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. Author manuscript; available in PMC: 2012 Feb 13.
Published in final edited form as: Nat Cell Biol. 2011 Feb 20;13(3):203–214. doi: 10.1038/ncb2163

Figure 5.

Figure 5

Srf-cKO early mitotic cells are defective in enriching and activating ERM proteins at the actin cortex and in recruiting myosin-IIA. (a,b) Immunofluorescence microscopy of E16.5 epidermis reveals an enrichment of cortically localized ERM in the basal layer and activation (phosphorylation) of ERM (pERM) in early mitotic cells. Both are perturbed in Srf-cKO skin. Dotted lines denote dermal–epidermal border. Thin line denotes boundary of mitotic cell at right. Scale bars,20 μm. (c) Quantifications reveal a threefold decrease in pERM (* indicates P =0.0007,n=3) at the cortex of early mitotic basal cells. (d) Immunoblot analyses=of total epidermal lysates. HPRT, hypoxanthine phosphoribosyl-transferase loading control. Note that despite reduction in ERM at basal cortex, overall levels of ERM seem to be similar. Note also that ERM activation is clearly reduced following Srf deficiency. (e) Treatment with 0.25 μM latrunculin achieves a similar effect to Srf deficiency with respect to pERM enrichment at the cortex of early mitotic cells (* indicates P =0.002,n=3). (f,g) Planar images are through the basal layer of E16.5 embryos, subjected to whole-mount fluorescence microscopy for F-actin (phalloidin), myosin-IIA and DAPI. Arrows indicate early mitotic cells, enriched in myosin-IIA in wild type, but not Srf-cKO. Insets, reconstructed X –Z view of the compressed Z -stack images to show that myosin-IIA is enriched throughout the cortex of wild-type, but not cKO, mitotic cells. Scale bars,10 μm. (g) Quantifications of fluorescence intensity ratios of F-actin and myosin-IIA from mitotic versus non-mitotic cells. * indicates P =0.0066,n=3. (h) Immunoblot analyses of total newborn epidermal lysates. Blots were probed with antibodies against myosin-IIA, pan-myosin heavy chain (MHC), myosin light chain (MLC) and HPRT. (i,j) E14.5 embryos were treated with DMSO or 0.25 μM latrunculin and stained for F-actin, myosin-IIA and DAPI. Shown are planar views of basal layer and quantifications of fluorescence intensity ratios of F-actin and myosin-IIA in mitotic versus neighbouring non-mitotic cells. indicates P =0.008,n=3. Scale bar,10 μm error bars represent s.d. Uncropped images of blots are shown in Supplementary Fig. S9.