Fig 2: Atypical PKCs control actin cytoskeleton via Cofilin activation. The Cofilin pathway is activated by atypical PKC via Slingshot, SSH. Phosphorylated Cofilin at serine-3 is inactive. Phosphatases such as SSH induces the dephosphorylation and activation of Cofilin. Activated Cofilin can then sever the mother filaments and produced free barbed ends leading to the elongation of newly formed actin filaments which are preferred for dendritic nucleation by ARP2/3 complex and globular actin. These newly formed structures push on the cell membrane to promote protrusion and motility.

Fig 3: 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.