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Available online at www.sciencedirect.com

journal homepage: www.elsevier.com/locate/yexcr

Review Article

Centrosome positioning in polarized cells: Common themes and variations Julien Elrica,b, Sandrine Etienne-Mannevillea,n a

Institut Pasteur - CNRS URA 2582, Cell Polarity, Migration and Cancer Unit, 25 rue du Dr Roux, 75724 Paris Cedex 15, France b Université Pierre et Marie Curie, Cellule Pasteur UPMC, rue du Dr Roux, 75015 Paris, France

article information

abstract

Article Chronology:

The centrosome position is tightly regulated during the cell cycle and during differentiated

Received 27 August 2014

cellular functions. Because centrosome organizes the microtubule network to coordinate both

Accepted 1 September 2014

intracellular organization and cell signaling, centrosome positioning is crucial to determine either

Available online 8 September 2014

the axis of cell division, the direction of cell migration or the polarized immune response of

Keywords:

lymphocytes. Since alteration of centrosome positioning seems to promote cell transformation

Polarity

and tumor spreading, the molecular mechanisms controlling centrosome movement in response

Centrosome

to extracellular and intracellular cues are under intense investigation. Evolutionary conserved

Nucleus

pathways involving polarity proteins and cytoskeletal rearrangements are emerging as common

Molecular motors

regulators of centrosome positioning in a wide variety of cellular contexts. & 2014 Elsevier Inc. All rights reserved.

Cytoskeleton

Contents 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Centrosome positioning during cell division . . . . . . . . . . . . . . . . . . . . . . . . . 3. Centrosome positioning during front-to-rear polarization of migrating cells 4. Centrosome positioning during immune synapse formation . . . . . . . . . . . . . 5. Conclusion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1.

Introduction

The centrosome, which is generally localized near the geometric cell center, displays a remarkably well-conserved structure among n

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distant organisms [8]. It consists of a pair of centrioles formed of nine-triplet microtubules. Surrounding the two centrioles, the pericentriolar material supports microtubule nucleation and microtubule minus end stabilization. In most eukaryotic cells, the

Corresponding author. Fax: þ33 1 4568 8548. E-mail address: [email protected] (S. Etienne-Manneville).

http://dx.doi.org/10.1016/j.yexcr.2014.09.004 0014-4827/& 2014 Elsevier Inc. All rights reserved.

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centrosome is the main microtubule-organizing center (MTOC) and thus participates in the control of cell shape, cell division and cell motility [60]. Beyond its function as MTOC the centrosome should also be considered as a signaling platform. This is well illustrated in the Caenorhabditis elegans one-cell embryo. The entry of the spermsupplied centrosome provides signaling molecules that initiate the anterior–posterior cell polarity axis [11,7]. In differentiating neurons, a centrosomal signaling involving CaMKIIβ controls dendrite retraction and pruning [73]. In 1888, Theodor Bovery, who coined the term centrosome, provided the first experimental data on the importance of this “dynamic center” in chromosome segregation and spindle maintenance. Following duplication, the so-called mother and daughter centrioles form the mother and daughter centrosomes. The position of the two centrosomes define the position of the mitotic spindle and hence the orientation and the position of the division plane [68]. The control of mitotic spindle orientation is essential for symmetric and asymmetric cell division. Spindle positioning depends on intracellular cues, in particular in single cell organisms such as budding yeast and in the C. elegans zygote. In multicellular organisms, extracellular signals resulting from cell adhesion to the extracellular matrix or from cell–cell interactions serve as major polarity cues. Spindle orientation relative to these environmental cues has a profound impact on tissue architecture. During epithelial morphogenesis, for instance, spindle orientation parallel to the plane of the epithelium drives symmetric cell divisions and tissue spreading [4,14,29], whereas spindle orientation along the baso-apical axis leads to asymmetric division and tissue thickening [55,72]. Defects in centrosome positioning will result in abnormal mitotic spindle orientation and defects in planar division [65]. In the case of asymmetric cell divisions, the orientation and the localization of the mitotic spindle insure that cell fate determinants are differently distributed between the daughter cells [51]. Moreover, the specific segregation of the two centrosomes in the daughter cells suggests that each centrosome detains specific functions during differentiation and that the regulation of centrosome positioning is associated with centriole specific factors. In Drosophila male germline stem cells and in mouse embryo neural progenitors, the mother centrosome is retained by the stem cells [101,95]. In contrast, in Drosophila neuroblasts, the daughter centrosome remains trapped near the neuroblast apical cortex while the differentiated Ganglion Mother Cell inherits the mother centrosome [47]. In this case, the daughter centriole, in which the protein centrobin is exclusively found, retains most of the pericentriolar material and most of the MTOC activity at the onset of mitosis [74,79]. In interphase cells, the centrosome is tightly associated with the nucleus. The interaction between the centrosome and the nucleus involves the cytoskeleton and multiple proteins of the nuclear envelope [38]. In non-polarized cells, the centrosome and the nucleus localize near the cell center with no preferential orientation of the nucleus–centrosome axis. In polarized cells, the relative position of the centrosome compared to the nucleus corresponds to the main cell polarity axis and has clear implications in cellular functions. In differentiated epithelial cells, the centrosome is localized near the apical surface above the nucleus and contributes to the formation of an apical–basal microtubule network and epithelial polarity [28]. During neuronal differentiation, the centrosome is located between the nucleus and a neurite that will become the single axon while all other neurites will differentiate into dendritic processes [16]. The Golgi apparatus

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localizes contiguously to the centrosome in a microtubule and dynein dependent manner [76]. Thus, the centrosome together with the Golgi apparatus promotes microtubule polymerization, membrane and protein delivery to favor axonal growth [16]. Because of its essential role in intracellular organization, the centrosome is precisely positioned in the cytoplasm and changes in centrosome positioning occur to facilitate specific cell functions, such as immune cell response or cell migration. When a T cell encounters a target antigen-presenting cell, the centrosome delocalizes to the newly formed immunological synapse (IS) [3]. The reorientation of the centrosome in front of the nucleus and its translocation to the membrane is essential to ensure the polarized delivery of secretory granules to the IS [91]. This event occurs within few minutes after T cell receptor (TCR) engagement and relies on major rearrangements of both the actin and the microtubule networks [3]. In migrating cells, the centrosome generally localizes between the nucleus and the leading edge [36,106]. The Golgi apparatus and the recycling compartment localize with the centrosome in front of the nucleus in the direction of migration. Microtubules emanating from the centrosome and the Golgi complex, extend toward the protrusion most likely to serve as delivery tracks for membrane components and signaling molecules [1,23]. Centrosome re-positioning in response to extracellular and intracellular cues is essential for centrosomal functions and delays in this process have been shown to directly impact how fast the first asymmetric division of C. elegans embryo, zebrafish neurulation, T cell immune response, or cell migration are achieved [94,12]. The central position of the centrosome in interphase cells may simply result from a balance of pushing or pulling forces reflecting the cell geometry [60,107]. Both microtubule dynamics and microtubule-associated motors anchored at the cell cortex can promote the centering of microtubule asters in microfabricated chambers [41,53]. The same force generators are likely to be involved in moving the centrosome to a specific location in response to extra or intracellular stimuli. However, the directed movement of the centrosome leading to its precise positioning necessitates the generation of polarized forces and therefore the localized regulation of the force generators. The intimate connection between the centrosome and microtubules has led to an intense investigation of the signaling cascades that control microtubule dynamics and cortical anchoring during centrosome positioning. More recently, the role of acto-myosin contraction has also emerged. In this review, we will examine the latest advances in the understanding of the different mechanisms that ensure correct centrosome positioning. Comparison of the mechanisms involved in various cellular functions gives evidence of redundant pathways that simultaneously control centrosome position and points to recurrent principles and cell-specific variations.

2.

Centrosome positioning during cell division

The positioning of the mitotic spindle poles requires the precise localization of both centrosomes and therefore the directed movement of at least one of the centrosomes. Forces generated by the microtubule minus-end directed motor dynein are crucial for correct spindle orientation in various organisms from budding yeast to mammalian cells [56,66,90]. The commonly accepted model involves a cortical pulling mechanism, in which dynein anchored at the cell

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cortex would pull on microtubules to promote centrosome movement. The control of mitotic spindle positioning has been essentially studied in asymmetric cell division, in which the spindle is offcentered and takes a specific orientation. In C. elegans one cell embryo, the mitotic spindle positioning is driven by a microtubule end-on capture-shrinkage mechanism [64]. This observation is consistent with more recent in silico study using a system with microfabricated barriers coated with dynein to reconstitute the cortical interaction of dynamic microtubules ends with dynein [53]. In this system, dynein binding to microtubule plus-ends increases the rate of microtubule catastrophes and disassembly. Dynein remains attached with the depolymerizing plus-ends, thereby generating forces up to several pico Newton. An even distribution of dyneingenerated forces from the cell periphery would thus promote centrosome centering. However, moving the two spindle poles to distinct and defined positions requires the generation of directed forces from specific sites at the cell cortex, suggesting that signaling

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cascades induced by intrinsic or extrinsic polarity cues converge to control dynein functions. A set of polarity proteins called Par proteins were initially identified in a genetic study of the asymmetric division of the C. elegans zygote. The antero–posterior asymmetric distribution of the different Par proteins controls the cortical pulling forces which position the mitotic spindle [37]. The asymmetric distribution of Par3, Par6 and of the atypical kinase C is a recurrent mechanism controlling centrosome positioning in multiple organisms [25]. The protein NuMA (Nuclear Mitotic Apparatus) serves as an evolutionary conserved bridge between the Par proteins and the dynein/dynactin complex [87,51] (Fig. 1A). NuMA provides a cortical anchor for dynein which generates pulling forces on astral microtubules [63]. In C. elegans aPKC (PKC-3) phosphorylates NuMA (LIN-5) at the anterior cell cortex. Although how phosphorylation exactly affects NuMA function remains to be elucidated, it is clear that it inhibits cortical pulling forces and promotes the posterior localization of the spindle [31]. Alternative

Fig. 1 – Multiple actin and microtubule-dependent processes that control centrosome positioning. A great diversity of molecular pathways regulates centrosome positioning during cell division (A), cell migration (B), or T cell activation (C). Green curved lines represent microtubules, blue straight lines acto-myosin fibers. The centrosome is shown as a purple dot, and chromosomes are in gray. Coordinated centrosome movements rely on microtubule-dependent as well as actin-dependent processes. The molecular mechanisms involved in centrosome movement are depicted in the right panels, in colored boxes. Their polarized distribution at the cell cortex is shown with the corresponding color on the drawing on the left. Despite the variety of cellular functions considered, several proteins such as Par proteins, PKCs, Dlg, and Cdc42, stand out as major regulators of the signaling pathways underlying centrosome positioning.

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pathways control NuMA and dynein recruitment to the cortex. LGN (leucine–glycine–asparagine repeat protein), in particular, binds to NuMA and plays a key role in its recruitment during asymmetric division. The Gαi subunit of heterotrimeric G proteins interacts with LGN and thus serves as a cortical anchor for NuMA and dynein (Fig. 1A). The Gαi/LGN/NuMA complex is conserved in vertebrates. Orthologs have been extensively studied in Drosophila (Gαi/Pins/Mud) and C. elegans (Gαi/GPR1/2/LIN-5) and have been shown to be essential for spindle orientation [5]. LGN also interacts with the Par complex. Par3 (Bazooka in Drosophila) interacts with the adapter protein mInsc (Inscuteable in Drosophila), a direct partner of LGN (Pins) [83,104,108]. Although LGN may not be able to bind simultaneously mInsc and NuMA [13,105], recent evidence suggests that LGN localization can be controlled by both the Par complex and by G proteins. During epidermal differentiation in mammals, Gαi3 and mInsc cooperate to promote the apical localization of LGN and nonplanar divisions [98]. Finally, ERM proteins (Ezrin, Radixin, Moesin) have been involved in the regulation of the LGN complex localization. ERM is a group of highly conserved proteins, which interact with transmembrane proteins and the cytoskeleton to organize membrane domains. In HeLa cells plated on adhesive micropatterns to control the orientation of the spindle axis, ERM proteins work in parallel of Gαi to ensure the polarized localization of LGN and NuMA and guide the spindle to its correct final orientation [59]. At the onset of mitosis, ERM proteins are phosphorylated by the SLK kinase. Activated ERM promote the local remodeling of cortical actin to facilitate the recruitment of the LGN/NuMA complex (Fig. 1A). The spatially restricted activation of ERM activation is essential for correct spindle orientation in HeLa cells in vitro and also in mouse apical progenitors in vivo [59]. Ezrin localization also tightly correlates with the position of the centrosome and the mitotic spindle during establishment of apico-basal polarity [39]. In this case, Merlin (also known as NF2 and closely related to ERM proteins), controls Ezrin cortical localization. In absence of Merlin, Ezrin is mislocalized and mitotic spindles are misoriented, leading to an aberrant epithelial architecture [39]. An alternative pathway involves moesin, the only ERM expressed in Drosophila. Moesin can directly bind to microtubules to influence their dynamics. By increasing the stability of microtubules at the cortex, moesin may locally facilitate the interaction between microtubules and NuMA to regulate mitotic spindle organization during metaphase [89]. Cell–cell interactions provide signals to guide the positioning of the mitotic spindle in epithelial sheets. NuMA localization is controlled, independently of LGN, by the planar cell polarity (PCP) pathway, which determines the orientation of the mitotic spindle along the antero–posterior or animal–vegetal tissue axis [84]. In Drosophila sensory organ precursors, Dsh interacts with NuMA (Mud) at the apical posterior cortex to position the posterior pole of the spindle, while the anterior LGN/NuMa complex pulls on the anterior spindle pole. This PCP–dependent Dsh-NuMA complex is involved in the control Drosophila asymmetric cell division and in symmetric cell division during zebrafish tissue morphogenesis [85]. Cadherin mediated cell–cell contacts have also been implicated in the control of centrosome positioning in astrocytes and epithelial cells, although the molecular pathway at play in these models is still unknown [18,21,22]. Herbert et al. [39] showed that α-catenin, a central component of adherens junctions, is required for the polarized distribution of Ezrin, suggesting that adherens junctions may regulate ERM recruitment to control centrosome and spindle positioning.

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Dynein-generated forces and astral microtubules are common players in a number of intracellular signals leading to spindle pole positioning. However, an alternative mechanism, independent of NuMA and dynein, has been revealed in Drosophila neuroblasts. LGN (Pins) binds to Dlg (Discs Large) to recruit the kinesin motor Kif13b (Khc73) and control microtubule anchoring [6,81,49]. Recent work studying the specific segregation of the mother and daughter centrosomes during the asymmetric division of Drosophila neuroblasts suggests that LGN (Pins) can act directly on the centrosome rather than at the cell cortex [48]. LGN (Pins) is required for the retention of the pericentriolar material in the daughter centriole and controls the movement of the daughter centrosome independently of NuMA (Mud) and Dlg. Finally, recent evidence indicates that the actin cytoskeleton may also be involved in mitotic spindle positioning. The role of the actin cytoskeleton in spindle orientation is well documented in yeast [52]. In MDCK cells, E-cadherin and α-catenin control spindle orientation without affecting the dynein complex [17]. In skin basal progenitors, inhibition of NuMA or dynein leads to a reorientation of the spindle from baso-apical to planar [97], suggesting the existence of a dynein-independent control of the planar orientation of the spindle. During the symmetric division of the epithelium in the early gastrula Xenopus embryo, the mitotic spindle is set up and maintained in the plane of the epithelium. This planar orientation relies on the balance between microtubules and Myosin 10-dependent forces oriented toward the basal side of the cell and acto-myosin flow oriented towards the apical surface [100]. Loss of one of these forces results in the baso-apical orientation of the spindle. It seems now essential to determine how polarity pathways coordinate the actin and microtubules functions during spindle positioning.

3. Centrosome positioning during front-to-rear polarization of migrating cells In migrating cells, the centrosome is generally localized between the nucleus and the leading edge and early studies in 2D showed that the centrosome constantly shifts as the lamellipodia changes direction to follow the direction of migration [27]. However, in 1D and 3D migration, fibroblasts exhibit a polarized microtubule network toward the direction of migration but the centrosome is located at the back of the cell [19]. The orientation of the centrosome–nucleus axis parallel to the front-to-rear polarity axis is thought to promote persistent directional migration by assuring a continuous trafficking to the leading edge [23]. Although this reorientation is seen in many cell types, the underlying mechanism(s) can vary from one cell type to another. In migrating astrocytes and neurons, the centrosome is actively repositioned in front of the nucleus [40]; SEM (unpublished results), whereas in fibroblasts, the centrosome remains at the center of the cell while the nucleus moves toward the cell rear [34]. In all cases, the centrosome localization is actively controlled and the process of centrosome positioning requires the integrity of the microtubule network. Hence, migrating cells cannot reorient their centrosome in the presence of microtubule depolymerizing drug such as nocodazole but will retrieve normal reorientation after wash out [27,24]. In fact, even a local disruption of microtubules triggered by a local application of nocodazole is sufficient to perturb centrosome positioning [9].

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The association of dynein with microtubules has long been implicated in centrosome positioning in migrating cells [42]. Cytoplasmic dynein is essential for centrosome reorientation and is concentrated at the leading edge of migrating astrocytes and fibroblasts [24,71,20]. In wound healing assays, activation of integrins at the wound edge is one of the first steps leading to cell polarization and migration. This activation leads to the spatially restricted activation of Cdc42 and to the recruitment of the Par6/ aPKC complex at the cell front [24,70,69]. Like in the context of cell division, the Par6/aPKC complex regulates centrosome positioning during cell migration in numerous organisms from Drosophila to mammalian cells [25]. In polarizing astrocytes, the activated Par6-aPKC complex induces the recruitment of adenomatous polyposis coli (APC) and Dlg1 at the cell front. APC, locally controlled by aPKC-dependent GSK3β inhibition, forms clusters at the plus-ends of leading microtubules. APC interaction with cortical Dlg1 promotes microtubule capture [26]. Dlg1 simultaneously associates with dynein via their common binding partner GKAP, leading to dynein accumulation on captured microtubules, to microtubule anchoring and to the forward movement of the centrosome [61]. Cdc42, Par6, PKCζ and GSK3β have also been involved in centrosome positioning and directed migration in neurons [99,40] (Fig. 1B). Although the centrosome is not actively moving in front of the nucleus in migrating NIH3T3 fibroblasts, dynein motor function is essential to maintain the centrosome central localization. The Par6/aPKC is also involved but, in this case, it acts in concert with the polarity protein PAR3 [82]. PAR3 localizes at cell–cell contacts where it can recruit the dynein complex by interacting with one of the dynein light intermediate chain (LIC2 but not LIC1) (Fig. 1B). The colocalization of dynein, Par3, and Par6/aPKC cannot be observed in migrating astrocytes (SEM, unpublished data), possibly because of differences in the composition of intercellular contacts in the two cell types. Junctional Par3 controls microtubule dynamics at cell–cell contacts to maintain the centrosome at the cell center. Par3 is also essential for centrosome positioning in vivo during zebrafish neurulation [43] and during epithelial organization in C. elegans intestinal cells [28]. Other proteins present at cell– cell contacts such as IQGAP or plus end-tracking proteins containing CAP-Gly microtubule-binding domains [30] are known to interact with microtubules and could potentially have a role in microtubule capture and centrosome positioning. Whether at the leading edge or at cell–cell contacts, dynein generates microtubulemediated pulling forces on the centrosome, which are likely to be coupled to the simultaneous depolymerization of the pulled microtubules. Kinesin-8 and kinesin-13 families have recently been implicated respectively in the regulation of astral microtubules in budding yeast and in chromosome segregation in vitro [67,93] and may contribute their depolymerizing activity to these processes in migrating cells. In migrating fibroblasts, the centrosome remains at the cell center while the nucleus is actively moved to the back of the cell, orientating the nucleus–centrosome axis towards the cell front. The nucleus rearward movement is linked to the retrograde flow of actin fibers that accompanies cell migration. Cdc42 is not only implicated in the Par6/aPKC recruitment but also in the activation of serine/threonin kinase MRCK which promotes myosin IIdependent actin retrograde flow to move the nucleus toward the rear of the cell [34,96]. The contact between the nucleus and actin structures is made through transmembrane actin-associated

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nuclear (TAN) lines, a nucleocytoskeletal structure composed of a linear array of Nesprin-2G and SUN2 proteins. Once coupled to actin cables, the nucleus moves with the actin flow toward the cell rear, behind the centrosome [58] (Fig. 1B). A pool of the LEMdomain protein emerin localizes at the outer nuclear envelope and modifies TAN line behavior, where it participates in centrosome orientation and nuclear movement. Emerin interacts with myosin IIB but not myosin IIA and organizes actin flow in migrating 3T3 fibroblasts. This activity of emerin does not require its actin binding site [10]. Emerin- or myosin IIB-depleted cells exhibit a non-directional nuclear movement and actin flow. Actomyosin contractions are also required to move the centrosome in migrating neurons. In this system, Par6α regulates myosin regulatory light chain (MLC2) phosphorylation that is necessary for myosin II activity [88]. In MDCK cells grown in 3D cysts, actomyosin contractility is also required for centrosome positioning and is mediated by Par-4/LKB1 via the RhoA–Rho kinase–myosin II pathway [77]. The impact of the nucleus position on centrosome positioning depends on the physical link between the nucleus and the centrosome. First identified in screens for neuronal migration defects [75,32], Lis1 and doublecortin are essential for the nucleus–centrosome coupling [103,35]. Taxol addition or dynein inhibition results in an increase of the centrosome–nucleus distance, which mimics the phenotype observed in Lis1 deficient cells. Doublecortin concentrates in a perinuclear area and is important for the stabilization of the perinuclear microtubule network [92]. Lis1 localizes predominantly at the centrosome and modulates dynein motor activity in neurons [33]. Lis1 interacts with dynein between the dynein ATPase and the microtubule binding domains. It prevents the communication between the two domains and increases the dynein attachment to microtubules [44]. Emerin which interacts with β-tubulin, can also participate in bridging the nucleus to the centrosome. The depletion of emerin in fibroblasts leads to an increased nucleus–centrosome distance [80]. It is tempting to speculate that regulating the coupling between the nucleus and the centrosome may be involved in controlling the relative position of these two structures. Despite cell type specificities, conserved molecular pathways acting on microtubule associated motors and on acto-myosin contractility lead to a balance between forces exerted on the centrosome and the nucleus in order to define and maintain a nucleus–centrosome axis parallel to the front-to-rear polarity axis of migrating cells (Fig. 2). In this context, small G proteins of the Rho family are emerging as essential players in the coordinated regulation of dynein and myosin generated forces required for the orientation of the centrosome–nucleus axis in migrating cells.

4. Centrosome positioning during immune synapse formation The activation of T cells triggered by an antigen-presenting cell induces the repositioning of the centrosome, which travels around the nucleus to reach the immunological synapse (IS). Dynein is enriched at the site of the immunological synapse [62]. Comparable to what happens in migrating cells, the mechanism at play involves Dlg1 [54]. In T cells, the localized accumulation of Dlg1 relies on its interaction with the FERM domain of the ERM protein

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Fig. 2 – The orientation of the centrosome–nucleus axis results from a balance of actin- and microtubule-mediated forces. The centrosome is depicted as a purple dot connected to the microtubule network (in green) which exerts dynein-mediated pulling forces from the cell cortex and from the nucleus. The acto-myosin network (in blue) generates pushing forces from the cell cortex and on the nucleus and contributes to the orientation of the centrosome–nucleus axis. Ezrin (Fig. 1C). Dlg1 silencing leads to a disorganization of the microtubule network and to defects in centrosome positioning. Yi et al. [102] developed an optical trap to gain spatial and temporal control over T cell activation by controlling the interaction between the antigen-presenting cell and the T cell . In this model, like during spindle positioning in dividing cells (see above), centrosome movement is driven by a microtubule end-on capture-shrinkage mechanism, combining pulling forces from cortical dynein and depolymerization of microtubules. The centrosome repositioning is biphasic, suggesting the involvement of several signaling pathways. The study of centrosome positioning in T cells also shed light on the potential role of microtubules post-translational modifications (PTM). Detyrosination and acetylation tend to characterize the most stable microtubules. The impact of PTMs on microtubule dynamics and functions remains unclear but may involve the preferential recruitment of specific microtubule motors [46]. In developing cortical neurons, the centrosomal protein Cep120 has been implicated in microtubule acetylation and centrosome localization [15]. In T cells, the main tubulin deacetylase HDAC6 concentrates at the IS where it modulates microtubule acetylation [86]. Over-expression of HDAC6 impairs centrosome translocation and the effect can be rescued by HDAC6 inhibition. Moreover, HDAC6 may also directly be involved in dynein recruitment [50]. Detyrosination seems equally important. Detyrosinated microtubules extend from the MTOC toward the IS and are required for

centrosome reorientation [86]. The detyrosination of a subset of microtubules requires the INF2 (inverted formin 2) as well as RAC1 and Cdc42 activity. Expression of Dia1 or FMNL1 is sufficient to restore detyrosinated tubulin levels and centrosome reorientation in INF2-depleted cells [2], suggesting that members of the formin family may be involved in the spatial and temporal regulation of microtubule stability. Although centrosome positioning in T cells is largely dependent on dynein, a recent study shed light on the additional role of myosin in this process [57]. Dynein and myosin II accumulate at opposite cell poles following TCR stimulation. While a dynein-dependent mechanism generates pulling forces at the contact site with the antigen-presenting cell, the asymmetric distribution of non-muscle myosin (NMII) creates pushing forces from the opposite pole. Diacylglycerol is produced at the IS and recruits PKCε, PKCη and PKCθ [57]. This leads to the phosphorylation of the non-muscle myosin regulatory light chain (MyoRLC) at Ser1 and Ser2 and to the inhibition of NMII. Rho-kinase (ROCK) phosphorylates MyoRLC on Thr18 and Ser19 to promote its local accumulation at the opposite cell pole (Fig. 1C). How exactly NMII participates in centrosome movement is not clear yet, but microtubules are required [57]. Similarly to what was described in budding yeast, microtubules could be directly pulled by myosin motors on actin cables [45].Silico study demonstrated that the association of strong dynein pulling forces and weaker myosin forces balanced with microtubules

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plus-end pushing forces on the cortex is sufficient to center the centrosome in interphase cells [107].

5.

Conclusion

In cell division, cell migration or T cell responses, the correct timing and the accuracy of centrosome positioning is crucial to define a polarity axis required for each of these cellular functions. Although signaling pathways controlling centrosome positioning vary with the nature of the signal regulating cell polarity, and with the cell type, the different systems show obvious similarity both in the signaling molecules involved and also in the mechanics responsible for directed intracytoplasmic movement of the centrosome. Hence, proteins such Cdc42, the LGN-NuMa complex, Dlg, and the Par complex are central elements in spindle pole localization, and in centrosome reorientation in migrating cells and activated T cells. Not surprisingly, dysfunction of several of the molecules involved in these processes results in tumor growth. In general, microtubules and their associated molecular motors generate forces directly on the centrosome. Even though it cannot solely control centrosome positioning, acto-myosin contraction takes part in the process. Coupling the two cytoskeletons through cross-regulation is a good way for the cell to maximize the efficiency of the repositioning. A number of proteins long described for regulating actin dynamics, such as Rho GTPases and formins, are now found to also function as microtubule regulators and are good candidates to coordinate the two cytoskeletons in a time and space dependent manner. Many other candidates such as APC, plectin, IQGAP [78] are also known to interact with both microtubules and actin and could mediate this regulation. The main challenge is now to dissect their specific implication with each cytoskeleton, and the mechanisms underlying the cytoskeletal interplay to gain a better understanding on how they can contribute to centrosome positioning.

Acknowledgments J. E. is funded by the University Paris VI. We thank J-B. Manneville and members of the Cell Polarity, Migration and Cancer lab. for their critical reading of the manuscript. This work was supported by the Institut National du Cancer (Grant no. N°2013-092), l’Association pour la Recherche contre le Cancer, and La Ligue contre le Cancer (Grant no. RS14/75-11).

references [1] A. Akhmanova, S.J. Stehbens, et al., Touch, grasp, deliver and control: functional cross-talk between microtubules and cell adhesions, Traffic 10 (3) (2009) 268–274. [2] L. Andres-Delgado, O.M. Anton, et al., INF2 promotes the formation of detyrosinated microtubules necessary for centrosome reorientation in T cells, J. Cell Biol. 198 (6) (2012) 1025–1037. [3] K.L. Angus, G.M. Griffiths, Cell polarisation and the immunological synapse, Curr. Opin. Cell Biol. 25 (1) (2013) 85–91. [4] L.A. Baena-Lopez, A. Baonza, et al., The orientation of cell divisions determines the shape of Drosophila organs, Curr. Biol. 15 (18) (2005) 1640–1644. [5] Y. Bellaiche, M. Gotta, Heterotrimeric G proteins and regulation of size asymmetry during cell division, Curr. Opin. Cell Biol. 17 (6) (2005) 658–663.

32 8 (2 014 ) 24 0 –24 8

[6] Y. Bellaiche, A. Radovic, et al., The partner of inscuteable/discslarge complex is required to establish planar polarity during asymmetric cell division in Drosophila, Cell 106 (3) (2001) 355–366. [7] D. Bienkowska, C.R. Cowan, Centrosomes can initiate a polarity axis from any position within one-cell C. elegans embryos, Curr. Biol. 22 (7) (2012) 583–589. [8] M. Bornens, J. Azimzadeh, Origin and evolution of the centrosome, Adv. Exp. Med. Biol. 607 (2007) 119–129. [9] A. Burakov, E. Nadezhdina, et al., Centrosome positioning in interphase cells, J. Cell Biol. 162 (6) (2003) 963–969. [10] W. Chang, E.S. Folker, et al., Emerin organizes actin flow for nuclear movement and centrosome orientation in migrating fibroblasts, Mol. Biol. Cell 24 (24) (2013) 3869–3880. [11] C.R. Cowan, A.A. Hyman, Asymmetric cell division in C. elegans: cortical polarity and spindle positioning, Annu. Rev. Cell Dev. Biol. 20 (2004) 427–453. [12] C.R. Cowan, A.A. Hyman, Centrosomes direct cell polarity independently of microtubule assembly in C. elegans embryos, Nature 431 (7004) (2004) 92–96. [13] S. Culurgioni, A. Alfieri, et al., Inscuteable and NuMA proteins bind competitively to Leu-Gly-Asn repeat-enriched protein (LGN) during asymmetric cell divisions, Proc. Natl. Acad. Sci. U S A 108 (52) (2011) 20998–21003. [14] S.M. da Silva, J.P. Vincent, Oriented cell divisions in the extending germband of Drosophila, Development 134 (17) (2007) 3049–3054. [15] F.C. de Anda, K. Meletis, et al., Centrosome motility is essential for initial axon formation in the neocortex, J. Neurosci. 30 (31) (2010) 10391–10406. [16] F.C. de Anda, G. Pollarolo, et al., Centrosome localization determines neuronal polarity, Nature 436 (7051) (2005) 704–708. [17] N. den Elzen, C.V. Buttery, et al., Cadherin adhesion receptors orient the mitotic spindle during symmetric cell division in mammalian epithelia, Mol. Biol. Cell 20 (16) (2009) 3740–3750. [18] R.A. Desai, L. Gao, et al., Cell polarity triggered by cell-cell adhesion via E-cadherin, J. Cell Sci. 122 (Pt 7) (2009) 905–911. [19] A.D. Doyle, F.W. Wang, et al., One-dimensional topography underlies three-dimensional fibrillar cell migration, J. Cell Biol. 184 (4) (2009) 481–490. [20] D.L. Dujardin, L.E. Barnhart, et al., A role for cytoplasmic dynein and LIS1 in directed cell movement, J. Cell Biol. 163 (6) (2003) 1205–1211. [21] I. Dupin, E. Camand, et al., Classical cadherins control nucleus and centrosome position and cell polarity, J. Cell Biol. 185 (5) (2009) 779–786. [22] I. Dupin, Y. Sakamoto, et al., Cytoplasmic intermediate filaments mediate actin-driven positioning of the nucleus, J. Cell Sci. 124 (Pt 6) (2011) 865–872. [23] S. Etienne-Manneville, Microtubules in cell migration, Annu. Rev. Cell Dev. Biol. 29 (2013) 471–499. [24] S. Etienne-Manneville, A. Hall, Integrin-mediated activation of Cdc42 controls cell polarity in migrating astrocytes through PKCzeta, Cell 106 (4) (2001) 489–498. [25] S. Etienne-Manneville, A. Hall, Cell polarity: Par6, aPKC and cytoskeletal crosstalk, Curr. Opin. Cell Biol. 15 (1) (2003) 67–72. [26] S. Etienne-Manneville, J.B. Manneville, et al., Cdc42 and Par6PKCzeta regulate the spatially localized association of Dlg1 and APC to control cell polarization, J. Cell Biol. 170 (6) (2005) 895–901. [27] U. Euteneuer, M. Schliwa, Mechanism of centrosome positioning during the wound response in BSC-1 cells, J. Cell Biol. 116 (5) (1992) 1157–1166. [28] J.L. Feldman, J.R. Priess, A role for the centrosome and PAR-3 in the hand-off of MTOC function during epithelial polarization, Curr. Biol. 22 (7) (2012) 575–582. [29] E.S. Fleming, M. Zajac, et al., Planar spindle orientation and asymmetric cytokinesis in the mouse small intestine, J. Histochem. Cytochem. 55 (11) (2007) 1173–1180.

E XP E RI M ENTA L CE L L R E S EA RC H

[30] N. Galjart, Plus-end-tracking proteins and their interactions at microtubule ends, Curr. Biol. 20 (12) (2010) R528–537. [31] M. Galli, J. Munoz, et al., aPKC phosphorylates NuMA-related LIN-5 to position the mitotic spindle during asymmetric division, Nat. Cell Biol. 13 (9) (2011) 1132–1138. [32] J.G. Gleeson, K.M. Allen, et al., Doublecortin, a brain-specific gene mutated in human X-linked lissencephaly and double cortex syndrome, encodes a putative signaling protein, Cell 92 (1) (1998) 63–72. [33] J.G. Gleeson, P.T. Lin, et al., Doublecortin is a microtubuleassociated protein and is expressed widely by migrating neurons, Neuron 23 (2) (1999) 257–271. [34] E.R. Gomes, S. Jani, et al., Nuclear movement regulated by Cdc42, MRCK, myosin, and actin flow establishes MTOC polarization in migrating cells, Cell 121 (3) (2005) 451–463. [35] P. Gonczy, J.M. Bellanger, et al., zyg-8, a gene required for spindle positioning in C. elegans, encodes a doublecortin-related kinase that promotes microtubule assembly, Dev. Cell 1 (3) (2001) 363–375. [36] A.I. Gotlieb, L.M. May, et al., Distribution of microtubule organizing centers in migrating sheets of endothelial cells, J. Cell Biol. 91 (2 Pt 1) (1981) 589–594. [37] S.W. Grill, P. Gonczy, et al., Polarity controls forces governing asymmetric spindle positioning in the Caenorhabditis elegans embryo, Nature 409 (6820) (2001) 630–633. [38] G.G. Gundersen, H.J. Worman, Nuclear positioning, Cell 152 (6) (2013) 1376–1389. [39] A.M. Hebert, B. DuBoff, et al., Merlin/ERM proteins establish cortical asymmetry and centrosome position, Genes Dev. 26 (24) (2012) 2709–2723. [40] H. Higginbotham, T. Tanaka, et al., GSK3beta and PKCzeta function in centrosome localization and process stabilization during slit-mediated neuronal repolarization, Mol. Cell Neurosci. 32 (1-2) (2006) 118–132. [41] T.E. Holy, M. Dogterom, et al., Assembly and positioning of microtubule asters in microfabricated chambers, Proc. Natl. Acad. Sci. U S A 94 (12) (1997) 6228–6231. [42] E.L. Holzbaur, R.B. Vallee, DYNEINS: molecular structure and cellular function, Annu. Rev. Cell Biol. 10 (1994) 339–372. [43] E. Hong, P. Jayachandran, et al., The polarity protein Pard3 is required for centrosome positioning during neurulation, Dev. Biol. 341 (2) (2010) 335–345. [44] J. Huang, A.J. Roberts, et al., Lis1 acts as a “clutch” between the ATPase and microtubule-binding domains of the dynein motor, Cell 150 (5) (2012) 975–986. [45] E. Hwang, J. Kusch, et al., Spindle orientation in Saccharomyces cerevisiae depends on the transport of microtubule ends along polarized actin cables, J. Cell Biol. 161 (3) (2003) 483–488. [46] C. Janke, J.C. Bulinski, Post-translational regulation of the microtubule cytoskeleton: mechanisms and functions, Nat. Rev. Mol. Cell Biol. 12 (12) (2011) 773–786. [47] J. Januschke, S. Llamazares, et al., Drosophila neuroblasts retain the daughter centrosome, Nat. Commun. 2 (2011) 243. [48] J. Januschke, J. Reina, et al., Centrobin controls mother-daughter centriole asymmetry in Drosophila neuroblasts, Nat. Cell Biol. 15 (3) (2013) 241–248. [49] C.A. Johnston, K. Hirono, et al., Identification of an Aurora-A/ PinsLINKER/Dlg spindle orientation pathway using induced cell polarity in S2 cells, Cell 138 (6) (2009) 1150–1163. [50] Y. Kawaguchi, J.J. Kovacs, et al., The deacetylase HDAC6 regulates aggresome formation and cell viability in response to misfolded protein stress, Cell 115 (6) (2003) 727–738. [51] J.A. Knoblich, Asymmetric cell division: recent developments and their implications for tumor biology, Nat. Rev. Mol. Cell Biol. 11 (12) (2010) 849–860. [52] P. Kunda, B. Baum, The actin cytoskeleton in spindle assembly and positioning, Trends Cell Biol. 19 (4) (2009) 174–179.

3 28 (2014) 24 0 –24 8

247

[53] L. Laan, N. Pavin, et al., Cortical dynein controls microtubule dynamics to generate pulling forces that position microtubule asters, Cell 148 (3) (2012) 502–514. [54] R. Lasserre, S. Charrin, et al., Ezrin tunes T-cell activation by controlling Dlg1 and microtubule positioning at the immunological synapse, EMBO J. 29 (14) (2010) 2301–2314. [55] T. Lechler, E. Fuchs, Asymmetric cell divisions promote stratification and differentiation of mammalian skin, Nature 437 (7056) (2005) 275–280. [56] Y.Y. Li, E. Yeh, et al., Disruption of mitotic spindle orientation in a yeast dynein mutant, Proc. Natl. Acad. Sci. U S A 90 (21) (1993) 10096–10100. [57] X. Liu, T.M. Kapoor, et al., Diacylglycerol promotes centrosome polarization in T cells via reciprocal localization of dynein and myosin II, Proc. Natl. Acad. Sci. U S A 110 (29) (2013) 11976–11981. [58] G.W. Luxton, E.R. Gomes, et al., TAN lines: a novel nuclear envelope structure involved in nuclear positioning, Nucleus 2 (3) (2011) 173–181. [59] M. Machicoane, C.A. de Frutos, et al., SLK-dependent activation of ERMs controls LGN-NuMA localization and spindle orientation, J. Cell Biol. 205 (6) (2014) 791–799. [60] J.B. Manneville, S. Etienne-Manneville, Positioning centrosomes and spindle poles: looking at the periphery to find the centre, Biol. Cell 98 (9) (2006) 557–565. [61] J.B. Manneville, M. Jehanno, et al., Dlg1 binds GKAP to control dynein association with microtubules, centrosome positioning, and cell polarity, J. Cell Biol. 191 (3) (2010) 585–598. [62] N.B. Martin-Cofreces, J. Robles-Valero, et al., MTOC translocation modulates IS formation and controls sustained T cell signaling, J. Cell Biol. 182 (5) (2008) 951–962. [63] A. Merdes, K. Ramyar, et al., A complex of NuMA and cytoplasmic dynein is essential for mitotic spindle assembly, Cell 87 (3) (1996) 447–458. [64] T. Nguyen-Ngoc, K. Afshar, et al., Coupling of cortical dynein and G alpha proteins mediates spindle positioning in Caenorhabditis elegans, Nat. Cell Biol. 9 (11) (2007) 1294–1302. [65] A. Noatynska, M. Gotta, et al., Mitotic spindle (DIS)orientation and DISease: cause or consequence?, J. Cell Biol. 199 (7) (2012) 1025–1035. [66] C.B. O’Connell, Y.L. Wang, Mammalian spindle orientation and position respond to changes in cell shape in a dynein-dependent fashion, Mol. Biol. Cell 11 (5) (2000) 1765–1774. [67] Y. Oguchi, S. Uchimura, et al., The bidirectional depolymerizer MCAK generates force by disassembling both microtubule ends, Nat. Cell Biol. 13 (7) (2011) 846–852. [68] S. Oliferenko, T.G. Chew, et al., Positioning cytokinesis, Genes Dev. 23 (6) (2009) 660–674. [69] N. Osmani, F. Peglion, et al., Cdc42 localization and cell polarity depend on membrane traffic, J. Cell Biol. 191 (7) (2010) 1261–1269. [70] N. Osmani, N. Vitale, et al., Scrib controls Cdc42 localization and activity to promote cell polarization during astrocyte migration, Curr. Biol. 16 (24) (2006) 2395–2405. [71] A.F. Palazzo, T.A. Cook, et al., mDia mediates Rho-regulated formation and orientation of stable microtubules, Nat. Cell Biol. 3 (8) (2001) 723–729. [72] N.D. Poulson, T. Lechler, Robust control of mitotic spindle orientation in the developing epidermis, J. Cell Biol. 191 (5) (2010) 915–922. [73] S.V. Puram, A.H. Kim, et al., A CaMKIIbeta signaling pathway at the centrosome regulates dendrite patterning in the brain, Nat. Neurosci. 14 (8) (2011) 973–983. [74] E. Rebollo, P. Sampaio, et al., Functionally unequal centrosomes drive spindle orientation in asymmetrically dividing Drosophila neural stem cells, Dev. Cell 12 (3) (2007) 467–474.

248

E XP E RI ME N TAL C E L L R ES E ARC H

[75] O. Reiner, R. Carrozzo, et al., Isolation of a Miller-Dieker lissencephaly gene containing G protein beta-subunit-like repeats, Nature 364 (6439) (1993) 717–721. [76] R.M. Rios, M. Bornens, The Golgi apparatus at the cell centre, Curr. Opin. Cell Biol. 15 (1) (2003) 60–66. [77] A.E. Rodriguez-Fraticelli, M. Auzan, et al., Cell confinement controls centrosome positioning and lumen initiation during epithelial morphogenesis, J. Cell Biol. 198 (6) (2012) 1011–1023. [78] O.C. Rodriguez, A.W. Schaefer, et al., Conserved microtubuleactin interactions in cell movement and morphogenesis, Nat. Cell Biol. 5 (7) (2003) 599–609. [79] N.M. Rusan, M. Peifer, A role for a novel centrosome cycle in asymmetric cell division, J. Cell Biol. 177 (1) (2007) 13–20. [80] G. Salpingidou, A. Smertenko, et al., A novel role for the nuclear membrane protein emerin in association of the centrosome to the outer nuclear membrane, J. Cell Biol. 178 (6) (2007) 897–904. [81] N. Sans, P.Y. Wang, et al., mPins modulates PSD-95 and SAP102 trafficking and influences NMDA receptor surface expression, Nat. Cell Biol. 7 (12) (2005) 1179–1190. [82] J. Schmoranzer, J.P. Fawcett, et al., Par3 and dynein associate to regulate local microtubule dynamics and centrosome orientation during migration, Curr. Biol. 19 (13) (2009) 1065–1074. [83] M. Schober, M. Schaefer, et al., Bazooka recruits inscuteable to orient asymmetric cell divisions in Drosophila neuroblasts, Nature 402 (6761) (1999) 548–551. [84] M. Segalen, Y. Bellaiche, Cell division orientation and planar cell polarity pathways, Semin. Cell Dev. Biol. 20 (8) (2009) 972–977. [85] M. Segalen, C.A. Johnston, et al., The Fz-Dsh planar cell polarity pathway induces oriented cell division via Mud/NuMA in Drosophila and zebrafish, Dev. Cell 19 (5) (2010) 740–752. [86] J.M. Serrador, J.R. Cabrero, et al., HDAC6 deacetylase activity links the tubulin cytoskeleton with immune synapse organization, Immunity 20 (4) (2004) 417–428. [87] K.H. Siller, C.Q. Doe, Spindle orientation during asymmetric cell division, Nat. Cell Biol. 11 (4) (2009) 365–374. [88] D.J. Solecki, N. Trivedi, et al., Myosin II motors and F-actin dynamics drive the coordinated movement of the centrosome and soma during CNS glial-guided neuronal migration, Neuron 63 (1) (2009) 63–80. [89] S. Solinet, K. Mahmud, et al., The actin-binding ERM protein Moesin binds to and stabilizes microtubules at the cell cortex, J. Cell Biol. 202 (2) (2013) 251–260. [90] L. Stevermann, D. Liakopoulos, Molecular mechanisms in spindle positioning: structures and new concepts, Curr. Opin. Cell Biol. 24 (6) (2012) 816–824. [91] J.C. Stinchcombe, E. Majorovits, et al., Centrosome polarization delivers secretory granules to the immunological synapse, Nature 443 (7110) (2006) 462–465. [92] T. Tanaka, F.F. Serneo, et al., Lis1 and doublecortin function with dynein to mediate coupling of the nucleus to the centrosome in neuronal migration, J. Cell Biol. 165 (5) (2004) 709–721.

32 8 (2 014 ) 24 0 –24 8

[93] R. Ten Hoopen, C. Cepeda-Garcia, et al., Mechanism for astral microtubule capture by cortical Bud6p priming spindle polarity in S. cerevisiae, Curr Biol 22 (12) (2012) 1075–1083. [94] M. Ueda, R. Graf, et al., Centrosome positioning and directionality of cell movements, Proc. Natl. Acad. Sci. U S A 94 (18) (1997) 9674–9678. [95] X. Wang, J.W. Tsai, et al., Asymmetric centrosome inheritance maintains neural progenitors in the neocortex, Nature 461 (7266) (2009) 947–955. [96] S. Wilkinson, H.F. Paterson, et al., Cdc42-MRCK and Rho-ROCK signalling cooperate in myosin phosphorylation and cell invasion, Nat. Cell Biol. 7 (3) (2005) 255–261. [97] S.E. Williams, S. Beronja, et al., Asymmetric cell divisions promote Notch-dependent epidermal differentiation, Nature 470 (7334) (2011) 353–358. [98] S.E. Williams, L.A. Ratliff, et al., Par3-mInsc and Galphai3 cooperate to promote oriented epidermal cell divisions through LGN, Nat. Cell Biol. 16 (8) (2014) 758–769. [99] K. Wong, X.R. Ren, et al., Signal transduction in neuronal migration: roles of GTPase activating proteins and the small GTPase Cdc42 in the Slit-Robo pathway, Cell 107 (2) (2001) 209–221. [100] S. Woolner, N. Papalopulu, Spindle position in symmetric cell divisions during epiboly is controlled by opposing and dynamic apicobasal forces, Dev. Cell 22 (4) (2012) 775–787. [101] Y.M. Yamashita, A.P. Mahowald, et al., Asymmetric inheritance of mother versus daughter centrosome in stem cell division, Science 315 (5811) (2007) 518–521. [102] J. Yi, X. Wu, et al., Centrosome repositioning in T cells is biphasic and driven by microtubule end-on capture-shrinkage, J. Cell. Biol. 202 (5) (2013) 779–792. [103] J. Yingling, Y.H. Youn, et al., Neuroepithelial stem cell proliferation requires LIS1 for precise spindle orientation and symmetric division, Cell 132 (3) (2008) 474–486. [104] F. Yu, X. Morin, et al., Analysis of partner of inscuteable, a novel player of Drosophila asymmetric divisions, reveals two distinct steps in inscuteable apical localization, Cell 100 (4) (2000) 399–409. [105] S. Yuzawa, S. Kamakura, et al., Structural basis for interaction between the conserved cell polarity proteins Inscuteable and Leu-Gly-Asn repeat-enriched protein (LGN), Proc. Natl. Acad. Sci. U S A 108 (48) (2011) 19210–19215. [106] A.M. Yvon, J.W. Walker, et al., Centrosome reorientation in wound-edge cells is cell type specific, Mol. Biol. Cell 13 (6) (2002) 1871–1880. [107] J. Zhu, A. Burakov, et al., Finding the cell center by a balance of dynein and myosin pulling and microtubule pushing: a computational study, Mol. Biol. Cell 21 (24) (2010) 4418–4427. [108] J. Zhu, W. Wen, et al., LGN/mInsc and LGN/NuMA complex structures suggest distinct functions in asymmetric cell division for the Par3/mInsc/LGN and Galphai/LGN/NuMA pathways, Mol. Cell 43 (3) (2011) 418–431.