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Ska3-microtubule interactions - Ska3 Ensures Timely Mitotic Progression by Interacting Directly With Microtubules and Ska1 Microtubule Binding Domain

ABSTRACT: The Ndc 80 and Ska complex are the major microtubule-binding factors of the kinetochore responsible for maintaining chromosome-microtubule coupling during chromosome segregation. We previously showed that the Ska1 subunit of the Skacomlex binds dynamic microtubules. This project demonstrated that Ska3 subunit is required to modulate the microtubule binding capability of the Ska complex by directly interacting with tubulin monomer and indirectly by interacting with tubulin contacting regions of Ska1.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Juan Zou

LAB HEAD: Prof. Juri Rappsilber

PROVIDER: PXD004952 | Pride | 2016-10-03

REPOSITORIES: Pride

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			Action	DRS
	Ska1_K155_K236_K245_Ska2_Ska3_MT.txt	Txt
	Ska1_K236_K245_Ska2_Ska3_MT.txt	Txt
	Ska1_Ska2_Ska3_MT.txt	Txt
	Zou_Rappsilber_JP_SkaComplex_HMB.xlsx	Xlsx
	Zou_Rappsilber_JP_SkaComplex_HMB.zip	Other

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Publications

Ska3 Ensures Timely Mitotic Progression by Interacting Directly With Microtubules and Ska1 Microtubule Binding Domain.

Abad Maria Alba MA Zou Juan J Medina-Pritchard Bethan B Nigg Erich A EA Rappsilber Juri J Santamaria Anna A Jeyaprakash A Arockia AA

Scientific reports 20160926

The establishment of physical attachment between the kinetochore and dynamic spindle microtubules, which undergo cycles of polymerization and depolymerization generating straight and curved microtubule structures, is essential for accurate chromosome segregation. The Ndc80 and Ska complexes are the major microtubule-binding factors of the kinetochore responsible for maintaining chromosome-microtubule coupling during chromosome segregation. We previously showed that the Ska1 subunit of the Ska co ...[more]

PMID: 27667719

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Ska3 Ensures Timely Mitotic Progression by Interacting Directly With Microtubules and Ska1 Microtubule Binding Domain.

Project description:The establishment of physical attachment between the kinetochore and dynamic spindle microtubules, which undergo cycles of polymerization and depolymerization generating straight and curved microtubule structures, is essential for accurate chromosome segregation. The Ndc80 and Ska complexes are the major microtubule-binding factors of the kinetochore responsible for maintaining chromosome-microtubule coupling during chromosome segregation. We previously showed that the Ska1 subunit of the Ska complex binds dynamic microtubules using multiple contact sites in a mode that allows conformation-independent binding. Here, we show that the Ska3 subunit is required to modulate the microtubule binding capability of the Ska complex (i) by directly interacting with tubulin monomers and (ii) indirectly by interacting with tubulin contacting regions of Ska1 suggesting an allosteric regulation. Perturbing either the Ska3-microtubule interaction or the Ska3-Ska1 interactions negatively influences microtubule binding by the Ska complex in vitro and affects the timely onset of anaphase in cells. Thus, Ska3 employs additional modulatory elements within the Ska complex to ensure robust kinetochore-microtubule attachments and timely progression of mitosis.

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Microtubule acetylation is required for mechanosensation in Drosophila

Project description:At the cellular level, α-tubulin acetylation alters the structure of microtubules to render them mechanically resistant to compressive forces. How this biochemical property of microtubule acetylation relates to mechanosensation remains unknown, though prior studies have shown that microtubule acetylation influences touch perception. Here, we identify the major Drosophila α-tubulin acetylase (dTAT) and show that it plays key roles in several forms of mechanosensation. dTAT is highly expressed in the larval peripheral nervous system (PNS), but is largely dispensable for neuronal morphogenesis. Mutation of the acetylase gene or the K40 acetylation site in α-tubulin impairs mechanical sensitivity in sensory neurons and behavioral responses to gentle touch, harsh touch, gravity, and vibration stimuli, but not noxious thermal stimulus. Finally, we show that dTAT is required for mechanically-induced activation of NOMPC, a microtubule-associated transient receptor potential channel, and functions to maintain integrity of the microtubule cytoskeleton in response to mechanical stimulation.

2018-09-22 | GSE120305 | GEO

Kinetochores read detyrosinated tubulin to sustain microtubule attachments

Project description:Post-translational cycles of α-tubulin detyrosination/tyrosination generate microtubule diversity, with broad physiological and clinical implications. However, the mechanistic understanding of how this microtubule diversity is decoded into specific cellular functions remains largely unknown. Here we show that the core microtubule-binding site at kinetochores ‘reads’ and responds to the α-tubulin detyrosination/tyrosination code. By combining CRISPR-Cas9 gene editing of enzymatic ‘writers’ of α-tubulin detyrosination/tyrosination in human cells, with biochemical purifications, mass spectrometry and super-resolution microscopy, we identified over a hundred proteins that preferentially associate with either detyrosinated or tyrosinated microtubules during mitosis. Among these, the conserved KNL-1/MIS12 complex/NDC80 complex (KMN) network at kinetochores showed a preference for tyrosinated microtubules. In addition to established roles in error correction and polar chromosome alignment, live-cell imaging uncovered a previously overlooked link between the α-tubulin detyrosination/tyrosination code and the formation of functional kinetochore-microtubule attachments, necessary for normal metaphase chromosome oscillations and timely spindle assembly checkpoint satisfaction. In vitro reconstitution experiments revealed that α-tubulin detyrosination specifically promotes Ndc80 diffusion along the microtubule lattice. We propose that the gradual accumulation of detyrosinated α-tubulin after initial microtubule capture at kinetochores facilitates the biased diffusion of core-binding proteins along the microtubule lattice, establishing a positive feedback loop that sustains kinetochore-microtubule attachments.

2024-10-03 | PXD047133 | Pride

Ciliopathy patient variants reveal organelle-specific functions for TUBB4B in axonemal microtubules

Project description:Tubulin, one of the most abundant cytoskeletal building blocks, exhibits extensive isotype diversity in humans. Displaying high similarity, whether these distinct isotypes form cell-type and context specific microtubule structures is poorly understood. Studying a cohort of 11 patients with the motile ciliopathy primary ciliary dyskinesia as well as mouse mutants, we report mutations in the TUBB4B isotype specifically perturb centriole and cilium biogenesis. We demonstrate that distinct TUBB4B mutations differentially affect microtubule dynamics and cilia formation in a dominant negative manner. Finally, structure-function studies reveal that different TUBB4B mutations disrupt distinct tubulin interfaces allowing clear stratification of patients into three classes of ciliopathic disease. These findings illustrate that specific tubulin isotypes have unique and non-redundant subcellular functions and establishes the missing link between human tubulinopathies and ciliopathies.

2024-02-04 | GSE246488 | GEO

MS-based proteomic characterization of assembled microtubules

Project description:Microtubules are dynamic polymers that interconvert between phases of growth and shrinkage, yet they somehow provide lasting structural stability to cells. Growth involves hydrolysis of GTP-tubulin to GDP-tubulin, which releases energy that is stored within the microtubule lattice and destabilizes it; a GTP cap at microtubule ends is thought to prevent GDP subunits from rapidly dissociating and causing catastrophe. We show that GDP-tubulin, usually considered as an inactive tubulin species, can itself assemble into microtubules preferentially at the minus end, and promote persistent growth. We characterized by mass spectrometry-based proteomics the protein content of tubulin purified from bovine brain (Total) and of microtubules grown from stable GMPCPP seeds in the presence of either GDP-tubulin or GTP-tubulin.

2024-04-30 | PXD049371 | Pride

Microtubules methylation LC-MSMS

Project description:Microtubules are critical for mitosis, cell motility, and protein and organelle transport, and are a validated target for anticancer drugs. However, tubulin regulation and recruitment in these cellular processes is less understood. Post-translational modifications of tubulin are proposed to regulate microtubule functions and dynamics. Although many such modifications have been investigated, tubulin methylations and enzymes responsible for methylation have only recently begun to be described. Here we report that N-lysine methyl transferase KMT5A (SET8/PR‑Set7), which methylates histone H4K20, also methylates α‑tubulin. Furthermore, the transcription factor LSF binds both tubulin and SET8, and enhances α-tubulin methylation in vitro, countered by FQI1, a specific small molecule inhibitor of LSF. Thus, the three SET8, LSF, and tubulin, all essential for mitotic progression, interact with each other. Overall, these results point to dual functions for both SET8 and LSF not only in chromatin regulation, but also for cytoskeletal modification.

2020-02-28 | PXD014257 | Pride

Distinct spectrum of ciliopathic diseases results from de novoTUBB4B variants through differential effects on organelle-specific microtubules

Project description:Microtubules are ancient and highly conserved polymers composed of alpha and beta tubulin heterodimers. Their dynamic remodelling drives diverse cellular processes from organelle trafficking and chromosome segregation to templating more stable structures like those of centrioles and ciliary axonemes. While organisms like Chlamydomonas reinhardtii only have a single - and -tubulin gene, TUBB4B, is one of 10 human -tubulin isotypes which can pair with one of the 9 isotypes of -tubulin. Whether this diversification permits discrete transcriptional units for cell type specific expression, or whether additional functional differences exist between isotypes remains unclear. Here, using human disease genetics, we reveal three distinct classes of ciliopathic disease in human patients carrying de novo mutations in TUBB4B. Using structure-function studies, we demonstrate these mutations differentially affect microtubule dynamics and cilia formation in a dominant negative manner. Importantly, using patient-modelled mutant as well as knockout Tubb4b mice we establish a necessary and non-redundant function for TUBB4B in building centrioles and axonemes of multiciliated cells. Our results suggest that a functional difference in TUBB4B is required to support the unique microtubule architecture in motile ciliated cells. It places primary ciliary dyskinesia and the ciliopathies more broadly into an expanding phenotypic and clinical spectrum of human tubulinopathies.

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