Journal: bioRxiv

Article Title: Spatiotemporal coordination of actin regulators generates invasive protrusions in cell-cell fusion

doi: 10.1101/2024.10.25.620345

Figure Lengend Snippet: F-actin enriched invasive protrusions mediate the fusion of mouse C2C12 cells and satellite cells (a) The gradual increase in the F-actin level during mouse myoblast differentiation. Wild-type C2C12 cells cultured in differentiation medium (DM) were immunostained with anti-MyoG, anti-MHC, and phalloidin at the indicated time points (e.g. DM0 – day 0, DM1 – day 1, DM2 – day2). Note the gradual increase in the F-actin intensity in cells over time. A few randomly selected F-actin high cells are indicated by arrowheads. (b) Quantification of the phalloidin signal intensity in cells shown in ( a ). MyoG - or MyoG + cells (n ≥ 18) at each indicated time point were measured for phalloidin intensity per experiment. n = 3 independent experiments were performed. Statistics source data can be found in “ Statistics Source Data-Extended Data Fig.1b ”. (c) MyoG expression increases F-actin level in U2OS cells. 80% confluent U2OS cells were infected by either empty lentiviral vector (EV) or lentivirus containing MyoG. Three days post-infection, the cells were incubated with SiR-Actin in culture medium for 30 minutes and fixed with 4% PFA. Note the increase in the F-actin level (indicated by the SiR-Actin intensity) in MyoG OE cells (bottom panel) compared with the negative control (top panel). n = 3 independent experiments were performed with similar results. (d) MyoG expression does not change the levels of total actin protein or induce MHC expression in U2OS cells. U2OS cells shown in ( c ) were collected and subjected to WB with anti-α-, β-, γ-, pan-actin, anti-MyoG, and anti-MHC at day three post MyoG expression. C2C12 cells at day three in DM was used as a positive control for MyoG and MHC expression. Note that none of the actin isoforms or the total actin level was affected by MyoG expression. MHC was not induced in U2OS cells expressing MyoG. (e) Quantification of protein expression as shown in ( d ). The band intensity of each protein was normalized against β-tubulin. For each protein, the y axis indicates the expression in MyoG OE cells relative to that in the EV cells. n = 3 independent experiments were performed. Statistics source data can be found in “ Statistics Source Data-Extended Data Fig.1e ”. (f) Schematic diagrams of live imaging C2C12 cell fusion in DM. C2C12 cells were first seeded at 80% confluency on micropatterns in growth medium (GM). Upon 100% confluency, the cells were cultured in DM for 48 hours and subjected to live imaging. (g) Still images from a time-lapse movie of a fusion event between F-tractin-mCherry-expressing C2C12 cells in DM. The F-tractin-mCherry - expressing C2C12 cells with or without cytosolic GFP were co-cultured, switched to DM, and subjected to live imaging as described in ( c ). Note that the F-actin-enriched membrane protrusion (1.5 min, arrowhead) led to fusion pore formation and GFP transfer from the top to the bottom cell (3 min, arrow) (see Supplemental Video 3 ). n = 23 fusion events were observed with similar results. (h) Still images from a time-lapse movie of a fusion event between two satellite cells cultured in DM. Low passage (≤ three passages) satellite cells were plated at 50% confluency on collagen type I-coated 6-well plates in satellite cell growth medium (SCGM) for 24 hours. The following day, the cells were infected by retrovirus containing LifeAct-mNG in SCGM. After 48 hours, the cells were trypsinized and plated at 40% confluence on fibronectin-coated cover glass in DM. After another 48 hours, the cells were subjected to live imaging. Note the presence of a prominent actin-enriched finger-like protrusion (56 min, arrowhead) prior to cell-cell fusion and LifeAct-mNG transfer from the invading cell to the receiving cell (58 min) (see Supplemental Video 4 ). n = 18 fusion events were observed with similar results. (i) Still images from a time-lapse movie of a fusion event between satellite cells cultured in GM with an Erk1/2 inhibitor. Low passage (≤ three passages) satellite cells were infected by a mixture of retroviruses containing LifeAct-mScar and Arp2-mNG in SCGM as described in ( g ). After 48 hours, the cells were trypsinized and plated at 40% confluence on fibronectin-coated cover glass in GM containing 1μM ERK1/2 inhibitor (SCH772984). After another 48 hours, the cells were subjected to live imaging. Note that the invading cell projected actin- and Arp2-enriched protrusions (40 min, arrowheads) prior to cell fusion ( Supplemental Video 5 ). n = 7 fusion events were observed with similar results. (j) Schematic diagrams of live imaging MyoD OE cell fusion in GM. C2C12 cells co-expressing LifeAct-mScar and FS-GFP were seeded at 30% confluency on fibronectin-coated cover glass in GM. After 24 hours, the cells were infected with retrovirus containing MyoD. 48 hours after MyoD overexpression, the culture medium was replaced with fresh GM and the cells were subjected to live imaging for 16 hours. (k) Zoomed-out view of the fusion event shown in . The boxed areas are enlarged in . ( a , c , i ) Max z-projection of 8-10 focal planes from the ventral plasma membrane (z-step size: 500nm). ( g , h , k ) Single plane confocal images. Mean ± s.d. values are shown in the line graph ( b ) and bar-dot plot ( e ), and significance was determined by two-tailed student’s t-test. **: p < 0.01, ****: p < 0.0001, ns: not significant. In ( g , h , i , k ), the cell boundaries are delineated by dotted lines. Scale bars: 50 μm ( a ), 200 μm ( c ), 10 μm ( h , top panel); 5 μm ( h , bottom panel) and 10 μm ( g , i , k ).

Article Snippet: At day two post MyoD overexpression, the wild-type C2C12 cells were immunostained with mouse anti-VASP (1:100, Santa Cruz; sc-46668) and Star Red-conjugated Phalloidin (1:200; Abberior; STRED-0100) overnight at 4 °C.

Techniques: Cell Culture, Expressing, Infection, Plasmid Preparation, Incubation, Negative Control, Positive Control, Imaging, Membrane, Over Expression, Two Tailed Test

Journal: bioRxiv

Article Title: Spatiotemporal coordination of actin regulators generates invasive protrusions in cell-cell fusion

doi: 10.1101/2024.10.25.620345

Figure Lengend Snippet: a , Still images of fusion events between SiR-Actin-labeled C2C12 cells cultured on a micropattern (schematic illustrated in Extended Data Fig. 1f). Three fusing myoblasts (arrowheads) are marked as 1, 2 and 3 ( Supplementary Video 1 ). b , Quantification of the fusion of SiR-Actin + vs. SiR-Actin - cells tracked by live imaging. n = 6 independent experiments ( Statistics Source Data-Fig.1b) . c , Still images of a fusion event between GFP + and GFP - C2C12 cells. Cell boundaries of the invading cell (inv) (arrowheads indicating the protrusions) and receiving cell (rec) are delineated by dotted lines except for the protrusive area ( Supplementary Video 2 ). A schematic diagram of this fusion event is at the bottom. n = 22 fusion events were observed with similar results. d , An electron micrograph of C2C12 cells cultured for 48 hours in DM. The invading cell is pseudo-colored in magenta. Invasive protrusions at the cell-cell contact site are indicated by arrows. n = 15 cell-cell contact sites were observed with similar results. e , Phalloidin staining of C2C12 cells at low cell density in GM at 72 hours post-overexpression of GFP, MyoD-P2A-GFP, or MyoG-P2A-GFP. f , Quantification of fusion index for experiments in e . n = 3 independent experiments ( Statistics Source Data-Fig.1f) . g , Still images of a fusion event between two MyoD OE cells (schematic and zoomed-out view shown in Extended Data Fig. 1j and 1k, respectively ) . Cell boundaries are delineated by dotted lines in merged channels ( Supplementary Video 6 ). n = 51 fusion events were observed with similar results. h , An electron micrograph of MyoD OE cells at 48 hours post MyoD overexpression. The invading cell is pseudo-colored in magenta. Invasive protrusions and dense actin bundles along the shaft of the protrusions are indicated by black and red arrowheads, respectively. n = 13 cell-cell contact sites were observed with similar results. ( a , c , g ) Max z-projection of 8-10 focal planes from ventral plasma membrane (z-step size: 500 nm). ( e ) Epifluorescence images. ( g ) Single focal plane confocal images. Mean ± s.d. values are shown in the dot plot ( b ) and bar-dot plot ( f ), and significance was determined by two-tailed student’s t-test. Scale bars: 55 μm ( a ), 5 μm ( c ), 500 nm ( d ), 100 μm ( e ), 3 μm ( g ), and 2 μm ( h ).

Article Snippet: At day two post MyoD overexpression, the wild-type C2C12 cells were immunostained with mouse anti-VASP (1:100, Santa Cruz; sc-46668) and Star Red-conjugated Phalloidin (1:200; Abberior; STRED-0100) overnight at 4 °C.

Techniques: Labeling, Cell Culture, Imaging, Staining, Over Expression, Membrane, Two Tailed Test

Journal: bioRxiv

Article Title: Spatiotemporal coordination of actin regulators generates invasive protrusions in cell-cell fusion

doi: 10.1101/2024.10.25.620345

Figure Lengend Snippet: (a) The sarcomeric (α-), but not the cytosolic (β- and γ-), actin level increases during C2C12 cell differentiation. Wild-type C2C12 cells were grown to 100% confluency in GM and then switched to DM. The cells were collected at the indicated time points during differentiation and subjected to western blot (WB) using antibodies specific for α-, β- and γ-actin, respectively. Note that α-actin expression significantly increased during differentiation, whereas the level of β- and γ-actin remained largely unchanged. (b) Quantification of protein expression shown in ( a ). The band intensity of each protein at each time point was normalized against β-tubulin. For each protein, the y axis indicates the expression relative to that in GM at the indicated time points. n = 3 independent experiments were performed. Statistics source data can be found in “ Statistics Source Data-Extended Data Fig.2b ”. (c , d) The invasive structure at fusogenic synapse contains all α-, β-, and γ-actin isoforms. Wild-type C2C12 cells were seeded at 60% confluence in GM. After 24 hours, the cells were infected by either empty retroviral vector (EV) or retrovirus containing MyoD. 48 hours post infection, the cells were fixed and subjected to immunostaining with anti-α-, β-, and γ-actin antibodies. The cell boundaries are delineated by dotted lines. Note that the fusogenic synapse (fs; arrowheads in c ) contained all three actin isoforms with cytosolic β- and γ-actin highly enriched, and that α-actin exhibited an overall high level in the cells ( c ). Within the fusogenic synapse, all three actin isoforms were enriched in the invasive protrusions (arrowheads in d ). n = 3 independent experiments were performed for each panel with similar results. (e) WB for α-, β-, and γ-actin in the doxycycline-induced conditional KD cells. Control (-Dox) and KD (+Dox) cells as described in Fig. 2a ,b were harvested at the indicated time points to examine the α-, β-, and γ-actin levels by WB. (f) Quantification of protein expression as shown in ( e ). The band intensity of each protein at each time point was normalized against β-tubulin. For each protein, the y axis indicates its expression in KD relative to control cells. n = 3 independent experiments were performed. Statistics source data can be found in “ Statistics Source Data-Extended Data Fig.2f ”. (g) WB for α-, β-, and γ-actin in the N-WASP -/- , WIP2 -/- , WAVE2 -/- KO, and Arp2 KD MyoD OE cells. The control (expressing a non-targeting sgRNA (sgNT) or a non-targeting shRNA (shNT), respectively), N-WASP -/- , WIP2 -/- , WAVE2 -/- KO, and Arp2 KD cells as described in were harvested at 48 hours post MyoD overexpression to examine the protein levels of α-, β- and γ-actin by WB. (h) The quantification of protein expression shown in ( g ). The band intensity of each protein was normalized against β-tubulin. For each protein, the y axis indicates the expression of each group relative to the control cells. n = 3 independent experiments were performed. Statistics source data can be found in “ Statistics Source Data-Extended Data Fig.2h ”. Mean ± s.d. values are shown in the line graph ( b , f ), and significance was determined by two-tailed student’s t-test. Three independent experiments were performed. *: p < 0.05, **: p < 0.01, ns: not significant. ( c ) Max z-projection of 8-10 focal planes from the ventral plasma membrane (z-step size: 500nm). ( d ) Single plane confocal images. Scale bars: 30 μm ( c ) and 3 μm ( d ).

Article Snippet: At day two post MyoD overexpression, the wild-type C2C12 cells were immunostained with mouse anti-VASP (1:100, Santa Cruz; sc-46668) and Star Red-conjugated Phalloidin (1:200; Abberior; STRED-0100) overnight at 4 °C.

Techniques: Cell Differentiation, Western Blot, Expressing, Infection, Retroviral, Plasmid Preparation, Immunostaining, Control, shRNA, Over Expression, Two Tailed Test, Membrane

Journal: bioRxiv

Article Title: Spatiotemporal coordination of actin regulators generates invasive protrusions in cell-cell fusion

doi: 10.1101/2024.10.25.620345

Figure Lengend Snippet: a , Schematic of conditional knockdown (cKD) of genes during myoblast fusion. b , Western blots showing the cKD of branched actin polymerization regulators during myoblast fusion. Control (-Dox) and KD (+Dox) cells as described in ( a ) were harvested at indicated time points to examine the protein levels of NPFs, the Arp2/3 complex, and muscle differentiation markers – MyoG and MHC. Quantification of MyoG and MHC protein levels at each indicated time point is shown in Extended Data Fig. 3m ( Statistics Source Data-Extended Data Fig. 3m). n = 3 independent experiments. c - d , Immunostaining with anti-MHC in the control and cKD cells described in ( b ) at day five of differentiation. Quantification of fusion index is shown in ( d ) ( Statistics Source Data-Fig.2d ). e , Western blots showing that depletion of N-WASP, WAVE2, or Arp2, but not WIP2, led to a decrease in the expression of other members of the respective complexes. Note that in WIP2 -/- cells, N-WASP expression remained unchanged. In rescued cells, expression of other members of the protein complex recovered to normal level. Quantification of MyoG and MHC protein levels is shown in Extended Data Fig. 3n (Statistics Source Data-Extended Data Fig. 3n ) . n = 3 independent experiments. EV: empty vector, sgNT: non-targeting sgRNA, shNT: non-targeting shRNA. f - g , Immunostaining with anti-MHC in the cells described in ( e ) at 72 hours post MyoD overexpression. Quantification of the fusion indexes is shown in ( g ) ( Statistics Source Data-Fig.2g ). h - i , Phalloidin staining of control or NPF KO cells describe in ( e ) at two days post MymK and MymX co-overexpression in GM. Quantification of fusion index is shown in ( i) ( Statistics Source Data-Fig.2i ). ( c , f , h ) Epifluorescence images. Mean ± s.d. values are shown in the bar-dot plot ( d , g , i ), and significance was determined by two-tailed student’s t-test. n = 3 independent experiments were performed. Scale bars: 100 μm ( c ) and 200 μm ( f , h ).

Article Snippet: At day two post MyoD overexpression, the wild-type C2C12 cells were immunostained with mouse anti-VASP (1:100, Santa Cruz; sc-46668) and Star Red-conjugated Phalloidin (1:200; Abberior; STRED-0100) overnight at 4 °C.

Techniques: Knockdown, Western Blot, Control, Immunostaining, Expressing, Plasmid Preparation, shRNA, Over Expression, Staining, Two Tailed Test

Journal: bioRxiv

Article Title: Spatiotemporal coordination of actin regulators generates invasive protrusions in cell-cell fusion

doi: 10.1101/2024.10.25.620345

Figure Lengend Snippet: a , Still images of live imaging of stable C2C12 cell lines co-expressing LifeAct-mScar and mNeongreen (mNG)-N-WASP (or mNG-WIP2, mNG-WAVE2, or Arp2-mNG) after 48 hours of MyoD overexpression. Cell boundaries are delineated by dotted lines in the merged panels ( Supplementary Video 8 ). n ≥ 15 fusogenic synapses (indicated by arrowheads) were imaged for each protein with similar results. b - c , Representative images of confocal microscopy ( b ) or structured illumination microscopy (SIM) ( c ) of C2C12 cell lines expressing 3×Flag-WIP2 or 3×Flag-N-WASP and immunostained with anti-WAVE2, anti-Flag, and phalloidin after 48 hours of MyoD overexpression. Single-channel images in ( b ) are shown in Extended Data Fig. 4f. WAVE2 enrichment are indicated by green arrows. N-WASP and WIP2 enrichment are indicated by arrowheads. n ≥ 20 cells were imaged for each panel with similar results. d - e , Immunostaining with anti-WAVE2, anti-VASP, and phalloidin in mNG-N-WASP-expressing C2C12 cells after 48 hours of MyoD overexpression. Note that the VASP - protrusions had enriched N-WASP in the shafts (arrowheads), and enriched WAVE2 at the leading edge of the invasive structure and at the tips of protrusions (arrows) ( d ), whereas the VASP tip filopodia (VASP enrichments indicated by hollow arrowheads) did not contain N-WASP in the shafts, but had N-WASP and WAVE2 enriched at the tips ( e ). n = 25 protrusions of each type were imaged with similar results. f - g , Representative images of stimulated emission depletion (STED) microscopy of C2C12 cells immunostained with anti-VASP and phalloidin at 48 hours post MyoD overexpression. Quantification of the protrusion diameters at the midpoint (indicated by brackets in ( f ) is shown in ( g ). Note that the VASP - invasive protrusions and VASP - free protrusions had similar diameters, which were significantly wider than that of the VASP tip free protrusions. Mean ± s.d. values are shown in the dot plot, and significance was determined by two-tailed student’s t-test. n = 23, 29 and 30 (up to down) protrusions for each type of protrusions were quantified ( Statistics Source Data-Fig.3g ). ( a ) Max z-projection of 8-10 focal planes from the ventral plasma membrane (z-step size: 500 nm) (single focal plane images are shown in Extended Data Fig. 4d). ( b - f ) Single plane confocal images. Scale bars: 5 μm ( a ), 2 μm ( b - e ) and 500 nm ( f ).

Article Snippet: At day two post MyoD overexpression, the wild-type C2C12 cells were immunostained with mouse anti-VASP (1:100, Santa Cruz; sc-46668) and Star Red-conjugated Phalloidin (1:200; Abberior; STRED-0100) overnight at 4 °C.

Techniques: Imaging, Expressing, Over Expression, Confocal Microscopy, Microscopy, Immunostaining, Two Tailed Test, Membrane

Journal: bioRxiv

Article Title: Spatiotemporal coordination of actin regulators generates invasive protrusions in cell-cell fusion

doi: 10.1101/2024.10.25.620345

Figure Lengend Snippet: (a,b) mNG-tagged N-WASP, WIP2, WAVE2, and Arp2 rescue the fusion defects in N-WASP-, WIP2-, WAVE2- and Arp2-KD cells, respectively (see Extended Data Fig. 3g,h for the KD efficiency and effects). The KD cells were infected by retrovirus containing the corresponding KD gene with an mNG tag. After two days, the cells were plated and allowed to grow in GM into 100% confluence, before switching into DM. At day five in DM, the cells were collected for WB ( a ) and immunostaining ( b ). Note the expression of mNG-tagged proteins shown in ( a ) and the rescue of fusion shown in ( b ). (c) Quantification of the cell fusion index of each genotype shown in ( b ). n = 3 independent experiments were performed. Mean ± s.d. values are shown in the bar-dot plot, and significance was determined by two-tailed student’s t-test. *: p < 0.05, **: p < 0.01, ns: not significant. Statistics source data can be found in “ Statistics Source Data-Extended Data Fig.4c ”. (d) Single focal plane stills from a time-lapse movie of the fusion events shown in . Note that the F-actin enrichments (arrowheads) at the fusogenic synapse are similar to those in (see Supplemental Video 8 ). (e) Testing the specificity of the WAVE2 antibody used in this study. The WAVE2 KD C2C12 cells as shown in Extended Data Fig. 3g were mixed with wild-type C2C12 cells and seeded at 30% confluence in GM. After 24 hours, the cells were immunostained with anti-WAVE2 and phalloidin. The WAVE2 KD cells were marked by mCherry expression and showed little anti-WAVE2 staining (the nuclear signal was non-specific). n = 3 experiments were performed with similar results. (f) Single-channel images for the merged images shown in Fig. 3b ,c . (g,h) Stills images from a time-lapse movie showing that N-WASP ( f ) and WIP2 ( g ) colocalized with F-actin along the shafts of straight protrusions. C2C12 cells co-expressing LifeAct-mScar and mNG-N-WASP (or mNG-WIP2) were infected with retrovirus containing MyoD. At 48 hours post MyoD overexpression, the cells were subjected to live imaging (see Supplemental Video 9 and 10 ). Single plane confocal images with F-actin enrichment are shown here. Arrowheads point to randomly selected protrusions. n = 3 experiments were performed with similar results. ( b ) Epifluorescence images. ( d,f,g,h ) Single plane confocal images. ( e ) Max z-projection of 8-10 focal planes from the ventral plasma membrane (z-step size: 500nm). Scale bars: 200 μm ( b ), 30 μm ( e ), 2 μm ( f ) and 5 μm ( d,g,h) .

Article Snippet: At day two post MyoD overexpression, the wild-type C2C12 cells were immunostained with mouse anti-VASP (1:100, Santa Cruz; sc-46668) and Star Red-conjugated Phalloidin (1:200; Abberior; STRED-0100) overnight at 4 °C.

Techniques: Infection, Immunostaining, Expressing, Two Tailed Test, Staining, Over Expression, Imaging, Membrane

Journal: bioRxiv

Article Title: Spatiotemporal coordination of actin regulators generates invasive protrusions in cell-cell fusion

doi: 10.1101/2024.10.25.620345

Figure Lengend Snippet: (a) Immunostaining with anti-VASP in the C2C12 cells expressing mNG-WIP2 at day two of MyoD overexpression. The cortical area of a randomly picked cell is shown. (b) Enlarged view of yellow-boxed area in (a) . Note that mNG-WIP2 was enriched in the shaft of the VASP - protrusion (invasive). (c) Enlarged view of red-boxed area in (a) . Note that mNG-WIP2 was not enriched in the shaft of VASP tip filopodia (non-invasive). (d) WIP2 is enriched in the shafts of VASP - , but not VASP tip , protrusions. The WIP2 enrichment was analyzed in the VASP - and VASP tip protrusions in mNG-WIP2-expressing cells at day two of MyoD overexpression. Note that the cells projected two major types of protrusions, ∼52% WIP2 shaft+ VASP - and ∼44% WIP2 shaft– VASP tip protrusions. The rest of the protrusions (4%) were short and WIP2 shaft– VASP - . n = 301 protrusions from ten randomly selected cells were analyzed. Mean ± s.d. values are shown in the dot plot. Statistics source data can be found in “ Statistics Source Data-Extended Data Fig.5d ”. ( e-g ) Mena and EVL are not enriched in the invasive protrusions. The mNG-WIP2-expressing cells was immunostained with anti-Mena or anti-EVL, together with anti-VASP and phalloidin at day two of MyoD overexpression. Randomly selected WIP2 shaft+ VASP - invasive protrusions (inv. pro.) and WIP2 shaft– VASP tip filopodia are shown for Mena ( e ) and EVL ( f ) labeling. Quantification of the percentage of Mena + and EVL + protrusions is shown in ( g ). Note that neither Mena or EVL was localized in the invasive protrusions (0 out of n ≥ 90 WIP2 shaft+ VASP - protrusions). EVL was co-enriched with VASP at the tips of ∼21% filopodia (41 out of n = 199 WIP2 shaft– VASP tip filopodia), and Mena was rarely observed at the tip of any filopodia (1 out of n = 186 WIP2 shaft– VASP tip filopodia). Mean ± s.d. values are shown in the dot plot ( g ). Statistics source data for g can be found in “ Statistics Source Data-Extended Data Fig.5g ”. (h) Ena/VASP family members are not enriched in the invasive protrusions at the fusogenic synaspe. After day two of MyoD overexpression, wild-type C2C12 cells were immunostained with anti-VASP, anti-Mena, anti-EVL, and phalloidin. Note that none of VASP, Mena, or EVL was enriched at the tips of the invasive protrusions at the fusogenic synapse (0 out of n ≥ 10 fusogenic synapse for each protein). Cell boundaries are delineated by dotted lines. ( a-c,e-g,i ) Single plane confocal images. Scale bars: 2 μm ( a,b,c ), 3 μm ( e ), 1 μm ( f,g ) and 10 μm ( i ).

Article Snippet: At day two post MyoD overexpression, the wild-type C2C12 cells were immunostained with mouse anti-VASP (1:100, Santa Cruz; sc-46668) and Star Red-conjugated Phalloidin (1:200; Abberior; STRED-0100) overnight at 4 °C.

Techniques: Immunostaining, Expressing, Over Expression, Labeling

Journal: bioRxiv

Article Title: Spatiotemporal coordination of actin regulators generates invasive protrusions in cell-cell fusion

doi: 10.1101/2024.10.25.620345

Figure Lengend Snippet: a , Still images of cortical protrusions of wild-type (Ctrl) ( a1 ), NPF KOs ( a2 - a4 ), and Arp2 KD ( a5 ) C2C12 cells at 48 hours post MyoD overexpression ( Supplementary Video 11 ). Randomly selected cortical regions containing finger-like protrusions (arrowheads) and lamellipodia (asterisks) are shown. Note the straight vs. bendy finger-like protrusions projected from the leading edge of the lamellipodia in Ctrl vs. N-WASP -/- cells; the loss of lamellipodia and the decreased number of protrusions in WAVE2 -/- cell; the bendy protrusions in WIP2 -/- cell; and no protrusions in Arp2 KD cells. b , Quantification of the number of newly formed finger-like protrusions in cells with the genotypes shown in ( a ). n = 86, 82, 90, 79 and 67 (left to right) cells from three independent experiments were measured for each genotype ( Statistics Source Data-Fig.4b ). c - g , Localization of N-WASP, WIP2, WAVE2, and VASP in individual protrusions of Ctrl and mutant MyoD OE cells. The mNG-N-WIP2-expressing Ctrl ( c ), mNG-N-WASP-expressing Ctrl ( d ), mNG-N-WASP-expressing WAVE2 -/- ( e ), N-WASP -/- ( f ), and mNG-N-WASP-expressing WIP2 -/- ( g ) cells were immunostained with anti-WAVE2, anti-VASP, and phalloidin at 48 hours post MyoD overexpression. h , Quantification of the percentage of VASP - protrusions in cells with the genotypes shown in ( c - g ). The Ctrl, WAVE2 -/- , N-WASP -/- , and WIP2 -/- cells were immunostained with anti-VASP and phalloidin at 48 hours post MyoD overexpression. n = 60 cells from three independent experiments were quantified for each genotype ( Statistics Source Data-Fig.4h ). ( a ) Max z-projection of 8-10 focal planes from the ventral plasma membrane (z-step size: 500 nm). ( c , g ) Single plane confocal images. Mean ± s.d. values are shown in the dot plot ( b , h ), and significance was determined by two-tailed student’s t-test. Scale bars: 10 μm ( a ), 3 μm ( e , f , g ), 2 μm ( c ) and 1.5 μm ( d ).

Article Snippet: At day two post MyoD overexpression, the wild-type C2C12 cells were immunostained with mouse anti-VASP (1:100, Santa Cruz; sc-46668) and Star Red-conjugated Phalloidin (1:200; Abberior; STRED-0100) overnight at 4 °C.

Techniques: Over Expression, Mutagenesis, Expressing, Membrane, Two Tailed Test

Journal: bioRxiv

Article Title: Spatiotemporal coordination of actin regulators generates invasive protrusions in cell-cell fusion

doi: 10.1101/2024.10.25.620345

Figure Lengend Snippet: a , Low-speed co-sedimentation assay for F-actin and mNG-WIP2-N. b , TIRF images of WIP2- and Shi ( Drosophila dynamin)-mediated actin bundling ( Supplementary Video 12 ). c , Still images of time-lapse TIRF imaging showing three examples of mNG-WIP2-mediated actin filament bundling ( Supplementary Video 13 ). The merging actin filaments are indicated by magenta or green dots. n = 3 independent imaging experiments were performed with similar results. d , SIM images of actin bundles assembled by Alexa Fluor 568-phalloidin (red) and mNG-WIP2-N (green). e , Electron micrographs of negatively stained actin filaments alone (top panel), WIP2-actin bundles (middle panels), and dynamin-actin bundles (bottom two panels). Three WIP2-actin bundles with different diameters are shown. A dynamin-actin single bundle and a dynamin-actin super bundle consisting of four parallel single bundles (each marked by an asterisk) are shown. A few mNG-WIP2-N patches are indicated by hollow arrowheads, and a few dynamin helical rungs are indicated by white arrowheads. f , Immunostaining with anti-VASP and phalloidin in wild-type cells (Ctrl), and WIP2 -/- cells expressing mNG-full-length (FL) WIP2, mNG-WIP2 ΔWH2-1 , mNG-WIP2 ΔWH2-2 , mNG-WIP2 ΔWH2-1&2 , or mNG after 48 hours of MyoD overexpression. The VASP - and VASP tip protrusions are indicated by arrowheads and arrows, respectively. Zoomed-out views of the cells are shown in Extended Data Fig. 6c. g , Phalloidin staining in cells with the genotypes shown in ( f ) at 72 hours post MyoD overexpression. h , Quantification of the percentage of VASP - protrusions for each genotype shown in ( f ). n = 60 cells for each genotype from three independent experiments were analyzed. i , Quantification of the bendiness of the protrusions in each genotype shown in ( f ). n = 55 protrusions from three independent experiments for each genotype were analyzed. j , Quantification of fusion index of cells in ( g ). n = 3 independent experiments were performed. ( f ) Single plane confocal images. ( g ) Epifluorescence images. In ( a , b , c , d , e ), n = 3 independent experiments were performed with similar results. Mean ± s.d. values are shown in the dot plot ( h , i ) and bar-dot plot ( j ), and significance was determined by two-tailed student’s t-test ( Statistics Source Data-Fig.5h-j ). Scale bars: 10 μm ( b ), 2 μm ( c ), 1 μm ( d ), 100 nm ( e ), 5 μm ( f ) and 50 μm ( g ).

Article Snippet: At day two post MyoD overexpression, the wild-type C2C12 cells were immunostained with mouse anti-VASP (1:100, Santa Cruz; sc-46668) and Star Red-conjugated Phalloidin (1:200; Abberior; STRED-0100) overnight at 4 °C.

Techniques: Sedimentation, Imaging, Staining, Immunostaining, Expressing, Over Expression, Two Tailed Test

Journal: bioRxiv

Article Title: Spatiotemporal coordination of actin regulators generates invasive protrusions in cell-cell fusion

doi: 10.1101/2024.10.25.620345

Figure Lengend Snippet: a-c , Dynamic enrichment of Arp2 and Dyn2 with newly polymerized branch actin filaments in C2C12 cells at 48 hours post MyoD overexpression ( Supplementary Video 14-16 ). A schematic diagram is shown at the bottom of each panel of still images. n = 3 independent experiments were performed with similar results. Actin “clouds” are outlined by dotted lines in the merged channel in ( a , b ). In ( a ), Arp2-mScar enrichment, actin clouds, actin bundles are indicated by red arrowheads, green arrowheads and green arrows, respectively. In ( b ), at 2 and 3 min, actin clouds and mScar-Dyn2 enrichment are indicated by green and red arrowheads, respectively. At 4 and 5 min, mScar-Dyn2 enrichment is indicated by hollow arrowheads. In ( c ), mScar-Dyn2 Arp2-mNG enrichment are indicated by red and green arrowheads, respectively. d , Still images of cortical protrusions of LifeAct-mNG-expressing C2C12 cells treated without (top) or with (bottom) 5 μM MiTMAB, at 48 hours post MyoD overexpression ( Supplementary Video 17 ). n = 3 independent experiments were performed with similar results. e , Localization of Dyn2 in the persistent actin clouds of MiTMAB-treated, LifeAct-mNG and mScar-Dyn2 co-expressing C2C12 cells at 48 hours post MyoD overexpression ( Supplementary Video 18 ). mScar-Dyn2 (red arrowheads; 6-12 min) was observed highly enriched in the persistent actin cloud (green arrowheads). n = 3 independent experiments were performed with similar results. f , MHC immunostaining of Ctrl and dynamin1/2/3 tKO MEFs expressing wild-type Shi or Shi 4RD at five days post MyoD overexpression. g , Quantification of fusion index of the cells shown in ( f ). n = 3 independent experiments were performed ( Statistics Source Data-Fig.6g ). h , Immunostaining with anti-VASP and phalloidin in the MEFs described in ( f ). The VASP - and VASP tip protrusions are indicated by red arrowheads and white arrows, respectively. i , Quantification of the percentage of VASP - protrusions shown in ( h ). n = 60 cells per genotype from three independent experiments were quantified ( Statistics Source Data-Fig.6i ). ( a-c , e , h ) Single plane confocal images. ( d ) Max z-projection of 8-10 focal planes from the ventral plasma membrane (z-step size: 500 nm). ( f ) Epifluorescence images. Mean ± s.d. values are shown in the dot plot ( j ) and bar-dot plot ( h ), and significance was determined by two-tailed student’s t-test. Sale bars: 3 μm ( a-c ), 2 μm ( d , e ), 5 μm ( h ) and 200 μm ( f ).

Article Snippet: At day two post MyoD overexpression, the wild-type C2C12 cells were immunostained with mouse anti-VASP (1:100, Santa Cruz; sc-46668) and Star Red-conjugated Phalloidin (1:200; Abberior; STRED-0100) overnight at 4 °C.

Techniques: Over Expression, Expressing, Immunostaining, Membrane, Two Tailed Test

Journal: bioRxiv

Article Title: Spatiotemporal coordination of actin regulators generates invasive protrusions in cell-cell fusion

doi: 10.1101/2024.10.25.620345

Figure Lengend Snippet: (a-c) The Dyn2 antibody (G-4) specifically recognizes Dyn2. ( a ) Tamoxifen-inducible dynamin 1/2/3 triple KO (tKO) MEFs were cultured with or without 2 μM 4-hydroxytamoxifen (4OH-TM) in the GM for 72 hours, and subsequently fixed for immunostaining with anti-Dyn2. ( b ) C2C12 cells were infected by lentivirus containing shRNAs against Dyn2. After 48 hours, the cells underwent puromycin selection for five days, and the viable cells were fixed for immunostaining ( b ) or harvested for WB ( c ) using anti-Dyn2. Note that anti-Dyn2 did not detect any signal in tKO cells and only greatly reduced signals in dyn2 KD cells, indicating its specificity. n = 3 independent experiments were performed with similar results. (d) Dyn2 and Arp2 are largely co-localized in the actin clouds. Arp2-mNG-expressing C2C12 cells were immunostained with anti-Dyn2 and phalloidin at day two of MyoD overexpression. Note the co-localization of Arp2 and Dyn2 within the actin clouds (arrowheads) at the periphery of actin bundles (arrows). n = 3 independent experiments were performed with similar results. (e) MiTMAB inhibits the disassembly of dynamin helices in the presence of GTP. F-actin pre-assembled from 1 μM G-actin was incubated with or without 1 μM dynamin (Shi) for 30 minutes at RT. Subsequently, 2mM GTP with or without 40 μM MiTMAB was added to the appropriate reaction mixtures and incubated for five minutes. The samples were then mixed with Alexa Fluor 488-conjugated phalloidin and subjected to confocal imaging on glass coverslips. Note that the dynamin-induced actin bundles were disassembled by GTP addition (top right panel). MiTMAB did not bundle actin by itself (bottom left panel) or inhibit dynamin-induced actin bundling (bottom middle panel), but it inhibited dynamin helix disassembly in the presence of GTP, thus leaving the actin bundles intact (bottom right panel). n = 3 independent experiments were performed with similar results. (f) Specificity test of the HA antibody used in the immunogold labeling experiments. Boxed area is enlarged on the right. Dyn2 KD C2C12 cells at day two of MyoD overexpression were stained with anti-HA for immunogold labeling, followed by PREM analysis. Note the absence of gold particles in these cells, demonstrating the specificity of the primary anti-HA antibody and the secondary antibody. n = 13 protrusions were imaged with similar results. (g) An additional example showing that WIP2 stabilizes actin bundles after dynamin helix disassembly. As described in , dynamin-actin bundles were incubated with 1mM GTP without (top panel) or with (bottom panel) mNG-WIP2-N for 30 min, and subjected to negative-stain EM. Note that GTP addition resulted in partial disassembly of the dynamin helix. Residual rungs (several indicated by arrowheads) of the dynamin helix are underlined by green dashed lines. The loosened actin bundle is indicated by a bracket (top panel), and the mNG-WIP2-N patches (light-colored) on the tightened bundle (red dashed lines) are indicated by arrows (bottom panel). ( a,b ) Epifluorescence images. ( d,e ) Single plane confocal images. Scale bars: 50 μm ( a,b,e ), 4 μm ( d ), 100 nm ( f ) and 300 nm ( g ).

Article Snippet: At day two post MyoD overexpression, the wild-type C2C12 cells were immunostained with mouse anti-VASP (1:100, Santa Cruz; sc-46668) and Star Red-conjugated Phalloidin (1:200; Abberior; STRED-0100) overnight at 4 °C.

Techniques: Cell Culture, Immunostaining, Infection, Selection, Expressing, Over Expression, Incubation, Imaging, Labeling, Staining