InteractiveFly: GeneBrief

Klp10A: Biological Overview | References

During anaphase identical sister chromatids separate and move towards opposite poles of the mitotic spindle. In the spindle, kinetochore microtubules have their plus ends embedded in the kinetochore and their minus ends at the spindle pole. Two models have been proposed to account for the movement of chromatids during anaphase. In the 'Pac-Man' model, kinetochores induce the depolymerization of kinetochore microtubules at their plus ends, which allows chromatids to move towards the pole by 'chewing up' microtubule tracks. In the 'poleward flux' model, kinetochores anchor kinetochore microtubules and chromatids are pulled towards the poles through the depolymerization of kinetochore microtubules at the minus ends. This study shows that two functionally distinct microtubule-destabilizing KinI kinesin enzymes (so named because they possess a kinesin-like ATPase domain positioned internally within the polypeptide) are responsible for normal chromatid-to-pole motion in Drosophila. One of them, KLP59C, is required to depolymerize kinetochore microtubules at their kinetochore-associated plus ends, thereby contributing to chromatid motility through a Pac-Man-based mechanism. The other, KLP10A, is required to depolymerize microtubules at their pole-associated minus ends, thereby moving chromatids by means of poleward flux (Rogers, 2004).

Although molecules involved in either Pac-Man- or poleward-flux-based motility mechanisms have not been identified previously, members of the KinI subfamily of kinesins are logical candidates for both (Hunter, 2000). These proteins bind directly to the ends of microtubules and promote their disassembly in vitro (Wordeman, 1995; Walczak, 1996; Desai, 1999). Moreover, the inhibition of KinI proteins in mitotic cells from various systems has been shown to disrupt mitosis (Maney, 1998; Kline-Smith, 2002; Walczak, 2002). To identify novel KinI family members in Drosophila, the Drosophila genome databases were searched for genes that share significant identity with the known KinI catalytic domains from other species. This revealed three different D. melanogaster genes predicted to encode products containing internal kinesin domains highly similar to vertebrate KinI (65% amino acid identity on average). These are referred to as KLP10A, KLP59C and KLP59D (kinesin-like protein at cytological regions 10A, 59C and 59D). Notably, it was observed that purified recombinant polypeptides corresponding to the catalytic domains of KLP10A and KLP59C destabilize microtubules in an ATP-dependent fashion. (Similar analysis of KLP59D has not yet been performed but the high degree of amino acid identity among the catalytic domains of all three Drosophila KinI proteins (66% on average) suggests that it will also possess microtubule-destabilizing activity.) Thus, Drosophila probably possesses three distinct microtubule-destabilizing KinI kinesins (Rogers, 2004).

Analysis of Drosophila S2 tissue culture cells depleted of each of the three KinI proteins (individually and in various combinations) using double-stranded RNA interference (RNAi) suggests that both KLP10A and KLP59C perform important but distinct mitotic functions. KLP10A depletion by RNAi treatment causes a marked perturbation of mitotic spindle architecture. In contrast, KLP59C RNAi treatment has no noticeable impact on mitotic spindle structure but does significantly elevate the frequency with which chromosome segregation defects are observed. Immunolocalization of these proteins in mitotic S2 cells also reveals distinctions that may provide insights into their specific mitotic mechanisms of action. Specifically, KLP10A, similarly to vertebrate KinI proteins, localizes to mitotic centrosomes, spindle poles and centromeres through metaphase. However, the centromeric localization of KLP10A diminishes markedly at the onset of anaphase, leaving the majority of KLP10A immunostaining on the spindle poles. KLP59C, in contrast, appears to be primarily restricted to centromeric regions of chromosomes during both metaphase and anaphase, and no KLP59C immunostaining is detectable on spindle poles throughout mitosis. Thus, during chromatid-to-pole motion in anaphase, KLP10A and KLP59C are positioned appropriately to interact with the opposite ends of kinetochore microtubules: KLP10A concentrates around the minus ends of kinetochore microtubules focused at the poles, whereas KLP59C is positioned to act on the plus ends of kinetochore microtubules embedded in the kinetochore. Finally, analyses of KLP59D do not support a role for this protein in spindle assembly or chromosome segregation (Rogers, 2004).

The specific mechanisms of action of mitotic KLP10A and KLP59C are impossible to discern from fixed samples. Therefore, living Drosophila syncytial blastoderm-stage embryos were used to elucidate the dynamic events leading to the mitotic phenotypes described in KLP10A- and KLP59C-deficient S2 cultures. These embryos are ideal for such analyses because they contain a monolayer of adjacent nuclei, within the same cytoplasm, that proceed through mitosis synchronously and relatively rapidly. Moreover, these cells lack a standard mitotic spindle checkpoint, which normally delays cells in prometaphase in response to spindle damage, allowing mitotic proteins that might function later in the cell cycle to be assessed. For these studies, soluble KinI inhibitors and fluorescent tubulin or histones were microinjected into living transgenic embryos expressing different green fluorescent protein (GFP)-tagged spindle proteins. This allows the dynamic behaviour of spindle components to be visualized in the presence and absence of specific KinI inhibitors (Rogers, 2004).

The introduction of KLP10A inhibitors into embryos rapidly alters the organization of mitotic spindle microtubules, as revealed by time-lapse imaging of anti-KLP10A antibody-injected transgenic embryos expressing GFP-tubulin as a microtubule marker. Within 2 min of antibody injection, the concentration of microtubules at centrosomes increases significantly compared with controls. The relative fluorescent intensities of GFP-tubulin in both prophase and metaphase domains were on average 1.4-fold higher in regions proximal to the injection site compared with mitotic domains at more distal sites. Thus, KLP10A normally limits the growth/density of microtubules at centrosomes. Subsequently, severe defects in mitotic spindle assembly become apparent. These can be categorized into two classes. Thirty per cent formed monopolar spindles, which assemble when centrosomes (partially separated during prophase) collapse back together at a rate of 0.019 microm s^-1 after nuclear envelope breakdown (class one). These structures may result from general defects in the organization of centrosomal microtubules. A total of 61% display abnormally long bipolar spindles, which often form adjacent to monopolar spindles (class two). These bipolar spindles elongate continuously during prometaphase through anaphase and attain metaphase lengths approximately twice that of controls. Notably, the rate of elongation of these spindles before anaphase is nearly identical to the rate of anaphase B spindle elongation in controls. Finally, during telophase, midbodies fail to form on these spindles and centrosomes 'snap-back' together (Rogers, 2004).

In addition to spindle defects, KLP10A inhibition in embryos perturbs the movement and segregation of chromosomes. Specifically, the motility of individual chromosomes during prometaphase is often incoherent in treated embryos and subsequently, during metaphase, the majority of chromosomes and kinetochores fail to align properly at the metaphase plate. Instead, they are scattered around the spindle equator. Finally, during anaphase, chromatids translocate towards spindle poles at rates significantly slower than controls. This results in a significant increase in severe chromosome segregation defects such as stretched or bi-lobed chromosome masses. These anaphase phenotypes are observed regardless of whether kinetochores congressed properly to the metaphase plate or not. Indistinguishable results were obtained using monovalent Fab fragments and were corroborated using a recombinant dominant/negative construct (Rogers, 2004).

In contrast to the findings for KLP10A, the introduction of KLP59C inhibitors into embryos has no apparent effect on the organization of mitotic spindles. However, KLP59C inhibition does produce significant defects in chromosome positioning and motility. Chromosomes and kinetochores fail to align tightly at the spindle equator during metaphase under these conditions. Moreover, KLP59C inhibition prevents normal chromosome segregation during anaphase. Failures in chromosome segregation do not seem to result from decondensation of lagging chromosomes at the metaphase plate, nor are they due to non-disjunction of sister chromatids, because kinetochores do segregate to opposite sides of the chromosomal mass. Instead, as with KLP10A, the rate of chromatid-to-pole motion is slowed significantly in KLP59C-inhibited embryos (Rogers, 2004).

The capacity of KinI kinesins to depolymerize microtubules in vitro, along with the observations that both KLP10A and KLP59C function in chromosome segregation, is consistent with the hypothesis that anaphase chromatid-to-pole motion requires the KinI-induced depolymerization of kinetochore microtubule ends. To test this hypothesis further, the behaviour of spindle microtubules was examined in the presence and absence of specific KLP10A and KLP59C inhibitors. Spindle microtubule dynamics were tracked using fluorescent speckle microscopy (FSM) -- a technique in which dilute levels of rhodamine-labelled tubulin are microinjected into cells, resulting in the random incorporation of fluorescent tubulin 'speckles' into the microtubule lattice. Speckles serve as markers that allow the direct observation of the dynamic behaviours of individual spindle microtubules. In controls, FSM reveals the persistent poleward movement of tubulin subunits within microtubule polymer lattices; that is, poleward flux (Rogers, 2004).

The simultaneous imaging of GFP-tagged kinetochores and tubulin speckles provides an in vivo method to distinguish between the depolymerization of kinetochore microtubule plus or minus ends; flux requires kinetochore microtubule minus-end depolymerization at spindle poles, and kinetochores that move poleward more rapidly than speckles must depolymerize kinetochore microtubules at their plus ends. It was found that the average rate of poleward flux is identical during metaphase and anaphase A and is consistently slower than chromatid-to-pole movement. This suggests that poleward flux can contribute approximately 40% to the rate of anaphase chromatid-to-pole motion whereas Pac-Man-based motility can contribute roughly 60%. Moreover, these data directly demonstrate that kinetochore microtubules simultaneously depolymerize at both plus and minus ends during anaphase in Drosophila embryos (Rogers, 2004).

To test the role of KLP10A in spindle microtubule depolymerization, FSM was performed in embryos injected with anti-KLP10A antibodies. In contrast to control embryos, fluorescent tubulin speckles either do not move poleward at detectable velocities or move at velocities that are significantly slowed. Kymograph analysis of speckle movements confirms the point tracking of speckles and spindle edges. Indeed, >90% of speckles in KLP10A-inhibited spindles either do not have measurable motion or move at a reduced rate. Thus, KLP10A is required for normal poleward flux, making it the first molecular component of this phenomenon identified so far. On the basis of the proximity of this protein to microtubule minus ends at poles, these findings suggest that KLP10A promotes flux by actively depolymerizing kinetochore microtubules at their minus ends (Rogers, 2004).

These data, along with the phenotypes observed after KLP10A inhibition, are notable in that they suggest roles for poleward flux in both anaphase chromatid-to-pole motion and in constraining the length of the mitotic spindle through metaphase. Although the focus of this study is on the former finding, the latter is also intriguing given the recent report that poleward flux ceases during anaphase B (which occurs after chromatids have moved completely poleward). Thus, inactivation of spindle-pole-associated KinI proteins may normally serve as a trigger for anaphase spindle elongation (Rogers, 2004).

In contrast to the KLP10A findings, KLP59C inhibition does not reduce the velocity of poleward tubulin speckle movement within spindles. However, kymographs comparing kinetochore movements to poleward flux reveal that, after KLP59C inhibition, kinetochores move poleward at the rate of flux but no faster, a decrease from control rates by roughly 60%. Indeed, under these conditions, kinetochores rarely bypass speckles on poleward-moving spindle microtubules, a phenomenon regularly observed in controls. Thus, KLP59C inhibition causes kinetochores to lose the ability to move along kinetochore microtubules and leaves them to behave solely as kinetochore microtubule anchors. Given the KLP59C localization to centromeres and its ability to destabilize microtubules in vitro, it is concluded that this protein actively depolymerizes kinetochore microtubule plus ends and thus, it is the first required molecular component of Pac-Man-based chromatid motility identified so far (Rogers, 2004).

Taken together, these findings support a mechanism for anaphase chromatid-to-pole movement in which poleward driving forces acting on chromosomes result from the simultaneous disassembly of kinetochore microtubules at both minus and plus ends. At the minus ends, KinI-induced disassembly allows chromosome-associated kinetochore microtubules to be driven back into the poles (poleward flux). This activity probably requires additional factors that slide microtubules poleward. At the other end of kinetochore microtubules, centromere-associated KinI proteins act to disassemble kinetochore microtubule plus ends, causing them to shorten (Pac-Man). It is proposed that the minus-end-directed motor, cytoplasmic dynein, participates in this process by feeding kinetochore microtubules into the kinetochore. It is tempting to speculate that this coupled, KinI-dependent, 'Pac-Man flux' model is conserved among phyla that contain KinI kinesins, although further work is required to establish this. If so, KinI motors will be promising targets for anti-tumour therapy. Although there are probably additional complexities to anaphase chromosome motility, the concerted action of flux and Pac-Man-based motility mechanisms provides the cell with a surprisingly dynamic means of segregating chromosomes (Rogers, 2004).

Functionally distinct kinesin-13 family members cooperate to regulate microtubule dynamics during interphase: KLP10A is deposited on microtubules by the plus-end tracking protein EB1

Regulation of microtubule polymerization and depolymerization is required for proper cell development. Two proteins of the Drosophila melanogaster kinesin-13 family, KLP10A and KLP59C, cooperate to drive microtubule depolymerization in interphase cells. Analyses of microtubule dynamics in S2 cells depleted of these proteins indicate that both proteins stimulate depolymerization, but alter distinct parameters of dynamic instability; KLP10A stimulates catastrophe (a switch from growth to shrinkage) whereas KLP59C suppresses rescue (a switch from shrinkage to growth). Moreover, immunofluorescence and live analyses of cells expressing tagged kinesins reveal that KLP10A and KLP59C target to polymerizing and depolymerizing microtubule plus ends, respectively. The data also suggest that KLP10A is deposited on microtubules by the plus-end tracking protein, EB1. These findings support a model in which these two members of the kinesin-13 family divide the labour of microtubule depolymerization (Mennella, 2005).

Cell cycle-dependent dynamics and regulation of mitotic kinesins in Drosophila S2 cells

Constructing a mitotic spindle requires the coordinated actions of several kinesin motor proteins. This study visualized the dynamics of five green fluorescent protein (GFP)-tagged mitotic kinesins (class 5, 6, 8, 13, and 14) in live Drosophila Schneider cell line (S2), after first demonstrating that the GFP-tag does not interfere with the mitotic functions of these kinesins using an RNA interference (RNAi)-based rescue strategy. Class 8 (Klp67A) and class 14 (Ncd) kinesin are sequestered in an active form in the nucleus during interphase and engage their microtubule targets upon nuclear envelope breakdown (NEB). Relocalization of Klp67A to the cytoplasm using a nuclear export signal resulted in the disassembly of the interphase microtubule array, providing support for the hypothesis that this kinesin class possesses microtubule-destabilizing activity. The interactions of Kinesin-5 (Klp61F) and -6 (Pavarotti) with microtubules, on the other hand, are activated and inactivated by Cdc2 phosphorylation, respectively, as shown by examining localization after mutating Cdc2 consensus sites. The actions of microtubule-destabilizing kinesins (class 8 and 13 [Klp10A]) seem to be controlled by cell cycle-dependent changes in their localizations. Klp10A, concentrated on microtubule plus ends in interphase and prophase, relocalizes to centromeres and spindle poles upon NEB and remains at these sites throughout anaphase. Consistent with this localization, RNAi analysis showed that this kinesin contributes to chromosome-to-pole movement during anaphase A. Klp67A also becomes kinetochore associated upon NEB, but the majority of the population relocalizes to the central spindle by the timing of anaphase A onset, consistent with the RNAi result showing no effect of depleting this motor on anaphase A. These results reveal a diverse spectrum of regulatory mechanisms for controlling the localization and function of five mitotic kinesins at different stages of the cell cycle (Goshima, 2005; full text of article).

A figure accompanying this article illustrates the diversity of mechanisms for controlling the mitotic kinesins during the cell cycle. The motor activities of two mitotic kinesins, Klp67A and Ncd, are not controlled by cell cycle kinases but are 'sequestered' by localization to the nucleus during interphase, shielding interphase microtubules from undesirable effects (destabilization by Klp67A and bundling by Ncd). NEB at prometaphase enables these motors to 'meet' and perform their actions on microtubules. Nuclear accumulation of vertebrate Ncd also occurs in cells, implicating the evolutional conservation of the motor inactivation mechanism. Nuclear import might require the importin protein, because tail domain of a Xenopus Kinesin-14 is shown to interact with importin to down-regulate microtubule binding activity of this domain (Goshima, 2005).

In contrast, Klp61F (and related Kinesin-5 motors) is constitutively inactive in the cytoplasm and requires activation by Cdc2 phosphorylation at a single site. Mislocalization of the T933A mutant agrees with studies of unphosphorylated Klp61F in the fly embryo and results of alanine substitution of the corresponding residue in Xenopus and human Eg5. However, previous studies did not demonstrate the necessity of this phosphorylation event for mitotic function. The current results extend these studies by showing that other Cdc2 sites are not needed for function and that phosphorylation of T933 is required not only for spindle localization but also for this motor's function in bipolar spindle formation. The mechanism by which incorporation of a single phosphate in the tail domain activates microtubule binding in the motor domain remains a fascinating issue. In contrast, Pav (and related Kinesin-6 motors) is inactivated by Cdc2 kinase before anaphase; its activation seems to require dephosphorylation of Cdc2 sites. The localization is more consistent with a mechanism of localization involving antiparallel microtubules (Goshima, 2005).

Microtubule plus end-tracking of Klp10A is regulated at the point of NEB. Plus end-tracking in interphase is mediated by the interaction with EB1 protein that is enriched at microtubule plus ends throughout the cell cycle. This implicates that NEB releases a factor, possibly derived from chromatin, which dissociates Klp10A from EB1 in prometaphase. The molecular mechanism of this dissociation is unknown. Klp67A relocalization, from kinetochores to central spindle, occurs during anaphase. The localization of Klp67A to the central spindle in late anaphase also is likely to serve a functional role, because mutations in the Klp67A gene cause cytokinesis failure in meiosis by central spindle deformation (Goshima, 2005).

The requirement of the above-mentioned regulatory mechanisms for mitotic kinesin function also seem to vary. The current results show that Cdc2 phosphorylation of Klp61F is absolutely required for function, because mutation of this phosphorylation site generates a monopolar phenotype as severe as the motor RNAi depletion. In contrast, interphase nuclear import of Pav, Klp67A, and Ncd are not essential for the subsequent mitotic functions of these motors, because the addition of a NES to these motors allows full rescue of RNAi phenotype. The regulation requirements for Kinesin-6 are more difficult to establish from this study and those in the literature. In S2 cells, interference with Cdc2 phosphorylation of Pav causes premature mislocalization to overlap zones of metaphase spindles, but this does not seem to cause problems in bipolar spindle formation, alignment of chromosomes, or cytokinesis. In contrast, it has been shown that mutations of two analogous Cdc2 phosphorylation sites (T8 and T450) in human Kinesin-6 (MKLP1) caused chromosome missegregation. The same group also showed that the T9A mutation of the C. elegans Kinesin-6 (ZEN-4) cannot rescue zen-4 null mutant. It is unlikely that Cdc2 phosphorylation of Pav is required for cytokinesis in any of these systems, because Cdc2 activity drops precipitously at the metaphase/anaphase transition. Rather, the above-mentioned differences may be due to the fact that premature spindle localization may cause dominant negative effects in some cell types but not in others. However, another possibility is that Drosophila Kinesin-6 has additional phosphorylation sites that contribute to the complete down-regulation of the motor activity (Goshima, 2005).

Chromosome-to-pole movement during anaphase A has been hypothesized to be driven by microtubule depolymerization. Two sites of depolymerization of kinetochore microtubules seem to contribute to this movement: minus end depolymerization at the centrosome region, leading to poleward flux of microtubule subunits; and plus end depolymerization at the kinetochore. The depolymerization of microtubules by Klp10A and other Kinesin-13 family members has been well established through in vitro studies. Studies in Drosophila embryos show that Klp10A drives microtubule depolymerization at the centrosome and that another Kinesin-13 member (Klp59C) is localized to the kinetochore and is responsible for depolymerization driving anaphase A movement. The class 8 kinesins not only have microtubule-translocating activity but also have been implicated in microtubule destabilization based upon the finding that longer than normal mitotic microtubules arise after genetic mutation or RNAi depletion of these kinesins (Garcia, 2002; West, 2002; Goshima, 2003; Gandhi, 2004; Savoian, 2004). Unlike Klp10A, the length increase is specific for kinetochore microtubules and noncentrosomal interpolar microtubules (Goshi, 2003). However, such an effect upon microtubules could be indirect. This study shows for the first time that expression of Klp67A in the interphase cytoplasm leads to a profound destabilization of the microtubule network. This result strongly suggests that the Kinesin-8 motors are microtubule-destabilizing proteins (Goshima, 2005).

Savoian (2004) recently found that anaphase chromosome-to-pole movement in meiosis I of a hypomorphic Klp67A mutant is significantly slower than wild type, demonstrating that this motor contributes to chromosome movement in the anaphase spindle. However, the slow movement could also be the consequence of unstable kinetochore-microtubule interaction in the Klp67A mutant (Savoian, 2004). This study shows by time-lapse imaging that subpopulation of Klp67A-GFP persistently localizes at the kinetochore, perhaps at outer region of the kinetochore where most of the K-fibers terminate, throughout anaphase A, which is undetected by immunofluorescent microscopy (Savoian, 2004). These results lead to a hypothesis that Klp67A might drive chromosome-to-pole movement by depolymerizing the plus ends of kinetochore microtubules. However, the rate of K-fiber shortening was unchanged after RNAi knockdown of Klp67A. To interpret this 'negative' result, it may be noted that RNAi method does not completely deplete the endogenous proteins and therefore some residual proteins might be sufficient to execute the depolymerizing function. However, Klp67A RNAi cells analyzed in this study had much longer K-fibers and interpolar microtubules than normal, which indicates that any residual small amount of Klp67A is insufficient to constrain metaphase microtubule length. Release of large population of Klp67A from kinetochores after mid-anaphase also may result in the little contribution of this motor-to-anaphase A depolymerization, which occurs from the middle of the anaphase in this cell line. Together, it is suggested that Klp67A is an essential preanaphase K-fiber depolymerase that controls the length of K-fibers but that is not involved in K-fiber shortening during anaphase, at least in S2 cells. The residual population of Klp67A in the anaphase kinetochore might be counterbalanced by kinetochore microtubule polymerase such as Mast/Orbit (CLASP) or inactivated by certain mechanisms such as phosphorylation/dephosphorylation (Goshima, 2005).

Regarding Klp10A, this study shows that in one-half of the Klp10A RNAi cells, K-fiber shortening did not take place for >5 min after sister chromatid separation and that shortening occurred with ~25% reduced velocity. These results indicate that Klp10A is a major contributor of anaphase A chromosome movement in S2 cells, although it is not possible to confirm in this study that its site of action is at the centrosome or kinetochore. Residual Klp10A might be contributing to the shortening in these cells, or other proteins also may participate in this process. Klp59C is the most reasonable candidate to depolymerize K-fiber from its plus end as shown in embryos. It also is reported that knockdown of this motor in S2 cells elevates the frequency with which lagging chromosomes are observed in anaphase. However, this study found no defects in the rate of K-fiber depolymerization in anaphase A of Klp59C RNAi cells. Other mechanisms also might contribute to chromosome-to-pole movement, such as inactivation of microtubule-stabilizing proteins (e.g., Mast/Orbit [CLASP]), other destabilizers (e.g., katanin) or dynein-dependent transport of kinetochores on microtubules. Such mechanisms might be possible to explore in S2 cells depleted of Klp10A by RNAi (Goshima, 2005).

Spindle pole organization in Drosophila S2 cells by dynein, abnormal spindle protein (Asp), and KLP10A

Dynein is a critical mitotic motor whose inhibition causes defects in spindle pole organization and separation, chromosome congression or segregation, and anaphase spindle elongation, but results differ in different systems. The functions of the dynein-dynactin complex was evaluated by using RNA interference (RNAi)-mediated depletion of distinct subunits in Drosophila S2 cells. A striking detachment of centrosomes from spindles, an increase in spindle length, and a loss of spindle pole focus were observed. RNAi depletion of Ncd, another minus-end motor, produced disorganized spindles consisting of multiple disconnected mini-spindles, a different phenotype consistent with distinct pathways of spindle pole organization. Two candidate dynein-dependent spindle pole organizers also were investigated. RNAi depletion of the abnormal spindle protein, Asp, which localizes to focused poles of control spindles, produced a severe loss of spindle pole focus, whereas depletion of the pole-associated microtubule depolymerase KLP10A increased spindle microtubule density. Depletion of either protein produced long spindles. After RNAi depletion of dynein-dynactin, subtle but significant mislocalization of KLP10A and Asp was observed, suggesting that dynein-dynactin, Asp, and KLP10A have complex interdependent functions in spindle pole focusing and centrosome attachment. These results extend recent findings from Xenopus extracts to Drosophila cultured cells and suggest that common pathways contribute to spindle pole organization and length determination (Morales-Mulia, 2005; full text of article).

Antagonistic activities of Klp10A and Orbit regulate spindle length, bipolarity and function in vivo

The metaphase-spindle steady-state length occurs as spindle microtubules 'flux', incorporating new subunits at their plus ends, while simultaneously losing subunits from their minus ends. Orbit/Mast/CLASP is required for tubulin subunit addition at kinetochores, and several kinesins regulate spindle morphology and/or flux by serving as microtubule depolymerases. This study used RNA interference in S2 cells to examine the relationship between Orbit and the four predicted kinesin-type depolymerases encoded by the Drosophila genome (Klp10A, Klp59C, Klp59D and Klp67A). Single depletion of Orbit results in monopolar spindles, mitotic arrest and a subsequent increase in apoptotic cells. These phenotypes are rescued by co-depleting Klp10A but none of the other three depolymerases. Spindle bipolarity is restored by preventing the spindle collapse seen in cells that lack Orbit, leading to functional spindles that are similar to controls in shape and length. It is concluded that Klp10A exclusively antagonises Orbit in the regulation of bipolar spindle formation and maintenance (Laycock, 2006).

Bipolar spindle formation and maintenance occur through the actions of multiple motor and microtubule dynamics-altering proteins. This study examined the interplay between Orbit, a protein needed for tubulin-dimer incorporation into kinetochore MTs, and each of the four microtubule depolymerising kinesins Klp67A, Klp10A, Klp59C and Klp59D. The co-depletion of Klp10A but not of the other KLP MT depolymerases diminished the number of apoptotic cells and prevented the spindle collapse associated with orbit knockdown. In contrast to individual downregulation of Orbit or Klp10A that resulted in abnormally short and/or monopolar or long spindles, respectively, spindles in double-deficient cells were bipolar and of an average length indistinguishable from controls. These spindles promoted chromosome alignment and anaphase entry, indicating that they were functional. Since both Orbit and Klp10A have been directly implicated in microtubule flux, these data suggest that this process is not required to determine mitotic spindle morphology or chromosome congression in Drosophila tissue culture cells (Laycock, 2006).

During the course of this work, it was reported that microtubule flux is not essential for bipolar spindle formation and chromosome congression in mitotic vertebrate cells. In contrast to the knocked down MT-stabilising and -depolymerising protein pair described in this study, the experiments in vertebrate used the co-depletion of two kinesin-13 depolymerases: Kif2A, the orthologue of Klp10A and Kif2C/MCAK/XKCM1, the vertebrate counterpart of Klp59C. Both Kif2A and Kif2C have overlapping localisations at centromeres and spindle poles and/or centrosomes and loss of either leads to a prometaphase spindle collapse. This monopolar spindle phenotype was rescued by simultaneously depleting both of these MT depolymerases, suggesting they form an antagonistic pair. However, an orthologous antagonism of Klp10A and Klp59C does not exist during mitosis in Drosophila, because loss of Klp59C function in tissue culture cells or syncitial embryos does not alter spindle morphology, and dual perturbations result in spindle abnormalities identical to that observed following single Klp10A disruptions (Laycock, 2006 ans references therein).

It is proposed that bipolar spindle formation occurs in at least two phases. During the first, MTs nucleated from the separated centrosomes invade the nuclear volume and make their initial interactions with the kinetochores. Time-lapse analyses indicate that this step is not affected by the depletion of either Orbit or Klp10A and is thus Orbit- and Klp10A-independent. The second phase, spindle stabilisation, occurs after chromosome bi-orientation and probably results from antagonistic pairs of molecules regulating the dynamics of the plus- and minus-ends of kinetochore MTs. It is at this stage that Orbit and Klp10A become engaged as evidenced by the collapse of the nascent spindle following their individual perturbations. Although the possibility that the spindle collapse results from a loss of interpolar MT integrity cannot be ruled out, this is thought to be unlikely. First, because the rate at which spindles collapse after orbit RNAi is similar to the rate of MT flux in Drosophila tissue culture cells, consistent with a flux depolymerase shortening kMTs in the absence of new tubulin-polymer growth at the kinetochore. Second, because kMTs that form independently from the centrosome and primary spindle axis (i.e., a mini-spindle) also shrink in the absence of Orbit. Since these bundles of kMTs are not present between two half spindles it unlikely that their shortening is the result of intervening interpolar MTs. Likewise, it has been previously demonstrated that the spindle collapse associated with the loss of Kif2A is kMT-dependent. Spindle bipolarity was restored in these cells by co-depleting the Nuf2 kinetochore protein, thereby preventing kMT formation without affecting any other spindle MTs. In the case of Orbit and Klp10A single-depleted cells, it is envisaged that the spindle collapse is due to an imbalance of regulatory components following the activation of the flux machinery during this second phase. Collapse could result, for example, as Klp10A depolymerises the non-polymerising kMTS that result from orbit downregulation. Conversely, because flux-generated tension has been proposed to promote tubulin subunit incorporate at kinetochores, depletion of Klp10A could also affect polymerisation of kMTs, which -- in the presence of other active phase two depolymerises -- would cause spindle collapse. In the absence of Orbit and Klp10A the flux machinery would not become engaged and spindle length would be determined by other antagonistic molecular pairs (Laycock, 2006).

The data in flies, in concert with that found in vertebrates, indicate that although microtubule flux is a characteristic of many animal cells it is not essential for pre-anaphase chromosome movements or spindle formation. Nevertheless, the plasticity it imparts is probably advantageous for spindle and kinetochore interactions, for example by promoting kMT polymerisation or by generating tension for satisfying the spindle checkpoint. It was found that cells simultaneously depleted of Klp10A and Orbit tended to spend variable but extremely prolonged periods of time in mitosis before entering anaphase. Since both fixed and live cell studies did not reveal an increase in non-equatorially positioned chromosomes compared with controls, it is believed that the prometaphase arrest was not due to activation of the checkpoint through unattached kinetochores. Although never fully relaxed, the centromeres of bi-oriented chromosomes in double-knockdown cells tended to be under diminished tension relative to controls. This corresponded to the retention of BubR1 at kinetochores. Cells depleted only of Klp10A also spent more time in prometaphase than their control counterparts, although this duration was substantially less than that observed for orbit and Klp10A double-RNAi cells. Despite decreased intra-centromeric tension in Klp10A downregulated cells, BubR1 was not observed on the kinetochores of congressed chromosomes. One explanation for this is that, even without flux, spindle MTs can still produce tension by transducing cortical forces. Here, the long spindles that form in the absence of Klp10A would position the asters in direct contact with the cortex where their component MTs would make increased numbers of contacts with cortical motor proteins such as cytoplasmic dynein. Just as cortex-based forces by this motor act along astral MTs for spindle positioning, astral pulling could generate tension across the centromeres and kinetochores of congressed chromosomes. If true, centromeric tension should correlate with the presence or absence of asters. This was found to be the case, and sister centromeres were separated to a greater extent when they were on bi-astral spindles than when attached to bipolar spindles with a single aster. Moreover, in those Klp10A-depleted cells displaying bipolar spindles capped at each end by an overgrown aster, the average intra-centromeric distance was greater than that seen in the controls (Laycock, 2006).

Together, these observations indicate that Orbit and Klp10A are an antagonistic molecular pair, consistent with their previous individual implicated roles in MT flux, a process that dispensable for bipolar spindle formation and chromosome congression. These data further suggest that astral-mediated pulling forces are involved in checkpoint satisfaction. The role that these forces may serve in the checkpoint has not been previously reported (Laycock, 2006).

Kinesin-13s form rings around microtubules

Kinesin is a superfamily of motor proteins that uses the energy of adenosine triphosphate hydrolysis to move and generate force along microtubules. A notable exception to this general description is found in the kinesin-13 family that actively depolymerizes microtubules rather than actively moving along them. This depolymerization activity is important in mitosis during chromosome segregation. It is still not fully clear by which mechanism kinesin-13s depolymerize microtubules. To address this issue, electron microscopy was used to investigate the interaction of kinesin-13s with microtubules. Surprisingly, it was found that proteins of the kinesin-13 family form rings and spirals around microtubules. This is the first report of this type of oligomeric structure for any kinesin protein. These rings may allow kinesin-13s to stay at the ends of microtubules during depolymerization (Tan, 2006).

Model for anaphase B: role of three mitotic motors in a switch from poleward flux to spindle elongation

It has been proposed that the suppression of poleward flux within interpolar microtubule (ipMT) bundles of Drosophila embryonic spindles couples outward forces generated by a sliding filament mechanism to anaphase spindle elongation. This study proposes (1) a molecular mechanism in which the bipolar kinesin KLP61F persistently slides dynamically unstable ipMTs outward, the MT depolymerase KLP10A acts at the poles to convert ipMT sliding to flux, and the chromokinesin KLP3A inhibits the depolymerase to suppress flux, thereby coupling ipMT sliding to spindle elongation; (2) KLP3A inhibitors were used to interfere with the coupling process, revealing an inverse linear relation between the rates of flux and elongation, supporting the proposed mechanism and demonstrating that the suppression of flux controls both the rate and onset of spindle elongation; and (3) a mathematical model was developed using force balance and rate equations to describe how motors sliding the highly dynamic ipMTs apart can drive spindle elongation at a steady rate determined by the extent of suppression of flux (Brust-Mascher, 2004; full text of article).

Search PubMed for articles about Drosophila Klp10A

Brust-Mascher, I., et al. (2004). Model for anaphase B: role of three mitotic motors in a switch from poleward flux to spindle elongation. Proc. Natl. Acad. Sci. 101(45): 15938-43. Medline abstract: 15522967

Desai, A., Verma, S., Mitchison, T. J. and Walczak, C. E. (1999). KinI kinesins are microtubule-destabilizing enzymes. Cell 96: 69-78. Medline abstract: 9989498

Gandhi, R., Bonaccorsi, S., Wentworth, D., Doxsey, S., Gatti, M., and Pereira, A. (2004). The Drosophila kinesin-like protein KLP67A is essential for mitotic and male meiotic spindle assembly. Mol. Biol. Cell 15: 121-131. Medline abstract: 13679514

Garcia, M. A., Koonrugsa, N., and Toda, T. (2002). Two kinesin-like Kin I family proteins in fission yeast regulate the establishment of metaphase and the onset of anaphase A. Curr. Biol. 12: 610-621. Medline abstract: 11967147

Goshima, G. and Vale, R. D. (2003). The roles of microtubule-based motor proteins in mitosis: comprehensive RNAi analysis in the Drosophila S2 cell line. J. Cell Biol. 162: 1003-1016. Medline abstract: 12975346

Goshima, G. and Vale, R. D. (2005). Cell cycle-dependent dynamics and regulation of mitotic kinesins in Drosophila S2 cells. Mol. Biol. Cell 16(8): 3896-907. Medline abstract: 15958489

Hunter, A. W. and Wordeman, L. (2000). How motor proteins influence microtubule polymerization dynamics. J. Cell Sci. 24: 4379-4389. Medline abstract: 11082031

Kline-Smith, S. L. and Walczak, C. E. (2002). The microtubule-destabilizing kinesin XKCM1 regulates microtubule dynamic instability in cells. Mol. Biol. Cell 13: 2718-2731. Medline abstract: 12181341

Laycock, J. E., Savoian, M. S. and Glover, D. M. (2006). Antagonistic activities of Klp10A and Orbit regulate spindle length, bipolarity and function in vivo. J. Cell Sci. 119(Pt 11): 2354-61. 16723741

Maney, T., Hunter, A. W., Wagenbach, M. and Wordeman, L. (1998). Mitotic centromere-associated kinesin is important for anaphase chromosome segregation. J. Cell Biol. 142: 787-801. Medline abstract: 9700166

Mennella, V., et al. (2005). Functionally distinct kinesin-13 family members cooperate to regulate microtubule dynamics during interphase. Nat. Cell Biol. 7(3): 235-45. Medline abstract: 15723056

Morales-Mulia, S. and Scholey, J. M. (2005). Spindle pole organization in Drosophila S2 cells by dynein, abnormal spindle protein (Asp), and KLP10A. Mol. Biol. Cell 16(7): 3176-86. Medline abstract: 15888542

Rogers, G. C., et al. (2004). Two mitotic kinesins cooperate to drive sister chromatid separation during anaphase. Nature. 427: 364-370. Medline abstract: 14681690

Savoian, M. S., Gatt, M. K., Riparbelli, M. G., Callaini, G., and Glover, D. M. (2004). Drosophila Klp67A is required for proper chromosome congression and segregation during meiosis I. J. Cell Sci. 117: 3669-3677

Tan, D., Asenjo, A. B., Mennella, V., Sharp, D. J. and Sosa, H. (2006). Kinesin-13s form rings around microtubules. J. Cell Biol. 175(1): 25-31. Medline abstract: 17015621

Walczak, C. E., Mitchison, T. J. and Desai, A. (1996). XKCM1: a Xenopus kinesin-related protein that regulates microtubule dynamics during mitotic spindle assembly. Cell 84: 37-47. Medline abstract: 8548824

Walczak, C. E., Gan, E. C., Desai, A., Mitchison, T. J. and Kline-Smith, S. L. (2002). The microtubule-destabilizing kinesin XKCM1 is required for chromosome positioning during spindle assembly. Curr. Biol. 12: 1885-1889. Medline abstract: 12419191

West, R. R., Malmstrom, T., and McIntosh, J. R. (2002). Kinesins klp5(+) and klp6(+) are required for normal chromosome movement in mitosis. J. Cell Sci. 115: 931-940. Medline abstract: 11870212

Wordeman, L. and Mitchison, T. J. (1995). Identification and partial characterization of mitotic centromere-associated kinesin, a kinesin-related protein that associates with centromeres during mitosis. J. Cell Biol. 128: 95-104. Medline abstract: 7822426

date revised: 17 January 2008

Home page: The Interactive Fly and#169; 2008 Thomas Brody, Ph.D.