This work reveals both the remarkable robustness of mitotic spindle assembly in response to extreme microtubule dynamics plasticity, and the requirement for strict control of microtubule dynamics across successive early embryonic divisions.
Abstract
Microtubules are dynamic, conserved cytoskeletal filaments that are essential for all eukaryotic cells. Microtubule dynamic properties are primarily characterized by measuring how fast they grow and shrink (growth and shrinkage rates) and how often they switch between assembly and disassembly (catastrophe and rescue frequencies). These four parameters measured for individual filaments can inform on the behavior of the entire microtubule network at the cell level. By comparing microtubule dynamics in Caenorhabditis elegans one-cell embryos using different genetically-encoded fluorescent probes, we observed an unexpected high variability in these parameters. Microtubule dynamics parameters were consistently higher in C. elegans strains expressing a fluorescently labelled microtubule end-binding protein than in strains relying on tubulin labelling, with microtubule growth rates differing by nearly a factor of two between the two conditions. This discrepancy was not limited to C. elegans, as we observed a similar effect in embryos of the tunicate Phallusia mammillata. Despite this, spindle size and assembly timing were only mildly affected. However, embryos expressing labelled end-binding protein exhibited higher frequency of mitotic defects and perturbed embryonic development upon exposure to various stresses such as elevated temperature or a compromised spindle assembly checkpoint. Thus, our work reveals both the remarkable robustness of mitotic spindle assembly in response to extreme microtubule dynamics plasticity, and the requirement for strict control of microtubule dynamics across successive early embryonic divisions. Our findings should also serve as a cautionary note when using tagged end-binding proteins to measure microtubule dynamics.
How embryos adapt their internal cellular machinery to reductions in cell size during development remains a fundamental question in cell biology. Here, we use high-resolution lattice light-sheet fluorescence microscopy and automated image analysis to quantify lineage-resolved mitotic spindle and chromosome segregation dynamics from the 2- to 64-cell stages in Caenorhabditis elegans embryos. While spindle length scales with cell size across both wild-type and size-perturbed embryos, chromosome segregation dynamics remain largely invariant, suggesting that distinct mechanisms govern these mitotic processes. Combining femtosecond laser ablation with large-scale electron tomography, we find that mid-spindle microtubules mediate chromosome segregation dynamics and remain uncoupled from cell size across all stages of early development. In contrast, spindle elongation is driven by cortically anchored motor proteins and astral microtubules, rendering it sensitive to cell size. Incorporating these experimental results into an extended stoichiometric model for both the spindle and chromosomes, we find that allowing only cell size and microtubule catastrophe rates to vary reproduces spindle pole-to-pole dynamics across development. The same model also accounts for centrosome separation and pronuclear positioning in the one-cell C. elegans embryo, spindle-length scaling across nematode species spanning ~100 million years of divergence, and spindle rotation in human cells. Thus, a unified stoichiometric framework provides a predictive, mechanistic account of spindle and nuclear dynamics across scales and species. Embryos must adapt to decreasing cell size. Although chromosome segregation. dynamics in C. elegans remain largely invariant, spindle length scales with cell. size and can be recapitulated by only varying cell size and microtubule catastrophe. rates.
Chukwuebuka William Okafornta, R. Farhadifar, G. Fabig et al.· Nature Communications· 0 citations
Dynein is an essential microtubule motor whose many roles in mitosis complicate efforts to resolve its spatiotemporally dynamic regulation. We previously established a method to classify single particles of the conserved cortical force generation complex (dynein-LIN-5NuMA-GPR-1/2LGN-GαGαi), as free or interacting with microtubules. Here, we report the results of depleting force generation complex components and regulators. Depleting LIS-1 reversed force asymmetry, while depletion of the conserved protein phosphatase regulatory subunit SUR-6PP2A-B55 significantly increased incidence of dynein trajectories with microtubule-interacting behavior during prophase. We next applied our classification scheme to the dynein anchor LIN-5. Microtubule-interacting LIN-5 trajectories were posteriorly enriched during anaphase, consistent with our dynein data and prior fluorescence studies. SUR-6PP2A-B55 depletion did not alter LIN-5 kinetics, suggesting regulation of a dynein specific function rather than regulation via LIN-5. By comparing and analyzing differences in LIN-5 trajectories, we found evidence that two distinct patterns of force generation complex behavior, microtubule interactions and cortical flows, emerge at the millisecond and second time scales, respectively. Our observations provide novel insight into the regulation of cortical dynein and the coupling among the cell membrane, actomyosin cortex, force generation complex, and microtubules that positions the mitotic spindle. [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text].
G. Alan Edwards, John B. Linehan, Amy Shaub Maddox et al.· Molecular Biology of the Cel...· 0 citations
Microtubules support neuronal morphology, intracellular transport, and neurite growth, but how microtubule turnover is regulated across neuronal compartments remains incompletely understood. To examine microtubule dynamics under physiological expression conditions, we expressed EGFP-tagged βIII-tubulin from the endogenous Tubb3 locus in cultured rat hippocampal neurons and measured tubulin turnover using fluorescence recovery after photobleaching (FRAP). FRAP analysis revealed pronounced spatial differences in microtubule dynamics at 9 days in vitro (DIV), with the highest turnover observed in growth cones and substantially slower turnover in dendrites, axons, and the axon initial segment (AIS). Microtubule turnover further decreased between 9 and 17 DIV in dendrites and axons, indicating progressive stabilization during neuronal maturation, whereas turnover in the AIS remained largely unchanged. Analysis of EB3 comet dynamics suggested that microtubule polymerization contributes substantially to tubulin turnover. In addition, manipulation of the dendritic microtubule-associated protein MAP-2 altered tubulin FRAP, indicating that MAP-2 contributes to the regulation of neuronal microtubule dynamics. Together, these results demonstrate that microtubule turnover is spatially and developmentally regulated in neurons.
Y. Sakai, Yuki Ogawa, Hiroaki Misonou· Neurosciences research· 0 citations
The possibility that microtubule architecture and dynamics evolved in Asgard archaea prior to eukaryogenesis is raised, as well as the broad occurrence of tubulins in Asgard archaea.
Jan Löwe, Andriko von Kügelgen, V. J. Planelles-Herrero et al.· Science Advances· 0 citations
Microtubules form dynamic cytoskeletal scaffolds essential for intracellular transport, organelle positioning, and spatial organization of signaling. Their architecture and function are continuously remodeled through the concerted actions of microtubule-associated proteins (MAPs), post-translational modifications (PTMs), and molecular motors. To precisely interrogate these processes in living systems, we developed a genetically encoded, single-component optogenetic platform for spatiotemporal control of microtubule organization and dynamics. By harnessing light-induced oligomerization to regulate microtubule association, this system supports reversible microtubule labeling and plus-end tracking, localized control of tubulin PTMs, optically regulated kinesin-driven cargo transport, and inducible microtubule severing within a unified design strategy. Using these tools, we reveal how local microtubule integrity governs lysosomal trafficking and endoplasmic reticulum (ER)-associated signaling dynamics. Collectively, this modular optogenetic toolkit bridges molecular design with cytoskeletal function, offering a versatile platform to dissect how dynamic cytoskeletal architectures coordinate intracellular organization, transport, and signaling.
Tien-Hung Lan, Guolin Ma, Xiaoxuan Liu et al.· Cell Reports Methods· 0 citations
Introduction The notion of microtubule (MT) dynamics relates to the changes in the length of MT polymers in living cells. They are governed by a stochastic process related to the rates and chances of adding or removing tubulin dimers at the ends of MT polymers, termed dynamic instability. The ability of each MT to swiftly switch between stages of adding or removing dimers at the tip of its lattice is critical for the overall success of the mitotic spindle, a molecular machinery built dynamically by MTs and associated molecular motor proteins, in properly segregating the duplicated DNA into the two daughter cells. When changes in the genetic and epigenetic regulation of the cell affect this ability, for example, by increasing the rates of hydrolysis of bound to tubulin dimers incorporated in the MT lattice, that results in segregation errors and is a hallmark of disease. Methods In cancer, the dysregulation of MT dynamics contributes to drug resistance. Measuring and modeling MT dynamics provides an insight into the regulation of the cell and its susceptibility to drug action. It can also characterize the function of patient immune cells and contribute to improving the success rate of cell therapy. Results Our investigation indicates that drug-resistant tumors exhibit particular changes in their regulation at the cellular level that expose cell state vulnerabilities, which can be targeted during therapy. We elucidate mechanisms of oncogenic activity in dividing cells and propose an approach for overcoming drug resistance. Conclusion Changes in MT regulation before and after drug treatment of patient cells are indicative of the susceptibility of tumors to particular drug regimens.
Alexandre Matov· Frontiers in Cell and Develo...· 0 citations
We use cookies to run the site and, with your consent, for analytics and to show ads.
See our Cookie Policy.
