Fig 2: Effect of functional loss of clusterin on actin cytoskeleton and IOP. siRNA-mediated knockdown of clusterin (siCLU) in serum-starved HTM cells compared to control scramble (siScr) shows significant decrease in (a). clusterin mRNA, (b) clusterin protein levels in WCL, and (c) in CM GAPDH was used as a loading control for WCL, and Ponceau S-band at 55 kDa was used for the CM. (d) Changes in cell shape due to siCLU (bottom panel) were compared to siScr (top panel) observed using light microscopy. Scale bar 100 microns. The inset panels represent the zoomed in image. Scale bar 20 microns. Histogram on the right represents the quantitative assessment of cell shape change based on the number of protrusions. (e) Reduced clusterin distribution (Alexa Flour 568/red) (bottom panel) due to siCLU compared with siScr (top panel). The nucleus was stained with DAPI in blue. Images were captured in z-stack in a confocal microscope, and stacks were orthogonally projected. Scale bar 20 microns. (f and g) Increased distribution of actin stress fibers stained by phalloidin (red) with (f). paxillin, and (g) vinculin (Alexa Flour 488/green) seen in siCLU compared with siScr. The nucleus was stained with DAPI in blue. Images were captured in z-stack in a confocal microscope, and stacks were orthogonally projected. Scale bar 100 microns. (h) and (i) Changes in the levels of actin cytoskeleton-associated proteins due to loss of clusterin expression. Western blot analysis showed significant increase in the PRKD1, LIMK1, PRK2, plectin, paxillin, and phosphorylated paxillin. Ratio of phosphorylated paxillin to total paxillin demonstrated an increasing trend with no significance. Integrin- INTaV, INTß5 showed significant increase in siCLU compared with siScr with no changes in INTß3. GAPDH was used as a loading control for immunoblotting analysis. Values represent the mean ± SEM, where n = 4 (biological replicates). *p = 0.05 was considered significant. (j) IOP values of wildtype (WT) and clusterin knockout (Clu -/- ) mice. Significant IOP elevation is observed in Clu -/- compared with WT by 70 days and continued over 90 and 105 days postnatal with no changes by 50 days. Data represented as mean ± SEM from 6 pairs of eyes (n = 6). *p = 0.05 indicates statistical significance. IOP, intraocular pressure; mRNA, messenger RNA; SEM, standard error of mean

Fig 3: PirB activates the RhoA/ROCK2/LIMK1/cofilin signalling pathway in CSR mice. a Western blot analysis of hippocampal RhoA, ROCK2, pLIMK1/LIMK1, and pcofilin/cofilin levels in control, CSR, PirB cKO, and PirB cKO + CSR mice. b–e Ratio of fold change from Western blot. The RhoA (n = 5) and ROCK2 (n = 5) levels were significantly higher in CSR mice than in control and PirB cKO + CSR mice. The phosphorylated LIMK1 (n = 5) and cofilin (n = 5) levels were significantly lower in CSR mice than in control and PirB cKO + CSR mice. f Western blot analysis of hippocampal ROCK2, pLIMK1/LIMK1, and pcofilin/cofilin levels in control and CSR mice after fasudil treatment. g–i Ratio of fold change from Western blot. The ROCK2 level was significantly lower in fasudil + CSR mice than in CSR mice (n = 5). The phosphorylated LIMK1 (n = 5) and cofilin (n = 5) levels were significantly higher in fasudil + CSR mice than in CSR mice. Data are presented as the means ± SD and were analysed by one-way ANOVA with Tukey’s test (*p < 0.05, **p < 0.01)

Fig 4: Ssh2 knockout (KO) spermatids display impaired COFILIN phospho-regulation that disrupts filamentous actin (F-actin) remodeling.(A) Immunoblotting against SSH2, p-COFILIN, COFILIN, LIMK1, and LIMK2 in wild-type (WT) and Ssh2 KO testes sampled on postnatal day (PD)82. GAPDH was used as the loading control. (B) Testicular sections of WT and Ssh2 KO mice stained with p-COFILIN (green) and DAPI (blue), n=3. The solid and hollow triangles point to the round spermatids and elongated spermatids of low p-COFILIN expression in seminiferous tubules of WT mice. The white arrow points to the round spermatids of strong p-COFILIN expression in seminiferous tubules of Ssh2 KO mice. The asterisk indicates a germ cell cluster. Scale bars: 20 µm. (C) Western blot analysis of SSH2, p-COFILIN, and F-ACTIN in lysates from 8-week-old WT and Ssh2 KO testes, n=3; GAPDH was used as the loading control. (D) Testicular sections of 8-week-old WT and Ssh2 KO mice stained for p-COFILIN (green) and F-actin (red), n=3. The solid and hollow triangles point to the round spermatids and elongated spermatids with intact F-actin organization in seminiferous tubules of WT mice. The white arrow points to the round spermatids of strong p-COFILIN expression with disorganized F-actin in seminiferous tubules of Ssh2 KO mice. The asterisk indicates a germ cell cluster. Scale bars: 20 µm. (E) Proposed model for the functional role of the SSH2-COFILIN pathway in acrosome biogenesis. SSH2 participates in F-actin remodeling by regulating the phosphorylation of COFILIN. Intact and organized F-actin dynamics enables the transport of proacrosomal vesicles from the Golgi apparatus to the apical pole of the nucleus of the spermatid. F-actin also participates in the fusion of Golgi-derived vesicles and extra-Golgi vesicles. Figure 7—source data 1.Original images/blots.