A viral fusogen hijacks the actin cytoskeleton to drive cell-cell fusion
- UC Berkeley–UC San Francisco Graduate Group in Bioengineering, United States
- Department of Bioengineering & Biophysics Graduate Group, University of California, Berkeley, United States
- Division of Biological Systems and Engineering, Lawrence Berkeley National Laboratory, United States
- Chan Zuckerberg Biohub, United States
Figures
Figure 1 with 1 supplement
Expression of p14 drives cell-cell fusion and is quantified with a splitYFP fluorescent assay.
(a) Schematic of fusion-associated small transmembrane protein, p14, in proportion to post-fusion trimeric structure of cell-cell fusogens, Eff-1 (PDB:4OJC) and Hap2 (PDB: 5MF1), on the plasma …
-
Figure 1—source data 1
Excel Spreadsheet of counts and distribution for p14-expressing HEK293T cells at 12 hr and 24 hr post transfection for Figure 1c and d.
- https://cdn.elifesciences.org/articles/51358/elife-51358-fig1-data1-v2.xlsx
Figure 1—figure supplement 1
Characterization of p14 cytoplasmic tail cleavage and the splitYFP cell-cell fusion assay.
(a) Schematic of hypothesized cleavage sites in p14 cytoplasmic tail based on molecular weights of cleaved fragments. (b) Membrane fractionation of p14 WT, p14 FVAI, Δcyto and GFP only, showing …
-
Figure 1—figure supplement 1—source data 1
Excel Spreadsheet of surface biotinylation of p14-WT-GFP and p14-Δcyto-GFP for Figure 1—figure supplement 1h.
- https://cdn.elifesciences.org/articles/51358/elife-51358-fig1-figsupp1-data1-v2.xlsx
Figure 2 with 1 supplement
p14 Y116 in the cytoplasmic tail is necessary for cell-cell fusion and is phosphorylated by c-src kinase.
(a) Schematic of predicted phosphotyrosines in p14 cytoplasmic tail (See also Figure 2—figure supplement 1a). (b) Western blot probed with α-phosphotyrosine confirming that p14 WT is phosphorylated. …
-
Figure 2—source data 1
Excel Spreadsheet of counts for p14-expressing SYF and SYF+c-src cells for Figure 2g.
- https://cdn.elifesciences.org/articles/51358/elife-51358-fig2-data1-v2.xlsx
Figure 2—figure supplement 1
Characterization of p14 cytoplasmic tail and Y116.
(a) Disorder probability of p14 cytoplasmic tail as calculated with DisEMBL (http://dis.embl.de/). (b) Representative field of view of HEK293T cells expressing p14 WT and p14 mutants with p14 …
Figure 3 with 1 supplement
p14 Y116 in the cytoplasmic tail binds to Grb2.
(a) Schematic of p14 mutants that disrupt predicted Grb2 binding motif. (b) Western blot of co-immunoprecipitation of p14 with Grb2 (lane 7) and p14 mutants, Y116F, N118A, FVAI, that does not bind …
-
Figure 3—source data 1
Excel Spreadsheet of plasma membrane enrichment of Grb2-GFP in cells expressing CA-src alone or with p14-WT-mcherry or with p14-FVAI mcherry.
- https://cdn.elifesciences.org/articles/51358/elife-51358-fig3-data1-v2.xlsx
Figure 3—figure supplement 1
Characterization of Grb2 binding to p14.
(a) Average biotinylated p14 FVAI to that of p14 WT. Error bars represent standard deviations from three independent transfections and biotinylation. P-values are two-tailed two-sample Student’s …
-
Figure 3—figure supplement 1—source data 1
Excel Spreadsheet of surface biotinylation of p14-WT-GFP and p14-FVAI-GFP for Figure 3—figure supplement 1a.
- https://cdn.elifesciences.org/articles/51358/elife-51358-fig3-figsupp1-data1-v2.xlsx
Figure 4 with 1 supplement
N-WASP-dependent assembly of branched actin network is necessary for cell-cell fusion.
(a) Schematic of Grb2 binding to two potential downstream effectors, SOS and N-WASP (b) Extent of cell-cell fusion quantified with splitYFP fluorescence assay of p14 expressing cells treated …
-
Figure 4—source data 1
Excel Spreadsheet of counts and distribution for p14-expressing HEK293T cells over-expressing Grb2 mutants for Figure 4c.
- https://cdn.elifesciences.org/articles/51358/elife-51358-fig4-data1-v2.xlsx
-
Figure 4—source data 2
Excel Spreadsheet of counts and distribution for p14-expressing N-WASP -/- or +/+ mouse embryonic fibroblasts for Figure 4d.
- https://cdn.elifesciences.org/articles/51358/elife-51358-fig4-data2-v2.xlsx
Figure 4—figure supplement 1
Characterization of over-expression of Grb2 mutants and purified components of actin motility assay.
(a) Western blot of surface biotinylated GFP-tagged p14 WT treated with eitherDMSO, Wiskostatin or CK-666. (b) Average biotinylated p14 WT treated with Wiskostatin and CK-666 normalized to that of …
-
Figure 4—figure supplement 1—source data 1
Excel Spreadsheet of surface biotinylation of p14-WT-GFP treated with wiskostatin and CK-666 for Figure 4—figure supplement 1b.
- https://cdn.elifesciences.org/articles/51358/elife-51358-fig4-figsupp1-data1-v2.xlsx
Figure 5 with 2 supplements
Branched actin assembly directly coupled to p14 cytoplasmic tail drives cell-cell fusion.
(a) Average fusion index of p14 truncation mutants normalized to that of p14 WT. P-values are two-tailed, two-sample Student’s t-test to p14 WT where *** = p<0.001. Error bars indicate standard …
-
Figure 5—source data 1
Excel Spreadsheet of counts and distribution for p14-expressing HEK293T cells over-expressing R47 constructs for Figure 5c.
- https://cdn.elifesciences.org/articles/51358/elife-51358-fig5-data1-v2.xlsx
Figure 5—figure supplement 1
Characterization of p14 Δectodomain and direct coupling of p14 to actin assembly.
(a) Average biotinylated p14 Δecto to that of p14 WT. Error bars represent standard deviations from three independent transfections and biotinylation. P-values are two-tailed two-sample Student’s …
-
Figure 5—figure supplement 1—source data 1
Excel Spreadsheet of surface biotinylation of p14-WT-GFP and p14-Δecto-GFP for Figure 5—figure supplement 1a.
- https://cdn.elifesciences.org/articles/51358/elife-51358-fig5-figsupp1-data1-v2.xlsx
Figure 5—figure supplement 2
Characterization of p14 at time and site of fusion.
(a) Representative fluorescence images of a fusion site 120 s prior to cytoplasmic mixing. Plasma membrane is marked with GPI-anchored pHluorin (green) and p14-WT mCherry in magenta. Dotted white …
-
Figure 5—figure supplement 2—source data 1
Excel Spreadsheet of normalized intensity of p14-WT-mCherry and SH2-GFP at fusion site ~200 s prior to fusion for Figure 5—figure supplement 2b and d.
- https://cdn.elifesciences.org/articles/51358/elife-51358-fig5-figsupp2-data1-v2.xlsx
Author response image 1
Surface biotinylation of p14, p14 mutants and cells treated with cytoskeletal drugs.
(a) Representative western blot probed with α-GFP of lysate and streptavidin eluate of surface biotinylated cells expressing p14 WT, p14 FVAI, p14 Δcyto, p14 Δecto, and p14 WT expressing cells …
Author response image 2
Extent of p14-mediated cell-cell fusion in Grb2 KD and KO cells.
(a) Western blot probed with α-Grb2 of HEK293T with Grb2 knockdown. (b) Extent of p14-mediated cell-cell fusion in Grb2 KD and wild-type HEK293T cells. (c) Western blot probed with α-Grb2 of clone 1 …
Author response image 3
Time-lapse imaging of p14 at sites of fusion using confocal microscopy.
SH2-GFP (green) re-localizes to the plasma membrane when co-expressed with p14-WT mCherry (not shown) and treated with pervanadate to increase phosphorylation of p14.
Author response image 4
Time-lapse imaging of p14 and actin at times and sites of fusion.
(a) Representative snapshots of a fusion site 90 sec prior to cytoplasmic mixing. Actin is visualized with Lifeact-GFP(green) and p14-WT mCherry in magenta. Boxed region marks the fusion site, where …
Author response image 5
Confocal image of HEK293T cell over-expressing SH2-GFP-ΔGBD-mDia2 and p14-WT-mCherry.
HEK293T cell expressing SH2-GFP- ΔGBD-mDia2 (green) and p14-WT-mCherry (magenta) with filopodia-like protrusions denoted with white arrows.
Videos
Video 1
Phase contrast timelapse of HEK293T cells expressing p14 WT showing extensive syncytium formation.
Video 2
Confocal timelapse of a HEK293T expressing p14-mcherry (magenta) fusing with a WT HEK293T cell that appears dark.
Plasma membrane is marked with gpi-anchored pHluorin (green). Scale bar is 5 μm.
Additional files
-
Supplementary file 1
Key resources table.
- https://cdn.elifesciences.org/articles/51358/elife-51358-supp1-v2.docx
-
Transparent reporting form
- https://cdn.elifesciences.org/articles/51358/elife-51358-transrepform-v2.docx
