QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online November 18, 2003
doi: 10.1242/10.1242/jcs.00823

This Article Summary Full Text Full Text (PDF) Movies Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Related articles in JCS Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Tsuruta, D. Articles by Jones, J. C. R. Search for Related Content PubMed PubMed Citation Articles by Tsuruta, D. Articles by Jones, J. C. R.

The vimentin cytoskeleton regulates focal contact size and adhesion of endothelial cells subjected to shear stress

Department of Cell and Molecular Biology, Northwestern University Medical School, Morton 4-616, 303 E. Chicago Avenue, Chicago, IL 60611, USA

View larger version (63K): [in a new window]   Fig. 1. Characterization of CFP-vimentin and YFP-ß3 integrin expression in TrHBMEC transfectants. (A) Extracts of transformed human bone marrow endothelial cells (TrHBMECs) co-transfected with expression vectors encoding CFP-vimentin and YFP-ß3 integrin were processed for western immunoblot analysis using antibodies against GFP (lane 1), ß3 integrin (lane 2) or vimentin (lane 3). Molecular masses are indicated on the left. The reactive species are indicated on the right. (C-I) Images of transfected TrHBMECs. Cells expressing CFP-vimentin (B,F) and YFP-ß3 integrin (C,G) were fixed and then processed for immunofluorescence microscopy using an anti-ß3 integrin subunit antiserum (D) or anti-vimentin antibodies (H). E and I are the merged images of B-D and F-H, respectively. YFP-ß3 integrin (C) colocalizes precisely with the endogenous vß3 integrin complexes in FC (D) (purple in the merged image in E). CFP-vimentin (B) colocalizes precisely with the endogenous vimentin intermediate filaments (H) (sky blue in the merged image in I). Scale bar: 10 µm. Vim, vimentin.

View larger version (60K): [in a new window]   Fig. 2. The dynamics of vimentin IFs and ß3 integrin-rich FCs in TrHBMEC transfectants. TrHBMECs co-expressing YFP-ß3 integrin and CFP-vimentin were followed over periods up to 60 minutes in the live state. A-C reflect different types of interactions and relative behaviors of vimentin IFs and FCs in double transfectants. (A,B) The signals for the three time points were coded blue, green and red, with the final panel reflecting all three images merged. In A, a YFP-ß3 integrin-containing FC moves centripetally in concert with vimentin IF (arrow). (B) Vimentin IFs elongate from the perinuclear zone towards an FC (arrow) at the edge of the cell. (C) A newly assembled FC (arrow) appears and establishes contact with vimentin IF. Scale bar: (A) 5 µm, (B,C) 3 µm. A, B and C can be viewed in video form (movies 1, 2 and 3, http://jcs.biologists.org/supplemental/).

View larger version (31K): [in a new window]   Fig. 3. The dynamics of vimentin IF and ß3 integrin-rich FCs following initiation of flow and during the subsequent 105 minutes. (A) TrHBMEC co-expressing YFP-ß3 integrin and CFP-vimentin were followed over a period of 105 minutes in the live state after the exposure to flowing medium at 12 dynes/cm2. The YFP images at 0, 45 and 105 minutes were coded in blue, green and red, respectively, and then overlaid. Note that FCs are dynamic during the viewing period as revealed by the rainbow color effect in D. Scale bar: 5 µm. This can be viewed in video form (movie 4, http://jcs.biologists.org/supplemental/).

View larger version (28K): [in a new window]   Fig. 4. Quantification of FC size and motility under static and flow conditions. (A) The size of FCs in cells was quantified by counting pixels of labeled FCs at 0, 60 and 120 minutes after the initiation of shear stress, in the form of flowing medium at 12 dynes/cm2. (B) Movement of FCs was assayed in non-treated (static) cells and in cells subjected to flow over periods 0-60 and 60-120 minutes after the initiation of shear stress. A total of 40 FCs in three cells were evaluated for each time point. *Indicates a significant difference (P<0.01).

View larger version (44K): [in a new window]   Fig. 5. The dynamics of vimentin filament and ß3 integrin-rich focal contacts 120-220 minutes after the initiation of flow. Populations of TrHBMECs co-expressing YFP-ß3 integrin and CFP-vimentin was subjected to flowing medium at 12 dynes/cm2 and then the live cells were viewed for 120-220 minutes after the initiation of shear stress. Images of a typical treated cell at 120, 170, and 220 minutes are shown. The YFP images at (A) 120, (B) 170 and (C) 220 minutes were coded in blue, green and red, respectively and then merged (D). The white colors of the large FCs in D indicate that they show minimal movement in the plane of the membrane. Note also that each FC in A-C shows extensive interaction with vimentin IF. Scale bar: 3 µm. This can be viewed in video form (movie 5, http://jcs.biologists.org/supplemental/).

View larger version (32K): [in a new window]   Fig. 6. (A) Western blot of cells transfected with control or vimentin siRNA showing silencing of vimentin in TrHBMECs Extracts of TrHBMECs transfected with control (lane 1) or vimentin siRNA (lane 2) were processed for immunoblot analyses using antibodies against vimentin (upper panel). The blot was re-probed with actin antibody as a loading control (lower panel). Molecular masses are indicated on the left. (B) The immunoblots in A were scanned and analyzed using MetaMorph software. The amount of vimentin in each sample was then normalized to the actin control. Vimentin expression in the control siRNA-treated cells is represented as 100% (white bar). Vimentin expression in the vimentin siRNA-treated cells is reduced by about 60% (black bar). (C-H) Immunofluorescence analyses of cytoskeletal networks and ß3 integrin subunits in vimentin siRNA treated TrHBMECs. TrHBMECs were transfected with control siRNA or vimentin siRNA and then 72 hours later were prepared for double label immunofluorescence confocal microscopy using the combinations of antibody or cytoskeleton probes as indicated at the left. The merged images of the two staining patterns are shown in each case. Note that there is a dramatic perturbation in the vimentin cytoskeleton in D, F and H. The FCs labeled by the ß3 integrin antibody probe are considerably smaller in D than those labeled by the same probe in C (also see Fig. 9). The vimentin siRNA treatment has minimal, if any, impact on the organization of the microtubule or microfilament networks (compare F and H with E and G). Scale bar: 5 µm.

View larger version (17K): [in a new window]   Fig. 7. Quantification of the size and dynamics of FCs in control siRNA and vimentin siRNA-treated TrHBMECs. TrHBMECs were transfected with control siRNA or vimentin siRNA and 72 hours later the size (A) and movement (B) of FCs in the treated cells was evaluated. In all 40 FCs in 3 cells were analyzed in each case. *Indicates a significant difference (P<0.01).

View larger version (52K): [in a new window]   Fig. 8. TrHBMECs treated with vimentin siRNA or cytochalasin D assemble small FCs. TrHBMECs, expressing YFP-ß3 integrin, were transfected with control siRNA (A) or vimentin siRNA (B) and after 72 hours were viewed in the live state. In C, TrHBMECs were treated with the 0.1 µM cytochalasin D for 30 minutes prior to observation. Note that FCs appear smaller in B and C than in A. Scale bar: 5 µm.

View larger version (41K): [in a new window]   Fig. 9. TrHBMECs treated with vimentin siRNA show reduced adhesion under flow conditions, while those treated with cytochalasin D do not. TrHBMECs were transfected with control siRNA (A,B) or vimentin siRNA (C,D) as indicated and after 72 hours the cells were subjected to flow stress. (E,F) Non-transfected cells were incubated with 0.1 µM cytochalasin D for 30 minutes prior to and during flow treatment. Medium was passed over the cells at 12 dynes/cm2 as before. Phase contrast images of the cells are shown at 0 and 50 minutes. Scale bar: (in F) 10 µm. (G) The number of cells attached after flow treatment for 50 minutes. Cells were treated with control siRNA, vimentin siRNA or 0.1 µM cytochalasin D (as above). The data were derived from observations of three separate trials involving at least 20 cells per trial.

View larger version (17K): [in a new window]   Fig. 10. Quantification of the size of FCs in 0.1 µM cytochalasin D treated TrHBMECs subjected to flow treatment. TrHBMECs were treated with 0.1 µM cytochalasin D for 30 minutes and were then exposed to flow stress for 120 minutes in the presence of the same amount of drug. The size of FCs was evaluated at the indicated time points. In all, 40 FCs in three cells were assayed at each time point. *Indicates a significant difference (P<0.01). Note that FCs show significant enlargement during flow treatment despite the presence of cytochalasin D.