine, Nanjing Medical University, Nanjing, China
| | - Lei-Lei Gao
- State Key Lab of Reproductive Medicine, Nanjing Medical University, Nanjing, China
| | - Wen-Tao Zeng
- Animal Core Facility, Nanjing Medical University, Nanjing, China
| | - Zhen-Bo Wang
- State Key Lab of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China.
| | - Qing-Yuan Sun
- Fertility Preservation Lab and Guangdong-Hong Kong Metabolism & Reproduction Joint Laboratory, Reproductive Medicine Center, Guangdong Second Provincial General Hospital, Guangzhou, China.
| | - Dong Zhang
- State Key Lab of Reproductive Medicine, Nanjing Medical University, Nanjing, China.
- Animal Core Facility, Nanjing Medical University, Nanjing, China.
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18
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Khanal P, Hotulainen P. Dendritic Spine Initiation in Brain Development, Learning and Diseases and Impact of BAR-Domain Proteins. Cells 2021; 10:cells10092392. [PMID: 34572042 PMCID: PMC8468246 DOI: 10.3390/cells10092392] [Citation(s) in RCA: 24] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/31/2021] [Revised: 09/08/2021] [Accepted: 09/09/2021] [Indexed: 02/08/2023] Open
Abstract
Dendritic spines are small, bulbous protrusions along neuronal dendrites where most of the excitatory synapses are located. Dendritic spine density in normal human brain increases rapidly before and after birth achieving the highest density around 2-8 years. Density decreases during adolescence, reaching a stable level in adulthood. The changes in dendritic spines are considered structural correlates for synaptic plasticity as well as the basis of experience-dependent remodeling of neuronal circuits. Alterations in spine density correspond to aberrant brain function observed in various neurodevelopmental and neuropsychiatric disorders. Dendritic spine initiation affects spine density. In this review, we discuss the importance of spine initiation in brain development, learning, and potential complications resulting from altered spine initiation in neurological diseases. Current literature shows that two Bin Amphiphysin Rvs (BAR) domain-containing proteins, MIM/Mtss1 and SrGAP3, are involved in spine initiation. We review existing literature and open databases to discuss whether other BAR-domain proteins could also take part in spine initiation. Finally, we discuss the potential molecular mechanisms on how BAR-domain proteins could regulate spine initiation.
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Affiliation(s)
- Pushpa Khanal
- Minerva Foundation Institute for Medical Research, Tukholmankatu 8, 00290 Helsinki, Finland;
- HiLIFE-Neuroscience Center, University of Helsinki, 00014 Helsinki, Finland
| | - Pirta Hotulainen
- Minerva Foundation Institute for Medical Research, Tukholmankatu 8, 00290 Helsinki, Finland;
- Correspondence:
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19
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Abstract
Cellular membranes are anything but flat structures. They display a wide variety of complex and beautiful shapes, most of which have evolved for a particular physiological reason and are adapted to accommodate certain cellular demands. In membrane trafficking events, the dynamic remodelling of cellular membranes is apparent. In clathrin-mediated endocytosis for example, the plasma membrane undergoes heavy deformation to generate and internalize a highly curved clathrin-coated vesicle. This process has become a model system to study proteins with the ability to sense and induce membrane curvature and over the last two decades numerous membrane remodelling molecules and molecular mechanisms have been identified in this process. In this review, we discuss the interaction of epsin1 ENTH domain with membranes, which is one of the best-studied examples of a peripheral and transiently membrane bending protein important for clathrin-mediated endocytosis.
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Affiliation(s)
- Claudia Steinem
- Institute for Organic and Biomolecular Chemistry, University of Göttingen, Tammannstr. 2, 37077 Göttingen, Germany
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20
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Bonazzi F, Hall CK, Weikl TR. Membrane morphologies induced by mixtures of arc-shaped particles with opposite curvature. SOFT MATTER 2021; 17:268-275. [PMID: 32270169 DOI: 10.1039/c9sm02476j] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/11/2023]
Abstract
Biological membranes are shaped by various proteins that either generate inward or outward membrane curvature. In this article, we investigate the membrane morphologies induced by mixtures of arc-shaped particles with coarse-grained modeling and simulations. The particles bind to the membranes either with their inward, concave side or their outward, convex side and, thus, generate membrane curvature of opposite sign. We find that small fractions of convex-binding particles can stabilize three-way junctions of membrane tubules, as suggested for the protein lunapark in the endoplasmic reticulum of cells. For comparable fractions of concave-binding and convex-binding particles, we observe lines of particles of the same type, and diverse membrane morphologies with grooves and bulges induced by these particle lines. The alignment and segregation of the particles is driven by indirect, membrane-mediated interactions.
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Affiliation(s)
- Francesco Bonazzi
- Department of Theory and Bio-Systems, Max Planck Institute of Colloids and Interfaces, Am Mühlenberg 1, 14476 Potsdam, Germany.
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21
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Yang CY, Huang WY, Chen LH, Liang NW, Wang HC, Lu J, Wang X, Wang TW. Neural tissue engineering: the influence of scaffold surface topography and extracellular matrix microenvironment. J Mater Chem B 2021; 9:567-584. [DOI: 10.1039/d0tb01605e] [Citation(s) in RCA: 31] [Impact Index Per Article: 7.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022]
Abstract
Strategies using surface topography, contact guidance and biomechanical cues in the design of scaffolds as an ECM support for neural tissue engineering.
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Affiliation(s)
- Chun-Yi Yang
- Institute for Regenerative Medicine and Biomimetic Materials
- School of Materials Science and Engineering
- Tsinghua University
- Beijing
- China
| | - Wei-Yuan Huang
- Department of Materials Science and Engineering
- National Tsing Hua University
- Hsinchu
- Taiwan
| | - Liang-Hsin Chen
- Department of Materials Science and Engineering
- National Tsing Hua University
- Hsinchu
- Taiwan
| | - Nai-Wen Liang
- Department of Materials Science and Engineering
- National Tsing Hua University
- Hsinchu
- Taiwan
| | - Huan-Chih Wang
- Division of Neurosurgery
- Department of Surgery
- National Taiwan University Hospital
- Taipei
- Taiwan
| | - Jiaju Lu
- Institute for Regenerative Medicine and Biomimetic Materials
- School of Materials Science and Engineering
- Tsinghua University
- Beijing
- China
| | - Xiumei Wang
- Institute for Regenerative Medicine and Biomimetic Materials
- School of Materials Science and Engineering
- Tsinghua University
- Beijing
- China
| | - Tzu-Wei Wang
- Department of Materials Science and Engineering
- National Tsing Hua University
- Hsinchu
- Taiwan
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22
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Induction of Rod-Shaped Structures by Herpes Simplex Virus Glycoprotein I. J Virol 2020; 94:JVI.00231-20. [PMID: 32581097 DOI: 10.1128/jvi.00231-20] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/11/2020] [Accepted: 06/15/2020] [Indexed: 02/05/2023] Open
Abstract
The envelope glycoprotein I (gI) of herpes simplex virus 1 (HSV-1) is a critical mediator of virus-induced cell-to-cell spread and cell-cell fusion. Here, we report a previously unrecognized property of this molecule. In transfected cells, the HSV-1 gI was discovered to induce rod-shaped structures that were uniform in width but variable in length. Moreover, the gI within these structures was conformationally different from the typical form of gI, as a previously used monoclonal antibody mAb3104 and a newly made peptide antibody to the gI extracellular domain (ECD) (amino acids [aa] 110 to 202) both failed to stain the long rod-shaped structures, suggesting the formation of a higher-order form. Consistent with this observation, we found that gI could self-interact and that the rod-shaped structures failed to recognize glycoprotein E, the well-known binding partner of gI. Further analyses by deletion mutagenesis and construction of chimeric mutants between gI and gD revealed that the gI ECD is the critical determinant, whereas the transmembrane domain served merely as an anchor. The critical amino acids were subsequently mapped to proline residues 184 and 188 within a conserved PXXXP motif. Reverse genetics analyses showed that the ability to induce a rod-shaped structure was not required for viral replication and spread in cell culture but rather correlated positively with the capability of the virus to induce cell fusion in the UL24syn background. Together, this work discovered a novel feature of HSV-1 gI that may have important implications in understanding gI function in viral spread and pathogenesis.IMPORTANCE The HSV-1 gI is required for viral cell-to-cell spread within the host, but the molecular mechanisms of how gI exactly works have remained poorly understood. Here, we report a novel property of this molecule, namely, induction of rod-shaped structures, which appeared to represent a higher-order form of gI. We further mapped the critical residues and showed that the ability of gI to induce rod-shaped structures correlated well with the capability of HSV-1 to induce cell fusion in the UL24syn background, suggesting that the two events may have an intrinsic link. Our results shed light on the biological properties of HSV-1 gI and may have important implications in understanding viral pathogenesis.
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23
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Molecular Biology of Escherichia Coli Shiga Toxins' Effects on Mammalian Cells. Toxins (Basel) 2020; 12:toxins12050345. [PMID: 32456125 PMCID: PMC7290813 DOI: 10.3390/toxins12050345] [Citation(s) in RCA: 33] [Impact Index Per Article: 6.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/17/2020] [Revised: 05/18/2020] [Accepted: 05/20/2020] [Indexed: 12/11/2022] Open
Abstract
Shiga toxins (Stxs), syn. Vero(cyto)toxins, are potent bacterial exotoxins and the principal virulence factor of enterohemorrhagic Escherichia coli (EHEC), a subset of Shiga toxin-producing E. coli (STEC). EHEC strains, e.g., strains of serovars O157:H7 and O104:H4, may cause individual cases as well as large outbreaks of life-threatening diseases in humans. Stxs primarily exert a ribotoxic activity in the eukaryotic target cells of the mammalian host resulting in rapid protein synthesis inhibition and cell death. Damage of endothelial cells in the kidneys and the central nervous system by Stxs is central in the pathogenesis of hemolytic uremic syndrome (HUS) in humans and edema disease in pigs. Probably even more important, the toxins also are capable of modulating a plethora of essential cellular functions, which eventually disturb intercellular communication. The review aims at providing a comprehensive overview of the current knowledge of the time course and the consecutive steps of Stx/cell interactions at the molecular level. Intervention measures deduced from an in-depth understanding of this molecular interplay may foster our basic understanding of cellular biology and microbial pathogenesis and pave the way to the creation of host-directed active compounds to mitigate the pathological conditions of STEC infections in the mammalian body.
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24
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Shi Y, Cai M, Zhou L, Wang H. Measurement of mechanical properties of naked cell membranes using atomic force microscope puncture test. Talanta 2020; 210:120637. [PMID: 31987211 DOI: 10.1016/j.talanta.2019.120637] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/25/2019] [Revised: 12/03/2019] [Accepted: 12/08/2019] [Indexed: 11/17/2022]
Abstract
Cell membrane defines the physical boundary of the cell, maintains their shape and volume, and meanwhile dominates the cells response to mechanical force. There are many limitations in the mechanical analysis of naked cell membrane, such as cell condition, the supporting of cytoskeleton and cytoplasm. To reduce these influences, non-supported membrane was prepared on high-ordered pore array silicon substrate. Rupture forces and Young's modulus of three non-supported membranes, red blood cell membrane, somatic cell (MDCK) membrane and phospholipids membrane, are obtained quantitatively using AFM puncture tests. Results indicate that the sequence of both rupture forces and Young's modulus of the three membranes is MDCK cell membrane > red blood cell membrane > phospholipids membrane. The determinant of naked cell membrane's mechanical properties is the constituent of membrane itself, including the quantity and distribution of membrane proteins. Focused on the distribution of membrane proteins, two proposed models of cell membrane-the semi-mosaic model of red blood cell membrane and the Protein Layer-Lipid-Protein Island (PLLPI) model of nucleated mammalian cell membrane can be used to explain the different mechanical properties of naked cell membranes.
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Affiliation(s)
- Yan Shi
- State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, Jilin, 130022, PR China
| | - Mingjun Cai
- State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, Jilin, 130022, PR China
| | - Lulu Zhou
- University of Chinese Academy of Sciences, Beijing, 100049, China
| | - Hongda Wang
- State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, Jilin, 130022, PR China.
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25
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Assoian RK, Bade ND, Cameron CV, Stebe KJ. Cellular sensing of micron-scale curvature: a frontier in understanding the microenvironment. Open Biol 2019; 9:190155. [PMID: 31640476 PMCID: PMC6833222 DOI: 10.1098/rsob.190155] [Citation(s) in RCA: 24] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022] Open
Abstract
The vast majority of cell biological studies examine function and molecular mechanisms using cells on flat surfaces: glass, plastic and more recently elastomeric polymers. While these studies have provided a wealth of valuable insight, they fail to consider that most biologically occurring surfaces are curved, with a radius of curvature roughly corresponding to the length scale of cells themselves. Here, we review recent studies showing that cells detect and respond to these curvature cues by adjusting and re-orienting their cell bodies, actin fibres and nuclei as well as by changing their transcriptional programme. Modelling substratum curvature has the potential to provide fundamental new insight into cell behaviour and function in vivo.
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Affiliation(s)
- Richard K Assoian
- Department of Systems Pharmacology and Translational Therapeutics, University of Pennsylvania, Philadelphia, PA 19104, USA.,Center for Engineering MechanoBiology, University of Pennsylvania, Philadelphia, PA 19104, USA
| | - Nathan D Bade
- Department of Chemical and Biomolecular Engineering, University of Pennsylvania, Philadelphia, PA 19104, USA
| | - Caroline V Cameron
- Department of Chemical and Biomolecular Engineering, University of Pennsylvania, Philadelphia, PA 19104, USA
| | - Kathleen J Stebe
- Department of Chemical and Biomolecular Engineering, University of Pennsylvania, Philadelphia, PA 19104, USA.,Center for Engineering MechanoBiology, University of Pennsylvania, Philadelphia, PA 19104, USA
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26
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Bonazzi F, Weikl TR. Membrane Morphologies Induced by Arc-Shaped Scaffolds Are Determined by Arc Angle and Coverage. Biophys J 2019; 116:1239-1247. [PMID: 30902368 DOI: 10.1016/j.bpj.2019.02.017] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/07/2018] [Revised: 01/13/2019] [Accepted: 02/19/2019] [Indexed: 10/27/2022] Open
Abstract
The intricate shapes of biological membranes such as tubules and membrane stacks are induced by proteins. In this article, we systematically investigate the membrane shapes induced by arc-shaped scaffolds such as proteins and protein complexes with coarse-grained modeling and simulations. We find that arc-shaped scaffolds induce membrane tubules at membrane coverages larger than a threshold of ∼40%, irrespective of their arc angle. The membrane morphologies at intermediate coverages below this tubulation threshold, in contrast, strongly depend on the arc angle. Scaffolds with arc angles of about 60°, akin to N-BAR domains, do not change the membrane shape at coverages below the tubulation threshold, whereas scaffolds with arc angles larger than about 120° induce double-membrane stacks at intermediate coverages. The scaffolds stabilize the curved membrane edges that connect the membrane stacks, as suggested for complexes of reticulon proteins. Our results provide general insights on the determinants of membrane shaping by arc-shaped scaffolds.
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Affiliation(s)
- Francesco Bonazzi
- Max Planck Institute of Colloids and Interfaces, Department of Theory and Bio-Systems, Potsdam, Germany
| | - Thomas R Weikl
- Max Planck Institute of Colloids and Interfaces, Department of Theory and Bio-Systems, Potsdam, Germany.
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27
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Directed Supramolecular Organization of N-BAR Proteins through Regulation of H0 Membrane Immersion Depth. Sci Rep 2018; 8:16383. [PMID: 30401832 PMCID: PMC6219572 DOI: 10.1038/s41598-018-34273-2] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/12/2018] [Accepted: 10/11/2018] [Indexed: 11/12/2022] Open
Abstract
Many membrane remodeling events rely on the ability of curvature-generating N-BAR membrane proteins to organize into distinctive supramolecular configurations. Experiments have revealed a conformational switch in N-BAR proteins resulting in vesicular or tubular membrane shapes, with shallow membrane immersion of the H0 amphipathic helices of N-BAR proteins on vesicles but deep H0 immersion on tubes. We develop here a minimal elastic model of the local thinning of the lipid bilayer resulting from H0 immersion. Our model predicts that the observed conformational switch in N-BAR proteins produces a corresponding switch in the bilayer-mediated N-BAR interactions due to the H0 helices. In agreement with experiments, we find that bilayer-mediated H0 interactions oppose N-BAR multimerization for the shallow H0 membrane immersion depths measured on vesicles, but promote self-assembly of supramolecular N-BAR chains for the increased H0 membrane immersion depths measured on tubes. Finally, we consider the possibility that bilayer-mediated H0 interactions might contribute to the concerted structural reorganization of N-BAR proteins suggested by experiments. Our results indicate that the membrane immersion depth of amphipathic protein helices may provide a general molecular control parameter for membrane organization.
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28
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FBP17 and CIP4 recruit SHIP2 and lamellipodin to prime the plasma membrane for fast endophilin-mediated endocytosis. Nat Cell Biol 2018; 20:1023-1031. [PMID: 30061681 DOI: 10.1038/s41556-018-0146-8] [Citation(s) in RCA: 68] [Impact Index Per Article: 9.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/19/2017] [Accepted: 06/20/2018] [Indexed: 12/29/2022]
Abstract
Endocytosis mediates the cellular uptake of micronutrients and the turnover of plasma membrane proteins. Clathrin-mediated endocytosis is the major uptake pathway in resting cells1, but several clathrin-independent endocytic routes exist in parallel2,3. One such pathway, fast endophilin-mediated endocytosis (FEME), is not constitutive but triggered upon activation of certain receptors, including the β1 adrenergic receptor4. FEME activates promptly following stimulation as endophilin is pre-enriched by the phosphatidylinositol-3,4-bisphosphate-binding protein lamellipodin4,5. However, in the absence of stimulation, endophilin foci abort and disassemble after a few seconds. Looking for additional proteins involved in FEME, we found that 20 out of 65 BAR domain-containing proteins tested colocalized with endophilin spots. Among them, FBP17 and CIP4 prime the membrane of resting cells for FEME by recruiting the 5'-lipid phosphatase SHIP2 and lamellipodin to mediate the local production of phosphatidylinositol-3,4-bisphosphate and endophilin pre-enrichment. Membrane-bound GTP-loaded Cdc42 recruits FBP17 and CIP4, before being locally deactivated by RICH1 and SH3BP1 GTPase-activating proteins. This generates the transient assembly and disassembly of endophilin spots, which lasts 5-10 seconds. This mechanism periodically primes patches of the membrane for prompt responses upon FEME activation.
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29
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A Flat BAR Protein Promotes Actin Polymerization at the Base of Clathrin-Coated Pits. Cell 2018; 174:325-337.e14. [PMID: 29887380 PMCID: PMC6057269 DOI: 10.1016/j.cell.2018.05.020] [Citation(s) in RCA: 82] [Impact Index Per Article: 11.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/07/2017] [Revised: 02/28/2018] [Accepted: 05/09/2018] [Indexed: 02/07/2023]
Abstract
Multiple proteins act co-operatively in mammalian clathrin-mediated endocytosis (CME) to generate endocytic vesicles from the plasma membrane. The principles controlling the activation and organization of the actin cytoskeleton during mammalian CME are, however, not fully understood. Here, we show that the protein FCHSD2 is a major activator of actin polymerization during CME. FCHSD2 deletion leads to decreased ligand uptake caused by slowed pit maturation. FCHSD2 is recruited to endocytic pits by the scaffold protein intersectin via an unusual SH3-SH3 interaction. Here, its flat F-BAR domain binds to the planar region of the plasma membrane surrounding the developing pit forming an annulus. When bound to the membrane, FCHSD2 activates actin polymerization by a mechanism that combines oligomerization and recruitment of N-WASP to PI(4,5)P2, thus promoting pit maturation. Our data therefore describe a molecular mechanism for linking spatiotemporally the plasma membrane to a force-generating actin platform guiding endocytic vesicle maturation.
FCHSD2 is a bona fide CME protein recruited to CCPs by intersectin Intersectin recruits FCHSD2 via an SH3-SH3 interaction FCHSD2 is a major activator of actin during CME FCHSD2 binds to the surrounding membrane around CCPs via its flat F-BAR domain
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30
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Increasing Diversity of Biological Membrane Fission Mechanisms. Trends Cell Biol 2018; 28:274-286. [DOI: 10.1016/j.tcb.2017.12.001] [Citation(s) in RCA: 34] [Impact Index Per Article: 4.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/02/2017] [Revised: 12/06/2017] [Accepted: 12/12/2017] [Indexed: 12/19/2022]
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31
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Bucher D, Frey F, Sochacki KA, Kummer S, Bergeest JP, Godinez WJ, Kräusslich HG, Rohr K, Taraska JW, Schwarz US, Boulant S. Clathrin-adaptor ratio and membrane tension regulate the flat-to-curved transition of the clathrin coat during endocytosis. Nat Commun 2018; 9:1109. [PMID: 29549258 PMCID: PMC5856840 DOI: 10.1038/s41467-018-03533-0] [Citation(s) in RCA: 91] [Impact Index Per Article: 13.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/09/2017] [Accepted: 02/15/2018] [Indexed: 12/03/2022] Open
Abstract
Although essential for many cellular processes, the sequence of structural and molecular events during clathrin-mediated endocytosis remains elusive. While it was long believed that clathrin-coated pits grow with a constant curvature, it was recently suggested that clathrin first assembles to form flat structures that then bend while maintaining a constant surface area. Here, we combine correlative electron and light microscopy and mathematical growth laws to study the ultrastructural rearrangements of the clathrin coat during endocytosis in BSC-1 mammalian cells. We confirm that clathrin coats initially grow flat and demonstrate that curvature begins when around 70% of the final clathrin content is acquired. We find that this transition is marked by a change in the clathrin to clathrin-adaptor protein AP2 ratio and that membrane tension suppresses this transition. Our results support the notion that BSC-1 mammalian cells dynamically regulate the flat-to-curved transition in clathrin-mediated endocytosis by both biochemical and mechanical factors. The sequence of structural and molecular events during clathrin-mediated endocytosis is unclear. Here the authors combine correlative microscopy and simple mathematical growth laws to demonstrate that the flat patch starts to curve when around 70% of the final clathrin content is reached.
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Affiliation(s)
- Delia Bucher
- Department of Infectious Diseases, Virology, University Hospital Heidelberg, Im Neuenheimer Feld 324, 69120, Heidelberg, Germany.,German Cancer Research Center (DKFZ), Im Neuenheimer Feld 581, 69120, Heidelberg, Germany
| | - Felix Frey
- BioQuant Center, Im Neuenheimer Feld 267, 69120, Heidelberg, Germany.,Institute for Theoretical Physics, Heidelberg University, Philosophenweg 19, 69120, Heidelberg, Germany
| | - Kem A Sochacki
- National Heart Lung and Blood Institute, National Institutes of Health, Bethesda, MD, 20892, USA
| | - Susann Kummer
- Department of Infectious Diseases, Virology, University Hospital Heidelberg, Im Neuenheimer Feld 324, 69120, Heidelberg, Germany
| | - Jan-Philip Bergeest
- German Cancer Research Center (DKFZ), Im Neuenheimer Feld 581, 69120, Heidelberg, Germany.,BioQuant Center, Im Neuenheimer Feld 267, 69120, Heidelberg, Germany.,Institute of Pharmacy and Molecular Biotechnology (IPMB), Department of Bioinformatics and Functional Genomics, Heidelberg University, Im Neuenheimer Feld 267, 69120, Heidelberg, Germany
| | - William J Godinez
- German Cancer Research Center (DKFZ), Im Neuenheimer Feld 581, 69120, Heidelberg, Germany.,BioQuant Center, Im Neuenheimer Feld 267, 69120, Heidelberg, Germany.,Institute of Pharmacy and Molecular Biotechnology (IPMB), Department of Bioinformatics and Functional Genomics, Heidelberg University, Im Neuenheimer Feld 267, 69120, Heidelberg, Germany
| | - Hans-Georg Kräusslich
- Department of Infectious Diseases, Virology, University Hospital Heidelberg, Im Neuenheimer Feld 324, 69120, Heidelberg, Germany
| | - Karl Rohr
- German Cancer Research Center (DKFZ), Im Neuenheimer Feld 581, 69120, Heidelberg, Germany.,BioQuant Center, Im Neuenheimer Feld 267, 69120, Heidelberg, Germany.,Institute of Pharmacy and Molecular Biotechnology (IPMB), Department of Bioinformatics and Functional Genomics, Heidelberg University, Im Neuenheimer Feld 267, 69120, Heidelberg, Germany
| | - Justin W Taraska
- National Heart Lung and Blood Institute, National Institutes of Health, Bethesda, MD, 20892, USA
| | - Ulrich S Schwarz
- BioQuant Center, Im Neuenheimer Feld 267, 69120, Heidelberg, Germany. .,Institute for Theoretical Physics, Heidelberg University, Philosophenweg 19, 69120, Heidelberg, Germany.
| | - Steeve Boulant
- Department of Infectious Diseases, Virology, University Hospital Heidelberg, Im Neuenheimer Feld 324, 69120, Heidelberg, Germany. .,German Cancer Research Center (DKFZ), Im Neuenheimer Feld 581, 69120, Heidelberg, Germany.
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32
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Abstract
Besides direct protein-protein interactions, indirect interactions mediated by membranes play an important role for the assembly and cooperative function of proteins in membrane shaping and adhesion. The intricate shapes of biological membranes are generated by proteins that locally induce membrane curvature. Indirect curvature-mediated interactions between these proteins arise because the proteins jointly affect the bending energy of the membranes. These curvature-mediated interactions are attractive for crescent-shaped proteins and are a driving force in the assembly of the proteins during membrane tubulation. Membrane adhesion results from the binding of receptor and ligand proteins that are anchored in the apposing membranes. The binding of these proteins strongly depends on nanoscale shape fluctuations of the membranes, leading to a fluctuation-mediated binding cooperativity. A length mismatch between receptor-ligand complexes in membrane adhesion zones causes repulsive curvature-mediated interactions that are a driving force for the length-based segregation of proteins during membrane adhesion.
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Affiliation(s)
- Thomas R Weikl
- Department of Theory and Bio-Systems, Max Planck Institute of Colloids and Interfaces, 14424 Potsdam, Germany;
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33
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Prévost C, Tsai FC, Bassereau P, Simunovic M. Pulling Membrane Nanotubes from Giant Unilamellar Vesicles. J Vis Exp 2017. [PMID: 29286431 DOI: 10.3791/56086] [Citation(s) in RCA: 20] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/27/2023] Open
Abstract
The reshaping of the cell membrane is an integral part of many cellular phenomena, such as endocytosis, trafficking, the formation of filopodia, etc. Many different proteins associate with curved membranes because of their ability to sense or induce membrane curvature. Typically, these processes involve a multitude of proteins making them too complex to study quantitatively in the cell. We describe a protocol to reconstitute a curved membrane in vitro, mimicking a curved cellular structure, such as the endocytic neck. A giant unilamellar vesicle (GUV) is used as a model of a cell membrane, whose internal pressure and surface tension are controlled with micropipette aspiration. Applying a point pulling force on the GUV using optical tweezers creates a nanotube of high curvature connected to a flat membrane. This method has traditionally been used to measure the fundamental mechanical properties of lipid membranes, such as bending rigidity. In recent years, it has been expanded to study how proteins interact with membrane curvature and the way they affect the shape and the mechanics of membranes. A system combining micromanipulation, microinjection, optical tweezers, and confocal microscopy allows measurement of membrane curvature, membrane tension, and the surface density of proteins, concurrently. From these measurements, many important mechanical and morphological properties of the protein-membrane system can be inferred. In addition, we lay out a protocol of creating GUVs in the presence of physiological salt concentration, and a method of quantifying the surface density of proteins on the membrane from fluorescence intensities of labeled proteins and lipids.
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Affiliation(s)
- Coline Prévost
- Laboratoire Physico Chimie Curie, Institut Curie, PSL Research University, CNRS UMR168; Department of Genetics and Complex Diseases, T. H. Chan School of Public Health, Harvard Medical School; Department of Cell Biology, Harvard Medical School
| | - Feng-Ching Tsai
- Laboratoire Physico Chimie Curie, Institut Curie, PSL Research University, CNRS UMR168; Sorbonne Universités, UPMC University Paris 06
| | - Patricia Bassereau
- Laboratoire Physico Chimie Curie, Institut Curie, PSL Research University, CNRS UMR168; Sorbonne Universités, UPMC University Paris 06;
| | - Mijo Simunovic
- Laboratoire Physico Chimie Curie, Institut Curie, PSL Research University, CNRS UMR168; Center for Studies in Physics and Biology, The Rockefeller University
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34
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Yamamoto H, Kondo A, Itoh T. A curvature-dependent membrane binding by tyrosine kinase Fer involves an intrinsically disordered region. Biochem Biophys Res Commun 2017; 495:1522-1527. [PMID: 29208465 DOI: 10.1016/j.bbrc.2017.12.009] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/12/2017] [Accepted: 12/01/2017] [Indexed: 11/18/2022]
Abstract
Tyrosine kinases are important enzymes that mediate signal transduction at the plasma membrane. While the significance of membrane localization of tyrosine kinases has been well evaluated, the role of membrane curvature in their regulation is unknown. Here, we demonstrate that an intrinsically disordered region in the tyrosine kinase Fer acts as a membrane curvature sensor that preferentially binds to highly curved membranes in vitro. This region forms an amphipathic α-helix upon interaction with curved membranes, aligning hydrophobic residues on one side of the helical structure. Further, the tyrosine kinase activity of Fer is significantly enhanced by the membrane in a manner dependent on curvature. We propose a model for the regulation of Fer based on an intramolecular interaction and the curvature-dependent membrane binding mediated by its intrinsically disordered region.
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Affiliation(s)
- Hikaru Yamamoto
- Division of Membrane Biology, Biosignal Research Center, Kobe University, 1-1 Rokkodai-cho, Nada-ku, Kobe, Hyogo, 657-8501, Japan
| | - Akihiro Kondo
- Department of Biochemistry and Molecular Biology, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe, Hyogo 650-0017, Japan
| | - Toshiki Itoh
- Division of Membrane Biology, Biosignal Research Center, Kobe University, 1-1 Rokkodai-cho, Nada-ku, Kobe, Hyogo, 657-8501, Japan; Department of Biochemistry and Molecular Biology, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe, Hyogo 650-0017, Japan.
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35
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Smith JA, Hall AE, Rose MD. Membrane curvature directs the localization of Cdc42p to novel foci required for cell-cell fusion. J Cell Biol 2017; 216:3971-3980. [PMID: 29066609 PMCID: PMC5716282 DOI: 10.1083/jcb.201703169] [Citation(s) in RCA: 17] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/23/2017] [Revised: 09/08/2017] [Accepted: 09/22/2017] [Indexed: 12/28/2022] Open
Abstract
Cell fusion is ubiquitous in eukaryotic fertilization and development. The highly conserved Rho-GTPase Cdc42p promotes yeast fusion through interaction with Fus2p, a pheromone-induced amphiphysin-like protein. We show that in prezygotes, Cdc42p forms a novel Fus2p-dependent focus at the center of the zone of cell fusion (ZCF) and remains associated with remnant cell walls after initial fusion. At the ZCF and during fusion, Cdc42p and Fus2p colocalized. In contrast, in shmoos, both proteins were near the cortex but spatially separate. Cdc42p focus formation depends on ZCF membrane curvature: mutant analysis showed that Cdc42p localization is negatively affected by shmoo-like positive ZCF curvature, consistent with the flattening of the ZCF during fusion. BAR-domain proteins such as the fusion proteins Fus2p and Rvs161p are known to recognize membrane curvature. We find that mutations that disrupt binding of the Fus2p/Rvs161p heterodimer to membranes affect Cdc42p ZCF localization. We propose that Fus2p localizes Cdc42p to the flat ZCF to promote cell wall degradation.
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Affiliation(s)
- Jean A Smith
- Department of Molecular Biology, Princeton University, Princeton, NJ
| | - Allison E Hall
- Department of Molecular Biology, Princeton University, Princeton, NJ
| | - Mark D Rose
- Department of Molecular Biology, Princeton University, Princeton, NJ
- Department of Biology, Georgetown University, Washington, DC
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36
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Cowling BS, Prokic I, Tasfaout H, Rabai A, Humbert F, Rinaldi B, Nicot AS, Kretz C, Friant S, Roux A, Laporte J. Amphiphysin (BIN1) negatively regulates dynamin 2 for normal muscle maturation. J Clin Invest 2017; 127:4477-4487. [PMID: 29130937 DOI: 10.1172/jci90542] [Citation(s) in RCA: 59] [Impact Index Per Article: 7.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/06/2016] [Accepted: 10/03/2017] [Indexed: 01/25/2023] Open
Abstract
Regulation of skeletal muscle development and organization is a complex process that is not fully understood. Here, we focused on amphiphysin 2 (BIN1, also known as bridging integrator-1) and dynamin 2 (DNM2), two ubiquitous proteins implicated in membrane remodeling and mutated in centronuclear myopathies (CNMs). We generated Bin1-/- Dnm2+/- mice to decipher the physiological interplay between BIN1 and DNM2. While Bin1-/- mice die perinatally from a skeletal muscle defect, Bin1-/- Dnm2+/- mice survived at least 18 months, and had normal muscle force and intracellular organization of muscle fibers, supporting BIN1 as a negative regulator of DNM2. We next characterized muscle-specific isoforms of BIN1 and DNM2. While BIN1 colocalized with and partially inhibited DNM2 activity during muscle maturation, BIN1 had no effect on the isoform of DNM2 found in adult muscle. Together, these results indicate that BIN1 and DNM2 regulate muscle development and organization, function through a common pathway, and define BIN1 as a negative regulator of DNM2 in vitro and in vivo during muscle maturation. Our data suggest that DNM2 modulation has potential as a therapeutic approach for patients with CNM and BIN1 defects. As BIN1 is implicated in cancers, arrhythmia, and late-onset Alzheimer disease, these findings may trigger research directions and therapeutic development for these common diseases.
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Affiliation(s)
- Belinda S Cowling
- Institut de Génétique et de Biologie Moléculaire et Cellulaire, Illkirch, France.,Institut National de la Santé et de la Recherche Médicale, U964, Illkirch, France.,Centre National de la Recherche Scientifique, UMR7104, Illkirch, France.,Université de Strasbourg, Illkirch, France
| | - Ivana Prokic
- Institut de Génétique et de Biologie Moléculaire et Cellulaire, Illkirch, France.,Institut National de la Santé et de la Recherche Médicale, U964, Illkirch, France.,Centre National de la Recherche Scientifique, UMR7104, Illkirch, France.,Université de Strasbourg, Illkirch, France
| | - Hichem Tasfaout
- Institut de Génétique et de Biologie Moléculaire et Cellulaire, Illkirch, France.,Institut National de la Santé et de la Recherche Médicale, U964, Illkirch, France.,Centre National de la Recherche Scientifique, UMR7104, Illkirch, France.,Université de Strasbourg, Illkirch, France
| | - Aymen Rabai
- Institut de Génétique et de Biologie Moléculaire et Cellulaire, Illkirch, France.,Institut National de la Santé et de la Recherche Médicale, U964, Illkirch, France.,Centre National de la Recherche Scientifique, UMR7104, Illkirch, France.,Université de Strasbourg, Illkirch, France
| | - Frédéric Humbert
- Biochemistry Department, University of Geneva, Geneva, Switzerland
| | - Bruno Rinaldi
- Department of Molecular and Cellular Genetics, UMR7156, Université de Strasbourg and CNRS, Strasbourg, France
| | - Anne-Sophie Nicot
- Institut de Génétique et de Biologie Moléculaire et Cellulaire, Illkirch, France.,Institut National de la Santé et de la Recherche Médicale, U964, Illkirch, France.,Centre National de la Recherche Scientifique, UMR7104, Illkirch, France.,Université de Strasbourg, Illkirch, France
| | - Christine Kretz
- Institut de Génétique et de Biologie Moléculaire et Cellulaire, Illkirch, France.,Institut National de la Santé et de la Recherche Médicale, U964, Illkirch, France.,Centre National de la Recherche Scientifique, UMR7104, Illkirch, France.,Université de Strasbourg, Illkirch, France
| | - Sylvie Friant
- Department of Molecular and Cellular Genetics, UMR7156, Université de Strasbourg and CNRS, Strasbourg, France
| | - Aurélien Roux
- Biochemistry Department, University of Geneva, Geneva, Switzerland.,Swiss National Centre of Competence in Research Programme Chemical Biology, Geneva, Switzerland
| | - Jocelyn Laporte
- Institut de Génétique et de Biologie Moléculaire et Cellulaire, Illkirch, France.,Institut National de la Santé et de la Recherche Médicale, U964, Illkirch, France.,Centre National de la Recherche Scientifique, UMR7104, Illkirch, France.,Université de Strasbourg, Illkirch, France
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37
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Johannes L. Shiga Toxin-A Model for Glycolipid-Dependent and Lectin-Driven Endocytosis. Toxins (Basel) 2017; 9:toxins9110340. [PMID: 29068384 PMCID: PMC5705955 DOI: 10.3390/toxins9110340] [Citation(s) in RCA: 57] [Impact Index Per Article: 7.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/28/2017] [Revised: 10/15/2017] [Accepted: 10/20/2017] [Indexed: 12/27/2022] Open
Abstract
The cellular entry of the bacterial Shiga toxin and the related verotoxins has been scrutinized in quite some detail. This is due to their importance as a threat to human health. At the same time, the study of Shiga toxin has allowed the discovery of novel molecular mechanisms that also apply to the intracellular trafficking of endogenous proteins at the plasma membrane and in the endosomal system. In this review, the individual steps that lead to Shiga toxin uptake into cells will first be presented from a purely mechanistic perspective. Membrane-biological concepts will be highlighted that are often still poorly explored, such as fluctuation force-driven clustering, clathrin-independent membrane curvature generation, friction-driven scission, and retrograde sorting on early endosomes. It will then be explored whether and how these also apply to other pathogens, pathogenic factors, and cellular proteins. The molecular nature of Shiga toxin as a carbohydrate-binding protein and that of its cellular receptor as a glycosylated raft lipid will be an underlying theme in this discussion. It will thereby be illustrated how the study of Shiga toxin has led to the proposal of the GlycoLipid-Lectin (GL-Lect) hypothesis on the generation of endocytic pits in processes of clathrin-independent endocytosis.
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Affiliation(s)
- Ludger Johannes
- Cellular and Chemical Biology Department, Institut Curie, PSL Research University, U1143 INSERM, UMR3666 CNRS, 26 rue d'Ulm, 75248 Paris CEDEX 05, France.
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38
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Sahu A, Sauer RA, Mandadapu KK. Irreversible thermodynamics of curved lipid membranes. Phys Rev E 2017; 96:042409. [PMID: 29347561 DOI: 10.1103/physreve.96.042409] [Citation(s) in RCA: 26] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/04/2017] [Indexed: 01/15/2023]
Abstract
The theory of irreversible thermodynamics for arbitrarily curved lipid membranes is presented here. The coupling between elastic bending and irreversible processes such as intramembrane lipid flow, intramembrane phase transitions, and protein binding and diffusion is studied. The forms of the entropy production for the irreversible processes are obtained, and the corresponding thermodynamic forces and fluxes are identified. Employing the linear irreversible thermodynamic framework, the governing equations of motion along with appropriate boundary conditions are provided.
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Affiliation(s)
- Amaresh Sahu
- Department of Chemical & Biomolecular Engineering, University of California at Berkeley, Berkeley, California 94720, USA
| | - Roger A Sauer
- Aachen Institute for Advanced Study in Computational Engineering Science (AICES), RWTH Aachen University, Templergraben 55, 52056 Aachen, Germany
| | - Kranthi K Mandadapu
- Department of Chemical & Biomolecular Engineering, University of California at Berkeley, Berkeley, California 94720, USA
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39
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Nonequilibrium fluctuations of lipid membranes by the rotating motor protein F 1F 0-ATP synthase. Proc Natl Acad Sci U S A 2017; 114:11291-11296. [PMID: 29073046 PMCID: PMC5664490 DOI: 10.1073/pnas.1701207114] [Citation(s) in RCA: 38] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022] Open
Abstract
The shape of biological membranes is constantly remodeled and maintained out of equilibrium by active proteins. The functional capacity of membrane deformation is mainly determined by the mechanical interplay between protein activity and bending elasticity. In our experiments, we find that ATP synthase, a rotating membrane protein that synthesizes the biochemical energy in cells through proton-pumping activity across the membrane, promotes localized nonequilibrium membrane fluctuations when reconstituted in giant lipid vesicles. The large membrane deformations emerge from the pumping action of rotating proteins clustered at specific emplacements in the membrane. Our results pave the way to new experimental realizations to explore the collective effects of rotating ATP synthases and their possible biological implications for biomembrane organization and protein functionality. ATP synthase is a rotating membrane protein that synthesizes ATP through proton-pumping activity across the membrane. To unveil the mechanical impact of this molecular active pump on the bending properties of its lipid environment, we have functionally reconstituted the ATP synthase in giant unilamellar vesicles and tracked the membrane fluctuations by means of flickering spectroscopy. We find that ATP synthase rotates at a frequency of about 20 Hz, promoting large nonequilibrium deformations at discrete hot spots in lipid vesicles and thus inducing an overall membrane softening. The enhanced nonequilibrium fluctuations are compatible with an accumulation of active proteins at highly curved membrane sites through a curvature−protein coupling mechanism that supports the emergence of collective effects of rotating ATP synthases in lipid membranes.
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40
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Delestre-Delacour C, Carmon O, Laguerre F, Estay-Ahumada C, Courel M, Elias S, Jeandel L, Rayo MV, Peinado JR, Sengmanivong L, Gasman S, Coudrier E, Anouar Y, Montero-Hadjadje M. Myosin 1b and F-actin are involved in the control of secretory granule biogenesis. Sci Rep 2017; 7:5172. [PMID: 28701771 PMCID: PMC5507975 DOI: 10.1038/s41598-017-05617-1] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/16/2017] [Accepted: 05/31/2017] [Indexed: 12/22/2022] Open
Abstract
Hormone secretion relies on secretory granules which store hormones in endocrine cells and release them upon cell stimulation. The molecular events leading to hormone sorting and secretory granule formation at the level of the TGN are still elusive. Our proteomic analysis of purified whole secretory granules or secretory granule membranes uncovered their association with the actomyosin components myosin 1b, actin and the actin nucleation complex Arp2/3. We found that myosin 1b controls the formation of secretory granules and the associated regulated secretion in both neuroendocrine cells and chromogranin A-expressing COS7 cells used as a simplified model of induced secretion. We show that F-actin is also involved in secretory granule biogenesis and that myosin 1b cooperates with Arp2/3 to recruit F-actin to the Golgi region where secretory granules bud. These results provide the first evidence that components of the actomyosin complex promote the biogenesis of secretory granules and thereby regulate hormone sorting and secretion.
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Affiliation(s)
- Charlène Delestre-Delacour
- Normandie Univ, UNIROUEN, INSERM, U1239, Laboratoire de Différenciation et Communication Neuronale et Neuroendocrine, Institut de Recherche et d'Innovation Biomédicale de Normandie, 76000, Rouen, France
| | - Ophélie Carmon
- Normandie Univ, UNIROUEN, INSERM, U1239, Laboratoire de Différenciation et Communication Neuronale et Neuroendocrine, Institut de Recherche et d'Innovation Biomédicale de Normandie, 76000, Rouen, France
| | - Fanny Laguerre
- Normandie Univ, UNIROUEN, INSERM, U1239, Laboratoire de Différenciation et Communication Neuronale et Neuroendocrine, Institut de Recherche et d'Innovation Biomédicale de Normandie, 76000, Rouen, France
| | - Catherine Estay-Ahumada
- Université de Strasbourg, CNRS UPR 3212, Institut des Neurosciences Cellulaires et Intégratives, 67000, Strasbourg, France
| | - Maïté Courel
- Normandie Univ, UNIROUEN, INSERM, U1239, Laboratoire de Différenciation et Communication Neuronale et Neuroendocrine, Institut de Recherche et d'Innovation Biomédicale de Normandie, 76000, Rouen, France.,CNRS-UPMC FRE3402, Pierre et Marie Curie University, 75252, Paris, Cedex 05, France
| | - Salah Elias
- Normandie Univ, UNIROUEN, INSERM, U1239, Laboratoire de Différenciation et Communication Neuronale et Neuroendocrine, Institut de Recherche et d'Innovation Biomédicale de Normandie, 76000, Rouen, France.,University of Oxford, Sir William Dunn School of Pathology, Oxford, Oxfordshire, United Kingdom
| | - Lydie Jeandel
- Normandie Univ, UNIROUEN, INSERM, U1239, Laboratoire de Différenciation et Communication Neuronale et Neuroendocrine, Institut de Recherche et d'Innovation Biomédicale de Normandie, 76000, Rouen, France
| | - Margarita Villar Rayo
- Instituto de Investigación en Recursos Cinegéticos, Proteomics Core Facility, 13071, Ciudad Real, Spain
| | - Juan R Peinado
- Laboratory of oxidative stress and neurodegeneration, Facultad de Medicina de Ciudad Real, 13071, Ciudad Real, Spain
| | - Lucie Sengmanivong
- Institut Curie - PSL Research University, Membrane Dynamics and Mechanics of Intracellular Signaling Laboratory, Nikon Imaging Centre, 75005, Paris, France
| | - Stéphane Gasman
- Université de Strasbourg, CNRS UPR 3212, Institut des Neurosciences Cellulaires et Intégratives, 67000, Strasbourg, France
| | - Evelyne Coudrier
- CNRS UMR 144 Cell Signaling and Morphogenesis, Institut Curie, 75005, Paris, France
| | - Youssef Anouar
- Normandie Univ, UNIROUEN, INSERM, U1239, Laboratoire de Différenciation et Communication Neuronale et Neuroendocrine, Institut de Recherche et d'Innovation Biomédicale de Normandie, 76000, Rouen, France.
| | - Maité Montero-Hadjadje
- Normandie Univ, UNIROUEN, INSERM, U1239, Laboratoire de Différenciation et Communication Neuronale et Neuroendocrine, Institut de Recherche et d'Innovation Biomédicale de Normandie, 76000, Rouen, France.
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41
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Abstract
Membrane deformation by proteins is a universal phenomenon that has been studied extensively in eukaryotes but much less in prokaryotes. In this study, we discovered a membrane-deforming activity of the phospholipid N-methyltransferase PmtA from the plant-pathogenic bacterium Agrobacterium tumefaciens PmtA catalyzes the successive three-step N-methylation of phosphatidylethanolamine to phosphatidylcholine. Here, we defined the lipid and protein requirements for the membrane-remodeling activity of PmtA by a combination of transmission electron microscopy and liposome interaction studies. Dependent on the lipid composition, PmtA changes the shape of spherical liposomes either into filaments or small vesicles. Upon overproduction of PmtA in A. tumefaciens, vesicle-like structures occur in the cytoplasm, dependent on the presence of the anionic lipid cardiolipin. The N-terminal lipid-binding α-helix (αA) is involved in membrane deformation by PmtA. Two functionally distinct and spatially separated regions in αA can be distinguished. Anionic interactions by positively charged amino acids on one face of the helix are responsible for membrane recruitment of the enzyme. The opposite hydrophobic face of the helix is required for membrane remodeling, presumably by shallow insertion into the lipid bilayer.IMPORTANCE The ability to alter the morphology of biological membranes is known for a small number of some bacterial proteins. Our study adds the phospholipid N-methyltransferase PmtA as a new member to the category of bacterial membrane-remodeling proteins. A combination of in vivo and in vitro methods reveals the molecular requirements for membrane deformation at the protein and phospholipid level. The dual functionality of PmtA suggests a contribution of membrane biosynthesis enzymes to the complex morphology of bacterial membranes.
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42
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Wei S, Bian Y, Zhao Q, Chen S, Mao J, Song C, Cheng K, Xiao Z, Zhang C, Ma W, Zou H, Ye M, Dai S. Salinity-Induced Palmella Formation Mechanism in Halotolerant Algae Dunaliella salina Revealed by Quantitative Proteomics and Phosphoproteomics. FRONTIERS IN PLANT SCIENCE 2017; 8:810. [PMID: 28588593 PMCID: PMC5441111 DOI: 10.3389/fpls.2017.00810] [Citation(s) in RCA: 29] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 01/23/2017] [Accepted: 04/30/2017] [Indexed: 05/05/2023]
Abstract
Palmella stage is critical for some unicellular algae to survive in extreme environments. The halotolerant algae Dunaliella salina is a good single-cell model for studying plant adaptation to high salinity. To investigate the molecular adaptation mechanism in salinity shock-induced palmella formation, we performed a comprehensive physiological, proteomics and phosphoproteomics study upon palmella formation of D. salina using dimethyl labeling and Ti4+-immobilized metal ion affinity chromatography (IMAC) proteomic approaches. We found that 151 salinity-responsive proteins and 35 salinity-responsive phosphoproteins were involved in multiple signaling and metabolic pathways upon palmella formation. Taken together with photosynthetic parameters and enzyme activity analyses, the patterns of protein accumulation and phosphorylation level exhibited the mechanisms upon palmella formation, including dynamics of cytoskeleton and cell membrane curvature, accumulation and transport of exopolysaccharides, photosynthesis and energy supplying (i.e., photosystem II stability and activity, cyclic electron transport, and C4 pathway), nuclear/chloroplastic gene expression regulation and protein processing, reactive oxygen species homeostasis, and salt signaling transduction. The salinity-responsive protein-protein interaction (PPI) networks implied that signaling and protein synthesis and fate are crucial for modulation of these processes. Importantly, the 3D structure of phosphoprotein clearly indicated that the phosphorylation sites of eight proteins were localized in the region of function domain.
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Affiliation(s)
- Sijia Wei
- Key Laboratory of Saline-Alkali Vegetation Ecology Restoration in Oil Field, Alkali Soil Natural Environmental Science Center, Ministry of Education, Northeast Forestry UniversityHarbin, China
| | - Yangyang Bian
- Key Laboratory of Separation Sciences for Analytical Chemistry, National Chromatographic R&A Center, Dalian Institute of Chemical Physics, Chinese Academy of SciencesDalian, China
| | - Qi Zhao
- College of Life and Environmental Sciences, Shanghai Normal UniversityShanghai, China
| | - Sixue Chen
- Department of Biology, Genetics Institute, Plant Molecular and Cellular Biology Program, Interdisciplinary Center for Biotechnology Research, University of FloridaGainesville, FL, Unites States
| | - Jiawei Mao
- Key Laboratory of Separation Sciences for Analytical Chemistry, National Chromatographic R&A Center, Dalian Institute of Chemical Physics, Chinese Academy of SciencesDalian, China
| | - Chunxia Song
- Key Laboratory of Separation Sciences for Analytical Chemistry, National Chromatographic R&A Center, Dalian Institute of Chemical Physics, Chinese Academy of SciencesDalian, China
| | - Kai Cheng
- Key Laboratory of Separation Sciences for Analytical Chemistry, National Chromatographic R&A Center, Dalian Institute of Chemical Physics, Chinese Academy of SciencesDalian, China
| | - Zhen Xiao
- College of Life and Environmental Sciences, Shanghai Normal UniversityShanghai, China
| | - Chuanfang Zhang
- College of Life and Environmental Sciences, Shanghai Normal UniversityShanghai, China
| | - Weimin Ma
- College of Life and Environmental Sciences, Shanghai Normal UniversityShanghai, China
| | - Hanfa Zou
- Key Laboratory of Separation Sciences for Analytical Chemistry, National Chromatographic R&A Center, Dalian Institute of Chemical Physics, Chinese Academy of SciencesDalian, China
| | - Mingliang Ye
- Key Laboratory of Separation Sciences for Analytical Chemistry, National Chromatographic R&A Center, Dalian Institute of Chemical Physics, Chinese Academy of SciencesDalian, China
- *Correspondence: Mingliang Ye
| | - Shaojun Dai
- Key Laboratory of Saline-Alkali Vegetation Ecology Restoration in Oil Field, Alkali Soil Natural Environmental Science Center, Ministry of Education, Northeast Forestry UniversityHarbin, China
- College of Life and Environmental Sciences, Shanghai Normal UniversityShanghai, China
- Shaojun Dai
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43
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Abstract
Replication of positive-strand RNA viruses occurs in tight association with reorganized host cell membranes. In a concerted fashion, viral and cellular factors generate distinct organelle-like structures, designated viral replication factories. These virus-induced compartments promote highly efficient genome replication, allow spatiotemporal coordination of the different steps of the viral replication cycle, and protect viral RNA from the hostile cytoplasmic environment. The combined use of ultrastructural and functional studies has greatly increased our understanding of the architecture and biogenesis of viral replication factories. Here, we review common concepts and distinct differences in replication organelle morphology and biogenesis within the Flaviviridae family, exemplified by dengue virus and hepatitis C virus. We discuss recent progress made in our understanding of the complex interplay between viral determinants and subverted cellular membrane homeostasis in biogenesis and maintenance of replication factories of this virus family.
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Affiliation(s)
- David Paul
- Department of Infectious Diseases, Molecular Virology, University of Heidelberg, 69120 Heidelberg, Germany; ,
| | - Ralf Bartenschlager
- Department of Infectious Diseases, Molecular Virology, University of Heidelberg, 69120 Heidelberg, Germany; , .,Division of Virus-Associated Carcinogenesis, German Cancer Research Center (DKFZ), 69120 Heidelberg, Germany
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44
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Klaus CJS, Raghunathan K, DiBenedetto E, Kenworthy AK. Analysis of diffusion in curved surfaces and its application to tubular membranes. Mol Biol Cell 2016; 27:3937-3946. [PMID: 27733625 PMCID: PMC5170615 DOI: 10.1091/mbc.e16-06-0445] [Citation(s) in RCA: 18] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/23/2016] [Revised: 09/08/2016] [Accepted: 10/04/2016] [Indexed: 11/11/2022] Open
Abstract
Diffusion of particles in curved surfaces is inherently complex compared with diffusion in a flat membrane, owing to the nonplanarity of the surface. The consequence of such nonplanar geometry on diffusion is poorly understood but is highly relevant in the case of cell membranes, which often adopt complex geometries. To address this question, we developed a new finite element approach to model diffusion on curved membrane surfaces based on solutions to Fick's law of diffusion and used this to study the effects of geometry on the entry of surface-bound particles into tubules by diffusion. We show that variations in tubule radius and length can distinctly alter diffusion gradients in tubules over biologically relevant timescales. In addition, we show that tubular structures tend to retain concentration gradients for a longer time compared with a comparable flat surface. These findings indicate that sorting of particles along the surfaces of tubules can arise simply as a geometric consequence of the curvature without any specific contribution from the membrane environment. Our studies provide a framework for modeling diffusion in curved surfaces and suggest that biological regulation can emerge purely from membrane geometry.
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Affiliation(s)
| | - Krishnan Raghunathan
- Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, TN 37232
| | | | - Anne K Kenworthy
- Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, TN 37232 .,Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, TN 37232
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45
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How curvature-generating proteins build scaffolds on membrane nanotubes. Proc Natl Acad Sci U S A 2016; 113:11226-11231. [PMID: 27655892 DOI: 10.1073/pnas.1606943113] [Citation(s) in RCA: 99] [Impact Index Per Article: 11.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
Bin/Amphiphysin/Rvs (BAR) domain proteins control the curvature of lipid membranes in endocytosis, trafficking, cell motility, the formation of complex subcellular structures, and many other cellular phenomena. They form 3D assemblies that act as molecular scaffolds to reshape the membrane and alter its mechanical properties. It is unknown, however, how a protein scaffold forms and how BAR domains interact in these assemblies at protein densities relevant for a cell. In this work, we use various experimental, theoretical, and simulation approaches to explore how BAR proteins organize to form a scaffold on a membrane nanotube. By combining quantitative microscopy with analytical modeling, we demonstrate that a highly curving BAR protein endophilin nucleates its scaffolds at the ends of a membrane tube, contrary to a weaker curving protein centaurin, which binds evenly along the tube's length. Our work implies that the nature of local protein-membrane interactions can affect the specific localization of proteins on membrane-remodeling sites. Furthermore, we show that amphipathic helices are dispensable in forming protein scaffolds. Finally, we explore a possible molecular structure of a BAR-domain scaffold using coarse-grained molecular dynamics simulations. Together with fluorescence microscopy, the simulations show that proteins need only to cover 30-40% of a tube's surface to form a rigid assembly. Our work provides mechanical and structural insights into the way BAR proteins may sculpt the membrane as a high-order cooperative assembly in important biological processes.
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46
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van der Mark VA, Ghiboub M, Marsman C, Zhao J, van Dijk R, Hiralall JK, Ho-Mok KS, Castricum Z, de Jonge WJ, Oude Elferink RPJ, Paulusma CC. Phospholipid flippases attenuate LPS-induced TLR4 signaling by mediating endocytic retrieval of Toll-like receptor 4. Cell Mol Life Sci 2016; 74:715-730. [PMID: 27628304 PMCID: PMC5272906 DOI: 10.1007/s00018-016-2360-5] [Citation(s) in RCA: 27] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/26/2016] [Revised: 08/31/2016] [Accepted: 09/06/2016] [Indexed: 01/01/2023]
Abstract
P4-ATPases are lipid flippases that catalyze the transport of phospholipids to create membrane phospholipid asymmetry and to initiate the biogenesis of transport vesicles. Here we show, for the first time, that lipid flippases are essential to dampen the inflammatory response and to mediate the endotoxin-induced endocytic retrieval of Toll-like receptor 4 (TLR4) in human macrophages. Depletion of CDC50A, the β-subunit that is crucial for the activity of multiple P4-ATPases, resulted in endotoxin-induced hypersecretion of proinflammatory cytokines, enhanced MAP kinase signaling and constitutive NF-κB activation. In addition, CDC50A-depleted THP-1 macrophages displayed reduced tolerance to endotoxin. Moreover, endotoxin-induced internalization of TLR4 was strongly reduced and coincided with impaired endosomal MyD88-independent signaling. The phenotype of CDC50A-depleted cells was also induced by separate knockdown of two P4-ATPases, namely ATP8B1 and ATP11A. We conclude that lipid flippases are novel elements of the innate immune response that are essential to attenuate the inflammatory response, possibly by mediating endotoxin-induced internalization of TLR4.
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Affiliation(s)
- Vincent A van der Mark
- Tytgat Institute for Liver and Intestinal Research, Academic Medical Center, Meibergdreef 69-71, 1105 BK, Amsterdam, The Netherlands
| | - Mohammed Ghiboub
- Tytgat Institute for Liver and Intestinal Research, Academic Medical Center, Meibergdreef 69-71, 1105 BK, Amsterdam, The Netherlands
| | - Casper Marsman
- Tytgat Institute for Liver and Intestinal Research, Academic Medical Center, Meibergdreef 69-71, 1105 BK, Amsterdam, The Netherlands
| | - Jing Zhao
- Tytgat Institute for Liver and Intestinal Research, Academic Medical Center, Meibergdreef 69-71, 1105 BK, Amsterdam, The Netherlands
| | - Remco van Dijk
- Tytgat Institute for Liver and Intestinal Research, Academic Medical Center, Meibergdreef 69-71, 1105 BK, Amsterdam, The Netherlands
| | - Johan K Hiralall
- Tytgat Institute for Liver and Intestinal Research, Academic Medical Center, Meibergdreef 69-71, 1105 BK, Amsterdam, The Netherlands
| | - Kam S Ho-Mok
- Tytgat Institute for Liver and Intestinal Research, Academic Medical Center, Meibergdreef 69-71, 1105 BK, Amsterdam, The Netherlands
| | - Zoë Castricum
- Tytgat Institute for Liver and Intestinal Research, Academic Medical Center, Meibergdreef 69-71, 1105 BK, Amsterdam, The Netherlands
| | - Wouter J de Jonge
- Tytgat Institute for Liver and Intestinal Research, Academic Medical Center, Meibergdreef 69-71, 1105 BK, Amsterdam, The Netherlands
| | - Ronald P J Oude Elferink
- Tytgat Institute for Liver and Intestinal Research, Academic Medical Center, Meibergdreef 69-71, 1105 BK, Amsterdam, The Netherlands
| | - Coen C Paulusma
- Tytgat Institute for Liver and Intestinal Research, Academic Medical Center, Meibergdreef 69-71, 1105 BK, Amsterdam, The Netherlands.
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47
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Myers MD, Ryazantsev S, Hicke L, Payne GS. Calmodulin Promotes N-BAR Domain-Mediated Membrane Constriction and Endocytosis. Dev Cell 2016; 37:162-73. [PMID: 27093085 DOI: 10.1016/j.devcel.2016.03.012] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/22/2015] [Revised: 02/15/2016] [Accepted: 03/16/2016] [Indexed: 10/21/2022]
Abstract
Membrane remodeling by BAR (Bin, Amphiphysin, RVS) domain-containing proteins, such as endophilins and amphiphysins, is integral to the process of endocytosis. However, little is known about the regulation of endocytic BAR domain activity. We have identified an interaction between the yeast Rvs167 N-BAR domain and calmodulin. Calmodulin-binding mutants of Rvs167 exhibited defects in endocytic vesicle release. In vitro, calmodulin enhanced membrane tubulation and constriction by wild-type Rvs167 but not calmodulin-binding-defective mutants. A subset of mammalian N-BAR domains bound calmodulin, and co-expression of calmodulin with endophilin A2 potentiated tubulation in vivo. These studies reveal a conserved role for calmodulin in regulating the intrinsic membrane-sculpting activity of endocytic N-BAR domains.
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Affiliation(s)
- Margaret D Myers
- Department of Biological Chemistry, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA
| | - Sergey Ryazantsev
- Department of Biological Chemistry, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA
| | - Linda Hicke
- Molecular Genetics and Microbiology, College of Natural Sciences, University of Texas at Austin, Austin, TX 78712, USA
| | - Gregory S Payne
- Department of Biological Chemistry, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA.
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48
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Phospholipase Cβ1 induces membrane tubulation and is involved in caveolae formation. Proc Natl Acad Sci U S A 2016; 113:7834-9. [PMID: 27342861 DOI: 10.1073/pnas.1603513113] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/09/2023] Open
Abstract
Lipid membrane curvature plays important roles in various physiological phenomena. Curvature-regulated dynamic membrane remodeling is achieved by the interaction between lipids and proteins. So far, several membrane sensing/sculpting proteins, such as Bin/amphiphysin/Rvs (BAR) proteins, are reported, but there remains the possibility of the existence of unidentified membrane-deforming proteins that have not been uncovered by sequence homology. To identify new lipid membrane deformation proteins, we applied liposome-based microscopic screening, using unbiased-darkfield microscopy. Using this method, we identified phospholipase Cβ1 (PLCβ1) as a new candidate. PLCβ1 is well characterized as an enzyme catalyzing the hydrolysis of phosphatidylinositol-4,5-bisphosphate (PIP2). In addition to lipase activity, our results indicate that PLCβ1 possessed the ability of membrane tubulation. Lipase domains and inositol phospholipids binding the pleckstrin homology (PH) domain of PLCβ1 were not involved, but the C-terminal sequence was responsible for this tubulation activity. Computational modeling revealed that the C terminus displays the structural homology to the BAR domains, which is well known as a membrane sensing/sculpting domain. Overexpression of PLCβ1 caused plasma membrane tubulation, whereas knockdown of the protein reduced the number of caveolae and induced the evagination of caveolin-rich membrane domains. Taken together, our results suggest a new function of PLCβ1: plasma membrane remodeling, and in particular, caveolae formation.
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49
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Abstract
Membrane curvature is an important parameter in defining the morphology of cells, organelles and local membrane subdomains. Transport intermediates have simpler shapes, being either spheres or tubules. The generation and maintenance of curvature is of central importance for maintaining trafficking and cellular functions. It is possible that local shapes in complex membranes could help to define local subregions. In this Cell Science at a Glance article and accompanying poster, we summarize how generating, sensing and maintaining high local membrane curvature is an active process that is mediated and controlled by specialized proteins using general mechanisms: (i) changes in lipid composition and asymmetry, (ii) partitioning of shaped transmembrane domains of integral membrane proteins or protein or domain crowding, (iii) reversible insertion of hydrophobic protein motifs, (iv) nanoscopic scaffolding by oligomerized hydrophilic protein domains and, finally, (v) macroscopic scaffolding by the cytoskeleton with forces generated by polymerization and by molecular motors. We also summarize some of the discoveries about the functions of membrane curvature, where in addition to providing cell or organelle shape, local curvature can affect processes like membrane scission and fusion as well as protein concentration and enzyme activation on membranes.
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Affiliation(s)
- Harvey T McMahon
- MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, UK
| | - Emmanuel Boucrot
- Institute of Structural and Molecular Biology, University College London & Birkbeck College, London WC1E 6BT, UK
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50
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Liu Y, Agudo-Canalejo J, Grafmüller A, Dimova R, Lipowsky R. Patterns of Flexible Nanotubes Formed by Liquid-Ordered and Liquid-Disordered Membranes. ACS NANO 2016; 10:463-74. [PMID: 26588094 DOI: 10.1021/acsnano.5b05377] [Citation(s) in RCA: 66] [Impact Index Per Article: 7.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/28/2023]
Abstract
Biological membranes form both intra- and intercellular nanotubes that are used for molecular sorting within single cells and for long-distance connections between different cells. Such nanotubes can also develop from synthetic lipid bilayers in their fluid state. Each nanotube has a large area-to-volume ratio and stably encloses a water channel that is thereby shielded from its surroundings. The tubes are rather flexible and can easily change both their length and their conformation. Here, we study nanotubes formed by liquid-ordered (Lo) and liquid-disordered (Ld) membranes with three lipid components exposed to aqueous mixtures of two polymers, polyethylene glycol (PEG) and dextran. Both types of membranes form striking patterns of nanotubes when we reduce the volume of giant vesicles by osmotic deflation, thereby exposing the two bilayer leaflets of the membranes to polymer solutions of different composition. With decreasing volume, three different patterns are observed corresponding to three distinct vesicle morphologies that reflect the interplay of spontaneous curvature and aqueous phase separation. We show that tube nucleation and growth is governed by two kinetic pathways and that the tubes undergo a novel shape transformation from necklace-like to cylindrical tubes at a certain critical tube length. We deduce the spontaneous curvature generated by the membrane-polymer interactions from the observed vesicle morphologies using three different and independent methods of image analysis. The spontaneous curvature of the Ld membranes is found to be 4.7 times larger than that of the Lo membranes. We also show that these curvatures are generated by weak PEG adsorption onto the membranes, with a binding affinity of about 1.6 kBT per chain. In this way, our study provides a direct connection between nanoscopic membrane shapes and molecular interactions. Our approach is rather general and can be applied to many other systems of interest such as polymersomes or membrane-bound proteins and peptides.
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Affiliation(s)
- Yonggang Liu
- Theory & Biosystems, Max Planck Institute of Colloids and Interfaces , 14424 Potsdam, Germany
- State Key Laboratory of Polymer Physics and Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences , 130022 Changchun, China
| | - Jaime Agudo-Canalejo
- Theory & Biosystems, Max Planck Institute of Colloids and Interfaces , 14424 Potsdam, Germany
| | - Andrea Grafmüller
- Theory & Biosystems, Max Planck Institute of Colloids and Interfaces , 14424 Potsdam, Germany
| | - Rumiana Dimova
- Theory & Biosystems, Max Planck Institute of Colloids and Interfaces , 14424 Potsdam, Germany
| | - Reinhard Lipowsky
- Theory & Biosystems, Max Planck Institute of Colloids and Interfaces , 14424 Potsdam, Germany
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