Figure 5.

hACTN4-M240T reexpression does not ameliorate actinin knockdown–associated phenotypes. (a,b) Representative micrographs of nephrocytes stained with (a) anti-Pyd and (b) quantification of the ND length. Nephrocytes derived from either control larvae, larvae with nephrocyte-specific knockdown of actinin (actn-RNAi2) as well as larvae reexpressing the indicated hACTN4-variant in the knockdown background. hACTN4-WT is able to partially rescue the actinin knockdown–associated reduction in ND length, whereas reexpression of hACTN-M240T does not lead to increased ND length. This is also true for FSGS-associated mutations FSGS-W59R and FSGS-K255E (gray dots indicate all nephrocytes measured, green dots show means of n = 3 independent experiments performed in 3 experimental crossings, error bars indicate SD, ∗∗∗P < 0.001, ∗P < 0.05, one-way analysis of variance with Tukey’s post hoc test). (c,d) Representative micrographs of nephrocytes subjected to (c) FITC-albumin tracer and (d) quantification of fluorescence intensity as a measure of uptake capacity. Nephrocytes were incubated in 0.2 mg/ml FITC-albumin solution for 30 seconds, and fluorescence intensity was quantified using Fiji. The data are presented as normalized to control levels. Reexpression of hACTN4-WT also leads to a significant increase in tracer uptake capacity, compared with hACTN4-M240T, hACTN4-W59R, and hACTN4-K255E, where a rescue of actinin knockdown–associated reduction of tracer uptake cannot be observed (gray dots indicate all nephrocytes measured, green dots show means of n = 3 independent experiments performed in 3 experimental crossings, error bars indicate SD, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001, one-way analysis of variance with Tukey’s post hoc test). Scale bars indicate 5 μm in (a) and 25 μm in (c). FITC, fluorescein isothiocyanate; ND, nephrocyte diaphragm; Pyd, polychaetoid; WT, wild-type.