Radial intercalation is regulated by the Par complex and the microtubule-stabilizing protein CLAMP/Spef1.
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ABSTRACT:
SUBMITTER: Werner ME
PROVIDER: S-EPMC4121976 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The PAR-3/PAR-6/atypical PKC (aPKC) complex is required for axon-dendrite specification of hippocampal neurons. However, the downstream effectors of this complex are not well defined. In this article, we report a role for microtubule affinity-regulating kinase (MARK)/PAR-1 in axon-dendrite specification. Knocking down MARK2 expression with small interfering RNAs induced formation of multiple axon-like neurites and promoted axon outgrowth. Ectopic expression of MARK2 caused phosphorylation of tau (S262) and led to loss of axons, and this phenotype was rescued by expression of PAR-3, PAR-6, and aPKC. In contrast, the polarity defects caused by an MARK2 mutant (T595A), which is not responsive to aPKC, were not rescued by the PAR-3/PAR-6/aPKC complex. Moreover, polarity was abrogated in neurons overexpressing a mutant of MARK2 with a deleted kinase domain but an intact aPKC-binding domain. Finally, suppression of MARK2 rescued the polarity defects induced by a dominant-negative aPKC mutant. These results suggest that MARK2 is involved in neuronal polarization and functions downstream of the PAR-3/PAR-6/aPKC complex. We propose that aPKC in complex with PAR-3/PAR-6 negatively regulates MARK(s), which in turn causes dephosphorylation of microtubule-associated proteins, such as tau, leading to the assembly of microtubules and elongation of axons.
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Project description:Nerve cells contain abundant subpopulations of cold-stable microtubules. We have previously isolated a calmodulin-regulated brain protein, STOP (stable tubule-only polypeptide), which reconstitutes microtubule cold stability when added to cold-labile microtubules in vitro. We have now cloned cDNA encoding STOP. We find that STOP is a 100.5-kDa protein with no homology to known proteins. The primary structure of STOP includes two distinct domains of repeated motifs. The central region of STOP contains 5 tandem repeats of 46 amino acids, 4 with 98% homology to the consensus sequence. The STOP C terminus contains 28 imperfect repeats of an 11-amino acid motif. STOP also contains a putative SH3-binding motif close to its N terminus. In vitro translated STOP binds to both microtubules and Ca2+-calmodulin. When STOP cDNA is expressed in cells that lack cold-stable microtubules, STOP associates with microtubules at 37 degrees C, and stabilizes microtubule networks, inducing cold stability, nocodazole resistance, and tubulin detyrosination on microtubules in transfected cells. We conclude that STOP must play an important role in the generation of microtubule cold stability and in the control of microtubule dynamics in brain.
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Project description:Post-translational modification of tubulin provides differential functions to microtubule networks. Here, we address the role of tubulin acetylation on the penetrative capacity of cells undergoing radial intercalation, which is the process by which cells move apically, insert between outer cells, and join an epithelium. There are opposing forces that regulate intercalation, namely, the restrictive forces of the epithelial barrier versus the penetrative forces of the intercalating cell. Positively and negatively modulating tubulin acetylation in intercalating cells alters the developmental timing such that cells with more acetylation penetrate faster. We find that intercalating cells preferentially penetrate higher-order vertices rather than the more prevalent tricellular vertices. Differential timing in the ability of cells to penetrate different vertices reveals that lower-order vertices represent more restrictive sites of insertion. We shift the accessibility of intercalating cells toward more restrictive junctions by increasing tubulin acetylation, and we provide a geometric-based mathematical model that describes our results.
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Project description:Mitotic spindles are primarily composed of microtubules (MTs), generated by polymerization of ?- and ?-Tubulin hetero-dimers. Tubulins undergo a series of protein folding and post-translational modifications in order to fulfill their functions. Defects in Tubulin polymerization dramatically affect spindle formation and disrupt chromosome segregation. We recently described a role for the product of the conserved misato (mst) gene in regulating mitotic MT generation in flies, but the molecular function of Mst remains unknown. Here, we use affinity purification mass spectrometry (AP-MS) to identify interacting partners of Mst in the Drosophila embryo. We demonstrate that Mst associates stoichiometrically with the hetero-octameric Tubulin Chaperone Protein-1 (TCP-1) complex, with the hetero-hexameric Tubulin Prefoldin complex, and with proteins having conserved roles in generating MT-competent Tubulin. We show that RNAi-mediated in vivo depletion of any TCP-1 subunit phenocopies the effects of mutations in mst or the Prefoldin-encoding gene merry-go-round (mgr), leading to monopolar and disorganized mitotic spindles containing few MTs. Crucially, we demonstrate that Mst, but not Mgr, is required for TCP-1 complex stability and that both the efficiency of Tubulin polymerization and Tubulin stability are drastically compromised in mst mutants. Moreover, our structural bioinformatic analyses indicate that Mst resembles the three-dimensional structure of Tubulin monomers and might therefore occupy the TCP-1 complex central cavity. Collectively, our results suggest that Mst acts as a co-factor of the TCP-1 complex, playing an essential role in the Tubulin-folding processes required for proper assembly of spindle MTs.
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Project description:Interphase microtubules are organized into a radial array with centrosome in the center. This organization is a subject of cellular regulation that can be driven by protein phosphorylation. Only few protein kinases that regulate microtubule array in interphase cells have been described. Ste20-like protein kinase LOSK (SLK) was identified as a microtubule and centrosome-associated protein. In this study we have shown that the inhibition of LOSK activity by dominant-negative mutant K63R-DeltaT or by LOSK depletion with RNAi leads to unfocused microtubule arrangement. Microtubule disorganization is prominent in Vero, CV-1, and CHO-K1 cells but less distinct in HeLa cells. The effect is a result neither of microtubule stabilization nor of centrosome disruption. In cells with suppressed LOSK activity centrosomes are unable to anchor or to cap microtubules, though they keep nucleating microtubules. These centrosomes are depleted of dynactin. Vero cells overexpressing K63R-DeltaT have normal dynactin "comets" at microtubule ends and unaltered morphology of Golgi complex but are unable to polarize it at the wound edge. We conclude that protein kinase LOSK is required for radial microtubule organization and for the proper localization of Golgi complex in various cell types.
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Project description:Epiboly is the first coordinated cell movement in most vertebrates and marks the onset of gastrulation. During zebrafish epiboly, enveloping layer (EVL) and deep cells spread over the vegetal yolk mass with a concomitant thinning of the deep cell layer. A prevailing model suggests that deep cell radial intercalations directed towards the EVL would drive deep cell epiboly. To test this model, we have globally recorded 3D cell trajectories for zebrafish blastomeres between sphere and 50% epiboly stages, and developed an image analysis framework to determine intercalation events, intercalation directionality, and migration speed for cells at specific positions within the embryo. This framework uses Voronoi diagrams to compute cell-to-cell contact areas, defines a feature-based spatio-temporal model for intercalation events and fits an anatomical coordinate system to the recorded datasets. We further investigate whether epiboly defects in MZspg mutant embryos devoid of Pou5f1/Oct4 may be caused by changes in intercalation behavior. In wild-type and mutant embryos, intercalations orthogonal to the EVL occur with no directional bias towards or away from the EVL, suggesting that there are no directional cues that would direct intercalations towards the EVL. Further, we find that intercalation direction is independent of the previous intercalation history of individual deep cells, arguing against cues that would program specific intrinsic directed migration behaviors. Our data support a dynamic model in which deep cells during epiboly migrate into space opening between the EVL and the yolk syncytial layer. Genetic programs determining cell motility may control deep cell dynamic behavior and epiboly progress.
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Project description:BACKGROUND:Astral microtubules emanating from the mitotic centrosomes play pivotal roles in defining cell division axis and tissue morphogenesis. Previous studies have demonstrated that human transforming acidic coiled-coil 3 (TACC3), the most conserved TACC family protein, regulates formation of astral microtubules at centrosomes in vertebrate cells by affecting ?-tubulin ring complex (?-TuRC) assembly. However, the molecular mechanisms underlying such function were not completely understood. RESULTS:Here, we show that Aurora A site-specific phosphorylation in TACC3 regulates formation of astral microtubules by stabilizing ?-TuRC assembly in human cells. Mutation of the most conserved Aurora A targeting site, Ser 558 to alanine (S558A) in TACC3 results in robust loss of astral microtubules and disrupts localization of the ?-tubulin ring complex (?-TuRC) proteins at the spindle poles. Under similar condition, phospho-mimicking S558D mutation retains astral microtubules and the ?-TuRC proteins in a manner similar to control cells expressed with wild type TACC3. Time-lapse imaging reveals that S558A mutation leads to defects in positioning of the spindle-poles and thereby causes delay in metaphase to anaphase transition. Biochemical results determine that the Ser 558- phosphorylated TACC3 interacts with the ?-TuRC proteins and further, S558A mutation impairs the interaction. We further reveal that the mutation affects the assembly of ?-TuRC from the small complex components. CONCLUSIONS:The results demonstrate that TACC3 phosphorylation stabilizes ?- tubulin ring complex assembly and thereby regulates formation of centrosomal asters. They also implicate a potential role of TACC3 phosphorylation in the functional integrity of centrosomes/spindle poles.
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Project description:Optical trapping has been instrumental for deciphering translocation mechanisms of the force-generating cytoskeletal proteins. However, studies of the dynamic interactions between microtubules (MTs) and MT-associated proteins (MAPs) with no motor activity are lagging. Investigating the motility of MAPs that can diffuse along MT walls is a particular challenge for optical-trapping assays because thermally driven motions rely on weak and highly transient interactions. Three-bead, ultrafast force-clamp (UFFC) spectroscopy has the potential to resolve static and diffusive translocations of different MAPs with sub-millisecond temporal resolution and sub-nanometer spatial precision. In this report, we present detailed procedures for implementing UFFC, including setup of the optical instrument and feedback control, immobilization and functionalization of pedestal beads, and preparation of MT dumbbells. Example results for strong static interactions were generated using the Kinesin-7 motor CENP-E in the presence of AMP-PNP. Time resolution for MAP-MT interactions in the UFFC assay is limited by the MT dumbbell relaxation time, which is significantly longer than reported for analogous experiments using actin filaments. UFFC, however, provides a unique opportunity for quantitative studies on MAPs that glide along MTs under a dragging force, as illustrated using the kinetochore-associated Ska complex.
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| S-EPMC2813359 | biostudies-literature
Project description:Radial intercalation is a fundamental process responsible for the thinning of multilayered tissues during large-scale morphogenesis; however, its molecular mechanism has remained elusive. Using amphibian epiboly, the thinning and spreading of the animal hemisphere during gastrulation, here we provide evidence that radial intercalation is driven by chemotaxis of cells toward the external layer of the tissue. This role of chemotaxis in tissue spreading and thinning is unlike its typical role associated with large-distance directional movement of cells. We identify the chemoattractant as the complement component C3a, a factor normally linked with the immune system. The mechanism is explored by computational modeling and tested in vivo, ex vivo, and in vitro. This mechanism is robust against fluctuations of chemoattractant levels and expression patterns and explains expansion during epiboly. This study provides insight into the fundamental process of radial intercalation and could be applied to a wide range of morphogenetic events.
| S-EPMC4865533 | biostudies-literature