Human Fidgetin is a microtubule severing the enzyme and minus-end depolymerase that regulates mitosis

Fidgetin is a member of the AAA protein superfamily with important roles in mammalian development. Here we show that human Fidgetin is a potent microtubule severing and depolymerizing the enzyme used to regulate mitotic spindle architecture, dynamics and anaphase A. In vitro, recombinant human Fidgetin severs taxol-stabilized microtubules along their length and promotes depolymerization, primarily from their minus-ends. In cells, human Fidgetin targets to centrosomes, and its depletion with siRNA significantly reduces the velocity of poleward tubulin flux and anaphase A chromatid-to-pole motion. In addition, the loss of Fidgetin induces a microtubule-dependent enlargement of mitotic centrosomes and an increase in the number and length of astral microtubules. Based on these data, we propose that human Fidgetin actively suppresses microtubule growth from and attachment to centrosomes.     

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.     

KL1 is a novel microtubule severing enzyme that regulates mitotic spindle architecture

Katanin is a microtubule severing enzyme with demonstrated roles in a variety of cellular activities including mitosis. Here we identify the closely related, but relatively uncharacterized human protein, Katanin-like 1 (KL1), as a novel mitotic regulator. Over expression of KL1 in tissue culture cells results in the complete disassembly of cellular microtubules strongly suggesting that it is an active microtubule severing protein. During mitosis, the localization of KL1 is restricted to spindle poles and is notably absent from centrosomes. This is in clear contrast to conventional Katanin whose localization extends from centrosomes onto poles. Consistent with its localization, siRNA depletion of KL1 from U2OS cells results in a specific and significant reduction in the density of microtubules at spindle poles and significantly increases spindle length. Depletion of KL1 also alters the distribution of gamma-tubulin at centrosomes/spindle poles. Despite its impact on spindle morphology, we could find no evidence that KL1 influences anaphase chromosome motility. Based on our findings, we propose that KL1-mediated microtubule severing is utilized to generate microtubule seeds within the poles and that loss of this activity alters the normal balance of motor-generated forces that determine spindle length.     

Ajuba: a new microtubule-associated protein that interacts with BUBR1 and Aurora B at kinetochores in metaphase

Background information. The role of the LIM‐domain‐containing protein Ajuba was initially described in cell adhesion and migration processes and recently in mitosis as an activator of the Aurora A kinase. Results. In the present study, we show that Ajuba localizes to centrosomes and kinetochores during mitosis. This localization is microtubule‐dependent and Ajuba binds microtubules in vitro. A microtubule regrowth assay showed that Ajuba follows nascent microtubules from centrosomes to kinetochores. Owing to its contribution to mitotic commitment and its microtubule‐dependent localization, Ajuba could also play a role during the metaphase—anaphase transition. We show that Ajuba interacts with Aurora B and BUBR1 [BUB (budding uninhibited by benomyl)‐related 1], two major components of the mitotic checkpoint. Inhibition of BUBR1 by siRNA (small interfering RNA) disrupts chromosome alignment at the metaphase plate and modifies Ajuba localization due to premature mitotic exit. Conclusions. Ajuba is a microtubule‐associated protein that collaborates with Aurora B and BUBR1 at the metaphase—anaphase transition and this may be important to ensure proper chromosome segregation.     

EB 1 immunofluorescence reveals an increase in growing astral microtubule length and number during anaphase in NRK-52E cells

Spindle positioning in animal cells is thought to rely upon the interaction of astral microtubules with the cell cortex. Information on the dynamics of astral microtubules during this process is scarce, in part because of the difficulty in visualising these microtubules by light microscopy. EB1 is a protein which specifically localises to growing microtubule distal tips. Immunostaining for EB1 therefore represents a powerful method for visualising the distribution of growing microtubule tips within cells. In this study we used EB1 immunostaining in mitotic NRK-52E cells to quantitatively analyse the length and number of growing astral microtubules during metaphase and anaphase. We observed a dramatic increase in growing astral microtubule length and number during anaphase. Furthermore, drug treatments which specifically destroyed astral microtubules resulted in an increase in misaligned anaphase but not metaphase spindles. We suggest that an anaphase-specific increase in growing astral microtubule length and number facilitates the maintenance of a correctly aligned spindle in mitotic NRK-52E cells.     

Building a spindle of the correct length in human cells requires the interaction between TPX2 and Aurora A

To assemble mitotic spindles, cells nucleate microtubules from a variety of sources including chromosomes and centrosomes. We know little about how the regulation of microtubule nucleation contributes to spindle bipolarity and spindle size. The Aurora A kinase activator TPX2 is required for microtubule nucleation from chromosomes as well as for spindle bipolarity. We use bacterial artificial chromosome-based recombineering to introduce point mutants that block the interaction between TPX2 and Aurora A into human cells. TPX2 mutants have very short spindles but, surprisingly, are still bipolar and segregate chromosomes. Examination of microtubule nucleation during spindle assembly shows that microtubules fail to nucleate from chromosomes. Thus, chromosome nucleation is not essential for bipolarity during human cell mitosis when centrosomes are present. Rather, chromosome nucleation is involved in spindle pole separation and setting spindle length. A second Aurora A-independent function of TPX2 is required to bipolarize spindles.     

Influence of M-phase chromatin on the anisotropy of microtubule asters

In many eukaryotic cells going through M-phase, a bipolar spindle is formed by microtubules nucleated from centrosomes. These microtubules, in addition to being "captured" by kinetochores, may be stabilized by chromatin in two different ways: short-range stabilization effects may affect microtubules in close contact with the chromatin, while long- range stabilization effects may "guide" microtubule growth towards the chromatin (e.g., by introducing a diffusive gradient of an enzymatic activity that affects microtubule assembly). Here, we use both meiotic and mitotic extracts from Xenopus laevis eggs to study microtubule aster formation and microtubule dynamics in the presence of chromatin. In "low-speed" meiotic extracts, in the presence of salmon sperm chromatin, we find that short-range stabilization effects lead to a strong anisotropy of the microtubule asters. Analysis of the dynamic parameters of microtubule growth show that this anisotropy arises from a decrease in the catastrophe frequency, an increase in the rescue frequency and a decrease in the growth velocity. In this system we also find evidence for long-range "guidance" effects, which lead to a weak anisotropy of the asters. Statistically relevant results on these long- range effects are obtained in "high-speed" mitotic extracts in the presence of artificially constructed chromatin stripes. We find that aster anisotropy is biased in the direction of the chromatin and that the catastrophe frequency is reduced in its vicinity. In this system we also find a surprising dependence of the catastrophe and the rescue frequencies on the length of microtubules nucleated from centrosomes: the catastrophe frequency increase and the rescue frequency decreases with microtubule length.     

Early endosomes, late endosomes, and lysosomes display distinct partitioning strategies of inheritance with similarities to Golgi-derived membranes

The pattern of inheritance of compartments of the endocytic pathway has been rarely reported, and the precise mechanism(s) are yet to be elucidated. We used antibodies reactive to early endosomes (anti-EEA1), late endosomes (anti-LBPA and anti-LAMP-1), lysosomes (anti-LAMP-1) and trans-Golgi network (TGN) (anti-GOLGA4) to examine the inheritance of these compartments in fixed human HEp-2 cells. Prior to entering M phase, these compartments display a perinuclear bias in their cytoplasmic distribution with areas of local accumulation juxtaposed to the centrosome. The location of these compartments during mitosis was examined relative to each other, the chromosomes, centrosomes and the microtubule network. During M phase early endosomes and TGN-derived compartments share overlapping subcellular distributions. A portion of these compartments display discernible clustering around the separated and migrating centrosomes in prophase. At metaphase these compartments co-localise with the mitotic spindle, are absent at the metaphase plate and do not overlay the astral microtubules. At anaphase these compartments are concentrated between shortening kinetochore microtubules and centrosomes. In addition, they appear distributed over the elongating polar microtubules in the body of the cell. From telophase and into cytokinesis these compartments concentrate around the minus ends of the constricted remnants of polar spindle microtubules and re-establish a prominent presence juxtaposed to the centrosome. In contrast, there is little evidence of movement of late endosomes and lysosomes with migrating centrosomes in prophase, and these compartments are excluded from the mitotic spindle at metaphase. However, by the end of telophase, the subcellular distribution of a portion of late endosomes and lysosomes share overlapping distributions with that of early endosomes. We conclude a portion of endosomal compartments and Golgi-derived membranes undergo ordered partitioning based on the centrosome and mitotic spindle.     

MCAK-independent functions of ch-Tog/XMAP215 in microtubule plus-end dynamics

The formation of a functional bipolar mitotic spindle is essential for genetic integrity. In human cells, the microtubule polymerase XMAP215/ch-Tog ensures spindle bipolarity by counteracting the activity of the microtubule-depolymerizing kinesin XKCM1/MCAK. Their antagonistic effects on microtubule polymerization confer dynamic instability on microtubules assembled in cell-free systems. It is, however, unclear if a similar interplay governs microtubule behavior in mammalian cells in vivo. Using real-time analysis of spindle assembly, we found that ch-Tog is required to produce or maintain long centrosomal microtubules after nuclear-envelope breakdown. In the absence of ch-Tog, microtubule assembly at centrosomes was impaired and microtubules were nondynamic. Interkinetochore distances and the lengths of kinetochore fibers were also reduced in these cells. Codepleting MCAK with ch-Tog improved kinetochore fiber length and interkinetochore separation but, surprisingly, did not rescue centrosomal microtubule assembly and microtubule dynamics. Our data therefore suggest that ch-Tog has at least two distinct roles in spindle formation. First, it protects kinetochore microtubules from depolymerization by MCAK. Second, ch-Tog plays an essential role in centrosomal microtubule assembly, a function independent of MCAK activity. Thus, the notion that the antagonistic activities of MCAK and ch-Tog determine overall microtubule stability is too simplistic to apply to human cells.     

Microtubule arrays present during the syncytial and cellular blastoderm stages of the early Drosophila embryo

The organization of microtubules within the surface caps of Drosophila embryos is described for the mitotic cycles of the syncytial blastoderm stage (particularly cycle 10), and for the subsequent cellularization process. Tubulin was labelled with the well characterized monoclonal antibody YL 1/2 (Kilmartin et al., J cell biol 93 (1982) 576). Each surface cap was found to contain an array of microtubules running around the nucleus. The microtubules originated at prominent centrosomes located close to the apical surface of each cap nucleus. During mitosis the spindle microtubules stained strongly for tubulin. A novel finding was that the spindle microtubules of the interzone region appeared to reduce their connections with the centrosomes at the end of anaphase. The spindle remnant remained in position during telophase but then became smaller in size, disappearing by interphase. At this phase of the cell cycle duplication of the aster centrosomes occurred. The cellular blastoderm stage was marked by a change in the main axis of microtubule orientation. The centrosomes of each cap separated somewhat and formed initiation centres for the development of a well developed basket of microtubules around each nucleus, but now perpendicular to the surface. The microtubule baskets were seen to extend in parallel with nuclear elongation, but not in concert with growth of the cell membranes, which extended some way beneath the bases of the nuclei.     

Downregulation of protein 4.1R, a mature centriole protein, disrupts centrosomes, alters cell cycle progression, and perturbs mitotic spindles and anaphase

Centrosomes nucleate and organize interphase microtubules and are instrumental in mitotic bipolar spindle assembly, ensuring orderly cell cycle progression with accurate chromosome segregation. We report that the multifunctional structural protein 4.1R localizes at centrosomes to distal/subdistal regions of mature centrioles in a cell cycle-dependent pattern. Significantly, 4.1R-specific depletion mediated by RNA interference perturbs subdistal appendage proteins ninein and outer dense fiber 2/cenexin at mature centrosomes and concomitantly reduces interphase microtubule anchoring and organization. 4.1R depletion causes G(1) accumulation in p53-proficient cells, similar to depletion of many other proteins that compromise centrosome integrity. In p53-deficient cells, 4.1R depletion delays S phase, but aberrant ninein distribution is not dependent on the S-phase delay. In 4.1R-depleted mitotic cells, efficient centrosome separation is reduced, resulting in monopolar spindle formation. Multipolar spindles and bipolar spindles with misaligned chromatin are also induced by 4.1R depletion. Notably, all types of defective spindles have mislocalized NuMA (nuclear mitotic apparatus protein), a 4.1R binding partner essential for spindle pole focusing. These disruptions contribute to lagging chromosomes and aberrant microtubule bridges during anaphase/telophase. Our data provide functional evidence that 4.1R makes crucial contributions to the structural integrity of centrosomes and mitotic spindles which normally enable mitosis and anaphase to proceed with the coordinated precision required to avoid pathological events.     

Chromatids segregate without centrosomes during Caenorhabditis elegans mitosis in a Ran- and CLASP-dependent manner

During mitosis, chromosomes are connected to a microtubule-based spindle. Current models propose that displacement of the spindle poles and/or the activity of kinetochore microtubules generate mechanical forces that segregate sister chromatids. Using laser destruction of the centrosomes during Caenorhabditis elegans mitosis, we show that neither of these mechanisms is necessary to achieve proper chromatid segregation. Our results strongly suggest that an outward force generated by the spindle midzone, independently of centrosomes, is sufficient to segregate chromosomes in mitotic cells. Using mutant and RNAi analysis, we show that the microtubule-bundling protein SPD-1/MAP-65 and BMK-1/kinesin-5 act as a brake opposing the force generated by the spindle midzone. Conversely, we identify a novel role for two microtubule-growth and nucleation agents, Ran and CLASP, in the establishment of the centrosome-independent force during anaphase. Their involvement raises the interesting possibility that microtubule polymerization of midzone microtubules is continuously required to sustain chromosome segregation during mitosis.     

CSPP and CSPP-L associate with centrosomes and microtubules and differently affect microtubule organization

We recently described the identification of a centrosome/spindle pole associated protein, CSPP, involved in cell cycle progression. Here we report a CSPP isoform denoted CSPP-L, with a 294 amino acids longer N-terminus and a 51 amino acids insertion located in the coiled-coil mid-domain. Expression analysis indicates an inverse cell cycle dependent regulation. CSPP mRNA expression is highest in G1 whereas CSPP-L expression is highest in G2/M. Ectopic expression of CSPP-L impairs cell cycle progression weaker in G1 than CSPP. Furthermore, normal mitotic phenotypes were observed in CSPP-L but not in CSPP transfectants. CSPP-L relocates from spindle microtubules and poles in metaphase to the mid-spindle in anaphase and concentrates at the mid-body in telophase/cytokinesis. CSPP-L high-expressing mitotic cells were predominantly characterized by lagging chromosomes or monopolar spindles, in contrast to the predominant multipolar spindles observed with CSPP expression. The different effects of CSPP and CSPP-L on microtubule organization in mitosis depend on the coiled-coil mid-domain insertion. The common C-terminal domain is required to repress that activity until mitosis. Notably, this C-terminal domain alone can associate with centrosomes in a microtubule independent manner. Taken together, CSPP and CSPP-L interact with centrosomes and microtubules and can differently affect microtubule organization.     

WDR62 localizes katanin at spindle poles to ensure synchronous chromosome segregation

Mutations in the WDR62 gene cause primary microcephaly, a pathological condition often associated with defective cell division that results in severe brain developmental defects. The precise function and localization of WDR62 within the mitotic spindle is, however, still under debate, as it has been proposed to act either at centrosomes or on the mitotic spindle. Here we explored the cellular functions of WDR62 in human epithelial cell lines using both short-term siRNA protein depletions and long-term CRISPR/Cas9 gene knockouts. We demonstrate that WDR62 localizes at spindle poles, promoting the recruitment of the microtubule-severing enzyme katanin. Depletion or loss of WDR62 stabilizes spindle microtubules due to insufficient microtubule minus-end depolymerization but does not affect plus-end microtubule dynamics. During chromosome segregation, WDR62 and katanin promote efficient poleward microtubule flux and favor the synchronicity of poleward movements in anaphase to prevent lagging chromosomes. We speculate that these lagging chromosomes might be linked to developmental defects in primary microcephaly.     

The chromosomal basis of meiotic acentrosomal spindle assembly and function in oocytes

Several aspects of meiosis are impacted by the absence of centrosomes in oocytes. Here, we review four aspects of meiosis I that are significantly affected by the absence of centrosomes in oocyte spindles. One, microtubules tend to assemble around the chromosomes. Two, the organization of these microtubules into a bipolar spindle is directed by the chromosomes. Three, chromosome bi-orientation and attachment to microtubules from the correct pole require modification of the mechanisms used in mitotic cells. Four, chromosome movement to the poles at anaphase cannot rely on polar anchoring of spindle microtubules by centrosomes. Overall, the chromosomes are more active participants during acentrosomal spindle assembly in oocytes, compared to mitotic and male meiotic divisions where centrosomes are present. The chromosomes are endowed with information that can direct the meiotic divisions and dictate their own behavior in oocytes. Processes beyond those known from mitosis appear to be required for their bi-orientation at meiosis I. As mitosis occurs without centrosomes in many systems other than oocytes, including all plants, the concepts discussed here may not be limited to oocytes. The study of meiosis in oocytes has revealed mechanisms that are operating in mitosis and will probably continue to do so.     

mini spindles: A gene encoding a conserved microtubule-associated protein required for the integrity of the mitotic spindle in Drosophila.

We describe a new Drosophila gene, mini spindles (msps) identified in a cytological screen for mitotic mutant. Mutation in msps disrupts the structural integrity of the mitotic spindle, resulting in the formation of one or more small additional spindles in diploid cells. Nucleation of microtubules from centrosomes, metaphase alignment of chromosomes, or the focusing of spindle poles appears much less affected. The msps gene encodes a 227-kD protein with high similarity to the vertebrate microtubule-associated proteins (MAPs), human TOGp and Xenopus XMAP215, and with limited similarity to the Dis1 and STU2 proteins from fission yeast and budding yeast. Consistent with their sequence similarity, Msps protein also associates with microtubules in vitro. In the embryonic division cycles, Msps protein localizes to centrosomal regions at all mitotic stages, and spreads over the spindles during metaphase and anaphase. The absence of centrosomal staining in interphase of the cellularized embryos suggests that the interactions between Msps protein and microtubules or centrosomes may be regulated during the cell cycle.     

Microtubule disassembly and inhibition of mitosis by a novel synthetic pharmacophore

Microtubule drugs, which block cell cycle progression through mitosis, have seen widespread use in cancer chemotherapies. Although microtubules are subject to regulation by signal transduction mechanisms, their pharmacological modulation has so far relied on compounds that bind to the tubulin subunit. A new microtubule pharmacophore, diphenyleneiodonium, causing disassembly of the microtubule cytoskeleton is described here. Although this synthetic compound does not affect the assembly state of purified microtubules, it profoundly suppresses microtubule assembly in vivo, causes paclitaxel-stabilized microtubules to cluster around the centrosomes, and selectively disassembles dynamic microtubules. Similar to other microtubule drugs, this new pharmacophore blocks mitotic spindle assembly and mitotic cell division.     

Regulated assembly of the mitotic spindle: a perspective from two ends

Chromosome segregration and cell division requires the regulated assembly of the mitotic spindle apparatus. This mitotic spindle is composed of condensed chromosomes attached to a dynamic array of microtubules. The microtubule array is nucleated by centrosomes and organized by associated structural and motor proteins. Mechanical linkages between sister chromatids and microtubules are critical for spindle assembly and chromosome segregation. Defects in either chromosome or centrosome segregation can lead to aneuploidy and are correlated with cancer progression. In this review, we discuss current models of how centrosomes and chromosomes organize the spindle for their equal distribution to each daughter cell.     

Plant microtubule studies: past and present

Here, I briefly review historical and morphological aspects of plant microtubule studies in land plants. Microtubules are formed from tubulins, and the polymeric configurations appear as singlet, doublet, and triplet microtubules. Doublet microtubules occur in the axoneme of cilia and flagella, and triplet microtubules occur in the basal bodies and centrosomes. Doublet and triplet microtubules are lost in all angiosperms and some gymnosperms that do not possess flagellated sperm. In land plants with flagellated sperm, centriolar centrosomes transform into basal bodies during spermatogenesis. In flowering plants, however, most male gametes (sperm) are conveyed to eggs without the benefit of cilia or flagella; thus, higher plants lack centriolar centrosome and doublet and triplet microtubules. The loss of centriolar centrosomes from the life cycle of flowering plants may have influenced the evolution of the plant microtubule system. Comparison of mitotic apparatuses in basal land plants and flowering plants illuminates the evolutionary transition from the centriolar microtubule system to the acentriolar microtubule system.     

ZYG-9, A Caenorhabditis elegans Protein Required for Microtubule Organization and Function, Is a Component of Meiotic and Mitotic Spindle Poles

We describe the molecular characterization of zyg-9, a maternally acting gene essential for microtubule organization and function in early Caenorhabditis elegans embryos. Defects in zyg-9 mutants suggest that the zyg-9 product functions in the organization of the meiotic spindle and the formation of long microtubules. One-cell zyg-9 embryos exhibit both meiotic and mitotic spindle defects. Meiotic spindles are disorganized, pronuclear migration fails, and the mitotic apparatus forms at the posterior, orients incorrectly, and contains unusually short microtubules. We find that zyg-9 encodes a component of the meiotic and mitotic spindle poles. In addition to the strong staining of spindle poles, we consistently detect staining in the region of the kinetochore microtubules at metaphase and early anaphase in mitotic spindles. The ZYG-9 signal at the mitotic centrosomes is not reduced by nocodazole treatment, indicating that ZYG-9 localization to the mitotic centrosomes is not dependent upon long astral microtubules. Interestingly, in embryos lacking an organized meiotic spindle, produced either by nocodazole treatment or mutations in the mei-1 gene, ZYG-9 forms a halo around the meiotic chromosomes. The protein sequence shows partial similarity to a small set of proteins that also localize to spindle poles, suggesting a common activity of the proteins.