The cortical protein Num1p is essential for dynein-dependent interactions of microtubules with the cortex

In budding yeast, the mitotic spindle moves into the neck between the mother and bud via dynein-dependent sliding of cytoplasmic microtubules along the cortex of the bud. How dynein and microtubules interact with the cortex is unknown. We found that cells lacking Num1p failed to exhibit dynein-dependent microtubule sliding in the bud, resulting in defective mitotic spindle movement and nuclear segregation. Num1p localized to the bud cortex, and that localization was independent of microtubules, dynein, or dynactin. These data are consistent with Num1p being an essential element of the cortical attachment mechanism for dynein-dependent sliding of microtubules in the bud.     

Astral Microtubule Dynamics in Yeast: A Microtubule-based Searching Mechanism for Spindle Orientation and Nuclear Migration into the Bud

Localization of dynein–green fluorescent protein (GFP) to cytoplasmic microtubules allowed us to obtain one of the first views of the dynamic properties of astral microtubules in live budding yeast. Several novel aspects of microtubule function were revealed by time-lapse, three-dimensional fluorescence microscopy. Astral microtubules, about four to six in number for each pole, exhibited asynchronous dynamic instability throughout the cell cycle, growing at 0.3–1.5 μm/min toward the cell surface then switching to shortening at similar velocities back to the spindle pole body (SPB). During interphase, a conical array of microtubules trailed the SPB as the nucleus traversed the cytoplasm. Microtubule disassembly by nocodozole inhibited these movements, indicating that the nucleus was pushed around the interior of the cell via dynamic astral microtubules. These forays were evident in unbudded G1 cells, as well as in late telophase cells after spindle disassembly. Nuclear movement and orientation to the bud neck in S/G2 or G2/M was dependent on dynamic astral microtubules growing into the bud. The SPB and nucleus were then pulled toward the bud neck, and further microtubule growth from that SPB was mainly oriented toward the bud. After SPB separation and central spindle formation, a temporal delay in the acquisition of cytoplasmic dynein at one of the spindle poles was evident. Stable microtubule interactions with the cell cortex were rarely observed during anaphase, and did not appear to contribute significantly to spindle alignment or elongation into the bud. Alterations of microtubule dynamics, as observed in cells overexpressing dynein-GFP, resulted in eventual spindle misalignment. These studies provide the first mechanistic basis for understanding how spindle orientation and nuclear positioning are established and are indicative of a microtubule-based searching mechanism that requires dynamic microtubules for nuclear migration into the bud.     

Cell division and the microtubular cytoskeleton]

Kinetochore microtubules result from an interaction between astral microtubules and the kinetochore of the chromosomes after breakdown of the nuclear envelope at the end of prophase. In this process, the end of a microtubule projecting from one of the polar regions contacts the primary constriction of a chromosome. The latter then undergoes rapid poleward movement. Concerning the mechanism of anaphase chromosome movement, the motive force for the chromosome-to-pole movement appears to be generated at the kinetochore or in the region very close to it. It has not been determined whether chromosomes propel themselves along stationary kinetochore microtubules by a motor at the kinetochore, or they are pulled poleward by a traction fiber consisting of kinetochore microtubules and associated motors. As chromosomes move poleward coordinate disassembly of kinetochore microtubules might occur from their kinetochore ends. In diatom and yeast spindles, elongation of the spindle in anaphase (anaphase B) may be explained by microtubule assembly at polar microtubule ends in the spindle mid-zone and sliding of the antiparallel microtubules from the opposite poles. The sliding force appears to be generated through an ATP-dependent microtubule motor. In isolated sea urchin spindles, the microtubule assembly at the equator alone might provide the force for spindle elongation, although, in addition, involvement of microtubule sliding by a GTP-requiring mechanochemical enzyme cannot be excluded. Discussions were made on possible participation in anaphase chromosome movement of such microtubule motors as dynein, kinesin, dynamin and the claret segregation protein.     

Dynein-Driven Mitotic Spindle Positioning Restricted to Anaphase by She1p Inhibition of Dynactin Recruitment

Dynein is a minus-end–directed microtubule motor important for mitotic spindle positioning. In budding yeast, dynein activity is restricted to anaphase when the nucleus enters the bud neck, yet the nature of the underlying regulatory mechanism is not known. Here, the microtubule-associated protein She1p is identified as a novel regulator of dynein activity. In she1Δ cells, dynein is activated throughout the cell cycle, resulting in aberrant spindle movements that misposition the spindle. We also found that dynactin, a cofactor essential for dynein motor function, is a dynamic complex whose recruitment to astral microtubules (aMTs) increases dramatically during anaphase. Interestingly, loss of She1p eliminates the cell-cycle regulation of dynactin recruitment and permits enhanced dynactin accumulation on aMTs throughout the cell cycle. Furthermore, localization of the dynactin complex to aMTs requires dynein, suggesting that dynactin is recruited to aMTs via interaction with dynein and not the microtubule itself. Lastly, we present evidence supporting the existence of an incomplete dynactin subcomplex localized at the SPB, and a complete complex that is loaded onto aMTs from the cytoplasm. We propose that She1p restricts dynein-dependent spindle positioning to anaphase by inhibiting the association of dynein with the complete dynactin complex.     

Kinesin-related KIP3 of Saccharomyces cerevisiae Is Required for a Distinct Step in Nuclear Migration

Spindle orientation and nuclear migration are crucial events in cell growth and differentiation of many eukaryotes. Here we show that KIP3, the sixth and final kinesin-related gene in Saccharomyces cerevisiae, is required for migration of the nucleus to the bud site in preparation for mitosis. The position of the nucleus in the cell and the orientation of the mitotic spindle was examined by microscopy of fixed cells and by time-lapse microscopy of individual live cells. Mutations in KIP3 and in the dynein heavy chain gene defined two distinct phases of nuclear migration: a KIP3-dependent movement of the nucleus toward the incipient bud site and a dynein-dependent translocation of the nucleus through the bud neck during anaphase. Loss of KIP3 function disrupts the unidirectional movement of the nucleus toward the bud and mitotic spindle orientation, causing large oscillations in nuclear position. The oscillatory motions sometimes brought the nucleus in close proximity to the bud neck, possibly accounting for the viability of a kip3 null mutant. The kip3 null mutant exhibits normal translocation of the nucleus through the neck and normal spindle pole separation kinetics during anaphase. Simultaneous loss of KIP3 and kinesin-related KAR3 function, or of KIP3 and dynein function, is lethal but does not block any additional detectable movement. This suggests that the lethality is due to the combination of sequential and possibly overlapping defects. Epitope-tagged Kip3p localizes to astral and central spindle microtubules and is also present throughout the cytoplasm and nucleus.     

Reorientation of mispositioned spindles in short astral microtubule mutant spc72Delta is dependent on spindle pole body outer plaque and Kar3 motor protein

Nuclear migration and positioning in Saccharomyces cerevisiae depend on long astral microtubules emanating from the spindle pole bodies (SPBs). Herein, we show by in vivo fluorescence microscopy that cells lacking Spc72, the SPB receptor of the cytoplasmic gamma-tubulin complex, can only generate very short (<1 microm) and unstable astral microtubules. Consequently, nuclear migration to the bud neck and orientation of the anaphase spindle along the mother-bud axis are absent in these cells. However, SPC72 deletion is not lethal because elongated but misaligned spindles can frequently reorient in mother cells, permitting delayed but otherwise correct nuclear segregation. High-resolution time-lapse sequences revealed that this spindle reorientation was most likely accomplished by cortex interactions of the very short astral microtubules. In addition, a set of double mutants suggested that reorientation was dependent on the SPB outer plaque and the astral microtubule motor function of Kar3 but not Kip2/Kip3/Dhc1, or the cortex components Kar9/Num1. Our observations suggest that Spc72 is required for astral microtubule formation at the SPB half-bridge and for stabilization of astral microtubules at the SPB outer plaque. In addition, our data exclude involvement of Spc72 in spindle formation and elongation functions.     

The role of the lissencephaly protein Pac1 during nuclear migration in budding yeast

During mitosis in Saccharomyces cerevisiae, the mitotic spindle moves into the mother-bud neck via dynein-dependent sliding of cytoplasmic microtubules along the cortex of the bud. Here we show that Pac1, the yeast homologue of the human lissencephaly protein LIS1, plays a key role in this process. First, genetic interactions placed Pac1 in the dynein/dynactin pathway. Second, cells lacking Pac1 failed to display microtubule sliding in the bud, resulting in defective mitotic spindle movement and nuclear segregation. Third, Pac1 localized to the plus ends (distal tips) of cytoplasmic microtubules in the bud. This localization did not depend on the dynein heavy chain Dyn1. Moreover, the Pac1 fluorescence intensity at the microtubule end was enhanced in cells lacking dynactin or the cortical attachment molecule Num1. Fourth, dynein heavy chain Dyn1 also localized to the tips of cytoplasmic microtubules in wild-type cells. Dynein localization required Pac1 and, like Pac1, was enhanced in cells lacking the dynactin component Arp1 or the cortical attachment molecule Num1. Our results suggest that Pac1 targets dynein to microtubule tips, which is necessary for sliding of microtubules along the bud cortex. Dynein must remain inactive until microtubule ends interact with the bud cortex, at which time dynein and Pac1 appear to be offloaded from the microtubule to the cortex.     

Dynein-dependent movements of the mitotic spindle in Saccharomyces cerevisiae Do not require filamentous actin

In budding yeast, the mitotic spindle is positioned in the neck between the mother and the bud so that both cells inherit one nucleus. The movement of the mitotic spindle into the neck can be divided into two phases: (1) Kip3p-dependent movement of the nucleus to the neck and alignment of the short spindle, followed by (2) dynein-dependent movement of the spindle into the neck and oscillation of the elongating spindle within the neck. Actin has been hypothesized to be involved in all these movements. To test this hypothesis, we disrupted the actin cytoskeleton with the use of mutations and latrunculin A (latrunculin). We assayed nuclear segregation in synchronized cell populations and observed spindle movements in individual living cells. In synchronized cell populations, no actin cytoskeletal mutant segregated nuclei as poorly as cells lacking dynein function. Furthermore, nuclei segregated efficiently in latrunculin-treated cells. Individual living cell analysis revealed that the preanaphase spindle was mispositioned and misaligned in latrunculin-treated cells and that astral microtubules were misoriented, confirming a role for filamentous actin in the early, Kip3p-dependent phase of spindle positioning. Surprisingly, mispositioned and misaligned mitotic spindles moved into the neck in the absence of filamentous actin, albeit less efficiently. Finally, dynein-dependent sliding of astral microtubules along the cortex and oscillation of the elongating mitotic spindle in the neck occurred in the absence of filamentous actin.     

Time-lapse video microscopy analysis reveals astral microtubule detachment in the yeast spindle pole mutant cnm67

Saccharomyces cerevisiae cnm67Delta cells lack the spindle pole body (SPB) outer plaque, the main attachment site for astral (cytoplasmic) microtubules, leading to frequent nuclear segregation failure. We monitored dynamics of green fluorescent protein-labeled nuclei and microtubules over several cell cycles. Early nuclear migration steps such as nuclear positioning and spindle orientation were slightly affected, but late phases such as rapid oscillations and insertion of the anaphase nucleus into the bud neck were mostly absent. Analyzes of microtubule dynamics revealed normal behavior of the nuclear spindle but frequent detachment of astral microtubules after SPB separation. Concomitantly, Spc72 protein, the cytoplasmic anchor for the gamma-tubulin complex, was partially lost from the SPB region with dynamics similar to those observed for microtubules. We postulate that in cnm67Delta cells Spc72-gamma-tubulin complex-capped astral microtubules are released from the half-bridge upon SPB separation but fail to be anchored to the cytoplasmic side of the SPB because of the absence of an outer plaque. However, successful nuclear segregation in cnm67Delta cells can still be achieved by elongation forces of spindles that were correctly oriented before astral microtubule detachment by action of Kip3/Kar3 motors. Interestingly, the first nuclear segregation in newborn diploid cells never fails, even though astral microtubule detachment occurs.     

The role of the proteins Kar9 and Myo2 in orienting the mitotic spindle of budding yeast

BACKGROUND: Two genetic 'pathways' contribute to the fidelity of nuclear segregation during the process of budding in the yeast Saccharomyces cerevisiae. An early pathway, involving Kar9p and other proteins, orients the mitotic spindle along the mother-bud axis. Upon the onset of anaphase, cytoplasmic dynein provides the motive force for nuclear movement into the bud. Loss of either pathway results in nuclear-migration defects; loss of both is lethal. Here, to visualize the functional steps leading to correct spindle orientation along the mother-bud axis, we imaged live yeast cells expressing Kar9p and dynein as green fluorescent protein fusions. RESULTS: Transport of Kar9p into the bud was found to require the myosin Myo2p. Kar9p interacted with microtubules through the microtubule-binding protein Bim1p and facilitated microtubule penetration into the bud. Once microtubules entered the bud, Kar9p provided a platform for microtubule capture at the bud cortex. Kar9p was also observed at sites of microtubule shortening in the bud, suggesting that Kar9p couples microtubule shortening to nuclear migration. CONCLUSIONS: Thus, Kar9p provides a key link between the actin cytoskeleton and microtubules early in the cell cycle. A cooperative mechanism between Kar9p and Myo2p facilitates the pre-anaphase orientation of the spindle. Later, Kar9p couples microtubule disassembly with nuclear migration.     

Astral Microtubule Pivoting Promotes Their Search for Cortical Anchor Sites during Mitosis in Budding Yeast

Positioning of the mitotic spindle is crucial for proper cell division. In the budding yeast Saccharomyces cerevisiae, two mechanisms contribute to spindle positioning. In the Kar9 pathway, astral microtubules emanating from the daughter-bound spindle pole body interact via the linker protein Kar9 with the myosin Myo2, which moves the microtubule along the actin cables towards the neck. In the dynein pathway, astral microtubules off-load dynein onto the cortical anchor protein Num1, which is followed by dynein pulling on the spindle. Yet, the mechanism by which microtubules target cortical anchor sites is unknown. Here we quantify the pivoting motion of astral microtubules around the spindle pole bodies, which occurs during spindle translocation towards the neck and through the neck. We show that this pivoting is largely driven by the Kar9 pathway. The microtubules emanating from the daughter-bound spindle pole body pivot faster than those at the mother-bound spindle pole body. The Kar9 pathway reduces the time needed for an astral microtubule inside the daughter cell to start pulling on the spindle. Thus, we propose a new role for microtubule pivoting: By pivoting around the spindle pole body, microtubules explore the space laterally, which helps them search for cortical anchor sites in the context of spindle positioning in budding yeast.     

Astral microtubules are not required for anaphase B in Saccharomyces cerevisiae

tub2-401 is a cold-sensitive allele of TUB2, the sole gene encoding beta-tubulin in the yeast, Saccharomyces cerevisiae. At 18 degrees C, tub2-401 cells are able to assemble spindle microtubules but lack astral microtubules. Under these conditions, movement of the spindle to the bud neck is blocked. However, spindle elongation and chromosome separation are unimpeded and occur entirely within the mother cell. Subsequent cytokinesis produces one cell with two nuclei and one cell without a nucleus. The anucleate daughter can not bud. The binucleate daughter proceeds through another cell cycle to produce a cell with four nuclei and another anucleate cell. With additional time in the cold, the number of nuclei in the nucleated cells continues to increase and the percentage of anucleate cells in the population rises. The results indicate that astral microtubules are needed to position the spindle in the bud neck but are not required for spindle elongation at anaphase B. In addition, cell cycle progression does not depend on the location or orientation of the spindle.     

Microtubule interactions with the cell cortex causing nuclear movements in Saccharomyces cerevisiae

During mitosis in budding yeast the nucleus first moves to the mother-bud neck and then into the neck. Both movements depend on interactions of cytoplasmic microtubules with the cortex. We investigated the mechanism of these movements in living cells using video analysis of GFP-labeled microtubules in wild-type cells and in EB1 and Arp1 mutants, which are defective in the first and second steps, respectively. We found that nuclear movement to the neck is largely mediated by the capture of microtubule ends at one cortical region at the incipient bud site or bud tip, followed by microtubule depolymerization. Efficient microtubule interactions with the capture site require that microtubules be sufficiently long and dynamic to probe the cortex. In contrast, spindle movement into the neck is mediated by microtubule sliding along the bud cortex, which requires dynein and dynactin. Free microtubules can also slide along the cortex of both bud and mother. Capture/shrinkage of microtubule ends also contributes to nuclear movement into the neck and can serve as a backup mechanism to move the nucleus into the neck when microtubule sliding is impaired. Conversely, microtubule sliding can move the nucleus into the neck even when capture/shrinkage is impaired.     

Bud morphogenesis and the actin and microtubule cytoskeletons during budding in the corn smut fungus,Ustilago maydis

Ustilago maydis is a dimorphic Basidiomycete fungus with a yeast-like form and a hyphal form. Here we present a comprehensive analysis of bud formation and the actin and microtubule cytoskeletons of the yeast-like form during the cell cycle. We show that bud morphogenesis entails a series of shape changes, initially a tubular or conical structure, culminating in a cigar-shaped cell connected to the mother cell by a narrow neck. Labelling of cells with concanavalin A demonstrated that growth occurs at bud tip. Indirect immunofluorescence studies revealed that the actin cytoskeleton consists of patches and cables that polarize to the presumptive bud site and the bud tip and an actin ring that forms at the neck region. Because the bud tip corresponds to the site of active cell wall growth, we hypothesize that actin is involved in secretion of cell wall components. The microtubule cytoskeleton has recently been shown to consist of a cytoplasmic network during interphase that disassembles at mitosis when a spindle and astral microtubules are formed. We have carried out studies of U. maydis cells synchronized by the microtubule-depolymerizing drug thiabendazole which allow us to construct a temporal sequence of steps in spindle formation and spindle elongation during the cell cycle. These studies suggest that astral microtubules may be involved in early stages of spindle orientation and migration of the nucleus into the bud and that the spindle pole bodies may be involved in reestablishment of the cytoplasmic microtubule network.     

Dynein-mediated pulling forces drive rapid mitotic spindle elongation in Ustilago maydis

Spindle elongation segregates chromosomes and occurs in anaphase, an essential step in mitosis. Dynein-mediated pulling forces position the spindle, but their role in anaphase is a matter of debate. Here, we demonstrate that dynein is responsible for rapid spindle elongation in the model fungus Ustilago maydis. We show that initial slow elongation is supported by kinesin-5, which is located in the spindle mid-zone. When the spindle reaches approximately 2 microm in length, the elongation rate increases four-fold. This coincides with the appearance of long and less-dynamic microtubules (MTs) at each pole that accumulate dynein at their tips. Laser-mediated nanosurgery revealed that these MTs exert pulling forces in control cells, but not in dynein mutants. In addition, dynein mutants undergo initial slow anaphase, but fail to establish less-dynamic MTs and do not perform rapid spindle elongation, suggesting that dynein drives anaphase B. This is most likely mediated by cortical sliding of astral MTs along stationary dynein, which is off-loaded from the MT plus-end to the cortex.     

The coordination of spindle‐positioning forces during the asymmetric division of the Caenorhabditis elegans zygote

In Caenorhabditis elegans zygote, astral microtubules generate forces essential to position the mitotic spindle, by pushing against and pulling from the cortex. Measuring microtubule dynamics there, we revealed the presence of two populations, corresponding to pulling and pushing events. It offers a unique opportunity to study, under physiological conditions, the variations of both spindle‐positioning forces along space and time. We propose a threefold control of pulling force, by polarity, spindle position and mitotic progression. We showed that the sole anteroposterior asymmetry in dynein on‐rate, encoding pulling force imbalance, is sufficient to cause posterior spindle displacement. The positional regulation, reflecting the number of microtubule contacts in the posterior‐most region, reinforces this imbalance only in late anaphase. Furthermore, we exhibited the first direct proof that dynein processivity increases along mitosis. It reflects the temporal control of pulling forces, which strengthens at anaphase onset following mitotic progression and independently from chromatid separation. In contrast, the pushing force remains constant and symmetric and contributes to maintaining the spindle at the cell centre during metaphase.     

Determinants of S. cerevisiae dynein localization and activation: implications for the mechanism of spindle positioning

BACKGROUND: During anaphase in budding yeast, dynein inserts the mitotic spindle across the neck between mother and daughter cells. The mechanism of dynein-dependent spindle positioning is thought to involve recruitment of dynein to the cell cortex followed by capture of astral microtubules (aMTs). RESULTS: We report the native-level localization of the dynein heavy chain and characterize the effects of mutations in dynein regulators on its intracellular distribution. Budding yeast dynein displays discontinuous localization along aMTs, with enrichment at the spindle pole body and aMT plus ends. Loss of Bik1p (CLIP-170), the cargo binding domain of Bik1p, or Pac1p (LIS1) resulted in diminished targeting of dynein to aMTs. By contrast, loss of dynactin or a mutation in the second P loop domain of dynein resulted in an accumulation of dynein on the plus ends of aMTs. Unexpectedly, loss of Num1p, a proposed dynein cortical anchor, also resulted in selective accumulation of dynein on the plus ends of anaphase aMTs. CONCLUSIONS: We propose that, rather than first being recruited to the cell cortex, dynein is delivered to the cortex on the plus ends of polymerizing aMTs. Dynein may then undergo Num1p-dependent activation and transfer to the region of cortical contact. Based on the similar effects of loss of Num1p and loss of dynactin on dynein localization, we suggest that Num1p might also enhance dynein motor activity or processivity, perhaps by clustering dynein motors.     

Spindle dynamics and cell cycle regulation of dynein in the budding yeast, Saccharomyces cerevisiae

We have used time-lapse digital- and video-enhanced differential interference contrast (DE-DIC, VE-DIC) microscopy to study the role of dynein in spindle and nuclear dynamics in the yeast Saccharomyces cerevisiae. The real-time analysis reveals six stages in the spindle cycle. Anaphase B onset appears marked by a rapid phase of spindle elongation, simultaneous with nuclear migration into the daughter cell. The onset and kinetics of rapid spindle elongation are identical in wild type and dynein mutants. In the absence of dynein the nucleus does not migrate as close to the neck as in wild-type cells and initial spindle elongation is confined primarily to the mother cell. Rapid oscillations of the elongating spindle between the mother and bud are observed in wild-type cells, followed by a slower growth phase until the spindle reaches its maximal length. This stage is protracted in the dynein mutants and devoid of oscillatory motion. Thus dynein is required for rapid penetration of the nucleus into the bud and anaphase B spindle dynamics. Genetic analysis reveals that in the absence of a functional central spindle (ndcl), dynein is essential for chromosome movement into the bud. Immunofluorescent localization of dynein-beta- galactosidase fusion proteins reveals that dynein is associated with spindle pole bodies and the cell cortex: with spindle pole body localization dependent on intact microtubules. A kinetic analysis of nuclear movement also revealed that cytokinesis is delayed until nuclear translocation is completed, indicative of a surveillance pathway monitoring nuclear transit into the bud.     

Dynamic reorganization of Eg5 in the mammalian spindle throughout mitosis requires dynein and TPX2

Kinesin-5 is an essential mitotic motor. However, how its spatial-temporal distribution is regulated in mitosis remains poorly understood. We expressed localization and affinity purification-tagged Eg5 from a mouse bacterial artificial chromosome (this construct was called mEg5) and found its distribution to be tightly regulated throughout mitosis. Fluorescence recovery after photobleaching analysis showed rapid Eg5 turnover throughout mitosis, which cannot be accounted for by microtubule turnover. Total internal reflection fluorescence microscopy and high-resolution, single-particle tracking revealed that mEg5 punctae on both astral and midzone microtubules rapidly bind and unbind. mEg5 punctae on midzone microtubules moved transiently both toward and away from spindle poles. In contrast, mEg5 punctae on astral microtubules moved transiently toward microtubule minus ends during early mitosis but switched to plus end-directed motion during anaphase. These observations explain the poleward accumulation of Eg5 in early mitosis and its redistribution in anaphase. Inhibition of dynein blocked mEg5 movement on astral microtubules, whereas depletion of the Eg5-binding protein TPX2 resulted in plus end-directed mEg5 movement. However, motion of Eg5 on midzone microtubules was not altered. Our results reveal differential and precise spatial and temporal regulation of Eg5 in the spindle mediated by dynein and TPX2.     

Mechanisms of spindle positioning

Accurate positioning of spindles is essential for asymmetric mitotic and meiotic cell divisions that are crucial for animal development and oocyte maturation, respectively. The predominant model for spindle positioning, termed “cortical pulling,” involves attachment of the microtubule-based motor cytoplasmic dynein to the cortex, where it exerts a pulling force on microtubules that extend from the spindle poles to the cell cortex, thereby displacing the spindle. Recent studies have addressed important details of the cortical pulling mechanism and have revealed alternative mechanisms that may be used when microtubules do not extend from the spindle to the cortex.Mitotic and meiotic spindles are precisely positioned within eukaryotic cells for several reasons. In animal cells, spindle position determines the location of contractile ring assembly (Green et al., 2012). Thus, placing a spindle in the center of the cell will result in daughter cells of equal size, whereas positioning the spindle asymmetrically results in daughter cells of different sizes. In oocytes, the extreme asymmetrical positioning of the meiotic spindle allows expulsion of three fourths of the chromosomes into two tiny polar bodies while preserving most of the cytoplasm in the egg for the developing zygote. In polarized cells, where proteins and RNAs are asymmetrically distributed before division, the orientation of the spindle relative to the polarity axis determines whether the daughter cells will have the same or different developmental fates. An excellent review of the developmental context of spindle positioning is provided by Morin and Bellaïche (2011). In budding yeast (Slaughter et al., 2009) and plants (Rasmussen et al., 2011), the site of cytokinesis is determined before the spindle forms. In these organisms, the spindle must be oriented relative to the predetermined division plane to ensure that both daughter cells receive a complete chromosome complement.The majority of research on the mechanisms of spindle positioning has focused on cell types that have “astral” microtubules. Astral microtubules have minus ends embedded in the spindle poles and plus ends extending outward, away from the spindle toward the cell cortex (Fig. 1, A and B). Astral microtubules have been proposed to mediate spindle positioning by generating pulling forces at the cortex or pulling forces against the cytoplasm. The minus ends of astral microtubules are embedded in the pericentriolar material that surrounds the centrioles of animal cells or in the spindle pole bodies of fungi. This attachment is essential for pulling forces on the astral microtubules to move the spindle. However, late stage oocytes of several animal phyla and all cells of flowering plants lack centrioles and lack obvious astral microtubules. Thus, these cell types have evolved alternative spindle positioning mechanisms. Here we review recent advances in both astral and nonastral spindle positioning mechanisms.Open in a separate windowFigure 1.Mitotic spindle movements in the C. elegans zygote. (A) Schematic diagram of a single-celled C. elegans embryo showing cortical pulling by cytoplasmic dynein during pronuclear centration and rotation. The nuclei move toward the anterior (left) so that the spindle assembles in the center of the embryo. (B) Schematic diagram of a single-celled C. elegans embryo showing cortical pulling by dynein during anaphase. The spindle moves to the posterior (right) so that cytokinesis generates two cells of different sizes. The squares highlight a dynein molecule that pulls toward the posterior before spindle displacement, then pulls toward the anterior after spindle displacement. (C) Schematic drawing of cytoplasmic pulling that contributes to centering the pronuclei. (D) Illustration of a spindle that was centered at metaphase but in which both poles moved all the way to the posterior end of the embryo. This occurs in zyg-8 mutants (Gönczy et al., 2001), cls-1,2 (RNAi) embryos (Espiritu et al., 2012), and zyg-9(ts) mutants shifted to a nonpermissive temperature at metaphase (Bellanger et al., 2007), possibly because astral microtubules are too short to reach force generators that would pull toward the anterior.

Cortical versus cytoplasmic pulling

The most prominent model of spindle positioning involves a cortical pulling mechanism. In this model, the minus end–directed microtubule motor protein, cytoplasmic dynein, is attached to the cell cortex and exerts pulling forces on the plus ends of astral microtubules that reach the cortex. In the single-celled Caenorhabditis elegans embryo at early prophase, complexes of GPR-1,2 (G protein regulator) and LIN-5 (abnormal cell lineage), positive regulators of cytoplasmic dynein, are more concentrated at the anterior cortex of the embryo (Fig. 1 A; Park and Rose, 2008), resulting in greater net pulling force toward the anterior. This results in net movement of the pronuclei to the center of the embryo and rotation of the centrosome–pronuclear complex so that the metaphase spindle forms in the center of the embryo with its poles oriented along the anterior-posterior axis of the embryo (Fig. 1 B). During metaphase and early anaphase, GPR-1,2 and LIN-5 become more concentrated at the posterior end of the embryo, resulting in movement of the spindle toward the posterior so that cytokinesis generates daughter cells of different sizes (Fig. 1 B; Grill et al., 2001, 2003). Depending on the relative distribution of active force generators at the cortex, this mechanism can also lead to centering the spindle within the cell to allow symmetric cytokinesis as occurs in HeLa cells (Kiyomitsu and Cheeseman, 2012) and LLC-Pk1 cells (Collins et al., 2012). Cortical pulling might involve pulling on the sides of microtubules that bend as they approach the cortex (Fig. 1 A, 1) or end-on interactions that require coupling of microtubule depolymerization with pulling (Fig. 1 A, 2), as occurs at kinetochores during anaphase A (McIntosh et al., 2010).Cortical pulling differs from a cytoplasmic pulling mechanism most clearly proposed by Kimura and Kimura (2011) and diagrammed in Fig. 1 C. In this cytoplasmic pulling mechanism, the viscous drag on membranous organelles transported toward the minus ends of astral microtubules by cytoplasmic dynein generates a force in the opposite direction, toward the cortex. Depletion of RAB-5, RAB-7, or RILP-1 (RAB-7 interacting lysosomal protein homologue) blocks dynein-dependent organelle transport and slows the velocity of pronuclear centration in C. elegans without affecting other dynein-dependent movements (Kimura and Kimura, 2011). Elimination of cortical pulling by depleting GPR-1,2 also slows pronuclear centration (Park and Rose, 2008), which suggests that cortical pulling and cytoplasmic pulling each contribute 50% of the velocity of pronuclear centration. Unlike end-on cortical pulling, cytoplasmic pulling force is proportional to the length of the astral microtubules because more organelles will be transported on a long microtubule than a short microtubule (Fig. 1 C). This generates a self-centering mechanism as the length of the astral microtubules equalize when the pronuclei reach the center of the zygote (Fig. 1 A; Hamaguchi and Hiramoto, 1986). Cytoplasmic pulling may predominate in very large zygotes where astral microtubules clearly do not reach the cortex but where both pronuclei and the mitotic spindle are centered (Mitchison et al., 2012).

Evidence for cortical pulling.

The cortical pulling mechanism requires that microtubules extend from the spindle to the cortex and form a contiguous structure that is mechanically robust enough that the force generators do not pull the minus ends of the microtubules out of the spindle pole or cause the plasma membrane to buckle inward. Experimental evidence for this contiguous mechanical linkage comes from experiments in oocytes of the marine annelid, Chaetopterus. Insertion of a glass needle into the meiotic spindle allowed pulling the spindle away from the cortex, which caused inward buckling of the cortex. Further pulling resulted in sudden release of the spindle from the cortex, restoration of cortical shape, and concomitant disappearance of a birefringent aster extending between the spindle and cortex (Lutz et al., 1988). The second requirement for a cortical pulling mechanism is that force is generated at the cortex. Using a laser to cut the central spindle of an early anaphase C. elegans embryo, Grill et al. (2001) showed that spindle poles are pulled from outside the spindle rather than pushed from inside the spindle during posterior spindle displacement. Fragmentation of centrosomes with a laser (Grill et al., 2003) revealed that astral microtubules freed from the spindle move outward toward the cortex. Either cortical pulling or cytoplasmic pulling could explain this result; however, the asymmetric distribution of fragment velocities is controlled by proteins that are localized at the cortex, GPR-1,2 (Grill et al., 2003) and LET-99 (lethal-99; Krueger et al., 2010). In a key experiment, Redemann et al. (2010) showed that microtubule plus ends pull tubular invaginations of the plasma membrane inward when cortical stiffness is partially reduced. This experiment showed that astral microtubules are pulling on the cortex during spindle displacement, which would not occur if forces were generated by movement of cytoplasmic organelles along the sides of microtubules.

End-on versus side-on microtubule–cortex interactions.

How do microtubules interact with force generators at the cortex? Astral microtubules might polymerize to the cortex then bend along it so that cortical motors interact with the side of the microtubule to generate force (Fig. 1 A, 1). This type of interaction would be consistent with the in vitro gliding motility of microtubules generated by cytoplasmic dynein immobilized on glass coverslips (Paschal et al., 1987; Vallee et al., 1988) and is the only type of dynein-dependent cortical microtubule interaction observed in budding yeast (Adames and Cooper, 2000). Studies of C. elegans embryos, however, support end-on microtubule contacts (Fig. 1 A, 2) as the functional contact with cortical force generators for posterior displacement of the early anaphase spindle. Live imaging of YFP-tubulin in optical sections at the surface of the embryo during anaphase revealed dots rather than lines, indicating that the microtubules contacting the cortex are <200 nm in length (Labbé et al., 2003; Kozlowski et al., 2007). When short microtubule fragments are generated by ectopic katanin activity, dynein-dependent gliding of microtubule “lines” on the cortex is frequently observed (Gusnowski and Srayko, 2011), indicating that cortical dynein is capable of moving microtubules along the cortex via side-on interactions in wild-type embryos but that it does not during posterior displacement of the anaphase spindle. A likely explanation comes from the finding that astral microtubule plus ends undergo catastrophe (switch to depolymerization) on average 1.4 s after polymerizing to the cortex (Kozlowski et al., 2007). Thus astral microtubule plus ends do not have time to polymerize along the cortex to establish extensive side-on contacts. Support for this idea comes from depletion of the conserved plasma membrane protein EFA-6 (exchange factor for Arf) from the C. elegans embryo. In the absence of EFA-6, the residence time of microtubule plus ends at the cortex increases fivefold, astral microtubules form extensive lateral contacts with the cortex, and centrosomes exhibit movements consistent with excessive dynein-dependent cortical pulling (O’Rourke et al., 2010). Side-on contacts of astral microtubules with the cortex occur later during telophase in the wild-type C. elegans embryo (Kozlowski et al., 2007), but the nature of this switch has not been addressed.The two distinct activities of cytoplasmic dynein, end-on pulling and walking along the side of a microtubule, have been genetically separated in C. elegans. Cortical pulling forces during early anaphase require the redundant cortical dynein activators GPR-1 and -2 (Grill et al., 2003), which bind to LIN-5 (Gotta et al., 2003). GPR-1,2/LIN-5 is anchored in the plasma membrane via the myristoyl and palmitoyl lipid modifications of the redundant Gα proteins GOA-1 and GPA-16 (Gotta et al., 2003; Park and Rose, 2008; Kotak et al., 2012). The complex of GPR-1 and LIN-5 interacts with the dynein light chain DYRB-1 (Couwenbergs et al., 2007) and the dynein regulator LIS-1 (human lissencephaly gene related; Nguyen-Ngoc et al., 2007). GPR-1 and -2, however, are not required for dynein-dependent gliding of severed microtubule fragments along the cortex (Gusnowski and Srayko, 2011), dynein-dependent transport of membranous organelles along the sides of microtubules (Kimura and Kimura, 2011), dynein-dependent positioning of the acentriolar C. elegans meiotic spindle (van der Voet et al., 2009), or dynein-dependent centration of the male pronucleus (Kimura and Kimura, 2011). These GPR-independent activities of cytoplasmic dynein likely do not require end-on pulling.Recently, end-on pulling by cytoplasmic dynein has been reconstituted in vitro with a purified preparation of artificially dimerized budding yeast cytoplasmic dynein. Laan et al. (2012) immobilized purified cytoplasmic dynein on microfabricated barriers and observed the interaction of centrosome-nucleated microtubules as they approached these dynein-coated barriers. Microtubule plus ends hitting a dynein-coated barrier switched to catastrophe with high frequency but the microtubule depolymerization rate after the catastrophe was reduced. The result was an extended period of interaction between the depolymerizing plus end and the dynein-coated barrier. Plus-end depolymerization pulled the centrosome toward the barrier, and in similar reactions the pulling force was measured as high as 5 pN. Side-on interactions with the barrier were not observed. Whereas ATP was required for this end-on pulling, it is not clear if the energy source is ATP hydrolysis–driven stepping by dynein or if the energy source is GTP hydrolysis–driven depolymerization of the microtubule. In the latter case, ATP might only be required to prevent rigor binding of dynein to the microtubule. Indeed, an artificial rigor binding of a streptavidin-coated bead to a depolymerizing biotinylated microtubule plus end resulted in a pulling force that is restricted to an extremely short distance (Grishchuk et al., 2005). In vitro reconstitution of pulling force coupled to depolymerizing microtubule plus ends was first demonstrated with beads coated with kinesin-1 or a nonmotile kinesin chimera (NK350; Lombillo et al., 1995). Like barrier-bound cytoplasmic dynein, kinesin-coated beads slowed the depolymerization rate of microtubule plus ends, whereas NK350-coated beads enhanced the depolymerization rate of bound plus ends. ATP enhanced depolymerization-coupled pulling for both kinesin-1 and NK350, just as it did for barrier-bound cytoplasmic dynein. The in vitro pulling reaction reconstituted by Laan et al. (2012) seems unlikely to be the same reaction that pulls on plus ends in the anaphase budding yeast cell, as these are through side-on interactions (Adames and Cooper, 2000). In vitro reconstitution of a GPR/LIN-5–dependent, end-on pulling reaction with purified metazoan cytoplasmic dynein may reveal mechanisms acting on spindles in vivo.Another interesting contrast between end-on versus side-on cortical pulling reactions is suggested by differences in the dependence on cortical F-actin. F-actin is required for cortical rigidity to prevent end-on microtubule contacts from pulling membrane tubules inward instead of moving the spindle pole outward (Redemann et al., 2010). Side-on pulling by cortical dynein in budding yeast, however, does not require F-actin (Theesfeld et al., 1999; Heil-Chapdelaine et al., 2000a). The curvature of microtubules gliding on the bud cortex indicates that the microtubule is engaged with multiple dynein molecules distributed over several microns of cortex, and this distribution of force might allow effective pulling against a less rigid cortex. Alternatively, rigidity of the yeast plasma membrane might be mediated by oligomers of BAR domain proteins like Num1 (nuclear migration; Tang et al., 2012) or eisosomes (Walther et al., 2006; Olivera-Couto et al., 2011), by osmotic pressure, or by attachment of the plasma membrane to the cell wall.

Why the spindle is not pulled all the way to the cortex with more active force generators.

What prevents the C. elegans centrosome–pronuclear complex from moving all the way to the anterior cortex where the concentration of cortical force generators is highest during prophase (Fig. 1 A), and what prevents the spindle from moving all the way to the posterior cortex, which has the highest concentration of active force generators during anaphase (Fig. 1, B and D)? Increasing the concentration of GPR-1,2/LIN-5 at the anterior cortex causes pronuclei to move further toward the anterior, but they still do not crash into the anterior cortex (Panbianco et al., 2008). During metaphase/anaphase (Fig. 1 B), weak cortical pulling on the anterior aster might oppose strong pulling on the posterior aster. Supporting this idea, spindle severing results in the posterior aster moving further posterior than when the spindle is intact (Grill et al., 2001), but the posterior pole still does not move all the way to the posterior cortex. Monopolar spindles move toward the posterior in a GPR1,2-dependent manner but then reverse direction and oscillate along the anterior-posterior axis (Krueger et al., 2010). Laan et al. (2012) found that centrosome-nucleated microtubule asters could accurately self-center within microchambers whose walls are coated with dynein. Their mathematical modeling suggested that self-centering was achieved because of a balance between cortical pulling by dynein and pushing by polymerizing microtubules (Dogterom and Yurke, 1997) that have not yet engaged a dynein molecule. Thus, cortical pushing by astral microtubules might buffer cortical pulling in vivo. Grill and Hyman (2005) suggested another simple solution. When a spindle pole moves to the posterior, it passes a subset of cortical force generators that were initially pulling toward the posterior (Fig. 1 B, box). After passage of the spindle pole, these force generators pull toward the anterior. Failure in any of these buffering mechanisms might explain why both spindle poles move to the posterior cortex (Fig. 1 D) in mutants that have either short astral microtubules or fewer astral microtubules reaching the cortex (Gönczy et al., 2001; Bellanger et al., 2007; Espiritu et al., 2012).Recent experiments in HeLa and LLC-Pk1 cells have revealed a more sophisticated feedback mechanism that effectively centers the mitotic spindle. Kiyomitsu and Cheeseman (2012) found that HeLa cell mitotic spindles oscillate back and forth along their pole-to-pole axis. They found that dynein/dynactin formed a lateral crescent on the cortex when the spindle was far from that lateral cortex (Fig. 2 A). As the spindle moved toward the dynein crescent, the dynein crescent disappeared as the spindle approached and a new crescent appeared on the opposite lateral cortex (Fig. 2 C). They found that polo kinase 1 (Plk1), which is concentrated on spindle poles, causes dynein/dynactin to dissociate from the LGN–NuMA–Gαi complex (Leu-Gly-Asn repeat enriched protein–nuclear mitotic apparatus protein–Gα; homologues of GPR-1,2–LIN-5–Gα), which explains why the dynein crescent disappears once the spindle pole gets close to the cortex. They also found that the GTP-Ran gradient produced by chromosome-bound RCC1 (regulator of chromosome condensation) was responsible for inhibiting LGN–NuMA association with the cortex, and hence dynein, above the central spindle (Fig. 2). The RCC1 pathway explains why the spindles move only along their pole–pole axis. The two pathways combine to center the spindle in two different axes. Similar spindle oscillations with dynein/dynactin crescents forming only when the spindle pole is far from the cortex were reported in LLC-Pk1 epithelial cells (Collins et al., 2012). Because spindles are small relative to the two-dimensional flattened area of these cells, the role of this pathway in centering spindles to allow symmetric cytokinesis is much more obvious than in HeLa cells.Open in a separate windowFigure 2.How to center a spindle. Schematic diagram of a metaphase HeLa cell where the spindle oscillates along its pole–pole axis to maintain a centered position to allow symmetric cytokinesis. (A) When the left spindle pole is close to the cortex, Plk1 on the pole (red) causes dynein (green) to dissociate from LGN–NuMA complexes (purple; human homologues of GPR-1,2/LIN-5). (B and C) The spindle moves to the right because of the higher concentration of LGN–NuMA–dynein complexes on the right cortex. When chromosomes are close to the cortex as in A, the GTP-Ran gradient from the chromosomes causes dissociation of LGN/NuMA from the cortex. This second system centers the spindle in the axis perpendicular to the pole–pole axis.There are normal situations where movement of one spindle pole all the way to the cortex occurs. This exaggerated movement that results in one set of astral microtubules being splayed onto the cortex is observed in fourth cleavage sea urchin embryos (Holy and Schatten, 1991) and for the female meiotic spindles of Chaetopterus (Lutz et al., 1988) and Spisula solidissima (Pielak et al., 2004).

The budding yeast model.

The S. cerevisiae mitotic spindle is positioned relative to a preformed bud neck by two sequential and partially redundant cortical pulling pathways to ensure that chromosomes are deposited into both mother and daughter cells. Spindle positioning is also monitored by a budding yeast–specific checkpoint (Caydasi et al., 2010). Deletion of genes in any one positioning pathway results in a viable yeast strain, whereas double mutants bearing deletions of genes in both positioning pathways or of genes in one positioning pathway plus the checkpoint results in lethality (Miller et al., 1998). In the early pathway, the microtubule plus-end tip tracking protein, Bim1 (binding to microtubules; EB1 homologue), binds to the yeast-specific adaptor protein Kar9 (karyogamy; Korinek et al., 2000; Lee et al., 2000; Miller et al., 2000), which binds to the yeast myosin V (Myo2; Yin et al., 2000). Myosin V transports the growing microtubule plus end toward the bud tip on polarized actin cables (Hwang et al., 2003). This results in a unique “sweeping” or “pivoting” of the growing astral microtubule toward the bud neck (Fig. 3 A; Adames and Cooper, 2000). Because Bim1 only binds to growing microtubule plus ends (Zimniak et al., 2009), this mechanism alone cannot bring the spindle to the bud neck because the polymerizing astral microtubule would push the spindle back into the mother cell. To prevent the astral microtubules from becoming too long, the kinesin-8 family member, Kip3, passively tracks the plus end until the plus end reaches the bud cortex (Fig. 3 A). Kip3 then switches to a plus-end depolymerase and shortens the astral microtubule to pull the spindle to the bud neck (Fig. 3, B and C; Gupta et al., 2006). Purified Kip3 has unique biochemical properties that contribute to its in vivo function. In vitro, Kip3 accumulates at plus-end tips via its plus end–directed motor activity. Longer microtubules accumulated more Kip3 at their plus ends than short microtubules simply because there are more lateral binding sites on a long microtubule and Kip3 switches to a plus-end depolymerase only when the microtubule has reached a threshold length (Varga et al., 2009). Plus-end pulling by the fission yeast homologue of Kip3 has also been reconstituted in vitro (Grissom et al., 2009). More recent work suggests that Kip3-dependent cortical pulling requires the concerted action of the cortical protein, Bud6 (BUD site selection), and the plus end tracker, Bim1, as well as cytoplasmic dynein (ten Hoopen et al., 2012).Open in a separate windowFigure 3.Spindle positioning in budding yeast. Schematic diagram of the two sequential spindle positioning pathways of budding yeast. In the early pathway (A–C), myosin V transports the plus end of an astral microtubule toward the bud tip on a polarized actin cable. Once the plus end has reached the bud cortex, the plus-end depolymerase, KIP3, is activated to allow pulling of the spindle pole toward the bud neck. In the late pathway (D–F), the plus end–directed microtubule motor Kip2 transports dynein to the plus ends of microtubules via the adaptor protein Bik1 (D). Dynein can be targeted to plus ends by two additional Bik1-dependent mechanisms (see text). When dynein reaches the bud cortex on a polymerizing microtubule plus end (E), contact with the cortical protein Num1 allows dynein to pull the spindle toward the bud (F). During early anaphase (D and E), dynein is not loaded onto microtubules in the mother cell. During late anaphase (F), dynein is loaded on microtubules in the mother cell to prevent movement of the spindle all the way into the bud.The late pathway is initiated when the yeast-specific dynein inhibitor She1 (sensitivity to high expression) is removed from astral microtubules at the metaphase–anaphase transition (Woodruff et al., 2009). She1 appears to act specifically by preventing recruitment of dynactin to microtubules (Bergman et al., 2012) and by inhibiting dynein motility (Markus et al., 2012). In the late pathway, cytoplasmic dynein is targeted to growing microtubule plus ends by the plus-end tracking protein Bik1 (bilateral karyogamy defect; Sheeman et al., 2003), which itself is targeted to plus ends by three partially redundant mechanisms. In the first mechanism, Bik1 is transported as cargo by the plus end–directed kinesin Kip2 (Carvalho et al., 2004). Cytoplasmic dynein is thus transported as cargo to the bud cortex by Bik1–Kip2 complexes (Fig. 3 D). In the absence of Kip2, Bik1 (and therefore dynein) can still track growing microtubule plus ends either through a second mechanism that requires the C-terminal tyrosine residue of α-tubulin or a third mechanism that requires the plus end–tracking protein Bim1 (Caudron et al., 2008). This is partially consistent with results of reconstitution experiments with purified proteins showing that the Bik1 homologue, CLIP170, tracks growing plus ends through a mechanism that involves binding to both the Bim1 homologue, EB1, and the C-terminal tyrosine-containing motif of α-tubulin (Bieling et al., 2008). When a microtubule plus end carrying dynein contacts the yeast-specific cortical protein Num1, Num1 apparently stimulates off-loading of the dynein tail onto the cortex so that the dynein motor domains engage the microtubule in a cortical pulling reaction (Fig. 3, E and F). Deletion of Num1 results in accumulation of inactive dynein at plus ends (Lee et al., 2003; Sheeman et al., 2003; Markus and Lee, 2011). In contrast with the GPR-dependent end-on cortical interactions observed in C. elegans, dynein-dependent cortical pulling is mediated by lateral sliding of astral microtubules along the yeast bud cortex (Fig. 3, E and F; Adames and Cooper, 2000). Also, unlike cortical GPR/LIN-5 in animal cells, cortical Num1 is distributed in patches throughout both mother and daughter cells (Heil-Chapdelaine et al., 2000b) and even participates in mitochondrial positioning and fission throughout the cell (Cerveny et al., 2007; Hammermeister et al., 2010). If there is no asymmetrically distributed cortical activator of dynein, how is dynein-mediated pulling directed specifically toward the bud cortex?

Why both spindle poles are not pulled to the same cortex in budding yeast.

The budding yeast spindle, rather than the cortex, is asymmetrical. At metaphase, Kar9 is asymmetrically localized on the spindle pole oriented toward the bud and on the plus ends of astral microtubules emanating from that bud-proximal spindle pole (Liakopoulos et al., 2003). This alone would explain why only one spindle pole moves toward the bud but then leaves the question of how Kar9 asymmetry is established. Cepeda-García et al. (2010) found that Kar9 at spindle poles became symmetrical and reduced in concentration after depolymerization of actin cables, depolymerization of microtubules, or disruption of the myosin V–Kar9 interaction. When microtubules were repolymerized, Kar9 was initially symmetrical on both spindle poles and quickly repolarized onto the first microtubule to make a functional cortical contact. These results suggested a positive feedback loop in which functional cortical pulling by Bim1–Kar9–Myo2 complexes causes loading of additional Kar9. Cytoplasmic dynein also accumulates preferentially on the plus-end tips that reach the bud neck first (Fig. 3, D and E) and on the bud-proximal spindle pole during metaphase. The asymmetrical accumulation of dynein on the bud-proximal microtubules and bud-proximal spindle pole requires kinases that are found at the bud neck. Thus, the asymmetry of dynein localization may be generated by a positive feedback loop, as suggested for Kar9 asymmetry. After the spindle is pulled into the bud neck, during anaphase, dynein becomes symmetrical on the plus ends emanating from both poles (Fig. 3 F; Grava et al., 2006). This regulation prevents both poles from moving toward the bud during metaphase and then prevents the spindle from being pulled all the way into the bud during anaphase. Asymmetric localization of dynein on spindle poles or microtubule plus ends has not yet been reported in animal cells.

Other spindle positioning mechanisms

In the examples of the C. elegans and budding yeast mitotic spindles, long astral microtubules are in contact with the cell cortex. In cells where spindles have no astral microtubules, other mechanisms must be at work. Female meiotic spindles are universally positioned with one spindle pole contacting the oocyte cortex so that one set of chromosomes can be eliminated in a polar body through an extremely asymmetrical division (Fabritius et al., 2011). Female meiotic spindles of at least three animal phyla (Chordata, Nematoda, and Arthropoda), however, have no centrioles in their spindle poles and no apparent astral microtubules. Work in C. elegans and mice suggests that different species have evolved different mechanisms for acentriolar meiotic spindle positioning.

Parallel metaphase meiotic spindles.

The metaphase I and metaphase II spindles of C. elegans (Fig. 4 A) and the metaphase II spindle of mouse (Fig. 4 B) are positioned at the cortex in a parallel orientation, with both poles equidistant from the cortex. In C. elegans, this parallel cortical positioning requires microtubules, kinesin-1, and a worm-specific kinesin-1–binding partner, KCA-1 (Yang et al., 2003, 2005), but is independent of F-actin (Yang et al., 2003). Kinesin-1 and microtubules are also required to move the nucleus to the cortex before germinal vesicle breakdown and to drive transport of yolk granules inward from the cortex, which results in a circular streaming pattern. It has been suggested that kinesin-1 may only move the germinal vesicle to the cortex and that an additional, unidentified pathway moves the spindle over the remaining distance to the cortex and establishes the parallel orientation (McNally et al., 2010). The mouse metaphase II spindle is maintained in a similar parallel orientation at the cortex, but this positioning requires the actin nucleator ARP2/3. ARP2/3 also drives streaming of actin filaments and cytoplasm in a pattern that has been proposed to push the spindle into the cortex to maintain parallel cortical position (Fig. 4 B; Yi et al., 2011).Open in a separate windowFigure 4.A plethora of nonastral spindle positioning mechanisms. (A) Metaphase C. elegans meiotic spindles are positioned in a parallel orientation at the cortex by microtubules and kinesin-1. (B) The mouse metaphase II spindle may be positioned by actin-dependent cytoplasmic streaming. Pole-first migration of the mouse meiosis I spindle to the cortex may be mediated by cargo transport on parallel actin filaments by spindle pole–bound myosin II (C), myosin II–based contraction of anti-parallel actin filaments (D), or pushing forces generated by polymerizing actin filaments nucleated by formin molecules on the spindle (E) or nucleated by formin molecules on the cortex (F). Red arrows indicate the pointed ends of actin filaments. (G) One spindle pole of the early anaphase C. elegans meiotic spindle may be transported to the cortex as cargo by dynein on polarized cytoplasmic microtubules.

Pole first migration of the mouse meiosis I spindle.

Unlike the C. elegans germinal vesicle, the mouse germinal vesicle is centered in the egg at germinal vesicle breakdown. Thus, the meiosis I spindle assembles near the center of the egg then migrates in a pole-first orientation to the nearest cortex so that it never adopts a parallel orientation (Fig. 4 C). This migration requires F-actin (Verlhac et al., 2000) and the actin nucleators Formin 2 (Dumont et al., 2007), Spire 1 and Spire 2 (Pfender et al., 2011), and ARP2/3 (Sun et al., 2011), but the mechanism of movement remains unclear. Schuh and Ellenberg (2008) demonstrated the existence of actin bundles extending between the spindle and an invagination of the cortex during spindle migration. The invagination indicated a pulling mechanism, and Schuh and Ellenberg (2008) suggested that myosin II on the spindle poles might walk on a discontinuous actin network with barbed ends oriented toward the cortex (Fig. 4 C). In support of this model, the Rho kinase inhibitor ML7 eliminated spindle pole staining by an antibody specific for phosphorylated myosin regulatory light chain and blocked spindle migration (Schuh and Ellenberg, 2008). However, Li et al. (2008) found that the myosin ATPase inhibitor, blebbistatin, had no effect on spindle migration even though it completely blocked polar body extrusion (cytokinesis). Because myosin II typically acts by forming bipolar thick filaments that exert contractile force on antiparallel actin filaments, a myosin II–based model would make more sense if myosin II was concentrated on antiparallel actin bundles extending between the spindle and cortex (Fig. 4 D). Myosin V is more typically associated with the transport of cargo on uniformly oriented actin filaments, and a recent study has shown that myosin V drives transport of secretory vesicles outward toward the cortex in germinal vesicle stage oocytes (Schuh, 2011). This study at least suggests that the cytoplasmic actin meshwork has a net polarity with barbed ends toward the cortex, a prerequisite for a myosin cargo transport model (Fig. 4 C).Li et al. (2008) proposed a completely different mechanism in which F-actin nucleated near the chromosomes generates a cloud of F-actin that pushes the spindle toward the cortex (Fig. 4 E) in a manner analogous to Listeria monocytogenes motility (Lambrechts et al., 2008). Support for this pushing model comes from imaging of an actin cloud behind the migrating spindle and localization of Formin 2 around the spindle (Li et al., 2008). In addition, Formin 2 overexpression causes invaginations in the nuclear envelope, which is consistent with inward pushing from the cortex, rather than protrusions that would be consistent with pulling forces from the cortex (Azoury et al., 2011). Formin 2 is symmetrical around the cortex during prophase but clears from the cortex in front of the migrating spindle, which is consistent with nucleation-based pushing from behind (Fig. 4 F; Azoury et al., 2011). As previously mentioned, cortical stiffness is a prerequisite for cortical pulling mechanisms because pulling on an unsupported plasma membrane should cause the membrane to invaginate inward instead of the spindle moving outward. Strikingly, cortical stiffness of the mouse oocyte decreases sixfold during spindle migration (Larson et al., 2010). Clearly, more work is required to resolve the mechanism of pole-first spindle migration in the mouse oocyte.

Rotation of the parallel metaphase spindle to a perpendicular anaphase spindle.

Activation of the anaphase-promoting complex results in rotation of the parallel metaphase meiotic spindle to a perpendicular orientation in both meiotic divisions of C. elegans, whereas fertilization induces rotation during meiosis II in mouse. In kinesin-1–depleted C. elegans embryos, the metaphase I spindle is far from the cortex and initiates pole-first migration to the cortex at the same time that wild-type rotation initiates (Yang et al., 2005). Both spindle rotation and late spindle migration in a kinesin mutant require cytoplasmic dynein (Ellefson and McNally, 2009, 2011). These results indicate that spindle rotation is simply migration of one spindle pole toward the cortex (analogous to spindle migration during mouse meiosis I). However, unlike dynein-dependent migration of one spindle pole toward the cortex during C. elegans mitosis or HeLa cell mitosis, C. elegans meiotic spindle rotation does not require GPR-1,2 (van der Voet et al., 2009). One possible model for C. elegans meiotic spindle rotation is that cytoplasmic dynein, which accumulates on both spindle poles just before and during rotation, transports one spindle pole as cargo on cytoplasmic microtubules with minus ends anchored at the cortex (Fig. 4 G). The orientation of these microtubules is inferred from the direction of kinesin-dependent yolk granule movement (McNally et al., 2010) and hook decoration in Xenopus laevis oocytes (Pfeiffer and Gard, 1999). This model is essentially the same as the myosin cargo transport model proposed for mouse meiosis I (Fig. 4 C). Rotation of the mouse meiosis II spindle is actin dependent (Maro et al., 1984) and myosin II dependent (Matson et al., 2006; Wang et al., 2011), but the mechanism is unknown.

Unifying themes and future directions

Work in HeLa cells and budding yeast suggests that negative feedback loops might generally lead to spindle centering and that positive feedback loops might generally lead to asymmetrical spindle positioning. Whereas the recent x-ray crystal structures of cytoplasmic dynein (Carter et al., 2011; Kon et al., 2012) have revealed great insights into how the motor walks, they have revealed little about how the motor is locally activated and anchored at the cortex or how GPR-1/LIN-5 switches the motor from a side-on motor to an end-on motor. More attention needs to be focused on the distinction between cortical stiffness and cortical anchoring required in any cortical pulling mechanism. The nonastral spindle positioning mechanisms acting in oocytes of mouse and C. elegans will require a much more detailed understanding of the polarity of cytoplasmic actin filaments and cytoplasmic microtubules.