Profilin2 regulates actin rod assembly in neuronal cells

Figures

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  Figure 1. Profilin2 is a component of actin rods formed in SMA. Actin rods were enriched according to earlier studies9,12. Cells were treated with siSmn and the lysate was separated on a 10%/15%-Optiprep gradient. (A) Fractions from the gradient were analyzed by Western blot. Actin rods (red box at fraction 10) were distinguished from cytoplasmic proteins (Fractions 1–5) by incubation of the blot with an anti-cofilin antibody. (B) 12.5% Coomassie-stained SDS-PAGE of the concentrated fraction 10 of (A). Labeled bands were analyzed by LC–MS. (C) Enrichment of proteins after identification by mass-spectrometry and classified by their molecular function using the PANTHER overrepresentation test (gene ontology database) and the mouse genome as reference. (D) Western blot of fractions after separation of SMN-depleted cell lysate on a 10%/40%-Optiprep gradient. Rods were detected by using an antibody against actin. In addition, fractions were probed for profilin1 (PFN1) and profilin2 (PFN2). (E) Confocal images of siSmn-treated NSC34 cells co-stained for PLXND1 and PFN1 or 2. Co-localization was analyzed by measuring the product of the differences of the mean (PDM) and those data are represented…

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  Figure 2. Profilin2 but not profilin1 is involved in actin rod formation. (A) Representative Western blots of cells treated with siPfn1 or siPfn2. Ponceau was used as loading control. (B) Quantification of PFN1 and PFN2 signal normalized to total protein (mean ± SEM, n = 3, unpaired two-tailed t-test, **p < 0.01). (C) Immunofluorescence detection of actin rods by PLXND1 antibody in NSC34 cells treated with scr siRNA, siPfn1 or siPfn2. Cells with actin rods are labeled with an asterisk. DAPI was used as nuclear counterstaining. Scale bar: 20 µm. (D) Quantification of actin rods in cells treated with scr siRNA, siPfn1 or siPfn2 (mean ± SEM, n = 5 independent biological replicates, (one-way ANOVA, Dunnett’s multiple comparison test, *p < 0.05). The following ranges represent different numbers of cells counted per condition as stated on the x-axis. Cell number counted for each replicate number was: #1: 374–503 cells; #2: 525–778 cells; #3: 406–612 cells; #4: 742–809 cells; #5: 648–698 cells. (E) Quantification of cells with rods after co-transfection with scr, siSmn and siPfn2 in combinations as indicated (mean ± SEM, n = 4 independent biological replicates, one-way ANOVA, Tukey’s multiple…

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  Figure 3. Single-site phosphorylation of profilin2 shows only minor effects on rod formation. (A) Primary structure of human PFN2 with putative phosphorylation sites (magenta). (B) Schematic drawing of a bicistronic plasmid for knock-down of endogenous mPFN2 and overexpression of hPFN2 WT and mutants, respectively. The EGFP gene was molecularly cloned under the control of an IRES sequence to function as a transfection control. (C) Confocal images of NSC34 cells transfected with the plasmid of (B) comprising the coding sequence of hPFN2 WT. Cells were stained with PFN2 and PLXND1 antibody. DAPI was used as nuclear staining. Scale bar: 20 µm. (D) Quantification of rod-containing cells transfected with the bicistronic plasmids comprising the coding sequences for hPFN2 phospho-mimetics (D-mutants) (mean ± SEM, n = 5 independent biological replicates, paired two-tailed t-tests in comparison to WT if not otherwise indicated, *p < 0.05). The following cell numbers were counted for each replicate: #1: 89–214 cells; #2: 108–238 cells; #3: 77–249 cells; #4: 95–235 cells; #5. 115–225 cells. (E) Quantification of cells with rods after co-transfection with siSmn and the bicistronic plasmids comprising…

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  Figure 4. RhoA-ROCK axis is involved in actin rod formation in SMN knock-down cells. Cells were treated with scr siRNA or siSmn and differentiated for three days. Activity of small G-proteins [(A) RhoA, (B) Rac1, (C) Cdc42] was measured by using G-LISA Activation Assays (Cytoskeleton) (mean ± SEM, n = 3, paired two-tailed t-test, **p < 0.01). (D) Efficacy of the ROCK inhibitor Y-27632 (50 µM for three days) shown by Western blot of p-cofilin and cofilin. Ponceau staining was used as loading control. (E) Representative immunofluorescence images of actin rods in NSC34 cells treated with scr siRNA, siSmn or siSmn + Y27632. Actin rods were detected by PLXND1 antibody and labeled with an asterisk. DAPI was used as nuclear counterstaining. Scale bar: 20 µm. (F) Quantification of actin rods in cells treated with scr or siSmn with and without Y27632. (Mean ± SEM, n = 4 independent biological replicates, two-way ANOVA, Sidak’s multiple comparisons test, **p < 0.01). The following cell numbers were counted for each replicate: #1: 388–737 cells; #2: 492–704 cells; #3: 680–741 cells; #4: 719–984 cells.