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PKA antagonizes CLASP-dependent microtubule stabilization to re-localize Pom1 and buffer cell size upon glucose limitation.

ABSTRACT: Cells couple growth with division and regulate size in response to nutrient availability. In rod-shaped fission yeast, cell-size control occurs at mitotic commitment. An important regulator is the DYRK-family kinase Pom1, which forms gradients from cell poles and inhibits the mitotic activator Cdr2, itself localized at the medial cortex. Where and when Pom1 modulates Cdr2 activity is unclear as Pom1 medial cortical levels remain constant during cell elongation. Here we show that Pom1 re-localizes to cell sides upon environmental glucose limitation, where it strongly delays mitosis. This re-localization is caused by severe microtubule destabilization upon glucose starvation, with microtubules undergoing catastrophe and depositing the Pom1 gradient nucleator Tea4 at cell sides. Microtubule destabilization requires PKA/Pka1 activity, which negatively regulates the microtubule rescue factor CLASP/Cls1/Peg1, reducing CLASP's ability to stabilize microtubules. Thus, PKA signalling tunes CLASP's activity to promote Pom1 cell side localization and buffer cell size upon glucose starvation.

SUBMITTER: Kelkar M

PROVIDER: S-EPMC4618306 | biostudies-literature |

REPOSITORIES: biostudies-literature

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CLASP stabilization of plus ends created by severing promotes microtubule creation and reorientation.

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Project description:Spatial regulation of microtubule (MT) dynamics contributes to cell polarity and cell division. MT rescue, in which a MT stops shrinking and reinitiates growth, is the least understood aspect of MT dynamics. Cytoplasmic Linker Associated Proteins (CLASPs) are a conserved class of MT-associated proteins that contribute to MT stabilization and rescue in vivo. We show here that the Schizosaccharomyces pombe CLASP, Cls1p, is a homodimer that binds an alphabeta-tubulin heterodimer through conserved TOG-like domains. In vitro, CLASP increases MT rescue frequency, decreases MT catastrophe frequency, and moderately decreases MT disassembly rate. CLASP binds stably to the MT lattice, recruits tubulin, and locally promotes rescues. Mutations in the CLASP TOG domains demonstrate that tubulin binding is critical for its rescue activity. We propose a mechanism for rescue in which CLASP-tubulin dimer complexes bind along the MT lattice and reverse MT depolymerization with their bound tubulin dimer.

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Stabilization of overlapping microtubules by fission yeast CLASP.

Project description:Many microtubule (MT) structures contain dynamic MTs that are bundled and stabilized in overlapping arrays. CLASPs are conserved MT-binding proteins implicated in the regulation of MT plus ends. Here, we show that the Schizosaccharomyces pombe CLASP, cls1p/peg1p, mediates the stabilization of overlapping MTs within the mitotic spindle and interphase bundles. cls1p localizes to these regions but not to interphase MT plus ends. Inactivation of cls1p leads to the rapid depolymerization of spindle midzone MTs. cls1p also stabilizes a subset of MTs within interphase bundles. cls1p prevents disassembly of the entire microtubule, while still allowing for plus-end growth. It has no measurable effects on MT nucleation, polymerization, catastrophe, or bundling. A direct interaction with ase1p (PRC1/MAP65) targets cls1p to regions of antiparallel MT overlap. These findings show how a MT-stabilizing factor attached to specific sites on MTs can help to generate MT structures that have both dynamic and stable components.

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CLASP Suppresses Microtubule Catastrophes through a Single TOG Domain.

Project description:The dynamic instability of microtubules plays a key role in controlling their organization and function, but the cellular mechanisms regulating this process are poorly understood. Here, we show that cytoplasmic linker-associated proteins (CLASPs) suppress transitions from microtubule growth to shortening, termed catastrophes, including those induced by microtubule-destabilizing agents and physical barriers. Mammalian CLASPs encompass three TOG-like domains, TOG1, TOG2, and TOG3, none of which bind to free tubulin. TOG2 is essential for catastrophe suppression, whereas TOG3 mildly enhances rescues but cannot suppress catastrophes. These functions are inhibited by the C-terminal domain of CLASP2, while the TOG1 domain can release this auto-inhibition. TOG2 fused to a positively charged microtubule-binding peptide autonomously accumulates at growing but not shrinking ends, suppresses catastrophes, and stimulates rescues. CLASPs suppress catastrophes by stabilizing growing microtubule ends, including incomplete ones, preventing their depolymerization and promoting their recovery into complete tubes. TOG2 domain is the key determinant of these activities.

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Articular Fibrocartilage-Targeted Therapy by Microtubule Stabilization

Project description:To investigate the fibrotic effect on BMSCs by CTGF and TGFβ1 treatment, and the anti-fibrotic effect of docetaxel on fibrotic chondrocytes. We established BMSCs primary culture and treated CTGF and TGFβ1, then treated by docetaxel.

2022-11-23 | GSE207148 | GEO

CLASP modulates microtubule-cortex interaction during self-organization of acentrosomal microtubules.

Project description:CLASP proteins associate with either the plus ends or sidewalls of microtubules depending on the subcellular location and cell type. In plant cells, CLASP's distribution along the full length of microtubules corresponds with the uniform anchorage of microtubules to the cell cortex. Using live cell imaging, we show here that loss of CLASP in Arabidopsis thaliana results in partial detachment of microtubules from the cortex. The detached portions undergo extensive waving, distortion, and changes in orientation, particularly when exposed to the forces of cytoplasmic streaming. These deviations from the normal linear polymerization trajectories increase the likelihood of intermicrotubule encounters that are favorable for subsequent bundle formation. Consistent with this, cortical microtubules in clasp-1 leaf epidermal cells are hyper-parallel. On the basis of these data, we identify a novel mechanism where modulation of CLASP activity governs microtubule-cortex attachment, thereby contributing to self-organization of cortical microtubules.

| S-EPMC2575154 | biostudies-literature

Multiparametric analysis of CLASP-interacting protein functions during interphase microtubule dynamics.

Project description:The microtubule (MT) plus-end tracking protein (+TIP) CLASP mediates dynamic cellular behaviors and interacts with numerous cytoplasmic proteins. While the influence of some CLASP interactors on MT behavior is known, a comprehensive survey of the proteins in the CLASP interactome as MT regulators is missing. Ultimately, we are interested in understanding how CLASP collaborates with functionally linked proteins to regulate MT dynamics. Here, we utilize multiparametric analysis of time-lapse MT +TIP imaging data acquired in Drosophila melanogaster S2R+ cells to assess the effects on individual microtubule dynamics for RNA interference-mediated depletion of 48 gene products previously identified to be in vivo genetic CLASP interactors. While our analysis corroborates previously described functions of several known CLASP interactors, its multiparametric resolution reveals more detailed functional profiles (fingerprints) that allow us to precisely classify the roles that CLASP-interacting genes play in MT regulation. Using these data, we identify subnetworks of proteins with novel yet overlapping MT-regulatory roles and also uncover subtle distinctions between the functions of proteins previously thought to act via similar mechanisms.

| S-EPMC3624261 | biostudies-literature

An isolated CLASP TOG domain suppresses microtubule catastrophe and promotes rescue.

Project description:Microtubules are heavily regulated dynamic polymers of αβ-tubulin that are required for proper chromosome segregation and organization of the cytoplasm. Polymerases in the XMAP215 family use arrayed TOG domains to promote faster microtubule elongation. Regulatory factors in the cytoplasmic linker associated protein (CLASP) family that reduce catastrophe and/or increase rescue also contain arrayed TOGs, but how CLASP TOGs contribute to activity is poorly understood. Here, using Saccharomyces cerevisiae Stu1 as a model CLASP, we report structural, biochemical, and reconstitution studies that clarify functional properties of CLASP TOGs. The two TOGs in Stu1 have very different tubulin-binding properties: TOG2 binds to both unpolymerized and polymerized tubulin, and TOG1 binds very weakly to either. The structure of Stu1-TOG2 reveals a CLASP-specific residue that likely confers distinctive tubulin-binding properties. The isolated TOG2 domain strongly suppresses microtubule catastrophe and increases microtubule rescue in vitro, contradicting the expectation that regulatory activity requires an array of TOGs. Single point mutations on the tubulin-binding surface of TOG2 ablate its anti-catastrophe and rescue activity in vitro, and Stu1 function in cells. Revealing that an isolated CLASP TOG can regulate polymerization dynamics without being part of an array provides insight into the mechanism of CLASPs and diversifies the understanding of TOG function.

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Microtubule-associated CLASP balances brassinosteroid signaling through negative feedback to sustain plant cell proliferation

Project description:The capacity for sustained cell division is a critical determinant of plant meristem development, and ultimately, organ size. This capacity is diminished in mutants lacking the microtubule-associated protein CLASP, and when brassinosteroid signaling is increased. Here, we discovered that CLASP is both targeted by and promotes activity of the brassinosteroid pathway in Arabidopsis root apical meristems. We show that enhanced brassinosteroid signalling reduces CLASP transcript and protein levels, and dramatically shifts microtubule organization to promote exit from the cell division cycle. Notably, CLASP sustains brassinosteroid signalling by fostering retrieval of endocytosed BRI1 receptors to the plasma membrane through the tethering of SNX1 vesicles to microtubules. clasp-1 null mutants have fewer BRI1 receptors, and impaired BR-mediated transcriptional activity and responses. Global transcript profiling confirmed the collapse of cell cycle activity in clasp-1 and revealed hormone crosstalk. Together, these findings reveal an unprecedented form of negative feedback that supports meristem homeostasis.

2017-05-01 | GSE85262 | GEO

pKD: re-designing protein pKa values.

Project description:The pK(a) values in proteins govern the pH-dependence of protein stability and enzymatic activity. A large number of mutagenesis experiments have been carried out in the last three decades to re-engineer the pH-activity and pH-stability profile of enzymes and proteins. We have developed the pKD webserver (http://polymerase.ucd.ie/pKa_Design), which predicts sets of point mutations that will change the pK(a) values of a set of target residues in a given direction, thus allowing for targeted re-design of the pH-dependent characteristics of proteins. The server provides the user with an interactive experience for re-designing pK(a) values by pre-calculating DeltapK(a) values from all feasible point mutations. Design solutions are found in less than 10 min for a typical design job for a medium-sized protein. Mutant DeltapK(a) values calculated by the pKD web server are in close agreement with those produced by comparing results from full-fledged pK(a) calculation methods.

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