WAVE facilitates polarized E-cadherin transport

Abstract

Cadherin dynamics drive morphogenesis, while defects in cadherin polarity contribute to diseases, including cancers. However, the forces polarizing cadherin membrane distribution are not well understood. We previously showed that WAVE-dependent branched actin polarizes cadherin distribution and suggested that one mechanism is protein transport. While previous studies suggested that WAVE is enriched at various endocytic organelles, the role of WAVE in protein traffic is understudied. Here we test the model that WAVE regulates cadherin by polarizing its transport. In support of this model we show that 1) endogenously tagged WAVE accumulates in vivo at several endocytic organelles, including recycling endosomes and at the Golgi; 2) likewise, cadherin protein accumulates at recycling endosomes and the Golgi; 3) loss of WAVE components reduces cadherin accumulation at apically directed RAB-11-positive recycling endosomes and increases accumulation at the Golgi. In addition, live imaging illustrates that dynamics and velocity of recycling endosomes enriched for RAB-11::GFP and RFP::RME-1 are reduced in animals depleted of WAVE components and RAB-11::GFP movements are misdirected, suggesting that WAVE powers and directs their movements. This in vivo study demonstrates the importance of WAVE in promoting polarized transport in epithelia and supports a model that WAVE promotes cell-cell adhesion and polarity by promoting cadherin transport.

FIGURE 1:

Cadherin localization depends on endosomal regulators and the WAVE complex. (A) Cartoon of C. elegans adult, focused on the intestine, a tube of 20 cells, linked anteriorly to the two-bulbed pharynx (gray), and posteriorly to the excretory apparatus. Imaging in all figures focuses on intestinal rings 2 and 3 and the lateral junction between them. The cross-section shows that most rings have only two cells. Because worms are imaged on their side, imaging at the surface shows the basal intestine, while focusing deeper, at the lumen, shows the apical region, the lateral junctions between cells, and, at the top and bottom, the basal regions of the cells. This “apical” focus is used for most of the images shown. (B) The levels of E-cadherin/HMR-1::GFP, endogenously tagged using CRISPR (Marston et al., 2016), were measured at apicolateral (red arrows) and basolateral (blue arrows) regions in L4 (larval stage 4) controls and animals depleted with null mutations or RNAi of gex-3 and endosomal regulators. Left image: dotted yellow lines of regions measured, 35 pixels long (3 μm). All images are shown at the same exposure, except that rme-1 (b1045) shows reduced exposure to better show localization differences. Graphs report the mean intensity and the ratio of apicolateral to basolateral. The numbers of animals measured were as follows: Controls (24), gex-3 (6), rab-10 (3), rab-7(10), rab-5 (12), rme-1 (5), and rab-11 (19). Each animal was measured once at each side. Statistical analysis used one-way ANOVA, with Holm–Sidak’s multiple comparisons tests, unless stated otherwise. Asterisks here and in all figures mark statistical significance: *p < 0.05, **p < 0.001, ***p < 0.0001, ****p < 0.00001. Error bars show 95% confidence intervals in all figures. (C) Apical F-actin (green signal at top, large white arrow) was measured in intestinal ring 3 (see cartoon). Dotted white line shows lateral and basolateral regions. Graph of mean intensity, apical GFP::ACT-5. At least five animals were measured per genotype, with at most two measurements (left and right signal) per animal. Statistical analysis used one-way ANOVA, with Dunnett’s multiple comparisons test. (D) HMR-1::GFP strain from B in controls and animals with reduced gex-3, cup-5, and the gex-3(RNAi) cup-5(ar465) double, measured as in B. ns = not significant. Statistical tests for multiple comparisons as in C, but pairwise comparisons used an unpaired t test with Welch’s correction.

FIGURE 2:

WVE-1 localizes to endosomes and Golgi. (A) WVE-1 endogenously tagged via CRISPR (GFP::WVE-1 or mKate2::WVE-1) is enriched apically along the lumen (large split arrow) and also in small puncta and larger rings. GFP::WVE-1 or mKate2::WVE-1 was combined with strains with endosomal markers. Colocalization was determined using line scans in ImageJ and saved using the ROI manager, as RGB images. Applying RGB Profile Plot generated graphs with the intensity per channel. Regions were eliminated from consideration if autofluorescence (blue, lysosomal) peaks overlapped other channels (marked by an asterisk, *). Regions were scored as colocalizing for GFP and RFP or mKate2 only if the peaks overlapped, well above background, and lacked autofluorescent (AUTO.) blue signal (marked in this figure and in Figure 3 by arrowheads). (B) Endogenously CRISPR-tagged WVE-1 is enriched at small puncta (yellow arrowheads) and also at large rings (white arrows in right panels). Some rings surround autofluorescent LROs (blue signal) while others surround nonfluorescent structures. (C) In each animal, a square region, 35 × 50 μm long, was analyzed, in the region around rings 2 and 3 of the intestine (cartoon). Graph shows WVE-1 enrichment at different endosomal organelles as scatterplots, where each dot indicates an individual worm. N = 3–6 worms per genotype. (D) Colocalization of WVE-1 with various recycling endosomes (GFP::RAB-11; RFP::RME-1, RFP::RAB-10), late endosomes (RFP::RAB-7), early endosomes (RFP::RAB-5), and Golgi (AMAN-2::GFP). Smaller crops show representative regions tested for colocalization by line scans. (E) Summary of vesicle analysis shows subcellular localization of endogenous WAVE/WVE-1. These data were used for the scatterplots in C.

FIGURE 3:

WAVE regulates levels of endosomal organelle and Golgi markers and cadherin accumulation at endosomes and Golgi. (A) Colocalization of HMR-1::GFP (green) or HMR-1::mKate2 (magenta) with endosomes enriched for distinct proteins in controls (top panels), and after depletion of the WAVE component gex-3 (bottom panels). Arrowheads mark representative areas of colocalized puncta in controls and mutants. Left panels show merged two-color images; yellow dotted box shows region enlarged in the insets. Insets are shown at higher contrast to illustrate regions of overlap. Blue signal is from intestinal autofluorescence, as explained in Figure 2. Middle and right panels are the grayscale images of HMR-1 and the endosomal organelle shown: apical recycling endosomes (RAB-11); Golgi (AMAN-2); recycling endosomes (RME-1, RAB-10), late endosomes (RAB-7), and early endosomes (RAB-5). Exposures were equally applied to controls and mutants except for the insets. All colocalizations were determined with individual line scans, as in Figure 2. Only regions significantly above background and without 405 nm autofluorescent signal (blue) were counted as colocalizing. (B) Cartoon shows the orientation and region used for the crops and the quantitation and the basal and apical regions in this tubular organ. (C) Quantitation of the mean intensity of puncta in the ring 2/3 region of the L4 larval intestine, measured using the Line tool of ImageJ. Statistical analysis used an unpaired t test with Welch’s correction. (D) Comparison of HMR-1/cadherin enrichment at different endosomal organelles, in controls and in animals depleted of WAVE component gex-3, shown as scatterplots, where each dot indicates an individual worm. N = 4–6 worms for each genotype. Statistical analysis here used one-way ANOVA with Holm–Sidak’s multiple comparisons tests. For the raw data used to generate the plots, see Supplemental Table S1.

FIGURE 4:

Proposed retrograde pathway uses WAVE and CDC-42 to transport cadherin from recycling endosomes to Golgi. (A) Region of intestine and orientation shown is the same as in the Figure 3B cartoon. The endosomal organelles most affected by WAVE loss were tested for effects by CDC-42 loss. Cadherin accumulation at RME-1, RAB-11, and the Golgi were compared in controls and in animals depleted of cdc-42 by RNAi. (B) Summary of results, as in Figure 3D and Supplemental Table S1. All statistical analysis in this figure used unpaired t test with Welch’s correction. (C) AMAN-2 levels at apical and basal regions in controls and in animals depleted of cdc-42 by RNAi. (D) Effect of blocking retromer, with snx-3(tm1595) mutation, on cadherin accumulation at the Golgi. Arrowheads indicate lateral membrane. Panel on the right is overexposed for GFP to show altered AMAN-2::GFP in the snx-3 mutant. (E) Effects of depleting the WAVE complex on transport of other cargoes, TGN-38:GFP and MIG-14/Wls::GFP, measured by Line Scans in ImageJ. (F) Overlap of MIG-14::GFP with the Golgi in controls and in animals depleted of gex-3.

FIGURE 5:

WAVE regulates vesicle dynamics and polarized movements. (A) The movements of endosomes were measured for velocity and directionality in apical and basal regions of the intestine using the ImageJ plug-in MTrackJ. Using the same strains as in Figure 3, controls and animals depleted of gex-3 via RNAi were imaged in one Z plane (either apical focus or basal focus) for 31 time points, as explained further in Materials and Methods. Numbered, colored marks indicate tracks of individual endosomes. Small white arrows show direction of the tracks over time. Large yellow arrow in bottom right panel indicates basal aggregates of GFP:RAB-11. All images shown are apical focus views of the intestine, except the bottom two of basal GFP::RAB-11, as explained in Figure 1A. (B) Graphs of velocity comparisons and tables of average velocity and mobile fraction. Movies of at least four animals were used for each genotype, with 100 to more than 500 individual movements tracked, depending on the strain. Statistical analysis used an unpaired t test. (C) Measurements of directionality toward apical or basal intestine. Graphs and polar plots show percentage of total movements toward apical or basal regions from the RFP::RME-1 and RAB-11::GFP movies, made with the apical focus view of the intestine. See Materials and Methods for details. See Supplemental Movies S2, S3, and S4 for representative movies for RFP::RAB-10, RFP::RME-1, and GFP::RAB-11. The apically oriented movements (red in the cartoon) and basally directed movements (blue in the cartoon) were plotted and statistically analyzed with a one-way ANOVA with Sidak’s multiple comparisons test.

FIGURE 6:

Model for how cadherin is transported in the presence, wild type, left, or the absence, right, of WAVE-dependent branched actin. Cadherin molecules that are endocytosed from the membrane can be sorted to late endosomes and then lysosomes for degradation or returned to the Golgi for refolding and modifications and recycled back to the apical membrane. Newly synthesized cadherin exits the Golgi with the help of WVE-1–dependent branched actin. In the absence of WAVE, 1) some retrograde transport steps are disrupted, trapping more cadherin at the Golgi and at some recycling endosomes; 2) apically directed transport of cadherin is reduced; and 3) exit of newly made cadherin molecules from the Golgi is also disrupted.