Direct Visualization of Large and Protein-Free Hemifusion Diaphragms
Fusion of cellular membranes is a ubiquitous biological process requiring remodeling of two phospholipid bilayers. We believe it is very likely that merging of membranes proceeds via similar sequential intermediates. Contacting membranes form a stalk between the proximal leaflets that expands radial...
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| Veröffentlicht in: | Biophysical journal 2010-04, Vol.98 (7), p.1192-1199 |
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| creator | Nikolaus, Jörg Stöckl, Martin Langosch, Dieter Volkmer, Rudolf Herrmann, Andreas |
| description | Fusion of cellular membranes is a ubiquitous biological process requiring remodeling of two phospholipid bilayers. We believe it is very likely that merging of membranes proceeds via similar sequential intermediates. Contacting membranes form a stalk between the proximal leaflets that expands radially into an hemifusion diaphragm (HD) and subsequently open to a fusion pore. Although considered to be a key intermediate in fusion, direct experimental verification of this structure is difficult due to its transient nature. Using confocal fluorescence microscopy we have investigated the fusion of giant unilamellar vesicles (GUVs) containing phosphatidylserine and fluorescent virus derived transmembrane peptides or membrane proteins in the presence of divalent cations. Time-resolved imaging revealed that fusion was preceded by displacement of peptides and fluorescent lipid analogs from the GUV-GUV adhesion region. A detailed analysis of this area being several μm in size revealed that peptides were completely sequestered as expected for an HD. Lateral distribution of lipid analogs was consistent with formation of an HD but not with the presence of two adherent bilayers. Formation and size of the HD were dependent on lipid composition and peptide concentration. |
| doi_str_mv | 10.1016/j.bpj.2009.11.042 |
| format | Article |
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We believe it is very likely that merging of membranes proceeds via similar sequential intermediates. Contacting membranes form a stalk between the proximal leaflets that expands radially into an hemifusion diaphragm (HD) and subsequently open to a fusion pore. Although considered to be a key intermediate in fusion, direct experimental verification of this structure is difficult due to its transient nature. Using confocal fluorescence microscopy we have investigated the fusion of giant unilamellar vesicles (GUVs) containing phosphatidylserine and fluorescent virus derived transmembrane peptides or membrane proteins in the presence of divalent cations. Time-resolved imaging revealed that fusion was preceded by displacement of peptides and fluorescent lipid analogs from the GUV-GUV adhesion region. A detailed analysis of this area being several μm in size revealed that peptides were completely sequestered as expected for an HD. Lateral distribution of lipid analogs was consistent with formation of an HD but not with the presence of two adherent bilayers. Formation and size of the HD were dependent on lipid composition and peptide concentration.</description><identifier>ISSN: 0006-3495</identifier><identifier>EISSN: 1542-0086</identifier><identifier>DOI: 10.1016/j.bpj.2009.11.042</identifier><identifier>PMID: 20371318</identifier><language>eng</language><publisher>United States: Elsevier Inc</publisher><subject>Animals ; Biophysics - methods ; Calcium - chemistry ; Cations ; Cells ; Fluorescence Recovery After Photobleaching ; Fluorescent Dyes - chemistry ; Lipid Bilayers - chemistry ; Lipids ; Lipids - chemistry ; Membrane ; Membranes ; Microscopy, Confocal - methods ; Microscopy, Fluorescence - methods ; Peptides ; Peptides - chemistry ; Phosphatidylserines - chemistry ; Phospholipids - chemistry ; Proteins ; Unilamellar Liposomes - chemistry ; Viruses ; Viruses - metabolism</subject><ispartof>Biophysical journal, 2010-04, Vol.98 (7), p.1192-1199</ispartof><rights>2010 Biophysical Society</rights><rights>Copyright (c) 2010 Biophysical Society. 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All rights reserved.</rights><rights>Copyright Biophysical Society Apr 7, 2010</rights><rights>2010 by the Biophysical Society. 2010 Biophysical Society</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c575t-8b7974766f2a8b605c0c6b1c5340f882c2ddf24679c0fca350868d58673078f93</citedby><cites>FETCH-LOGICAL-c575t-8b7974766f2a8b605c0c6b1c5340f882c2ddf24679c0fca350868d58673078f93</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC2849057/pdf/$$EPDF$$P50$$Gpubmedcentral$$H</linktopdf><linktohtml>$$Uhttps://www.sciencedirect.com/science/article/pii/S0006349509060007$$EHTML$$P50$$Gelsevier$$Hfree_for_read</linktohtml><link.rule.ids>230,314,723,776,780,881,3537,27901,27902,53766,53768,65306</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/20371318$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Nikolaus, Jörg</creatorcontrib><creatorcontrib>Stöckl, Martin</creatorcontrib><creatorcontrib>Langosch, Dieter</creatorcontrib><creatorcontrib>Volkmer, Rudolf</creatorcontrib><creatorcontrib>Herrmann, Andreas</creatorcontrib><title>Direct Visualization of Large and Protein-Free Hemifusion Diaphragms</title><title>Biophysical journal</title><addtitle>Biophys J</addtitle><description>Fusion of cellular membranes is a ubiquitous biological process requiring remodeling of two phospholipid bilayers. 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Lateral distribution of lipid analogs was consistent with formation of an HD but not with the presence of two adherent bilayers. Formation and size of the HD were dependent on lipid composition and peptide concentration.</description><subject>Animals</subject><subject>Biophysics - methods</subject><subject>Calcium - chemistry</subject><subject>Cations</subject><subject>Cells</subject><subject>Fluorescence Recovery After Photobleaching</subject><subject>Fluorescent Dyes - chemistry</subject><subject>Lipid Bilayers - chemistry</subject><subject>Lipids</subject><subject>Lipids - chemistry</subject><subject>Membrane</subject><subject>Membranes</subject><subject>Microscopy, Confocal - methods</subject><subject>Microscopy, Fluorescence - methods</subject><subject>Peptides</subject><subject>Peptides - chemistry</subject><subject>Phosphatidylserines - chemistry</subject><subject>Phospholipids - chemistry</subject><subject>Proteins</subject><subject>Unilamellar Liposomes - chemistry</subject><subject>Viruses</subject><subject>Viruses - metabolism</subject><issn>0006-3495</issn><issn>1542-0086</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2010</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkU1v1DAQhi0EokvhB3BBERdOCeNvR0hIqEsp0kpwAK6W49hbR0m82Ekl-PV42VIBBzjNYZ55NTMPQk8xNBiweDk03WFoCEDbYNwAI_fQBnNGagAl7qMNAIiaspafoUc5DwCYcMAP0RkBKjHFaoO225CcXaovIa9mDN_NEuJcRV_tTNq7ysx99THFxYW5vkzOVVduCn7NR2gbzOE6mf2UH6MH3ozZPbmt5-jz5dtPF1f17sO79xdvdrXlki-16mQrmRTCE6M6AdyCFR22nDLwShFL-t4TJmRrwVtDeblC9VwJSUEq39Jz9PqUe1i7yfXWzUsyoz6kMJn0TUcT9J-dOVzrfbzRRLEWuCwBL24DUvy6urzoKWTrxtHMLq5ZS8ZEKwRR_ycpVQIYPmY-_4sc4prm8gdNMBctZwwXCJ8gm2LOyfm7pTHoo0s96OJSH11qjHVxWWae_X7t3cQveQV4dQJc-flNcElnG9xsXf_Tqe5j-Ef8DyRsrd8</recordid><startdate>20100407</startdate><enddate>20100407</enddate><creator>Nikolaus, Jörg</creator><creator>Stöckl, Martin</creator><creator>Langosch, Dieter</creator><creator>Volkmer, Rudolf</creator><creator>Herrmann, Andreas</creator><general>Elsevier Inc</general><general>Biophysical Society</general><general>The Biophysical Society</general><scope>6I.</scope><scope>AAFTH</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7QO</scope><scope>7QP</scope><scope>7TK</scope><scope>7TM</scope><scope>7U9</scope><scope>8FD</scope><scope>FR3</scope><scope>H94</scope><scope>K9.</scope><scope>P64</scope><scope>7X8</scope><scope>5PM</scope></search><sort><creationdate>20100407</creationdate><title>Direct Visualization of Large and Protein-Free Hemifusion Diaphragms</title><author>Nikolaus, Jörg ; 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| source | MEDLINE; Cell Press Free Archives; Elsevier ScienceDirect Journals; Elektronische Zeitschriftenbibliothek - Frei zugängliche E-Journals; PubMed Central |
| subjects | Animals Biophysics - methods Calcium - chemistry Cations Cells Fluorescence Recovery After Photobleaching Fluorescent Dyes - chemistry Lipid Bilayers - chemistry Lipids Lipids - chemistry Membrane Membranes Microscopy, Confocal - methods Microscopy, Fluorescence - methods Peptides Peptides - chemistry Phosphatidylserines - chemistry Phospholipids - chemistry Proteins Unilamellar Liposomes - chemistry Viruses Viruses - metabolism |
| title | Direct Visualization of Large and Protein-Free Hemifusion Diaphragms |
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