Precise segregation of chromosomes during mitosis requires assembly of a bipolar mitotic spindle followed by correct attachment of microtubules to the kinetochores. This highly spatiotemporally organized process is controlled by various mitotic kinases and molecular motors. We have recently shown that Casein Kinase 1 (CK1) promotes timely progression through mitosis by phosphorylating FAM110A leading to its enrichment at spindle poles. However, the mechanism by which FAM110A exerts its function in mitosis is unknown. Using structure prediction and a set of deletion mutants, we mapped here the interaction of the N- and C-terminal domains of FAM110A with actin and tubulin, respectively. Next, we found that the FAM110A-Δ40-61 mutant deficient in actin binding failed to rescue defects in chromosomal alignment caused by depletion of endogenous FAM110A. Depletion of FAM110A impaired assembly of F-actin in the proximity of spindle poles and was rescued by expression of the wild-type FAM110A, but not the FAM110A-Δ40-61 mutant. Purified FAM110A promoted binding of F-actin to microtubules as well as bundling of actin filaments in vitro. Finally, we found that the inhibition of CK1 impaired spindle actin formation and delayed progression through mitosis. We propose that CK1 and FAM110A promote timely progression through mitosis by mediating the interaction between spindle microtubules and filamentous actin to ensure proper mitotic spindle formation.

• Klíčová slova
• actin, microtubules, mitosis, mitotic spindle, protein kinase,
• MeSH
• aktiny metabolismus   MeSH
• aparát dělícího vřeténka * metabolismus   MeSH
• HeLa buňky MeSH
• kaseinkinasa I metabolismus   genetika   MeSH
• lidé MeSH
• mikrofilamenta * metabolismus   MeSH
• mikrotubuly * metabolismus   MeSH
• mitóza * MeSH
• proteiny buněčného cyklu metabolismus   genetika   MeSH
• vazba proteinů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• aktiny MeSH
• FAM110A protein, human MeSH Prohlížeč
• kaseinkinasa I MeSH
• proteiny buněčného cyklu MeSH

Microtubule doublets (MTDs) comprise an incomplete microtubule (B-tubule) attached to the side of a complete cylindrical microtubule. These compound microtubules are conserved in cilia across the tree of life; however, the mechanisms by which MTDs form and are maintained in vivo remain poorly understood. Here, we identify microtubule-associated protein 9 (MAP9) as an MTD-associated protein. We demonstrate that C. elegans MAPH-9, a MAP9 homolog, is present during MTD assembly and localizes exclusively to MTDs, a preference that is in part mediated by tubulin polyglutamylation. We find that loss of MAPH-9 causes ultrastructural MTD defects, including shortened and/or squashed B-tubules with reduced numbers of protofilaments, dysregulated axonemal motor velocity, and perturbed cilia function. Because we find that the mammalian ortholog MAP9 localizes to axonemes in cultured mammalian cells and mouse tissues, we propose that MAP9/MAPH-9 plays a conserved role in regulating ciliary motors and supporting the structure of axonemal MTDs.

• Klíčová slova
• C. elegans, MAP9, axoneme, cilia, dynein, kinesin, microtubule, microtubule doublet, microtubule-associated protein, polyglutamylation,
• MeSH
• axonema * metabolismus   ultrastruktura   MeSH
• Caenorhabditis elegans * metabolismus   MeSH
• cilie metabolismus   MeSH
• mikrotubuly metabolismus   MeSH
• myši MeSH
• pohyb MeSH
• savci MeSH
• tubulin metabolismus   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• Map9 protein, mouse MeSH Prohlížeč
• tubulin MeSH

Spatial light modulators have become an essential tool for advanced microscopy, enabling breakthroughs in 3D, phase, and super-resolution imaging. However, continuous spatial-light modulation that is capable of capturing sub-millisecond microscopic motion without diffraction artifacts and polarization dependence is challenging. Here we present a photothermal spatial light modulator (PT-SLM) enabling fast phase imaging for nanoscopic 3D reconstruction. The PT-SLM can generate a step-like wavefront change, free of diffraction artifacts, with a high transmittance and a modulation efficiency independent of light polarization. We achieve a phase-shift > π and a response time as short as 70 µs with a theoretical limit in the sub microsecond range. We used the PT-SLM to perform quantitative phase imaging of sub-diffractional species to decipher the 3D nanoscopic displacement of microtubules and study the trajectory of a diffusive microtubule-associated protein, providing insights into the mechanism of protein navigation through a complex microtubule network.

• MeSH
• časové faktory MeSH
• interferenční mikroskopie metody   statistika a číselné údaje   MeSH
• kovové nanočástice ultrastruktura   MeSH
• lidé MeSH
• mikroskopie atomárních sil MeSH
• mikroskopie fázově kontrastní metody   statistika a číselné údaje   MeSH
• mikrotubuly metabolismus   ultrastruktura   MeSH
• nanotechnologie MeSH
• nanotrubičky ultrastruktura   MeSH
• optické jevy MeSH
• počítačová simulace MeSH
• proteiny asociované s mikrotubuly metabolismus   MeSH
• proteiny buněčného cyklu metabolismus   MeSH
• Schizosaccharomyces pombe - proteiny metabolismus   MeSH
• světlo MeSH
• tubulin metabolismus   MeSH
• zlato MeSH
• zobrazování trojrozměrné metody   statistika a číselné údaje   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• Ase1 protein, S pombe MeSH Prohlížeč
• PRC1 protein, human MeSH Prohlížeč
• proteiny asociované s mikrotubuly MeSH
• proteiny buněčného cyklu MeSH
• Schizosaccharomyces pombe - proteiny MeSH
• tubulin MeSH
• zlato MeSH

Form and function of the mitotic spindle depend on motor proteins that crosslink microtubules and move them relative to each other. Among these are kinesin-14s, such as Ncd, which interact with one microtubule via their non-processive motor domains and with another via their diffusive tail domains, the latter allowing the protein to slip along the microtubule surface. Little is known about the influence of the tail domains on the protein's performance. Here, we show that diffusive anchorage of Ncd's tail domains impacts velocity and force considerably. Tail domain slippage reduced velocities from 270 nm s-1 to 60 nm s-1 and forces from several piconewtons to the sub-piconewton range. These findings challenge the notion that kinesin-14 may act as an antagonizer of other crosslinking motors, such as kinesin-5, during mitosis. It rather suggests a role of kinesin-14 as a flexible element, pliantly sliding and crosslinking microtubules to facilitate remodeling of the mitotic spindle.

• MeSH
• aparát dělícího vřeténka metabolismus   MeSH
• kineziny izolace a purifikace   metabolismus   MeSH
• mikrotubuly metabolismus   MeSH
• mitóza fyziologie   MeSH
• optická pinzeta MeSH
• proteinové domény MeSH
• proteiny asociované s mikrotubuly genetika   izolace a purifikace   metabolismus   MeSH
• proteiny Drosophily izolace a purifikace   metabolismus   MeSH
• rekombinantní proteiny genetika   izolace a purifikace   metabolismus   MeSH
• Saccharomyces cerevisiae - proteiny genetika   izolace a purifikace   metabolismus   MeSH
• vazba proteinů fyziologie   MeSH
• zelené fluorescenční proteiny genetika   izolace a purifikace   metabolismus   MeSH
• Publikační typ
• audiovizuální média MeSH
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• Ase1 protein, S cerevisiae MeSH Prohlížeč
• kineziny MeSH
• ncd protein, Drosophila MeSH Prohlížeč
• proteiny asociované s mikrotubuly MeSH
• proteiny Drosophily MeSH
• rekombinantní proteiny MeSH
• Saccharomyces cerevisiae - proteiny MeSH
• zelené fluorescenční proteiny MeSH

Microtubule-crosslinking motor proteins, which slide antiparallel microtubules, are required for the remodeling of microtubule networks. Hitherto, all microtubule-crosslinking motors have been shown to slide microtubules at a constant velocity until no overlap remains between them, leading to the breakdown of the initial microtubule geometry. Here, we show in vitro that the sliding velocity of microtubules, driven by human kinesin-14 HSET, decreases when microtubules start to slide apart, resulting in the maintenance of finite-length microtubule overlaps. We quantitatively explain this feedback using the local interaction kinetics of HSET with overlapping microtubules that cause retention of HSET in shortening overlaps. Consequently, the increased HSET density in the overlaps leads to a density-dependent decrease in sliding velocity and the generation of an entropic force that antagonizes the force exerted by the motors. Our results demonstrate that a spatial arrangement of microtubules can regulate the collective action of molecular motors through the local alteration of their individual interaction kinetics.

• MeSH
• kinetika MeSH
• kineziny chemie   metabolismus   MeSH
• lidé MeSH
• mikrotubuly chemie   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• kineziny MeSH