Cortical control of microtubule stability and polarization Gregg G Gundersen1, Edgar R Gomes and Ying Wen In both dividing and interphase cells, microtubules are remodeled in response to signal transduction pathways triggered by a variety of stimuli. Members of the Rho family of small GTPases have emerged as key intermediates in transmitting signals to cortical factors that mediate capture of dynamic microtubules at specific sites. The specificity of cortical capture appears to be controlled by microtubule tip proteins and cortical receptors that bind these proteins. Recent studies suggest that some of the proteins interacting with microtubule tips behave as bridging proteins between the microtubule tip proteins and their cortical receptors. Such bridging proteins may enhance cortical capture of microtubules directly or indirectly through interactions with the actin cytoskeleton. Addresses Department of Anatomy & Cell Biology, Columbia University, Black Building 1217, 630 W. 168th Street, New York, NY 10032, USA 1 e-mail: [email protected]

Current Opinion in Cell Biology 2004, 16:106–112 This review comes from a themed issue on Cell structure and dynamics Edited by John A Cooper and Margaret A Titus 0955-0674/$ – see front matter ß 2003 Elsevier Ltd. All rights reserved. DOI 10.1016/j.ceb.2003.11.010

Abbreviations APC adenomatous polyposis coli protein CLASP CLIP-associated protein CLIP-170 cytoplasmic linker protein 170 LPA lysophosphatidic acid MT microtubule MTOC microtubule organizing center PKC protein kinase C SPB spindle pole body

Introduction How microtubule (MT) arrays are remodeled into specific arrays that contribute to cell division, migration and differentiation is a central question in cell biology. The intrinsic capability of MTs to grow and shrink, termed dynamic instability, is important for remodeling of MT arrays. Several factors can alter the intrinsic dynamic instability of MTs and so enhance the remodeling of MT arrays (reviewed in [1]). Dynamic instability has long been considered to give MTs the ability to search or sample the three-dimensional space of the cell for sites of interaction or attachment that contribute to the formation of specific arrays Current Opinion in Cell Biology 2004, 16:106–112

necessary for a particular cell function. In the selective stabilization hypothesis, Kirschner and Mitchison proposed that external signals would locally active cortical factors to stabilize dynamic MTs that happened to encounter the activated cortical factors [2]. One essential aspect of this model, namely the need for dynamic MTs, has been supported by abundant evidence that dynamic MTs are necessary for cell division, cell migration and cell differentiation [3,4]. However, evidence for signalmediated changes in MTs and the identity of cortical factors that mediate MT interactions with the cortex has been more difficult to obtain. The past two years, which is our focus in this review, has been a time of dramatic advances in understanding how signals are transmitted through intermediates to bring about changes in MT stability and polarization at cortical sites. In essence, these studies provide evidence for the first signal transduction pathways that regulate MT remodeling. These studies also point to a more extensive repertoire of cortical interactions than was envisioned by the original selective stabilization model. In this review, we consider the signals that stimulate MT remodeling, the role of Rho GTPases as signaling intermediates for the MT–cortex interactions, and the functions of MT- and cortex-associated proteins that act to mediate the interactions of MTs with the cortex. Throughout, we refer to interactions of MTs with the cortex as MT capture, by analogy with MT capture at the kinetochore. Direct evidence for MT capture at the cortex has been obtained in several systems by observing changes in the dynamic behavior of MTs at cortical sites when specific signaling pathways are activated [5,6,7,8]. Some confusion has arisen over the exact role that the proteins that are found at MT ends, which are called MT tip proteins [9,10], play in MT capture. MT tip proteins are localized selectively at the ends of growing MTs and are maintained there by unknown mechanisms [10,11]. We propose that, in addition to MT tip proteins and their cortical receptors, there may be a third class of proteins, which we call bridging proteins, that function to link the MT tip proteins to their cortical receptors.

Systems and signals The systems that have been particularly useful for studying MT–cortex interactions fall into two groups: first, those that involve spindle MTs and asymmetric cell divisions, and second, those that involve signal-induced remodeling of interphase or cytoplasmic MTs (Table 1). For the first group, the overall function of the MT remodeling is clear: as the spindle determines the plane www.sciencedirect.com

Cortical control of microtubule stability and polarization Gundersen, Gomes and Wen 107

Table 1 Summary of the signals and their molecular targets for regulating MT–cortex interaction in different systems. Systems

Signal

Small G protein

Effector

Effect of cortical interaction

References

Dividing cells S. cerevisiae (budding) C. elegans embryos (one-cell stage)

Bud site determinants; cell cycle Sperm entry?

Cdc42/Rho Cdc42

Bni1 Par6

MT capture and shrinkage MT pausing

[5,32,62] [22,23,63,64]

LPA EGF, HGF LPA Woundinga Shear stress Cell–cell interaction

Rho Cdc42/Rac Cdc42 Cdc42 Cdc42 Cdc42

mDia IQGAP Par6 ? Par6 Par6 ?

MT stabilization –Long term –Short term MTOC reorientation MTOC reorientation MTOC reorientation MTOC reorientation

[6,25] [7] [19] [20] [21] [18]

Interphase cells Fibroblasts

Astrocytes Endothelial cells T cells

? indicates a possible or unknown candidate. Wounding refers to the scratching of a strip of cells from a confluent monolayer.

a

of the cleavage furrow, asymmetrically positioning the spindle results in asymmetric daughter cells. In budding yeast, which is a special case of asymmetric cell division, cytoplasmic MTs move the intranuclear spindle to a preformed bud to ensure that both mother and bud receive a full complement of chromosomes. For the second group, the remodeled MT arrays may aid polarized secretion or delivery of factors to specific sites [12]. Remodeling of MTs in many of these systems has the characteristics of MT capture, but only in a few cases (budding and fission yeast, fibroblasts and C. elegans embryos) has MT capture been demonstrated by directly observing a change in MTs during activation of specific factors [5,6,7,8,13].

indication that Rho GTPases regulate MTs was the finding that Cdc42 was involved in MT organizing center (MTOC) reorientation during interactions between T helper cells and their targets [18]. Subsequently, RhoA was found to regulate the formation of a subset of stabilized MTs in the leading edge of migrating fibroblasts [6]. Rho appeared to regulate MT capture at the cell cortex, as movies showed that Rho activation induced a subset of MTs near the leading edge to pause for long periods without affecting parameters of dynamic instability [6].

Rho family GTPases: central regulators of MT–cortex interactions

Recent work has substantially extended these early studies. Cdc42 has now been shown to regulate MTOC reorientation in migrating fibroblasts [19], astrocytes [20] and endothelial cells [21], and to contribute to asymmetric spindle position in C. elegans embryos [22,23]. Dynein and dynactin are necessary for positioning the MTOC and spindle in many of these systems and appear to function downstream of Cdc42, although how Cdc42 regulates dynein or dynactin is unknown. In migrating cells, Cdc42 regulates MTOC reorientation by binding to its effector Par6 to activate PKCz [20,21]. Activation of PKCz in astrocytes inactivates GSK3b and this appears to be important for MTOC reorientation [24]. Targets of GSK3b are not yet known. The GSK3b substrate adenomatous polyposis coli protein (APC) is a candidate as dominantnegative versions of APC inhibited MTOC reorientation in astrocytes [24]. However, it is not yet clear whether dynein or dynactin are downstream of APC in this system.

Members of the family of Ras-related Rho GTPases have emerged as key intermediaries between the initial membrane signals and the cortical factors that are involved in controlling MT–cortex interactions. Rho GTPases are activated by GTP exchange factors in response to membrane receptors (such as the LPA receptor) and other factors [17]. In the active GTP-bound state, Rho GTPases interact with and activate effectors that directly or indirectly effect cortical capture of MT. The first

Par6 may not be the only effector for Cdc42-regulated MTOC reorientation in migrating cells. Another Cdc42 effector, IQGAP, also regulates MT capture in fibroblasts [7]. IQGAP interacts directly with cytoplasmic linker protein 170 (CLIP-170), a MT tip protein; in cells with activated IQGAP, MTs that reach the leading edge where IQGAP is localized exhibit transient stabilization. This cortical interaction is clearly distinct from the

There are a variety of signals that initiate MT remodeling in these systems, including soluble factors, cell–cell interactions, cell-cycle factors and even mechanical stimuli (Table 1). In most cases, it appears that diffusible signaling factors combine with cellular landmarks to establish localized cortical control of MTs. Thus, in budding yeast, bud-site determinants combine with cell-cycle signals to limit MT capture to the bud and bud neck [14,15]. In migrating fibroblasts, Rho activation by lysophosphatidic acid (LPA), combined with signals from newly engaged integrins, limits MT capture and stabilization to the leading edge [16].

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dynein-dependent sliding seen in yeast [5] and may serve to concentrate MTs toward the leading edge. Whether IQGAP and CLIP-170 also participate in MTOC reorientation is unknown; however, expression of a mutant of IQGAP that does not interact with CLIP-170 results in cells with multiple leading edges [7]. A Rho effector domain screen identified the formin mDia as the effector involved in the selective stabilization of MTs in migrating fibroblasts [25]. MTs stabilized by mDia neither grow nor shrink and are thought to be capped on their plus ends to give them long term (>1 hour) stability [26]. In budding yeast, the RhoGTPase-regulated formin Bni1 was previously shown to regulate end-on capture of MTs at the bud tip [5,27,28]. The MTs captured at the bud in yeast exhibit controlled shrinkage and do not persist for hours like those in mammalian cells; nonetheless, this raises the possibility that there is a conserved Rho–formin pathway to regulate end-on cortical MT capture and stability [29]. Further evidence for this comes from the finding that mammalian orthologs of other proteins in the yeast ‘capture and shrinkage’ pathway play a role in MT capture and stabilization in mammalian cells (see below). Rho GTPases may not solely regulate MTs through their action on MT capture. Rac can regulate MT dynamic instability by activating its effector Pak to phosphorylate the MT-destabilizing protein stathmin [30,31].

Cortical MT receptors, bridging proteins and MT tip proteins In the simplest scenario for MT capture, MTs interact with prepositioned cortical receptors through MT tip proteins [10,32]. A good example of this is in yeast, where the putative cortical receptor, Kar9, interacts with the MT

tip protein Bim1/Yeb1 to mediate the capture and shrinkage of MTs at the bud tip [33,34]. As there are several MT tip proteins and putative cortical receptors, this could explain the varied responses of captured MTs. Indeed, captured MTs can become stabilized (either transiently or more permanently), undergo controlled shrinkage or growth while remaining attached to the receptor or slide laterally along the cortex [35]. The role of Rho GTPases in such a model would be to regulate, either directly or indirectly, the cortical receptors and activate them for interaction with the tip proteins. Although there is support for such a two-component model, recent results call this model into question. Below we examine this new data and suggest a new model for the interaction of MTs with the cortex which involves intermediate ‘bridging proteins’ that function between MT tip proteins and the cortical receptors. These bridging proteins may act to bring the MT tip proteins and cortical receptors together (‘direct bridging’) and/or may fulfil this function by using actin filaments to guide MTs to their cortical receptors (‘indirect bridging’) (Figure 1, Table 2). Earlier studies suggested that Kar9 could be considered to be a cortical receptor for cytoplasmic MTs in yeast, as it was localized to the bud tip, interacted with the MT tip protein Bim1/Yeb1, and was necessary for capture and shrinkage of MTs at the bud tip [33,34,36]. However, Kar9 was later found to interact with the type-V myosin motor Myo2, which was also necessary for the spindle orientation mediated by this type of MT capture [37]. As Myo2 is responsible for moving cargoes to the bud along polarized actin cables that are anchored there [38], this raised the possibility that Myo2, through Kar9, was responsible for moving MTs to the bud. Results from

Figure 1

Indirect bridging

Tip Protein

Bridging protein

Direct bridging

Cortical receptor

Microtubule

F-Actin

Current Opinion in Cell Biology

Models for the role of bridging proteins in MT capture. In the indirect model, the MT tip protein interacts with a bridging protein that guides the MT to the cortical receptor by interacting with an actin filament. In the direct model, the bridging protein interacts with the MT tip protein and the cortical receptor. Current Opinion in Cell Biology 2004, 16:106–112

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Cortical control of microtubule stability and polarization Gundersen, Gomes and Wen 109

Table 2 Summary of cortical receptors, bridging proteins and MT tip proteins. Systems

Cortical receptor

Proposed bridging protein

MT tip protein

References

S. cerevisiae (bud tip)

Bni1? Num1 ApsA Mod5 Par3/Par6/aPKC? mDia IQGAP Par6/aPKC? Par6/aPKC?

Kar9 Dynein Dynein Tea1 Dynein? APC CLASP? Dynein Dynein, APC?

Bim1/Yeb1 Bik1, Pac1 NUDF Tip1 Dynactin? EB1 CLIP-170, CLIP-115 Dynactin Dynactin

[5,14,15,32,39,62] [40,41,42,43] [48,49] [8,53,54] [65–69] [25] (Wen and Gundersen, unpublished) [7,52] [19,21,51] [20,24]

Aspergillus nidulans S. pombe C. elegans embryos Fibroblasts Fibroblasts and endothelial cells Astrocytes ? indicates a possible candidate.

three recent studies support this idea and suggest that Kar9 may act as a bridging protein rather than a cortical receptor. Kar9 is first loaded on the spindle pole body (SPB) and then is repositioned to the ends of growing MTs before they reach the bud [14,15]. Also, Kar9tipped MTs, emerging at high angles from the SPB, are oriented toward the bud rapidly (in seconds) in a process dependent on actin and Myo2 [15]. Kar9 function in this spindle orientation pathway can be substituted with a chimeric protein comprised of Bim1 and Myo2 [39]. These results point to a role for Kar9 in mediating interaction between MTs and the actin cytoskeleton and place Kar9 activity before capture at the bud. A new question to emerge from these studies is whether Kar9 is involved in the ultimate capture at bud sites. The formin Bni1, which is responsible for nucleating the actin cables, is positioned at the bud tip and is a candidate for capturing MTs that are brought there by Myo2 and Kar9. To date there is no evidence for interactions between Bni1 and Kar9 or Bim1/Yeb1 in yeast. However, a recent study of the related formin pathway mediating MT capture and stabilization in mammalian cells has found that EB1, the mammalian ortholog of Bim1/Yeb1, and APC, a functional homolog of Kar9, both interact with the formin mDia and are involved in MT stabilization (Wen and Gundersen, unpublished). Thus, both Kar9 and APC may act as bridging proteins during MT capture to mediate or enhance the interaction with formins, which act as the cortical receptors (Table 2). Analogous results have been obtained for the MTcapture-and-sliding pathway in yeast. This pathway works in conjunction with the capture-and-shrinkage pathway to position the spindle in the bud neck and is known to be mediated by dynein and its regulator, dynactin, and Num1, a cortical protein with a PH domain [5,40,41]. Dynein has been proposed to be localized in the bud cortex and to slide MTs to move the nucleus through the bud neck. However, dynein has not been detected in the bud cortex and recent studies show that endogenous www.sciencedirect.com

dynein is localized at the ends of growing MTs in yeast in a fashion that depends on the proteins Pac1 and Bik1 [42,43]. Bik1 and Pac1 are the yeast orthologs of mammalian MT tip proteins CLIP-170 [44] and Lis1 [45] and are localized on MT tips in yeast [42,46]. Thus, Bik1 and/or Pac1 may be the tip molecules for dynein, which may act through them to bridge the MT end with the cortical receptor Num1. Consistent with this, dynein interacts with Lis-1 [45] and Lis-1/Pac1 interacts with CLIP-170/Bik1 in mammalian cells [47] and yeast [43]. Further evidence for such a bridging role for dynein comes from Aspergillus, where dynein is also localized at the ends of growing MTs, along with NUDF, the Lis-1 ortholog [48]. Aspergillus also has a protein related to Num1, namely ApsA, which may be the cortical receptor [49]. In mammalian cells, dynein may also act as a bridging molecule between MT tip proteins and cortical receptors. Dynactin is a good candidate for the tip protein in mammalian cell as it is localized at the ends of growing MTs [50], interacts with dynein, and functions with dynein downstream of Cdc42 during MTOC reorientation in migrating cells [19,20]. Dynein is less frequently found at the ends of MTs [50]; however, it is localized with dynactin on MT ends in the leading edge of migrating fibroblasts where it may mediate MT capture, to reorient the MTOC [51]. The identity of the cortical receptor in this case is unknown, but the Cdc42 effector Par6, which acts upstream of dynein in MTOC reorientation in migrating cells, may be a candidate as it is also localized in the leading edge [20]. Does MT capture always require a bridging molecule? In fibroblasts the MT tip protein, CLIP-170, interacts directly with the Cdc42 effector IQGAP and this results in the transient stabilization of captured MTs at the leading edge of fibroblasts [7]. Because other Rho effectors may be cortical receptors (above) and IQGAP is prominently localized in the cortex, IQGAP may function as a cortical receptor. CLIP-170 also interacts with CLASPs (CLLIP-asssociated proteins) at the ends of Current Opinion in Cell Biology 2004, 16:106–112

110 Cell structure and dynamics

MTs and CLASPs contribute to MT stabilization in fibroblasts [52], suggesting that CLASPs may be bridging proteins for CLIP-170. The interaction of CLASPs with IQGAP has not yet been tested. The concept of bridging proteins may also help to explain MT capture in Saccharomyces pombe. In these cells, the tip protein Tip1, an ortholog of CLIP-170, controls the localization of Tea1, a kelch domain protein that accumulates at cell ends [8,53]. Tea 1 may act as a bridging molecule as its accumulation at cell ends is dependent on the recently identified cortical receptor Mod5 [54]. The MT capture mediated by these proteins is transient, with the MTs persisting at cell ends for less than two minutes, similar to the transient MT stabilization by CLIP-170 and IQGAP in mammalian cells. We have raised the idea that there may be bridging proteins that enhance the capture of MTs by cortical receptors. These bridging proteins share the ability to interact with both MT tip proteins and cortical capture receptors. Considered as a group, they exhibit distributions in cells that have been confusing: sometimes they are localized on MTs, usually at their ends, at other times they exhibit cortical localizations, and they have been found at both locations. We think these varied locations may reflect different states of their bridging activity. We propose that the function of these bridging proteins is to enhance the probability that rare MT capture events will occur. At the simplest level this may just be due to their ability to tether directly the MT tip protein to the cortical receptor (Figure 1). However, they may also enhance the probability that MTs will encounter their cortical receptors. In the best-documented case, that of Kar9 in yeast, the bridging molecule interacts with the actin cytoskeleton (via Myo2) and this enhances the probability that the MT will find the bud tip and be available for capture. Intriguingly, the actin filament that directs the MT to the capture site is nucleated by a formin (Bni1), whose orthologue in mammalian cells, mDia, is thought to be involved in MT capture. In mammalian cells, a similar bridging function may be served by APC. APC is known to interact with the actin cytoskeleton, perhaps with a myosin motor [55], and is moved to the ends of some MTs where it may direct MTs to capture sites [56]. In contrast to the relatively simple actin structures in yeast, the actin cytoskeleton in mammalian cells forms diverse arrays, ranging from dense meshworks to long stress fibers. If bridging proteins interact with the actin cytoskeleton in mammalian cells, they may be important to enhance penetration of MTs through dense actin arrays, or to enhance delivery of MTs to cortical receptors from greater distances. Indeed, MTs are known to be directed toward focal adhesions, sites which anchor stress fibers [57,58], and there is new evidence that MTs are moved by the actin cytoskeleton Current Opinion in Cell Biology 2004, 16:106–112

in fibroblasts and neurons [59,60]. Some of the proteins that we have proposed to exhibit bridging activity are not known to be associated with myosins, but interact with other molecules associated with the actin cytoskeleton. For example, dynein interacts with b-catenin [61], a molecule that indirectly interacts with the actin cytoskeleton. So it may be that most bridging proteins make use of the actin cytoskeleton to enhance MT capture.

Conclusions We have surveyed the systems that have begun to yield important new information about how MT arrays are remodeled in cells, focusing on the major actors in the processes by which signals activate the capture of MTs in the cell cortex. We have raised the possibility that bridging proteins, in addition to MT tip proteins and cortical proteins, are involved in this process. If bridging proteins can indeed link MT tip proteins to cortical receptors and also to the actin cytoskeleton, this may be an important way in which the two cytoskeletal elements ‘crosstalk’.

References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as:  of special interest  of outstanding interest 1.

Howard J, Hyman AA: Dynamics and mechanics of the microtubule plus end. Nature 2003, 422:753-758.

2.

Kirschner M, Mitchison T: Beyond self-assembly: from microtubules to morphogenesis. Cell 1986, 45:329-342.

3.

Liao G, Nagasaki T, Gundersen GG: Low concentrations of nocodazole interfere with fibroblast locomotion without significantly affecting microtubule level: implications for the role of dynamic microtubules in cell locomotion. J Cell Sci 1995, 108:3473-3483.

4.

Tanaka E, Ho T, Kirschner MW: The role of microtubule dynamics in growth cone motility and axonal growth. J Cell Biol 1995, 128:139-155.

5.

Adames NR, Cooper JA: Microtubule interactions with the cell cortex causing nuclear movements in Saccharomyces cerevisiae. J Cell Biol 2000, 149:863-874.

6.

Cook TA, Nagasaki T, Gundersen GG: Rho guanosine triphosphatase mediates the selective stabilization of microtubules induced by lysophosphatidic acid. J Cell Biol 1998, 141:175-185.

7. 

Fukata M, Watanabe T, Noritake J, Nakagawa M, Yamaga M, Kuroda S, Matsuura Y, Iwamatsu A, Perez F, Kaibuchi K: Rac1 and Cdc42 capture microtubules through IQGAP1 and CLIP-170. Cell 2002, 109:873-885. Rac1/Cdc42, IQGAP and CLIP-170 form a tripartite complex to capture and transiently stabilize MTs. Disruption of the interaction results in altered MT arrays or cells with multiple leading edges. 8.

Brunner D, Nurse P: CLIP170-like tip1p spatially organizes microtubular dynamics in fission yeast. Cell 2000, 102:695-704.

9.

Schuyler SC, Pellman D: Microtubule ‘plus-end-tracking proteins’: the end is just the beginning. Cell 2001, 105:421-424.

10. Carvalho P, Tirnauer JS, Pellman D: Surfing on microtubule ends. Trends Cell Biol 2003, 13:229-237. 11. Galjart N, Perez F: A plus-end raft to control microtubule dynamics and function. Curr Opin Cell Biol 2003, 15:48-53. 12. Wittmann T, Waterman-Storer CM: Cell motility: can Rho GTPases and microtubules point the way? J Cell Sci 2001, 114:3795-3803. www.sciencedirect.com

Cortical control of microtubule stability and polarization Gundersen, Gomes and Wen 111

13. Labbe JC, Maddox PS, Salmon ED, Goldstein B: PAR proteins  regulate microtubule dynamics at the cell cortex in C. elegans. Curr Biol 2003, 13:707-714. MT dynamics in the anterior and posterior cortex during anaphase B are determined by measuring the time that a MT remains at the cortex. In wild-type embryos, MTs are more dynamic in the posterior cortex and depletion of Par-1 by RNAi does not affect this asymmetry. On the other hand, analysis of Par-2, Par-3 and Par-2/Par-3 (RNAi) mutants suggests that Par-3 stabilizes MT and that the effect of Par-2 on MT dynamics is due to the displacement of Par-3. Goa1 and Goa-16 depletion disrupt the asymmetry in MT dynamics, independently of Par-2 and Par-3. 14. Maekawa H, Usui T, Knop M, Schiebel E: Yeast Cdk1 translocates  to the plus end of cytoplasmic microtubules to regulate bud cortex interactions. EMBO J 2003, 22:438-449. This paper, along with [15], shows that Kar9 is loaded asymmetrically onto one of the SPBs. Unlike [15], they find Cdc28 localized on both SPBs and on MT tips. Transportation of both Cdc28 and Kar9 to MT tips requires a plus-end-directed kinesin Kip2. 15. Liakopoulos D, Kusch J, Grava S, Vogel J, Barral Y: Asymmetric  loading of Kar9 onto spindle poles and microtubules ensures proper spindle alignment. Cell 2003, 112:561-574. Kar9 associates with budward-directed SPBs and MTs. Photobleaching experiments show that Kar9 localization at MT tips is due to Kap 9 loading at SPBs rather than from cortical sites. Cdc28–Clb3 and –Clb4 phosphorylate Kar9. Clb-4 is localized at the mother-bound SPB so activated Cdc28 will prevent Kar9 association. 16. Palazzo AF, Eng CE, Schlaepfer DD, Marcantonio EE,  Gundersen GG: Localized stabilization of miicrotubules by integrin and FAK facilitated Rho signaling. Science 2003, in press. This paper shows that integrins and FAK regulate the ability of Rho to activate mDia to induce MT stabilization. FAK appears to regulate mDia activation indirectly by regulating the trafficking of a GM1 (ganglioside) lipid domain at the leading edge of migrating cells. 17. Etienne-Manneville S, Hall A: Rho GTPases in cell biology. Nature 2002, 420:629-635. 18. Stowers L, Yelon D, Berg LJ, Chant J: Regulation of the polarization of T cells toward antigen-presenting cells by Ras-related GTPase CDC42. Proc Natl Acad Sci U S A 1995, 92:5027-5031. 19. Palazzo AF, Joseph HL, Chen YJ, Dujardin DL, Alberts AS, Pfister KK, Vallee RB, Gundersen GG: Cdc42, dynein, and dynactin regulate MTOC reorientation independent of Rho-regulated microtubule stabilization. Curr Biol 2001, 11:1536-1541. 20. Etienne-Manneville S, Hall A: Integrin-mediated activation of Cdc42 controls cell polarity in migrating astrocytes through PKCf. Cell 2001, 106:489-498. 21. Tzima E, Kiosses WB, del Pozo MA, Schwartz MA:  Localized cdc42 activation, detected using a novel assay, mediates microtubule organizing center positioning in endothelial cells in response to fluid shear stress. J Biol Chem 2003, 278:31020-31023. Shear stress polarizes Cdc42 activation in the direction of flow to induce MTOC reorientation in endothelial cells. 22. Gotta M, Abraham MC, Ahringer J: CDC-42 controls early cell polarity and spindle orientation in C. elegans. Curr Biol 2001, 11:482-488. 23. Kay AJ, Hunter CP: CDC-42 regulates PAR protein localization and function to control cellular and embryonic polarity in C. elegans. Curr Biol 2001, 11:474-481. 24. Etienne-Manneville S, Hall A: Cdc42 regulates GSK-3b and  adenomatous polyposis coli to control cell polarity. Nature 2003, 421:753-756. Cdc42 activation of Par6/PKCz is shown to regulate MTOC reorientation in migrating astrocytes by inhibiting GSK3b. GSK3b may regulate MTOC reorientation through its action on APC.

27. Kohno H, Tanaka K, Mino A, Umikawa M, Imamura H, Fujiwara T, Fujita Y, Hotta K, Qadota H, Watanabe T et al.: Bni1p implicated in cytoskeletal control is a putative target of Rho1p small GTP binding protein in Saccharomyces cerevisiae. EMBO J 1996, 15:6060-6068. 28. Lee L, Klee SK, Evangelista M, Boone C, Pellman D: Control of mitotic spindle position by the Saccharomyces cerevisiae formin Bni1p. J Cell Biol 1999, 144:947-961. 29. Gundersen GG: Evolutionary conservation of microtubulecapture mechanisms. Nat Rev Mol Cell Biol 2002, 3:296-304. 30. Daub H, Gevaert K, Vandekerckhove J, Sobel A, Hall A: Rac/Cdc42 and p65PAK regulate the microtubule-destabilizing protein stathmin through phosphorylation at serine 16. J Biol Chem 2001, 276:1677-1680. 31. Wittmann T, Bokoch GM, Waterman-Storer CM: Regulation of  leading edge microtubule and actin dynamics downstream of Rac1. J Cell Biol 2003, 161:845-851. Rac1 is shown to promote MT plus end growth and turnover in PtK1 cells. 32. Bloom K: It’s a kar9ochore to capture microtubules. Nat Cell Biol 2000, 2:E96-E98. 33. Lee L, Tirnauer JS, Li J, Schuyler SC, Liu JY, Pellman D: Positioning of the mitotic spindle by a cortical-microtubule capture mechanism. Science 2000, 287:2260-2262. 34. Korinek WS, Copeland MJ, Chaudhuri A, Chant J: Molecular linkage underlying microtubule orientation toward cortical sites in yeast. Science 2000, 287:2257-2259. 35. Gundersen GG: Microtubule capture: IQGAP and CLIP-170 expand the repertoire. Curr Biol 2002, 12:R645-R647. 36. Miller RK, Rose MD: Kar9p is a novel cortical protein required for cytoplasmic microtubule orientation in yeast. J Cell Biol 1998, 140:377-390. 37. Yin H, Pruyne D, Huffaker TC, Bretscher A: Myosin V orientates the mitotic spindle in yeast. Nature 2000, 406:1013-1015. 38. Bretscher A: Polarized growth and organelle segregation in yeast: the tracks, motors, and receptors. J Cell Biol 2003, 160:811-816. 39. Hwang E, Kusch J, Barral Y, Huffaker TC: Spindle orientation in  Saccharomyces cerevisiae depends on the transport of microtubule ends along polarized actin cables. J Cell Biol 2003, 161:483-488. This paper and [14,15] show that Kar9-associated MTs do not search for the bud cortex; rather, they bind to Myo2 and are actively transported on actin cables to the bud tip. Myo2 localizes to MT plus ends. Cells with Myo2 mutants with a slower velocity also show reduced cytoplasmic MT movement. 40. Heil-Chapdelaine RA, Oberle JR, Cooper JA: The cortical protein Num1p is essential for dynein-dependent interactions of microtubules with the cortex. J Cell Biol 2000, 151:1337-1344. 41. Farkasovsky M, Kuntzel H: Cortical Num1p interacts with the dynein intermediate chain Pac11p and cytoplasmic microtubules in budding yeast. J Cell Biol 2001, 152:251-262. 42. Lee W-L, Oberle JR, Cooper JA: The role of the lissencephaly  protein Pac1 during nuclear migration in budding yeast. J Cell Biol 2003, 160:355-364. Dynein is localized on dynamic MT tips (see also [43]). The localization is lost in Pac1 (LIS1)-deleted cells and enhanced in cells lacking Num1 or Arp1 (a dynactin component). This suggests that dynein is delivered on MTs to the cortex where it generates forces for MT sliding.

25. Palazzo AF, Cook TA, Alberts AS, Gundersen GG: mDia mediates Rho-regulated formation and orientation of stable microtubules. Nat Cell Biol 2001, 3:723-729.

43. Sheeman B, Carvalho P, Sagot I, Geiser J, Kho D, Hoyt MA,  Pellman D: Determinants of S. cerevisiae dynein localization and activation. Implications for the mechanism of spindle positioning. Curr Biol 2003, 13:364-372. Dynein is localized on the tips of dynamic MTs. The authors also show that Bik1 (CLIP-170) is required for dynein localization on MTs. Deletion of dynactin components (p50, p150Glued) results in enhanced dynein association with MTs.

26. Infante AS, Stein MS, Zhai Y, Borisy GG, Gundersen GG: Detyrosinated (Glu) microtubules are stabilized by an ATPsensitive plus-end cap. J Cell Sci 2000, 113:3907-3919.

44. Perez F, Diamantopoulos GS, Stalder R, Kreis TE: CLIP-170 highlights growing microtubule ends in vivo. Cell 1999, 96:517-527.

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45. Faulkner NE, Dujardin DL, Tai CY, Vaughan KT, O’Connell CB, Wang Y, Vallee RB: A role for the lissencephaly gene LIS1 in mitosis and cytoplasmic dynein function. Nat Cell Biol 2000, 2:784-791. 46. Lin H, de Carvalho P, Kho D, Tai CY, Pierre P, Fink GR, Pellman D: Polyploids require Bik1 for kinetochore–microtubule attachment. J Cell Biol 2001, 155:1173-1184. 47. Coquelle FM, Caspi M, Cordelieres FP, Dompierre JP, Dujardin DL,  Koifman C, Martin P, Hoogenraad CC, Akhmanova A, Galjart N et al.: LIS1, CLIP-170’s key to the dynein/dynactin pathway. Mol Cell Biol 2002, 22:3089-3102. The MT tip protein CLIP-170 is shown to interact with dynein/dynactin through LIS1. 48. Han G, Liu B, Zhang J, Zuo W, Morris NR, Xiang X: The Aspergillus cytoplasmic dynein heavy chain and NUDF localize to microtubule ends and affect microtubule dynamics. Curr Biol 2001, 11:719-724. 49. Suelmann R, Sievers N, Fischer R: Nuclear traffic in fungal hyphae: in vivo study of nuclear migration and positioning in Aspergillus nidulans. Mol Microbiol 1997, 25:757-769. 50. Vaughan KT, Tynan SH, Faulkner NE, Echeverri CJ, Vallee RB: Colocalization of cytoplasmic dynein with dynactin and CLIP170 at microtubule distal ends. J Cell Sci 1999, 112:1437-1447. 51. Dujardin DL, Barnhart LE, Stehman S, Gomes ER, Gundersen GG,  Vallee RB: A role for cytoplasmic dynein in directed cell movement. J Cell Biol 2003, in press. Dynein, dynactin and Lis1 are localized at the leading edge of a migrating cell. Disruption of dynein or dynactin inhibits cell migration. 52. Akhmanova A, Hoogenraad CC, Drabek K, Stepanova T, Dortland B, Verkerk T, Vermeulen W, Burgering BM, De Zeeuw CI, Grosveld F, Galjart N: CLASPS are CLIP-115 and -170 associating proteins involved in the regional regulation of microtubule dynamics in motile fibroblasts. Cell 2001, 104:923-935. 53. Mata J, Nurse P: tea1 and the microtubular cytoskeleton are important for generating global spatial order within the fission yeast cell. Cell 1997, 89:939-949. 54. Snaith HA, Sawin KE: Fission yeast mod5p regulates polarized  growth through anchoring of tea1p at cell tips. Nature 2003, 423:647-651. Mod5 is identified as a cortical receptor for tea1. In mod5-deletion cells, tea1 is transported efficiently to MT tips, but it does not accumulate normally at cell tips. 55. Allan V, Nathke IS: Catch and pull a microtubule: getting a grasp on the cortex. Nat Cell Biol 2001, 3:E226-E228. 56. Mimori-Kiyosue Y, Shiina N, Tsukita S: Adenomatous polyposis coli (APC) protein moves along microtubules and concentrates at their growing ends in epithelial cells. J Cell Biol 2000, 148:505-518. 57. Kaverina I, Krylyshkina O, Small JV: Microtubule targeting of substrate contacts promotes their relaxation and dissociation. J Cell Biol 1999, 146:1033-1044.

Current Opinion in Cell Biology 2004, 16:106–112

58. Krylyshkina O, Anderson KI, Kaverina I, Upmann I, Manstein DJ,  Small JV, Toomre DK: Nanometer targeting of microtubules to focal adhesions. J Cell Biol 2003, 161:853-859. Using total-internal-reflection fluorescence microscopy, polymerizing MTs are shown to approach within 50 nm of focal adhesions. Shrinking MTs quickly move away from the cortex, suggesting that they may track along cortical elements during MT growth. 59. Salmon WC, Adams MC, Waterman-Storer CM: Dual-wavelength  fluorescent speckle microscopy reveals coupling of microtubule and actin movements in migrating cells. J Cell Biol 2002, 158:31-37. See also [60]. Fluorescence speckle microscopy of the actin and MT cytoskeletons shows that the two polymers are coordinately moved rearward in the cell body. 60. Schaefer AW, Kabir N, Forscher P: Filopodia and actin arcs guide  the assembly and transport of two populations of microtubules with unique dynamic parameters in neuronal growth cones. J Cell Biol 2002, 158:139-152. Similar analysis to [59] of coordinated movements of actin and MTs, but in neuronal growth cones. 61. Ligon LA, Karki S, Tokito M, Holzbaur EL: Dynein binds to b-catenin and may tether microtubules at adherens junctions. Nat Cell Biol 2001, 3:913-917. 62. Heil-Chapdelaine RA, Adames NR, Cooper JA: Formin’ the connection between microtubules and the cell cortex. J Cell Biol 1999, 144:809-811. 63. Wallenfang MR, Seydoux G: Polarization of the anterior– posterior axis of C. elegans is a microtubule-directed process. Nature 2000, 408:89-92. 64. Grill SW, Gonczy P, Stelzer EH, Hyman AA: Polarity controls forces governing asymmetric spindle positioning in the Caenorhabditis elegans embryo. Nature 2001, 409:630-633. 65. Etemad-Moghadam B, Guo S, Kemphues KJ: Asymmetrically distributed PAR-3 protein contributes to cell polarity and spindle alignment in early C. elegans embryos. Cell 1995, 83:743-752. 66. Hung TJ, Kemphues KJ: PAR-6 is a conserved PDZ domain-containing protein that colocalizes with PAR-3 in Caenorhabditis elegans embryos. Development 1999, 126:127-135. 67. Tabuse Y, Izumi Y, Piano F, Kemphues KJ, Miwa J, Ohno S: Atypical protein kinase C cooperates with PAR-3 to establish embryonic polarity in Caenorhabditis elegans. Development 1998, 125:3607-3614. 68. Skop AR, White JG: The dynactin complex is required for cleavage plane specification in early Caenorhabditis elegans embryos. Curr Biol 1998, 8:1110-1116. 69. Gonczy P, Pichler S, Kirkham M, Hyman AA: Cytoplasmic dynein is required for distinct aspects of MTOC positioning, including centrosome separation, in the one cell stage Caenorhabditis elegans embryo. J Cell Biol 1999, 147:135-150.

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