pone.0200487.g007.tif (3.71 MB)

AIF1L knockout clones exhibit an impaired actomyosin contractility reserve.

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posted on 12.07.2018, 17:50 by Mako Yasuda-Yamahara, Manuel Rogg, Kosuke Yamahara, Jasmin I. Maier, Tobias B. Huber, Christoph Schell

(a-c) Western blot experiments and quantification by densitometry demonstrated decreased levels of MYL9 as well as MYH9 in respective AIF1L knockout clones, whereas UNC45A or MYPT were not affected (n = 6 WT and KO WB intensities out of 3 independent experiments; *** p<0.001; **** p<0.0001). (d-g) Treatment of wild type cells and AIF1L knockout clones with the myosin-II inhibitor blebbistatin resulted in a more rapid dissolution of FA complexes in conditions of AIF1L loss, indicating a decreased actomyosin contractility reserve (n = 29 WT, 27 KO-A and 29 KO-B podocytes out of 3 independent experiments were analyzed; * p<0.05; **** p<0.0001). (h-j) Washout experiments for blebbistatin showed that in conditions of reconstituting actomyosin contractility podocytes show a high generation rate of filopodial protrusions (note that these structures are associated with FA sites at the filopodial basis indicated by white arrows; white dashed boxes indicate areas of magnification;(n = 20 podocytes per condition were analyzed; n.s.–non significant, **** p<0.0001)).