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.
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.
Project description:In this study, we use Cross-linking Mass Spectrometry to identify the interaction sites of the microtubule binding N-terminal domain of the companion of cellulose synthase 1 protein (CC1∆C223) from Arabidopsis thaliana with tubulin. We consistently detected four cross-linked peptides of CC1∆C223 to β-tubulin (K40 to E111, K94 to E111, K96 to E111 and K96 to E158; letters and numbers indicate specific amino acids in the protein sequence of CC1∆C223 and β-tubulin, respectively) and one cross-linked peptide of CC1∆C223 to α-tubulin (K40 to D327).
Project description:Microtubules (MTs) are fundamental to cellular architecture, function and organismal development. They are nucleated from microtubule organizing centres by the evolutionary conserved ?-tubulin ring complex (?TuRC). However, the molecular mechanism of nucleation remains elusive. Here, we used cryo-electron tomography (cryo-ET) to determine the structure of the native ?TuRC capping the minus end of a MT in the context of enriched budding yeast spindles. In our structure, ?TuRC presents a ring of ?-tubulin subunits to seed nucleation of exclusively 13-protofilament microtubules, and it adopts an active closed conformation to function as a perfect geometric template for MT nucleation. Our cryo-ET reconstruction also revealed that a novel coiled-coil protein staples the first row of ?/?-tubulin molecules to alternating positions along the ?-tubulin ring. This positioning of ?/?-tubulin onto ?TuRC suggests a role for the coiled-coil protein in augmenting ?TuRC-mediated microtubule nucleation. Based on our results we describe a molecular model for budding yeast ?TuRC activation and MT nucleation.
Project description:Microtubule cytoskeleton, built from heterodimers of α and β-tubulins, is critically important to physically organize cells, mediate intracellular transport and power cell division. The availability of soluble αβ-tubulins influences the biomechanical properties and functions of microtubules. When present in excess, soluble αβ-tubulins induce degradation of their encoding mRNAs. This process involves the activation of a specificity factor TTC5, which recognizes nascent tubulins and recruits effectors that degrade the mRNA. But how TTC5 activity is regulated is unknown. Our biochemical and structural proteomic approaches reveal that soluble αβ-tubulins at steady-state levels sequester TTC5, repressing its activity. The carboxy-terminal domain of TTC5 acts as a molecular switch, toggling between soluble αβ-tubulin-bound and nascent tubulin-bound states. Loss of sequestration by soluble αβ-tubulins constitutively activates TTC5, leading to diminished tubulin mRNA levels and compromised microtubule-dependent chromosome segregation during cell division. Our findings provide a paradigm for how cells regulate the activity of a specificity factor to adapt posttranscriptional regulation of gene expression to cellular needs.
Project description:Microtubules (MTs) are built from alpha/beta-tubulin dimers and used as tracks by kinesin and dynein motors to transport a variety of cargos, such as mRNAs, proteins, and organelles, within the cell. Tubulins are subjected to several post-translational modifications (PTMs). Glutamylation is one of them, and it is responsible for adding one or more glutamic acid residues as branched peptide chains to the C-terminal tails of both alpha- and beta-tubulin. However, very little is known about the specific modifications found on the different tubulin isoforms in vivo and the role of these PTMs in cargo transport along MTs in vivo. In this study, we found that in Drosophila, glutamylation of the alpha-tubulin isoforms occurs specifically on the C-terminal ends of TBA1 and TBA3 in the ovaries. In contrast, the ovarian isoform TBA4 is not glutamylated. The C-terminal ends of TBA1 and TBA3 are glutamylated at several glutamyl side chains in various combinations. Drosophila TTLL5 is required for the mono- and polyglutamylation of ovarian TBA1 and 3. Furthermore, glutamylation of the alpha-tubulin is essential for the efficient localization of Staufen/osk mRNA and to give directionality to the fast ooplasmic streaming, two processes known to depend on kinesin mediated processes during oogenesis. In the nervous system, the kinesin-dependent neuronal transport of mitochondria also depends on TTLL5. Additionally, alpha-tubulin glutamylation affects the pausing of the transport of individual mitochondria in the axons. Our results demonstrate the in vivo role of TTLL5 in differential glutamylation of alpha-tubulin isoforms and point to the in vivo importance of alpha-tubulin glutamylation for kinesin-dependent processes.
Project description:Doublecortin (DCX) is a microtubule (MT) associated protein that regulates MT structure and function during neuronal development and mutations in DCX lead to a spectrum of neurological disorders. The structural properties of MT-bound DCX remain poorly resolved. Here, we describe the molecular architecture of the DCX-MT complex through an integrative modeling approach that combines data from X-ray crystallography, cryo-EM and a high-fidelity chemical crosslinking method. We demonstrate that DCX interacts with MTs through its N-terminal domain and induces a lattice-dependent self-association involving both the C-terminal structured domain and the C-tails, in a conformation that favors an open, domain-swapped state. The networked state is shown to accommodate multiple different attachment points on the MT lattice, all of which orient the C-tails away from the lattice. As numerous disease mutations cluster in the C-terminus and regulatory phosphorylations cluster in the C-tail, our study shows that lattice-driven self-assembly is an important property of DCX.
Project description:Exposure to organophosphorus pesticides (OP) can have chronic adverse effects that are independent of inhibition of acetylcholinesterase, the classic target for acute OP toxicity. In pure proteins, the organophosphorus pesticide chlorpyrifos oxon induces a crosslink between lysine and glutamate (or aspartate) with loss of water. Tubulin is particularly sensitive to OP-induced crosslinking. Our goal was to explore OP-induced crosslinking in a complex protein sample, MAP-rich tubulin from Sus scrofa, and to test 8 OP for their capacity to catalyze isopeptide crosslinking. We treated 100 µg of MAP-rich tubulin with 100 µM chlorpyrifos, chlorpyrifos oxon, methamidophos, paraoxon, diazinon, diazoxon, monocrotophos, or dichlorvos. Each sample was separated on SDS PAGE and stained with Coomassie blue. Five gel slices (at about 30, 50, 150, and 300 kDa, and the top of the separating gel) were removed from the lanes for each of the eight OP samples and from untreated control lanes. These gel slices were subjected to in-gel trypsin digestion. MSMS fragmentation spectra of the tryptic peptides were examined for isopeptide crosslinks. Sixteen spectra yielded convincing evidence for isopeptide crosslinked peptides. Ten were from the chlorpyrifos oxon reaction, 1 from dichlorvos, 1 from paraoxon, 1 from diazinon, and 3 from diazoxon. It was concluded that catalysis of protein crosslinking is a general property of organophosphorus pesticides and pesticide metabolites.