Tropomyosin Regulates Cell Migration during Skin Wound Healing

Precise orchestration of actin polymer into filaments with distinct characteristics of stability, bundling, and branching underpins cell migration. A key regulator of actin filament specialization is the tropomyosin family of actin-associating proteins. This multi-isoform family of proteins assemble into polymers that lie in the major groove of polymerized actin filaments, which in turn determine the association of molecules that control actin filament organization. This suggests that tropomyosins may be important regulators of actin function during physiological processes dependent on cell migration, such as wound healing. We have therefore analyzed the requirement for tropomyosin isoform expression in a mouse model of cutaneous wound healing. We find that mice in which the 9D exon from the TPM3/γTm tropomyosin gene is deleted (γ9D -/-) exhibit a more rapid wound-healing response 7 days after wounding compared with wild-type mice. Accelerated wound healing was not associated with increased cell proliferation, matrix remodeling, or epidermal abnormalities, but with increased cell migration. Rac GTPase activity and paxillin phosphorylation are elevated in cells from γ9D -/- mice, suggesting the activation of paxillin/Rac signaling. Collectively, our data reveal that tropomyosin isoform expression has an important role in temporal regulation of cell migration during wound healing.(NHMRC) grant 51225

Paxillin phosphorylation is increased in NEDD9 −/− MEFs.

<p>A. Paxillin (pax) and phospho-paxillin (ppax) immunostaining. Merged images show colour overlays of paxillin (green) and phospho-paxillin (red). Right hand panels show ratio images of paxillin phosphorylation. Red hues reflect regions of highest phosphorylated paxillin. Boxed insets shows magnified focal adhesions. B. Ratio of phosphorylated paxillin at focal adhesions in WT (n = 197 from 10 individual cells) and NEDD9 −/− MEFs (n = 201 from 10 individual cells). ***p<0.0001, Students' <i>t</i>-test.</p

Adhesion signalling pathways are differentially activated in 2D versus 3D.

<p>A. Western blot analysis of lysates extracted from WT MEF and NEDD9−/− MEF grown on tissue culture plastic dishes (2D) or in collagen gels (3D). Blots were probed with the indicated antibodies to total proteins and phosphorylated proteins (-p-). Note that total Src was immunoprecipitated (IP) prior to probing with anti-p-Src antibodies. Blots were probed with Hsp70 or tubulin as a loading control. B. Histograms showing densitometric measurements of the level of phosphorylated protein divided by the matching total protein amount and expressed relative to the level in WT MEFs under 2D culture conditions. Data represent the average of triplicate repeats on separate days and error bars show the S.E.M. *<i>p</i><0.05, ** <i>p</i><0.001, NS = not significant, Students' <i>t</i>-test.</p

Reduced stress fibres and activated β1 in NEDD9−/− MEFs in 3D.

<p>A. Activated β1 integrin (detected using β1 antibody clone 9EG7) and actin (detected with fluorescently-tagged phalloidin) immunostaining of cells grown in 2D conditions. Merged images show colour overlays of β1 (green) and actin (red). Arrows point to examples of active β1 integrin associated with the ends of actin stress fibres. Cells were imaged by wide-field microscopy. B. Activated β1 integrin and actin immunostaining of cells grown in 3D collagen gels. Boxed insets shows magnified regions containing actin stress fibres. Arrow heads point to actin stress fibres. Images represent single confocal z-slices.</p

NEDD9 increases 3D migration speed.

<p>A. Western blot analysis of cell extracts following treatment with siRNA targeting NEDD9 or control siRNA sequences. Blots were probed with antibodies to NEDD9 which detects 105 kD and 115 kD NEDD9 phospho-forms and cross-reacted with p130Cas as indicated. Probing with anti-tubulin antibodies confirmed equal loading. B. MSD calculated from trajectories of WT MEFs (black squares, average of 3 independent experiments), NEDD9−/− MEFs (white squares, average of 3 independent experiments n>22 cells per experiment), MEFs treated with control siRNA (black circles, average of n = 38 cells) and NEDD9 siRNA (white circles, average of n = 25 cells). Note that identical values were obtained for the WT MEFs and WT MEFs treated with control siRNA; these data points are therefore super-imposed on the graph. C. Cell speed is significantly reduced in NEDD9−/− MEFs and in NEDD9 siRNA treated cells relative to the matched controls. Horizontal bars indicate the average speed for all cells tracked. D. Cell persistence is significantly reduced in NEDD9−/− and NEDD9 siRNA treated MEFs. Histogram shows the average persistence for all cells tracked. *<i>p</i><0.05, ** <i>p</i><0.001, *** <i>p</i><0.0001, Students' <i>t</i>-test.</p

NEDD9 −/− MEFs have increased migration speed in 2D cell migration assays.

<p>A. MSD calculated from trajectories of WT (black squares), and NEDD9−/− (white squares) MEFs in 2D cell migration assays. Data are the average values from 3 independent experiments, n>45 cells tracked per experiment. B. Cell speed is significantly increased in NEDD9−/− MEFs (WT n = 150, −/− n = 143). C. MSD calculated from the trajectories of GFP-transfected NEDD9−/− MEFs (black squares) and GFP.NEDD9 transfected NEDD9−/− MEFs (white squares). D. GFP.NEDD9 expression significantly reduces the migration speed of NEDD9−/− MEFs. (n = 53 each). *** <i>p</i><0.0001, Students' <i>t</i>-test.</p

Focal adhesion turnover is more rapid in the absence of NEDD9 expression.

<p>A. Western blot analysis of extracts of wild-type (WT) and NEDD9 knockout (−/−) MEFs. Blots were probed with antibodies to NEDD9 which detects the 105 kD and 115 kD phospho-forms of NEDD9 and p130Cas antibodies as indicated. Blots were then probed with anti-tubulin or anti-HSP70 antibodies to confirm equal loading. B. WT and NEDD9 −/− MEFs immunostained with antibodies to paxillin (pax) to detect focal adhesions (white arrows) and TRITC-phalloidin to detect actin stress fibres. C. Examples of YFP-paxillin positive focal adhesions in transfected WT (72 minute lifetime) and NEDD9−/− MEFs (52 minute lifetime) are shown. D. Focal adhesion assembly rate constants (<i>k</i>) for cells transfected with YFP-paxillin (n = 32 focal adhesions per cell line). Horizontal bars show the average value per cell line. E. As for D, except data showing the disassembly rate constants (<i>k</i>). (WT n = 26, −/− n = 33). *p<0.01, N.S. = not significant.</p

Tropomyosin regulates cell migration during skin wound healing

Precise orchestration of actin polymer into filaments with distinct characteristics of stability, bundling, and branching underpins cell migration. A key regulator of actin filament specialization is the tropomyosin family of actin-associating proteins. This multi-isoform family of proteins assemble into polymers that lie in the major groove of polymerized actin filaments, which in turn determine the association of molecules that control actin filament organization. This suggests that tropomyosins may be important regulators of actin function during physiological processes dependent on cell migration, such as wound healing. We have therefore analyzed the requirement for tropomyosin isoform expression in a mouse model of cutaneous wound healing. We find that mice in which the 9D exon from the TPM3/γTm tropomyosin gene is deleted (γ9D -/-) exhibit a more rapid wound-healing response 7 days after wounding compared with wild-type mice. Accelerated wound healing was not associated with increased cell proliferation, matrix remodeling, or epidermal abnormalities, but with increased cell migration. Rac GTPase activity and paxillin phosphorylation are elevated in cells from γ9D -/- mice, suggesting the activation of paxillin/Rac signaling. Collectively, our data reveal that tropomyosin isoform expression has an important role in temporal regulation of cell migration during wound healing.Justin G. Lees, Yu Wooi Ching, Damian H. Adams, Cuc T.T. Bach, Michael S. Samuel, Anthony J. Kee, Edna C. Hardeman, Peter Gunning, Allison J. Cowin and Geraldine M. O’Neil