Patronin/Shot Cortical Foci Assemble the Noncentrosomal Microtubule Array that Specifies the Drosophila Anterior-Posterior Axis.
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ABSTRACT:
SUBMITTER: Nashchekin D
PROVIDER: S-EPMC4943857 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Tubulin assembles into microtubule polymers that have distinct plus and minus ends. Most microtubule plus ends in living cells are dynamic; the transitions between growth and shrinkage are regulated by assembly-promoting and destabilizing proteins. In contrast, minus ends are generally not dynamic, suggesting their stabilization by some unknown protein. Here, we have identified Patronin (also known as ssp4) as a protein that stabilizes microtubule minus ends in Drosophila S2 cells. In the absence of Patronin, minus ends lose subunits through the actions of the Kinesin-13 microtubule depolymerase, leading to a sparse interphase microtubule array and short, disorganized mitotic spindles. In vitro, the selective binding of purified Patronin to microtubule minus ends is sufficient to protect them against Kinesin-13-induced depolymerization. We propose that Patronin caps and stabilizes microtubule minus ends, an activity that serves a critical role in the organization of the microtubule cytoskeleton.
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Project description:Rationale: Emerging evidence suggests that noncentrosomal microtubules play an essential role in intracellular transport, cell polarity and cell motility. Whether these noncentrosomal microtubules exist or function in cancer cells remains unclear. Methods: The expression and prognostic values of CAMSAP2 and its functional targets were analyzed by immunohistochemistry in two independent HCC cohorts. Immunofluorescence and co-immunoprecipitation were used for detection of CAMSAP2-decorated noncentrosomal microtubule. Chromatin immunoprecipitation and luciferase report assays were used to determine the c-Jun binding sites in HDAC6 promoter region. In vitro migration and invasion assays and in vivo orthotopic metastatic models were utilized to investigate invasion and metastasis. Results: We reported a microtubule minus‑end‑targeting protein, CAMSAP2, is significantly upregulated in hepatocellular carcinoma (HCC) and correlated with poor prognosis. CAMSAP2 was specifically deposited on microtubule minus ends to serve as a "seed" for noncentrosomal microtubule outgrowth in HCC cells. Upon depletion of CAMSAP2, the noncentrosomal microtubule array was transformed into a completely radial centrosomal pattern, thereby impairing HCC cell migration and invasion. We further demonstrated that CAMSAP2 cooperates with EB1 to regulate microtubule dynamics and invasive cell migration via Trio/Rac1 signaling. Strikingly, both immunofluorescence staining and western blotting showed that CAMSAP2 depletion strongly reduced the abundance of acetylated microtubules in HCC cells. Our results revealed that HDAC6, a promising target for cancer therapy, was inversely downregulated in HCC and uniquely endowed with tumor-suppressive activity by regulation CAMSAP2-mediated microtubule acetylation. Mechanistically, CAMSAP2 activates c-Jun to induce transrepression of HDAC6 through Trio-dependent Rac1/JNK pathway. Furthermore, NSC23766, a Rac1-specific inhibitor significantly inhibited CAMSAP2-mediated HCC invasion and metastasis. Conclusions: CAMSAP2 is functionally, mechanistically, and clinically oncogenic in HCC. Targeting CAMSAP2-mediated noncentrosomal microtubule acetylation may provide new therapeutic strategies for HCC metastasis.
| S-EPMC7069094 | biostudies-literature
Project description:The microtubule (MT) cytoskeleton plays an essential role in mitosis, intracellular transport, cell shape, and cell migration. The assembly and disassembly of MTs, which can occur through the addition or loss of subunits at the plus- or minus-ends of the polymer, is essential for MTs to carry out their biological functions. A variety of proteins act on MT ends to regulate their dynamics, including a recently described family of MT minus-end binding proteins called calmodulin-regulated spectrin-associated protein (CAMSAP)/Patronin/Nezha. Patronin, the single member of this family in Drosophila, was previously shown to stabilize MT minus-ends against depolymerization in vitro and in vivo. Here, we show that all three mammalian CAMSAP family members also bind specifically to MT minus-ends and protect them against kinesin-13-induced depolymerization. However, these proteins differ in their abilities to suppress tubulin addition at minus-ends and to dissociate from MTs. CAMSAP1 does not interfere with polymerization and tracks along growing minus-ends. CAMSAP2 and CAMSAP3 decrease the rate of tubulin incorporation and remain bound, thereby creating stretches of decorated MT minus-ends. By using truncation analysis, we find that somewhat different minimal domains of CAMSAP and Patronin are involved in minus-end localization. However, we find that, in both cases, a highly conserved C-terminal domain and a more variable central domain cooperate to suppress minus-end dynamics in vitro and that both regions are required to stabilize minus-ends in Drosophila S2 cells. These results show that members of the CAMSAP/Patronin family all localize to and protect minus-ends but have evolved distinct effects on MT dynamics.
| S-EPMC4000804 | biostudies-literature
Project description:Axon formation is the initial step in establishing neuronal polarity. We examine here the role of microtubule dynamics in neuronal polarization using hippocampal neurons in culture. We see increased microtubule stability along the shaft in a single neurite before axon formation and in the axon of morphologically polarized cells. Loss of polarity or formation of multiple axons after manipulation of neuronal polarity regulators, synapses of amphids defective (SAD) kinases, and glycogen synthase kinase-3beta correlates with characteristic changes in microtubule turnover. Consistently, changing the microtubule dynamics is sufficient to alter neuronal polarization. Application of low doses of the microtubule-destabilizing drug nocodazole selectively reduces the formation of future dendrites. Conversely, low doses of the microtubule-stabilizing drug taxol shift polymerizing microtubules from neurite shafts to process tips and lead to the formation of multiple axons. Finally, local stabilization of microtubules using a photoactivatable analogue of taxol induces axon formation from the activated area. Thus, local microtubule stabilization in one neurite is a physiological signal specifying neuronal polarization.
| S-EPMC2234250 | biostudies-literature
Project description:Microtubule minus ends are thought to be stable in cells. Surprisingly, in Drosophila and zebrafish neurons, we observed persistent minus end growth, with runs lasting over 10 min. In Drosophila, extended minus end growth depended on Patronin, and Patronin reduction disrupted dendritic minus-end-out polarity. In fly dendrites, microtubule nucleation sites localize at dendrite branch points. Therefore, we hypothesized minus end growth might be particularly important beyond branch points. Distal dendrites have mixed polarity, and reduction of Patronin lowered the number of minus-end-out microtubules. More strikingly, extra Patronin made terminal dendrites almost completely minus-end-out, indicating low Patronin normally limits minus-end-out microtubules. To determine whether minus end growth populated new dendrites with microtubules, we analyzed dendrite development and regeneration. Minus ends extended into growing dendrites in the presence of Patronin. In sum, our data suggest that Patronin facilitates sustained microtubule minus end growth, which is critical for populating dendrites with minus-end-out microtubules.
| S-EPMC6605808 | biostudies-literature
Project description:Microtubule biogenesis initiates at various intracellular sites, including the centrosome, the Golgi apparatus, the nuclear envelope, and preexisting microtubules. Similarly, in the fission yeast Schizosaccharomyces pombe, interphase microtubules are nucleated at the spindle pole body (SPB), the nuclear envelope, and preexisting microtubules, depending on Mto1 activity. Despite the essential role of Mto1 in promoting microtubule nucleation, how distribution of Mto1 in different sites is regulated has remained elusive. Here, we show that the J-domain cochaperone Rsp1 interacts with Mto1 and specifies the localization of Mto1 to non-SPB nucleation sites. The absence of Rsp1 abolishes the localization of Mto1 to non-SPB nucleation sites, with concomitant enrichment of Mto1 to the SPB and the nuclear envelope. In contrast, Rsp1 overexpression impairs the localization of Mto1 to all microtubule organization sites. These findings delineate a previously uncharacterized mechanism in which Rsp1-Mto1 interaction orchestrates non-SPB microtubule formation.
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Project description:Using a pangenomic loss-of-function screening strategy, we have previously identified 76 potent modulators of paclitaxel responsiveness in non-small-cell lung cancer. The top hit isolated from this screen, symplekin, is a well-established component of the mRNA polyadenylation machinery. Here, we performed a high-resolution phenotypic analysis to reveal the mechanistic underpinnings by which symplekin depletion collaborates with paclitaxel. We find that symplekin supports faithful mitosis by contributing to the formation of a bipolar spindle apparatus. Depletion of symplekin attenuates microtubule polymerization activity as well as expression of the critical microtubule polymerization protein CKAP5 (TOGp). Depletion of additional members of the polyadenylation complex induces similar phenotypes, suggesting that polyadenylation machinery is intimately coupled to microtubule function and thus mitotic spindle formation. Importantly, tumor cells depleted of symplekin display reduced fecundity, but the mitotic defects that we observe are not evident in immortalized cells. These results demonstrate a critical connection between the polyadenylation machinery and mitosis and suggest that tumor cells have an enhanced dependency on these components for spindle assembly.
| S-EPMC2953045 | biostudies-literature
Project description:Synapses are fundamental components of the animal nervous system. Synaptic cytoskeleton is essential for maintaining proper neuronal development and wiring. Perturbations in neuronal microtubules (MTs) are correlated with numerous neuropsychiatric disorders. Despite discovering multiple synaptic MT regulators, the importance of MT stability, and particularly the polarity of MT in synaptic function, is still under investigation. Here, we identify Patronin, an MT minus-end-binding protein, for its essential role in presynaptic regulation of MT organization and neuromuscular junction (NMJ) development. Analyses indicate that Patronin regulates synaptic development independent of Klp10A. Subsequent research elucidates that it is short stop (Shot), a member of the Spectraplakin family of large cytoskeletal linker molecules, works synergistically with Patronin to govern NMJ development. We further raise the possibility that normal synaptic MT polarity contributes to proper NMJ morphology. Overall, this study demonstrates an unprecedented role of Patronin, and a potential involvement of MT polarity in synaptic development.
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Project description:The characteristic geometry of the unicellular chlorophyte Chlamydomonas reinhardtii has contributed to its adoption as a model system for cellular asymmetry and organelle positioning. The eyespot, a photosensitive organelle, is localized asymmetrically in the cell at a precisely defined position relative to the flagella and cytoskeletal microtubule rootlets. We have isolated a mutant, named pey1 for posterior eyespot, with variable microtubule rootlet lengths. The length of the acetylated daughter four-membered (D4) microtubule rootlet correlates with the position of the eyespot, which appears in a posterior position in the majority of cells. The correlation of rootlet length with eyespot positioning was also observed in the cmu1 mutant, which has longer acetylated microtubules, and the mlt1 mutant, in which the rootlet microtubules are shorter. Observation of eyespot positioning after depolymerization of rootlet microtubules indicated that eyespot position is fixed early in eyespot development and becomes independent of the rootlet. Our data demonstrate that the length of the D4 rootlet is the major determinant of eyespot positioning on the anterior-posterior axis and are suggestive that the gene product of the PEY1 locus is a novel regulator of acetylated microtubule length.
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Project description:Spectroscopic ellipsometry is a potent method that is widely adopted for the measurement of thin film thickness and refractive index. Most conventional ellipsometers utilize mechanically rotating polarizers and grating-based spectrometers for spectropolarimetric detection. Here, we demonstrated a compact metasurface array-based spectroscopic ellipsometry system that allows single-shot spectropolarimetric detection and accurate determination of thin film properties without any mechanical movement. The silicon-based metasurface array with a highly anisotropic and diverse spectral response is combined with iterative optimization to reconstruct the full Stokes polarization spectrum of the light reflected by the thin film with high fidelity. Subsequently, the film thickness and refractive index can be determined by fitting the measurement results to a proper material model with high accuracy. Our approach opens up a new pathway towards a compact and robust spectroscopic ellipsometry system for the high throughput measurement of thin film properties.
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