Biomedical subjects

Michael Glotzer

Publications and source records attributed to Michael Glotzer.

At least 19 recordsLinked to original sources

PubMed2006

Structural analysis of the ZEN-4/CeMKLP1 motor domain and its interaction with microtubules.

The centralspindlin complex is required for the assembly and maintenance of the central spindle during late anaphase and the completion of cytokinesis. It is composed of two copies each of the kinesin-like protein ZEN-4, a Caenorhabditis elegans MKLP-1 (Kinesin-6 family), and the RhoGAP CYK-4. By using cryo-electron microscopy and helical 3D reconstruction, we are investigating the structural features of the interactions between monomeric and dimeric motor domain constructs of ZEN-4 and microtubules. We have calculated helically averaged 3D maps of microtubules decorated with ZEN-4 motor domain in the presence of AMP-PNP, ADP, ADP-AlF(4)(-), and nucleotide-free conditions. We used statistical difference mapping to compare these maps among each other and to related maps obtained from microtubules decorated with a well-characterized Kinesin-1 motor domain from Neurospora crassa. Thereby, we found distinct structural features in microtubule-ZEN-4 complexes that may directly relate to the functional properties of ZEN-4 and centralspindlin. Furthermore, we investigated the location, structure, and function of a highly conserved extension of approximately 50 residues unique to the Kinesin-6 subfamily, located in the motor core loop6/beta4 region.

Adenosine Diphosphate↗

PubMed2005

Cytokinesis: welcome to the Rho zone.

Cytokinesis follows nuclear division and generates two distinct daughter cells, each replete with a full complement of the genome and cytoplasmic organelles. Members of the Rho family of GTPases are crucial regulators of this process in a wide variety of species. In many cell types, cytokinesis is mediated by a discretely localized contractile ring that is rich in actin and myosin. In this article (which is part of the Cytokinesis series), we review recent studies in animal cells that have shown that local assembly of the contractile ring is mediated by a discrete pool of GTP-bound, active RhoA. Advances in detecting the active pool of RhoA have allowed insights into the mechanisms and the molecules that promote the accumulation of active RhoA at the correct time and place in the cell.

Actins↗

PubMed2005

An ECT2-centralspindlin complex regulates the localization and function of RhoA.

In anaphase, the spindle dictates the site of contractile ring assembly. Assembly and ingression of the contractile ring involves activation of myosin-II and actin polymerization, which are triggered by the GTPase RhoA. In many cells, the central spindle affects division plane positioning via unknown molecular mechanisms. Here, we dissect furrow formation in human cells and show that the RhoGEF ECT2 is required for cortical localization of RhoA and contractile ring assembly. ECT2 concentrates on the central spindle by binding to centralspindlin. Depletion of the centralspindlin component MKLP1 prevents central spindle localization of ECT2; however, RhoA, F-actin, and myosin still accumulate on the equatorial cell cortex. Depletion of the other centralspindlin component, CYK-4/MgcRacGAP, prevents cortical accumulation of RhoA, F-actin, and myosin. CYK-4 and ECT2 interact, and this interaction is cell cycle regulated via ECT2 phosphorylation. Thus, central spindle localization of ECT2 assists division plane positioning and the CYK-4 subunit of centralspindlin acts upstream of RhoA to promote furrow assembly.

Actins↗

PubMed2005

Phosphorylation of ZEN-4/MKLP1 by aurora B regulates completion of cytokinesis.

The central spindle regulates the formation and positioning of the contractile ring and is essential for completion of cytokinesis [1]. Central spindle assembly begins in early anaphase with the bundling of overlapping, antiparallel, nonkinetochore microtubules [2, 3], and these bundles become compacted and mature into the midbody. Prominent components of the central spindle include aurora B kinase and centralspindlin, a complex containing a Kinesin-6 protein (ZEN-4/MKLP1) and a Rho family GAP (CYK-4/MgcRacGAP) that is essential for central spindle assembly [4]. Centralspindlin localization depends on aurora B kinase [5]. Aurora B concentrates in the midbody and persists between daughter cells. Here, we show that in C. elegans embryos and in cultured human cells, respectively, ZEN-4 and MKLP1 are phosphorylated by aurora B in vitro and in vivo on conserved C-terminal serine residues. In C. elegans embryos, a nonphosphorylatable mutant of ZEN-4 localizes properly but does not efficiently support completion of cytokinesis. In mammalian cells, an inhibitor of aurora kinase acutely attenuates phosphorylation of MKLP1. Inhibition of aurora B in late anaphase causes cytokinesis defects without disrupting the central spindle. These data indicate a conserved role for aurora-B-mediated phosphorylation of ZEN-4/MKLP1 in the completion of cytokinesis.

Animals↗

PubMed2005

The molecular requirements for cytokinesis.

After anaphase onset, animal cells build an actomyosin contractile ring that constricts the plasma membrane to generate two daughter cells connected by a cytoplasmic bridge. The bridge is ultimately severed to complete cytokinesis. Myriad techniques have been used to identify proteins that participate in cytokinesis in vertebrates, insects, and nematodes. A conserved core of about 20 proteins are individually involved with cytokinesis in most animal cells. These components are found in the contractile ring, on the central spindle, within the RhoA pathway, and on vesicles that expand the membrane and sever the bridge. Cytokinesis involves additional proteins, but they, or their requirement in cytokinesis, are not conserved among animal cells.

Actin Cytoskeleton↗

PubMed2004

Comparative analysis of cytokinesis in budding yeast, fission yeast and animal cells.

Cytokinesis is a temporally and spatially regulated process through which the cellular constituents of the mother cell are partitioned into two daughter cells, permitting an increase in cell number. When cytokinesis occurs in a polarized cell it can create daughters with distinct fates. In eukaryotes, cytokinesis is carried out by the coordinated action of a cortical actomyosin contractile ring and targeted membrane deposition. Recent use of model organisms with facile genetics and improved light-microscopy methods has led to the identification and functional characterization of many proteins involved in cytokinesis. To date, this analysis indicates that some of the basic components involved in cytokinesis are conserved from yeast to humans, although their organization into functional machinery that drives cytokinesis and the associated regulatory mechanisms bear species-specific features. Here, we briefly review the current status of knowledge of cytokinesis in the budding yeast Saccharomyces cerevisiae, the fission yeast Schizosaccharomyces pombe and animal cells, in an attempt to highlight both the common and the unique features. Although these organisms diverged from a common ancestor about a billion years ago, there are eukaryotes that are far more divergent. To evaluate the overall evolutionary conservation of cytokinesis, it will be necessary to include representatives of these divergent branches. Nevertheless, the three species discussed here provide substantial mechanistic diversity.

Animals↗

PubMed2004

Cell cycle regulation of central spindle assembly.

The bipolar mitotic spindle is responsible for segregating sister chromatids at anaphase. Microtubule motor proteins generate spindle bipolarity and enable the spindle to perform mechanical work. A major change in spindle architecture occurs at anaphase onset when central spindle assembly begins. This structure regulates the initiation of cytokinesis and is essential for its completion. Central spindle assembly requires the centralspindlin complex composed of the Caenorhabditis elegans ZEN-4 (mammalian orthologue MKLP1) kinesin-like protein and the Rho family GAP CYK-4 (MgcRacGAP). Here we describe a regulatory mechanism that controls the timing of central spindle assembly. The mitotic kinase Cdk1/cyclin B phosphorylates the motor domain of ZEN-4 on a conserved site within a basic amino-terminal extension characteristic of the MKLP1 subfamily. Phosphorylation by Cdk1 diminishes the motor activity of ZEN-4 by reducing its affinity for microtubules. Preventing Cdk1 phosphorylation of ZEN-4/MKLP1 causes enhanced metaphase spindle localization and defects in chromosome segregation. Thus, phosphoregulation of the motor domain of MKLP1 kinesin ensures that central spindle assembly occurs at the appropriate time in the cell cycle and maintains genomic stability.

Adenosine Triphosphatases↗

PubMed2004

Cleavage furrow positioning.

To complete the cell cycle, the cleavage furrow draws the plasma membrane toward the cell center, pinching the cytoplasm into two lobes that are subsequently separated into two cells. The position of the cleavage furrow is induced by the mitotic spindle during early anaphase. Although the mechanism of cleavage furrow positioning is not understood at a molecular level, recent results suggest that it might be mediated by local relief from the inhibitory effects of microtubules.

Animals↗

PubMed2003

Cytokinesis: a logical GAP.

Two complexes localize to the central spindle and regulate completion of cytokinesis: one, centralspindlin, contains a kinesin-like protein and a Rho-family GAP; the second contains Aurora B kinase. Aurora B kinase is known to regulate localization of centralspindlin and may regulate the activity of the RhoGAP component of centralspindlin.

Amino Acid Sequence↗

PubMed2003

CSC-1: a subunit of the Aurora B kinase complex that binds to the survivin-like protein BIR-1 and the incenp-like protein ICP-1.

The Aurora B kinase complex is a critical regulator of chromosome segregation and cytokinesis. In Caenorhabditis elegans, AIR-2 (Aurora B) function requires ICP-1 (Incenp) and BIR-1 (Survivin). In various systems, Aurora B binds to orthologues of these proteins. Through genetic analysis, we have identified a new subunit of the Aurora B kinase complex, CSC-1. C. elegans embryos depleted of CSC-1, AIR-2, ICP-1, or BIR-1 have identical phenotypes. CSC-1, BIR-1, and ICP-1 are interdependent for their localization, and all are required for AIR-2 localization. In vitro, CSC-1 binds directly to BIR-1. The CSC-1/BIR-1 complex, but not the individual subunits, associates with ICP-1. CSC-1 associates with ICP-1, BIR-1, and AIR-2 in vivo. ICP-1 dramatically stimulates AIR-2 kinase activity. This activity is not stimulated by CSC-1/BIR-1, suggesting that these two subunits function as targeting subunits for AIR-2 kinase.

Amino Acid Sequence↗

PubMed2003

Cytokinesis: progress on all fronts.

Cell multiplication requires sequestration of the duplicated and segregated genome into two daughter cells. The mitotic spindle is critical for orchestrating sister chromatid separation and division plane positioning. During anaphase, spindle microtubules become bundled to form the central spindle, which is essential for completion of cytokinesis. Central spindle assembly is mediated by a microtubule-associated protein and a kinesin-RhoGAP complex, both of which are regulated by phosphorylation/dephosphorylation. The central spindle also plays a role in cleavage furrow positioning, which appears to involve activation of RhoA. New results have provided some initial clues as to how furrow positioning is achieved. Particularly notable is the discovery that a protein activated by RhoA, formin, has actin nucleation activity.

Actins↗

PubMed2003

Kleisins: a superfamily of bacterial and eukaryotic SMC protein partners.

We describe a superfamily of eukaryotic and prokaryotic proteins (kleisins) that includes ScpA, Scc1, Rec8, and Barren. Scc1 interacts with SMC proteins through N- and C-terminal domains to form a ring-like structure. Since these are the only domains conserved among kleisins, we suggest that ring formation with SMC proteins may define this family.

Adenosine Triphosphatases↗

PubMed2003

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.

Animals↗

PubMed2002

GTP binding induces filament assembly of a recombinant septin.

The septins are a family of GTPases involved in cytokinesis in budding yeast, Drosophila, and vertebrates (see for review). Septins are associated with a system of 10 nm filaments at the S. cerevisiae bud neck, and heteromultimeric septin complexes have been isolated from cell extracts in a filamentous state. A number of septins have been shown to bind and hydrolyze guanine nucleotide. However, the role of GTP binding and hydrolysis in filament formation has not been elucidated. Furthermore, several lines of evidence suggest that not all the subunits of the septin complex are required for all aspects of septin function. To address these questions, we have reconstituted filament assembly in vitro by using a recombinant Xenopus septin, Xl Sept2. Filament assembly is GTP dependent; moreover, the coiled-coil domain common to most septins is not essential for filament formation. Septin polymerization is preceded by a lag phase, suggesting a cooperative assembly mechanism. The slowly hydrolyzable GTP analog, GTP-gamma-S, also induces polymerization, indicating that polymerization does not require GTP hydrolysis. If the properties of Xl Sept2 filaments reflect those of native septin complexes, these results imply that the growth or stability of septin filaments, or both, is regulated by the state of bound nucleotide.

Animals↗

PubMed2002

The CeCDC-14 phosphatase is required for cytokinesis in the Caenorhabditis elegans embryo.

In all eukaryotic organisms, the physical separation of two nascent cells must be coordinated with chromosome segregation and mitotic exit. In Saccharomyces cerevisiae and Schizosaccharomyces pombe this coordination depends on a number of genes that cooperate in intricate regulatory pathways termed mitotic exit network and septum initiation network, respectively. Here we have explored the function of potentially homologous genes in a metazoan organism, Caenorhabditis elegans, using RNA-mediated interference. Of all the genes tested, only depletion of CeCDC-14, the C. elegans homologue of the budding yeast dual-specificity phosphatase Cdc14p (Clp1/Flp1p in fission yeast), caused embryonic lethality. We show that CeCDC-14 is required for cytokinesis but may be dispensable for progression of the early embryonic cell cycles. In response to depletion of CeCDC-14, embryos fail to establish a central spindle, and several proteins normally found at this structure are mislocalized. CeCDC-14 itself localizes to the central spindle in anaphase and to the midbody in telophase. It colocalizes with the mitotic kinesin ZEN-4, and the two proteins depend on each other for correct localization. These findings identify the CDC14 phosphatase as an important regulator of central spindle formation and cytokinesis in a metazoan organism.

Anaphase↗

PubMed2002

A ubiquitin C-terminal hydrolase is required to maintain osmotic balance and execute actin-dependent processes in the early C. elegans embryo.

In the early Caenorhabditis elegans embryo, establishment of cell polarity and cytokinesis are both dependent upon reorganization of the actin cytoskeleton. Mutations in the cyk-3 gene cause maternal effect embryonic lethality. Embryos produced by homozygous cyk-3 mutant animals become multinucleate. We have further analyzed the cyk-3 mutant phenotype and have found that cyk-3 mutant embryos fail to properly polarize the actin cytoskeleton and fail to segregate germline determinants. In addition, they fail to assemble an intact cleavage furrow. However, we have found that cyk-3 mutant embryos are intrinsically defective in osmotic regulation and that the cytokinesis defects can be partially rescued by providing osmotic support. The cyk-3 gene has been identified and found to encode a ubiquitin C-terminal hydrolase that is active against model substrates. These data indicate that the deubiquitination of certain substrates by CYK-3 is crucial for cellular osmoregulation. Defects in osmoregulation appear to indirectly affect actin-dependent processes.

Actin Cytoskeleton↗

PubMed2002

The aurora B kinase AIR-2 regulates kinetochores during mitosis and is required for separation of homologous Chromosomes during meiosis.

BACKGROUND: Mitotic chromosome segregation depends on bi-orientation and capture of sister kinetochores by microtubules emanating from opposite spindle poles and the near synchronous loss of sister chromatid cohesion. During meiosis I, in contrast, sister kinetochores orient to the same pole, and homologous kinetochores are captured by microtubules emanating from opposite spindle poles. Additionally, mechanisms exist that prevent complete loss of cohesion during meiosis I. These features ensure that homologs separate during meiosis I and sister chromatids remain together until meiosis II. The mechanisms responsible for orienting kinetochores in mitosis and for causing asynchronous loss of cohesion during meiosis are not well understood. RESULTS: During mitosis in C. elegans, aurora B kinase, AIR-2, is not required for sister chromatid separation, but it is required for chromosome segregation. Condensin recruitment during metaphase requires AIR-2; however, condensin functions during prometaphase, independent of AIR-2. During metaphase, AIR-2 promotes chromosome congression to the metaphase plate, perhaps by inhibiting attachment of chromatids to both spindle poles. During meiosis in AIR-2-depleted oocytes, congression of bivalents appears normal, but segregation fails. Localization of AIR-2 on meiotic bivalents suggests this kinase promotes separation of homologs by promoting the loss of cohesion distal to the single chiasma. Inactivation of the phosphatase that antagonizes AIR-2 causes premature separation of chromatids during meiosis I, in a separase-dependent reaction. CONCLUSIONS: Aurora B functions to resolve chiasmata during meiosis I and to regulate kinetochore function during mitosis. Condensin mediates chromosome condensation during prophase, and condensin-independent pathways contribute to chromosome condensation during metaphase.

Adenosine Triphosphatases↗

PubMed2002

Cytokinesis: regulated by destruction.

Important transitions in the cell cycle are regulated by ubiquitin-dependent degradation of specific proteins. Recent data indicate that cytokinesis is also regulated by proteolysis.

Anaphase-Promoting Complex-Cyclosome↗