Fusogenic Pairings of Vesicle-Associated Membrane Proteins (VAMPs) and Plasma Membrane t-SNAREs – VAMP5 as the Exception
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{"title"=>"Fusogenic pairings of vesicle-associated membrane proteins (VAMPs) and plasma membrane t-SNAREs - VAMP5 as the exception", "type"=>"journal", "authors"=>[{"first_name"=>"Nazarul", "last_name"=>"Hasan", "scopus_author_id"=>"13102912800"}, {"first_name"=>"Deborah", "last_name"=>"Corbin", "scopus_author_id"=>"37021305700"}, {"first_name"=>"Chuan", "last_name"=>"Hu", "scopus_author_id"=>"8264552800"}], "year"=>2010, "source"=>"PLoS ONE", "identifiers"=>{"issn"=>"19326203", "scopus"=>"2-s2.0-78650124252", "sgr"=>"78650124252", "pui"=>"360158272", "isbn"=>"1932-6203 (Electronic)\\r1932-6203 (Linking)", "pmid"=>"21151919", "doi"=>"10.1371/journal.pone.0014238"}, "id"=>"86f0d5b7-42cb-32e2-aefa-7c0bc7822deb", "abstract"=>"BACKGROUND: Intracellular vesicle fusion is mediated by the interactions of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins on vesicles (v-SNAREs) and on target membranes (t-SNAREs). The vesicle-associated membrane proteins (VAMPs) are v-SNAREs that reside in various post-Golgi vesicular compartments. To fully understand the specific role of each VAMP in vesicle trafficking, it is important to determine if VAMPs have differential membrane fusion activities.\\n\\nMETHODOLOGY/PRINCIPAL FINDINGS: In this study, we developed a cell fusion assay that quantifies SNARE-mediated membrane fusion events by activated expression of β-galactosidase, and examined fusogenic pairings between the seven VAMPs, i.e., VAMPs 1, 2, 3, 4, 5, 7 and 8, and two plasma membrane t-SNARE complexes, syntaxin1/SNAP-25 and syntaxin4/SNAP-25. VAMPs 1, 2, 3, 4, 7 and 8 drove fusion efficiently, whereas VAMP5 was unable to mediate fusion with the t-SNAREs. By expressing VAMPs 1, 3, 4, 7 and 8 at the same level, we further compared their membrane fusion activities. VAMPs 1 and 3 had comparable and the highest fusion activities, whereas VAMPs 4, 7 and 8 exhibited 30-50% lower fusion activities. Moreover, we determined the dependence of cell fusion activity on VAMP1 expression level. Analysis of the dependence data suggested that there was no cooperativity of VAMP proteins in the cell fusion reaction.\\n\\nCONCLUSIONS/SIGNIFICANCE: These data indicate that VAMPs have differential membrane fusion capacities, and imply that with the exception of VAMP5, VAMPs are essentially redundant in mediating fusion with plasma membrane t-SNAREs.", "link"=>"http://www.mendeley.com/research/fusogenic-pairings-vesicleassociated-membrane-proteins-vamps-plasma-membrane-tsnares-vamp5-exception", "reader_count"=>24, "reader_count_by_academic_status"=>{"Professor > Associate Professor"=>1, "Researcher"=>10, "Student > Doctoral Student"=>1, "Student > Ph. D. Student"=>7, "Other"=>1, "Student > Bachelor"=>2, "Lecturer"=>1, "Professor"=>1}, "reader_count_by_user_role"=>{"Professor > Associate Professor"=>1, "Researcher"=>10, "Student > Doctoral Student"=>1, "Student > Ph. D. Student"=>7, "Other"=>1, "Student > Bachelor"=>2, "Lecturer"=>1, "Professor"=>1}, "reader_count_by_subject_area"=>{"Biochemistry, Genetics and Molecular Biology"=>5, "Agricultural and Biological Sciences"=>15, "Medicine and Dentistry"=>2, "Neuroscience"=>1, "Computer Science"=>1}, "reader_count_by_subdiscipline"=>{"Medicine and Dentistry"=>{"Medicine and Dentistry"=>2}, "Neuroscience"=>{"Neuroscience"=>1}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>15}, "Computer Science"=>{"Computer Science"=>1}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>5}}, "reader_count_by_country"=>{"United States"=>2, "Japan"=>1, "France"=>1}, "group_count"=>2}

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/814181"], "description"=>"<p>To express VAMPs 1, 3, 4, 5, 7 and 8 at same level at the cell surface, and to express syntaxin1/SNAP-25 and syntaxin4/SNAP-25 at the same level, plasmids were transfected at the concentrations described in the legend of <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014238#pone-0014238-g004\" target=\"_blank\">Fig. 4B</a>. (A and C) 24 h after mixing the v- and t-cells, cell fusion was quantified using the enzymatic fusion assay. Error bars represent standard deviation of four independent experiments. **<i>P</i><0.01 vs. VAMP1; *** <i>P</i><0.001 vs. VAMP1. (B and D) The fusion activities (OD<sub>420</sub>) of the control cells expressing the empty vector (-VAMP) and the v-cells expressing VAMPs 3, 4, 5, 7 or 8 were normalized to the fusion activity of the v-cells expressing VAMP1. Error bars represent standard deviation of the four independent experiments. (E) Time course of cell fusion. 6, 12 or 24 h after combining the v-cells expressing different VAMPs with the t-cells expressing syntaxin1/SNAP-25, cell fusion was quantified. The time-course curve of the VAMP5-expressing cells overlapped completely with the time-course curve of the control cells. Shown is a representative of two independent experiments.</p>", "links"=>[], "tags"=>["fusion", "activities"], "article_id"=>484560, "categories"=>["Cell Biology", "Biochemistry"], "users"=>["Nazarul Hasan", "Deborah Corbin", "Chuan Hu"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0014238.g005", "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Comparison_of_fusion_activities_of_VAMPs_/484560", "title"=>"Comparison of fusion activities of VAMPs.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2010-12-06 01:16:00"}
  • {"files"=>["https://ndownloader.figshare.com/files/813787"], "description"=>"<p>(A) In COS-7 cells that expressed v-SNARE proteins on the surface (v-cells), flipped v-SNARE was cotransfected with a plasmid that encodes the tetracycline-controlled transactivator (tTA). In COS-7 cells that expressed t-SNARE proteins on the surface (t-cells), flipped t-SNAREs were cotransfected with a reporter plasmid that encodes β-galactosidase under control of the tetracycline-response element (TRE-<i>LacZ</i>). 24 h after transfection, v-cells were detached from cell culture dishes, and then overlaid on t-cells. Fusion of the v- and t-cells led to the binding of tTA to TRE and expression of β-galactosidase. (B) Cell fusion depends on the interactions of v- and t-SNAREs. v-cells that expressed tTA and VAMP2 were incubated with t-cells that expressed TRE-<i>LacZ</i>, syntaxin1 and SNAP-25. After 24 h, the cells were lysed and the activity of β-galactosidase in the lysates was determined using a colorimetric method by absorbance at 420 nm. Only baseline β-galactosidase activity was detected when either flipped VAMP2 or SNAP-25 was omitted from transfection. Error bars represent standard deviation of four independent experiments.</p>", "links"=>[], "tags"=>["fusion"], "article_id"=>484163, "categories"=>["Cell Biology", "Biochemistry"], "users"=>["Nazarul Hasan", "Deborah Corbin", "Chuan Hu"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0014238.g001", "stats"=>{"downloads"=>1, "page_views"=>5, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Enzymatic_cell_fusion_assay_/484163", "title"=>"Enzymatic cell fusion assay.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2010-12-06 01:09:23"}
  • {"files"=>["https://ndownloader.figshare.com/files/406105"], "description"=>"<div><h3>Background</h3><p>Intracellular vesicle fusion is mediated by the interactions of SNARE (<u>s</u>oluble <em><u>N</u></em>-ethylmaleimide-sensitive factor <u>a</u>ttachment protein <u>re</u>ceptor) proteins on vesicles (v-SNAREs) and on target membranes (t-SNAREs). The vesicle-associated membrane proteins (VAMPs) are v-SNAREs that reside in various post-Golgi vesicular compartments. To fully understand the specific role of each VAMP in vesicle trafficking, it is important to determine if VAMPs have differential membrane fusion activities.</p><h3>Methodology/Principal Findings</h3><p>In this study, we developed a cell fusion assay that quantifies SNARE-mediated membrane fusion events by activated expression of β-galactosidase, and examined fusogenic pairings between the seven VAMPs, <em>i.e.</em>, VAMPs 1, 2, 3, 4, 5, 7 and 8, and two plasma membrane t-SNARE complexes, syntaxin1/SNAP-25 and syntaxin4/SNAP-25. VAMPs 1, 2, 3, 4, 7 and 8 drove fusion efficiently, whereas VAMP5 was unable to mediate fusion with the t-SNAREs. By expressing VAMPs 1, 3, 4, 7 and 8 at the same level, we further compared their membrane fusion activities. VAMPs 1 and 3 had comparable and the highest fusion activities, whereas VAMPs 4, 7 and 8 exhibited 30–50% lower fusion activities. Moreover, we determined the dependence of cell fusion activity on VAMP1 expression level. Analysis of the dependence data suggested that there was no cooperativity of VAMP proteins in the cell fusion reaction.</p><h3>Conclusions/Significance</h3><p>These data indicate that VAMPs have differential membrane fusion capacities, and imply that with the exception of VAMP5, VAMPs are essentially redundant in mediating fusion with plasma membrane t-SNAREs.</p></div>", "links"=>[], "tags"=>["fusogenic", "pairings", "vesicle-associated", "membrane", "proteins", "plasma", "t-snares", "vamp5"], "article_id"=>140231, "categories"=>["Cell Biology", "Biochemistry"], "users"=>["Nazarul Hasan", "Deborah Corbin", "Chuan Hu"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0014238", "stats"=>{"downloads"=>2, "page_views"=>10, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/Fusogenic_Pairings_of_Vesicle_Associated_Membrane_Proteins_VAMPs_and_Plasma_Membrane_t_SNAREs_VAMP5_as_the_Exception/140231", "title"=>"Fusogenic Pairings of Vesicle-Associated Membrane Proteins (VAMPs) and Plasma Membrane t-SNAREs – VAMP5 as the Exception", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2010-12-06 00:03:51"}
  • {"files"=>["https://ndownloader.figshare.com/files/814081"], "description"=>"<p>24 h after cotransfection with tTA and the empty vector, flipped VAMPs 1, 3, 4, 5, 7 or 8 (v-cells), or 24 h after cotransfection with TRE-<i>LacZ</i>, flipped SNAP-25 and syntaxins 1 or 4 (t-cells), unpermeabilized COS-7 cells were stained with an anti-Myc antibody, and then analyzed by flow cytometry. (A) Representative FACS profiles of the cells transfected with the empty vector, flipped VAMP1 or syntaxin4/SNAP-25. (B) To express the v- and t-SNAREs at same level at the cell surface, flipped SNARE plasmids were transfected at the following concentrations (per 10 cm<sup>2</sup> growth area, <i>i.e.</i>, per well in 6-well plates): VAMP1, 0.2 µg; VAMP3, 0.5 µg; VAMP4, 0.5 µg; VAMP5, 0.05 µg; VAMP7, 1.0 µg; VAMP8, 0.1 µg; syntaxin1, 0.5 µg; syntaxin4, 0.05 µg. tTA, TRE-<i>LacZ</i> and flipped SNAP-25 were cotransfected at 1 µg per 10 cm<sup>2</sup> growth area. The mean fluorescence intensity of staining of the SNAREs was determined by FACS analysis. Error bars represent standard deviation of four independent experiments.</p>", "links"=>[], "tags"=>["snare"], "article_id"=>484459, "categories"=>["Cell Biology", "Biochemistry"], "users"=>["Nazarul Hasan", "Deborah Corbin", "Chuan Hu"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0014238.g004", "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_FACS_analysis_of_SNARE_expression_at_the_cell_surface_/484459", "title"=>"FACS analysis of SNARE expression at the cell surface.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2010-12-06 01:14:19"}
  • {"files"=>["https://ndownloader.figshare.com/files/813997"], "description"=>"<p>24 h after transfection, v-cells that expressed tTA and VAMPs 1, 2, 3, 4, 5, 7 or 8 were combined with t-cells that expressed TRE-<i>LacZ</i> and (A) syntaxin1/SNAP-25 or (B) syntaxin4/SNAP-25. After 24 h, cell fusion was quantified using the enzymatic cell fusion assay. Control cells (-VAMP) were cotransfected with the empty vector and the plasmid encoding tTA. Only baseline β-galactosidase activity was detected when the control cells were incubated with the t-cells. The flipped SNARE plasmids were transfected at the same concentration. Error bars represent standard deviation of three independent experiments. * <i>P</i><0.05 vs. VAMP1.</p>", "links"=>[], "tags"=>["fusion", "vamps", "plasma", "membrane"], "article_id"=>484368, "categories"=>["Cell Biology", "Biochemistry"], "users"=>["Nazarul Hasan", "Deborah Corbin", "Chuan Hu"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0014238.g003", "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Cell_fusion_by_VAMPs_and_plasma_membrane_t_SNAREs_/484368", "title"=>"Cell fusion by VAMPs and plasma membrane t-SNAREs.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2010-12-06 01:12:48"}
  • {"files"=>["https://ndownloader.figshare.com/files/813905"], "description"=>"<p>(A) Domain structure of flipped VAMPs. The preprolactin signal sequence (SS) was fused to the N-termini of VAMPs 1, 3, 4, 5, 7 and 8. A Myc tag was inserted between the signal sequence and the VAMP proteins. (B) COS-7 cells were cotransfected with tTA and the empty vector pcDNA3.1(+), flipped VAMPs 1, 3, 4, 5, 7 or 8. 24 h after transfection, unpermeabilized cells were stained with an anti-Myc monoclonal antibody to detect the expression of VAMPs at the cell surface. (C) COS-7 cells were cotransfected with TRE-<i>LacZ</i>, flipped SNAP-25 and syntaxins 1 or 4. 24 h after transfection, unpermeabilized cells were dual labeled with antibodies. Syntaxins 1 and 4 were labeled with the anti-Myc monoclonal antibody (green), and SNAP-25 was labeled with a polyclonal antibody (red). Representative confocal microscopy images of four independent experiments are shown. Scale bar, 20 µm.</p>", "links"=>[], "tags"=>["flipped", "snare", "proteins"], "article_id"=>484276, "categories"=>["Cell Biology", "Biochemistry"], "users"=>["Nazarul Hasan", "Deborah Corbin", "Chuan Hu"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0014238.g002", "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Expression_of_flipped_SNARE_proteins_at_the_cell_surface_/484276", "title"=>"Expression of flipped SNARE proteins at the cell surface.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2010-12-06 01:11:16"}
  • {"files"=>["https://ndownloader.figshare.com/files/814295"], "description"=>"<p>v-cells expressing VAMPs 4 or 5 were labeled by the green fluorescent protein EGFP. t-cells expressing syntaxin1/SNAP-25, syntaxin4/SNAP-25, syntaxin1 alone, or syntaxin4 alone were labeled by the red fluorescent protein DsRed2. The v- and t-cells were combined for 24 h. EGFP and DeRed2 were imaged sequentially on a confocal microscope before merging the images. Representative confocal images of three independent experiments are shown. Arrows indicate fused cells with a yellow cytoplasm. Scale bar, 100 µm.</p>", "links"=>[], "tags"=>["mediate", "membrane", "fusion", "plasma"], "article_id"=>484663, "categories"=>["Cell Biology", "Biochemistry"], "users"=>["Nazarul Hasan", "Deborah Corbin", "Chuan Hu"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0014238.g006", "stats"=>{"downloads"=>3, "page_views"=>5, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_VAMP5_does_not_mediate_membrane_fusion_with_plasma_membrane_t_SNAREs_/484663", "title"=>"VAMP5 does not mediate membrane fusion with plasma membrane t-SNAREs.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2010-12-06 01:17:43"}
  • {"files"=>["https://ndownloader.figshare.com/files/814422"], "description"=>"<p>(A) v-cells were cotransfected with tTA (1 µg per 10 cm<sup>2</sup> growth area) and increasing amount of the flipped VAMP1 plasmid (per 10 cm<sup>2</sup> growth area): 0.01, 0.02, 0.05, 0.1, 0.2 and 0.5 µg. After the v-cells were combined with the t-cells expressing syntaxin1/SNAP-25 for 24 h, cell fusion was quantified. In parallel experiments, the expression level of VAMP1 proteins at surface of the v-cells was determined by staining with the anti-Myc antibody and FACS analysis. A polynomial regression curve was generated to model the relationship between cell fusion activity (OD<sub>420</sub>) and mean fluorescence intensity of VAMP1 staining. Error bars represent standard deviation of three independent experiments. (B) Log-log plot of cell fusion activity vs. mean fluorescence intensity of VAMP1 staining. The solid line shows a linear regression, which yields a slope of 0.52 with a correlation coefficient of 0.901.</p>", "links"=>[], "tags"=>["fusion"], "article_id"=>484795, "categories"=>["Cell Biology", "Biochemistry"], "users"=>["Nazarul Hasan", "Deborah Corbin", "Chuan Hu"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0014238.g007", "stats"=>{"downloads"=>0, "page_views"=>3, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Dependence_of_cell_fusion_activity_on_cell_surface_density_of_VAMP1_/484795", "title"=>"Dependence of cell fusion activity on cell surface density of VAMP1.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2010-12-06 01:19:55"}

PMC Usage Stats | Further Information

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