Loss of mDia1/3 in the Sertoli cell results in impaired actomyosin bundles and adherens junction.

(A) TIRF N-STORM imaging of F-actin bundles of primary cultured Sertoli cells. Magnified images of the corresponding white box areas are shown below. Note that the F-actin bundle is a structure continuous with cortical actin filaments meshwork. Arrows indicate the bending F-actin bundles observed in mDia1/3 DKO primary cultured Sertoli cells. Scale bars, 500 nm. (B) Primary cultured WT and mDia1/3 DKO Sertoli cells stained with phalloidin (magenta) and pMLC (green). Note the reduced and discontinuity of pMLC staining on the F-actin bundles in the mDia1/3 DKO Sertoli cell. Scale bars, 20 μm and 5 μm. White boxes are magnified and shown below the low magnification images. (C) Quantification of pMLC staining intensity. Staining intensity was normalized to the average intensity of control WT cells. Result is a sum of three independent experiments. Data are represented as mean ± SEM (n = 33 and 30 for WT cells and mDia1/3 DKO cells, respectively). ***P t test). (D) Primary cultured WT and mDia1/3 DKO Sertoli cells stained with phalloidin (magenta) and espin1 (green). Note increased espin1 staining overlapped with F-actin staining in mDia1/3 DKO Sertoli cell. Scale bars, 20 μm and 5 μm. White boxes are magnified and shown below the low magnification images. (E) Quantification of espin1-positive actin bundles per cell. Result is a sum of three independent experiments. Data are represented as mean ± SEM (n = 25 for WT Sertoli cells and n = 2 for mDia1/3 DKO Sertoli cells). ***P t test). (F) Schematic representation of Sertoli cell–germ cell coculture experiment. Green circle indicates germ cell isolated from 3-wk-old EGFP transgenic mouse testes, and gray ellipse indicates WT or mDia1/3 DKO Sertoli cells. Red lines indicate actin filaments of the Sertoli cell, which surround the germ cell. (G) Percentage of germ cells formed adherens junction with Sertoli cells, as judged by N-cadherin staining on WT or mDia1/3 DKO Sertoli cells in Sertoli cell–germ cell coculture experiments. Germ cells that had N-cadherin signals surrounding the germ cells were counted as adhesive cells. Result is the average of three independent experiments. Data are represented as mean ± SEM (n = 124 and 89 cells for WT and mDia1/3 DKO, respectively). *P P = 0.0306, Student t test). (H) Representative images of a germ cell judged as an adhesive cell with a WT Sertoli cell. (I) SDSRM images of Sertoli cell–germ cell in cocultured experiment stained with phalloidin. Images consist of 10 pairs of serial planes (z-step = 0.5 μm from bottom to top). Note that, while the germ cell attached to WT Sertoli cell is surrounded by actin filaments, such actin filaments are impaired in the germ cell attached to mDia1/3 DKO Sertoli cell. (J) Quantification of the percentage of germ cells with actin filaments surrounding them in Sertoli cell–germ cell cocultured experiment. Result is from three independent experiments. Data are represented as mean ± SEM (n = 157 and 147 cells for WT and mDia1/3 DKO, respectively). ***P P = 0.0005, Student t test). (K) Quantification of the cortical F-actin intensity underneath the germ cell of the WT or mDia1/3 DKO Sertoli cell in cocultured experiment. Staining intensity was normalized to the average intensity of control WT cells. Result is from two independent experiments. Data are represented as mean ± SEM (n = 23 and 17 cells for WT and m1/3 DKO, respectively). ***P P = 0.0001, Student t test). DKO, double knockout; EGFP, enhanced green fluorescent protein; F-actin, filamentous actin; mDia1/3, mammalian diaphanous homolog1/3; N-cad, neural cadherin; N-cadherin, neural cadherin; Pha, phalloidin; pMLC, phosphorylated myosin light chain; SDSRM, spinning disk superresolution microscopy; TIRF N-STORM, total internal reflection N-stochastic optical reconstruction microscopy; WT, wild-type.