Kinesin-like protein at 10A: Biological Overview | References
Gene name - Kinesin-like protein at 10A
Cytological map position-10A6-10A6
Symbol - Klp10A
FlyBase ID: FBgn0030268
Genetic map position - X: 11,024,269..11,031,056 [-]
Classification - Kinesin motor domain
Cellular location - cytoplasmic
|Recent literature||Chatterjee, C., Benoit, M. P., DePaoli, V., Diaz-Valencia, J. D., Asenjo, A. B., Gerfen, G. J., Sharp, D. J. and Sosa, H. (2016). Distinct interaction modes of the Kinesin-13 motor domain with the microtubule. Biophys J 110: 1593-1604. PubMed ID: 27074684
Kinesins-13s are members of the kinesin superfamily of motor proteins that depolymerize microtubules (MTs) and have no motile activity. Instead of generating unidirectional movement over the MT lattice, like most other kinesins, kinesins-13s undergo one-dimensional diffusion (ODD) and induce depolymerization at the MT ends. To understand the mechanism of ODD and the origin of the distinct kinesin-13 functionality, ensemble and single-molecule fluorescence polarization microscopy was used to analyze the behavior and conformation of Drosophila melanogaster kinesin-13 KLP10A protein constructs bound to the MT lattice. KLP10A was found to interact with the MT in two coexisting modes: one in which the motor domain binds with a specific orientation to the MT lattice and another where the motor domain is very mobile and able to undergo ODD. By comparing the orientation and dynamic behavior of mutated and deletion constructs it is concluded that 1) the Kinesin-13 class specific neck domain and loop-2 help orienting the motor domain relative to the MT. 2) During ODD the KLP10A motor-domain changes orientation rapidly (rocks or tumbles). 3) The motor domain alone is capable of undergoing ODD. 4) A second tubulin binding site in the KLP10A motor domain is not critical for ODD. 5) The neck domain is not the element preventing KLP10A from binding to the MT lattice like motile kinesins.
|Chen, C., Inaba, M., Venkei, Z. G. and Yamashita, Y. M. (2016). Klp10A, a stem cell centrosome-enriched kinesin, balances asymmetries in Drosophila male germline stem cell division. Elife 5. PubMed ID: 27885983
Asymmetric stem cell division is often accompanied by stereotypical inheritance of the mother and daughter centrosomes. However, it remains unknown whether and how stem cell centrosomes are uniquely regulated and how this regulation may contribute to stem cell fate. This study identifies Klp10A, a microtubule-depolymerizing kinesin of the kinesin-13 family, as the first protein enriched in the stem cell centrosome in Drosophila male germline stem cells (GSCs). Depletion of klp10A results in abnormal elongation of the mother centrosomes in GSCs, suggesting the existence of a stem cell-specific centrosome regulation program. Concomitant with mother centrosome elongation, GSCs form asymmetric spindle, wherein the elongated mother centrosome organizes considerably larger half spindle than the other. This leads to asymmetric cell size, yielding a smaller differentiating daughter cell. It is proposed that klp10A functions to counteract undesirable asymmetries that may result as a by-product of achieving asymmetries essential for successful stem cell divisions.
|Gottardo, M., Callaini, G. and Riparbelli, M. G. (2016). Klp10A modulates the localization of centriole-associated proteins during Drosophila male gametogenesis. Cell Cycle [Epub ahead of print] PubMed ID: 27764551
Mutations in Klp10A, a microtubule-depolymerising Kinesin-13, lead to overly long centrioles in Drosophila male germ cells. This study has demonstrated that the loss of Klp10A modifies the distribution of typical proteins involved in centriole assembly and function. In the absence of Klp10A the distribution of Drosophila pericentrin-like protein (Dplp), Sas-4 and Sak/Plk4 that are restricted in control testes to the proximal end of the centriole increase along the centriole length. Remarkably, the cartwheel is lacking or it appears abnormal in mutant centrioles, suggesting that this structure may spatially delimit protein localization. Moreover, the parent centrioles that in control cells have the same dimensions grow at different rates in mutant testes with the mother centrioles longer than the daughters. Daughter centrioles have often an ectopic position with respect to the proximal end of the mothers and failed to recruit Dplp.
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).
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).
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).
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).
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 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).
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).
Chromosomes move toward mitotic spindle poles by a Pacman-flux mechanism linked to microtubule depolymerization: chromosomes actively depolymerize attached microtubule plus ends (Pacman) while being reeled in to spindle poles by the continual poleward flow of tubulin subunits driven by minus-end depolymerization (flux). Pacman-flux in Drosophila melanogaster incorporates the activities of three different microtubule severing enzymes, Spastin, Fidgetin, and Katanin. Spastin and Fidgetin are utilized to stimulate microtubule minus-end depolymerization and flux. Both proteins concentrate at centrosomes, where they catalyze the turnover of γ-tubulin, consistent with the hypothesis that they exert their influence by releasing stabilizing γ-tubulin ring complexes from minus ends. In contrast, Katanin appears to function primarily on anaphase chromosomes, where it stimulates microtubule plus-end depolymerization and Pacman-based chromatid motility. Collectively, these findings reveal novel and significant roles for microtubule severing within the spindle and broaden the understanding of the molecular machinery used to move chromosomes (Zhang, 2007).
The results of this study show that three closely related MT severing enzymes, Dm-Kat60, Spastin, and Fidgetin, are important for mitosis in D. melanogaster S2 cells. Interestingly, the activity of these proteins is segregated both spatially and temporally, allowing them to perform complementary functions throughout the spindle. This is most apparent during anaphase A, when all three are integrated into the Pacman-flux machinery used to move chromosomes (Zhang, 2007).
Spastin and Fidgetin emerge from this study as new regulators of poleward MT flux. Specifically, inhibition of either protein results in a significant reduction in flux velocity. In addition, it was found that both proteins similarly promote the turnover of γ-tubulin at spindle poles and γ-tubulin at centrosomes. In sum, these data are consistent with a general model for flux and chromosome motility, in which Spastin and Fidgetin function to release MT minus ends from their nucleating γ-TuRCs, which are believed to cap and stabilize MT ends. In turn, severing exposes minus ends to depolymerization by spindle pole-associated Kinesin-13 (KLP10A in D. melanogaster), which has been shown to also contribute to flux. During anaphase A, the MT minus-end depolymerization of flux 'reels in' chromosomes to the poles (Zhang, 2007).
Based on the proposal that Spastin and Fidgetin work in concert with KLP10A to promote flux, one would expect many similarities in the phenotypes resulting from the inhibition of these proteins. Indeed, as in Spastin or Fidgetin RNAi-treated cells, depletion of KLP10A also inhibits flux and slows anaphase A. A notable difference, however, is that KLP10A RNAi induces spindle elongation, whereas Spastin or Fidgetin RNAi does not. One possible explanation for this apparent inconsistency stems from the fact that spindles probably elongate as a result of continued plus-end polymerization and MT sliding when minus-end depolymerization (i.e., flux) is decreased after KLP10A RNAi. Thus, the puzzling absence of spindle elongation after Spastin or Fidgetin RNAi might be explained by the observation that plus-end polymerization is also significantly decreased in these cells (Zhang, 2007).
Although these data demonstrate roles for Spastin and Fidgetin in regulating flux and MT-centrosome interaction (i.e., catalyzing the turnover of γ-tubulin and regulating abnormal spindle-mediated attachments with MT minus ends), it is currently unclear whether centrosomes are the sole or even primary site of action of these proteins in the spindle. Indeed, the presence of centrosomes is not required for spindle assembly, flux, and chromosome segregation in some D. melanogaster cell types and other systems such as oocyte spindles. Additionally, even in centrosome-containing cells, the majority of MT minus ends are often positioned at a distance from centrosomes, and many spindle MTs are thought to arise from noncentrosomal sources (e.g., chromosomes/kinetochores). These MTs may still be capped by cytoplasmic γ-TuRCs, and it is conceivable that severing within the spindle (i.e., away from centrosomes) is required for their normal dynamics and flux. Thus, although this model depicts Spastin and Fidgetin as functioning only at centrosomes (where these proteins concentrate), this may be an oversimplification (Zhang, 2007).
Why both Spastin and Fidgetin would be used for the same task is unclear. At present, there is no clear evidence for a functional or physical interaction between these proteins. Each protein might sever a distinct subset of centrosomal MTs, but this would be unprecedented, and it is therefore considered unlikely. Unfortunately, coinhibition of these proteins by RNAi causes a high degree of cell death, making it difficult to assess this possibility. Alternatively, a degree of functional redundancy may explain why a small portion of D. melanogaster carrying null mutations in the spastin gene survive to adulthood. Genetic analysis of the relationship between these proteins should be revealing and may help answer this question (Zhang, 2007).
It is notable that Dm-Kat60 also localizes to centrosomes but performs no obvious function there, at least based on the assays used in this study. However, Dm-Kat60 RNAi does impact the mitotic index, which is likely indicative of subtle preanaphase Dm-Kat60 activities, which are beyond the sensitivity of current visualization techniques (Zhang, 2007).
Although Dm-Kat60 does not appear to function at centrosomes, the data indicate that this protein plays an important role in moving anaphase chromosomes (McNally, 1993). Dm-Kat60, which localizes to both chromosome arms and kinetochores, functions during anaphase to stimulate the depolymerization of MT plus ends, thereby moving chromosomes by a Pacman mechanism. It is proposed that Dm-Katanin functions in this regard by uncapping MT plus ends -- much the same as Spastin and Fidgetin do at minus ends -- and exposing them to depolymerization by centromere/kinetochore-associated Kinesin-13, which is also required for Pacman. Although Pacman-inhibiting plus-end caps have not been identified, several MT-stabilizing microtubule-associated proteins (such as the plus-end tracking proteins CLASP, EB1, and CLIP-190) associate with kinetochore-associated MT plus ends. Whether the association of these proteins with plus ends inhibits depolymerization by Kinesin-13s is unknown (Zhang, 2007).
Additionally, severing by Katanin could uncap plus ends associated with chromosome arms. A vertebrate kinesin, XKLP1, which targets to chromosome arms, has been shown to bind and stabilize MT plus ends and would probably resist Pacman motility. The D. melanogaster genome encodes several potential XKLP1 homologues, and it will be interesting to see whether Katanin has an antagonistic relationship with any of these (Zhang, 2007).
The possibility cannot be ruled out that D. melanogaster Katanin directly stimulates the depolymerization of kinetochore-associated MT plus ends. Indeed, it could conceivably supplant chromosome-associated Kinesin-13s in some systems, potentially explaining why the Kinesin-13 KLP59C does not appear to play a direct role in chromosome motility in S2 cells even though it drives Pacman in D. melanogaster embryos. However, another Kinesin-13 that is needed for Pacman in S2 cells has been identified (unpublished data of Zhang, 2007), making it unlikely that Dm-Katanin directly depolymerizes plus ends (Zhang, 2007).
It is notable that Spastin and Fidgetin also target to chromosomes before anaphase, where they may function similarly to Dm-Katanin. FRAP analysis indicates that both proteins normally enhance the turnover of chromosome-associated plus ends on preanaphase spindles. Why the chromosome activity of these proteins is down-regulated at the onset of anaphase while Katanin, which associates with chromosome throughout mitosis, is up-regulated is unclear. The loss of Spastin and Fidgetin from chromosomes may result from the underlying dependence of this targeting on MTs. Both are released from chromosomes in the presence of colchicine, and alterations in MT dynamics that accompany the onset of anaphase may have the same effect. Alternatively, Katanin's activity may be up- or down-regulated by phosphorylation. Indeed, the primary sequence of Dm-Kat60 contains several putative CDK1 phosphorylation motifs. Finally, Katanin's severing activity may be negatively regulated by MT-coating microtubule-associated proteins (Zhang, 2007).
Interestingly, Katanin does not appear to target to chromosomes or kinetochores in many cell types. In fact, the only system besides D. melanogaster in which a Katanin homologue has been reported to associate with chromosomes is C. elegans, which does not use Katanin for mitosis. This raises the question of whether the mitotic functions of MT severing proteins, particularly Katanin, are conserved throughout phylogeny. In this regard, it is noted that several additional Katanin p60 homologues whose functions have not yet been analyzed have been identified within vertebrate and invertebrate genomes. Any of these could target to chromosomes and stimulate Pacman-based anaphase A. Moreover, a recent yeast two-hybrid study has shown that Fidgetin associates with the protein kinase A anchoring protein, AKAP95, which targets to chromosomes throughout mitosis. D. melanogaster contain no obvious AKAP95 homologue, perhaps explaining why Fidgetin does not impact Pacman in this system. Future studies to examine the possible mitotic functions of vertebrate Fidgetin and Katanin homologues would address this question (Zhang, 2007).
In closing, this study suggests a general mechanism in which appropriately positioned and tightly regulated MT severing proteins provide a means to rapidly create free MT ends, which are then exposed to the actions of other regulatory proteins. During anaphase, such an activity works in close coordination with Kinesin-13s, stimulating poleward chromatid motility by a combined Pacman-flux mechanism. In other instances, the creation of free ends could have a very different impact on MT behaviors. Future analyses examining the interactions between severing proteins and Kinesin-13s, as well as other regulators of MT dynamics, will help test this proposal (Zhang, 2007).
Regulation of microtubule dynamics at the cell cortex is important for cell motility, morphogenesis and division. This study shows that the Drosophila katanin Dm-Kat60 functions to generate a dynamic cortical-microtubule interface in interphase cells. Dm-Kat60 concentrates at the cell cortex of S2 Drosophila cells during interphase, where it suppresses the polymerization of microtubule plus-ends, thereby preventing the formation of aberrantly dense cortical arrays. Dm-Kat60 also localizes at the leading edge of migratory D17 Drosophila cells and negatively regulates multiple parameters of their motility. Finally, in vitro, Dm-Kat60 severs and depolymerizes microtubules from their ends. On the basis of these data, it is proposed that Dm-Kat60 removes tubulin from microtubule lattice or microtubule ends that contact specific cortical sites to prevent stable and/or lateral attachments. The asymmetric distribution of such an activity could help generate regional variations in microtubule behaviours involved in cell migration (Zhang, 2011).
Microtubules form complex and dynamic arrays with pivotal roles in the development and function of eukaryotic cells. Although microtubules are intrinsically dynamic, their cellular behaviours are tightly regulated by a host of other factors. Thus, the microtubule cytoskeleton is responsive to a variety of cues and can locally adapt its dynamic properties accordingly. These regulatory inputs seem to be particularly relevant at the cell cortex, where localized alterations in microtubule dynamics and organization are central to cell migration, polarization, morphogenesis and division (Zhang, 2011).
Katanin is a phylogenetically conserved enzyme that uses the energy of ATP hydrolysis to generate microtubule breakage in vitro (Roll-Mecak, 2010; see Schematic representation of the domain architecture of microtubule-severing enzymes). Katanin was originally purified from sea urchin eggs as a heterodimer of p60, a catalytic subunit of relative molecular mass 60,000 (Mr 60,K) and p80, a targeting and regulatory subunit Mr (K) 80 (McNally, 1993). Katanin p60 and p80 homologues have now been identified in evolutionarily diverse systems and many organisms contain several genes encoding distinct p60 and/or p80 proteins. Functional analyses reveal diverse roles for katanin in mitosis and meiosis, in neuronal morphogenesis, and in the assembly and disassembly of cilia and flagella. In addition, a katanin in higher plants has been shown to regulate the assembly of cortical microtubule arrays, which, in turn, determine the directional deposition of cellulose and thus impact cell morphogenesis. In this context, katanin releases new microtubules nucleated from the walls of pre-existing microtubules (Roll-Mecak, 2010; Nakamura, 2010; Zhang, 2011 and references therein).
Previous studies have found that the Drosophila katanin p60, Dm-Kat60, associates with mitotic chromosomes and stimulates the depolymerization of kinetochore-associated microtubule plus-ends during anaphase A (Zhang, 2007). The present study tested the hypothesis that Dm-Kat60 also functions to regulate microtubule dynamics during interphase -- a topic that has not been addressed in any other animal system (Zhang, 2011).
The results identify Dm-Kat60 as an important regulator of microtubule dynamics and cell migration. The human katanin KATNA1 behaves similarly. In addition to its cellular roles, in vitro analyses indicate that Dm-Kat60 has the capacity to function as both a microtubule-severing enzyme and a microtubule end depolymerase. On the basis of its cortical localization, RNAi phenotypes and catalytic activity, it is proposed that Dm-Kat60 (and the human p60-like protein KATNA1) contributes to the generation of a dynamic interface between the microtubule cytoskeleton and the interphase cortex by removing tubulin subunits from any region of the microtubule making contact with Dm-Kat60-rich cortical sites (Zhang, 2011).
Among the more unexpected outcomes of this study is the observation that Dm-Kat60 induces microtubule end depolymerization in vitro. However, given present models of the interaction of katanin with the microtubule, such a finding is not entirely surprising. Biophysical and biochemical studies have indicated that severing by katanin is mediated by the transient hexamerization of p60 proteins at the C terminus of a single tubulin within the microtubule (see Roll-Mecak, 2010). ATP hydrolysis and/or the subsequent disassembly of the hexamer is believed to generate a mechanical force, which, through multiple iterations, induces the removal of the tubulin from the lattice. If katanin works by 'tugging' on a single tubulin heterodimer, then the exposed tubulins at the microtubule end are likely to be the easiest to remove because they lack a longitudinal contact. However, the possibility cannot bevrule out that Dm-Kat60-mediated end depolymerization is a manifestation of multiple severing events occurring very near the tip (Zhang, 2011).
Within the cell, the severing and depolymerase activities of Dm-Kat60 probably remain under very tight spatial constraints. In this regard, the recruitment of Dm-Kat60 to the cell cortex seems to be central to its interphase functions. Although the data indicate that this process is reliant on the presence of actin, but not microtubules, the specific mechanisms that deliver Dm-Kat60 to the cortex remain a mystery. One appealing hypothesis is that Dm-Kat60 is directly or indirectly linked to the cortical actin array through Drosophila p80. The p80 subunit contains repeated WD40 motifs known to mediate protein-protein interactions. Similar motifs have been identified in some actin-binding proteins. The WD40 repeats of p80 are essential for the centrosomal targeting of katanin in other organisms. It has also been suggested that p60 acts independently of p80 in some circumstances. The identification of the binding partners of Dm-Kat60 represents an important next step in understanding its cellular activities (Zhang, 2011).
At the cortex, Dm-Kat60 suppresses microtubule growth primarily by inducing plus-end catastrophes and transitions from growth to pause. Although other classes of proteins are known to induce microtubule depolymerization, in vitro, the presumptive ability of Dm-Kat60 to remove tubulins from any region of the microtubule -- end or lattice -- may be particularly useful in the more complex cellular environment. For example, such an activity could enable Dm-Kat60 to prevent sustained microtubule growth along the cortex regardless of whether the microtubule contacts the cortex end-on or side-on. The newly created plus-end at the cortical-microtubule interface would then initiate catastrophe or enter the pause state depending on its association with other microtubule-binding proteins. Moreover, the ends of polymerizing microtubules in cells are often 'capped' by plus-end-binding proteins such as EB1. Dm-Kat60 could remove these by severing the microtubule at the base of the EB1 'cap' and/or directly removing EB1-bound tubulins from the plus-end. The acidic tail of EB1 could mimic the C terminus of tubulin, thereby providing a substrate for Dm-Kat60 (Zhang, 2011 and references therein).
Of course, Dm-Kat60 is not alone in its ability to stimulate the catastrophe of microtubule plus-ends near the cortex of interphase S2 cells, as the Drosophila kinesin-13 KLP10A also shows this activity. However, aside from the ability of both proteins to promote catastrophes, the activities of Dm-Kat60 and KLP10A are quite distinct. The most notable difference is that KLP10A does not concentrate on the cortex, but instead binds to the ends of polymerizing microtubules to which it is recruited by EB1. Intriguingly, recent work indicates that EB1 can inhibit the depolymerase activity of kinesin-13 proteins by shielding the plus-end. If Dm-Kat60 were to generate plus-ends lacking EB1, then it could relieve this inhibition (Zhang, 2011).
It is proposed that Dm-Kat60 and KLP10A work together as follows. (1) Dm-Kat60 removes tubulins (EB1 bound or otherwise) from regions of the microtubule that come in close proximity to the cortex, thereby creating a free plus-end at the cortical interface. Many of these newly created plus-ends immediately enter a paused state (depletion of EB1 has been shown to strongly promote pause). (2) Next, KLP10A, which has already been accumulated near the end by EB1, promotes the transition of this end from pause to shrinkage -- this transition can occur rapidly and may often appear as a catastrophe. The present study also indicates that KLP10A increases the rate of plus-end depolymerization and thus a small, difficult-to-detect, portion of the protein may remain associated with the microtubule end as it depolymerizes. Why such an effect was not noted in the initial analysis of KLP10A is unknown, but may be due to the more limited region of the cortex analysed in that study (Zhang, 2011 and references therein).
The finding that Dm-Kat60 targets the leading edge of motile D17 cells and alters their migration provides a broader biological context through which the current findings can be viewed and interpreted. Although the depletion of Dm-Kat60 had no obvious influence on the establishment of cell polarity, it did increase both the frequency and displacement of membrane protrusions, at least in S2 cells. The localized suppression of protrusions at the leading edge of motile D17 cells could exert negative control over the rate and persistence of cell movement (Zhang, 2011).
The observation that Dm-Kat60 RNAi results in faster and more persistent migration seems consistent with other studies demonstrating that growing microtubules stimulate the GTPase Rac at the leading edge, which may promote adhesion-complex remodelling, needed to drive and sustain protrusions. In addition, because Dm-Kat60 depletion at the leading edge of migratory cells should decrease catastrophes, microtubules could become unusually persistent and abundant in the extending protrusion, which may intensify other processes that favour protrusion, such as increased kinesin-mediated delivery of vesicles to the protrusion zone (Zhang, 2011).
A recent study examining haemocyte migration in developing Drosophila embryos, for which D17 cells may be a model, showed that haemocytes migrate less efficiently in response to guidance cues following the disruption of microtubule dynamics. Under these conditions, an increase in cell velocities and a decrease in directional persistence were observed, similar to D17 cells depleted of Dm-Kat60 by RNAi. Thus, Dm-Kat60 may modulate microtubule dynamics at the leading edge to 'fine-tune' cell migration by suppressing protrusions (Zhang, 2011).
The findings of this study uncover unexpected roles for the Drosophila katanin p60 Dm-Kat60 in the regulation of cortical microtubule dynamics and provide insights into how the microtubule cytoskeleton affects cell migration. The ability of cells to move and change shape is central to organismal development. Defects in these processes have been linked to human diseases such as cancer. Thus, the finding that human KATNA1 has many of the same functions as Dm-Kat60 suggests the former as a potentially useful therapeutic target (Zhang, 2011).
Klp10A is a kinesin-13 of Drosophila melanogaster that depolymerizes cytoplasmic microtubules. In interphase, it promotes microtubule catastrophe; in mitosis, it contributes to anaphase chromosome movement by enabling tubulin flux. This study shows that Klp10A also acts as a microtubule depolymerase on centriolar microtubules to regulate centriole length. Thus, in both cultured cell lines and the testes, absence of Klp10A leads to longer centrioles that show incomplete 9-fold symmetry at their ends. These structures and associated pericentriolar material undergo fragmentation. It was also show that in contrast to mammalian cells where depletion of CP110 leads to centriole elongation, in Drosophila cells it results in centriole length diminution that is overcome by codepletion of Klp10A to give longer centrioles than usual. How loss of centriole capping by CP110 might have different consequences for centriole length in mammalian and insect cells is discussed, and also these findings are related to the functional interactions between mammalian CP110 and another kinesin-13, Kif24, that in mammalian cells regulates cilium formation (Delgehyr, 2012).
To study Klp10A's roles at the spindle poles, flies with reduced Klp10A expression were examined that showed male sterility resulting from a P element insertion. Meiotic cells in Klp10A testes had supernumerary asters and abnormal, frequently multipolar meiotic spindles. Consistently, 79% of elongating spermatid bundles contained fewer than the normal 64 nuclei, and flagella were immotile and shorter than in control flies. Electron microscopy revealed that elongating spermatids had missing or incomplete axonemes. Moreover, Klp10A adults were uncoordinated and needed increased time to recover from mechanical shock, a hallmark of centriole defects (Delgehyr, 2012).
These findings led to an examination of centrioles in 16-cell cysts of primary spermatocytes in late G2 or in meiosis. Most such cells (>70%) were found to have more than two centrosomes together with additional rod- and dot-like structures that stained with anti-Dplp (Drosophila pericentrin-like protein). Moreover, whereas wild-type spermatocytes had only a few centrioles (2.2%) with no detectable Sas6 (spindle assembly abnormal protein 6) at their distal part, Klp10A spermatocytes showed an increase of such centrioles, suggesting they were incomplete or fragmented. Spd2 (spindle defective 2), which is closely associated with spermatocyte centrioles otherwise lacking pericentriolar material (PCM), also associated with centriolar fragments in Klp10A mutants, often fraying out from their ends. By the end of G2, Klp10A mutant centrioles displayed discontinuities in the outer microtubules of both basal body-like structures and the short primary cilia that they nucleate. Finally, Klp10A was present along the length of wild-type centrioles, being more concentrated near both ends, but was barely detectable in mutant centrioles (Delgehyr, 2012).
To understand how defective centrioles and centrosomes might arise in Klp10A mutant testes, earlier mitotic stages of spermatogenesis were examined. These showed significantly more cells with either more than (>20% of the cells) or fewer than two Dplp (centriole marker) bodies. Not only the number but also the size of Dplp bodies was compromised: whereas in wild-type spermatogonia, Dplp bodies were of uniform size, Klp10A mutants showed a significant increase in both shorter and longer Dplp bodies. Similar findings were obtained using anti-Spd2 labeling. Examination of spermatogonia by electron microscopy confirmed that Klp10A centrioles were both longer and shorter than wild-type; longer centrioles were generally not uniformly elongated, and shorter ones often appeared tenuously attached to them. This excessive elongation did not, however, seem to prevent formation of procentrioles, indicating that centriole duplication could occur. To better correlate the immunofluorescence observations with electron microscopy, structured illumination microscopy was carried out on anti-Spd2-stained spermatogonial cells. This revealed both shorter and longer centrioles 'frayed' at their ends in Klp10A spermatogonia. Apparent segments of centriolar walls were found both associated with the frayed ends or free in the cytoplasm, suggesting that the elongated structures could become fragmented (Delgehyr, 2012).
To test Klp10A's role in somatic centriole biogenesis, RNA interference (RNAi) was used to efficiently deplete the protein from cultured Dmel cells. Previous studies of the functional requirements for Klp10A for spindle microtubules have not examined centrosome behavior per se. After Klp10A RNAi, cells either lacking or having numerous Dplp bodies were detected. The increase in cells without Dplp bodies was however quite modest, around twice the control. Moreover, the proportion of such cells did not increase following several rounds of Klp10A depletion. This was a quite different outcome from that following depletion of Plk4, a core component of the centrosome duplication pathway, where virtually 100% of cells lose their centrosomes. Accordingly, it was found that centrosomes could reform after Plk4 RNAi treatment in either the presence or absence of Klp10A. Under these conditions, depletion of Sas6, which is known to be essential for centriole duplication, would block de novo centriole formation. Together, these results indicate that the centriole duplication pathway is independent of Klp10A function (Delgehyr, 2012).
It was noticed that Klp10A-depleted cells contained both weakly and brightly fluorescing Dplp bodies. Because it was difficult to measure the length of these bodies (centrioles are less than 0.2 μm in these cells), their fluorescence intensity was measured. Dplp is found at both centrioles and PCM, giving a combined indication of PCM size and centriole length. It was found that Klp10A RNAi resulted in a doubling of both very bright and very weak dots. By contrast, cells depleted for the microtubule depolymerases Klp59C or Klp59D (kinesin-13 family) or Klp67A (kinesin-8) did not show an increase in brightly fluorescent Dplp bodies, suggesting that changes in centrosome size are not a general consequence of cytoplasmic microtubule depolymerization (Delgehyr, 2012).
To understand how these different centrosomal bodies might be generated, video microscopy was carried out to follow centrosomes in cells constitutively expressing GFP-Spd2 from a weak promoter. Because centrosome number is notoriously variable in Dmel cells, cells with two GFP-Spd2 punctae were examined at interphase. In control cells, the two punctae became brighter on mitotic entry as centrosomes recruited PCM and separated to form the spindle poles. Centriole disengagement occurred in telophase, and the daughters had two centrosomal punctae. In Klp10A-depleted cells, the two centrosomes coalesced upon mitotic entry with the apparent collapse of the spindle and bipolarity was recovered; the coalesced centrosomes split into several punctae at one pole, whereas there were none at the other. Centrosomes then dispersed into numerous scattered dots. Thus, in the absence of Klp10A, fragmentation in M phase appears to account for the weak Dplp bodies (Delgehyr, 2012).
It is considered that the weak Dplp bodies could arise by PCM dispersion or centriole fragmentation per se. The former possibility was addressed by staining to reveal Spd2, which makes a greater contribution to PCM, in addition to Dplp. Klp10A depletion led to an increase in small punctae positive for Spd2 but not Dplp, consistent with some PCM fragmentation. In the absence of good antibodies to label centrioles, the possibility of centriole fragmentation was addressed by applying structured illumination immunofluorescence microscopy and electron microscopy to examine centrioles in control and Klp10A-depleted cells. Structured illumination microscopy resolved the punctae staining in control cells into circular structures of ∼0.4 μm outer diameter that, when rotated through 90°, revealed the rod-like structures of centrioles. In Klp10A-depleted cells, these rods could be the same length or longer than in control cells. What appeared to be segments of the centriolar wall was also observed, suggesting that centriole fragmentation could also contribute to the weak Dplp bodies seen by conventional immunofluorescence (Delgehyr, 2012).
Electron microscopy revealed an increase in centrioles longer than 0.18 μm from 15.4% in control cells to 72.1% in Klp10A-depleted cells and a less pronounced increase in centrioles shorter than 0.15 μm (from 12.8% to 17.6%). Moreover, 10.5% of centrioles in Klp10A-depleted cells had an incomplete complement of microtubules. It is possible that the small centrioles represent intermediates in centriole duplication. If so, the relatively small increase in their number suggests that centriole duplication cannot account for the almost doubling of weak Dplp bodies after Klp10A RNAi. Together, these results therefore suggest that Klp10A depletion leads to centriole elongation and fragmentation associated with dispersion of the PCM (Delgehyr, 2012).
Centrosome separation during mitosis is mainly asymmetric in Klp10A-depleted cells: one cell inherits both centrosomes, and the other inherits none. Because the latter cell might be expected to engage in de novo centriole formation, it was wondered whether formation of longer centrioles following Klp10A depletion could be a consequence of this. Therefore centriole reformation was examined after inducing their loss by extensive Plk4 depletion, and it was found that such centrioles were no longer than in control cells. This accords with the normal morphology of centrioles formed de novo in unfertilized eggs overexpressing Plk4. Thus, the long fragmented centrioles seen after Klp10A depletion are unlikely to be a consequence of de novo centriole formation (Delgehyr, 2012).
It was previously found that injection of anti-polyglutamylated tubulin antibody into HeLa cells in G2 resulted in centriole loss and scattering of PCM in a dynein-dependent manner. This has been widely taken to indicate that polyglutamylation might stabilize centrosomes to forces exerted upon them in mitosis. Unlike in mammalian cells, tubulin is not polyglutamylated in most Drosophila tissues, and accordingly this modification could not be detected in Drosophila centrosome preparations or on centrioles of primary spermatocytes or Dmel cells. It was therefore wondered whether centrosomes might scatter following Klp10A downregulation in a dynein-dependent process. To test this, it was decided to partially release tension on centrosomes by depleting dynein (Dhc64, dynein heavy chain 64) in the presence or absence of Klp10A. Because dynein depletion leads to inequitable heritance of centrosomes, their detachment from spindle poles, and cell death, around 50% of the protein was depleted. This did not lead to defects in centrosome number or staining intensity. When Klp10A was extensively depleted and dynein was partially depleted, numbers of weak Dplp bodies decreased at the expense of an increase in bright Dplp bodies as compared to cells depleted for Klp10A alone. Thus, partial depletion of dynein apparently rescues centrosome fragmentation resulting from Klp10A depletion. Electron microscopy revealed that codepletion of Klp10A and dynein resulted in long centrioles, some exceeding the size observed after Klp10A depletion alone. Although the possibility cannot be completely excluded that dynein depletion directly affects the centriole per se, it is more likely that dynein depeletion results in reduced forces upon the centriole, thus protecting the long centrioles of Klp10A-depleted cells from fragmentation (Delgehyr, 2012).
To determine whether Klp10A's microtubule-depolymerizing properties are needed for its centrosomal functions, mutations were generated in two conserved class-specific motifs of its motor domain shown to be essential for microtubule depolymerization in other organisms. These mutations changed residues KVD (aa 317–319) or KEC (aa 546–548) of Klp10A each to three alanines. In accord with the known properties of Klp10A, it was found that high levels of wild-type protein depolymerized interphase microtubules, whereas cells expressing similar levels of the mutant forms had interphase microtubules of normal length. Thus, both mutations abolish Klp10A's microtubule depolymerization activity. Lines expressing either wild-type or mutant forms of GFP-tagged Klp10A were also generated from the endogenous Klp10A promoter that had the 5′ but not the 3′ untranslated region (UTR) of Klp10A. Both wild-type and mutant forms were expressed at levels comparable to the endogenous protein and were associated with microtubules and centrosomes or spindle poles in interphase and mitosis. Expression of the tagged wild-type protein at these low levels did not result in depolymerization of cytoplasmic microtubules. Then endogenous Klp10A was depleted using RNAi directed at the 3′ UTR and the effects upon centrosomes were assessed. This led to an increase in cells either without centrosomes or with both weak and bright Dplp bodies in RNAi-treated cells expressing GFP (control) or GFP-tagged mutant forms of Klp10A, but not in cells expressing GFP-tagged wild-type protein. Thus, generation of both larger centrosomes and centrosome fragments requires loss of the microtubule-depolymerizing activity of Klp10A (Delgehyr, 2012).
Because human CP110 or Centrobin (required for CP110's centriolar localization) results in centriole elongation, it was intriguing to find Drosophila CP110 enriched at the distal ends of the centrioles of primary spermatocytes in a region similar to Klp10A. Moreover, CP110 colocalized with Klp10A in interphase Dmel cells, and other evidence suggested that CP110 and Klp10A can physically interact. It was found that several rounds of CP110 depletion led to an increase in Dmel cells lacking centrosomes, CP110 depletion affects centrosome biogenesis. However, centrosomes could reform after their elimination by Plk4 RNAi treatment, even in the absence of CP110, although this was prevented by further depletion of Sas6, required for bona fide centriolar duplicatio. Thus, there is no absolute requirement for CP110 for centriole duplication. Interestingly, however, there was an increase in weakly fluorescing Dplp bodies in CP110-depleted cells, and electron microscopy revealed that 50% of centrioles were shorter than 0.15 μm, compared to 12.8% in control cells, with most being about 0.11 μm long. This is unlikely to represent an increase in procentrioles, because total centriole number decreased. Centrioles of about 0.11 μm are similar in length to the unit cartwheel made mainly of three or four tiers in Chlamydomonas reinhardtii and may represent the smallest stable centriole structures. Thus, it is concluded that, in contrast to depletion of CP110 in mammalian cells , depletion of CP110 in Drosophila leads to centriole shortening and destabilization (Delgehyr, 2012).
It was then asked whether centriole shortening following CP110 depletion might be rescued by sequentially codepleting Klp10A. It was found that this resulted in the reappearance of long centrioles: 62.5% of the centrioles exceeded 0.18 μm, compared to 15.4% in control cells. When Klp10A was depleted first, followed by codepletion with CP110, the reappearance of longer centrioles was observed. Thus, in the absence of Klp10A, centrioles increase in length regardless of whether CP110 is present or absent. The destabilization of Dplp bodies after CP110 depletion appeared to be enhanced by overexpression of GFP-Klp10A that led to an increased proportion of cells lacking centrosomes after 3 days of CP110 RNAi. Together, these results suggest that CP110 might provide a barrier to prevent Klp10A-mediated depolymerization of centriolar microtubules, and indeed, it forms a plug-like structure at the distal part of the centriole. However, CP110 has no effect on microtubule elongation in the absence of Klp10A. Thus, Klp10A can restrict centriole length regardless of whether or not CP110 is present, and so their physical interaction is not required for Klp10A's recruitment and/or microtubule-depolymerizing activity. Indeed Klp10A may be recruited directly on the centrioles, because it is known to have affinity for the microtubule lattice in vitro (Delgehyr, 2012).
Klp10A is the first kinesin-13 demonstrated to regulate centriole length. Other family members have, however, been shown to regulate the length of flagella and more recently formation of cilia. In contrast to Klp10A, however, the mammalian kinesin-13 Kif24 is further limited to acting from the mother centriole on microtubules of cilia. Thus, Kif24 depletion leads to aberrant cilia formation in cycling cells but does not promote growth of centrioles in nonciliated cells. Although Drosophila Klp10A and mammalian Kif24 act differently, both are able to interact with CP110. However, the interaction of CP110 and Kif24 in mammalian cells affects only the process of cilium formation. These functional differences between kinesin-13:CP110 complexes could reflect a regulative adaptation by mammals associated with evolution of the ability to generate primary cilia in many cell types. Thus, whereas in Drosophila, where few cells are ciliated, the capping function of CP110 might help to fix the length of the centriole by blocking Klp10A-mediated microtubule depolymerization, in mammals it has the additional function of blocking cilium formation until the correct phase of the cell cycle. Whether in mammals another kinesin-13 might play the role of Klp10A in regulating centriole length remains an open question (Delgehyr, 2012).
During cell division, a bipolar array of microtubules forms the spindle through which the forces required for chromosome segregation are transmitted. Interestingly, the spindle as a whole is stable enough to support these forces even though it is composed of dynamic microtubules, which are constantly undergoing periods of growth and shrinkage. Indeed, the regulation of microtubule dynamics is essential to the integrity and function of the spindle. A member of an important class of microtubule-depolymerizing kinesins, KLP10A, is required for the proper organization of the acentrosomal meiotic spindle in Drosophila oocytes. In the absence of KLP10A, microtubule length is not controlled, resulting in extraordinarily long and disorganized spindles. In addition, the interactions between chromosomes and spindle microtubules are disturbed and can result in the loss of contact. These results indicate that the regulation of microtubule dynamics through KLP10A plays a critical role in restricting the length and maintaining bipolarity of the acentrosomal meiotic spindle and in promoting the contacts that the chromosomes make with microtubules required for meiosis I segregation (Radford, 2012).
During cell division, a stable bipolar spindle is crucial for the accurate distribution of genetic material to daughter cells. How the stability of this structure is achieved when the spindle is composed of dynamically unstable microtubules is an important question. This study has shown that KLP10A, a member of the kinesin-13 family of microtubule-depolymerizing proteins, is essential to the organization of the acentrosomal meiotic spindle in Drosophila oocytes. An interesting feature of acentrosomal meiosis is that microtubule ends appear to be distributed throughout the spindle. This has implications for the regulation of microtubule dynamics by kinesin-13 family members, which have been shown in vitro to act at the ends of microtubules to induce depolymerization. KLP10A localizes throughout the meiotic spindle at metaphase I. This distribution may reflect the binding of KLP10A along the entire length of microtubules. Kinesin-13s are known to bind along the length of microtubules in vitro, diffusing to the ends before becoming active. This seems unlikely, however, given the propensity for kinesin-13s in general, and KLP10A specifically, to localize to the regions of the mitotic spindle with the highest concentrations of microtubule ends -- spindle poles and kinetochores (Radford, 2012).
Instead, it is suggested that KLP10A localizes to microtubule ends that are present throughout the spindle, which implies that microtubule depolymerization occurring throughout the meiotic spindle may be a normal part of spindle assembly and stability. Indeed, Domnitz (2012) has shown that MCAK activity at the tips of nonkinetochore microtubules regulates mitotic spindle length. Alternatively, KLP10A may be present at ends throughout the spindle, but maintained in an inactive state in most locations. If the microtubule end to which KLP10A is bound is near a spindle location where depolymerization is needed, such as near the chromosomes or poles, then KLP10A may become active. There is a large body of evidence that the activity and localization of kinesin-13s are regulated during mitotic cell division by phosphorylation and protein interactions (reviewed in Ems-McClung, 2010), but whether these mechanisms are active during meiotic cell division remains to be examined (Radford, 2012).
Surprisingly, this study found no evidence for enrichment of KLP10A at centromeres as observed in a previous study in S2 cells. It should be noted that the interpretation of centromere localization in this previous study was not confirmed with a centromere marker. The finding that its localization is enriched towards the poles is consistent with the conclusion that KLP10A is required for poleward flux, which depends on microtubule depolymerization at the poles. Thus, while it cannot be ruled out that KLP10A localizes to the centromeres during female meiosis, there is no conclusive evidence for it (Radford, 2012).
The results show that loss of KLP10A dramatically impacts spindle organization. The spindles assembled in the absence of KLP10A present widely varying organizational arrangements, which may result from an imbalance in the dynamic nature of microtubules during spindle assembly and maintenance. In wild-type Drosophila oocytes, a bipolar spindle assembles, and its organization and length is stably maintained in metaphase I for extended periods of time. In contrast, in mutants that affect spindle organization, the spindle can dramatically change shape over the course of live imaging. It is proposed that the deregulation of microtubule dynamics in Klp10A germline mutants results in the formation of unstable meiotic spindles because of the loss of the ability to shorten microtubules. This implies that the regulation of microtubule dynamics by KLP10A is required to maintain a stable bipolar spindle in Drosophila oocytes (Radford, 2012).
The central spindle comprises a band of antiparallel microtubules that extends across the chromosomes to connect the two half spindles. While many spindles from Klp10A germline mutants are severely disorganized, even in some cases of only mild spindle disorganization, the central spindle is missing. This suggests that the integrity of the central spindle depends on the regulation of microtubule dynamics. Several proteins including the chromosomal passenger complex (CPC) and Subito localize to the central spindle and are required for meiotic spindle assembly and bipolarity, respectively. Thus, the central spindle is important for organizing the meiotic spindle, and the instability of this structure in the absence of KLP10A may contribute to the spindle organization defects. Loss of the central spindle cannot explain all of the spindle defects, however, because the central spindle is absent in subito mutants, but this results primarily in monopolar and tripolar spindles with no effect on spindle length (Radford, 2012).
Interestingly, the spindle defects observed in Klp10A germline mutants differ from previous kinesin-13 loss-of-function studies. Knockdown of kinesin-13 homologs in human cells, Xenopus laevis egg extracts, and Drosophila S2 cells primarily results in monopolar spindles, chromosome congression and segregation defects, and long astral microtubules. RNAi knockdown of KLP10A in Drosophila S2 cells does result in an increase in spindle length; however, the magnitude of the effect is modest in comparison to the effect on microtubule length that was observed in Klp10A germline mutants. One obvious explanation for the different effects is the different organization of the centrosomal mitotic and acentrosomal meiotic spindles. The need for KLP10A to maintain spindle length in mitotic spindles may be tempered by the presence of centrosomes and astral microtubules, whereas in acentrosomal spindles, the determination of spindle length is dominated by a balance between microtubule depolymerization by KLP10A and spindle elongation by a mechanism that is not yet known. At this point, however, it is possible that there are other differences between the mitotic and oocyte spindles that make acentrosomal spindle length hypersensitive to loss of KLP10A. Whether KLP10A plays an additional role in spindle organization or whether the spindle disorganization in Klp10A germline mutants results from overgrowth of microtubules also remains to be determined (Radford, 2012).
The loss of KLP10A also impacts the interaction of the spindle with the chromosomes. KLP10A could be required to regulate interactions between microtubules and chromosomes because kinesin-13s have been shown to play an important role in correcting improper kinetochore-microtubule attachments in mitosis. In Drosophila female meiosis, however, KLP10A appears to have the opposite effect, promoting or maintaining contact between chromosomes and spindle poles, suggesting that the mechanism by which KLP10A regulates chromosome-microtubule attachments differs in mitotic vs. acentrosomal meiotic spindles. Interestingly, Domnitz (2012) argues that MCAK depolymerizing activity at the tips of microtubules actually promotes robust kinetochore attachments. It is also possible, however, that this function could be indirect, through the maintenance of microtubule length and spindle organization. Importantly, however, these results demonstrate that the microtubule-depolymerizing kinesin KLP10A is essential for the establishment of an acentrosomal meiotic spindle with the capacity to properly segregate homologous chromosomes (Radford, 2012).
During asymmetric division, fate determinants at the cell cortex segregate unequally into the two daughter cells. It has recently been shown that Sara (Smad anchor for receptor activation) signalling endosomes in the cytoplasm also segregate asymmetrically during asymmetric division. Biased dispatch of Sara endosomes mediates asymmetric Notch/Delta signalling during the asymmetric division of sensory organ precursors in Drosophila. In flies, this has been generalized to stem cells in the gut and the central nervous system, and, in zebrafish, to neural precursors of the spinal cord. However, the mechanism of asymmetric endosome segregation is not understood. This study shows that the plus-end kinesin motor Klp98A targets Sara endosomes to the central spindle, where they move bidirectionally on an antiparallel array of microtubules. The microtubule depolymerizing kinesin Klp10A and its antagonist Patronin generate central spindle asymmetry. This asymmetric spindle, in turn, polarizes endosome motility, ultimately causing asymmetric endosome dispatch into one daughter cell. This mechanism was demonstrated by inverting the polarity of the central spindle by polar targeting of Patronin using nanobodies (single-domain antibodies). This spindle inversion targets the endosomes to the wrong cell. These data uncover the molecular and physical mechanism by which organelles localized away from the cellular cortex can be dispatched asymmetrically during asymmetric division (Derivery, 2015).
Klp98A was first identified as the kinesin mediating Sara endosome motility during sensory organ precursor (SOP) division. Klp98A is the Drosophila homologue of mammalian KIF16B, an early endosomal kinesin containing a phosphatidylinositol 3-phosphate-binding PX domain. Indeed, Klp98A localizes to Sara-positive early endosomes (Derivery, 2015).
During SOP division, Klp98A-GFP-positive Sara endosomes segregate to the pIIa daughter, but not the pII. Sara endosomes were monitored by following Delta 20 min after internalization (iDelta20) through an improved antibody internalization assay. iDelta20 parallels Sara endosome dynamics in the controls and mutants studied here (in vivo and primary cultures. Like KIF16B, purified Klp98A binds specifically to phosphatidylinositol 3-phosphate and is a plus-end-directed motor whose velocity is 0.76 ± 0.02 μm s-1 (Derivery, 2015).
To study Klp98A function, deletions within the motor domain (Klp98AΔ6, Klp98AΔ7 and Klp98AΔ8, 6, 7 and 8-base-pair deletions, respectively) and a clean coding sequence deletion (Klp98AΔ47). Except Klp98AΔ6, all are protein nulls. In Klp98A-, Sara endosomes move diffusively. Therefore, Klp98A mediates Sara endosome motility (Derivery, 2015).
In wild-type cells, Sara endosomes move on microtubules to the Pavarotti-positive central spindle and, late in cytokinesis, to pIIa. Spindle microtubule plus-ends are oriented towards the equator, explaining central spindle endosomal targeting by a plus-end motor. Indeed, Sara endosome central spindle targeting fails in Klp98A- mutants. Importantly, in Klp98A- mutants and upon RNAi-mediated Klp98A knockdown, endosomes are symmetrically dispatched (Derivery, 2015).
Klp98A-mediated motility contributes to cell fate assignation through asymmetric Notch signalling, but this activity is redundantly covered by Neuralized and Numb. Indeed, Klp98A-;pnr > neurRNAidouble mutants show a synergistic fate assignation phenotype: the notum is largely void of bristles. Conversely, Klp98A;Numb double mutants strongly suppress the diagnostic Numb- multiple socket phenotype. Therefore, having established the role of Klp98A motility in Notch signalling, this study focused on the mechanisms orchestrating asymmetric motility (Derivery, 2015).
Central spindle targeting of Sara endosomes precedes asymmetric segregation to pIIa. Focus was therefore placed on Sara endosome motility with respect to the central spindle reference frame. The central spindle is composed of the Pavarotti-positive core (containing antiparallel microtubules) plus the microtubules emanating from it. The Pavarotti core was automatically tracked, defining a 2D cartesian reference frame whose origin is the Pavarotti centroid and whose x axis is the pIIb-pIIa axis. This also defines a Pavarotti width (PW) and length (PL, the length of the microtubule antiparallel array (Derivery, 2015).
Sara endosomes were tracked with respect to this reference frame (with 160 nm accuracy. Automatic tracking and spatio-temporal registration provided a large data set (2,897 traces) from which a spatio-temporal density plot of endosomes at the central spindle was generated. For 500 s, endosomes remain mostly within the Pavarotti region. Remarkably, at the central spindle, motility along the x axis is bidirectional. Motility along the y axis merely follows PW contraction, consistent with motility along central spindle microtubules, parallel to the x axis. Velocities are similar towards pIIa and pIIb and slower than in vitro, possibly due to crowding by microtubule-associated proteins (Derivery, 2015).
Confinement within the Pavarotti region and bidirectional movement are both consistent with a plus-end motor switching direction on antiparallel microtubules. On single microtubules, Klp98A-bound quantum dots always maintain their directionality when resuming after a pause. It was asked whether Klp98A could switch direction in an antiparallel bundle. In an in vitro reconstitution assay, Klp98A-bound quantum dots move bidirectionally within antiparallel MAP65-1-mediated microtubule arrays. 68% tracks change direction after pausing. Therefore, Klp98A supports bidirectional motility in antiparallel array (Derivery, 2015).
Notably, in vivo, bidirectional endosome motility is asymmetric: the residence time in pIIa is 1.8-fold longer than in pIIb. Consistently, the spatio-temporal density plot is asymmetric. Furthermore, tracks overshoot beyond the Pavarotti region more frequently into pIIa (Derivery, 2015).
Eventually, endosomes depart from central spindle microtubules into the cytoplasm and therefore move also on the y axis. The longer pIIa residence time and higher pIIa overshoot frequency make this final departure asymmetric, explaining the biased segregation into pIIa. Therefore, asymmetric endosome motility at the central spindle underlies asymmetric dispatch to pIIa (Derivery, 2015).
It was then asked whether the central spindle itself is asymmetric. Using Pavarotti spatio-temporal registration, an 'average cell' was generated to map the densities of the microtubule markers Jupiter and SiR-tubulin (microtubule markers), Patronin (minus-end), and Pavarotti (plus-ends/antiparallel overlap). This 'average cell' reveals a polarity map of the central spindle consistent with electron microscopy reports: plus-ends are in the middle and minus-ends on the outer side. Microtubule densities in general, and Patronin in particular, are ~20% higher on the pIIb side. This asymmetry depends on Par complex activity, and is absent in neighbouring cells dividing symmetrically, but this seems independent of central spindle or endosomal asymmetry (Derivery, 2015).
Microtubule asymmetry builds up during anaphaseB, concomitant with biased endosome motility, while, earlier, the metaphase spindle is symmetrical. During anaphaseB, the central spindle shrinks by microtubule depolymerization through depolymerizing kinesins like Klp10A, among other factors. Depolymerization dynamics are asymmetric: microtubule loss is faster in pIIa. This could be explained by Patronin enrichment in the central spindle pIIb outer side where it binds to minus-ends, counteracting Klp10A-mediated depolymerization (Goodwin, 2010; Wang, 2013; Hendershott, 2014; Jiang, 2014; Derivery, 2015 and references therein).
Indeed, Klp10A/Patronin control asymmetric microtubule depolymerization: their depletion abolishes spindle asymmetry. In Patronin-knockdown cells, both sides exhibit low microtubule densities characteristic of pIIa, consistent with Patronin pIIb enrichment in wild type and its activity against depolymerization. Conversely, upon knockdown of Klp10A, both sides exhibit high microtubule densities resembling pIIb (Derivery, 2015).
The parallelism between central spindle asymmetry and asymmetric endosome motility suggests that spindle asymmetry causes biased motility. Indeed, endosome motility at the central spindle and, therefore, segregation become symmetric in Klp10A- and Patronin-knockdowns, while early central spindle targeting is normal. This uncovers a quantitative correlation between spindle and endosomal asymmetry (Derivery, 2015).
Together, a plus-end motor and microtubule plus-ends facing the centre explain why a higher pIIb microtubule density (~20% enrichment) targets endosomes to pIIa (~80% pIIa, that is, >300% enrichment). In other words, endosomes move away from higher microtubule densities in pIIb (Derivery, 2015).
Based on a theoretical model of plus-end endosomal motility on an antiparallel, asymmetric microtubule overlap, the steady-state endosome distribution is
Where PpIIa, PpIIb, the probabilities for an endosome to be on either side of the antiparallel overlap; ρa, ρb, microtubule densities in pIIa/pIIb, respectively; kon, koff, microtubule association/dissociation constants of the motor, respectively; v, the endosome motor-driven velocity; D, the diffusion coefficient of endosomes detached from microtubules; and l, the antiparallel overlap length (Derivery, 2015).
To generate this inverted spindle, 'nanobody assay' was established based on GFP-binding-peptide (GBP)-Pon, a nanobody fused to the Pon localization domain. GBP-Pon traps GFP-Patronin away from the spindle at the pIIb cortex thereby reducing, specifically in pIIb, Patronin-dependent protection against central spindle depolymerization. This inverts spindle asymmetry, which consequently inverts endosomal asymmetry. SiR-Tubulin and endogenous acetyl-tubulin stainings confirmed this spindle inversion (Derivery, 2015).
Interestingly, this assay generates a phenotypic series of different levels of spindle reversal and their corresponding endosomal reversals. These data fall on the theoretical curve obtained with independently measured parameters. Therefore these results uncover the quantitative dependence of asymmetric endosome targeting on spindle asymmetry (Derivery, 2015).
This study has identified Klp10A/Patronin as the machinery generating spindle asymmetry, which is read out by Klp98A to achieve asymmetric targeting of signalling endosomes. Asymmetric endosomal targeting contributes in turn to asymmetric cell fate assignation, confirming previous reports in flies and fish. These data thus uncover a mechanism by which intracellular cargoes in general, and signalling endosomes in particular, can be targeted to one of the daughter cells during asymmetric division (Derivery, 2015).
How could then other cargoes segregate symmetrically, if the spindle is asymmetric? Asymmetric targeting would only be efficient if kon, koff and v are optimized to amplify the mild asymmetry of the spindle, otherwise concealed by noise sources in the cell. More generally, plus- and minus-end motors are present simultaneously in the same vesicle and thereby may counteract each other to achieve symmetrical dispatch (a sort of 'tug of war'). Therefore, the precise landscape of microtubule polarity trails combined with the right cocktail of motors in vesicles provides the plasticity required to generate the plethora of molecular spatial patterns observed in polarized cells (Derivery, 2015).
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. PubMed ID: 15522967
Delgehyr, N., et al. (2012). Klp10A, a microtubule-depolymerizing kinesin-13, cooperates with CP110 to control Drosophila centriole length. Curr Biol. 22(6): 502-9. PubMed ID: 22365849
Derivery, E., Seum, C., Daeden, A., Loubery, S., Holtzer, L., Julicher, F. and Gonzalez-Gaitan, M. (2015). Polarized endosome dynamics by spindle asymmetry during asymmetric cell division. Nature 528: 280-285. PubMed ID: 26659188
Desai, A., Verma, S., Mitchison, T. J. and Walczak, C. E. (1999). KinI kinesins are microtubule-destabilizing enzymes. Cell 96: 69-78. PubMed ID: 9989498
Domnitz, S. B., Wagenbach, M., Decarreau, J. and Wordeman, L. (2012). MCAK activity at microtubule tips regulates spindle microtubule length to promote robust kinetochore attachment. J Cell Biol 197: 231-237. PubMed ID:22492725
Ems-McClung, S. C. and Walczak, C. E. (2010). Kinesin-13s in mitosis: Key players in the spatial and temporal organization of spindle microtubules. Semin Cell Dev Biol 21: 276-282. PubMed ID:20109574
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. PubMed ID: 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. PubMed ID: 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. PubMed ID: 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. PubMed ID: 15958489
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date revised: 22 December 2018
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