SUMOylation of α-tubulin is a novel modification regulating microtubule dynamics.
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ABSTRACT:
SUBMITTER: Feng W
PROVIDER: S-EPMC8104938 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Dynamic instability is a critical property of microtubules (MTs). By regulating the rate of tubulin polymerization and depolymerization, cells organize the MT cytoskeleton to accommodate their specific functions. Among many processes, posttranslational modifications of tubulin are implicated in regulating MT functions. Here we report a novel tubulin acetylation catalyzed by acetyltransferase San at lysine 252 (K252) of ?-tubulin. This acetylation, which is also detected in vivo, is added to soluble tubulin heterodimers but not tubulins in MTs. The acetylation-mimicking K252A/Q mutants were incorporated into the MT cytoskeleton in HeLa cells without causing any obvious MT defect. However, after cold-induced catastrophe, MT regrowth is accelerated in San-siRNA cells while the incorporation of acetylation-mimicking mutant tubulins is severely impeded. K252 of ?-tubulin localizes at the interface of ?-/?-tubulins and interacts with the phosphate group of the ?-tubulin-bound GTP. We propose that the acetylation slows down tubulin incorporation into MTs by neutralizing the positive charge on K252 and allowing tubulin heterodimers to adopt a conformation that disfavors tubulin incorporation.
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Project description:Microtubules polymerize and depolymerize stochastically, a behavior essential for cell division, motility, and differentiation. While many studies advanced our understanding of how microtubule-associated proteins tune microtubule dynamics in trans, we have yet to understand how tubulin genetic diversity regulates microtubule functions. The majority of in vitro dynamics studies are performed with tubulin purified from brain tissue. This preparation is not representative of tubulin found in many cell types. Here we report the 4.2-Å cryo-electron microscopy (cryo-EM) structure and in vitro dynamics parameters of ?1B/?I+?IVb microtubules assembled from tubulin purified from a human embryonic kidney cell line with isoform composition characteristic of fibroblasts and many immortalized cell lines. We find that these microtubules grow faster and transition to depolymerization less frequently compared with brain microtubules. Cryo-EM reveals that the dynamic ends of ?1B/?I+?IVb microtubules are less tapered and that these tubulin heterodimers display lower curvatures. Interestingly, analysis of EB1 distributions at dynamic ends suggests no differences in GTP cap sizes. Last, we show that the addition of recombinant ?1A/?III tubulin, a neuronal isotype overexpressed in many tumors, proportionally tunes the dynamics of ?1B/?I+?IVb microtubules. Our study is an important step toward understanding how tubulin isoform composition tunes microtubule dynamics.
| S-EPMC5706985 | biostudies-literature
Project description:LLCPK-1 cells were transfected with a green fluorescent protein (GFP)-alpha tubulin construct and a cell line permanently expressing GFP-alpha tubulin was established (LLCPK-1alpha). The mitotic index and doubling time for LLCPK-1alpha were not significantly different from parental cells. Quantitative immunoblotting showed that 17% of the tubulin in LLCPK-1alpha cells was GFP-tubulin; the level of unlabeled tubulin was reduced to 82% of that in parental cells. The parameters of microtubule dynamic instability were compared for interphase LLCPK-1alpha and parental cells injected with rhodamine-labeled tubulin. Dynamic instability was very similar in the two cases, demonstrating that LLCPK-1alpha cells are a useful tool for analysis of microtubule dynamics throughout the cell cycle. Comparison of astral microtubule behavior in mitosis with microtubule behavior in interphase demonstrated that the frequency of catastrophe increased twofold and that the frequency of rescue decreased nearly fourfold in mitotic compared with interphase cells. The percentage of time that microtubules spent in an attenuated state, or pause, was also dramatically reduced, from 73.5% in interphase to 11.4% in mitosis. The rates of microtubule elongation and rapid shortening were not changed; overall dynamicity increased 3.6-fold in mitosis. Microtubule release from the centrosome and a subset of differentially stable astral microtubules were also observed. The results provide the first quantitative measurements of mitotic microtubule dynamics in mammalian cells.
| S-EPMC32280 | biostudies-literature
Project description:Microtubules are dynamic polymers of ??-tubulin that form diverse cellular structures, such as the mitotic spindle for cell division, the backbone of neurons, and axonemes. To control the architecture of microtubule networks, microtubule-associated proteins (MAPs) and motor proteins regulate microtubule growth, shrinkage, and the transitions between these states. Recent evidence shows that many MAPs exert their effects by selectively binding to distinct conformations of polymerized or unpolymerized ??-tubulin. The ability of ??-tubulin to adopt distinct conformations contributes to the intrinsic polymerization dynamics of microtubules. ??-Tubulin conformation is a fundamental property that MAPs monitor and control to build proper microtubule networks.
| S-EPMC4226729 | biostudies-literature
Project description:Posttranslational modifications (PTMs) on microtubules differentiate these cytoskeletal elements for a variety of cellular functions. We recently identified SETD2 as a dual-function histone and microtubule methyltransferase, and methylation as a new microtubule PTM that occurs on lysine 40 of ?-tubulin, which is trimethylated (?-TubK40me3) by SETD2. In the course of these studies, we generated polyclonal (?-TubK40me3 pAb) and monoclonal (?-TubK40me3 mAb) antibodies to a methylated ?-tubulin peptide (GQMPSD-Kme3-TIGGGDC). Here, we characterize these antibodies, and the specific mono-, di- or tri-methylated lysine residues they recognize. While both the pAb and mAb antibodies recognized lysines methylated by SETD2 on microtubules and histones, the clone 18 mAb was more specific for methylated microtubules, with little cross-reactivity for methylated histones. The clone 18 mAb recognized specific subsets of microtubules during mitosis and cytokinesis, and lacked the chromatin staining seen by immunocytochemistry with the pAb. Western blot analysis using these antibodies revealed that methylated ?-tubulin migrated faster than unmethylated ?-tubulin, suggesting methylation may be a signal for additional processing of ?-tubulin and/or microtubules. As the first reagents that specifically recognize methylated ?-tubulin, these antibodies are a valuable tool for studying this new modification of the cytoskeleton, and the function of methylated microtubules.
| S-EPMC5098444 | biostudies-literature
Project description:Acetylation of α-tubulin lysine 40 (αK40) contributes to microtubule (MT) stability and is essential for neuronal development and function, whereas excessive αK40 deacetylation is observed in neurodegenerative disorders including Alzheimer's disease (AD). Here we identified inhibitor of DNA binding 2 (Id2) as a novel MT-binding partner that interacts with α-tubulin and enhances αK40 acetylation, leading to MT polymerization in the neurons. Commensurate with our finding that the low levels of Id2 expression along with a reduced αK40 acetylation in the postmortem human AD patient and 5X-FAD, AD model mice brain, Id2 upregulation in the hippocampus of 5X-FAD, which exhibit high levels of Sirt2 expression, increased αK40 acetylation and reconstitutes axon growth. Hence our study suggests that Id2 is critical for maintaining MT stability during neural development and the potential of Id2 to counteract pathogenic Sirt2 activity in AD.
| S-EPMC8455547 | biostudies-literature
Project description:Acetylation of K40 in α-tubulin is the sole posttranslational modification to mark the luminal surface of microtubules. It is still controversial whether its relationship with microtubule stabilization is correlative or causative. We have obtained high-resolution cryo-electron microscopy (cryo-EM) reconstructions of pure samples of αTAT1-acetylated and SIRT2-deacetylated microtubules to visualize the structural consequences of this modification and reveal its potential for influencing the larger assembly properties of microtubules. We modeled the conformational ensembles of the unmodified and acetylated states by using the experimental cryo-EM density as a structural restraint in molecular dynamics simulations. We found that acetylation alters the conformational landscape of the flexible loop that contains αK40. Modification of αK40 reduces the disorder of the loop and restricts the states that it samples. We propose that the change in conformational sampling that we describe, at a location very close to the lateral contacts site, is likely to affect microtubule stability and function.
| S-EPMC6535015 | biostudies-literature
Project description:The dynamic instability of microtubules has long been understood to depend on the hydrolysis of GTP bound to beta-tubulin, an event stimulated by polymerization and necessary for depolymerization. Crystallographic studies of tubulin show that GTP is bound by beta-tubulin at the longitudinal dimer-dimer interface and contacts particular alpha-tubulin residues in the next dimer along the protofilament. This structural arrangement suggests that these contacts could account for assembly-stimulated GTP hydrolysis. As a test of this hypothesis, we examined, in yeast cells, the effect of mutating the alpha-tubulin residues predicted, on structural grounds, to be involved in GTPase activation. Mutation of these residues to alanine (i.e., D252A and E255A) created poisonous alpha-tubulins that caused lethality even as minor components of the alpha-tubulin pool. When the mutant alpha-tubulins were expressed from the galactose-inducible promoter of GAL1, cells rapidly acquired aberrant microtubule structures. Cytoplasmic microtubules were largely bundled, spindle assembly was inhibited, preexisting spindles failed to completely elongate, and occasional, stable microtubules were observed unattached to spindle pole bodies. Time-lapse microscopy showed that microtubule dynamics had ceased. Microtubules containing the mutant proteins did not depolymerize, even in the presence of nocodazole. These data support the view that alpha-tubulin is a GTPase-activating protein that acts, during microtubule polymerization, to stimulate GTP hydrolysis in beta-tubulin and thereby account for the dynamic instability of microtubules.
| S-EPMC60769 | biostudies-literature
Project description:Vascular supply of tumors is one of the main targets for cancer therapy. Here, we investigated if plocabulin (PM060184), a novel marine-derived microtubule-binding agent, presents antiangiogenic and vascular-disrupting activities.The effects of plocabulin on microtubule network and dynamics were studied on HUVEC endothelial cells. We have also studied its effects on capillary tube structures formation or destabilization in three-dimensional collagen matrices. In vivo experiments were performed on different tumor cell lines.In vitro studies show that, at picomolar concentrations, plocabulin inhibits microtubule dynamics in endothelial cells. This subsequently disturbs the microtubule network inducing changes in endothelial cell morphology and causing the collapse of angiogenic vessels, or the suppression of the angiogenic process by inhibiting the migration and invasion abilities of endothelial cells. This rapid collapse of the endothelial tubular network in vitro occurs in a concentration-dependent manner and is observed at concentrations lower than that affecting cell survival. The in vitro findings were confirmed in tumor xenografts where plocabulin treatment induced a large reduction in vascular volume and induction of extensive necrosis in tumors, consistent with antivascular effects.Altogether, these data suggest that an antivascular mechanism is contributing to the antitumor activities of plocabulin.
| S-EPMC5803861 | biostudies-other
Project description:Microtubules play a key role in cell division, motility, and intracellular trafficking. Microtubule lattices are generally regarded as stable structures that undergo turnover through dynamic instability of their ends [1]. However, recent evidence suggests that microtubules also exchange tubulin dimers at the sites of lattice defects, which can be induced by mechanical stress, severing enzymes, or occur spontaneously during polymerization [2-6]. Tubulin incorporation can restore microtubule integrity; moreover, "islands" of freshly incorporated GTP-tubulin can inhibit microtubule disassembly and promote rescues [3, 4, 6-8]. Microtubule repair occurs in vitro in the presence of tubulin alone [2-6, 9]. However, in cells, it is likely to be regulated by specific factors, the nature of which is currently unknown. CLASPs are interesting candidates for microtubule repair because they induce microtubule nucleation, stimulate rescue, and suppress catastrophes by stabilizing incomplete growing plus ends with lagging protofilaments and promoting their conversion into complete ones [10-17]. Here, we used in vitro reconstitution assays combined with laser microsurgery and microfluidics to show that CLASP2? indeed stimulates microtubule lattice repair. CLASP2? promoted tubulin incorporation into damaged lattice sites, thereby restoring microtubule integrity. Furthermore, it induced the formation of complete tubes from partial protofilament assemblies and inhibited microtubule softening caused by hydrodynamic-flow-induced bending. The catastrophe-suppressing domain of CLASP2?, TOG2, combined with a microtubule-tethering region, was sufficient to stimulate microtubule repair, suggesting that catastrophe suppression and lattice repair are mechanistically similar. Our results suggest that the cellular machinery controlling microtubule nucleation and growth can also help to maintain microtubule integrity.
| S-EPMC7280784 | biostudies-literature