Zyxin Is Involved in Fibroblast Rigidity Sensing and Durotaxis

Abstract

Focal adhesions (FAs) are specialized structures that enable cells to sense their extracellular matrix rigidity and transmit these signals to the interior of the cells, bringing about actin cytoskeleton reorganization, FA maturation, and cell migration. It is known that cells migrate towards regions of higher substrate rigidity, a phenomenon known as durotaxis. However, the underlying molecular mechanism of durotaxis and how different proteins in the FA are involved remain unclear. Zyxin is a component of the FA that has been implicated in connecting the actin cytoskeleton to the FA. We have found that knocking down zyxin impaired NIH3T3 fibroblast's ability to sense and respond to changes in extracellular matrix in terms of their FA sizes, cell traction stress magnitudes and F-actin organization. Cell migration speed of zyxin knockdown fibroblasts was also independent of the underlying substrate rigidity, unlike wild type fibroblasts which migrated fastest at an intermediate substrate rigidity of 14 kPa. Wild type fibroblasts exhibited durotaxis by migrating toward regions of increasing substrate rigidity on polyacrylamide gels with substrate rigidity gradient, while zyxin knockdown fibroblasts did not exhibit durotaxis. Therefore, we propose zyxin as an essential protein that is required for rigidity sensing and durotaxis through modulating FA sizes, cell traction stress and F-actin organization.

Keywords: durotaxis; focal adhesion; mechanotransduction; rigidity sensing; zyxin.

FIGURE 1

Knocking down zyxin in NIH3T3 cells does not affect levels of other FA proteins (A) 1) Western blot and 2) its quantification showing levels of zyxin in NIH3T3 cells transfected with a control siRNA (siLuc) and zyxin siRNA (siZyx). β-actin levels are used as loading control (n = 3) (B) immunofluorescence staining for paxillin (red) and zyxin (green) in 1) control siLuc-NIH3T3 and 2) zyxin knockdown siZyx-NIH3T3 cells on a stiff polyacrylamide gel (61 kPa). Right panels show the magnified view of the yellow boxes and the white arrows denote the FA stained by paxillin (red) (C) Quantification of zyxin colocalization with paxillin in siLuc-NIH3T3 and siZyx-NIH3T3 cells on the stiff polyacrylamide substrate (61 kPa). Pearson’s coefficient of 1 indicates perfect colocalization, 0 indicates no colocalization (n = 8). Error bars represent standard error of the mean. *** represents p < 0.005.

FIGURE 2

Zyxin-knockdown NIH3T3 cells do not show increased cell-substrate adhesion and actin polarization in response to increasing substrate rigidity (A) Representative F-actin (green) and (B) paxillin (red) immunofluorescence staining images for siLuc- and siZxy- NIH3T3 cells at different substrate rigidity (C) Graph of cell area vs substrate rigidity for siLuc- and siZyx- NIH3T3 cells (D) Graph of focal adhesion (FA) area vs substrate rigidity for siLuc- and siZyx- NIH3T3 cells (E) Graph of F-actin coherency vs substrate rigidity for siLuc- and siZyx- NIH3T3 cells. n = 12, 16, 19 for siLuc NIH3T3 6 kPa, 14 and 31 kPa respectively. n = 10, 15, 11 for siZyx NIH3T3 6 kPa, 14 and 31 kPa respectively. Error bars represent standard error of the mean. **** represents p < 0.001, * represents p < 0.05 and n.s represents not significant.

FIGURE 3

Zyxin-knockdown NIH3T3 cells do not increase cell traction stress in response to increasing substrate rigidity (A–F) Traction stress maps of (A–C) siLuc-NIH3T3 cells and (D–F) siZyx-NIH3T3 cells on substrates of rigidity 6 kPa, 14 and 31 kPa. Arrows denote the direction and magnitude of the traction stress in the x- and y-directions (G) Average traction stress magnitudes of siLuc- and siZyx- NIH3T3 cells on substrates of rigidity 6, 14, and 31 kPa, n = 11, 22, 19 for siLuc-NIH3T3 6 kPa, 14 and 31 kPa, respectively. n = 17, 25, 12 for siZyx-NIH3T3 6 kPa, 14 and 31 kPa, respectively. Error bars represent standard error of the mean. **** represents p < 0.001 and * represents p < 0.05.

FIGURE 4

Graph of cell speed vs substrate rigidity for siLuc- and siZyx- NIH3T3 cells. n = 62, 161, and 54 for siLuc-NIH3T3 6 kPa, 14 kPa, and 31 kPa, respectively. n = 80, 81, and 85 for siZyx-, NIH3T3 6 kPa, 14 kPa, and 31 kPa respectively. Error bars represent standard error of the mean. **** represents p < 0.001 and n.s represents not significant.

FIGURE 5

Zyxin-knockdown NIH3T3 cells do not exhibit durotaxis (A) Illustration of method used to fabricate gradient polyacrylamide gels (B) Young’s modulus of the polyacrylamide gradient gel as a function of distance. Trajectories of NIH3T3 cells transfected with (C) control siLuc (n = 15) and (D) siZyx1 siRNA (n = 16) over period of 6 h. Different colors represent the cell trajectories for each cell. Rose plot showing direction cell migration of NIH3T3 cells transfected with (E) control siLuc and (F) siZyx siRNA.

FIGURE 6

Zyxin facilitates substrate rigidity sensing and impacts on durotaxis. Cells with high zyxin levels can sense substrate rigidity through modulating actin, focal adhesion, and traction stress, allowing the cells to move up rigidity gradients (left panel). Cells with low zyxin levels (right panel) are not able to sense substrate rigidity because they have lost the ability to tune cellular actin, focal adhesion, and traction stress in response to changes on substrate stiffness. Therefore, these cells are not able to perform durotaxis and they migrate randomly in all directions.