Dynamin 2 orchestrates the global actomyosin cytoskeleton for epithelial maintenance and apical constriction

Abstract

The mechanisms controlling cell shape changes within epithelial monolayers for tissue formation and reorganization remain unclear. Here, we investigate the role of dynamin, a large GTPase, in epithelial morphogenesis. Depletion of dynamin 2 (Dyn2), the only dynamin in epithelial cells, prevents establishment and maintenance of epithelial polarity, with no junctional formation and abnormal actin organization. Expression of Dyn2 mutants shifted to a non-GTP state, by contrast, causes dramatic apical constriction without disrupting polarity. This is due to Dyn2's interactions with deacetylated cortactin and downstream effectors, which cause enhanced actomyosin contraction. Neither inhibitors of endocytosis nor GTP-shifted Dyn2 mutants induce apical constriction. This suggests that GTPase-dependent changes in Dyn2 lead to interactions with different effectors that may differentially modulate endocytosis and/or actomyosin dynamics in polarized cells. We propose this enables Dyn2 to coordinate, in a GTPase-dependent manner, membrane recycling and actomyosin contractility during epithelial morphogenesis.

Fig. 1.

Dyn2-targeted siRNA inhibits epithelial monolayer formation and maintenance. (A) Blot analysis of cell lysate from MDCK cells transfected with Dyn2 or control siRNA. 1× or 2× indicates the number of times cells were subjected to siRNA transfection. (B) Maximum-intensity projection images of monolayers formed after transfection of MDCK cells in suspension with either control (Upper) or Dyn2 (Lower) siRNA and stained for ZO-1 (green) and actin (red). Note the severely disrupted tight junctions, increase in actin lateral staining, and lack of stress fibers in Dyn2 siRNA-treated cells. (C) Maximum intensity projection images of MDCK cells transfected in preformed monolayers with either control (Upper) or Dyn2 siRNA (Lower) and stained for ZO-1 (red) and Dynamin (green). Note the severely disrupted tight junctions in cells that do not stain for dynamin (Lower).

Fig. 2.

Apical constriction is induced by Dyn2K44A expression. (A–C) Maximum intensity projection images of monolayers transfected with Dyn2wt-GFP (A Top), GFP (A Middle), or Dyn2K44A-GFP (A Bottom, B, and C) and stained for ZO-1 (A), occludin (B), or claudin (C). Note constriction of the junctional belt in A Bottom, B, and C. (D) Schematic diagram of epithelial cells having undergone apical constriction (Left) or with normal morphology (Right). Ratio (AP/L) = the ratio of the apical area relative to an area of the lateral cross-section 1–2 μm below the AJC. (E) Distributions of individual ratio values in cells expressing Dyn2wt-GFP, Dyn2K44A-GFP, or GFP from three separate experiments. (F) Mean ratio (AP/L) in cells transfected with Dyn2wt-GFP, Dyn2K44A-GFP, or GFP from eight separate experiments. **, P < 0.001.

Fig. 3.

Apical constriction is not due to inhibiting endocytosis, and dynamin is enriched at the apical junctional region. (A) Maximum intensity projection images of cells expressing Dyn2wt-GFP (Left Upper, green) and Dyn2K44A-GFP (Left Lower, green) that were incubated with TxRed-transferrin (red) and stained for ZO-1 (blue). (B) Single plane images taken below AJC of Dyn2K44A-GFP-expressing cells subjected to TxRed-dextran applied either apically or basolaterally and imaged live. (C) Mean ratio (AP/L) for cells expressing Dyn2wt-GFP, Dyn2K44A-GFP, Eps15wt-GFP, Eps15Δ95/295-GFP, FLAG-Cav1wt, FLAG-Cav1Y14F, or Eps15Δ95/295-GFP and FLAG-Cav1Y14F in at least three separate experiments. **, P < 0.001. (D) Mean ratio (AP/L) for cells expressing Dyn2wt-GFP, Dyn2K44A-GFP, Dyn2K44A-CeFP, and Eps15Δ95/295-GFP, or Dyn2K44A-GFP and FLAG-Cav1Y14F from two independent experiments. (E) Single-plane images of a monolayer stained for dynamin (green) and ZO-1 (red) at the AJC (Upper) and 1 μm below the AJC (Lower). Note dynamin enrichment (arrows) at the AJC but not at the basolateral membranes. (F) XZ sections of cells expressing Dyn2wt-GFP (Left) and Dyn2K44A-GFP (Right) before and after treatment with 0.01% digitonin. Note the enrichment of Dyn2K44A-GFP relative to Dyn2wt-GFP (arrows). (G) FRAP recovery kinetics for Dyn2wt-GFP and Dyn2K44A-GFP in cells expressing an individual chimera.

Fig. 4.

The actomyosin contractile system is involved in Dyn2K44A-induced apical constriction. (A) Maximum intensity projection images of cells expressing Dyn2wt-GFP (Left Upper, green) or Dyn2K44A-GFP (Left Lower, green) and stained with TxRed-phalloidin (red) and occludin (blue). Note an increase in actin density at the AJC in Dyn2K44A-GFP-expressing cells. (B) Single plane images through the AJC of cells expressing Dyn2wt-GFP or Dyn2K44A-GFP and stained for occludin (red) and myosin II (gray). The apical junctional plane of the Dyn2K44A-GFP-expressing cell is above those of untransfected cells. (C) Mean ratio (AP/L) of no treatment, cytochalasin D-treated, or jasplakinolide-treated Dyn2K44A-GFP-expressing cells from three independent experiments. **, P < 0.001. (D) Mean ratio (AP/L) of Dyn2K44A-GFP-expressing cells incubated in 4 °C for 1 h, followed by no treatment, nocodazole treatment, or demecolcine treatment from three independent experiments. (E) Maximum intensity projection images of DMSO- or blebbistatin-treated Dyn2K44A-GFP-expressing cells that are stained for claudin (red). Note that inhibiting myosin II with blebbistatin relieves apical constriction. (F) Mean ratio (AP/L) of DMSO- or blebbistatin-treated, Dyn2K44A-GFP-expressing cells from four separate experiments. **, P < 0.001. (G) Mean ratio (AP/L) of DMSO-, Y27632-, or ML7-treated Dyn2K44A-GFP-expressing cells from three independent experiments. **, P < 0.001.

Fig. 5.

A shift in nucleotide status dictates dynamin's involvement in apical constriction. (A) Maximum intensity projection images of cells expressing Dyn2K44A-GFP, Dyn2S45N-CeFP, and Dyn2G146S-CeFP at 32 °C and Dyn2G146S-CeFP at 40 °C and stained for occludin (red). (B) Maximum intensity projection images of cells expressing Dyn2wt-GFP, Dyn2S61D-CeFP, and Dyn2G273D at 32 °C and Dyn2G273D-CeFP at 40 °C (green), and stained for occludin (red). (C) Mean ratio (AP/L) of cells expressing Dyn2wt-GFP, Dyn2K44A-GFP, Dyn2S45N-CeFP, and Dyn2S61D-CeFP. (D) Mean ratio (AP/L) of cells expressing Dyn2K44A-GFP and Dyn2K44A-I684K-CeFP. (E) Mean ratio (AP/L) of DMSO- or dynasore-treated, Dyn2K44A-GFP-expressing cells. **, P < 0.001.

Fig. 6.

Testing the role of cortactin and deacetylase in apical constriction induced by Dyn2K44A expression. (A) Maximum intensity projection images of Dyn2K44A-GFP-expressing cells cotransfected with FLAG-cortactinW525K (Upper) or FLAG-cortactinW22A (Lower) and stained for the FLAG tag (red) and occludin (blue). (B) Mean ratio (AP/L) for cells expressing Dyn2wt-GFP, Dyn2K44A-GFP, FLAG-cortactinW525K, FLAG-cortactinW22A, Dyn2K44A-GFP and FLAG-cortactinW525K, or Dyn2K44A-GFP and FLAG-cortactinW22A in three independent experiments. **, P < 0.001. Note that coexpression of Dyn2K44A-GFP and FLAG-cortactinW525K relieves apical constriction induced by Dyn2K44A. (C) Mean ratio (AP/L) for cells expressing Dyn2K44A-RFP only or Dyn2K44A-RFP and Dyn2ΔPRD-GFP in three independent experiments. **, P < 0.001. (D) Mean ratio (AP/L) for cells expressing Dyn2wt-GFP, Dyn2K44A-GFP, FLAG-Cort9KQ, Dyn2K44A-GFP and FLAG-Cort9KQ, FLAG-Cort9KR, or Dyn2K44A-GFP and FLAG-Cort9KR in two independent experiments. **, P < 0.001. Note that coexpression of Dyn2K44A and FLAG-Cort9KQ relieved apical constriction induced by Dyn2K44A. (E) Mean ratio (AP/L) for DMSO-treated, TSA-treated (5 h), or TSA-treated (overnight) cells expressing Dyn2K44A-GFP in two independent experiments. **, P < 0.001.