Three microtubule severing enzymes contribute to the "Pacman-flux" machinery that moves chromosomes

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). We report that 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 gamma-tubulin, consistent with the hypothesis that they exert their influence by releasing stabilizing gamma-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 our understanding of the molecular machinery used to move chromosomes.     

Plus-end motors override minus-end motors during transport of squid axon vesicles on microtubules

Plus- and minus-end vesicle populations from squid axoplasm were isolated from each other by selective extraction of the minus-end vesicle motor followed by 5'-adenylyl imidodiphosphate (AMP-PNP)- induced microtubule affinity purification of the plus-end vesicles. In the presence of cytosol containing both plus- and minus-end motors, the isolated populations moved strictly in opposite directions along microtubules in vitro. Remarkably, when treated with trypsin before incubation with cytosol, purified plus-end vesicles moved exclusively to microtubule minus ends instead of moving in the normal plus-end direction. This reversal in the direction of movement of trypsinized plus-end vesicles, in light of further observation that cytosol promotes primarily minus-end movement of liposomes, suggests that the machinery for cytoplasmic dynein-driven, minus-end vesicle movement can establish a functional interaction with the lipid bilayers of both vesicle populations. The additional finding that kinesin overrides cytoplasmic dynein when both are bound to bead surfaces indicates that the direction of vesicle movement could be regulated simply by the presence or absence of a tightly bound, plus-end kinesin motor; being processive and tightly bound, the kinesin motor would override the activity of cytoplasmic dynein because the latter is weakly bound to vesicles and less processive. In support of this model, it was found that (a) only plus-end vesicles copurified with tightly bound kinesin motors; and (b) both plus- and minus-end vesicles bound cytoplasmic dynein from cytosol.     

A microtubule-destabilizing kinesin motor regulates spindle length and anchoring in oocytes

The kinesin-13 motor, KLP10A, destabilizes microtubules at their minus ends in mitosis and binds to polymerizing plus ends in interphase, regulating spindle and microtubule dynamics. Little is known about kinesin-13 motors in meiosis. In this study, we report that KLP10A localizes to the unusual pole bodies of anastral Drosophila melanogaster oocyte meiosis I spindles as well as spindle fibers, centromeres, and cortical microtubules. We frequently observe the pole bodies attached to cortical microtubules, indicating that KLP10A could mediate spindle anchoring to the cortex via cortical microtubules. Oocytes treated with drugs that suppress microtubule dynamics exhibit spindles that are reoriented more vertically to the cortex than untreated controls. A dominant-negative klp10A mutant shows both reoriented and shorter oocyte spindles, implying that, unexpectedly, KLP10A may stabilize rather than destabilize microtubules, regulating spindle length and positioning the oocyte spindle. By altering microtubule dynamics, KLP10A could promote spindle reorientation upon oocyte activation.     

Microtubule plus-end dynamics in Xenopus egg extract spindles

Microtubule dynamics underlie spindle assembly, yet we do not know how the spindle environment affects these dynamics. We developed methods for measuring two key parameters of microtubule plus-end dynamic instability in Xenopus egg extract spindles. To measure plus-end polymerization rates and localize growing plus ends, we used fluorescence confocal imaging of EB1. This revealed plus-end polymerization throughout the spindle at approximately 11 microm/min, similar to astral microtubules, suggesting polymerization velocity is not regionally regulated by the spindle. The ratio of EB1 to microtubule fluorescence revealed an enrichment of polymerizing ends near the spindle middle, indicating enhanced nucleation or rescue there. We measured depolymerization rates by creating a front of synchronized depolymerization in spindles severed with microneedles. This front could be tracked by polarization and fluorescence microscopy as it advanced from each cut edge toward the associated pole. Both imaging modalities revealed rapid depolymerization ( approximately 30 microm/min) superimposed on a subset of microtubules stable to depolymerization. Larger spindle fragments contained a higher percentage of stable microtubules, which we believe were oriented with their minus ends facing the cut. Depolymerization was blocked by the potent microtubule stabilizing agent hexylene glycol, but was unaffected by alpha-MCAK antibody and AMPPNP, which block catastrophe and kinesin motility, respectively. These measurements move us closer to understanding the complete life history of a spindle microtubule.     

Posterior localization of dynein and dorsal-ventral axis formation depend on kinesin in Drosophila oocytes

To establish the major body axes, late Drosophila oocytes localize determinants to discrete cortical positions: bicoid mRNA to the anterior cortex, oskar mRNA to the posterior cortex, and gurken mRNA to the margin of the anterior cortex adjacent to the oocyte nucleus (the "anterodorsal corner"). These localizations depend on microtubules that are thought to be organized such that plus end-directed motors can move cargoes, like oskar, away from the anterior/lateral surfaces and hence toward the posterior pole. Likewise, minus end-directed motors may move cargoes toward anterior destinations. Contradicting this, cytoplasmic dynein, a minus-end motor, accumulates at the posterior. Here, we report that disruption of the plus-end motor kinesin I causes a shift of dynein from posterior to anterior. This provides an explanation for the dynein paradox, suggesting that dynein is moved as a cargo toward the posterior pole by kinesin-generated forces. However, other results present a new transport polarity puzzle. Disruption of kinesin I causes partial defects in anterior positioning of the nucleus and severe defects in anterodorsal localization of gurken mRNA. Kinesin may generate anterodorsal forces directly, despite the apparent preponderance of minus ends at the anterior cortex. Alternatively, kinesin I may facilitate cytoplasmic dynein-based anterodorsal forces by repositioning dynein toward microtubule plus ends.     

A determinant for directionality of organelle transport in Drosophila embryos

BACKGROUND: Motor-driven transport along microtubules is a primary cellular mechanism for moving and positioning organelles. Many cargoes move bidirectionally by using both minus and plus end-directed motors. How such cargoes undergo controlled net transport is unresolved. RESULTS: Using a combination of genetics, molecular biology, and biophysics, we have identified Halo, a novel regulator of lipid droplet transport in early Drosophila embryos. In embryos lacking Halo, net transport of lipid droplets, but not that of other cargoes, is specifically altered; net transport is minus-end directed at developmental stages when it is normally plus-end directed. This reversal is due to an altered balance of motion at the level of individual organelles; without Halo, travel distances and stall forces are reduced for plus-end and increased for minus-end motion. During development, halo mRNA is highly upregulated just as net plus-end transport is initiated (phase II), and its levels drop precipitously shortly before transport becomes minus-end directed (phase III). Exogenously provided Halo prevents the switch to net minus-end transport in phase III in wild-type embryos and induces net plus-end transport during phase II in halo mutant embryos. This mechanism of regulation is likely to be of general importance because the Drosophila genome encodes a family of related proteins with similar sequences, each transiently expressed in distinct domains. CONCLUSIONS: We conclude that Halo acts as a directionality determinant for embryonic droplet transport and is the first member of a new class of transport regulators.     

Cytoplasmic dynein is required for poleward chromosome movement during mitosis in Drosophila embryos

The movement of chromosomes during mitosis occurs on a bipolar, microtubule-based protein machine, the mitotic spindle. It has long been proposed that poleward chromosome movements that occur during prometaphase and anaphase A are driven by the microtubule motor cytoplasmic dynein, which binds to kinetochores and transports them toward the minus ends of spindle microtubules. Here we evaluate this hypothesis using time-lapse confocal microscopy to visualize, in real time, kinetochore and chromatid movements in living Drosophila embryos in the presence and absence of specific inhibitors of cytoplasmic dynein. Our results show that dynein inhibitors disrupt the alignment of kinetochores on the metaphase spindle equator and also interfere with kinetochore- and chromatid-to-pole movements during anaphase A. Thus, dynein is essential for poleward chromosome motility throughout mitosis in Drosophila embryos.     

A homotetrameric kinesin-5, KLP61F, bundles microtubules and antagonizes Ncd in motility assays

BACKGROUND: Mitosis depends upon the cooperative action of multiple microtubule (MT)-based motors. Among these, a kinesin-5, KLP61F, and the kinesin-14, Ncd, are proposed to generate antagonistic-sliding forces that control the spacing of the spindle poles. We tested whether purified KLP61F homotetramers and Ncd homodimers can generate a force balance capable of maintaining a constant spindle length in Drosophila embryos. RESULTS: Using fluorescence microscopy and cryo-EM, we observed that purified full-length, motorless, and tailless KLP61F tetramers (containing a tetramerization domain) and Ncd dimers can all cross-link MTs into bundles in MgATP. In multiple-motor motility assays, KLP61F and Ncd drive plus-end and minus-end MT sliding at 0.04 and 0.1 microm/s, respectively, but the motility of either motor is decreased by increasing the mole fraction of the other. At the "balance point," the mean velocity was zero and MTs paused briefly and then oscillated, taking approximately 0.3 microm excursions at approximately 0.02 microm/s toward the MT plus end and then the minus end. CONCLUSIONS: The results, combined with quantitative analysis, suggest that these motors could act as mutual brakes to modulate the rate of pole-pole separation and could maintain a prometaphase spindle displaying small fluctuations in its steady-state length.     

Antagonistic microtubule-sliding motors position mitotic centrosomes in Drosophila early embryos

The positioning of centrosomes, or microtubule-organizing centres, within cells plays a critical part in animal development. Here we show that, in Drosophila embryos undergoing mitosis, the positioning of centrosomes within bipolar spindles and between daughter nuclei is determined by a balance of opposing forces generated by a bipolar kinesin motor, KLP61F, that is directed to microtubule plus ends, and a carboxy-terminal kinesin motor, Ncd, that is directed towards microtubule minus ends. This activity maintains the spacing between separated centrosomes during prometaphase and metaphase, and repositions centrosomes and daughter nuclei during late anaphase and telophase. Surprisingly, we do not observe a function for KLP61F in the initial separation of centrosomes during prophase. Our data indicate that KLP61F and Ncd may function by crosslinking and sliding antiparallel spindle microtubules in relation to one another, allowing KLP61F to push centrosomes apart and Ncd to pull them together.     

Coordination of opposite-polarity microtubule motors

Many cargoes move bidirectionally, frequently reversing course between plus- and minus-end microtubule travel. For such cargoes, the extent and importance of interactions between the opposite-polarity motors is unknown. In this paper we test whether opposite-polarity motors on lipid droplets in Drosophila embryos are coordinated and avoid interfering with each other's activity, or whether they engage in a tug of war. To this end we impaired the minus-end transport machinery using dynein and dynactin mutations, and then investigated whether plus-end motion was improved or disrupted. We observe a surprisingly severe impairment of plus-end motion due to these alterations of minus-end motor activity. These observations are consistent with a coordination hypothesis, but cannot be easily explained with a tug of war model. Our measurements indicate that dynactin plays a crucial role in the coordination of plus- and minus-end-directed motors. Specifically, we propose that dynactin enables dynein to participate efficiently in bidirectional transport, increasing its ability to stay "on" during minus-end motion and keeping it "off" during plus-end motion.     

K-fibre minus ends are stabilized by a RanGTP-dependent mechanism essential for functional spindle assembly

Chromosome segregation requires the formation of K-fibres, microtubule bundles that attach sister kinetochores to spindle poles. Most K-fibre microtubules originate around the chromosomes through a non-centrosomal RanGTP-dependent pathway and become oriented with the plus ends attached to the kinetochore and the minus ends focused at the spindle poles. The capture and stabilization of microtubule plus ends at the kinetochore has been extensively studied but very little is known on how their minus-end dynamics are controlled. Here we show that MCRS1 is a RanGTP-regulated factor essential for non-centrosomal microtubule assembly. MCRS1 localizes to the minus ends of chromosomal microtubules and K-fibres, where it protects them from depolymerization. Our data reveal the existence of a mechanism that stabilizes the minus ends of chromosomal microtubules and K-fibres, and is essential for the assembly of a functional bipolar spindle.     

Kinesin-13 regulates flagellar, interphase, and mitotic microtubule dynamics in Giardia intestinalis

Microtubule depolymerization dynamics in the spindle are regulated by kinesin-13, a nonprocessive kinesin motor protein that depolymerizes microtubules at the plus and minus ends. Here we show that a single kinesin-13 homolog regulates flagellar length dynamics, as well as other interphase and mitotic dynamics in Giardia intestinalis, a widespread parasitic diplomonad protist. Both green fluorescent protein-tagged kinesin-13 and EB1 (a plus-end tracking protein) localize to the plus ends of mitotic and interphase microtubules, including a novel localization to the eight flagellar tips, cytoplasmic anterior axonemes, and the median body. The ectopic expression of a kinesin-13 (S280N) rigor mutant construct caused significant elongation of the eight flagella with significant decreases in the median body volume and resulted in mitotic defects. Notably, drugs that disrupt normal interphase and mitotic microtubule dynamics also affected flagellar length in Giardia. Our study extends recent work on interphase and mitotic kinesin-13 functioning in metazoans to include a role in regulating flagellar length dynamics. We suggest that kinesin-13 universally regulates both mitotic and interphase microtubule dynamics in diverse microbial eukaryotes and propose that axonemal microtubules are subject to the same regulation of microtubule dynamics as other dynamic microtubule arrays. Finally, the present study represents the first use of a dominant-negative strategy to disrupt normal protein function in Giardia and provides important insights into giardial microtubule dynamics with relevance to the development of antigiardial compounds that target critical functions of kinesins in the giardial life cycle.     

Directionality of individual kinesin-5 Cin8 motors is modulated by loop 8, ionic strength and microtubule geometry

Kinesin-5 motors fulfil essential roles in mitotic spindle morphogenesis and dynamics as slow, processive microtubule (MT) plus-end directed motors. The Saccharomyces cerevisiae kinesin-5 Cin8 was found, surprisingly, to switch directionality. Here, we have examined directionality using single-molecule fluorescence motility assays and live-cell microscopy. On spindles, Cin8 motors mostly moved slowly (～25 nm/s) towards the midzone, but occasionally also faster (～55 nm/s) towards the spindle poles. In vitro, individual Cin8 motors could be switched by ionic conditions from rapid (380 nm/s) and processive minus-end to slow plus-end motion on single MTs. At high ionic strength, Cin8 motors rapidly alternated directionalities between antiparallel MTs, while driving steady plus-end relative sliding. Between parallel MTs, plus-end motion was only occasionally observed. Deletion of the uniquely large insert in loop 8 of Cin8 induced bias towards minus-end motility and affected the ionic strength-dependent directional switching of Cin8 in vitro. The deletion mutant cells exhibited reduced midzone-directed motility and efficiency to support spindle elongation, indicating the importance of directionality control for the anaphase function of Cin8.     

TPX2 phosphorylation maintains metaphase spindle length by regulating microtubule flux

A steady-state metaphase spindle maintains constant length, although the microtubules undergo intensive dynamics. Tubulin dimers are incorporated at plus ends of spindle microtubules while they are removed from the minus ends, resulting in poleward movement. Such microtubule flux is regulated by the microtubule rescue factors CLASPs at kinetochores and depolymerizing protein Kif2a at the poles, along with other regulators of microtubule dynamics. How microtubule polymerization and depolymerization are coordinated remains unclear. Here we show that TPX2, a microtubule-bundling protein and activator of Aurora A, plays an important role. TPX2 was phosphorylated by Aurora A during mitosis. Its phospho-null mutant caused short metaphase spindles coupled with low microtubule flux rate. Interestingly, phosphorylation of TPX2 regulated its interaction with CLASP1 but not Kif2a. The effect of its mutant in shortening the spindle could be rescued by codepletion of CLASP1 and Kif2a that abolished microtubule flux. Together we propose that Aurora A–dependent TPX2 phosphorylation controls mitotic spindle length through regulating microtubule flux.     

Drosophila EB1 is important for proper assembly,dynamics, and positioning of the mitotic spindle

EB1 is an evolutionarily conserved protein that localizes to the plus ends of growing microtubules. In yeast, the EB1 homologue (BIM1) has been shown to modulate microtubule dynamics and link microtubules to the cortex, but the functions of metazoan EB1 proteins remain unknown. Using a novel preparation of the Drosophila S2 cell line that promotes cell attachment and spreading, we visualized dynamics of single microtubules in real time and found that depletion of EB1 by RNA-mediated inhibition (RNAi) in interphase cells causes a dramatic increase in nondynamic microtubules (neither growing nor shrinking), but does not alter overall microtubule organization. In contrast, several defects in microtubule organization are observed in RNAi-treated mitotic cells, including a drastic reduction in astral microtubules, malformed mitotic spindles, defocused spindle poles, and mispositioning of spindles away from the cell center. Similar phenotypes were observed in mitotic spindles of Drosophila embryos that were microinjected with anti-EB1 antibodies. In addition, live cell imaging of mitosis in Drosophila embryos reveals defective spindle elongation and chromosomal segregation during anaphase after antibody injection. Our results reveal crucial roles for EB1 in mitosis, which we postulate involves its ability to promote the growth and interactions of microtubules within the central spindle and at the cell cortex.     

Quantitative analysis of an anaphase B switch: predicted role for a microtubule catastrophe gradient

Anaphase B in Drosophila embryos is initiated by the inhibition of microtubule (MT) depolymerization at spindle poles, which allows outwardly sliding interpolar (ip) MTs to drive pole-pole separation. Using fluorescence recovery after photobleaching, we observed that MTs throughout the preanaphase B spindle are very dynamic and display complete recovery of fluorescence, but during anaphase B, MTs proximal to the poles stabilize and therefore display lower recovery than those elsewhere. Fluorescence microscopy of the MT tip tracker EB1 revealed that growing MT plus ends localize throughout the preanaphase B spindle but concentrate in the overlap region of interpolar MTs (ipMTs) at anaphase B onset. None of these changes occurred in the presence of nondegradable cyclin B. Modeling suggests that they depend on the establishment of a spatial gradient of MT plus-end catastrophe frequencies, decreasing toward the equator. The resulting redistribution of ipMT plus ends to the overlap zone, together with the suppression of minus-end depolymerization at the poles, could constitute a mechanical switch that initiates spindle elongation.     

The polarity and dynamics of microtubule assembly in the budding yeast Saccharomyces cerevisiae

Microtubule assembly in Saccharomyces cerevisiae is initiated from sites within spindle pole bodies (SPBs) in the nuclear envelope. Microtubule plus ends are thought to be organized distal to the SPBs, while minus ends are proximal. Several hypotheses for the function of microtubule motor proteins in force generation and regulation of microtubule assembly propose that assembly and disassembly occur at minus ends as well as at plus ends. Here we analyse microtubule assembly relative to the SPBs in haploid yeast cells expressing green fluorescent protein fused to alpha-tubulin, a microtubule subunit. Throughout the cell cycle, analysis of fluorescent speckle marks on cytoplasmic astral microtubules reveals that there is no detectable assembly or disassembly at minus ends. After laser-photobleaching, metaphase spindles recover about 63% of the bleached fluorescence, with a half-life of about 1 minute. After anaphase onset, photobleached marks in the interpolar spindle are persistent and do not move relative to the SPBs. In late anaphase, the elongated spindles disassemble at the microtubule plus ends. These results show for astral and anaphase interpolar spindle microtubules, and possibly for metaphase spindle microtubules, that microtubule assembly and disassembly occur at plus, and not minus, ends.     

Arabidopsis thaliana protein,ATK1, is a minus-end directed kinesin that exhibits non-processive movement

The microtubule cytoskeleton forms the scaffolding of the meiotic spindle. Kinesins, which bind to microtubules and generate force via ATP hydrolysis, are also thought to play a critical role in spindle assembly, maintenance, and function. The A. thaliana protein, ATK1 (formerly known as KATA), is a member of the kinesin family based on sequence similarity and is implicated in spindle assembly and/or maintenance. Thus, we want to determine if ATK1 behaves as a kinesin in vitro, and if so, determine the directionality of the motor activity and processivity character (the relationship between molecular "steps" and microtubule association). The results show that ATK1 supports microtubule movement in an ATP-dependent manner and has a minus-end directed polarity. Furthermore, ATK1 exhibits non-processive movement along the microtubule and likely requires at least four ATK1 motors bound to the microtubule to support movement. Based on these results and previous data, we conclude that ATK1 is a non-processive, minus-end directed kinesin that likely plays a role in generating forces in the spindle during meiosis.     

A minus-end-directed kinesin with plus-end tracking protein activity is involved in spindle morphogenesis

Diverse kinesin motor proteins are involved in spindle function; however, the mechanisms by which they are targeted to specific sites within spindles are not well understood. Here, we show that a fusion between yellow fluorescent protein (YFP) and a minus-end-directed Kinesin-14 (C-terminal family) from Arabidopsis, ATK5, localizes to mitotic spindle midzones and regions rich in growing plus-ends within phragmoplasts. Notably, in Arabidopsis interphase cells, YFP::ATK5 localizes to microtubules with a preferential enrichment at growing plus-ends; indicating ATK5 is a plus-end tracking protein (+TIP). This +TIP activity is conferred by regions outside of the C-terminal motor domain, which reveals the presence of independent plus-end tracking and minus-end motor activities within ATK5. Furthermore, mitotic spindles of atk5 null mutant plants are abnormally broadened. Based on these data, we propose a model in which ATK5 uses plus-end tracking to reach spindle midzones, where it then organizes microtubules via minus-end-directed motor activity.     

Inhibition of Cell Migration and Cell Division Correlates with Distinct Effects of Microtubule Inhibiting Drugs

Drugs that target microtubules are thought to inhibit cell division and cell migration by suppressing dynamic instability, a “search and capture” behavior that allows microtubules to probe their environment. Here, we report that subtoxic drug concentrations are sufficient to inhibit plus-end microtubule dynamic instability and cell migration without affecting cell division or microtubule assembly. The higher drug concentrations needed to inhibit cell division act through a novel mechanism that generates microtubule fragments by stimulating microtubule minus-end detachment from their organizing centers. The frequency of microtubule detachment in untreated cells increases at prophase suggesting that it is a regulated cellular process important for spindle assembly and function. We conclude that drugs produce differential dose-dependent effects at microtubule plus and minus-ends to inhibit different microtubule-mediated functions.