The RabGAPs EPI64A and EPI64B regulate the apical structure of epithelial cells †

Abstract

Here we report on the related TBC/RabGAPs EPI64A and EPI64B and show that they function to organize the apical aspect of epithelial cells. EPI64A binds the scaffolding protein EBP50/NHERF1, which itself binds active ezrin in epithelial cell microvilli. Epithelial cells additionally express EPI64B that also localizes to microvilli. However, EPI64B does not bind EBP50 and both proteins are shown to have a microvillar localization domain that spans the RabGAP domains. CRISPR/Cas9 was used to inactivate expression of each protein individually or both in Jeg-3 and Caco2 cells. In Jeg-3 cells, loss of EPI64B resulted in a reduction of apical microvilli, and a further reduction was seen in the double knockout, mostly likely due to misregulation of Rab8 and Rab35. In addition, apical junctions were partially disrupted in cells lacking EPI64A and accentuated in the double knockout. In Caco2 loss of EPI64B resulted in wavy junctions, whereas loss of both EPI64A and EPI64B had a severe phenotype often resulting in cells with a stellate apical morphology. In the knockout cells, the basal region of the cell remained unchanged, so EPI64A and EPI64B specifically localize to and regulate the morphology of the apical domain of polarized epithelial cells.

FIGURE 1:

EPI64A and EPI64B both localize to apical microvilli. (A) Schematic of EPI64A and EPI64B domains with percentage identities and the TBC/RabGAP arginine residue necessary for the RabGAP activity indicated. (B) Western blots with antibodies to EPI64B and tubulin of Jeg-3 cell lysates after treatment with the indicated siRNAs to EPI64B. (C) Western blot with antibodies to EPI64B on cell lysates from several cultured cell lines. (D) SIM microscopy of Jeg-3 cells transfected with GFP-EPI64A (top block of panels) or GFP-EPI64B (bottom block of panels). Cells were transfected to express GFP-tagged constructs (green) and stained for ezrin (red) and actin (blue). Arrows indicate the localization of GFP-EPI64A to the base of microvilli. Scale bars: top panels, 10 µm; bottom panels, 10 µm.(E) Quantitation of GFP-EPI64A and GFP-EPI64B to localize to microvilli in wild-type cells.

FIGURE 2:

EPI64A and EPI64B can localize to microvilli independently of the scaffolding protein EBP50 (A) Jeg-3 cells transfected with 3xFLAG-EBP50 were co-transfected with either GFP-EPI64A or GFP-EPI64B. The GFP-tagged proteins were immunoprecipitated with GFP-Trap beads and the immunoprecipitates blotted for FLAG and GFP. (B) Confocal imaging of microvillar localization of GFP-EPI-64A and GFP-EPI64A-LA, which cannot bind EBP50, in Jeg-3 cells. Scale bar 10 µm. (C) Western blot of cell lysates of Jeg-3 wild type, or CRISPR-modified EBP50 deletion cell line, blotted for ezrin, EBP50, and tubulin. Scale bar: 10 µm (D) Localization of ezrin and actin in Jeg-3 cells lacking endogenous EBP50. Scale bars: 10 µm. (E) Confocal imaging of GFP-EPI64A or GFP-EPI64B in Jeg-3 cells lacking EBP50. Scale bars: 10 µm.

FIGURE 3:

EPI64A contains a localization domain spanning its TBC domain. (A) Schematic of the EPI64 constructs used in B and C and summary of results shown in this figure (B) Confocal imaging of GFP-EPI64A-314-508, which contains the C-terminal -DTYL sequence, in Jeg-3 wild-type cells and Jeg-3 cells lacking EBP50. (C) Confocal images showing the localization of GFP-tagged deletion constructs of GFP-EPI64A. Scale bar 10 µm. (D) Immunolocalization of two HA-tagged constructs, the top one containing the minimal region that localizes to microvilli (HA-EPI64A-61-408) and the bottom one (HA-71-408) that does not localize. Scale bar: 10 µm. (E) GFP-trap pull down: Jeg-3 cells were transfected with either GFP or GFP-Arf6 together with the indicated HA-EPI64A constructs. The GFP or GFP-Arf6 were recovered and analyzed for the presence of the HA-EPI64A constructs by immunoblotting.

FIGURE 4:

EPI64B also contains a localization domain spanning the TBC domain. (A) Schematic of GFP-EP64B constructs used, and summary of results shown in this figure. Confocal imaging of (B) the N-terminal GFP-EPI64AB-1-275, (C) GFP-EPI64B-276-808, (D) GFP-EPI64B-310-657, the region homologous to EPI64A-61-408, and (E) the C-terminal GFP-EPI64B-651-808. Scale bars all: 10µm.

FIGURE 5:

Jeg-3 cells lacking EPI64A and EPI64B lack microvilli (A) Western blot with antibodies to EPI64A, EPI64B, and tubulin on whole cell lysates of Jeg-3 cells genetically modified to lack EPI64A, EPI64B, or both proteins (DKO). (B) Confocal imaging showing localization of ezrin and actin in wild-type Jeg-3 cells and the single (A-KO, B-KO) and double knockout cells. Scale bar 10 µm. (C) Quantitation of the percentage of indicated cells stained for ezrin that express surface microvilli. Normal defined >50% coverage of the apical surface with microvilli. One-way analysis of variance gave the indicated p values. (D) EPI64A/B double knockout cells were transfected to express the indicated constructs and the percentage of ezrin-stained cells (total for either untransfected or GFP-expressing for transfected cells) that express normal apical microvilli. One-way analysis of variance gave the indicated p values. (E) Localization of tight junction ZO-1 in wild-type and knockout Jeg-3 cells. Scale bar: 10 µm.

FIGURE 6:

Dominant negative Rab8A and Rab35A can restore microvilli to EPI64A/B double knockout cells. (A) Wild-type Jeg-3 cells were transfected to express GFP or the indicated GFP-Rab proteins and the percentage of cells (total for either untransfected or GFP-expressing for transfected cells) determined that express apical microvilli. (B) Jeg-3 EPI64A/B double knockout cells were transfected with GFP or the indicated GFP-Rab proteins and the percentage of cells (total for either untransfected or GFP-expressing for transfected cells) expressing microvilli scored. One-way analysis of variance gave the indicated p values.

FIGURE 7:

Caco-2 cells lacking EPI64A and EPI64B have microvilli (A) Western blots of whole cell lysates from Caco-2 BBE1 wild-type, EPI64A, and EPI64B single knockout and double knockout cells blotted for EPI64A, EPI64B, and tubulin. (B) Confocal imaging showing localization of ezrin in the apical region of wild-type and knockout cells. Scale bar: 10 µm. (C) Localization of GFP-EPI64A and GFP-EPI64B expressed in double knockout cells. Scale bar 10 µm.

FIGURE 8:

Caco-2 cells lacking EPI64A and EPI64B have aberrant apical junctions (A) Fields of wild-type, EPI64A and EPI64B single knockout and EPI64A/B double knockout cells stained for actin and the tight junction marker ZO-1. The phenotypes seen were variable, so the most wild–type-looking regions of cells are shown (Normal) and contrasted with regions where the normal polygonal organization is disrupted (Severe). Scale bar: 10 µm. (B) Percentage of wild-type and knockout cells in which one or more of its junctions shows a reflex angle (>180°). One-way analysis of variance gave the indicated p values. (C) Example of stellate knockout cell stained for ezrin, myosin IIA, and actin XY-dimensions (top panels) and YZ-dimensions (bottom panel). (D) Localization of vinculin, actin, and myosin IIA in the apical (top panels) and basal (bottom panels) sections of wild-type and double knockout Caco-2 cells. Scale bars 10 µm.