NuMA is a component of an insoluble matrix at mitotic spindle poles

NuMA associates with microtubule motors during mitosis to perform an essential role in organizing microtubule minus ends at spindle poles. Using immunogold electron microscopy, we show that NuMA is a component of an electron-dense material concentrated at both mitotic spindle poles in PtK1 cells and the core of microtubule asters formed through a centrosome-independent mechanism in cell-free mitotic extracts. This NuMA-containing material is distinct from the peri-centriolar material and forms a matrix that appears to anchor microtubule ends at the spindle pole. In stark contrast to conventional microtubule-associated proteins whose solubility is directly dependent on microtubules, we find that once NuMA is incorporated into this matrix either in vivo or in vitro, it becomes insoluble and this insolubility is no longer dependent on microtubules. NuMA is essential for the formation of this insoluble matrix at the core of mitotic asters assembled in vitro because the matrix is absent from mitotic asters assembled in a cell-free mitotic extract that is specifically depleted of NuMA. These physical properties are consistent with NuMA being a component of the putative mitotic spindle matrix in vertebrate cells. Furthermore, given that NuMA is essential for spindle pole organization in vertebrate systems, it is likely that this insoluble matrix plays an essential structural function in anchoring and/or stabilizing microtubule minus ends at spindle poles in mitotic cells.     

Dynamic instability of microtubules

Recent evidence shows that dynamic instability is the dominant mechanism for the assembly of pure tubulin in vitro and for the great majority of microtubules in the mitotic spindle and the interphase cytoplasmic microtubule complex. The basic concepts of this model provide a framework for future characterization of the molecular basis of spatial and temporal regulation of microtubule dynamics in the cell and the function of microtubule dynamics in motile processes such as chromosome movement.     

Centrosome separation and central spindle assembly act in redundant pathways that regulate microtubule density and trigger cleavage furrow formation

The mitotic spindle provides the spatial cue that coordinates cytokinesis with nuclear division. However, the specific property of the mitotic spindle that mediates this spatial regulation remains obscure, in part because different aspects of the mitotic spindle appear to have furrow inducing activity in different systems. We show that in C. elegans embryos, although the central spindle is usually dispensable for furrow initiation, it becomes essential for furrow formation when the extent of centrosome separation during anaphase is reduced. Measurements of microtubule density demonstrate that furrow formation occurs in the vicinity of a local minimum of microtubule density. Reduction of the extent of spindle elongation or disruption of the central spindle causes delayed formation of the cleavage furrow. These data suggest that reduced microtubule density triggers cleavage furrow initiation and demonstrate that redundant mechanisms direct efficient formation of the cleavage furrow.     

Katanin controls mitotic and meiotic spindle length

Accurate control of spindle length is a conserved feature of eukaryotic cell division. Lengthening of mitotic spindles contributes to chromosome segregation and cytokinesis during mitosis in animals and fungi. In contrast, spindle shortening may contribute to conservation of egg cytoplasm during female meiosis. Katanin is a microtubule-severing enzyme that is concentrated at mitotic and meiotic spindle poles in animals. We show that inhibition of katanin slows the rate of spindle shortening in nocodazole-treated mammalian fibroblasts and in untreated Caenorhabditis elegans meiotic embryos. Wild-type C. elegans meiotic spindle shortening proceeds through an early katanin-independent phase marked by increasing microtubule density and a second, katanin-dependent phase that occurs after microtubule density stops increasing. In addition, double-mutant analysis indicated that gamma-tubulin-dependent nucleation and microtubule severing may provide redundant mechanisms for increasing microtubule number during the early stages of meiotic spindle assembly.     

Cep192 and the generation of the mitotic spindle

The cellular mechanisms used to generate sufficient microtubule polymer mass to drive the assembly and function of the mitotic spindle remain a matter of great interest. As the primary microtubule nucleating structures in somatic animal cells, centrosomes have been assumed to figure prominently in spindle assembly. At the onset of mitosis, centrosomes undergo a dramatic increase in size and microtubule nucleating capacity, termed maturation, which is likely a key event in mitotic spindle formation. Interestingly, however, spindles can still form in the absence of centrosomes calling into question the specific mitotic role of these organelles. Recent work has shown that the human centrosomal protein, Cep192, is required for both centrosome maturation and spindle assembly thus providing a molecular link between these two processes. In this article, we propose that Cep192 does so by forming a scaffolding on which proteins involved in microtubule nucleation are sequestered and become active in mitotic cells. Normally, this activity is largely confined to centrosomes but in their absence continues to function but is dispersed to other sites within the cell.     

Requirements for NuMA in maintenance and establishment of mammalian spindle poles

Microtubules of the mitotic spindle in mammalian somatic cells are focused at spindle poles, a process thought to include direct capture by astral microtubules of kinetochores and/or noncentrosomally nucleated microtubule bundles. By construction and analysis of a conditional loss of mitotic function allele of the nuclear mitotic apparatus (NuMA) protein in mice and cultured primary cells, we demonstrate that NuMA is an essential mitotic component with distinct contributions to the establishment and maintenance of focused spindle poles. When mitotic NuMA function is disrupted, centrosomes provide initial focusing activity, but continued centrosome attachment to spindle fibers under tension is defective, and the maintenance of focused kinetochore fibers at spindle poles throughout mitosis is prevented. Without centrosomes and NuMA, initial establishment of spindle microtubule focusing completely fails. Thus, NuMA is a defining feature of the mammalian spindle pole and functions as an essential tether linking bulk microtubules of the spindle to centrosomes.     

A Rae1-containing ribonucleoprotein complex is required for mitotic spindle assembly

Centrosome-independent microtubule polymerization around chromosomes has been shown to require a local gradient of RanGTP, which discharges mitotic cargoes from the nuclear import receptor importin beta. Here, we have used an activity-based assay in Xenopus egg extracts to purify the mRNA export protein Rae1 as a spindle assembly factor regulated by this pathway. Rae1 is a microtubule-associated protein that binds directly to importin beta. Depletion of Rae1 from extracts or cells severely inhibits mitotic spindle assembly. A purified Rae1 complex stabilizes microtubules in egg extracts in a RanGTP/importin beta-regulated manner. Interestingly, Rae1 exists in a large ribonucleoprotein complex, which requires RNA for its activity to control microtubule dynamics in vitro. Furthermore, we provide evidence that RNA associates with the mitotic spindle and that it plays a direct, translation-independent role in spindle assembly. Our studies reveal an unexpected function for RNA in spindle morphogenesis.     

The astrin-kinastrin/SKAP complex localizes to microtubule plus ends and facilitates chromosome alignment

Astrin is a mitotic spindle-associated protein required for the correct alignment of all chromosomes at the metaphase plate. Astrin depletion delays chromosome alignment and causes the loss of normal spindle architecture and sister chromatid cohesion before anaphase onset. Here we describe an astrin complex containing kinastrin/SKAP, a novel kinetochore and mitotic spindle protein, and three minor interaction partners: dynein light chain, Plk1, and Sgo2. Kinastrin is the major astrin-interacting protein in mitotic cells, and is required for astrin targeting to microtubule plus ends proximal to the plus tip tracking protein EB1. Cells overexpressing or depleted of kinastrin mislocalize astrin and show the same mitotic defects as astrin-depleted cells. Importantly, astrin fails to localize to and track microtubule plus ends in cells depleted of or overexpressing kinastrin. These findings suggest that microtubule plus end targeting of astrin is required for normal spindle architecture and chromosome alignment, and that perturbations of this pathway result in delayed mitosis and nonphysiological separase activation.     

Spindle pole mechanics studied in mitotic asters: dynamic distribution of spindle forces through compliant linkages

During cell division, chromosomes must faithfully segregate to maintain genome integrity, and this dynamic mechanical process is driven by the macromolecular machinery of the mitotic spindle. However, little is known about spindle mechanics. For example, spindle microtubules are organized by numerous cross-linking proteins yet the mechanical properties of those cross-links remain unexplored. To examine the mechanical properties of microtubule cross-links we applied optical trapping to mitotic asters that form in mammalian mitotic extracts. These asters are foci of microtubules, motors, and microtubule-associated proteins that reflect many of the functional properties of spindle poles and represent centrosome-independent spindle-pole analogs. We observed bidirectional motor-driven microtubule movements, showing that microtubule linkages within asters are remarkably compliant (mean stiffness 0.025 pN/nm) and mediated by only a handful of cross-links. Depleting the motor Eg5 reduced this stiffness, indicating that Eg5 contributes to the mechanical properties of microtubule asters in a manner consistent with its localization to spindle poles in cells. We propose that compliant linkages among microtubules provide a mechanical architecture capable of accommodating microtubule movements and distributing force among microtubules without loss of pole integrity—a mechanical paradigm that may be important throughout the spindle.     

Poleward Transport of TPX2 in the Mammalian Mitotic Spindle Requires Dynein,Eg5, and Microtubule Flux

TPX2 is a Ran-regulated spindle assembly factor that is required for kinetochore fiber formation and activation of the mitotic kinase Aurora A. TPX2 is enriched near spindle poles and is required near kinetochores, suggesting that it undergoes dynamic relocalization throughout mitosis. Using photoactivation, we measured the movement of PA-GFP-TPX2 in the mitotic spindle. TPX2 moves poleward in the half-spindle and is static in the interzone and near spindle poles. Poleward transport of TPX2 is sensitive to inhibition of dynein or Eg5 and to suppression of microtubule flux with nocodazole or antibodies to Kif2a. Poleward transport requires the C terminus of TPX2, a domain that interacts with Eg5. Overexpression of TPX2 lacking this domain induced excessive microtubule formation near kinetochores, defects in spindle assembly and blocked mitotic progression. Our data support a model in which poleward transport of TPX2 down-regulates its microtubule nucleating activity near kinetochores and links microtubules generated at kinetochores to dynein for incorporation into the spindle.     

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

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

Augmin: a protein complex required for centrosome-independent microtubule generation within the spindle

Since the discovery of gamma-tubulin, attention has focused on its involvement as a microtubule nucleator at the centrosome. However, mislocalization of gamma-tubulin away from the centrosome does not inhibit mitotic spindle formation in Drosophila melanogaster, suggesting that a critical function for gamma-tubulin might reside elsewhere. A previous RNA interference (RNAi) screen identified five genes (Dgt2-6) required for localizing gamma-tubulin to spindle microtubules. We show that the Dgt proteins interact, forming a stable complex. We find that spindle microtubule generation is substantially reduced after knockdown of each Dgt protein by RNAi. Thus, the Dgt complex that we name "augmin" functions to increase microtubule number. Reduced spindle microtubule generation after augmin RNAi, particularly in the absence of functional centrosomes, has dramatic consequences on mitotic spindle formation and function, leading to reduced kinetochore fiber formation, chromosome misalignment, and spindle bipolarity defects. We also identify a functional human homologue of Dgt6. Our results suggest that an important mitotic function for gamma-tubulin may lie within the spindle, where augmin and gamma-tubulin function cooperatively to amplify the number of microtubules.     

GCP-WD is a gamma-tubulin targeting factor required for centrosomal and chromatin-mediated microtubule nucleation

The gamma-tubulin ring complex (gammaTuRC) is a large multi-protein complex that is required for microtubule nucleation from the centrosome. Here, we show that the GCP-WD protein (originally named NEDD1) is the orthologue of the Drosophila Dgrip71WD protein, and is a subunit of the human gammaTuRC. GCP-WD has the properties of an attachment factor for the gammaTuRC: depletion or inhibition of GCP-WD results in loss of the gammaTuRC from the centrosome, abolishing centrosomal microtubule nucleation, although the gammaTuRC is intact and able to bind to microtubules. GCP-WD depletion also blocks mitotic chromatin-mediated microtubule nucleation, resulting in failure of spindle assembly. Mitotic phosphorylation of GCP-WD is required for association of gamma-tubulin with the spindle, separately from association with the centrosome. Our results indicate that GCP-WD broadly mediates targeting of the gammaTuRC to sites of microtubule nucleation and to the mitotic spindle, which is essential for spindle formation.     

The forces that position a mitotic spindle asymmetrically are tethered until after the time of spindle assembly

Regulation of the mitotic spindle's position is important for cells to divide asymmetrically. Here, we use Caenorhabditis elegans embryos to provide the first analysis of the temporal regulation of forces that asymmetrically position a mitotic spindle. We find that asymmetric pulling forces, regulated by cortical PAR proteins, begin to act as early as prophase and prometaphase, even before the spindle forms and shifts to a posterior position. The spindle does not shift asymmetrically during these early phases due to a tethering force, mediated by astral microtubules that reach the anterior cell cortex. We show that this tether is normally released after spindle assembly and independently of anaphase entry. Monitoring microtubule dynamics by photobleaching segments of microtubules during anaphase revealed that spindle microtubules do not undergo significant poleward flux in C. elegans. Together with the known absence of anaphase A, these data suggest that the major forces contributing to chromosome separation during anaphase originate outside the spindle. We propose that the forces positioning the mitotic spindle asymmetrically are tethered until after the time of spindle assembly and that these same forces are used later to drive chromosome segregation at anaphase.     

The stabilization of microtubules in isolated spindles by tubulin-colchicine complex

We have analyzed the effect of colchicine and tubulin dimer-colchicine complex (T-C) on microtubule assembly in mitotic spindles. Cold- and calcium-labile mitotic spindles were isolated from embryos of the sea urchin Lytechinus variegatus employing EGTA/glycerol stabilization buffers. Polarization microscopy and measurements of spindle birefringent retardation (BR) were used to record the kinetics of microtubule assembly-disassembly in single spindles. When isolated spindles were perfused out of glycerol stabilizing buffer into a standard in vitro microtubule reassembly buffer (0.1 M Pipes, pH 6.8, 1 mM EGTA, 0.5 mM MgCl2, and 0.5 mM GTP) lacking glycerol, spindle BR decreased with a half-time of 120 s. Colchicine at 1 mM in this buffer had no effect on the rate of spindle microtubule disassembly. Inclusion of 20 microM tubulin or microtubule protein, purified from porcine brain, in this buffer resulted in an augmentation of spindle BR. Interestingly, in the presence of 20 microM T-C, spindle BR did not increase, but was reversibly stabilized; subsequent perfusion with reassembly buffer without T-C resulted in depolymerization. This behavior is striking in contrast to the rapid depolymerization of spindle microtubules induced by colchicine and T-C in vivo. These results support the current view that colchicine does not directly promote microtubule depolymerization. Rather, it is T-C complex that alters microtubule assembly, by reversibly binding to microtubules and inhibiting elongation. In vivo, colchicine can induce depolymerization of nonkinetochore spindle microtubules within 20 s. In vitro, colchicine blocks further microtubule assembly, but does not induce rapid disassembly.(ABSTRACT TRUNCATED AT 250 WORDS)     

Balanced regulation of microtubule dynamics during the cell cycle: a contemporary view

Assembly of mitotic and meiotic spindles into an elliptical bipolar shape is an example of morphogenetic processes that involve local chromosomal regulation of microtubule dynamics for proper spatial microtubule assembly. Global microtubule dynamics during the cell cycle and local microtubule dynamics during spindle assembly are regulated by a balance between microtubule stabilizing and destabilizing factors. How a chromosome-induced phosphorylation gradient may be generated and modulate spindle microtubule assembly through balanced regulation of the activity of microtubule-associated proteins and Stathmin/Op 18 is analyzed.     

Involvement of the telomeric protein Pin2/TRF1 in the regulation of the mitotic spindle

Pin2/TRF1 was independently identified as a telomeric DNA-binding protein (TRF1) that regulates telomere length, and as a protein (Pin2) that can bind the mitotic kinase NIMA and suppress its lethal phenotype. We have previously demonstrated that Pin2/TRF1 levels are cell cycle-regulated and its overexpression induces mitotic arrest and then apoptosis. This Pin2/TRF1 activity can be potentiated by microtubule-disrupting agents, but suppressed by phosphorylation of Pin2/TRF1 by ATM; this negative regulation is critical in mediating for many, but not all, ATM-dependent phenotypes. Interestingly, Pin2/TRF1 specifically localizes to mitotic spindles in mitotic cells and affects the microtubule polymerization in vitro. These results suggest a role of Pin2/TRF1 in mitosis. However, nothing is known about whether Pin2/TRF1 affects the spindle function in mitotic progression. Here we characterized a new Pin2/TRF1-interacting protein, EB1, that was originally identified in our yeast two-hybrid screen. Pin2/TRF1 bound EB1 both in vitro and in vivo and they also co-localize at the mitotic spindle in cells. Furthermore, EB1 inhibits the ability of Pin2/TRF1 to promote microtubule polymerization in vitro. Given that EB1 is a microtubule plus end-binding protein, these results further confirm a specific interaction between Pin2/TRF1 and the mitotic spindle. More importantly, we have shown that inhibition of Pin2/TRF1 in ataxia-telangiectasia cells is able to fully restore their mitotic spindle defect in response to microtubule disruption, demonstrating for the first time a functional involvement of Pin2/TRF1 in mitotic spindle regulation.     

Substrate degradation by the anaphase promoting complex occurs during mitotic slippage

Microtubule targeting drugs are successful in chemotherapy because they indefinitely activate the spindle assembly checkpoint. The spindle assembly checkpoint monitors proper attachment of all kinetochores to microtubules and tension between the kinetochores of sister chromatids to prevent premature anaphase entry. To this end, the activated spindle assembly checkpoint suppresses the E3 ubiquitin ligase activity of the anaphase-promoting complex (APC). In the continued presence of conditions that activate the spindle assembly checkpoint, cells eventually escape from mitosis by "slippage". It has not been directly tested whether APC activation accompanies slippage. Using cells blocked in mitosis with the microtubule assembly inhibitor nocodazole, we show that mitotic APC substrates are degraded upon mitotic slippage. To confirm that APC is normally activated upon mitotic slippage we have found that knockdown of Cdc20 and Cdh1, two mitotic activators of APC, prevents the degradation of APC substrates during mitotic slippage. Knockdown of Cdc20 and Cdh1 prevents the degradation of APC substrates during mitotic slippage. We provide the first direct demonstration that despite conditions that activate the spindle checkpoint, APC is indeed activated upon mitotic slippage of cells to interphase cells. Activation of the spindle checkpoint by microtubule targeting drugs used in chemotherapy may not indefinitely prevent APC activation.     

The ciliopathy protein CCDC66 controls mitotic progression and cytokinesis by promoting microtubule nucleation and organization

Precise spatiotemporal control of microtubule nucleation and organization is critical for faithful segregation of cytoplasmic and genetic material during cell division and signaling via the primary cilium in quiescent cells. Microtubule-associated proteins (MAPs) govern assembly, maintenance, and remodeling of diverse microtubule arrays. While a set of conserved MAPs are only active during cell division, an emerging group of MAPs acts as dual regulators in dividing and nondividing cells. Here, we elucidated the nonciliary functions and molecular mechanism of action of the ciliopathy-linked protein CCDC66, which we previously characterized as a regulator of ciliogenesis in quiescent cells. We showed that CCDC66 dynamically localizes to the centrosomes, the bipolar spindle, the spindle midzone, the central spindle, and the midbody in dividing cells and interacts with the core machinery of centrosome maturation and MAPs involved in cell division. Loss-of-function experiments revealed its functions during mitotic progression and cytokinesis. Specifically, CCDC66 depletion resulted in defective spindle assembly and orientation, kinetochore fiber stability, chromosome alignment in metaphase as well as central spindle and midbody assembly and organization in anaphase and cytokinesis. Notably, CCDC66 regulates mitotic microtubule nucleation via noncentrosomal and centrosomal pathways via recruitment of gamma-tubulin to the centrosomes and the spindle. Additionally, CCDC66 bundles microtubules in vitro and in cells by its C-terminal microtubule-binding domain. Phenotypic rescue experiments showed that the microtubule and centrosome-associated pools of CCDC66 individually or cooperatively mediate its mitotic and cytokinetic functions. Collectively, our findings identify CCDC66 as a multifaceted regulator of the nucleation and organization of the diverse mitotic and cytokinetic microtubule arrays and provide new insight into nonciliary defects that underlie ciliopathies.

The ciliopathy-linked protein CCDC66 is only known for its ciliary functions. This study reveals that CCDC66 also has extensive non-ciliary functions, localizing to the spindle poles, spindle midzone, central spindle and midbody throughout cell division, where it regulates mitosis and cytokinesis by promoting microtubule nucleation and organization.     

A mutation in aspergillus nidulans that blocks the transition from interphase to prophase

In order to develop a method for obtaining mitotic synchrony in aspergillus nidulans, we have characterized previously isolated heat-sensitive nim mutations that block the nuclear division cycle in interphase at restrictive temperature. After 3.5 h at restrictive temperature the mitotic index of a strain carrying one of these mutations, nimA5, was 0, but when this strain was subsequently shifted from restrictive to permissive temperature the mitotic index increased rapidly, reaching a maximum of 78 percent after 7.5 min. When this strain was examined electron-microscopically, mitotic spindles were absent at restrictive temperature. From these data we conclude that at restrictive temperature nimA5 blocks the nuclear division cycle at a point immediately preceding the initiation of chromosomal condensation and mitotic microtubule assembly, and upon shifting to permissive control over the initiation of microtubule assembly and chromosomal condensation in vivo through a simple temperature shift and, consequently, nimA5 should be a powerful tool for studying these processes. Electron-microscopic examination of spindles of material synchronized in this manner reveals that spindle formation, although very rapid, is gradual in the sense that spindle microtubule numbers increase as spindle formation proceeds.