Augmin-dependent microtubule nucleation at microtubule walls in the spindle

© The Author(s), 2013. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Journal of Cell Biology 202 (2013): 25-33, doi:10.1083/jcb.201304031.The formation of a functional spindle requires microtubule (MT) nucleation from within the spindle, which depends on augmin. How augmin contributes to MT formation and organization is not known because augmin-dependent MTs have never been specifically visualized. In this paper, we identify augmin-dependent MTs and their connections to other MTs by electron tomography and 3D modeling. In metaphase spindles of human cells, the minus ends of MTs were located both around the centriole and in the body of the spindle. When augmin was knocked down, the latter population of MTs was significantly reduced. In control cells, we identified connections between the wall of one MT and the minus end of a neighboring MT. Interestingly, the connected MTs were nearly parallel, unlike other examples of end–wall connections between cytoskeletal polymers. Our observations support the concept of augmin-dependent MT nucleation at the walls of existing spindle MTs. Furthermore, they suggest a mechanism for maintaining polarized MT organization, even when noncentrosomal MT initiation is widespread.This work was supported by the Next Generation grant (Japan Society for the Promotion of Science), Human Frontier Science Program, James A. and Faith Miller Memorial Fund (to G. Goshima), the Hori Sciences and Arts Foundation, the Sasakawa Scientific Research Grant, the Kazato Research foundation (to T. Kamasaki), and the National Institutes of Health (8P41GM103431-42 to A. Hoenger). T. Kamasaki was a recipient of the Japan Society for the Promotion of Science postdoctoral fellowship.2014-01-0

Microtubule depolymerization by the kinesin-8 motor Kip3p: a mathematical model

Proteins from the kinesin-8 family promote microtubule (MT) depolymerization, a process thought to be important for the control of microtubule length in living cells. In addition to this MT shortening activity, kinesin 8s are motors that show plus-end directed motility on MTs. Here we describe a simple model that incorporates directional motion and destabilization of the MT plus end by kinesin 8. Our model quantitatively reproduces the key features of length-vs-time traces for stabilized MTs in the presence of purified kinesin 8, including length-dependent depolymerization. Comparison of model predictions with experiments suggests that kinesin 8 depolymerizes processively, i.e., one motor can remove multiple tubulin dimers from a stabilized MT. Fluctuations in MT length as a function of time are related to depolymerization processivity. We have also determined the parameter regime in which the rate of MT depolymerization is length dependent: length-dependent depolymerization occurs only when MTs are sufficiently short; this crossover is sensitive to the bulk motor concentration.Comment: 34 pages, 11 figure

Decreased Expression of Semaphorin-3A, a Neurite-Collapsing Factor, is Associated With Itch in Psoriatic Skin

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Spatial control of translation repression and polarized growth by conserved NDR kinase Orb6 and RNA-binding protein Sts5

© The Author(s), 2016. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in eLife 5 (2016): e14216, doi:10.7554/eLife.14216.RNA-binding proteins contribute to the formation of ribonucleoprotein (RNP) granules by phase transition, but regulatory mechanisms are not fully understood. Conserved fission yeast NDR (Nuclear Dbf2-Related) kinase Orb6 governs cell morphogenesis in part by spatially controlling Cdc42 GTPase. Here we describe a novel, independent function for Orb6 kinase in negatively regulating the recruitment of RNA-binding protein Sts5 into RNPs to promote polarized cell growth. We find that Orb6 kinase inhibits Sts5 recruitment into granules, its association with processing (P) bodies, and degradation of Sts5-bound mRNAs by promoting Sts5 interaction with 14-3-3 protein Rad24. Many Sts5-bound mRNAs encode essential factors for polarized cell growth, and Orb6 kinase spatially and temporally controls the extent of Sts5 granule formation. Disruption of this control system affects cell morphology and alters the pattern of polarized cell growth, revealing a role for Orb6 kinase in the spatial control of translational repression that enables normal cell morphogenesis.Work in FV’s laboratory is supported by the National Institutes of Health R01 grant number GM095867. Part of this work was also supported by NSF grant 0745129. TT was supported by Japan Society for the Promotion of Science grants 16H02503 and 16K14672 and by Cancer Research UK

Inducible fluorescent speckle microscopy

The understanding of cytoskeleton dynamics has benefited from the capacity to generate fluorescent fiducial marks on cytoskeleton components. Here we show that light-induced imprinting of three-dimensional (3D) fluorescent speckles significantly improves speckle signal and contrast relative to classic (random) fluorescent speckle microscopy. We predict theoretically that speckle imprinting using photobleaching is optimal when the laser energy and fluorophore responsivity are related by the golden ratio. This relation, which we confirm experimentally, translates into a 40% remaining signal after speckle imprinting and provides a rule of thumb in selecting the laser power required to optimally prepare the sample for imaging. This inducible speckle imaging (ISI) technique allows 3D speckle microscopy to be performed in readily available libraries of cell lines or primary tissues expressing fluorescent proteins and does not preclude conventional imaging before speckle imaging. As a proof of concept, we use ISI to measure metaphase spindle microtubule poleward flux in primary cells and explore a scaling relation connecting microtubule flux to metaphase duration.H. Maiato is funded by the seventh framework program grant PRE CISE from the European Research Council, FLAD Life Science 2020, and the Louis-Jeantet Young Investigator Career Award

Biophysically Realistic Filament Bending Dynamics in Agent-Based Biological Simulation

An appealing tool for study of the complex biological behaviors that can emerge from networks of simple molecular interactions is an agent-based, computational simulation that explicitly tracks small-scale local interactions – following thousands to millions of states through time. For many critical cell processes (e.g. cytokinetic furrow specification, nuclear centration, cytokinesis), the flexible nature of cytoskeletal filaments is likely to be critical. Any computer model that hopes to explain the complex emergent behaviors in these processes therefore needs to encode filament flexibility in a realistic manner. Here I present a numerically convenient and biophysically realistic method for modeling cytoskeletal filament flexibility in silico. Each cytoskeletal filament is represented by a series of rigid segments linked end-to-end in series with a variable attachment point for the translational elastic element. This connection scheme allows an empirically tuning, for a wide range of segment sizes, viscosities, and time-steps, that endows any filament species with the experimentally observed (or theoretically expected) static force deflection, relaxation time-constant, and thermal writhing motions. I additionally employ a unique pair of elastic elements – one representing the axial and the other the bending rigidity– that formulate the restoring force in terms of single time-step constraint resolution. This method is highly local –adjacent rigid segments of a filament only interact with one another through constraint forces—and is thus well-suited to simulations in which arbitrary additional forces (e.g. those representing interactions of a filament with other bodies or cross-links / entanglements between filaments) may be present. Implementation in code is straightforward; Java source code is available at www.celldynamics.org

Microtubule sliding activity of a kinesin-8 promotes spindle assembly and spindle length control

Molecular motors play critical roles in the formation of mitotic spindles, either through controlling the stability of individual microtubules, or by cross-linking and sliding microtubule arrays. Kinesin-8 motors are best known for their regulatory roles in controlling microtubule dynamics. They contain microtubule-destabilizing activities, and restrict spindle length in a wide variety of cell types and organisms. Here, we report for the first time on an anti-parallel microtubule-sliding activity of the budding yeast kinesin-8, Kip3. The in vivo importance of this sliding activity was established through the identification of complementary Kip3 mutants that separate the sliding activity and microtubule destabilizing activity. In conjunction with kinesin-5/Cin8, the sliding activity of Kip3 promotes bipolar spindle assembly and the maintenance of genome stability. We propose a “slide-disassemble” model where Kip3’s sliding and destabilizing activity balance during pre-anaphase. This facilitates normal spindle assembly. However, Kip3’s destabilizing activity dominates in late anaphase, inhibiting spindle elongation and ultimately promoting spindle disassembly

Kank Is an EB1 Interacting Protein that Localises to Muscle-Tendon Attachment Sites in Drosophila

Little is known about how microtubules are regulated in different cell types during development. EB1 plays a central role in the regulation of microtubule plus ends. It directly binds to microtubule plus ends and recruits proteins which regulate microtubule dynamics and behaviour. We report the identification of Kank, the sole Drosophila orthologue of human Kank proteins, as an EB1 interactor that predominantly localises to embryonic attachment sites between muscle and tendon cells. Human Kank1 was identified as a tumour suppressor and has documented roles in actin regulation and cell polarity in cultured mammalian cells. We found that Drosophila Kank binds EB1 directly and this interaction is essential for Kank localisation to microtubule plus ends in cultured cells. Kank protein is expressed throughout fly development and increases during embryogenesis. In late embryos, it accumulates to sites of attachment between muscle and epidermal cells. A kank deletion mutant was generated. We found that the mutant is viable and fertile without noticeable defects. Further analysis showed that Kank is dispensable for muscle function in larvae. This is in sharp contrast to C. elegans in which the Kank orthologue VAB-19 is required for development by stabilising attachment structures between muscle and epidermal cells

Motor domain phosphorylation and regulation of the Drosophila kinesin 13, KLP10A

Microtubule (MT)-destabilizing kinesin 13s perform fundamental roles throughout the cell cycle. In this study, we show that the Drosophila melanogaster kinesin 13, KLP10A, is phosphorylated in vivo at a conserved serine (S573) positioned within the α-helix 5 of the motor domain. In vitro, a phosphomimic KLP10A S573E mutant displays a reduced capacity to depolymerize MTs but normal affinity for the MT lattice. In cells, replacement of endogenous KLP10A with KLP10A S573E dampens MT plus end dynamics throughout the cell cycle, whereas a nonphosphorylatable S573A mutant apparently enhances activity during mitosis. Electron microscopy suggests that KLP10A S573 phosphorylation alters its association with the MT lattice, whereas molecular dynamics simulations reveal how KLP10A phosphorylation can alter the kinesin–MT interface without changing important structural features within the motor’s core. Finally, we identify casein kinase 1α as a possible candidate for KLP10A phosphorylation. We propose a model in which phosphorylation of the KLP10A motor domain provides a regulatory switch controlling the time and place of MT depolymerization