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J. Biol. Chem., Vol. 278, Issue 33, 31020-31023, August 15, 2003

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Localized Cdc42 Activation, Detected Using a Novel Assay, Mediates Microtubule Organizing Center Positioning in Endothelial Cells in Response to Fluid Shear Stress^*

Eleni Tzima , William B. Kiosses , Miguel A. del Pozo  and Martin Alexander Schwartz ¶

From the Department of Cell Biology, Division of Vascular Biology, The Scripps Research Institute, La Jolla, California 92037

Received for publication, February 3, 2003 , and in revised form, April 28, 2003.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Fluid flow regulates morphology, physiology, and pathophysiology of vascular endothelial cells (reviewed in Ref. 1). The small GTPase Cdc42 mediates polarity in several systems including migrating cells and early embryos, which involve reorientation of the microtubule organizing center (MTOC) and Golgi apparatus toward the direction of movement. Here, we show that Cdc42 is activated by fluid shear stress and that activation is a consequence of integrins binding to extracellular matrix. A novel fluorescence energy transfer assay to visualize Cdc42 activation in single cells shows that Cdc42 activity is polarized in the direction of flow. Localized activation of Cdc42 as well as the activity of Par6 and protein kinase C direct the reorientation of the MTOC to a position on the downstream side of the nucleus relative to the direction of flow. Thus, shear-stimulated integrin dynamics induce polarized Cdc42 activity, which induces MTOC localization through the Par6-protein kinase C complex.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Shear stress induces cell elongation and alignment of stress fibers and microtubules in the direction of flow (2, 3). Previous work has shown that the MTOC¹ in vascular endothelial cells is oriented toward the heart in vivo and in vitro (4–6). The MTOC also localizes in front of the nucleus toward the direction of migration in EC sheets (7). Reorientation of the MTOC similar to that in endothelial cells (ECs) has been reported in other cell types and is an important determinant of polarized movement (8, 9). In a number of systems, Cdc42 has been implicated in MTOC reorientation via a pathway involving PAR3, PAR6, atypical protein kinase C, and dynactin complexes (10–12). Interaction of MT plus ends at the cortex through IQGAP1 and CLIP-170 also appears to participate (13). As shear stress regulates activation of Rho (14) and Rac (15), we examined the regulation of Cdc42 activity by shear stress and investigated its role in shear stress-dependent MTOC reorientation.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Cell Culture, Shear Stress, and Transfections—Bovine aortic endothelial cells (BAECs) were maintained in Dulbecco's modified Eagle's medium with 10% fetal bovine serum, 1% penicillin/streptomycin, and 2 mM L-glutamine (Invitrogen) in a humidified 5% CO₂, 95% air incubator at 37 °C. BAECs on 38 x 76-mm slides at confluence were subjected to shear stress at 12 dynes/cm² in a parallel plate flow chamber (14, 15). For transient transfections, 2.5 µg of GFP-N17Cdc42, GFP-V12Cdc42, GFP-WTCdc42, and GFP-N19Rho or 2.25 µg of WTPKC, DNPKC, WTPar6, NtPar6, PDZPar6, and Cdc42-binding domain and 0.25 µg of GFP using Effectene according to the manufacturer's instructions (Qiagen) were used. Transfections for FRET assays used 0.5 µg of GFP-WTCdc42. After 10h in growth medium, cells were starved overnight in 0.5% serum prior to stimulation.

GTPase Assays—Pull-down assays for activated Cdc42 were carried out as described for Rac (15, 16) but were probed with an antibody against Cdc42 (Santa Cruz Biotechnology). Cdc42 FRET assays were performed as described previously (15). BAECs were microinjected as described previously (17) with GFP-WTCdc42, GFP-V12Cdc42, GFP-WTRac + Zizimin CAAX, and GFP-N17Cdc42 GFP (all at 50 µg/ml with the exception of N17 Cdc42 GFP, which was 200 µg/ml) and were maintained at 37 °C for 2 h. GFP-positive cells received a second microinjection of Alexa-p21-binding domain of PAK1 (PBD) (50 µg/ml) into the cytoplasm. Cells were then fixed in 2% formaldehyde. For shear stress Cdc42 FRET experiments, BAECs transiently transfected with GFP-WTCdc42 were serum-starved overnight in 0.5% serum-containing medium, loaded with Alexa-PBD as described previously (18), plated on fibronectin (FN)-coated glass slides, and allowed to adhere for 2 h and then were subjected to shear. Images of fixed cells were acquired using a Bio-Rad 1024 confocal microscope. Calculations to account for bleed-through and background were performed as described previously (19). The resultant corrected 8-bit FRET images typically had a fluorescence intensity range of 30–110 for shear flow experiments and 30–100 for re-plating on FN experiments and were displayed using pseudocolor where blue was closest to 0 and red was closest to 110/100.

Fibronectin was blocked by the addition of Fab fragments of antibody 16G3, which blocks both the V3 and 51 binding sites for fibronectin. Alternatively, cultures were treated antibody 11E5, which is non-function blocking (a generous gift from Dr. K. M. Yamada). These cultures were added at 10 µg/ml to well spread cells on FN for 15 min before applying shear as previously described (14, 15).

Fluorescence Microscopy—Cells were fixed for 10 min in methanol at –20 °C, permeabilized in 0.2% Triton X-100/PBS, and blocked with 10% goat serum. Cells were stained with an antibody against -tubulin (Sigma) followed by TRITC-conjugated goat anti-rabbit IgG (Sigma). Nuclei were stained with TOTO-3 (Molecular Probes). Confocal serial sectioned images were acquired using a Bio-Rad 1024 MRC scanning confocal microscope.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
To investigate the role of Cdc42 in flow-induced MT organization, we first asked whether shear stress could activate Cdc42. Pull-down assays were carried out in which Cdc42 GTP loading was determined by specific binding of the active GTPase to the PBD fused to glutathione S-transferase (20, 21). Serum-starved BAECs subjected to shear stress for the indicated times showed an increase in Cdc42 activity that was detectable at 5 min and maximal at 30 min after shear stress. Activity decreased to basal levels 1–2 h after shear stress (Fig. 1). A previous study (22) showed that shear stress induced translocation of Cdc42 to the membrane fraction on a somewhat faster time scale. Whereas differences in technique cannot be excluded, the finding that membrane translocation can be regulated separately from biochemical activation (16, 23) may account for this discrepancy.

View larger version (27K): [in this window] [in a new window]   FIG. 1. Shear stress activates Cdc42 and requires new integrin-ligand connections. A, BAECs were subjected to shear stress for the indicated times. Cdc42 activity is indicated by the amount of PBD-bound Cdc42 normalized to total Cdc42 in whole cell lysates. B, quantitation of Cdc42 activity relative to cells at time 0. Values are means ± S.E. from four independent experiments, each of which was performed in duplicate. C, BAECS were plated on fibronectin for 2 h. Cells were incubated with anti-FN Fab fragments for 15 min and subjected to shear stress or kept under static conditions for 30 min. Cdc42 activation was assayed. Values are means ± S.E. from three independent experiments, each of which was performed in duplicate.

Shear stress triggers integrin activation followed by new integrin binding to extracellular matrix (ECM) (14, 24). Additionally, integrins can activate Cdc42 (11, 25). Moreover, the time course of Cdc42 activation (Fig. 1) is consistent with it being downstream of integrin binding to ECM, which is detectable at 2 min and maximal at 30 min (14). To test whether Cdc42 activation requires new integrin binding, ECs fully spread on FN were briefly treated with anti-FN antibodies. This treatment blocks excess unligated FN to prevent formation of new integrin-ligand connections without disrupting existing adhesions (14, 15, 24). Shear stress was then applied, and Cdc42 activity was assayed. The blocking antibody 16G3 strongly inhibited the shear stress-induced increase in Cdc42 activity, whereas the non-blocking antibody 11E5 had no effect (Fig. 1C). Thus, the activation of Cdc42 is downstream of new integrin binding to ECM in this system.

Whether Cdc42 conveys spatial information to the MTOC or is simply required for MTOC movement is presently unclear in any system (10, 11). To address this question, we monitored the subcellular localization of Cdc42 nucleotide state by adapting a recently developed assay for localized Rac activity in which a GFP-tagged GTPase is introduced into cells together with an Alexa546-labeled effector domain from PAK (PBD) (19, 27). Activation of the GTPase leads to the binding of the PBD and increased FRET between the two fluorophores. Because PAK binds equally well to Cdc42, we expressed GFP-Cdc42 and introduced Alexa546-PBD into cells by shear loading (18). Images of GFP, Alexa, and FRET were taken for each cell, and FRET images were corrected for bleed-through as described previously (19). Control experiments to validate the assay showed that cells expressing dominant-negative N17Cdc42 that does not bind PBD had only low levels of FRET that we take as the base line (Fig. 2). In unstimulated cells containing WTCdc42, FRET was usually high in regions close to the nucleus and, in some cases, a weak positive FRET signal was present near cell edges. Constitutively active GFP-V12Cdc42 produced much stronger FRET at cell edges, often at the base of small protrusions that may be nascent filopodia, whereas perinuclear FRET was unchanged. To test whether WT protein can be activated, GFP-WTCdc42 was expressed together with the Cdc42 nucleotide exchange factor zizimin1 (28). Zizimin1 induced a substantial increase in the Cdc42 FRET near cell edges in the vicinity of small protrusions. We noticed that Cdc42 activation was often lower at the base of large filopodia, suggesting that Cdc42 activity is more important for formation than maintenance of these structures. These results demonstrate the Cdc42 FRET assay shows the correct behavior.

View larger version (65K): [in this window] [in a new window]   FIG. 2. Cdc42 Assay. BAECs were microinjected with cDNAs encoding the indicated GFP-Cdc42 fusion proteins and then with Alexa-PBD protein. Representative GFP and corrected FRET images are shown. Similar results were obtained in four independent experiments. A color scale with the equivalent numerical values for the intensity of FRET for all of the images is displayed. Red represents a high FRET signal, and blue represents a low signal.

To visualize Cdc42 activation in response to shear stress, BAECs containing GFP-WTCdc42 and Alexa-PBD were subjected to fluid flow for various times and then fixed and assessed. We observed a marked increase in FRET near cell edges after 30 min, which returned to base line by 2 h (Fig. 3). FRET results were therefore consistent with the biochemical assay. Importantly, shear stress preferentially induced FRET in the downstream side relative to the direction of flow. To quantify polarity, images were analyzed by dividing cells in half relative to the direction of flow and the upstream or downstream localization of the FRET signal was assessed. When scored by eye, shear stress for 30 min induced a dramatic increase in the fraction of cells in which FRET was predominantly down-stream (Fig. 3). Polarization dissipated at later times of shear stress. Second, to quantify in a completely objective way, the number of pixels and pixel intensity in both halves were quantified. The total signal above the background was calculated for both upstream and downstream areas of cells. The results demonstrated higher Cdc42 activity in the downstream half in cells subjected to shear stress for 30 min. In contrast, Cdc42 activity was randomly distributed in unsheared cells and cells sheared for 2 h. Thus, shear stress activates Cdc42 preferentially downstream of flow.

View larger version (99K): [in this window] [in a new window]   FIG. 3. Spatio-temporal localization of shear stress-induced Cdc42 activity. BAECs transiently transfected with GFP-WTCdc42 were shear-loaded with Alexa-PBD, plated on fibronectin for 2 h, and subjected to shear stress or kept under static conditions for 30 min. Cdc42 activation (FRET) is shown. Graphs, cells were first scored for the presence of a FRET signal that was above background. Those cells showing a positive signal were then scored for whether the signal was predominantly localized to the downstream edge, i.e. flow was from left to right, and the strongest FRET signals were present at right (left graph). Cells were divided in half relative to the direction of flow, and the sum of pixel intensities within these areas was calculated. The fraction of the total FRET signal within the upstream and downstream areas was calculated for each cell and expressed as percent of the total (right graph). Values are means ± S.E., n = 3, >100 cells were scored/condition.

Cdc42 is required for lysophosphatic acid-induced MTOC reorientation (10) as well as MTOC reorientation in wounded astrocyte monolayers (11). Therefore, we tested whether shear stress has an effect on MTOC orientation in confluent monolayers of BAECs subjected to fluid shear stress. MTOC orientation was assessed in fixed preparations stained for -tubulin and nuclei. Cells were scored for whether the MTOC position relative to the nucleus was within 120^o relative to the direction of flow. 16 h of flow induced MTOC reorientation in the direction of flow in the majority of the cells compared with control cells where MTOC position was random (Fig. 4). To investigate the role of Cdc42 in this process, BAECs were transiently transfected with DNA constructs for wild type, dominant negative (N17Cdc42), or dominant active Cdc42 (V12Cdc42). Polarization of the MTOC was unaffected by WTCdc42 but dramatically inhibited by N17Cdc42. Importantly, expression of constitutively active V12Cdc42 also blocked reorientation of the MTOC in response to shear stress, indicating that correct spatial activation of Cdc42 rather than activity per se is essential for localization of the MTOC after shear stress. Expression of the Cdc42-binding domain of Wiscott-Aldrich Syndrome protein (29) also abolished reorientation of the MTOC.

View larger version (46K): [in this window] [in a new window]   FIG. 4. MTOC reorientation in response to shear stress requires correct spatial activation of Cdc42. A, BAECs transiently transfected with GFP-WTCdc42, GFP-N17Cdc42, GFP-V12Cdc42, GFP-WTRho, GFP-N19Rho, GFP-WTRac, GFP-N17Rac, WTPKC, DNPKC, WTPar6, NtPar6, PDZPar6, and Cdc42-binding domain (CBD), and GFP were subjected to shear stress or kept as static controls for 16 h. Cells were then fixed and stained with antibody against -tubulin to visualize the MTOC and with TOTO-3 to label the nuclei. Circles are drawn around the MTOCs of transfected cells. Dotted lines separate the nuclei into upstream and downstream halves relative to flow. B, histogram showing number of cells in which MTOCs are localized downstream relative to the direction of flow.

To investigate the role of other members of the Rho GTPase family in the microtubule-dependent establishment of polarity, cells were transfected with dominant negative Rho and Rac. Polarization of the MTOC was unaffected by dominant negative Rac (N17Rac) or Rho (N19Rho). Previous investigators reported that the Par6-PKC complex controls cell polarity (11, 30). Expression of the PDZ-mutated mPar6 or an N-terminal deletion mutant abolished MTOC reorientation in response to shear stress. By contrast, WTPAR6 had no effect. The N-terminal domain of mPar6 interacts with atypical PKCs (31). To identify whether an atypical PKC is involved, we expressed a kinase-dead version of PKC in cells. This construct but not WTPKC blocked MTOC polarization in response to shear stress. We conclude that the mPar6/PKC is required for Cdc42-dependent orientation of the MTOC.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Shear stress promotes integrin activation followed by formation of new integrin-ECM interactions, leading to changes in Shc, Rho, and Rac signaling pathways (14, 15, 24). Additionally, integrins can activate Cdc42 (14, 15, 25, 32). We now report that shear stress transiently activates Cdc42 and that blockade of new integrin-ligand connections blocks this event. Integrin-ligand binding therefore mediates activation of Cdc42 by shear stress. Previous studies showed that fluid flow induces formation of focal adhesions preferentially at the down-stream edge of the cells (33). This finding is consistent with the preferential activation of Cdc42 in a directional polarized manner downstream relative to the direction of fluid flow. Importantly, Cdc42 activation per se is not sufficient for MTOC reorientation since expression of V12Cdc42 also blocks MTOC orientation. Thus, polarized activity of Cdc42 is critical for correct orientation of MTOC. This result is particularly interesting in light of the role of Rac in shear stress-induced alignment. The correct spatial activation of Rac is required for the orientation of stress fibers with the direction of flow (15). In addition, although Rho, Rac, and Cdc42 all control the organization of the actin cytoskeleton (34), only Cdc42 was responsible for shear stress-induced MTOC reorientation. In agreement with our findings, high Cdc42 activity was also observed at the leading edge of migrating cells using the Raichu-Cdc42 probe (35); however, this probe is constitutively membrane-bound. Thus the effect of Rho-guanidine dissociation inhibitor-dependent membrane-cytoplasm dynamics, which is an important aspect of GTPase localization (23), could not be monitored.

The results presented here provide a mechanism through which Cdc42 regulates the microtubule-dependent establishment of cell polarity under shear stress as the Cdc42 target protein, PAR6, and its associated protein, PKC, are essential for the reorientation of the MTOC. PAR proteins were identified as key regulators of cell polarity in early Caenorhabditis elegans development (36). Our data show that PAR6 and PKC are involved in a signaling pathway directly responsible for the reorientation of the MTOC in response to shear stress.

In conclusion, our results define a signal transduction pathway controlled by Cdc42 that links integrin activation and engagement to the establishment of polarity in cells subjected to shear stress. The formation of new integrin-ligand connections seems to be the first polarity signal, which leads to localized activation of Cdc42. Spatially restricted activation of Cdc42 then establishes and maintains polarity by promoting PAR6/PKC-dependent reorientation of the MTOC in the direction of flow. MTs are the first cytoskeletal components to assume orientation during cell shape change (37). They play a central role in alignment in the direction of flow as disruption of MTs blocks EC alignment by flow and prevents the induction of actin stress fibers (26). The functional link among integrins, Cdc42, and MTs is therefore essential to the endothelial response to fluid shear stress.

	FOOTNOTES

 
^* This work was supported by U. S. Public Health Service Grant PO1 HL48728. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Both authors contributed equally to this work.

A Leukemia and Lymphoma Society Special Fellow (Grant 3347-02).

^¶ To whom correspondence should be addressed: Depts. of Microbiology and Biomedical Engineering, Cardiovascular Research Center, Mellon Prostate Cancer Research Center, University of Virginia, 415 Lane Rd., Charlottesville, VA 22908. E-mail: maschwartz{at}virginia.edu.

¹ The abbreviations used are: MTOC, microtubule organizing center; EC, endothelial cell; WT, wild type; BAECs, bovine aortic endothelial cells; GFP, green fluorescent protein; PKC, protein kinase C; DN, dominant negative; PBD, p21-binding domain of PAK1; FN, fibronectin; TRITC, tetramethylrhodamine isothiocyanate; ECM, extracellular matrix; MT, microtubule; FRET, fluorescence resonance energy transfer.

	ACKNOWLEDGMENTS

 
We are grateful to Nazilla Alderson for technical assistance. We thank Dr. I. Macara for supplying us with PAR6 and PKC constructs, Dr. Ken Yamada for generously providing anti-fibronectin monoclonal antibodies, and Dr. Klaus Hahn for advice in the development of Cdc42 FRET assays.

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TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

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