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

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

Eg5 is static in bipolar spindles relative to tubulin: evidence for a static spindle matrix

We used fluorescent speckle microscopy to probe the dynamics of the mitotic kinesin Eg5 in Xenopus extract spindles, and compared them to microtubule dynamics. We found significant populations of Eg5 that were static over several seconds while microtubules flux towards spindle poles. Eg5 dynamics are frozen by adenylimidodiphosphate. Bulk turnover experiments showed that Eg5 can exchange between the spindle and the extract with a half life of <55 s. Eg5 distribution in spindles was not perturbed by inhibition of its motor activity with monastrol, but was perturbed by inhibition of dynactin with p50 dynamitin. We interpret these data as revealing the existence of a static spindle matrix that promotes Eg5 targeting to spindles, and transient immobilization of Eg5 within spindles. We discuss alternative interpretations of the Eg5 dynamics we observe, ideas for the biochemical nature of a spindle matrix, and implications for Eg5 function.     

Nuclear envelope‐associated dynein drives prophase centrosome separation and enables Eg5‐independent bipolar spindle formation

The microtubule motor protein kinesin‐5 (Eg5) provides an outward force on centrosomes, which drives bipolar spindle assembly. Acute inhibition of Eg5 blocks centrosome separation and causes mitotic arrest in human cells, making Eg5 an attractive target for anti‐cancer therapy. Using in vitro directed evolution, we show that human cells treated with Eg5 inhibitors can rapidly acquire the ability to divide in the complete absence of Eg5 activity. We have used these Eg5‐independent cells to study alternative mechanisms of centrosome separation. We uncovered a pathway involving nuclear envelope (NE)‐associated dynein that drives centrosome separation in prophase. This NE‐dynein pathway is essential for bipolar spindle assembly in the absence of Eg5, but also functions in the presence of full Eg5 activity, where it pulls individual centrosomes along the NE and acts in concert with Eg5‐dependent outward pushing forces to coordinate prophase centrosome separation. Together, these results reveal how the forces are produced to drive prophase centrosome separation and identify a novel mechanism of resistance to kinesin‐5 inhibitors.     

RHAMM is a centrosomal protein that interacts with dynein and maintains spindle pole stability

The receptor for hyaluronan-mediated motility (RHAMM), an acidic coiled coil protein, has previously been characterized as a cell surface receptor for hyaluronan, and a microtubule-associated intracellular hyaluronan binding protein. In this study, we demonstrate that a subset of cellular RHAMM localizes to the centrosome and functions in the maintenance of spindle integrity. We confirm a previous study showing that the amino terminus of RHAMM interacts with microtubules and further demonstrate that a separate carboxy-terminal domain is required for centrosomal targeting. This motif overlaps the defined hyaluronan binding domain and bears 72% identity to the dynein interaction domain of Xklp2. RHAMM antibodies coimmunprecipitate dynein IC from Xenopus and HeLa extracts. Deregulation of RHAMM expression inhibits mitotic progression and affects spindle architecture. Structure, localization, and function, along with phylogenetic analysis, suggests that RHAMM may be a new member of the TACC family. Thus, we demonstrate a novel centrosomal localization and mitotic spindle-stabilizing function for RHAMM. Moreover, we provide a potential mechanism for this function in that RHAMM may cross-link centrosomal microtubules, through a direct interaction with microtubules and an association with dynein.     

Functional interaction between dynein light chain and intermediate chain is required for mitotic spindle positioning

Cytoplasmic dynein is a large multisubunit complex involved in retrograde transport and the positioning of various organelles. Dynein light chain (LC) subunits are conserved across species; however, the molecular contribution of LCs to dynein function remains controversial. One model suggests that LCs act as cargo-binding scaffolds. Alternatively, LCs are proposed to stabilize the intermediate chains (ICs) of the dynein complex. To examine the role of LCs in dynein function, we used Saccharomyces cerevisiae, in which the sole function of dynein is to position the spindle during mitosis. We report that the LC8 homologue, Dyn2, localizes with the dynein complex at microtubule ends and interacts directly with the yeast IC, Pac11. We identify two Dyn2-binding sites in Pac11 that exert differential effects on Dyn2-binding and dynein function. Mutations disrupting Dyn2 elicit a partial loss-of-dynein phenotype and impair the recruitment of the dynein activator complex, dynactin. Together these results indicate that the dynein-based function of Dyn2 is via its interaction with the dynein IC and that this interaction is important for the interaction of dynein and dynactin. In addition, these data provide the first direct evidence that LC occupancy in the dynein motor complex is important for function.     

Kinesin-5 is not essential for mitotic spindle elongation in Dictyostelium

The proper assembly and operation of the mitotic spindle is essential to ensure the accurate segregation of chromosomes and to position the cytokinetic furrow during cell division in eukaryotes. Not only are dynamic microtubules required but also the concerted actions of multiple motor proteins are necessary to effect spindle pole separation, chromosome alignment, chromatid segregation, and spindle elongation. Although a number of motor proteins are known to play a role in mitosis, there remains a limited understanding of their full range of functions and the details by which they interact with other spindle components. The kinesin-5 (BimC/Eg5) family of motors is largely considered essential to drive spindle pole separation during the initial and latter stages of mitosis. We have deleted the gene encoding the kinesin-5 member in Dictyostelium, (kif13), and find that, in sharp contrast with results found in vertebrate, fly, and yeast organisms, kif13(-) cells continue to grow at rates indistinguishable from wild type. Phenotype analysis reveals a slight increase in spindle elongation rates in the absence of Kif13. More importantly, there is a dramatic, premature separation of spindle halves in kif13(-) cells, suggesting a novel role of this motor in maintaining spindle integrity at the terminal stages of division.     

Probing spindle assembly mechanisms with monastrol, a small molecule inhibitor of the mitotic kinesin, Eg5

Monastrol, a cell-permeable small molecule inhibitor of the mitotic kinesin, Eg5, arrests cells in mitosis with monoastral spindles. Here, we use monastrol to probe mitotic mechanisms. We find that monastrol does not inhibit progression through S and G2 phases of the cell cycle or centrosome duplication. The mitotic arrest due to monastrol is also rapidly reversible. Chromosomes in monastrol-treated cells frequently have both sister kinetochores attached to microtubules extending to the center of the monoaster (syntelic orientation). Mitotic arrest-deficient protein 2 (Mad2) localizes to a subset of kinetochores, suggesting the activation of the spindle assembly checkpoint in these cells. Mad2 localizes to some kinetochores that have attached microtubules in monastrol-treated cells, indicating that kinetochore microtubule attachment alone may not satisfy the spindle assembly checkpoint. Monastrol also inhibits bipolar spindle formation in Xenopus egg extracts. However, it does not prevent the targeting of Eg5 to the monoastral spindles that form. Imaging bipolar spindles disassembling in the presence of monastrol allowed direct observations of outward directed forces in the spindle, orthogonal to the pole-to-pole axis. Monastrol is thus a useful tool to study mitotic processes, detection and correction of chromosome malorientation, and contributions of Eg5 to spindle assembly and maintenance.     

TPX2 regulates the localization and activity of Eg5 in the mammalian mitotic spindle

Mitotic spindle assembly requires the regulated activity of numerous spindle-associated proteins. In mammalian cells, the Kinesin-5 motor Eg5 interacts with the spindle assembly factor TPX2, but how this interaction contributes to spindle formation and function is not established. Using bacterial artificial chromosome technology, we generated cells expressing TPX2 lacking the Eg5 interaction domain. Spindles in these cells were highly disorganized with multiple spindle poles. The TPX2-Eg5 interaction was required for kinetochore fiber formation and contributed to Eg5 localization to spindle microtubules but not spindle poles. Microinjection of the Eg5-binding domain of TPX2 resulted in spindle elongation, indicating that the interaction of Eg5 with TPX2 reduces motor activity. Consistent with this possibility, we found that TPX2 reduced the velocity of Eg5-dependent microtubule gliding, inhibited microtubule sliding, and resulted in the accumulation of motor on microtubules. These results establish a novel function of TPX2 in regulating the location and activity of the mitotic motor Eg5.     

Dynactin-dependent cortical dynein and spherical spindle shape correlate temporally with meiotic spindle rotation in Caenorhabditis elegans

Oocyte meiotic spindles orient with one pole juxtaposed to the cortex to facilitate extrusion of chromosomes into polar bodies. In Caenorhabditis elegans, these acentriolar spindles initially orient parallel to the cortex and then rotate to the perpendicular orientation. To understand the mechanism of spindle rotation, we characterized events that correlated temporally with rotation, including shortening of the spindle in the pole-to pole axis, which resulted in a nearly spherical spindle at rotation. By analyzing large spindles of polyploid C. elegans and a related nematode species, we found that spindle rotation initiated at a defined spherical shape rather than at a defined spindle length. In addition, dynein accumulated on the cortex just before rotation, and microtubules grew from the spindle with plus ends outward during rotation. Dynactin depletion prevented accumulation of dynein on the cortex and prevented spindle rotation independently of effects on spindle shape. These results support a cortical pulling model in which spindle shape might facilitate rotation because a sphere can rotate without deforming the adjacent elastic cytoplasm. We also present evidence that activation of spindle rotation is promoted by dephosphorylation of the basic domain of p150 dynactin.     

NUDEL is a novel Cdk5 substrate that associates with LIS1 and cytoplasmic dynein

Disruption of one allele of the LIS1 gene causes a severe developmental brain abnormality, type I lissencephaly. In Aspergillus nidulans, the LIS1 homolog, NUDF, and cytoplasmic dynein are genetically linked and regulate nuclear movements during hyphal growth. Recently, we demonstrated that mammalian LIS1 regulates dynein functions. Here we characterize NUDEL, a novel LIS1-interacting protein with sequence homology to gene products also implicated in nuclear distribution in fungi. Like LIS1, NUDEL is robustly expressed in brain, enriched at centrosomes and neuronal growth cones, and interacts with cytoplasmic dynein. Furthermore, NUDEL is a substrate of Cdk5, a kinase known to be critical during neuronal migration. Inhibition of Cdk5 modifies NUDEL distribution in neurons and affects neuritic morphology. Our findings point to cross-talk between two prominent pathways that regulate neuronal migration.     

Docking and rolling, a model of how the mitotic motor Eg5 works

Whereas kinesin I is designed to transport cargoes long distances in isolation, a closely related kinesin motor, Eg5, is designed to generate a sustained opposing force necessary for proper mitotic spindle formation. Do the very different roles for these evolutionarily related motors translate into differences in how they generate movement? We have addressed this question by examining when in the ATPase cycle the Eg5 motor domain and neck linker move through the use of a series of novel spectroscopic probes utilizing fluorescence resonance energy transfer, and we have compared our results to kinesin I. Our results are consistent with a model in which movement in Eg5 occurs in two sequential steps, an ATP-dependent docking of the neck linker, followed by a rotation or "rolling" of the entire motor domain on the microtubule surface that occurs with ATP hydrolysis. These two forms of movement are consistent with the functions of a motor designed to generate sustained opposing force, and hence, our findings support the argument that the mechanochemical features of a molecular motor are shaped more by the demands placed on it than by its particular family of origin.     

The KinI kinesin Kif2a is required for bipolar spindle assembly through a functional relationship with MCAK

Although the microtubule-depolymerizing KinI motor Kif2a is abundantly expressed in neuronal cells, we now show it localizes to centrosomes and spindle poles during mitosis in cultured cells. RNAi-induced knockdown of Kif2a expression inhibited cell cycle progression because cells assembled monopolar spindles. Bipolar spindle assembly was restored in cells lacking Kif2a by treatments that altered microtubule assembly (nocodazole), eliminated kinetochore-microtubule attachment (loss of Nuf2), or stabilized microtubule plus ends at kinetochores (loss of MCAK). Thus, two KinI motors, MCAK and Kif2a, play distinct roles in mitosis, and MCAK activity at kinetochores must be balanced by Kif2a activity at poles for spindle bipolarity. These treatments failed to restore bipolarity to cells lacking the activity of the kinesin Eg5. Thus, two independent pathways contribute to spindle bipolarity, with the Eg5-dependent pathway using motor force to drive spindle bipolarity and the Kif2a-dependent pathway relying on microtubule polymer dynamics to generate force for spindle bipolarity.     

Prion disease incubation time is not affected in mice heterozygous for a dynein mutation

A mechanism for transmission of the infectious prions from the peripheral nerve ends to the central nervous system is thought to involve neuronal anterograde and retrograde transport systems. Cytoplasmic dynein is the major retrograde transport molecular motor whose function is impaired in the Legs at odd angles (Loa) mouse due to a point mutation in the cytoplasmic dynein heavy chain subunit. Loa is a dominant trait which causes neurodegeneration and progressive motor function deficit in the heterozygotes. To investigate the role of cytoplasmic dynein in the transmission of prions within neurons, we inoculated heterozygous Loa and wild type littermates with mouse-adapted scrapie prions intracerebrally and intraperitonially, and determined the incubation period to onset of clinical prion disease. Our data indicate that the dynein mutation in the heterozygous state does not affect prion disease incubation time or its neuropathology in Loa mice.     

The kinesin-related protein, HSET, opposes the activity of Eg5 and cross-links microtubules in the mammalian mitotic spindle.

We have prepared antibodies specific for HSET, the human homologue of the KAR3 family of minus end-directed motors. Immuno-EM with these antibodies indicates that HSET frequently localizes between microtubules within the mammalian metaphase spindle consistent with a microtubule cross-linking function. Microinjection experiments show that HSET activity is essential for meiotic spindle organization in murine oocytes and taxol-induced aster assembly in cultured cells. However, inhibition of HSET did not affect mitotic spindle architecture or function in cultured cells, indicating that centrosomes mask the role of HSET during mitosis. We also show that (acentrosomal) microtubule asters fail to assemble in vitro without HSET activity, but simultaneous inhibition of HSET and Eg5, a plus end-directed motor, redresses the balance of forces acting on microtubules and restores aster organization. In vivo, centrosomes fail to separate and monopolar spindles assemble without Eg5 activity. Simultaneous inhibition of HSET and Eg5 restores centrosome separation and, in some cases, bipolar spindle formation. Thus, through microtubule cross-linking and oppositely oriented motor activity, HSET and Eg5 participate in spindle assembly and promote spindle bipolarity, although the activity of HSET is not essential for spindle assembly and function in cultured cells because of centrosomes.     

Prion disease incubation time is not affected in mice heterozygous for a dynein mutation

A mechanism for transmission of the infectious prions from the peripheral nerve ends to the central nervous system is thought to involve neuronal anterograde and retrograde transport systems. Cytoplasmic dynein is the major retrograde transport molecular motor whose function is impaired in the Legs at odd angles (Loa) mouse due to a point mutation in the cytoplasmic dynein heavy chain subunit. Loa is a dominant trait which causes neurodegeneration and progressive motor function deficit in the heterozygotes. To investigate the role of cytoplasmic dynein in the transmission of prions within neurons, we inoculated heterozygous Loa and wild type littermates with mouse-adapted scrapie prions intracerebrally and intraperitonially, and determined the incubation period to onset of clinical prion disease. Our data indicate that the dynein mutation in the heterozygous state does not affect prion disease incubation time or its neuropathology in Loa mice.     

The time-dependent distribution of phosphorylated intermediates in native sarcoplasmic reticulum Ca2+-ATPase from skeletal muscle is not compatible with a linear kinetic model

Quenched-flow mixing was used to characterize the kinetic behavior of the intermediate reactions of the skeletal muscle sarcoplasmic reticulum (SR) Ca-ATPase (SERCA1) at 2 and 21 degrees C. At 2 degrees C, phosphorylation of SR Ca-ATPase with 100 microM ATP labeled one-half of the catalytic sites with a biphasic time dependence [Mahaney, J. E., Froehlich, J. P., and Thomas, D. D. (1995) Biochemistry 34, 4864-4879]. Chasing the phosphoenzyme (EP) with 1.66 mM ADP 10 ms after the start of phosphorylation revealed mostly ADP-insensitive E2P (95% of EP(total)), consistent with its rapid formation from ADP-sensitive E1P. The consecutive relationship of the phosphorylated intermediates predicts a decrease in the proportion of E1P ([E1P]/[EP(total)]) with increasing phosphorylation time. Instead, after 10 ms the proportion of E1P increased and that of E2P decreased until they reached a constant 1:1 stoichiometry ([E1P]:[E2P] approximately 1). At 21 degrees C, phosphorylation displayed a transient overshoot associated with an inorganic phosphate (P(i)) burst, reflecting increased turnover of E2P at the higher temperature. The P(i) burst exceeded the decay of the EP overshoot, suggesting that rephosphorylation of the enzyme occurs before the recycling step (E2 --> E1). This behavior and the reversed order of accumulation of phosphorylated intermediates at 2 degrees C are not compatible with the conventional linear consecutive reaction mechanism: E1 + ATP --> E1.ATP --> E1P + ADP --> E2P --> E2.P(i) --> E1 + P(i). Solubilization of the Ca-ATPase into monomers using the nonionic detergent C(12)E(8) gave a pattern of phosphorylation in which E1P and E2P behave like consecutive intermediates. Kinetic modeling of the C(12)E(8)-solubilized SR Ca-ATPase showed that it behaves according to the conventional Ca-ATPase reaction mechanism, consistent with monomeric catalytic function. We conclude that the nonconforming features of native SERCA1 arise from oligomeric protein conformational interactions that constrain the subunits to a staggered or out-of-phase mode of operation.     

In vivo functional antagonism between isoproterenol and bronchoconstrictants in the dog

The functional antagonism between isoproterenol and methacholine, histamine and serotonin, as described in vitro in respiratory smooth muscle was explored in vivo in a canine model. Infusions of isoproterenol were administered during brief peaks of bronchospasm produced by aerosolized methacholine and histamine, or during sustained bronchospasm produced by infused serotonin. In eight mongrel dogs anesthetized with pentobarbital sodium, the mean protection by infused isoproterenol against methacholine challenge decreased from 60.6 to 29.1% as the mean lung resistance (RL) was increased from 78 to 232% over base line by a fourfold increase in methacholine (P less than 0.002). In six dogs, the mean protection by isoproterenol against histamine decreased from 55.5 to 26.9% as the opposing RL increased from 80 to 182% over base line with a fourfold increase in histamine (P less than 0.02). However, with serotonin infusions there was only a small 18% mean decrease in protection (P = 0.05), associated with a correspondingly small 37% mean increase in dose of serotonin despite a 269% mean increase in resistance (P = 0.02). In all cases, the loss of protection correlated more closely with the dose of constrictant than the resistance increase over base line. These findings demonstrate in vivo functional antagonism between isoproterenol and the dose of bronchoconstrictant but not necessarily resistance increase per se.     

A functional relationship between NuMA and kid is involved in both spindle organization and chromosome alignment in vertebrate cells

We examined spindle morphology and chromosome alignment in vertebrate cells after simultaneous perturbation of the chromokinesin Kid and either NuMA, CENP-E, or HSET. Spindle morphology and chromosome alignment after simultaneous perturbation of Kid and either HSET or CENP-E were no different from when either HSET or CENP-E was perturbed alone. However, short bipolar spindles with organized poles formed after perturbation of both Kid and NuMA in stark contrast to splayed spindle poles observed after perturbation of NuMA alone. Spindles were disorganized if Kid, NuMA, and HSET were perturbed, indicating that HSET is sufficient for spindle organization in the absence of Kid and NuMA function. In addition, chromosomes failed to align efficiently at the spindle equator after simultaneous perturbation of Kid and NuMA despite appropriate kinetochore-microtubule interactions that generated chromosome movement at normal velocities. These data indicate that a functional relationship between the chromokinesin Kid and the spindle pole organizing protein NuMA influences spindle morphology, and we propose that this occurs because NuMA forms functional linkages between kinetochore and nonkinetochore microtubules at spindle poles. In addition, these data show that both Kid and NuMA contribute to chromosome alignment in mammalian cells.     

Evidence that a globular conformation is not compatible with FhaC-mediated secretion of the Bordetella pertussis filamentous haemagglutinin

The 220 kDa Bordetella pertussis filamentous haemagglutinin (FHA) is the major extracellular protein of this organism. It is exported using a signal peptide-dependent pathway, and its secretion depends on one specific outer membrane accessory protein, FhaC. In this work, we have investigated the influence of conformation on the FhaC-mediated secretion of FHA using an 80 kDa N-terminal FHA derivative, Fha44. In contrast to many signal peptide-dependent secretory proteins, no soluble periplasmic intermediate of Fha44 could be isolated. In addition, cell-associated Fha44 synthesized in the absence of FhaC did not remain competent for extracellular secretion upon delayed expression of FhaC, indicating that the translocation steps across the cytoplasmic and the outer membrane might be coupled. A chimeric protein, in which the globular B subunit of the cholera toxin, CtxB, was fused at the C-terminus of Fha44, was not secreted in B. pertussis or in Escherichia coli expressing FhaC. The hybrid protein was only secreted when both disulphide bond-forming cysteines of CtxB were replaced by serines or when it was produced in DsbA^?E. coli. The Fha44 portion of the secretion-incompetent hybrid protein was partly exposed on the cell surface. These results argue that the Fha44–CtxB hybrid protein transited through the periplasmic space, where disulphide bond formation is specifically catalysed, and that secretion across the outer membrane was initiated. The folded CtxB portion prevented extracellular release of the hybrid, in contrast to the more flexible CtxB domain devoid of cysteines. We propose a secretion model whereby Fha44 transits through the periplasmic space on its way to the cell surface and initiates its translocation through the outer membrane before being released from the cytoplasmic membrane. Coupling of Fha44 translocation across both membranes would delay the acquisition of its folded structure until the protein emerges from the outer membrane. Such a model would be consistent with the extensive intracellular proteolysis of FHA derivatives in B. pertussis.     

Cytoplasmic dynein/dynactin drives kinetochore protein transport to the spindle poles and has a role in mitotic spindle checkpoint inactivation.

We discovered that many proteins located in the kinetochore outer domain, but not the inner core, are depleted from kinetochores and accumulate at spindle poles when ATP production is suppressed in PtK1 cells, and that microtubule depolymerization inhibits this process. These proteins include the microtubule motors CENP-E and cytoplasmic dynein, and proteins involved with the mitotic spindle checkpoint, Mad2, Bub1R, and the 3F3/2 phosphoantigen. Depletion of these components did not disrupt kinetochore outer domain structure or alter metaphase kinetochore microtubule number. Inhibition of dynein/dynactin activity by microinjection in prometaphase with purified p50 "dynamitin" protein or concentrated 70.1 anti-dynein antibody blocked outer domain protein transport to the spindle poles, prevented Mad2 depletion from kinetochores despite normal kinetochore microtubule numbers, reduced metaphase kinetochore tension by 40%, and induced a mitotic block at metaphase. Dynein/dynactin inhibition did not block chromosome congression to the spindle equator in prometaphase, or segregation to the poles in anaphase when the spindle checkpoint was inactivated by microinjection with Mad2 antibodies. Thus, a major function of dynein/dynactin in mitosis is in a kinetochore disassembly pathway that contributes to inactivation of the spindle checkpoint.