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. 2016 Aug 3;7:12426.
doi: 10.1038/ncomms12426.

Cingulin and Actin Mediate Midbody-Dependent Apical Lumen Formation During Polarization of Epithelial Cells

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Free PMC article

Cingulin and Actin Mediate Midbody-Dependent Apical Lumen Formation During Polarization of Epithelial Cells

Anthony J Mangan et al. Nat Commun. .
Free PMC article

Abstract

Coordinated polarization of epithelial cells is a key step during morphogenesis that leads to the formation of an apical lumen. Rab11 and its interacting protein FIP5 are necessary for the targeting of apical endosomes to the midbody and apical membrane initiation site (AMIS) during lumenogenesis. However, the machinery that mediates AMIS establishment and FIP5-endosome targeting remains unknown. Here we identify a FIP5-interacting protein, Cingulin, which localizes to the AMIS and functions as a tether mediating FIP5-endosome targeting. We analysed the machinery mediating AMIS recruitment to the midbody and determined that both branched actin and microtubules are required for establishing the site of the nascent lumen. We demonstrate that the Rac1-WAVE/Scar complex mediates Cingulin recruitment to the AMIS by inducing branched actin formation, and that Cingulin directly binds to microtubule C-terminal tails through electrostatic interactions. We propose a new mechanism for apical endosome targeting and AMIS formation around the midbody during epithelial lumenogenesis.

Figures

Figure 1
Figure 1. CGN is an AMIS-associated FIP5-binding protein.
(a) Schematic model showing the formation of a nascent apical lumen. Rab11/FIP5 endosomes are transported along central spindle microtubules and fuse to the AMIS present at the midbody during late telophase. (b) List of proteins identified in FIP5 immunoprecipitate by mass spectroscopy analysis. Proteins known to be present in the same complex are highlighted together. (c) FIP5 co-immunoprecipitation with GFP-CGN from MDCK lysates. Images shown are the immunoblots after probing with anti-CGN (top gel) or anti-FIP5 (bottom gel) antibodies. (d) Co-immunoprecipitation of purified 6His-CGN with 6His-FIP5. Images shown are the immunoblots after probing with anti-CGN (top gel) or anti-FIP5 (bottom gel) antibodies. (e) Mapping 6His-FIP5 binding domain using glutathione bead pull-down assays. Image shown is Coomassie stained SDS–PAGE gel.
Figure 2
Figure 2. CGN is required for the formation of a single apical lumen.
MDCK cells stably expressing CGN shRNA were embedded in 3D Matrigel and allowed to grow for 4 days (ad) or 24 h (el) in the presence (dox+) or absence (dox−) of doxycycline. Cells were then fixed and stained with phalloidin-Alexa594 (ac,eh,k,l; red), anti-CGN (ac and eh; green), anti-tubulin (i,j; red) or anti-FIP5 (i,j; green) antibodies. Asterisks in (g,h,j) mark ectopic lumen formation sites. Arrows in (k) point to the AMIS. (d) shows quantification of cysts with one, two, or multiple lumens. Data shown are means and s.d. of three independent experiments. Consecutive images without an individual letter label show the same cell.
Figure 3
Figure 3. Components of the WAVE/Scar complex and branched actin filaments are present at the midbody during lumen formation.
(a) List of proteins identified in CGN immunoprecipitate by mass spectroscopy. Proteins known to interact as a complex are highlighted together. (b) Schematic representation of the WAVE/Scar complex. (ci) MDCK cells were embedded in 3D Matrigel and allowed to grow for 24 h. Cells were then fixed and stained with phalloidin-Alexa594 (gi; red), anti-CGN (f,g,i), anti-acetylated tubulin (c; red and h; green) or anti-Nap1 (ce; green) antibodies. In (f), cell is expressing Arp3-GFP. White arrows identify structures as labelled. Consecutive images without an individual letter label show the same cell.
Figure 4
Figure 4. Rac1 is activated at the AMIS.
(ad) MDCK cells transiently expressing CFP-Rac1 (green) were embedded in 3D Matrigel and allowed to grow for 24 h. Cells were then fixed and stained with phalloidin-Alexa594 (a,b,d; red) or anti-CGN (c; red) antibodies. (eg) MDCK cells transiently expressing FRET-based Rac1 biosensor were embedded in 3D Matrigel and imaged either at metaphase (e) or telophase (f). (g) shows quantification of FRET signal at the AMIS (area #1) and cell periphery (area #2) during telophase. Data shown are the means and s.d. derived from six cells. (h) MDCK cells transiently expressing CFP-Arf6 (red) were embedded in 3D Matrigel and allowed to grow for 24 h. Cells were then fixed and stained with anti-acetylated tubulin (red) antibodies. Arrows mark the midbody. Consecutive images without an individual letter label show the same cell.
Figure 5
Figure 5. Rac1 inhibition affects midbody-associated AMIS formation.
(ad) MDCK cells were embedded in 3D Matrigel and allowed to grow for 24 h. Where indicated, cells were treated with 200 μM Rac1 inhibitor (bd). Cells were then fixed and stained with phalloidin-Alexa594 (red) and anti-CGN (green) antibodies. White boxes indicate area zoomed in and shown in far right box. White arrows point to nascent lumen, black arrows point to areas of actin concentration. Consecutive images without an individual letter label show the same cell. (e,f) MDCK cells stably expressing GFP-CGN were embedded in 3D Matrigel. Untreated (e) or Rac1 inhibitor treated (f) cells were then imaged using time-lapse microscopy. In all cases, cell progression from metaphase to abscission was analysed. Arrow in (e) points to the midbody associated GFP-CGN.
Figure 6
Figure 6. Rac1 is required for the gp135 targeting during apical lumen formation.
(ad) MDCK cells transiently expressing GFP-Abi2 were embedded in 3D Matrigel and allowed to grow for 24 h. Where indicated, cells were treated with 200 μM Rac1 inhibitor (b,d). Cells (a,b) were then fixed and stained with anti-acetylated tubulin (red) antibodies. White arrows (a,b) point to the midbody. (eh) MDCK cells were embedded in 3D Matrigel and allowed to grow for 24 h. Where indicated, cells were treated with 200 μM Rac1 inhibitor (g,h). Cells were then fixed and stained with anti-gp135 (green) and anti-CGN (red) antibodies. White arrow in (e) points to the midbody. White arrow in (g) points to misplaced CGN. (i,j) MDCK cells were embedded in 3D Matrigel and allowed to grow for 2 days. Cells were treated with 200 μM Rac1 inhibitor for the first 12 h of the incubation. Cells were then fixed and stained with anti-gp135 (green) and anti-CGN (red) antibodies. White arrow in (j) points to misplaced CGN. Consecutive images without an individual letter label show the same cell.
Figure 7
Figure 7. Rac1 is required for the formation of a single apical lumen.
(a) Schematic representation showing timing of 12 h treatment with Rac1 inhibitor. (be) MDCK cells were embedded in 3D Matrigel and allowed to grow for 4 days in the absence (c) or presence (d,e) of Rac1 inhibitor. The timing of inhibitor addition is shown in (a). Cells were then fixed and stained with phalloidin-Alexa594 (red) and anti-CGN (green) antibodies. Consecutive images without an individual letter label show the same cell. (b) shows quantification of cells with single or multiple lumens. Data shown are the means and s.d. derived from three independent experiments.
Figure 8
Figure 8. Electrostatic interactions mediate CGN binding to microtubule CTTs.
(a) Microtubule binding assay showing amount of pelleted GST-CGN(1–406) and GST-CGN(1015–1203) when incubated with (+) and without (−) taxol-stabilized microtubules (tubulin). Anti-GST western blot (top gel), and Coomassie stained SDS–PAGE gel (bottom gel) are shown. (b) Diagram depicting positioning of α- and β-tubulin CTTs within polymerized microtubule. (c) Amino acid sequence alignment comparing CTTs of human and yeast β-tubulins. (d) Microtubule binding assay comparing amount of CGN(1–406) that pellets with wild-type tubulin and subtilisin-digested tubulin. Coomassie stained SDS–PAGE gel is shown. (e) Microtubule binding assay comparing CGN(1–406) pelleting with wild-type yeast tubulin and mutant yeast tubulin containing no β-CTT. Anti-tubulin and anti-CGN western blot is shown. (f) Amino acid sequence alignment comparing CGN basic patch in different vertebrate species. Boxes mark the sites mutated to alanine for binding analysis shown in (g). (g) Quantification comparing microtubule binding of wild-type CGN(1–406), CGN-M1 (R36/37A) and CGN-M2 (R40/41A). Data shown are the means and s.d. derived from four independent binding experiments.
Figure 9
Figure 9. Mutation of basic patch disrupts subcellular CGN targeting.
MDCK-CGN-KO cells were transfected with either wild-type CGN-GFP (a,b) or CGN-M1/M2-GFP mutant (R36/37/40/41A) (ce). Cells were embedded in Matrigel, incubated for 12 h, fixed and stained with phalloidin-Alexa596. Arrows in (a) point to actin rich lumen surrounded by CGN-GFP-containing TJs. Arrows in (c) point to actin accumulation at the neck of bud-like structures. Arrows in (d) point to ectopic accumulation of actin and CGN rings. Finally, arrows in (e) point to intracellular formation of mini-lumens in CGN-KO cells.
Figure 10
Figure 10. CGN interaction with midbody microtubules and Rac1-induced branched actin cytoskeleton is required for AMIS formation and apical lumen initiation.
Model depicting pathways that lead to CGN recruitment and AMIS formation at the midbody as well as the targeting/tethering of Rab11/FIP5 apical endosomes to form a single apical lumen.

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