Fig 2: Arp3 isoforms confer different cellular properties to Arp2/3. (A) Immunofluorescence images of vaccinia-induced actin tails labeled with Alexa Fluor 568 phalloidin (magenta) together with quantification of their length and number at 8 h after infection in HeLa cells stably expressing RNAi-resistant GFP-tagged Arp3 (blue) or Arp3B (red) and treated with Arp3 siRNA. (B) Immunoblot analysis of GFP-Trap pulldowns from HeLa cells lysates stably expressing GFP, GFP-Arp3, or GFP-Arp3B demonstrates that Arp3 and Arp3B associate with all Arp2/3 complex subunits. The Arp3 antibody does not detect Arp3B. (C) Immunoblot analysis of Arp2/3 complex subunits in HeLa cells treated with Arp3 and Arp3B siRNAs (Fig. S1 A shows RT-PCR analysis of the level of Arp3 and Arp3B mRNA relative to control in HeLa cells after knockdown with the individual siRNA from the siGenome pools against Arp3 and Arp3B). (D) Immunofluorescence images of virus (magenta) induced actin tails labeled with Alexa Fluor 488 phalloidin (green) together with quantification of their length in cells treated with the indicated siRNAs. All error bars represent SEM from n = 3 independent experiments in which the length of 100 tails or their number in six cells was analyzed per condition. Tukey’s multiple comparisons test was used to determine statistical significance; ****, P < 0.0001; ***, P < 0.001; *, P < 0.05. Scale bars = 5 µm.

Fig 3: Changes in the distribution of F-actin, actin binding/regulatory proteins, and adhesion proteins at the apical ES following CRB3 knockdown in the testis in vivo that impede spermatid transport and polarity in the adluminal compartment.Frozen sections of testes following transfection with CRB3 siRNA duplexes vs. non-targeting negative control duplexes were fluorescently stained for F-actin (green), branched actin polymerization protein Arp3 (green), actin barbed end capping and bundling protein Eps8 (red) vs. adhesion proteins ß1-integrin and laminin-?3 at the apical ES. Cell nuclei were visualized by DAPI. (A) In stage VII tubules, F-actin was prominently localized to the concave (ventral) side of spermatid heads in control testes transfected with non-targeting negative control siRNA duplexes. However, CRB3 KD led to changes in actin organization in which F-actin no longer prominently localized to the concave side of spermatid heads but mostly to the convex (dorsal) side of spermatid heads. These changes thus caused defects in spermatid polarity in which many spermatid heads no longer pointing towards the basement membrane but deviated by at least 90° from the intended orientation (annotated by yellow arrowheads). These changes in F-actin organization were the result of changes in the spatiotemporal expression of Arp3 and Eps8 in which these actin regulatory proteins no longer restrictively expressed at the concave side of spermatid heads, but considerably diminished in particular in spermatids displaying defects in polarity (annotated by yellow arrowheads). Scale bar, 20 µm. (B) Adhesion proteins at the apical ES, such as ß1-integrin (an integral membrane protein exclusively expressed by Sertoli cells at the apical ES) no longer tightly localized to the convex side of spermatid heads, but it was detaching from the site and its expression was considerably weakened, similar to the spatiotemporal expression of laminin-?3 (a spermatid-specific apical ES adhesion protein) which prominently expressed at the spermatid head in control was considerably diminished after CRB3 KD. Scale bar, 20 µm.

Fig 4: CRB3 knockdown causes disorganization of actin microfilaments in Sertoli cells mediated by changes in the spatiotemporal expression of actin binding and regulatory proteins.(A) After CRB3 KD, the F-actin (green or gray) network in Sertoli cells became truncated and branched, illustrating changes in their organization in which actin microfilaments no longer stretched across the entire Sertoli cell as noted in control cells, supporting the possibility of a reduced actin bundling capability. Scale bar, 20 µm. (B) A study by actin bundling assay supported the notion that CRB3 KD led to considerable reduction in actin bundling capability. In this biochemical assay, pellet contained bundled F-actin, whereas supernatant contained unbundled and linear as well as truncated actin microfilaments. Each bar in the histogram is a mean ± SD of n = 4 experiments. **P < 0.01 by Student’s t-test. (C) Changes in actin microfilament organization following CRB3 KD were shown to be mediated by changes in the spatiotemporal expression and/or localization of branched actin polymerization protein Arp3, which was internalized from near the cell surface, relocated to cell cytosol, thereby facilitating the organization of actin microfilaments into a branched network. Furthermore, actin barbed end capping and bundling protein Eps8 no longer localized to the cell-cell interface to maintain actin microfilament bundles to support the barrier function, instead, Eps8 became scattered across the cytosol. The actin cross-linking/bundling protein palladin distribution remained somewhat unaltered which continued to support the shorter actin microfilaments in Sertoli cells following CRB3 KD, whereas barbed end actin nucleation protein formin 1 was considerably down-regulated, consistent with immunoblotting data shown in Fig. 5B. Sertoli cell nuclei were visualized by DAPI (blue). siGLO transfection indicator (red) illustrated successful transfection. Scale bar, 20 µm. (D) To confirm re-distribution of actin regulatory proteins, such as Eps8, through its re-location from near the cell surface to cell cytosol, Eps8 before and after CRB3 knockdown was co-localized with a TJ-integral membrane protein CAR vs. a TJ adaptor protein ZO-1. Scale bar, 25 µm.

Fig 5: FCHSD2 Is a Bona Fide CME Protein, Related to Figure 1(A) Graphs for the recruitment dynamics of FCHSD2 versus ClathrinLC, Dynamin1, ITSN1L and ARP3. Number of events measured are shown at the top left of each graph. Data are presented as mean ± SD.(B) Immunoblots for FCHSD2 showing both FCHSD2 CRISPR/Cas9 knockout clones used and the rescue cell line (FCHSD2 KO-1 stably expressing untagged FCHSD2).(C) Increased surface and total transferrin receptor (TFR) in FCHSD2 knockout cells shown by immunofluorescence (left), immunoblots (center) and flow cytometry (right). Each value represents median fluorescence from at least 5000 cells (n = 3, mean ± SD). **p > 0.01, *p > 0.05. One-way ANOVA with Tukey’s post hoc analysis.(D) Immunoblots showing FCHSD2 knockdown in BSC1 AP2s2-GFP cells.(E) Immunoblots showing ARP3 knockdown in wild-type and FCHSD2 KO cells.(F) Immunoblots showing ARP3 knockdown in BSC1 AP2s2-GFP cells.