Devitt CC et al. (2021), Twinfilin1 controls lamellipodial protrusive ac...

XB-ART-58363

J Cell Sci 2021 Jul 15;13414:. doi: 10.1242/jcs.254011.

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Twinfilin1 controls lamellipodial protrusive activity and actin turnover during vertebrate gastrulation.

Devitt CC , Cox RM , Papoulas O , Alvarado J , Shekhar S , Marcotte EM , Wallingford JB .

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The dynamic control of the actin cytoskeleton is a key aspect of essentially all animal cell movements. Experiments in single migrating cells and in vitro systems have provided an exceptionally deep understanding of actin dynamics. However, we still know relatively little of how these systems are tuned in cell-type-specific ways, for example in the context of collective cell movements that sculpt the early embryo. Here, we provide an analysis of the actin-severing and depolymerization machinery during vertebrate gastrulation, with a focus on Twinfilin1 (Twf1) in Xenopus. We find that Twf1 is essential for convergent extension, and loss of Twf1 results in a disruption of lamellipodial dynamics and polarity. Moreover, Twf1 loss results in a failure to assemble polarized cytoplasmic actin cables, which are essential for convergent extension. These data provide an in vivo complement to our more-extensive understanding of Twf1 action in vitro and provide new links between the core machinery of actin regulation and the specialized cell behaviors of embryonic morphogenesis.

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Species referenced: Xenopus laevis
Genes referenced: cap1 cap2 cfl2 myh9 twf1
GO keywords: gastrulation [+]

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	Figure 1. Lamellipodia in the Xenopus DMZ. (A) Schematic showing CE cell movements and DMZ explant method. Cells are unpolarized at the start of gastrulation and over time take on a bipolar polarity and intercalate to form a longer, narrower array. (B) Image showing elongated DMZ explants, close-up is shown at right. Scale bars: 1 mm. (C) Still from time-lapse confocal microscopy showing a single round of lamellipodial extension and retraction from the mediolateral end of a single DMZ cell. (D) DMSO does not affect DMZ lamellipodia. (E) Typical effect of CK-666 on DMZ lamellipodia. (F) Quantification of effects of CK-666 on protrusion frequency. n=9 DMSO protrusions from four embryos; n=7 CK666 protrusions from three embryos. *P<0.001 for DMSO vs CK-666 (Mann–Whitney U-test). (G) Quantification of effects of CK-666 on protrusion size. n=43 DMSO-treated protrusions from four embryos, n=49 CK-666-treated protrusions from three embryos. *P<0.001 for DMSO vs CK-666 (Mann–Whitney U-test). Graphs in F, G are violin plots with the median being highlighted by a dashed line and quartiles with dotted lines. Scale bars: 10 μm.
	Figure 2. Localization of lamellipodial markers. (A) Interaction partners of Cfl2 were identified based on their enrichment in APMS of the Cfl2–GFP-tagged bait protein (vertical axes) relative to APMS of the untagged GFP controls (horizontal axes). Confidence values were calculated by one-sided Z-test (see Materials and Methods). A pseudocount of 1 PSM was added to each protein for visualization on a log–log plot. (B) Schematic showing the quantification scheme used here. b″, red dotted line indicates line-plot measurement taken along protrusion length; b‴, trace of actin intensity along protrusion length. Red line highlights one example trace, black dots are representative of several line plots. a.u., arbitrary units. (C,c′,c″) Pip3 localization and quantification; LifeAct in the alternate channel reports actin localization. (D,d′,d″) Myh9 localization and quantification. (E,e′,e″) Localization and quantification of Cfl2; LifeAct in the alternate channel reports actin localization. (F,f′,f″) Localization and quantification of Twf1. (G,g′,g″) Localization and quantification of Cap2. Dashed lines highlight cell edges. au, arbitrary units. Images shown are representative of at least three experiments. Scale bars: 10 μm.
	Figure 3. Twf1 is required for axis elongation and convergent extension. (A) Quantification of axis elongation (as length/width ratio) for control, Twf1 morphants, and Twf1 mRNA rescued embryos, along with sgRNA control and Twf1 crispants (inset images show representative examples; see more embryos in Fig. S3C), n=60 control embryos, n=88 morphant embryos, n=43 rescue embryos, n=155 sgRNA control, n=119 Twf1 Crispr. *P<0.001; P<0.0001; n.s., not significant (Kruskal–Wallis test). Scale bars: 1 mm. (B) Quantification of explant elongation (as length/width ratio) for isolated DMZs from control and Twf1 morphant embryos. n=28, control; n=46, 10 ng; n=41, 20 ng; n=17, 40 ng. P<0.0001 for control vs 10 ng, 20 ng, 40 ng, n.s., not significant (P=0.0966) for 20 ng versus 40 ns (one-way Kruskal–Wallis test). (C) Quantification of cell shapes for control versus Twf1 morphant, and sgRNA control and Twf1 crispant DMZs. n=168 control cells from six embryos, n=156 Twf1 morphant cells from six embryos, n=169 sgRNA control cells from 11 embryos, n=294 Twf1 Crispr cells from 14 embryos, three experiments (representative images in Fig. S3C). **P<0.0001 (Mann–Whitney U-test). Graphs in A–C are violin plots with the median being highlighted by a dashed line and quartiles with dotted lines. (D,E) Representative image (D) and rose diagram (E) showing quantification of cell axis polarity in control DMZ. Scale bar: 50 μm. (F,G) Representative image (F) and rose diagram (G) showing disrupted cell axis polarity in Twf1 morphant. Scale bar: 50 μm. E,G shows control vs Twf1 morphant, n=387 control cells from nine embryos, n=522 Twf1 morphant cells from 15 embryos. P<0.0001 (Kolmogorov–Smirnov test). (H) Confocal image of mosaically labeled cell in control DMZ. Scale bar: 10 μm. (I) Rose diagram showing the normal mediolateral polarization of lamellipodia in the control DMZ. (J) Confocal image of mosaically labeled cell in Twf1 morphant DMZ. Scale bar: 10 μm. (K) Rose diagram showing the disrupted mediolateral polarization of lamellipodia in the morphant. I,K show control vs Twf1 morphant, n=119 control protrusions from five embryos, n=194 Twf1 morphant protrusions from nine embryos. P<0.0001 (Kolmogorov–Smirnov test).
	Figure 4. Twinfilin is required for actin node and cable formation. (A,B) TIRF images of actin cables in control and Twf1 morphant DMZ cells. In right-hand panels, color reflects orientation of cables as indicated by the legend in C. (C) Quantification of actin cable organization in control and Twf1 morphants. n=6 control embryos, n=11 Twf1 morphant embryos. P<0.0001 for control vs Twf1 morphant (Kolmogorov–Smirnov test). Scale bars: 10 μm.
	Figure 5. Twf1 controls lamellipodial dynamics in the Xenopus DMZ. (A,B) Confocal images of single cells in a LifeAct–GFP-expressing control and Twf1 morphant DMZ. Scale bars: 10 μm. (C) Quantification of lamellipodial protrusion frequency for control and Twf1 morphants. Control versus Twf1 morphant, n=33 control protrusions from two embryos; DMSO, n=44 Twf1 morphant protrusions from four embryos. **P<0.0001 (Mann–Whitney U-test). (D) Quantification of lamellipodial extension rate. n=17 control protrusions from four embryos; n=23 Twf1 morphant protrusions from five embryos. n.s., not significant for control vs Twf1 morphant (P=0.516; Mann–Whitney U-test). (E) Quantification of lamellipodial extension and retraction lifetimes. n=42 control protrusions from four embryos; n=22 Twf1 morphant protrusions from six embryos. P<0.01 for control vs Twf1 morphant, extension lifetime; n.s., not significant, for control vs Twf1 morphant retraction lifetime (Mann–Whitney U-test). (F) Quantification of lamellipodial area for control and Twf1 morphants. n=144 control protrusions from three embryos, n=127 Twf1 morphant protrusions from six embryos. ****P<0.0001 for control vs Twf1 morphant (Mann–Whitney U-test). Graphs in C–F are violin plots with the median being highlighted by a dashed line and quartile with dotted lines.
	Figure 6. Twinfilin regulates actin turnover in protrusions during CE. (A) Schematic of fluorescent speckle imaging. (B,C) Kymograph from TIRF movies showing fluorescent actin speckles in lamellipodia in control and Twf1 morphant Xenopus DMZs. Scale (x, distance): 5 μm, scale (y, time): 50 s. (D) Quantification of actin speckle lifetimes. n=15 control protrusions from three embryos; n=15 Twf1 morphant protrusions from six embryos. P<0.01 for control vs Twf1 morphant (Mann–Whitney U-test). Graph is violin plots with the median being highlighted by a dashed line and quartiles with dotted lines. (E) Cap localization in control protrusion; LifeAct in the alternate channel reports actin localization. Red line indicates line-plot measurement taken along protrusion length. (F) Quantification of distally restricted Cap localization in control protrusion. Red line highlighting one example trace, black dots representative of several line plots. (G) Cap localization in Twf1 morphant protrusion. (H) Quantification showing expansion of distal bias of Cap localization in Twf1 morphant cells. Scale bars: 10 μm, unless otherwise noted. (G,H) Cap1 distribution in Control vs Twf1 morphant was significantly different. n=36 protrusions from eight control embryos; n=28 cells from nine Twf1 morphant embryos. **P<0.0001 (Kolmogorov–Smirnov test). a.u., arbitrary units.

References [+] :

Bibeau, Clusters of a Few Bound Cofilins Sever Actin Filaments. 2021, Pubmed

	Figure 1. Lamellipodia in the Xenopus DMZ. (A) Schematic showing CE cell movements and DMZ explant method. Cells are unpolarized at the start of gastrulation and over time take on a bipolar polarity and intercalate to form a longer, narrower array. (B) Image showing elongated DMZ explants, close-up is shown at right. Scale bars: 1 mm. (C) Still from time-lapse confocal microscopy showing a single round of lamellipodial extension and retraction from the mediolateral end of a single DMZ cell. (D) DMSO does not affect DMZ lamellipodia. (E) Typical effect of CK-666 on DMZ lamellipodia. (F) Quantification of effects of CK-666 on protrusion frequency. n=9 DMSO protrusions from four embryos; n=7 CK666 protrusions from three embryos. *P<0.001 for DMSO vs CK-666 (Mann–Whitney U-test). (G) Quantification of effects of CK-666 on protrusion size. n=43 DMSO-treated protrusions from four embryos, n=49 CK-666-treated protrusions from three embryos. *P<0.001 for DMSO vs CK-666 (Mann–Whitney U-test). Graphs in F, G are violin plots with the median being highlighted by a dashed line and quartiles with dotted lines. Scale bars: 10 μm.
	Figure 2. Localization of lamellipodial markers. (A) Interaction partners of Cfl2 were identified based on their enrichment in APMS of the Cfl2–GFP-tagged bait protein (vertical axes) relative to APMS of the untagged GFP controls (horizontal axes). Confidence values were calculated by one-sided Z-test (see Materials and Methods). A pseudocount of 1 PSM was added to each protein for visualization on a log–log plot. (B) Schematic showing the quantification scheme used here. b″, red dotted line indicates line-plot measurement taken along protrusion length; b‴, trace of actin intensity along protrusion length. Red line highlights one example trace, black dots are representative of several line plots. a.u., arbitrary units. (C,c′,c″) Pip3 localization and quantification; LifeAct in the alternate channel reports actin localization. (D,d′,d″) Myh9 localization and quantification. (E,e′,e″) Localization and quantification of Cfl2; LifeAct in the alternate channel reports actin localization. (F,f′,f″) Localization and quantification of Twf1. (G,g′,g″) Localization and quantification of Cap2. Dashed lines highlight cell edges. au, arbitrary units. Images shown are representative of at least three experiments. Scale bars: 10 μm.
	Figure 3. Twf1 is required for axis elongation and convergent extension. (A) Quantification of axis elongation (as length/width ratio) for control, Twf1 morphants, and Twf1 mRNA rescued embryos, along with sgRNA control and Twf1 crispants (inset images show representative examples; see more embryos in Fig. S3C), n=60 control embryos, n=88 morphant embryos, n=43 rescue embryos, n=155 sgRNA control, n=119 Twf1 Crispr. *P<0.001; P<0.0001; n.s., not significant (Kruskal–Wallis test). Scale bars: 1 mm. (B) Quantification of explant elongation (as length/width ratio) for isolated DMZs from control and Twf1 morphant embryos. n=28, control; n=46, 10 ng; n=41, 20 ng; n=17, 40 ng. P<0.0001 for control vs 10 ng, 20 ng, 40 ng, n.s., not significant (P=0.0966) for 20 ng versus 40 ns (one-way Kruskal–Wallis test). (C) Quantification of cell shapes for control versus Twf1 morphant, and sgRNA control and Twf1 crispant DMZs. n=168 control cells from six embryos, n=156 Twf1 morphant cells from six embryos, n=169 sgRNA control cells from 11 embryos, n=294 Twf1 Crispr cells from 14 embryos, three experiments (representative images in Fig. S3C). **P<0.0001 (Mann–Whitney U-test). Graphs in A–C are violin plots with the median being highlighted by a dashed line and quartiles with dotted lines. (D,E) Representative image (D) and rose diagram (E) showing quantification of cell axis polarity in control DMZ. Scale bar: 50 μm. (F,G) Representative image (F) and rose diagram (G) showing disrupted cell axis polarity in Twf1 morphant. Scale bar: 50 μm. E,G shows control vs Twf1 morphant, n=387 control cells from nine embryos, n=522 Twf1 morphant cells from 15 embryos. P<0.0001 (Kolmogorov–Smirnov test). (H) Confocal image of mosaically labeled cell in control DMZ. Scale bar: 10 μm. (I) Rose diagram showing the normal mediolateral polarization of lamellipodia in the control DMZ. (J) Confocal image of mosaically labeled cell in Twf1 morphant DMZ. Scale bar: 10 μm. (K) Rose diagram showing the disrupted mediolateral polarization of lamellipodia in the morphant. I,K show control vs Twf1 morphant, n=119 control protrusions from five embryos, n=194 Twf1 morphant protrusions from nine embryos. P<0.0001 (Kolmogorov–Smirnov test).
	Figure 4. Twinfilin is required for actin node and cable formation. (A,B) TIRF images of actin cables in control and Twf1 morphant DMZ cells. In right-hand panels, color reflects orientation of cables as indicated by the legend in C. (C) Quantification of actin cable organization in control and Twf1 morphants. n=6 control embryos, n=11 Twf1 morphant embryos. P<0.0001 for control vs Twf1 morphant (Kolmogorov–Smirnov test). Scale bars: 10 μm.
	Figure 5. Twf1 controls lamellipodial dynamics in the Xenopus DMZ. (A,B) Confocal images of single cells in a LifeAct–GFP-expressing control and Twf1 morphant DMZ. Scale bars: 10 μm. (C) Quantification of lamellipodial protrusion frequency for control and Twf1 morphants. Control versus Twf1 morphant, n=33 control protrusions from two embryos; DMSO, n=44 Twf1 morphant protrusions from four embryos. **P<0.0001 (Mann–Whitney U-test). (D) Quantification of lamellipodial extension rate. n=17 control protrusions from four embryos; n=23 Twf1 morphant protrusions from five embryos. n.s., not significant for control vs Twf1 morphant (P=0.516; Mann–Whitney U-test). (E) Quantification of lamellipodial extension and retraction lifetimes. n=42 control protrusions from four embryos; n=22 Twf1 morphant protrusions from six embryos. P<0.01 for control vs Twf1 morphant, extension lifetime; n.s., not significant, for control vs Twf1 morphant retraction lifetime (Mann–Whitney U-test). (F) Quantification of lamellipodial area for control and Twf1 morphants. n=144 control protrusions from three embryos, n=127 Twf1 morphant protrusions from six embryos. ****P<0.0001 for control vs Twf1 morphant (Mann–Whitney U-test). Graphs in C–F are violin plots with the median being highlighted by a dashed line and quartile with dotted lines.
	Figure 6. Twinfilin regulates actin turnover in protrusions during CE. (A) Schematic of fluorescent speckle imaging. (B,C) Kymograph from TIRF movies showing fluorescent actin speckles in lamellipodia in control and Twf1 morphant Xenopus DMZs. Scale (x, distance): 5 μm, scale (y, time): 50 s. (D) Quantification of actin speckle lifetimes. n=15 control protrusions from three embryos; n=15 Twf1 morphant protrusions from six embryos. P<0.01 for control vs Twf1 morphant (Mann–Whitney U-test). Graph is violin plots with the median being highlighted by a dashed line and quartiles with dotted lines. (E) Cap localization in control protrusion; LifeAct in the alternate channel reports actin localization. Red line indicates line-plot measurement taken along protrusion length. (F) Quantification of distally restricted Cap localization in control protrusion. Red line highlighting one example trace, black dots representative of several line plots. (G) Cap localization in Twf1 morphant protrusion. (H) Quantification showing expansion of distal bias of Cap localization in Twf1 morphant cells. Scale bars: 10 μm, unless otherwise noted. (G,H) Cap1 distribution in Control vs Twf1 morphant was significantly different. n=36 protrusions from eight control embryos; n=28 cells from nine Twf1 morphant embryos. **P<0.0001 (Kolmogorov–Smirnov test). a.u., arbitrary units.