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Address correspondence to Clare M. Waterman-Storer, The Scripps Research Institute, Dept. of Cell Biology, CB 163, 10550 North Torrey Pines Rd., La Jolla, CA 92037. Tel.: (858) 784-9764. Fax: (858) 784-9779. E-mail: waterman{at}scripps.edu
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Abstract |
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Key Words: cell motility; cytoskeleton; tubulin; Rho GTPases; p21-activated kinase
* Abbreviations used in this paper: MT, microtubule; Pak, p21-activated kinase; PBD/ID(H83L), mutated p21-binding and autoinhibitory domains of Pak1.
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Introduction |
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In interphase cells, MTs are organized by the centrosome where many minus ends are anchored, whereas MT plus ends extend into the periphery. MT plus ends exhibit a nonequilibrium polymerization behavior referred to as dynamic instability (Desai and Mitchison, 1997), the stochastic switching between phases of growth and shortening. This is described by four parameters: the rates of growth and shortening and the transition frequencies from growth to shortening (catastrophe frequency) and from shortening to growth (rescue frequency). In vivo, MT-associated proteins, MT plus-end binding proteins, and soluble factors regulate dynamic instability. In migrating cells, the MT cytoskeleton is polarized. In fibroblasts and endothelial cells, the centrosome and most MTs are oriented toward the leading edge (Wittmann and Waterman-Storer, 2001), and in epithelial cells, MT plus ends close to the leading edge often grow more persistently than MTs further back in the cell body (Waterman-Storer and Salmon, 1997; Wadsworth, 1999; Ballestrem et al., 2000).
Recent evidence suggests that MTs can also be targets of Rho GTPase regulation. Cdc42 mediates the cytoplasmic dynein-dependent orientation of the centrosome toward the leading edge (Etienne-Manneville and Hall, 2001; Palazzo et al., 2001a), whereas RhoA and its effector mDia regulate the formation of stable MTs (Palazzo et al., 2001b). In addition, a recent report showed that stimulation of HeLa cells with EGF results in phosphorylation of Op18/stathmin in a Rac1- and p21-activated serine/threonine kinase-dependent manner (Daub et al., 2001; Bokoch, 2003). Because Op18/stathmin is an inhibitor of MT polymerization that is inactivated by phosphorylation, we used time-lapse fluorescence microscopy to investigate whether signaling through Rac1 and p21-activated kinases (Paks) could regulate MT dynamic instability in vivo. We find that constitutively active Rac1(Q61L) promotes Pak-dependent MT growth and turnover. Similarly, Pak also mediates lamellipodial actin polymerization and retrograde flow downstream of Rac1(Q61L), demonstrating a coordinated regulation of MT and actin dynamics in the leading edge of migrating cells.
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Results and discussion |
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Similar to our current finding that Rac1-dependent increases in MT retrograde flow promote MT turnover, pharmacological treatment of newt lung cells that resulted in an increased rate of retrograde flow also increased MT breakage and turnover (Gupton et al., 2002). This supports the idea that retrograde flowinduced MT breakage may be a major pathway for MT turnover in migrating cells.
Pak inhibition blocks Rac1(Q61L)-induced MT growth
To determine if Paks mediated these dramatic Rac1 effects on MT plus-end growth and retrograde flow, we analyzed individual MT dynamics in control and Rac1(Q61L)-expressing cells injected with a mutated fragment of Pak1, PBD/ID(H83L), that inhibits the kinase activity of Paks, but cannot sequester activated Rac1 (Zenke et al., 1999). In PtK1 cells, Pak inhibition often caused complete retraction of the lamellum (Fig. 4, A and C; Video 6), but did not have this effect in cells expressing constitutively active Rac1(Q61L) (Fig. 4, B and D; Video 7). However, either Pak inhibition alone or in combination with Rac1(Q61L) expression resulted in MTs spending less time growing and exhibiting an increased catastrophe frequency, resulting in decreased MT net growth as compared with MTs in Rac1(Q61L)-expressing cells or pioneer MTs in control cells (Fig. 4, E and F; Table I). In addition, Pak inhibition blocked Rac1(Q61L)-accelerated MT retrograde flow, buckling, and breakage, which returned the total MT polymer level (68 ± 6%, n = 35) to what we observed in control cells, indicating that Pak is required for Rac1(Q61L)-mediated increase in MT turnover. As a control, we found that an inactivated Pak inhibitor, PBD/ID(H83L, L107F), did not affect MT dynamics in Rac1(Q61L)-expressing cells. Thus, Pak activation is required for the promotion of pioneer MT dynamics downstream of Rac1.
To determine if Paks were the sole regulators of MT dynamics downstream of Rac1, we examined the effects of various constitutively active forms of Pak1, Pak1(T423E), or Pak1(H83L, H86L) (Sells et al., 1999) and their membrane-targeted CAAX-box fusions, on the dynamics of individual MTs in PtK1 cells. However, we could not observe a consistent effect on MT organization or dynamic behavior as compared with control cells (unpublished data), indicating that although Pak activation is necessary for Rac1-induced MT plus-end growth, constitutively active Pak1 alone is not sufficient. This suggests that Pak-mediated phosphorylation and inactivation of Op18/stathmin might not be the major regulatory mechanism for leading edge MT dynamics induced by Rac1 (Daub et al., 2001). However, expression of constitutively active Pak1 likely caused high Pak1 activity globally throughout the cell, but local regulation of cytoskeletal dynamics at the leading edge may require local activation of Paks (Sells et al., 2000). Thus, constitutively active Pak1 could have simply caused a shift to a new global equilibrium in which MT plus-end dynamics might be similar to control cells. In addition, constitutively active Pak1 did not promote obvious changes of cell morphology or lamellipodial protrusion as it has been observed in fibroblasts (Sells et al., 1999). This indicates that multiple pathways regulating cytoskeletal dynamics downstream of Rac1 must exist, and is consistent with the apparently complicated role of Paks in the regulation of actin contraction and polymerization dynamics (Bokoch, 2003).
Paks regulate actin retrograde flow downstream of Rac1
Because altering the activity of Rac1 and downstream inhibition of Paks affected MT retrograde flow, we hypothesized that this was due to coupling of MT movements to those of the actin cytoskeleton (Gupton et al., 2002; Salmon et al., 2002). Therefore, we examined actin organization and dynamics in cells expressing constitutively active Rac1(Q61L), as compared with such cells additionally injected with the Pak inhibitor PBD/ID(H83L). First, we fixed and stained cells with fluorescent phalloidin. Rac1(Q61L) expression induced the formation of a dense F-actin meshwork in the lamellipodium and of transverse bundles at the base of the lamellum (Fig. 5 A). When PBD/ID(H83L) was injected into Rac1(Q61L)-expressing cells, the transverse actin bundles were lost in 90% of the cells and the width of the lamellipodium was reduced significantly (1.9 ± 0.4 µm versus 0.9 ± 0.2 µm; Fig. 5 D). In addition, extensive parallel actin bundles that stretched from the cell body to the lamellipodium formed in >60% of injected cells (Fig. 5 A) (Zhao et al., 1998). The F-actin organization in control PtK1 cells appeared to be a hybrid of these two phenotypes.
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Together, these results indicate that Pak activity is required for Rac1-induced changes in actin and MT organization and dynamics. In this paper, we have shown that Rac1 activation promotes Pak-dependent MT growth in epithelial cells. However, the underlying molecular mechanism remains to be defined, and is likely to involve more than one MT regulatory protein. In addition, dynamic interactions between MTs and the actin cytoskeleton might be required for pioneer MT behavior to occur. Our previous work has shown that MT growth can activate Rac1 in tissue cells by a yet unknown mechanism (Waterman-Storer et al., 1999). Thus, a decrease in Rac1 activity could be responsible for the inhibition of protrusion in the absence of MTs. Together with our current results, this suggests that Rac1 and MT growth might constitute a positive feedback loop in which MT growth promotes Rac1 activation and Rac1 promotes further MT growth to reinforce the polarization of a migrating cell in the absence of extracellular signals.
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Materials and methods |
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Microscopy and image analysis
Cells were maintained on the microscope stage in aluminum slide chambers in culture medium containing 0.51.0 U oxyrase per ml (Oxyrase, Inc.) to inhibit photobleaching and kept at 3537°C with an air stream incubator (Nevtek). To measure MT dynamic instability parameters, epifluorescence image series were acquired on an inverted microscope (model TE300; Nikon) equipped with electronically controlled shutters, filter wheels, and a 14-bit cooled CCD camera (Orca II; Hamamatsu Corporation) controlled by MetaMorph® software (Universal Imaging Corp.) at 5-s intervals using a 60x/1.4 NA Plan Apo objective lens (Nikon). Actin fluorescent speckle time-lapse series were acquired on a spinning disk confocal microscope (Adams et al., 2003) with a 100x/1.4 NA Plan Apo objective lens, and images were processed as described previously (Salmon et al., 2002).
Images in MT time-lapse sequences were registered relative to fiduciary marks on the MT lattices such that tracking of the MT ends accurately measured plus end dynamics without the bias of retrograde MT flow. Approximately 10 MTs that remained visible for several minutes were tracked in each cell using the MetaMorph® "track points" function, and data were analyzed in Excel (Microsoft) with a custom-written macro. Only MT length changes exceeding the optical resolution limit of 0.25 µm per frame (3 µm/min) were considered growth or shortening events, and rates were calculated as instantaneous velocities for each pair of consecutive image frames. Transition events from pause to shortening (or growth) were only classified as catastrophes (or rescues) if growth (or shortening) preceded the pause. The catastrophe (or rescue) frequency was defined as number of catastrophes (or rescues) divided by the time the MT spent growing (or shortening). Images for presentation and movie sequences were contrast inverted and subjected to an "unsharp mask" filter.
Online supplemental material
Video 1 shows MTs in a control PtK1 cell (Fig. 1). Video 2 shows MTs in a constitutively active Rac1(Q61L)-expressing PtK1 cell (Fig. 2). Video 3 shows MTs in a dominant-negative Rac1(T17N)-expressing PtK1 cell (Fig. 3). Video 4 shows direct comparison of MT dynamics in PtK1 cells expressing Rac1(Q61L) or Rac1(T17N). Video 5 shows MT fragments exhibiting net treadmilling in a Rac1(Q61L)-expressing PtK1 cell (Fig. 2 D). Video 6 shows MTs in a PtK1 cell injected with the Pak inhibitory fragment PBD/ID(H83L) (Fig. 4, A and C). Video 7 shows MTs in a PBD/ID(H83L)-injected, Rac1(Q61L)-expressing PtK1 cell (Fig. 4, B and D). Video 8 shows actin dynamics in a Rac1(Q61L)-expressing PtK1 cell (Fig. 5 B). Video 9 shows actin dynamics in a PBD/ID(H83L)-injected, Rac1(Q61L)-expressing PtK1 cell (Fig. 5 B). Online supplemental material available at http://www.jcb.org/cgi/content/full/jcb.200303082/DC1.
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Acknowledgments |
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This work was funded by National Institutes of Health grants GM61804 to C.M. Waterman-Storer, GM39434 to G.M. Bokoch, and an EMBO Long Term fellowship to T. Wittmann.
Submitted: 12 March 2003
Revised: 24 April 2003
Accepted: 28 April 2003
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References |
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