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First published online 12 September 2007
doi: 10.1242/jcs.006049

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mDia2 regulates actin and focal adhesion dynamics and organization in the lamella for efficient epithelial cell migration

¹ Department of Cell Biology, The Scripps Research Institute, La Jolla, CA 92037, USA
² Van Andel Research Institute, 333 Bostwick Avenue, Grand Rapids, MI 49503, USA
³ Department of Cell Biology, The Scripps Research Institute, La Jolla, CA 92037, and Cell Biology and Physiology Center, National Heart, Lung and Blood Institute, Building 50 South Drive, Bethesda, MD 20892-8019, USA

View larger version (69K): [in this window] [in a new window]   Fig. 1. PtK1 cells express mDia2, which localizes in the lamella and cell center and not to the lamellipodium. Whole cell lysates from HEK 293T cells expressing either YFP-fused mDia1 (A) or Myc-tagged mDia2 (B), as well as HeLa and Ptk1 cell lysates (A-C) were prepared. Lysates were immunoblotted with antibodies directed against mDia1 (A), mDia2 (B), or mDia3 (C). (D) Fluorescent phalloidin staining of F-actin and immunofluorescence of tubulin and mDia2. In the merged image, tubulin is blue, mDia2 is green, and F-actin is red. mDia2 partially colocalizes with microtubules in the lamella and is excluded from near the cell edge. The cell edge as determined from phase-contrast images (not shown) is outlined in white in this and subsequent images. (E) Immunofluorescent staining for Arp3 and mDia2 in PtK1 cells. In the merged image, Arp3 is in red, and mDia2 is in green. Arp3 concentrates in a thin band along the cell edge in the lamellipodium where mDia2 is depleted. (F) Immunofluorescent staining for myosin II regulatory light chain phosphorylated at Ser-19 (pMLC) and mDia2. Merged image shows pMLC in red and mDia2 in green. mDia2 is present in the lamella where pMLC is concentrated. (G) Paxillin and mDia2 immunofluorescence, paxillin in red, mDia2 is in green. mDia2 and paxillin do not colocalize.

View larger version (35K): [in this window] [in a new window]   Fig. 2. mDia2 forms a stable pool of cortical actin in the lamella. (A) Example of a TIR-FRAP experiment; X-Rhodamine actin TIRF images taken from a time-lapse movie of a PtK1 control cell prior to and after fluorescence photobleaching of 150 nm of the ventral cell cortex, which occurs at t=0. Lamellipodium measurements were taken at the extreme cell edge (red ovals). Green ovals indicate regions where fluorescence intensity measurements were taken in the lamella. (B) t1/2 of fluorescence recovery of F-actin in the lamellipodium of control (n=5) and mDia2 antibody (anti-mDia2, n=5) -injected cells. (C) Example of actin fluorescence recovery data (diamonds) fit to a single term exponential (squares) in the lamellipodium of a control PtK1 cell. (D) Percentage of fluorescence recovery after photobleaching of F-actin in the lamella of control (n=5) and mDia2 inhibited cells (n=5, ± s.d.). mDia2 antibody inhibition mobilizes a stable, non-recovering pool of fluorescent actin. (E,F) Examples of fluorescence recovery data (diamonds) fit to a single term exponential (black line) obtained from the lamella of (E) a control PtK1 cell and (F) an mDia2 antibody-injected cell. (G) Data from F (diamonds) fit to a two-term exponential (black line), revealing that F-actin in the lamella of mDia2 antibody-injected cells has a different mechanism of fluorescence recovery than controls, in which the data was fit by a single exponential.

View larger version (94K): [in this window] [in a new window]   Fig. 3. mDia2 is necessary for the segregation of distinct dynamic F-actin behavior of the lamellipodium and lamella. Phase-contrast (A) and FSM (B) images of X-Rhodamine actin in control and mDia2 antibody-injected (anti-mDia2) cells. (C) Kymographs taken along the axis of F-actin flow, indicated by the lines in B. Three kymographs were taken from each of five cells per treatment for the analyses shown in G and I. In C, lines indicate the F-actin speckle flow rate in the lamellipodium (LP) and lamella (LA); arrowheads indicate lack of the rapid retrograde flow typical of the LP. (D) qFSM maps of F-actin polymerization (red) and depolymerization (green) rates in control and mDia2 antibody-injected (anti-mDia2) cells. Brightness indicates the relative magnitude of the rate. The arrow indicates a wide region of rapid F-actin polymerization along the cell edge (LP), and the arrowhead indicates a region of no rapid F-actin polymerization at the cell edge (no LP). (E) qFSM maps of the speed of F-actin flow in control and mDia2 antibody-injected (anti-mDia2) cells; the arrow indicates a shallow speed gradient. The arrowhead indicates a region of no rapid retrograde flow at the cell edge. (F) qFSM speckle velocity from regions indicated by boxes in E. (G) Average rates of F-actin retrograde (–) or anterograde flow (+) in the LP and LA, determined from kymographs of FSM movies (± s.e.m.). (H) Rate of F-actin flow as a function of distance from the cell edge in control and anti-mDia2 antibody injected cells. F-actin flow speed at all points in the cell, as determined by qFSM, was averaged parallel to the cell edge in 1 µm intervals behind the cell edge for five control and anti-mDia2 antibody-injected cells. (± s.e.m.). (I) Percentage of time in which fast flow of lamellipodium was present, as analyzed from kymographs (± s.e.m.). The asterisk in I indicates statistical significance (P<0.05) between control and mDia2 antibody-injected cells. (J,K) qFSM maps of (J) the speed of F-actin flow and (K) of F-actin polymerization (red) and depolymerization (green) rates in a PtK1 cell expressing a dominant negative FH2 (FH2) construct.

View larger version (62K): [in this window] [in a new window]   Fig. 4. mDia2 is necessary for normal focal adhesion morphometry. (A) Paxillin immunofluorescence in control, mDia2 antibody-injected (anti-mDia2), dominant negative mutant expressing (FH2), and mDia1 antibody-injected (anti-mDia1) cells. (B) Control and mDia2 antibody-injected cells expressing GFP-paxillin. (C,D) Average number per cell (C), and size (D) of paxillin stained focal adhesions ± s.e.m., n=12 cells per treatment.

View larger version (103K): [in this window] [in a new window]   Fig. 5. mDia2 is necessary for normal focal adhesion dynamics. (A) Example of focal adhesion dynamics seen in frames from a time-lapse spinning disk confocal microscopy image series of GFP-paxillin in control and anti-mDia2 antibody-injected cells, arrows and arrowheads indicate focal adhesion assembly and disassembly respectively. (B) The average rate constants of FA assembly and disassembly measured from 5-20 focal adhesions per cell in four to five cells per condition (± s.d.). (C) Example of a TIRF-FRAP experiment; GFP-paxillin TIRF images were taken from a time-lapse of a PtK1 cell prior to and after fluorescence photobleaching of the ventral cell cortex, which occurs at t=0. (D) t1/2 of fluorescent recovery after photobleaching of GFP-paxillin in focal adhesions in control and mDia2 antibody-injected cells, n=5 cells per treatment. (E) Percentage of fluorescent recovery after photobleaching of GFP-paxillin in focal adhesions in control and mDia2 antibody-injected cells.

View larger version (73K): [in this window] [in a new window]   Fig. 6. mDia2 maintains free filament barbed ends at focal adhesions. Barbed-end actin incorporation (green) and fluorescent phalloidin staining of F-actin (red) in a control cell (A) and mDia2 antibody-injected cell (anti-mDia2; B). To find mDia2 antibody-injected cells after permeabilization, mDia2 antibody was co-microinjected with fluorescent tubulin that can be seen incorporated into microtubules that are easily distinguishable from actin structures. Arrows indicate plaques at the termini of actin bundles (arrowheads). (C) Intensity ratio of fluorescent actin incorporation marking free barbed filament ends relative to total F-actin (phalloidin) at the terminal 2 µm of actin bundles (n=10 cells per treatment, 5-10 bundles/cell). (D) Intensity ratio of fluorescent actin incorporation marking free barbed filament ends relative to total F-actin (phalloidin) (± s.e.m.) from the leading-edge into the cell center is not altered by mDia2 inhibition, n=10 cell per treatment, three regions per cell. (E,F) Fluorescent phalloidin, barbed-end actin incorporation, and paxillin immunofluorescence in a control cell (E) and an mDia2 antibody-injected cell (F). In a control cell, actin incorporates at focal adhesions that are positive for paxillin (arrowheads), but this does not occur in mDia2 antibody-injected cells. Note the altered focal adhesion morphology in cells injected with mDia2 antibody as seen by paxillin immunofluorescence (F), suggesting that antibody inhibition renders focal adhesions labile to the pre-permeabilization procedure required for localization of free barbed ends.

View larger version (79K): [in this window] [in a new window]   Fig. 7. mDia2 is necessary for concerted leading edge protrusions and retractions and rapid cell migration. (A) Control and mDia2 antibody-injected (anti-mDia2) cells were microinjected with X-Rhodamine actin and filmed by time-lapse spinning-disk confocal microscopy. Using qFSM software, the position of the cell edge was extracted from each image and are shown as changing from warm to cool colors over time. The edge of control cells are smooth and protrude and adjacent regions retract in a concerted fashion, whereas mDia2 antibody-injected cells displayed a much more ragged cell edge. (B) Kymographs taken from phase-contrast time-lapse images used to measure parameters of leading-edge dynamics. (C) The distance, rate and frequency of protrusions and retractions were not affected by mDia2 inhibition; n=10 cells per treatment, three measurements per cell. (D) The rate of PtK1 cell migration was significantly reduced by mDia2 inhibition, n=30 cells per treatment.