Microtubule Regulation in Mitosis: Tubulin Phosphorylation by the Cyclin-dependent Kinase Cdk1 (original) (raw)
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Dynamic Recruitment of Cdc2 to Specific Microtubule Structures during Mitosis
THE PLANT CELL ONLINE, 2001
A-type cyclin-dependent kinases (CDKs), also known as cdc2, are central to the orderly progression of the cell cycle. We made a functional Green Fluorescent Protein (GFP) fusion with CDK-A (Cdc2-GFP) and followed its subcellular localization during the cell cycle in tobacco cells. During interphase, the Cdc2-GFP fusion protein was found in both the cytoplasm and the nucleus, where it was highly resistant to extraction. In premitotic cells, a bright and narrow equatorial band appeared on the cell surface, resembling the late preprophase band, which disintegrated within 10 min as followed by time-lapse images. Cdc2-GFP was not found on prophase spindles but left the chromatin soon after this stage and associated progressively with the metaphase spindle in a microtubule-dependent manner. Arresting cells in mitosis through the stabilization of microtubules by taxol further enhanced the spindle-localized pool of Cdc2-GFP. Toward the end of mitosis, Cdc2-GFP was found at the midzone of the anaphase spindle and phragmoplast; eventually, it became focused at the midline of these microtubule structures. In detergent-extracted cells, the Cdc2-GFP remained associated with mitotic structures. Retention on spindles was prevented by pretreatment with the CDK-specific inhibitor roscovitine and was enhanced by the protein phosphatase inhibitor okadaic acid. Furthermore, we demonstrate that both the endogenous CDK-A and Cdc2-GFP were cosedimented with taxol-stabilized plant microtubules from cell extracts and that Cdc2 activity was detected together with a fraction of polymerized tubulin. These data provide evidence that the A-type CDKs associate physically with mitotic structures in a microtubule-dependent manner and may be involved in regulating the behavior of specific microtubule arrays throughout mitosis.
Cellular regulation of microtubule organization
The Journal of cell biology, 1984
Microtubules are constituents of axonemes, mitotic spindles, and elaborate arrays in interphase cells, and, with intermediate filaments and microfilaments, are among the most prevalent structures visualized in the cytomatrix (22, 44). With the exception ofthe A microtubule ofcilia and flagella, the lattice geometry ofmicrotubules is highly conserved. However, each of the major subunits of microtubules, a-and a-tubulin, shows heterogeneity. The number ofa-and f3-tubulin subspecies differs among tissues and organisms, and a number of types of analysis are used to examine how these tubulin variants are related to specific cell functions (1, 9-11, 33, 40). Investigations of the number and complexity of genes coding for these polypeptides have also been initiated (see reference 13 for review). However, the mechanisms that regulate the posttranslational compartmentalization of subunits, the spatial and temporal assembly of subunits into microtubules, and the integration of microtubules in various cellular events are still largely unknown. There are many levels at which the formation and organization of microtubules might be determined. A postulate originating from early analyses of mitotic spindle formation (32) was that a pool of subunits existed in equilibrium with formed microtubules; increases in the subunit concentration could therefore result in a net increase in polymer. With few exceptions, however, a rapid increase in the total tubulin pool does not appear to occur before the elaboration of more extensive microtubule arrays. For example, our studies (42, 50) have demonstrated that mouse neuroblastoma cells possessing microtubule-filled neurites contain four to five times more tubulin polymer than rounded, nondifferentiated cells, but the total tubulin content of these two cell types is the same. On the basis of volume calculations, the equilibrium concentration of subunits in the nondifferentiated cells is at least twice that in differentiated cells. Data such as this indicate that a simple equilibrium between subunit and polymer cannot account for the changes in microtubule formation coordinated with certain cellular events. In addition, recent findings show that an increase in the subunit concentration in cells, brought about either by drug treatment (15) or injection of tubulin (16), results in a depression of tubulin synthesis and the loss of tubulin mRNA. These data suggest that cells autoregulate the total tubulin pool and that this may be effected by "monitoring" of the monomer concentration (14).
γ-Tubulin 2 Nucleates Microtubules and Is Downregulated in Mouse Early Embryogenesis
PLoS ONE, 2012
c-Tubulin is the key protein for microtubule nucleation. Duplication of the c-tubulin gene occurred several times during evolution, and in mammals c-tubulin genes encode proteins which share ,97% sequence identity. Previous analysis of Tubg1 and Tubg2 knock-out mice has suggested that c-tubulins are not functionally equivalent. Tubg1 knock-out mice died at the blastocyst stage, whereas Tubg2 knock-out mice developed normally and were fertile. It was proposed that c-tubulin 1 represents ubiquitous c-tubulin, while c-tubulin 2 may have some specific functions and cannot substitute for c-tubulin 1 deficiency in blastocysts. The molecular basis of the suggested functional difference between c-tubulins remains unknown.
2000
Microtubules are dynamic polymers that move stochastically between periods of growth and shrinkage, a property known as dynamic instability. Here, to investigate the mechanisms regulating microtubule dynamics in Xenopus egg extracts, we have cloned the complementary DNA encoding the microtubule-associated protein XMAP215 and investigated the function of the XMAP215 protein. Immunodepletion of XMAP215 indicated that it is a major microtubule-stabilizing factor in Xenopus egg extracts. During interphase, XMAP215 stabilizes microtubules primarily by opposing the activity of the destabilizing factor XKCM1, a member of the kinesin superfamily. These results indicate that microtubule dynamics in Xenopus egg extracts are regulated by a balance between a stabilizing factor, XMAP215, and a destabilizing factor, XKCM1.
Cytoskeleton, 2014
Protein kinase C (PKC) engenders motility through phosphorylation of a-tubulin at Ser-165 in nontransformed MCF-10A cells. Live cell imaging explored the impact of PKC-mediated phosphorylation on microtubule (MT) dynamics. MTs fluorescently labeled with GFP-a-tubulin were treated with diacylglycerol (DAG)lactone (a membrane-permeable PKC activator), or cotransfected with a pseudophosphorylated S165D-a6tubulin mutant. Each condition increased the dynamicity of MTs by stimulating the rate and duration of the growth phase and decreasing the frequency of catastrophe. In MDA-MB-231 metastatic breast cells where the intrinsic PKC activity is high, these MT growth parameters were also high but could be suppressed by expression of phosphorylation-resistant S165N-a6-tubulin or by treatment with a pan-PKC inhibitor (bis-indoleylmaleimide). Subcellular fractionation and immunofluorescence of MCF-10A cells showed that phosphorylation (via DAG-lactone) or pseudophosphorylation of a6-tubulin increased its partitioning into MTs as compared to controls, and produced longer, more stable MTs. Following expression of the plusend binding protein GFP-EB1, DAG-lactone accelerated the formation and increased the number of nascent MTs. Expression of S165D-a6-tubulin promoted Rac1 activation and Rac1-dependent cell motility.
SnapShot: Microtubule Regulators II
Cell, 2009
Dynamic remodeling of the microtubule cytoskeleton is essential for many cell processes including division, migration and differentiation. Microtubules are dynamic polymers of α/β-tubulin dimers and transition stochastically between phases of growth (polymerization) and shortening (depolymerization). Intracellular microtubule organization is controlled by the activity and distribution of nucleation sites, proteins that directly influence polymerization dynamics, proteins that cut or bundle existing microtubules, and proteins that indirectly stabilize microtubules. Classical microtubule-associated proteins are mainly found in neuronal cells. Tau and MAP1B is specific for axons and MAP2 is predominantly localized to dendrites. These proteins bind along the length of microtubules and protect neurite microtubule arrays from depolymerization. Although many microtubule-associated proteins bundle microtubules when overexpressed in cells, true bundling activity has only been demonstrated for few protein families. Homotetrameric motor proteins of the kinesin-5 family slide antiparallel microtubules, and are required for spindle formation and spindle pole separation. MAP65-related proteins have been shown to promote antiparallel microtubule bundling, and yeast Ase1 is required for spindle midzone formation. MAP65 proteins are particularly numerous in plants. Together with additional plant-specific proteins such as WVD2, MAP65 proteins are involved in the formation of cortical microtubule bundles in plant cells. In addition to direct regulation of polymerization dynamics, microtubules can be stabilized by interactions with other intracellular structures. For example, CLASPs and spectraplakins mediate microtubule interactions with actin cables and adhesion sites. Because microtubules are the primary component of the mitotic spindle and essential for accurate chromosome segregation during cell division, it is not surprising that a number of microtubule-regulatory proteins function predominantly during spindle assembly. Many mitosis-specific microtubule stabilizers such as TPX2, NuMA, RHAMM, and HURP are segregated into the nucleus during interphase and are activated in a Ran•GTP dependent manner around mitotic chromatin. Tektins are a group of highly specialized microtubulestabilizing proteins necessary for the assembly of cilia and flagella in all eukaryotic cells. The specific functions of many other microtubule-associated and/or stabilizing proteins are poorly understood. Microtubule-based motor proteins that have no documented effects on microtubule dynamics are not included in this table.
Biochemical Pharmacology, 2009
15-deoxi-Δ 12,14 -prostaglandin J 2 (15d-PGJ 2 ) is known to play an important role in the pathophysiology of carcinogenesis, however the molecular mechanisms underlying these effects are not yet fully understood. Recently, we have shown that 15d-PGJ 2 is a potent inducer of breast cancer cell death and that this effect is associated with a disruption of the microtubule cytoskeletal network. Here, we show that treatment of the MCF-7 breast cancer cell line with 15d-PGJ 2 induces an accumulation of cells in the G 2 /M compartment of the cell cycle and a marked disruption of the microtubule network. 15d-PGJ 2 treatment causes mitotic abnormalities that consist of failure to form a stable metaphase plate, incapacity to progress through anaphase, and failure to complete cytokinesis. 15d-PGJ 2 binds to tubulin through the formation of a covalent adduct with at least four cysteine residues in and -tubulin, as detected by hybrid triple-quadrupole mass spectrometry analysis. Overall, these results support the hypothesis that microtubule disruption and mitotic arrest, as a consequence of the binding of 15d-PGJ 2 to tubulin, can represent one important pathway leading to breast cancer cell death.