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CLIC4 Regulates Apical Exocytosis and Renal Tube Luminogenesis Through Retromer- And Actin-Mediated Endocytic Trafficking

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CLIC4 Regulates Apical Exocytosis and Renal Tube Luminogenesis Through Retromer- And Actin-Mediated Endocytic Trafficking

Szu-Yi Chou et al. Nat Commun.

Abstract

Chloride intracellular channel 4 (CLIC4) is a mammalian homologue of EXC-4 whose mutation is associated with cystic excretory canals in nematodes. Here we show that CLIC4-null mouse embryos exhibit impaired renal tubulogenesis. In both developing and developed kidneys, CLIC4 is specifically enriched in the proximal tubule epithelial cells, in which CLIC4 is important for luminal delivery, microvillus morphogenesis, and endolysosomal biogenesis. Adult CLIC4-null proximal tubules display aberrant dilation. In MDCK 3D cultures, CLIC4 is expressed on early endosome, recycling endosome and apical transport carriers before reaching its steady-state apical membrane localization in mature lumen. CLIC4 suppression causes impaired apical vesicle coalescence and central lumen formation, a phenotype that can be rescued by Rab8 and Cdc42. Furthermore, we show that retromer- and branched actin-mediated trafficking on early endosome regulates apical delivery during early luminogenesis. CLIC4 selectively modulates retromer-mediated apical transport by negatively regulating the formation of branched actin on early endosomes.

Figures

Figure 1
Figure 1. Expression of CLIC4 in embryonic kidney and characterization of CLIC4-KO metanephros.
(a-d) E15 WT metanephros labelled for CLIC4, cytokeratin (CK) and LTA. DAPI: blue. Arrows and arrowheads point to the lumens of cytokeratin and LTA-labelled tubules, respectively. (e-i) Electron micrographs of pre-PT cells of WT (e-g) and CLIC4-KO (h-i) E15 metanephros labelled with CLIC4 immunogolds. The metanephros lumen (L) in WT is filled with electron-dense ECM. (f,g) Enlarged views of the boxed area in (e). CLIC4 immunogolds in MV (red arrows in f), EE (green arrowheads in g) and RE (blue arrows in g). The inset highlights the CLIC4 immunogolds (red arrows) on electron-dense RE tubules. N: nuclei. (h) A mutant PT without detectable central lumen. (i) A hypomorphic mutant PT containing an electron-dense ECM-devoid lumen. The inset shows the enlarged view of the boxed luminal surface demonstrating the presence of residual CLIC4 immunogolds on the short, irregular MV-like processes. (j,k) PAS staining of longitudinally sectioned E15 metanephros of WT (j) and CLIC4-KO (k) mice. Black brackets indicate the cortical areas. Enlarged views of boxed areas highlight the primitive tubules (arrows) and condensed mesenchyme clusters (arrowheads) found in the WT and KO, respectively. Scale bars, 50 μm (d); 5 μm (e,h,i); 2 μm (f); 1 μm (g, inset of i); 200 μm (j,k).
Figure 2
Figure 2. Lumen formation and maturation defect of CLIC4-KO developing PT.
(a,b) PAS-stained renal cortical sections of PN0 WT (a) and CLIC4-KO (b) mice. Arrows in (a) point to the PTs displaying intense PAS+ luminal PM signals. Arrows and arrowheads in (b) point to the cell clusters and the primitive tubes, respectively; both of them contained PAS-positive granules. (c,d) LTA and megalin staining of PTs in PN0 WT(c) and CLIC4-KO (d) mice. Prominent luminal LTA and subluminal megalin signals (arrow) were seen in the WT. Mutant PTs displayed abundant LTA-labelled vacuoles; some contained mislocalized megalin (arrowheads). The inset shows the magnified view of the box area. (e,f) Immunolabelling of Rab11a in PN0 WT (e) and KO (f) PTs. An arrow points to the (sub)luminal lining pattern of Rab11a staining in WT. An arrowhead in (f) points to the granular/vacuolar staining pattern of the remaining Rab11a in mutant PT. (g-j) Representative electron micrographs of PN0 PTs in WT (g) and CLIC4-KO (h-j) mice. Red brackets indicate the apical cytoplasmic regions containing RE (red arrows), which are electron-dense tubules. Mutant PT cells did not develop typical MVs (white arrow, h,i), but contained unusually high numbers of LE-, autophagosome- (black arrows in h,j) and lysosome- (black arrowheads in i) like structures. Scale bars, 50 μm (a,b); 20 μm (c-f); 1 μm (g,h,j); 3 μm (i).
Figure 3
Figure 3. Adult CLIC4-KO PTs have dilated lumens and abnormal MV morphogenesis.
(a,b) PAS staining of 3-week-old WT (a) and CLIC4-KO (b) renal cortical sections. An arrow in (a) points to strong PAS-labelled brush borders facing a PT lumen. (c,d) EM analysis of 3-week-old WT (c) and CLIC4-KO (d) mouse PT. Red brackets indicate the RE-containing apical cytoplasmic regions. Arrows in (d) point to the large vacuoles in the mutant PT. Inset in (d) shows a low-magnification view of a dilated PT that failed to develop a typical MV-lining lumen. (e,f) Megalin immunostaining of adult WT (e) and mutant (f) PTs. Megalin had a lumen outlining pattern (arrows) in WT. Mutant PTs displayed weak megalin signals (arrowheads). (g) Immunoblots of lysates of LLC-PK1(CL4) cells without (WT) or with (KD) doxycycline-induced CLIC4-shRNA expression. (h,i) LLC-PK1(CL4) cells, without (h) or with (i) expression of CLIC4-shRNA/RFP, stained with megalin and β-catenin. Representative confocal images of x-y (upper panel) and x-z (two lower panels; arrows point to the apical side of cells) views are shown. Note that in (i) the ‘red' CLIC4-shRNA-expressing cells had selectively reduced megalin signal. Scale bars, 100 μm (a,b); 2 μm (c,d); 50 μm (e,f); 10 μm (h,i).
Figure 4
Figure 4. Expression and functional analyses of CLIC4 in MDCK luminogenesis.
(a) A model illustrating the stepwise de novo lumen formation in a MDCK 3D cyst. Red and gray represent the membranes containing apical and basolateral molecules, respectively. Blue: nuclei. (b-g) The expression pattern of endogenous CLIC4 and gp135 in cysts formed by MDCK cells stably expressing mCherry-Rab11a. Images were taken from 12 h-2 day (early aggregates to PAP) and 3 days (open lumen) cultures. Arrows in (b,c) point to overlapping signals of CLIC4, gp135 and Rab11a. In more developed cysts (d-g), CLIC4, gp135 (arrows) and Rab11a (arrowheads) are spatially resolvable. L: lumen. (h) Immunoblots of lysates obtained from MDCK (WT) cells and a representative CLIC4-KD stable line. A single ∼28-kDa band of CLIC4 was specifically detected in WT MDCK lysates. CLIC4, but not the control protein. α-Tubulin (α-tub) was drastically diminished in the CLIC4-KD lysates. (i) Two representative examples of CLIC4-KD (2-day) cysts immunolabelled for gp135 and Rab11a. Enlarged views of the boxed areas show the accumulation of these two molecules (Pearson's coefficient=0.64±0.05; n=6) in the ectopic lumens/vacuoles (dash lines). (j,k) Immunolabelling of PTEN and gp135 in WT (j) and CLIC4-KD (k) cysts of different stages (1-2-day). Enlarged views of the boxed areas are also shown. In WT, PTEN was first enriched at AMIS/PAP (bracket) and then TJ (arrowheads). Similar enrichment was absent from the KD cysts. (l) Representative images of WT and CLIC4-KD (3-day) cysts labelled for F-actin, β-catenin, and ZO-1. (m) The percentages of the (3-day) cysts containing a signal lumen. Error bars represent standard deviation; P value by t-test; n>380; 3 repeats. Scale bars, 10 μm (b-g,i-k); 100 μm (l).
Figure 5
Figure 5. Mechanistic characterization of CLIC4-regulated central lumen formation.
(a) Examples of the luminal PM location of transfected Flag-Rab8 in WT (arrows) and CLIC4-KD (arrowheads) 3-day cysts. (b) A representative CLIC4-KD 3-day cyst transfected with GFP-Rab11a. Arrows point to the overlapping Rab11a and gp135 signals in the ectopic lumen. (c) Quantitation of the rescue effect exerted by indicated Rab GTPases on single-lumen formation. The fraction of 3-day cysts containing a single lumen in WT cells was considered as 100%. Error bars represent standard deviation; P value by t-test; n>150; 3 repeats. (d) Different stages (1-3-day) of 3D cultures formed by GFP-Cdc42-transfected WT and CLIC4-KD cells. Strong Cdc42 signals were continuously present in gp135-labelled luminal PM in WT cysts (arrows; top panel). GFP-Cdc42 had a diffuse, cytosolic expression pattern in early cysts formed by CLIC4-KD cells (arrowheads, bottom panel); GFP-Cdc42 reappeared on the gp135-enriched luminal PM (arrows, bottom panel). (e) Quantification of 3-day cysts formed by CLIC4-KD cells transfected with the indicated plasmids. The fraction of cysts containing a single lumen in WT cells was considered as 100%. Error bars represent standard deviation; P value by t-test; n>170; 3 repeats. (f,g) Electron micrographs of low- (insets) and high-power views of MDCK WT (f) and CLIC4-KD (g) cysts cultured for 6 days. The red bracket in (f) marks the subapical cytoplasm of WT cyst containing many endosome-like membrane structures. The red bracket in (g) shows that the thin subapical region CLIC4-KD cyst is largely devoid of tubular membrane structures. Ly: electron-dense lysosome. On the surfaces facing both central and ectopic lumens, the mutant cysts developed significantly shorter and sparse MV-like processes. Scale bars, 10 μm (a,b,d); 500 nm (f,g); 2 μm (inset of f,g).
Figure 6
Figure 6. CLIC4 regulates retromer-mediated apical sorting on EE.
(a) A representative confocal image shows an early (1-day) WT cyst labelled for CLIC4 and Vps35. Enlarged views of the boxed areas (1-4) on the right show the close association between CLIC4 and Vps35 on EE. Pearson's coefficient=0.35±0.04 (n=8). (b) A representative frame of the time-lapse video taken from a transfected MEF expressing low levels of GFP-CLIC4 and Vps29-mCherry (see Supplementary Movie 1). Enlarged views of the boxed area demonstrate the extensive colocalization of these two molecules. Pearson's coefficient=0.64±0.16 (n=11). (c) Immunoblots of total membrane lysates of MDCK cells and immunoprecipitates pulled down by using either the host species-matched control (cont.) IgG or anti-CLIC4 antibody. (d) A representative image shows an early (1-day) WT cyst triply labelled for PTEN, Vps35 and gp135. Enlarged view of a boxed area highlights the close association between PTEN and Vps35, which was frequently seen in the apical cytoplasm (arrows). Arrowheads show the steady-state PTEN location at TJs. gp135 labelled the nascent luminal PM. Pearson's coefficient (PTEN versus Vps35)=0.28±0.09 (n=4). (e) Examples of triple-labelled early cysts (1-day) formed by MDCK cells transfected with vector alone or Vps35-shRNA. Enlarged views of the boxed areas are shown. Note the reduction of Vps35 labelling in the Vps35-shRNA transfected cells, as well as the disappearance of PTEN at TJs surrounding the nascent lumen. (f) Percentage of 3-day cysts containing a single lumen. Error bars represent standard deviation; P value by t-test; n>450; 3 repeats. (g) Immunolabelling of Rab11a. Instead of enriching as the perinulcear punctate in WT MEFs, Rab11a appeared diffuse in the cytoplasm in CLIC4-KO MEFs. (h) Percentage of cells (WT MEF, CLIC-KO MEF without (−) or with (+) the transfection of Flag-CLIC4) displaying perinuclear enrichment of Rab11a labelling. Error bars represent standard deviation; P value by t-test; n>250; 3 repeats. (i) Similar TGN distribution of CI-MPR was found in WT and CLIC4-KD MDCK cells. Scale bars, 10 μm (a,b,d,e,g,i).
Figure 7
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).

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