Actomyosin and microtubule cytoskeleton organization the MC. (A-C) Single plane confocal images of cryosectioned ovaries displaying the temporal changes in the localization of Myl12.1-GFP at the animal cortex of the oocyte (N=2, n=34). During stage III, a zone with strongly reduced Myl12.1-GFP forms (white bracket in B,C). (D-E′) Orthogonal views of confocal stacks. Whole-mount follicles stained with an anti-pNMII and Rhodamine Phalloidin (n=27) at the indicated stages of oogenesis. Arrows in E,E’ indicate the localization of pNMII and actin at the lateral membranes of the MC. Rhodamine Phalloidin is used to independently mark the MC membranes (D′-F′). (G-I) Whole-mount follicles stained with an anti-acetylated tubulin antibody to detect stable microtubules in the MC (arrow in H and I) (n=16). (J-O) Whole-mount follicles immunostained with Krt18 (J-K, see Fig. 7 and Results) or acetylated Tubulin (AcTub, N-O) antibodies and counterstained with Rhodamine Phalloidin (RhPh) and DAPI after either cytochalasin D treatment (K, DMSO control in J) (N=2, control n=19, treated n=19) or cold treatment (O, control in N) (N=3, control n=26, treated n=25). (L,M) Snapshots of 3D reconstruction of MCs (see Materials and Methods) from follicles treated with cytochalasin D (M) or with DMSO (L) (see also Movie 2). (P,Q) Quantification of the area of contact between the MC and the oocyte surface (P, P=0.032) or the angle formed at this point (Q, dotted line in N,O; P<0.0001, unpaired two-tailed t-tests). N, number of experiments; n, number of samples. Scale bars: 10 μm in A-O.
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