Assembly and reorientation of stress fibers drives morphological changes to endothelial cells exposed to shear stress

Abstract

Fluid shear stress greatly influences the biology of vascular endothelial cells and the pathogenesis of atherosclerosis. Endothelial cells undergo profound shape change and reorientation in response to physiological levels of fluid shear stress. These morphological changes influence cell function; however, the processes that produce them are poorly understood. We have examined how actin assembly is related to shear-induced endothelial cell shape change. To do so, we imposed physiological levels of shear stress on cultured endothelium for up to 96 hours and then permeabilized the cells and exposed them briefly to fluorescently labeled monomeric actin at various time points to assess actin assembly. Alternatively, monomeric actin was microinjected into cells to allow continuous monitoring of actin distribution. Actin assembly occurred primarily at the ends of stress fibers, which simultaneously reoriented to the shear axis, frequently fused with neighboring stress fibers, and ultimately drove the poles of the cells in the upstream and/or downstream directions. Actin polymerization occurred where stress fibers inserted into focal adhesion complexes, but usually only at one end of the stress fiber. Neither the upstream nor downstream focal adhesion complex was preferred. Changes in actin organization were accompanied by translocation and remodeling of cell-substrate adhesion complexes and transient formation of punctate cell-cell adherens junctions. These findings indicate that stress fiber assembly and realignment provide a novel mode by which cell morphology is altered by mechanical signals.

Figure 1

Postconfluency affects actin organization in endothelial cell responses to shear stress. Endothelial cells that have just achieved confluence (top left) display randomly oriented microfilament bundles and actin staining fails to delineate individual cells. At 2 days after confluence, a dense peripheral band of actin assembles at the cell-cell junctions and only sparse central stress fibers are seen. When shear is initiated at 2 days after confluence, no changes in cell shape or actin distribution are detectable at 4 hours and only modest and sporadic loss of the dense peripheral band of actin is detected at 8 hours. Cell elongation and actin alignment with shear stress becomes apparent during the next 8 hours and cells complete shape change after 24 hours of shear stress.

Figure 2

Shear-induced changes to endothelium. Cell membranes of endothelial cells in static cultures (left) or after 16 hours of shear stress (right) were labeled with the DiI. Areas of cell-cell overlap are brighter because multiple layers of plasma membrane are detected. a: In static cultures, linear cell-cell junctions were interrupted by pad-like regions of cell overlap (arrow) that were separated by linear cell-cell junctions (arrowhead). b: Shear induced elongation of endothelial cells in the direction of shear vector (right to left) that occurred between 8 hours and 24 hours. During elongation, regions of cell-cell overlap that are neither filopodia-like or lamellipodia-like were observed; instead, triangle-shaped projections of cell membranes became oriented in the upstream and downstream directions (arrow). Each finding is representative of at least three replicate experiments.

Figure 3

Shear stress induces assembly of actin at the ends of stress fibers. Endothelial cells were incubated briefly with fluorescently labeled G-actin (green) after exposure to shear stress for 0 hours (A), 8 hours (B), 16 hours (C), or 96 hours (D). Cells were then fixed and filamentous actin was stained with rhodamine phalloidin (red). Little or no actin assembly occurred in postconfluent cells not exposed to shear stress (A). In contrast, exposure to shear stress induced actin assembly at the ends of stress fibers (B–D). Initially, actin assembly was randomly oriented, but assembly became aligned with shear stress by 16 to 24 hours. Shear stress is directed from right to left. Each finding is representative of at least three replicate experiments.

Figure 4

Actin assembly occurs at focal adhesion complexes at one end of stress fibers and localizes to subdomains of these complexes. Endothelial cells were exposed to fluorescently labeled G-actin (green) after exposure to shear stress for 8 hours (A) or 16 hours (B), then stained for filamentous actin with rhodamine phalloidin (red) and immunostained for vinculin (blue). Actin assembly usually occurred at only one end of stress fibers (arrows in A). At these sites, newly assembled actin was excluded from subdomains of the focal adhesion complex, as indicated by overlap of vinculin and F-actin only (fuscia, insets). Shear stress is directed from right to left. Findings are representative of three replicate experiments.

Figure 5

Shear stress determines orientation but not direction of actin assembly. Graph displays percentage of cells in which actin assembly was predominantly at the upstream, downstream, or both ends (bipolar) of stress fibers, or when assembly was directed away from the nucleus at both ends of the cells (anti-podal). Numbers at the base of each bar indicate hours of shear stress. Also shown is the percentage of cells in which assembly was randomly distributed between the two ends of the stress fibers, which never occurred after 24 hours. The only other statistically significant temporal change was an increase in bipolar assembly at 48 hours.

Figure 6

VASP does not mediate shear-induced actin assembly. Shear-induced stress fiber assembly was not VASP (green immunostaining) localized to focal adhesion complexes in endothelium (A) but was dislodged from these sites by a zyxin peptide sequence designed to interfere with zyxin-VASP interaction (B). VASP association with ends of stress fibers remained suppressed at 1 hour (not shown). Incorporation of Alexa 488-labeled monomeric actin (green) into the end of stress fibers (red) after 16 hours of shear stress (C) was not suppressed by peptides that disrupt association of VASP with focal adhesion complexes (D), or by control (scrambled) peptide (not shown).

Figure 7

Stress fiber extension protrudes the endothelial cell membrane at the cell poles. Endothelial cells were microinjected with fluorescently labeled G-actin 24 hours before exposing the cells to shear stress for ∼20 hours. Top horizontal line joins the ends of stress fibers that do not advance during the observation time. The middle line joins stress fibers that advance between t = 20:15 hours and t = 21:45 hours. The bottom line is in the vicinity of three stress fibers that advance substantially throughout the period of observation. Note the advance of the cell membrane in the latter two cases. Frequently, two or more adjacent stress fibers fused throughout the period of observation (arrows). Shear stress is directed from top to bottom. Findings are representative of three replicate experiments.

Figure 8

Focal adhesion complexes become extended and linear under the influence of shear stress. The figure shows vinculin and paxillin immunostaining after 0 hours, 16 hours, and 96 hours of shear stress, which is directed from right to left. Shear stress induces the formation of long, linear adhesion complexes that are aligned with shear stress. Findings are representative of three replicate experiments.

Figure 9

Focal adhesion complexes become depleted of tensin under the influence of shear stress. Endothelium in postconfluent cultures was immunostained for tensin and viewed by confocal microscopy. Tensin localized to focal adhesion complexes in static cultures (left) but dramatic decreases in tensin contents of focal adhesions were observed at 24 hours of shear (middle) as well as at 8 hours, 16 hours, and 48 hours (not shown). Recovery of tensin at focal adhesions was detected at 96 hours. Images were captured in the same microscopy session with identical settings on the microscope and identical conditions for figure production. Shear stress is directed from right to left. Findings are representative of three replicate experiments.

Figure 10

A punctate distribution of adherens junctions in shear-stressed endothelium is transitory and dependent on actin microfilaments. A: Confocal micrograph shows F-actin (red), exogenous fluorescent actin (newly assembled actin, green), and the adherens junction protein, α-catenin, after 48 hours of shear stress. Note the discontinuous distribution of adherens junctions (adherens plaques). (The α-catenin signal alone is reproduced as online Figure 6 so that the discontinuous nature of adherens conjunctions can be easily visualized.) B: This distribution was dependent on filamentous actin because cytochalasin, which disperses microfilaments, induced the redistribution of adherens junctions into a linear array at 48 hours of shear stress. C: The punctate pattern that adherens junctions assumed under shear stress was transitory because these structures spontaneously resumed a linear distribution at the cell-cell junction at 96 hours, a distribution that mimicked that observed for β-catenin in unmanipulated rabbit carotid arteries in situ (D).