Figure 6. New protein complex between Gpsm2 and whirlin.
(a–c) COS-7 cells transiently transfected with GFP–Myo15, DsRed-whirlin, myc-Gpsm2FL, untagged Gαi3 or DsRed as indicated. (a) Co-expression of myosin 15 and whirlin leads to the formation of actin-rich filopodial structures with Gpsm2 accumulating at the tip with the two proteins. Absence of whirlin (DsRed only) markedly reduces the colocalization at the tips. (b) Gαi3 also accumulates at the tip of filopodia with myosin 15 and whirlin. Transfections were repeated more than three times. Scale bars, 4 μm. (c) Percentage of filopodia tips (±s.e.m.) displaying colocalization of Gpsm2, myosin 15 and whirlin in the two contexts. n=number of filopodia tips from three independent experiments. ***P<0.001 with Mann–Whitney test. (d) Schematic representation of Gpsm2FL and whirlin proteins. Four variants identified in CMCS patients, including Gpsm2R318RfsX8 are indicated. (e) Whirlin co-immunoprecipitates with myc-Gpsm2FL but not with non-immune IgG. (f) The interaction is maintained, though reduced, with the Gpsm2R318RfsX8 (Gpsm2R318R) variant. Representative images of n>3 independent experiments. (g) A GST-pull-down assay indicates that the unstructured region of whirlin between aa 672 and aa 810 binds to Gpsm2R318RfsX8. Representative image of n=3 independent experiments. (h,i) Increasing amounts of myc-Gpsm2FL-encoding cDNA over constant amounts of whirlin leads to a net increase in whirlin expression levels, whereas increasing doses of whirlin had no significant effect on Gpsm2 levels. Five independent experiments presented as whisker box plots (min/max) combined with dot plot (blue bars representing the mean values) ***P<0.001, **P<0.01, *P<0.05 (for h,i) with one simple t-test or one-way ANOVA (post-hoc Bonferonni’s test). NS, not significant. (j) Gpsm2FL significantly increased the length of filopodia generated by eGFP–myosin 15 and DsRed-whirlin (control) in COS-7 cells. Constructs bearing CMCS mutations led to shorter filopodia, compared with control or to Gpsm2FL. Data are presented as whisker box plots (min/max) from three independent experiments (n=220, 207, 345, 228 and 188 filopodia, in the order indicated on the histogram’s x axis) (blue bars represent the mean values). ***P<0.001, **P<0.01 with Kruskal–Wallis test (post-hoc Dunn's Multiple Comparison Test). NS, not significant.