Fig 2: Loss of T-Plastin reduces protrusion width and length but has no effect on actin turnover.a Sparsely plated WT, KO1, and KO1 HUVEC stably expressing a CRISPR-resistant T-Plastin (KO1 + T-Plastin) stained for F-actin and Arp3. Bars. 10 µm. b Quantification of protrusion widths based on their Arp3 staining in cells as shown in (a). Black bars indicate the mean and 95% confidence intervals for WT (blue, n protrusions = 88), KO1 (red, n protrusions = 101), KO1 + T-Plastin (gray, n protrusions = 96) taken from two independent replicates. *P < 0.05, **P < 0.01, ****P < 0.0001, analyzed using a one-way ANOVA with Tukey’s multiple comparison test. c Quantification of photoactivated protrusive actin intensities in WT (blue, n cells = 14) and KO1 (red, n cells = 17) cells as shown in (d). As a control, cells were treated with a mixture of Jasplakinolide, Latrunculin-B, and Y-27632 (JLY, black, n cells = 8); taken from three independent replicates. Means are shown as solid lines, transparent regions indicate 95% confidence intervals. d Photoactivation images of WT and KO1 HUVEC expressing PAGFP-Actin (green) and F-tractin-mRuby3 (magenta). A small region of the protrusion was activated and measured (white boxes). Bar, 5 µm. e Sparsely plated WT and T-Plastin KO cell lines (KO1-3) were fixed and stained for F-actin (green) and paxillin (magenta). The areas containing nascent and mature focal adhesions are marked by cyan and yellow lines, respectively. Bars, 5 µm.

Fig 3: Ultrastructural analysis showing that T-Plastin localizes to and strengthens protrusive actin structures.a PREM images of WT and T-Plastin KO1 HUVEC protrusions. Top, low magnification images of cell protrusions, regions in yellow boxes correspond to magnified regions shown in middle. Bars, 5 µm. Middle, magnified areas from top showing the leading edge. Cyan boxes correspond to the further magnified regions shown in bottom. The depth of the branched protrusive network is highlighted in yellow. Bars, 200 nm. Bottom, Highly magnified areas from middle showing the arrangement of actin filaments at the very leading edge. Bars, 25 nm. b Quantification of protrusion depth, the distance the branched actin network persists from the leading edge of protrusions. Black bars represent the mean and 95% confidence intervals, analyzed using unpaired t-test, **** P < 0.0001 (n protrusions WT = 43, KO = 100; taken from =3 independent replicates). c STED super-resolution images of WT and KO1 HUVEC stained for T-Plastin (green) and F-actin (magenta). Regions of high and low actin density in a WT protrusion are magnified in orange and cyan boxes as indicated. Bars 2 µm and 500 nm, respectively. Yellow lines indicate extent of the protrusion. d Violin plot of intensity correlation quotients between T-Plastin and F-actin (purple), quantified in protrusion and cytoplasmic regions (see Fig. S8b, for example). As a control (cyan) the values for F-actin were scrambled. Black dots indicate individual values for different images (n cells = 4, representative of three independent replicates), medians are shown as black dotted line within colored violins.

Fig 4: Deregulation of cytoskeletal proteins in Cul3 mutant embryonic forebrain tissue.a Sample preparation for proteomic analysis (n(Cul3+/-) = 5 per genotype, n(Cul3+/fl Emx1-Cre) = 3 per genotype, n(Cul3fl/fl Emx1-Cre) = 3 per genotype; male mutant and control littermate pairs). b–d, Volcano plot of deregulated proteins at 10% FDR cut-off in the Cul3+/- developing cortex (b), Cul3+/fl Emx1-Cre (c), and Cul3fl/fl Emx1-Cre (d) developing forebrain; cytoskeleton related proteins (purple), proteins differently regulated in more than one data set are underlined, Cul3 (red) (b–d) (details: Supplementary Data 3–5). e DAVID functional annotation identified down- (left) and up- (right) regulated proteins at 20% FDR of the Cul3fl/fl Emx1-Cre forebrain involved in regulating the activity of RNA polymerase II, the proteasome core complex, neurogenesis and actin and microtubule cytoskeletal organization (selected GO-terms, significant GO-terms in red: RNA polymerase II core complex, GO: 0005665, adj. P-value = 0.002; proteasome core complex GO: 0005839, adj. P-value= 4.9e-07; details: Supplementary Data 6). f, g Comparison of ratios of up- and downregulated proteins in the Cul3+/-, Cul3+/fl Emx1-Cre and Cul3fl/fl Emx1-Cre cortex at log 0.1 change (details: Supplementary Data 7). h, Fitting the mean raw expression levels of Pls3 and INA to a linear regression model indicates that these proteins follow a genotype-dependent dose-response (Pls3: R2 = 0.999; INA: R2 = 0.932; Cul3+/fl < Cul3+/fl Emx1-Cre < Cul3fl/fl Emx1-Cre; boxplot shows the median value and 25–75th percentile, whiskers show minimum and maximum). i Western blot (left) and analysis of Gapdh normalized intensities (right) confirm increased levels of the cytoskeletal proteins Pls3 and INA in Cul3+/- lysates (n = 5 per genotype; *P = 0.04, ***P < 0.001; unpaired two-tailed t-tests). j Western blot and analysis of Gapdh normalized intensities confirm increased levels of cytoskeletal proteins Nisch, Pls3, and INA in Cul3fl/fl Emx1-Cre cortical lysates (samples on the same membrane cut for better visualization, n(Cul3+/fl) = 5, n(Cul3fl/fl Emx1-Cre) = 3; **P = 0.003, ***P < 0.001; unpaired two-tailed t-tests). k Quantitative real-time PCR analysis of mRNA levels of Pls1, Pls3, and INA normalized to wild-type levels (?Cq expression values; n = 5 per genotype; *P = 0.02; unpaired two-tailed t-tests). Data presented as or as box and whiskers, min to max (h) and mean ± SEM in i–k. Detailed statistics are provided in Supplementary Data 1.

Fig 5: T-Plastin enhances protrusion dynamics.a HUVEC expressing F-tractin-mCitrine were treated with control or T-Plastin siRNA, sparsely plated, and imaged every 10 s for ~33 min. Masks were generated of the cell edge boundaries and time-projected with each line representing a 50 s interval, the parula colormap represents the 0 min time as blue and 33.3 min time as yellow. Straight yellow lines over F-tractin images indicate regions used to generate kymographs shown in (b). Bars, 10 µm. b Kymographs generated from F-tractin images shown in (a), bars 2.5 µm in the x-direction. The y-direction is time with each pixel as 10 s, the entire length equaling 33.3 min. Protrusions can be seen moving outward from the cell body (toward the right) and retractions inward toward the left. c Quantification of protrusion rates generated from kymographs such as those shown in (b). (n of protrusions are: siControl = 38, siT-Plastin #1 = 34, siT-Plastin #2 = 30, taken from two independent replicates). d Quantification of retraction rates generated from kymographs such as those shown in (b). (n of retractions are: siControl = 28, siT-Plastin #1 = 28, siT-Plastin #2 = 20, taken from two independent replicates). Bars represent means, error bars represent 95% CI, each siRNA targeting T-Plastin was compared to siControl using a one-way ANOVA with Dunnett’s multiple comparison test **P < 0.01, ***P < 0.001.