The stereotypical function of kinesin superfamily motors is to transport cargo along microtubules. However, some kinesins also shape the microtubule track by regulating microtubule assembly and disassembly. Recent work has shown that the kinesin-8 family of motors are key regulators of cellular microtubule length. The studied kinesin-8s are highly processive motors that walk towards the microtubule plus-end. Once at plus-ends, they have complex effects on polymer dynamics: kinesin-8s either destabilize or stabilize microtubules, depending on the context. This review will focus on the mechanisms underlying kinesin-8-microtubule interactions and microtubule length control. We will compare and contrast kinesin-8s with the other major microtubule-regulating kinesins (kinesin-4 and kinesin-13), to survey the current understanding of the diverse ways that kinesins control microtubule dynamics.
kinesin-8; kinesin-13; kinesin-4; microtubule dynamics; microtubule depolymerase
During the oocyte-to-embryo transition in Drosophila, degradation of the Polo kinase inhibitor, Matrimony, depends on Cortex, a meiosis-specific form of the Anaphase Promoting Complex/Cyclosome that is required for the oocyte's normal transition from meiosis to mitosis.
Oocytes are stockpiled with proteins and mRNA that are required to drive the initial mitotic divisions of embryogenesis. But are there proteins specific to meiosis whose levels must be decreased to begin embryogenesis properly? The Drosophila protein Cortex (Cort) is a female, meiosis-specific activator of the Anaphase Promoting Complex/Cyclosome (APC/C), an E3 ubiquitin ligase. We performed immunoprecipitation of Cortex followed by mass spectrometry, and identified the Polo kinase inhibitor Matrimony (Mtrm) as a potential interactor with Cort. In vitro binding assays showed Mtrm and Cort can bind directly. We found Mtrm protein levels to be reduced dramatically during the oocyte-to-embryo transition, and this downregulation did not take place in cort mutant eggs, consistent with Mtrm being a substrate of APCCort. We showed that Mtrm is subject to APCCort-mediated proteasomal degradation and have identified a putative APC/C recognition motif in Mtrm that when mutated partially stabilized the protein in the embryo. Furthermore, overexpression of Mtrm in the early embryo caused aberrant nuclear divisions and developmental defects, and these were enhanced by decreasing levels of active Polo. These data indicate APCCort ubiquitylates Mtrm at the oocyte-to-embryo transition, thus preventing excessive inhibition of Polo kinase activity due to Mtrm's presence.
Despite their many differences, the meiotic and mitotic divisions of the early embryo take place within the same cytoplasmic space. The oocyte-to-embryo transition is the process by which an oocyte, which initially undergoes meiosis, becomes “adapted” to support the rapid mitotic divisions of embryogenesis. This involves fertilization as well as the stockpiling of proteins and mRNA for the transcriptionally silent early embryo. The Anaphase Promoting Complex/Cyclosome (APC/C) is a large protein complex that is active during both mitosis and meiosis and is responsible for targeting certain proteins for degradation. The discovery of the existence of APC/C activators that are present only during meiosis hinted at the possibility that this complex also functions to regulate protein degradation during the oocyte-to-embryo transition. Here we study Cortex, a female- and meiosis-specific activator of the APC/C in the fruit fly Drosophila melanogaster. We find that Cortex activity is necessary for the degradation of Matrimony, a key regulator of female meiosis in Drosophila. Matrimony itself inhibits Polo kinase, another important regulator of both mitosis and meiosis that also functions in chromosome segregation, centrosome dynamics, and cytokinesis. When excess Matrimony protein is not removed from the early embryo, developmental defects arise. Together our findings demonstrate that the precise regulation of Matrimony levels in the egg is necessary for the switch from meiosis to mitosis.
The dynein partner dynactin not only binds to microtubules, but is found to potently influence microtubule dynamics in neurons.
Regulation of microtubule dynamics in neurons is critical, as defects in the microtubule-based transport of axonal organelles lead to neurodegenerative disease. The microtubule motor cytoplasmic dynein and its partner complex dynactin drive retrograde transport from the distal axon. We have recently shown that the p150Glued subunit of dynactin promotes the initiation of dynein-driven cargo motility from the microtubule plus-end. Because plus end-localized microtubule-associated proteins like p150Glued may also modulate the dynamics of microtubules, we hypothesized that p150Glued might promote cargo initiation by stabilizing the microtubule track. Here, we demonstrate in vitro using assembly assays and TIRF microscopy, and in primary neurons using live-cell imaging, that p150Glued is a potent anti-catastrophe factor for microtubules. p150Glued alters microtubule dynamics by binding both to microtubules and to tubulin dimers; both the N-terminal CAP-Gly and basic domains of p150Glued are required in tandem for this activity. p150Glued is alternatively spliced in vivo, with the full-length isoform including these two domains expressed primarily in neurons. Accordingly, we find that RNAi of p150Glued in nonpolarized cells does not alter microtubule dynamics, while depletion of p150Glued in neurons leads to a dramatic increase in microtubule catastrophe. Strikingly, a mutation in p150Glued causal for the lethal neurodegenerative disorder Perry syndrome abrogates this anti-catastrophe activity. Thus, we find that dynactin has multiple functions in neurons, both activating dynein-mediated retrograde axonal transport and enhancing microtubule stability through a novel anti-catastrophe mechanism regulated by tissue-specific isoform expression; disruption of either or both of these functions may contribute to neurodegenerative disease.
Microtubules are polymers of tubulin that undergo successive cycles of growth and shrinkage so that the cell can maintain a stable yet adaptable cytoskeleton. In neurons, the microtubule motor protein dynein and its partner complex dynactin drive retrograde transport along microtubules from the distal axon towards the cell body. In addition to binding to dynein, the p150Glued subunit of dynactin independently binds directly to microtubules. We hypothesized that by binding to microtubules, p150Glued might also alter microtubule dynamics. We demonstrate using biochemistry and microscopy in vitro and in cells that p150Glued stabilizes microtubules by inhibiting the transition from growth to shrinkage. We show that specific domains of p150Glued encoded by neuronally enriched splice-forms are necessary for this activity. Although depletion of p150Glued in nonpolarized cells does not alter microtubule dynamics, depletion of endogenous p150Glued in neurons leads to dramatic microtubule instability. Strikingly, a mutation in p150Glued known to cause the neurodegenerative disorder Perry syndrome abolishes this activity. In summary, we identified a previously unappreciated function of dynactin in direct regulation of the microtubule cytoskeleton. This activity may enhance generic microtubule stability in the cell, but could be especially important in specific areas of the cell where dynactin and dynein are loaded onto microtubules.
Cellular defects that impair the fidelity of mitosis promote chromosome missegregation and aneuploidy. Increasing evidence reveals that errors in mitosis can also promote the direct and indirect acquisition of DNA damage and chromosome breaks. Consequently, deregulated cell division can devastate the integrity of the normal genome and unleash a variety of oncogenic stimuli that may promote transformation. Recent work has shed light on the mechanisms that link abnormal mitosis with the development of DNA damage, how cells respond to such affronts, and the potential impact on tumorigenesis.
The septin-associated kinase Gin4 is required for the localization and activation of Bnr1, and the septin Shs1 is essential for Bnr1 activation. The loss of Gin4 or Shs1 phenocopies the loss of Bnr1; these defects are suppressed by constitutive activation of Bnr1. The data reveal novel regulatory links between the actin and septin cytoskeletons.
Formin-family proteins promote the assembly of linear actin filaments and are required to generate cellular actin structures, such as actin stress fibers and the cytokinetic actomyosin contractile ring. Many formin proteins are regulated by an autoinhibition mechanism involving intramolecular binding of a Diaphanous inhibitory domain and a Diaphanous autoregulatory domain. However, the activation mechanism for these Diaphanous-related formins (DRFs) is not completely understood. Although small GTPases play an important role in relieving autoinhibition, other factors likely contribute. Here we describe a requirement for the septin Shs1 and the septin-associated kinase Gin4 for the localization and in vivo activity of the budding yeast DRF Bnr1. In budding yeast strains in which the other formin, Bni1, is conditionally inactivated, the loss of Gin4 or Shs1 results in the loss of actin cables and cell death, similar to the loss of Bnr1. The defects in these strains can be suppressed by constitutive activation of Bnr1. Gin4 is involved in both the localization and activation of Bnr1, whereas the septin Shs1 is required for Bnr1 activation but not its localization. Gin4 promotes the activity of Bnr1 independently of the Gin4 kinase activity, and Gin4 lacking its kinase domain binds to the critical localization region of Bnr1. These data reveal novel regulatory links between the actin and septin cytoskeletons.
The kinesin-8 family of microtubule motors plays a critical role in microtubule length control in cells. These motors have complex effects on microtubule dynamics: they destabilize growing microtubules yet stabilize shrinking microtubules. The budding yeast kinesin-8, Kip3, accumulates on plus ends of growing but not shrinking microtubules. Here we identify an essential role of the tail domain of Kip3 in mediating both its destabilizing and stabilizing activities. The Kip3-tail promotes Kip’s accumulation at the plus ends and facilitates the destabilizing effect of Kip3. However, the Kip3-tail also inhibits microtubule shrinkage and is required for promoting microtubule rescue by Kip3. These effects of the tail domain are likely to be mediated by the tubulin- and microtubule-binding activities that we describe. We propose a concentration-dependent model for the coordination of the destabilizing and stabilizing activities of Kip3 and discuss its relevance to cellular microtubule organization.
Whether whole-chromosome aneuploidy promotes tumorigenesis has been controversial, in large part because of the paucity of insight into underlying mechanisms. Here we identify a mechanism by which mitotic chromosome segregation errors generate DNA breaks via the formation of structures called micronuclei. Whole chromosome-containing micronuclei form when mitotic errors produce lagging chromosomes. We tracked the fate of newly generated micronuclei and found that they undergo defective and asynchronous DNA replication, resulting in DNA damage and frequently pulverization of the chromosome in the micronucleus. Micronuclei can persist in cells over several generations but the chromosome in the micronucleus can also be distributed to daughter nuclei. Thus, chromosome segregation errors potentially lead to mutations and chromosome rearrangements that can integrate into the genome. Pulverization of chromosomes in micronuclei may also be one explanation for “chromothripsis” in cancer and developmental disorders, where isolated chromosomes or chromosome arms undergo massive local DNA breakage and rearrangement.
Centrioles are microtubule-derived structures that are essential to form centrosomes, cilia and flagella. The centrosome is the major microtubule organiser in animal cells, participating in a variety of processes from cell polarization to cell division, while cilia and flagella contribute to several mechanisms in eukaryotic cells from motility to sensing. Although it was suggested more than a century ago that these microtubule-derived structures are involved in human disease, the molecular bases of this association have only recently been discovered. Surprisingly, there is very little overlap between the genes affected in the different diseases, suggesting there are tissue-specific requirements for these microtubule-derived structures. Knowledge of these requirements and disease mechanisms has opened new avenues for therapeutical strategies. Here, we give an overview of recent developments in this field focusing on cancer, diseases of brain development and ciliopathies.
Members of chromosome passenger complex BIR1 and SLI15 suppress the chromosome segregation defect of bub1Δ and sgo1Δ. Neither Bub1 nor Sgo1 is required for CPC activity. This study found genetic interaction between Mps1 and Sgo1. Mps1 governs localization of Sgo1. Bub1, Sgo1, and Mps1 function in parallel to the CPC in correction of syntelic attachments.
The conserved mitotic kinase Bub1 performs multiple functions that are only partially characterized. Besides its role in the spindle assembly checkpoint and chromosome alignment, Bub1 is crucial for the kinetochore recruitment of multiple proteins, among them Sgo1. Both Bub1 and Sgo1 are dispensable for growth of haploid and diploid budding yeast, but they become essential in cells with higher ploidy. We find that overexpression of SGO1 partially corrects the chromosome segregation defect of bub1Δ haploid cells and restores viability to bub1Δ tetraploid cells. Using an unbiased high-copy suppressor screen, we identified two members of the chromosomal passenger complex (CPC), BIR1 (survivin) and SLI15 (INCENP, inner centromere protein), as suppressors of the growth defect of both bub1Δ and sgo1Δ tetraploids, suggesting that these mutants die due to defects in chromosome biorientation. Overexpression of BIR1 or SLI15 also complements the benomyl sensitivity of haploid bub1Δ and sgo1Δ cells. Mutants lacking SGO1 fail to biorient sister chromatids attached to the same spindle pole (syntelic attachment) after nocodazole treatment. Moreover, the sgo1Δ cells accumulate syntelic attachments in unperturbed mitoses, a defect that is partially corrected by BIR1 or SLI15 overexpression. We show that in budding yeast neither Bub1 nor Sgo1 is required for CPC localization or affects Aurora B activity. Instead we identify Sgo1 as a possible partner of Mps1, a mitotic kinase suggested to have an Aurora B–independent function in establishment of biorientation. We found that Sgo1 overexpression rescues defects caused by metaphase inactivation of Mps1 and that Mps1 is required for Sgo1 localization to the kinetochore. We propose that Bub1, Sgo1, and Mps1 facilitate chromosome biorientation independently of the Aurora B–mediated pathway at the budding yeast kinetochore and that both pathways are required for the efficient turnover of syntelic attachments.
Similar to clustering of extra centrosomes in cancer cells, HURP promotes microtubule stability and sorts MTOCs into distinct poles during meiosis.
In contrast to somatic cells, formation of acentriolar meiotic spindles relies on the organization of microtubules (MTs) and MT-organizing centers (MTOCs) into a stable bipolar structure. The underlying mechanisms are still unknown. We show that this process is impaired in hepatoma up-regulated protein (Hurp) knockout mice, which are viable but female sterile, showing defective oocyte divisions. HURP accumulates on interpolar MTs in the vicinity of chromosomes via Kinesin-5 activity. By promoting MT stability in the spindle central domain, HURP allows efficient MTOC sorting into distinct poles, providing bipolarity establishment and maintenance. Our results support a new model for meiotic spindle assembly in which HURP ensures assembly of a central MT array, which serves as a scaffold for the genesis of a robust bipolar structure supporting efficient chromosome congression. Furthermore, HURP is also required for the clustering of extra centrosomes before division, arguing for a shared molecular requirement of MTOC sorting in mammalian meiosis and cancer cell division.
Mps1, a dual-specificity kinase, is required for the proper functioning of the spindle assembly checkpoint and the maintenance of chromosomal stability. As Mps1 function has been implicated in numerous phases of the cell cycle, it is expected the development of a potent, selective small molecule inhibitor of Mps1 would greatly facilitate dissection of Mps1-related biology. We describe the cellular effects and Mps1 co-crystal structures of novel, selective small molecule inhibitors of Mps1. Consistent with RNAi studies, chemical inhibition of Mps1 leads to defects in Mad1 and Mad2 establishment at unattached kinetochores, decreased Aurora B kinase activity, premature mitotic exit, and gross aneuploidy, without any evidence of centrosome duplication defects. However, in U2OS cells possessing extra centrosomes, an abnormality found in some cancers, Mps1 inhibition increases the frequency of multipolar mitoses. Lastly, Mps1 inhibitor treatment resulted in a decrease in cancer cell viability.
Fanconi anemia (FA) is a genomic instability disorder characterized by bone marrow failure and cancer predisposition. FA is caused by mutations in any one of several genes that encode proteins cooperating in a repair pathway and is required for cellular resistance to DNA crosslinking agents. Recent studies suggest that the FA pathway may also play a role in mitosis, since FANCD2 and FANCI, the 2 key FA proteins, are localized to the extremities of ultrafine DNA bridges (UFBs), which link sister chromatids during cell division. However, whether FA proteins regulate cell division remains unclear. Here we have shown that FA pathway–deficient cells display an increased number of UFBs compared with FA pathway–proficient cells. The UFBs were coated by BLM (the RecQ helicase mutated in Bloom syndrome) in early mitosis. In contrast, the FA protein FANCM was recruited to the UFBs at a later stage. The increased number of bridges in FA pathway–deficient cells correlated with a higher rate of cytokinesis failure resulting in binucleated cells. Binucleated cells were also detectable in primary murine FA pathway–deficient hematopoietic stem cells (HSCs) and bone marrow stromal cells from human patients with FA. Based on these observations, we suggest that cytokinesis failure followed by apoptosis may contribute to bone marrow failure in patients with FA.
A growing body of evidence indicates that early mitotic inhibitor 1 (Emi1) is essential for genomic stability, but how this function relates to embryonic development and cancer pathogenesis remains unclear. We have identified a zebrafish mutant line in which deficient emi1 gene expression results in multilineage hematopoietic defects and widespread developmental defects that are p53 independent. Cell cycle analyses of Emi1-depleted zebrafish or human cells showed chromosomal rereplication, and metaphase preparations from mutant zebrafish embryos revealed rereplicated, unsegregated chromosomes and polyploidy. Furthermore, EMI1-depleted mammalian cells relied on topoisomerase IIα-dependent mitotic decatenation to progress through metaphase. Interestingly, the loss of a single emi1 allele in the absence of p53 enhanced the susceptibility of adult fish to neural sheath tumorigenesis. Our results cast Emi1 as a critical regulator of genomic fidelity during embryogenesis and suggest that the factor may act as a tumor suppressor.
Mammalian Bicaudal D2 is the missing molecular link between cytoplasmic motor proteins and the nucleus during nuclear positioning prior to the onset of mitosis.
BICD2 is one of the two mammalian homologues of the Drosophila Bicaudal D, an evolutionarily conserved adaptor between microtubule motors and their cargo that was previously shown to link vesicles and mRNP complexes to the dynein motor. Here, we identified a G2-specific role for BICD2 in the relative positioning of the nucleus and centrosomes in dividing cells. By combining mass spectrometry, biochemical and cell biological approaches, we show that the nuclear pore complex (NPC) component RanBP2 directly binds to BICD2 and recruits it to NPCs specifically in G2 phase of the cell cycle. BICD2, in turn, recruits dynein-dynactin to NPCs and as such is needed to keep centrosomes closely tethered to the nucleus prior to mitotic entry. When dynein function is suppressed by RNA interference-mediated depletion or antibody microinjection, centrosomes and nuclei are actively pushed apart in late G2 and we show that this is due to the action of kinesin-1. Surprisingly, depletion of BICD2 inhibits both dynein and kinesin-1-dependent movements of the nucleus and cytoplasmic NPCs, demonstrating that BICD2 is needed not only for the dynein function at the nuclear pores but also for the antagonistic activity of kinesin-1. Our study demonstrates that the nucleus is subject to opposing activities of dynein and kinesin-1 motors and that BICD2 contributes to nuclear and centrosomal positioning prior to mitotic entry through regulation of both dynein and kinesin-1.
Bidirectional microtubule-based transport is responsible for the positioning of a large variety of cellular organelles, but the molecular mechanisms underlying the recruitment of microtubule-based motors to their cargoes and their activation remain poorly understood. In particular, the molecular players involved in the important processes of nuclear and centrosomal positioning prior to the onset of cell division are not known. In this study we focus on the function of one of the mammalian homologues of Drosophila Bicaudal D, an adaptor for the microtubule minus-end-directed dynein-dynactin motor complex. Previously, Drosophila Bicaudal D and its mammalian homologues were shown to act as linkers between the dynein motor and mRNP complexes or secretory vesicles. Here, we identify a new cargo for mammalian Bicaudal D2 (BICD2)–the nucleus. We show that BICD2 specifically binds to nuclear pore complexes in cells in G2 phase of the cell division cycle. We also show that this interaction is required for G2-specific recruitment of dynein to the nuclear envelope and thus for proper positioning of the nucleus relative to centrosomes prior to the onset of mitosis. Further, our findings demonstrate that the motor protein kinesin-1 opposes dynein's activity during this process and requires BICD2 for its activity. Our study therefore reveals BICD2 as the critical molecular adaptor that allows molecular motors to regulate nuclear and centrosomal positioning before cell division.
Chromosome instability (CIN) is a hallmark of many tumors and correlates with the presence of extra centrosomes1-4. However, a direct mechanistic link between extra centrosomes and CIN has not been established. It has been proposed that extra centrosomes generate CIN by promoting multipolar anaphase, a highly abnormal division that produces 3 or more aneuploid daughter cells. Here, we use long-term live-cell imaging to demonstrate that cells with multiple centrosomes rarely undergo multipolar cell divisions, and the progeny of these divisions are typically inviable. Thus, multipolar divisions cannot explain observed rates of CIN. By contrast, we observe that CIN cells with extra centrosomes routinely undergo bipolar cell divisions, but display a significantly elevated frequency of lagging chromosomes during anaphase. To define the mechanism underlying this mitotic defect, we generated cells that differ only in their centrosome number. We demonstrate that extra centrosomes alone are sufficient to promote chromosome missegregation during bipolar cell division. These segregation errors are a consequence of cells passing through a transient ‘multipolar spindle intermediate’ in which merotelic kinetochore-microtubule attachment errors accumulate prior to centrosome clustering and anaphase. These findings provide a direct mechanistic link between extra centrosomes and CIN, two common characteristics of solid tumors. We propose that this mechanism may be a common underlying cause of CIN in human cancer.
syntelic; tetraploid; aneuploid; mitosis
Aneuploidy is a hallmark of cancer cells and is assumed to play a causative role. This relationship is dissected in a yeast, with results that show that anueploidy can be removed, but cells maintain their proliferative advantage.
Cancer cells have acquired mutations that alter their growth. Aneuploidy that typify cancer cells are often assumed to contribute to the abnormal growth characteristics. Here we test the idea of a link between aneuploidy and mutations allowing improved growth, using Saccharomyces cerevisiae containing a mcm4 helicase allele that was shown to cause cancer in mice. Yeast bearing this mcm4 allele are prone to undergoing a “hypermutable phase” characterized by a changing karyotype, ultimately yielding progeny with improved growth properties. When such progeny are returned to a normal karyotype by mating, their improved growth remains. Genetic analysis shows their improved growth is due to mutations in just a few loci. In sum, the effects of the mcm4 allele in mice are recapitulated in yeast, and the aneuploidy is not required to maintain improved growth.
Aneuploidy, an abnormality in chromosome number and structure, occurs commonly in cancers and has been suggested to be required to maintain accelerated cell proliferation. However, this hypothesis remains untested as it is not possible to selectively remove the acquired aneuploidy in cells that already have altered growth. Using a yeast model bearing mcm4Chaos3, an allele that causes mammary tumors in mice, these technical hurdles in animal cells can be overcome. We show that aneuploidy is not responsible for accelerated proliferation in yeast but mutations in just a few loci are. This study provides an excellent example of how a complex disease can be dissected in a simple model organism, and that the information extracted from yeast may be used to guide mammalian studies.
Self-regulated movement of Polo-like kinase 1 to the midzone of the mitotic spindle initiates a local signaling cascade that activates the cell division machinery at the cell's equator.
Animal cells initiate cytokinesis in parallel with anaphase onset, when an actomyosin ring assembles and constricts through localized activation of the small GTPase RhoA, giving rise to a cleavage furrow. Furrow formation relies on positional cues provided by anaphase spindle microtubules (MTs), but how such cues are generated remains unclear. Using chemical genetics to achieve both temporal and spatial control, we show that the self-organized delivery of Polo-like kinase 1 (Plk1) to the midzone and its local phosphorylation of a MT-bound substrate are critical for generating this furrow-inducing signal. When Plk1 was active but unable to target itself to this equatorial landmark, both cortical RhoA recruitment and furrow induction failed to occur, thus recapitulating the effects of anaphase-specific Plk1 inhibition. Using tandem mass spectrometry and phosphospecific antibodies, we found that Plk1 binds and directly phosphorylates the HsCYK-4 subunit of centralspindlin (also known as MgcRacGAP) at the midzone. At serine 157, this modification creates a major docking site for the tandem BRCT repeats of the Rho GTP exchange factor Ect2. Cells expressing only a nonphosphorylatable form of HsCYK-4 failed to localize Ect2 at the midzone and were severely impaired in cleavage furrow formation, implying that HsCYK-4 is Plk1's rate-limiting target upstream of RhoA. Conversely, tethering an inhibitor-resistant allele of Plk1 to HsCYK-4 allowed furrows to form despite global inhibition of all other Plk1 molecules in the cell. Our findings illuminate two key mechanisms governing the initiation of cytokinesis in human cells and illustrate the power of chemical genetics to probe such regulation both in time and space.
During mitosis, the separation of duplicated chromosomes and subsequent cytokinesis (cell division) are tightly coupled processes. Cytokinesis must occur not only after chromosomes have separated but also in the physical space between the chromosomes, so that each daughter cell inherits the appropriate genetic material. The mechanisms responsible for this cellular choreography are poorly understood, however. We used chemical genetics to dissect the role of a key regulator of cell division, Polo-like kinase 1 (Plk1) in human cells. We show that, contrary to previous models, the ability of Plk1 to seek out microtubules that lie between the separated chromosomes (so-called midzone microtubules) provides the cell with an affirmative command to divide. Once assembled at this landmark, Plk1 phosphorylates HsCYK-4, a component of the centralspindlin complex (so named because it assembles at the spindle midzone) and enables binding between HsCYK-4 and Ect2, another regulator of cell division. Bound Ect2 then communicates with the machinery that assembles the actin- and myosin-based contractile ring, leading to division of the cell into two daughters. Our work therefore reveals new insights into how Plk1 temporally and spatially orchestrates division of human cells.
Polo-like kinase 1 promotes assembly of the contractile ring that divides a cell in two by creating a docking site for the RhoA activator Ect2 on the Cyk-4-containing centralspindlin complex at the midzone of the mitotic spindle.
To complete cell division with high fidelity, cytokinesis must be coordinated with chromosome segregation. Mammalian Polo-like kinase 1, Plk1, may function as a critical link because it is required for chromosome segregation and establishment of the cleavage plane following anaphase onset. A central spindle–localized pool of the RhoGEF Ect2 promotes activation of the small GTPase RhoA, which drives contractile ring assembly at the equatorial cortex. Here, we have investigated how Plk1 promotes the central spindle recruitment of Ect2. Plk1 phosphorylates the noncatalytic N terminus of the RhoGAP HsCyk-4 at the central spindle, creating a phospho-epitope recognized by the BRCA1 C-terminal (BRCT) repeats of Ect2. Failure to phosphorylate HsCyk-4 blocks Ect2 recruitment to the central spindle and the subsequent induction of furrowing. Microtubules, as well as the microtubule-associated protein (MAP) Prc1, facilitate Plk1 phosphorylation of HsCyk-4. Characterization of a phosphomimetic version of HsCyk-4 indicates that Plk1 promotes Ect2 recruitment through multiple targets. Collectively, our data reveal that formation of the HsCyk-4-Ect2 complex is subject to multiple layers of regulation to ensure that RhoA activation occurs between the segregated sister chromatids during anaphase.
The plane of cell division in animal cells is determined by the position of the mitotic spindle during early anaphase, but the molecular signaling that leads to proper formation of the division plane is not fully understood. The actin- and myosin-rich contractile ring, which physically divides a cell in two, localizes to the presumptive division plane through the local activation of a molecular switch protein, RhoA. RhoA is activated by Ect2, which binds to the protein complex centralspindlin found on microtubules in the vicinity of the division plane (the midzone microtubules). One critical component of centralspindlin is Cyk-4, a putative negative regulator of RhoA. Here, we have analyzed the mechanisms that are responsible for targeting the RhoA activator Ect2 to the midzone microtubules. We show that Polo-like kinase 1 (Plk1), in part through the microtubule-associated protein Prc1, phosphorylates Cyk-4. Ect2 binds to phosphorylated Cyk-4 and is then able to activate RhoA and induce proper formation of the contractile ring. Our study therefore has elucidated important details of the signaling cascade in animal cells that ensures proper division-plane formation.
Mad2 is a key component of the spindle assembly checkpoint, a safety device ensuring faithful sister chromatid separation in mitosis. The target of Mad2 is Cdc20, an activator of the anaphase-promoting complex/cyclosome (APC/C). Mad2 binding to Cdc20 is a complex reaction that entails the conformational conversion of Mad2 from an open (O-Mad2) to a closed (C-Mad2) conformer. Previously, it has been hypothesized that the conversion of O-Mad2 is accelerated by its conformational dimerization with C-Mad2. This hypothesis, known as the Mad2-template hypothesis, is based on the unproven assumption that the natural conversion of O-Mad2 required to bind Cdc20 is slow. Here, we provide evidence for this fundamental assumption and demonstrate that conformational dimerization of Mad2 accelerates the rate of Mad2 binding to Cdc20. On the basis of our measurements, we developed a set of rate equations that deliver excellent predictions of experimental binding curves under a variety of different conditions. Our results strongly suggest that the interaction of Mad2 with Cdc20 is rate limiting for activation of the spindle checkpoint. Conformational dimerization of Mad2 is essential to accelerate Cdc20 binding, but it does not modify the equilibrium of the Mad2:Cdc20 interaction, i.e., it is purely catalytic. These results surpass previously formulated objections to the Mad2-template model and predict that the release of Mad2 from Cdc20 is an energy-driven process.
Mitosis, the partition of chromosomes from a mother cell to the two daughter cells, is based on the formation of attachments between chromosomes and the mitotic spindle. Cells enter mitosis with replicated chromosomes (sister chromatids) that are held together by a cohesive force. Upon attachment of the sister chromatids to the mitotic spindle, the cohesive force that holds them is removed, and the sisters are parted to opposite poles of the spindle. It is essential for the long-term viability of cells that chromosomes not be lost in the process. For this purpose, cells have evolved a molecular device (the spindle assembly checkpoint or SAC), which prevents loss of sister chromatid cohesion until all sister chromatids are properly attached to the mitotic spindle. An outstanding question concerns the way the SAC signal is amplified away from chromosomes that are not yet attached to the spindle. Such an amplification mechanism has been predicted on the fact that as few as a single unattached kinetochore is able to prevent sister chromatid cohesion. In this paper, we show that the properties of the SAC protein Mad2 are ideally suited to provide a mechanism of amplification to the SAC.
The reconstitution in vitro of key reactions of the spindle assembly checkpoint reveals the presence of catalysis and autocatalysis during checkpoint activation.
Chromosome segregation requires sister chromatid resolution. Condensins are essential for this process since they organize an axial structure where topoisomerase II can work. How sister chromatid separation is coordinated with chromosome condensation and decatenation activity remains unknown. We combined four-dimensional (4D) microscopy, RNA interference (RNAi), and biochemical analyses to show that topoisomerase II plays an essential role in this process. Either depletion of topoisomerase II or exposure to specific anti-topoisomerase II inhibitors causes centromere nondisjunction, associated with syntelic chromosome attachments. However, cells degrade cohesins and timely exit mitosis after satisfying the spindle assembly checkpoint. Moreover, in topoisomerase II–depleted cells, Aurora B and INCENP fail to transfer to the central spindle in late mitosis and remain tightly associated with centromeres of nondisjoined sister chromatids. Also, in topoisomerase II–depleted cells, Aurora B shows significantly reduced kinase activity both in S2 and HeLa cells. Codepletion of BubR1 in S2 cells restores Aurora B kinase activity, and consequently, most syntelic attachments are released. Taken together, our results support that topoisomerase II ensures proper sister chromatid separation through a direct role in centromere resolution and prevents incorrect microtubule–kinetochore attachments by allowing proper activation of Aurora B kinase.
Successful cell division requires that chromosomes are properly condensed and that each sister chromatid is self-contained by the time the sister pairs are segregated into separate daughter cells. It is also essential that the kinetochores at the centromeres of each pair of sister chromatids bind microtubules from opposite spindle poles. Topoisomerase II is a highly conserved enzyme that removes interlinks from DNA and is known to be essential to proper chromosome segregation during cell division. In this work, we have used state-of-the-art four-dimensional fluorescent microscopy to follow progression through mitosis in living cells depleted of topoisomerase II. We find that when the enzyme is absent, the two sister centromeres do not separate, and chromosomes missegregate. Moreover, the inappropriate centromere structure that results prevents the correct activation of the Aurora B kinase, which forms part of a regulatory mechanism that monitors correct segregation of chromosomes; as a result, cells exit mitosis abnormally.
Analysis of cells lacking topoisomerase II reveals that the enzyme has an essential role in the segregation of chromosomes, and specifically centromeres, at anaphase-telophase of mitosis: it prevents non-disjunction and allows activation of the Aurora B kinase, so as to correct improper attachments between microtubules and the kinetochore.
Cell fate can be determined by asymmetric segregation of gene expression regulators. In the budding yeast Saccharomyces cerevisiae, the transcription factor Ace2 accumulates specifically in the daughter cell nucleus, where it drives transcription of genes that are not expressed in the mother cell. The NDR/LATS family protein kinase Cbk1 is required for Ace2 segregation and function. Using peptide scanning arrays, we determined Cbk1′s phosphorylation consensus motif, the first such unbiased approach for an enzyme of this family, showing that it is a basophilic kinase with an unusual preference for histidine −5 to the phosphorylation site. We found that Cbk1 phosphorylates such sites in Ace2, and that these modifications are critical for Ace2′s partitioning and function. Using proteins marked with GFP variants, we found that Ace2 moves from isotropic distribution to the daughter cell nuclear localization, well before cytokinesis, and that the nucleus must enter the daughter cell for Ace2 accumulation to occur. We found that Cbk1, unlike Ace2, is restricted to the daughter cell. Using both in vivo and in vitro assays, we found that two critical Cbk1 phosphorylations block Ace2′s interaction with nuclear export machinery, while a third distal modification most likely acts to increase the transcription factor's activity. Our findings show that Cbk1 directly controls Ace2, regulating the transcription factor's activity and interaction with nuclear export machinery through three phosphorylation sites. Furthermore, Cbk1 exhibits a novel specificity that is likely conserved among related kinases from yeast to metazoans. Cbk1 is functionally restricted to the daughter cell, and cannot diffuse from the daughter to the mother. In addition to providing a mechanism for Ace2 segregation, these findings show that an isotropically distributed cell fate determinant can be asymmetrically partitioned in cytoplasmically contiguous cells through spatial segregation of a regulating protein kinase.
Cells can differentiate by segregating molecules that direct expression of specific sets of genes to one of the two cells produced by division. This generally occurs by direct mechanical movement or asymmetric anchoring of these molecules, which act after division to influence gene expression. In this study, we define a different mechanism by which the budding yeast transcription regulator Ace2 is asymmetrically partitioned. We show that Ace2 moves from uniform distribution to strong accumulation in the daughter nucleus while mother and daughter cells are still connected, and that the enzyme Cbk1 directly controls this segregation by attaching phosphate to specific sites on Ace2. We also demonstrate that Cbk1 is restricted to the daughter cell. Using both biochemical and live-cell experiments, we show that the Cbk1-mediated modifications activate Ace2 and block its interaction with nuclear export machinery, trapping it in the daughter cell nucleus. In addition to demonstrating Cbk1′s remarkable biochemical similarity to related enzymes in multicellular organisms, our analysis shows that a uniformly distributed regulator of gene expression can be made asymmetrically active in connected cells through the direct action of a localized modifying enzyme.
A conserved protein kinase, Cbk-1, produces different gene expression programs in cytoplasmically connected cells by directly blocking nuclear export of the transcription factor Ace2.
Rho1p is an essential small GTPase that plays a key role in the morphogenesis of Saccharomyces cerevisiae. We show here that the activation of Rho1p is regulated by a cyclin-dependent kinase (CDK). Rho1p is activated at the G1/S transition at the incipient-bud sites by the Cln2p (G1 cyclin) and Cdc28p (CDK) complex, in a process mediated by Tus1p, a guanine nucleotide exchange factor for Rho1p. Tus1p interacts physically with Cln2p/Cdc28p and is phosphorylated in a Cln2p/Cdc28p-dependent manner. CDK phosphorylation consensus sites in Tus1p are required for both Cln2p-dependent activation of Rho1p and polarized organization of the actin cytoskeleton. We propose that Cln2p/Cdc28p-dependent phosphorylation of Tus1p is required for appropriate temporal and spatial activation of Rho1p at the G1/S transition.
Deficiencies in the SBDS gene result in Shwachman-Diamond syndrome (SDS), an inherited bone marrow failure syndrome associated with leukemia predisposition. SBDS encodes a highly conserved protein previously implicated in ribosome biogenesis. Using human primary bone marrow stromal cells (BMSCs), lymphoblasts, and skin fibroblasts, we show that SBDS stabilized the mitotic spindle to prevent genomic instability. SBDS colocalized with the mitotic spindle in control primary BMSCs, lymphoblasts, and skin fibroblasts and bound to purified microtubules. Recombinant SBDS protein stabilized microtubules in vitro. We observed that primary BMSCs and lymphoblasts from SDS patients exhibited an increased incidence of abnormal mitoses. Similarly, depletion of SBDS by siRNA in human skin fibroblasts resulted in increased mitotic abnormalities and aneuploidy that accumulated over time. Treatment of primary BMSCs and lymphoblasts from SDS patients with nocodazole, a microtubule destabilizing agent, led to increased mitotic arrest and apoptosis, consistent with spindle destabilization. Conversely, SDS patient cells were resistant to taxol, a microtubule stabilizing agent. These findings suggest that spindle instability in SDS contributes to bone marrow failure and leukemogenesis.
The forkhead transcription factor FoxM1 has been reported to regulate, variously, proliferation and/or spindle formation during the G2/M transition of the cell cycle. Here we define specific functions of FoxM1 during brain development by the investigation of FoxM1 loss-of-function mutations in the context of Sonic hedgehog (Shh)-induced neuroproliferation in cerebellar granule neuron precursors (CGNP). We show that FoxM1 is expressed in the cerebellar anlagen as well as in postnatal proliferating CGNP and that it is upregulated in response to activated Shh signaling. To determine the requirements for FoxM1 function, we used transgenic mice carrying conventional null alleles or conditionally targeted alleles in conjunction with specific Cre recombinase expression in CGNP or early neural precursors driven by Math1 or Nestin enhancers. Although the overall cerebellar morphology was grossly normal, we observed that the entry into mitosis was postponed both in vivo and in Shh-treated CGNP cultures. Cell cycle analysis and immunohistochemistry with antibodies against phosphorylated histone H3 indicated a significant delay in the G2/M transition. Consistent with this, FoxM1-deficient CGNP showed decreased levels of the cyclin B1 and Cdc25b proteins. Furthermore, the loss of FoxM1 resulted in spindle defects and centrosome amplification. These findings indicate that the functions of FoxM1 in Shh-induced neuroproliferation are restricted to the regulation of the G2/M transition in CGNP, most probably through transcriptional effects on target genes such as those coding for B-type cyclins.
Actin polymerization-driven protrusion of the leading edge is a key element of cell motility. The important actin nucleators formins and the Arp2/3 complex are believed to have nonoverlapping functions in inducing actin filament bundles in filopodia and dendritic networks in lamellipodia, respectively. We tested this idea by investigating the role of mDia2 formin in leading-edge protrusion by loss-of-function and gain-of-function approaches. Unexpectedly, mDia2 depletion by short interfering RNA (siRNA) severely inhibited lamellipodia. Structural analysis of the actin network in the few remaining lamellipodia suggested an mDia2 role in generation of long filaments. Consistently, constitutively active mDia2 (ΔGBD-mDia2) induced accumulation of long actin filaments in lamellipodia and increased persistence of lamellipodial protrusion. Depletion of mDia2 also inhibited filopodia, whereas expression of ΔGBD-mDia2 promoted their formation. Correlative light and electron microscopy showed that ΔGBD-mDia2–induced filopodia were formed from lamellipodial network through gradual convergence of long lamellipodial filaments into bundles. Efficient filopodia induction required mDia2 targeting to the membrane, likely through a scaffolding protein Abi1. Furthermore, mDia2 and Abi1 interacted through the N-terminal regulatory sequences of mDia2 and the SH3-containing Abi1 sequences. We propose that mDia2 plays an important role in formation of lamellipodia by nucleating and/or protecting from capping lamellipodial actin filaments, which subsequently exhibit high tendency to converge into filopodia.
Cell motility is a cyclic process, with the protrusion of the leading edge followed by retraction of the rear. Protrusion is driven by polymerization of actin filaments, with the spatial organization of these filaments determining the shape of the protrusions. For example, the spike-like filopodia contain bundles of long actin filaments, whereas the sheet-like lamellipodia contain branched actin networks. In biochemical assays, two stimulators of actin polymerization, Arp2/3 complex and formins, induce branched or individual filaments, respectively. In cells, Arp2/3 complex and formins also appear to be implicated in the formation of lamellipodia and filopodia, respectively. However, when we investigated the role of mDia2 formin by functional approaches, we unexpectedly found that it is essential, not only for filopodia, but also for lamellipodia. Moreover, functions of mDia2 in lamellipodia and filopodia appeared intimately linked. We recorded behavior of cells by light microscopy and then used electron microscopy to study actin architecture in the same cells. We found that an activated form of mDia2 was first recruited to lamellipodia, where it induced many long, unbranched filaments, and from there, drove formation of filopodia through gradual convergence of these lamellipodial filaments into bundles. These data demonstrate a strong relationship between structurally different actin filament arrays and molecular machineries involved in their formation.
Formin mDia2 was believed to function mainly in the generation of filopodia in migrating cells. We unexpectedly found that mDia2 is also important for lamellipodia and induces filopodia in association with lamellipodia.