Figure 7: CLIC4-cortactin association modulates actin assembly on EE surface.

(a) Low- (insets) and high-power views of MEFs stained for cortactin and EEA1. Arrows point to EEs displaying one cortacin-labelled ‘patch’ in WT. Arrowheads point to enlarged EEs decorated with multiple cortactin-labelled ‘patches’ in KO. (b) Fractions of MEFs (WT, CLIC4-KO without or with transfected Flag-CLIC4) decorated with 1-4 (or more) cortactin-positive patches on EE. Error bars represent standard deviation; P value by t-test; n>145; 3 repeats. (c) Representative images of triply labelled EE of WT and CLIC4-KO MEF. (d) MEFs expressing low levels of transfected Flag-Rab5Q79L and mCherry-LifeAct. Arrows and arrowheads point to the actin patches on the EE of WT and KO cells, respectively. (e) Total cell lysates (TCL) from the CLIC4-shRNA inducible MDCK cells treated with (KD) or without (WT) doxycycline were subjected to immunoprecipitation using the anti-WASH1 or anti-Vps35 antibody. Host species-matched IgGs were used for negative controls (cont.). (f) Immunoprecipitation of HEK cells transfected with mCherry-cortactin and/or Flag-CLIC4 using the anti-Flag antibody, and subsequent immunoblottings using anti-DsRed (crossreacts with mCherry) and anti-CLIC4 antibodies. (g) Quantification of CLIC4-KO MEF (without or with cortactin-shRNA transfection) that expressed perinuclear enrichment of Rab11a. Error bars represent standard deviation; P value by t-test; n>100; 3 repeats. (h) Quantification of the single-lumen formation in (3-day) MDCK cysts transfected with various shRNA. The cortactin-shRNA (or control vector) was transfected into either the WT or the CLIC4-KD MDCK line described in (e). Error bars represent standard deviation; P value by t-test; n>550; 3 repeats. (i) Working model. CLIC4 regulates apical trafficking by modulating the EE’s surface branched actin, required for organelle microdomain remodeling, cargo molecule sorting and/or tubular fission. Endosomal vesicles harbouring apical cargo may directly fuse onto, or transit through RE en route to apical PM (AMIS). CLIC4 depletion abnormally increases branched actin on EE surface, and interferes with membrane remodelling, cargo sorting and apical vesicular delivery. These abnormalities together may enhance the membrane’s involution, rendering more LE formation; apical elements crucial for RE genesis tend to be shunted to the LE/lysosomal degradation pathway. Scale bars, 2 μm (a), 1 μm (c), 5 μm (d).