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First published online August 29, 2005
doi: 10.1242/10.1242/jcs.02523

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Polarized downregulation of the paxillin-p130^CAS-Rac1 pathway induced by shear flow

Department of Molecular Cell Biology, The Weizmann Institute of Science, Rehovot 76100, Israel

View larger version (158K): [in a new window]   Fig. 1. Flow inhibits the upstream migration of endothelial cells in an in-vitro wound. A confluent PAEC monolayer was wounded, and 4 hours later placed under perpendicular flow (from top to bottom in all figures). Migration of cells into the wound was monitored by time-lapse phase-contrast microscopy. (A) Image of a wound, 1 hour after the application of flow (25 dyn/cm2), with the white lines marking the contour of the front of the cell at time 0 and the black lines marking the front after 60 minutes. Cells migrating downstream advanced on average more than 15 µm, whereas cells facing upstream did not advance but retracted. (B) A kymograph of two cells, located at either side of a wound, depicting the dynamics of the leading edge at 16-second resolution over a period of 70 minutes under flow (20 dyn/cm2). (C) The relationship between migration speed and flow rate (average values, based on 40 cells). Empty and solid circles represent cells migrating downstream and upstream, respectively. (D) Immunolabeling of cells after 30 minutes of flow (20 dyn/cm2) for actin and phosphotyrosine (PY). Bar, 10 µm (A,B); 5 µm (D).

View larger version (98K): [in a new window]   Fig. 2. Inhibition of upstream protrusion leads to polarization of single endothelial cells under flow. (A) Frames from a phase-contrast time-lapse movie of a single PAE cell exposed to flow (arrow, 20 dyn/cm2). (B) PAE cells from cultures under stationary condition (control) or after 5 minutes of flow (arrow, 20 dyn/cm2), stained for actin with phalloidin. Arrowheads mark protrusions. (C) Quantitative analysis of the number and direction of protrusions in control cells and cells exposed to flow. The average number of protrusions in each of eight directions is displayed. Notice that polarization under flow is primarily due to inhibition of upstream protrusions, rather than to increase in downstream protrusion. Bar, 5 µm.

View larger version (144K): [in a new window]   Fig. 3. Inhibition of upstream protrusion is not due to increased RhoA activity and/or acto-myosin contractility. (A) Sparse PAE cells were treated for 30 minutes with 10 µM Y27632, a ROCK inhibitor, then exposed to flow for 5 minutes before being fixed and stained for actin. Notice the reduction in stress fibers, yet maintenance of polarized protrusion under flow (panel with arrow). (B) Kymograph demonstrating the effect of the ROCK/myosin light chain kinase inhibitor H7 (300 µM) on PAE cells in a wound, exposed to flow (arrow, 18 dyn/cm2). Notice that although the addition of H7 had a significant effect on downstream migration, it did not affect the flow-induced inhibition of upstream migration. (C,D) Sparse PAE cells expressing a dominant-negative form of RhoA (N19) (C) or C3-toxin (D) were exposed to flow for 5 minutes before being fixed and stained for actin. (E) At least 60 cells in each treatment were scored for the number and direction of protrusions. To visualize the degree of cell polarization the mean number of protrusions in the upstream or downstream directions (±45°) is presented. Error bars indicate s.e.m. Bar, 5 µm.

View larger version (90K): [in a new window]   Fig. 4. Rac1 activity is inhibited in the upstream region of endothelial cells under flow. (A) Rac1 activity in live PAECs was monitored using the FRET reporter Raichu-Rac. The negative control (Raichu-RacY40C) shows stable and low FRET levels. With no flow, Raichu-Rac shows a relatively high level of activity throughout the entire cell, and specifically in the leading lamella. With the application of flow (arrow, 20 dyn/cm2) a dramatic decrease in Rac1 activity is observed in the upstream region of the cell. Rac FRET activity levels in upstream or downstream sides of three cells, normalized to their values 2.5 minutes before flow onset, are shown in the graph. Error bars denote s.d. (B) A cell expressing constitutively active Rac1-L61 (asterisk) along the edge of a wound. Kymographs I and II correspond to the lines in the movie frame, where line I crosses the cell expressing active Rac. Arrows in the kymographs denote initiation of flow. Note that the transfected cell retracts and then advances upstream, despite the flow (20 dyn/cm2) whereas its neighbors only retract (see also supplementary material Movie 3). (C) Single PAE cell, expressing Rac1-L61 under no flow conditions, or after five minutes of flow (arrow, 20 dyn/cm2). Under both conditions, lamellar protrusions are formed in all directions. Bar, 10 µm (B); 5 µm (C).

View larger version (92K): [in a new window]   Fig. 5. p130CAS and Paxillin are dephosphorylated in upstream focal adhesions in response to flow. (A) Sparse PAECs were exposed to shear flow for five minutes (arrow, 20 dyn/cm2), and then immediately fixed and double stained for paxillin, p130CAS and FAK (left column) and the corresponding phosphorylated forms, namely, pY118, pY165 and pY397, respectively (middle column). Ratio images (right column) highlight the relative levels of the phosphorylated proteins compared to total protein. Images of p130CAS and FAK under no flow conditions can be seen in supplementary material Fig. S2. (B) Quantification of the average intensity of total and phosphorylated forms of paxillin, p130CAS and FAK in upstream and downstream focal adhesions was calculated for more than a thousand focal adhesions from 20 different cells, under no flow conditions or after 15 minutes of flow. The average intensity after 15 minutes of flow, normalized by the intensity under no flow conditions, is presented. Bars indicate s.e.m. Bar, 5 µm.

View larger version (77K): [in a new window]   Fig. 6. DOCK180/ELMO overexpression can overcome flow-induced cell polarization, whereas Tiam1 overexpression does not. Single PAE cells overexpressing both subunits of the Rac1 GEF DOCK180/ELMO (A) or Tiam1 (B) were placed under control or flow conditions (arrow, 20 dyn/cm2) for 10 minutes before being fixed and stained for actin. Notice the large upstream lamellipodium in the DOCK/ELMO expressing cell. (C) Quantification of degree of polarity for these cells, as well as cells expressing active Rac1 was performed as described in Fig. 3. Bar, 5 µm.

View larger version (81K): [in a new window]   Fig. 7. Shear stress induces growth of upstream focal adhesions and reduction of downstream focal adhesion area. PAECs expressing CFP-vinculin were monitored at 2-minute intervals before and after application of flow (arrow, 20 dyn/cm2). (A) Frames from such a movie, before and after 10 minutes of flow, and a ratio image of the two time points. In this ratio image, extension of focal adhesions appears red whereas loss of focal adhesion area appears blue. (B) Gain and loss of focal adhesion area are quantified by examination of a large number of focal adhesions (80 for flow, 60 for no flow controls). For each focal adhesion the area and intensity at two time points, 6-9 minutes apart were compared. Here, the average area `lost' or `gained' is presented, normalized to the area of the focal adhesion at time 0, in addition to the change in average intensity. Error bars indicate s.e.m. Bar, 5 µm.

View larger version (75K): [in a new window]   Fig. 8. Differential effect of flow on focal adhesion dynamics depends on focal adhesion orientation and not on their subcellular localization. PAECs expressing both CFP-vinculin and YFP-actin, were monitored, at two-minute intervals, before, and after application of flow (arrow, 20 dyn/cm2). (A) The last frame before application of flow is shown at the top left. To visualize flow-induced changes in focal adhesion structure, ratio images were generated for vinculin by dividing frame t+X by frame t (top right). For three vertical focal adhesion pairs (roman numbers) plus one perpendicular pair (P), a series of ratio images (2 minutes apart) is shown. Arrows and arrowheads indicate the upstream and downstream focal adhesions, respectively. (B) The change in area of each focal adhesion, as a function of time under flow (normalized to pre-flow values) is shown for the three pairs in A along with another nine pairs from other cells. The same color is used in both graphs to identify paired focal adhesions. Notice that the overall trend (marked by a black line) is growth of upstream focal adhesions and reduction is size of downstream focal adhesions. Bar, 5 µm.