Drosophila kinesin-8 stabilizes the kinetochore–microtubule interaction

© The Authors, 2019. This article is distributed under the terms of the Creative Commons Attribution-Noncommercial-Share Alike 4.0 International License. The definitive version was published in Journal of Cell Biology 218(2), (2019): 474-488, doi: 10.1083/jcb.201807077.Kinesin-8 is required for proper chromosome alignment in a variety of animal and yeast cell types. However, it is unclear how this motor protein family controls chromosome alignment, as multiple biochemical activities, including inconsistent ones between studies, have been identified. Here, we find that Drosophila kinesin-8 (Klp67A) possesses both microtubule (MT) plus end–stabilizing and –destabilizing activity, in addition to kinesin-8's commonly observed MT plus end–directed motility and tubulin-binding activity in vitro. We further show that Klp67A is required for stable kinetochore–MT attachment during prometaphase in S2 cells. In the absence of Klp67A, abnormally long MTs interact in an “end-on” fashion with kinetochores at normal frequency. However, the interaction is unstable, and MTs frequently become detached. This phenotype is rescued by ectopic expression of the MT plus end–stabilizing factor CLASP, but not by artificial shortening of MTs. We show that human kinesin-8 (KIF18A) is also important to ensure proper MT attachment. Overall, these results suggest that the MT-stabilizing activity of kinesin-8 is critical for stable kinetochore–MT attachment.We thank Kosuke Ariga for helping data analysis, Tomoko Nishiyama for technical support, Tomomi Kiyomitsu for valuable comments on the manuscript, and Elsa Tungadi for proofreading. This work was funded by Japan Society for the Promotion of Science (JSPS) KAK ENHI (15KT0077 and 17H01431) and Laura and Arthur Colwin Endowed Summer Research Fellowship Fund (2015) of the Marine Biological Laboratory to G. Goshima. T. Edzuka is a recipient of a JSPS pre-doctoral fellowship (16J02807)

Five factors can reconstitute all three phases of microtubule polymerization dynamics

© The Author(s), 2016. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Journal of Cell Biology 215 (2016): 357, doi:10.1083/jcb.201604118.Cytoplasmic microtubules (MTs) undergo growth, shrinkage, and pausing. However, how MT polymerization cycles are produced and spatiotemporally regulated at a molecular level is unclear, as the entire cycle has not been recapitulated in vitro with defined components. In this study, we reconstituted dynamic MT plus end behavior involving all three phases by mixing tubulin with five Drosophila melanogaster proteins (EB1, XMAP215Msps, Sentin, kinesin-13Klp10A, and CLASPMast/Orbit). When singly mixed with tubulin, CLASPMast/Orbit strongly inhibited MT catastrophe and reduced the growth rate. However, in the presence of the other four factors, CLASPMast/Orbit acted as an inducer of pausing. The mitotic kinase Plk1Polo modulated the activity of CLASPMast/Orbit and kinesin-13Klp10A and increased the dynamic instability of MTs, reminiscent of mitotic cells. These results suggest that five conserved proteins constitute the core factors for creating dynamic MTs in cells and that Plk1-dependent phosphorylation is a crucial event for switching from the interphase to mitotic mode.This work was supported by the Grants-in-Aid for Scientific Research (Japan Society for the Promotion of Science KAK ENHI; 15H01317 and 15KT0077).2017-04-3

The roles of microtubule-based motor proteins in mitosis: comprehensive RNAi analysis in the Drosophila S2 cell line

Kinesins and dyneins play important roles during cell division. Using RNA interference (RNAi) to deplete individual (or combinations of) motors followed by immunofluorescence and time-lapse microscopy, we have examined the mitotic functions of cytoplasmic dynein and all 25 kinesins in Drosophila S2 cells. We show that four kinesins are involved in bipolar spindle assembly, four kinesins are involved in metaphase chromosome alignment, dynein plays a role in the metaphase-to-anaphase transition, and one kinesin is needed for cytokinesis. Functional redundancy and alternative pathways for completing mitosis were observed for many single RNAi knockdowns, and failure to complete mitosis was observed for only three kinesins. As an example, inhibition of two microtubule-depolymerizing kinesins initially produced monopolar spindles with abnormally long microtubules, but cells eventually formed bipolar spindles by an acentrosomal pole-focusing mechanism. From our phenotypic data, we construct a model for the distinct roles of molecular motors during mitosis in a single metazoan cell type

Clustering of a kinesin-14 motor enables processive retrograde microtubule-based transport in plants

Author Posting. © The Author(s), 2015. This is the author's version of the work. It is posted here by permission of Nature Publishing Group for personal use, not for redistribution. The definitive version was published in Nature Plants 1 (2015): 15087, doi:10.1038/nplants.2015.87.The molecular motors kinesin and dynein drive bidirectional motility along microtubules (MTs) in most eukaryotic cells1,2. Land plants, however, are a notable exception, since they contain a large number of kinesins but lack cytoplasmic dynein, the foremost processive retrograde transporter3,4. It remains unclear how plants achieve retrograde cargo transport without dynein. Here, we have analyzed the motility of the six members of minus-end-directed kinesin-14 motors in the moss Physcomitrella patens and found that none are processive as native dimers. However, when artificially clustered into as little as dimer of dimers, the type-VI kinesin-14 (a homologue of Arabidopsis KCBP [kinesin-like calmodulin binding protein]) exhibited highly processive and fast motility (up to 0.6 μm/s). Multiple kin14-VI dimers attached to liposomes also induced transport of this membrane cargo over several microns. Consistent with these results, in vivo observations of GFP-tagged kin14-VI in moss cells revealed fluorescent punctae that moved processively towards the minus ends of the cytoplasmic MTs. These data suggest that clustering of a kinesin-14 motor serves as a dynein-independent mechanism for retrograde transport in plants.This work was supported by the Human Frontier Science Program, the James A. and Faith Miller Memorial Fund (MBL), the Laura and Arthur Colwin Endowed Summer Research Fellowship Fund (MBL), the TORAY Science Foundation, Grants-in-Aid for Scientific Research (15K14540, MEXT) (G.G), and the NIH (38499; R.D.V).2015-12-2

Human centromere chromatin protein hMis12, essential for equal segregation, is independent of CENP-A loading pathway

Kinetochores are the chromosomal sites for spindle interaction and play a vital role for chromosome segregation. The composition of kinetochore proteins and their cellular roles are, however, poorly understood in higher eukaryotes. We identified a novel kinetochore protein family conserved from yeast to human that is essential for equal chromosome segregation. The human homologue hMis12 of yeast spMis12/scMtw1 retains conserved sequence features and locates at the kinetochore region indistinguishable from CENP-A, a centromeric histone variant. RNA interference (RNAi) analysis of HeLa cells shows that the reduced hMis12 results in misaligned metaphase chromosomes, lagging anaphase chromosomes, and interphase micronuclei without mitotic delay, while CENP-A is located at kinetochores. Further, the metaphase spindle length is abnormally extended. Spindle checkpoint protein hMad2 temporally localizes at kinetochores at early mitotic stages after RNAi. The RNAi deficiency of CENP-A leads to a similar mitotic phenotype, but the kinetochore signals of other kinetochore proteins, hMis6 and CENP-C, are greatly diminished. RNAi for hMis6, like that of a kinetochore kinesin CENP-E, induces mitotic arrest. Kinetochore localization of hMis12 is unaffected by CENP-A RNAi, demonstrating an independent pathway of CENP-A in human kinetochores

RNAi screening identifies the armadillo repeat-containing kinesins responsible for microtubule-dependent nuclear positioning in Physcomitrella patens

Author Posting. © The Author(s), 2015. This is the author's version of the work. It is posted here by permission of Oxford University Press for personal use, not for redistribution. The definitive version was published in Plant and Cell Physiology 56 (2015): 737-749, doi:10.1093/pcp/pcv002.Proper positioning of the nucleus is critical for the functioning of various cells. Actin and myosin have been shown to be crucial for the localisation of the nucleus in plant cells, whereas microtubule (MT)-based mechanisms are commonly utilised in animal and fungal cells. In this study, we combined live cell microscopy with RNA interference (RNAi) screening or drug treatment and showed that MTs and a plant-specific motor protein, armadillo repeat-containing kinesin (kinesin-ARK), are required for nuclear positioning in the moss Physcomitrella patens. In tip-growing protonemal apical cells, the nucleus was translocated to the centre of the cell after cell division in an MT-dependent manner. When kinesin-ARKs were knocked down using RNAi, the initial movement of the nucleus towards the centre took place normally; however, before reaching the centre, the nucleus was moved back to the basal edge of the cell. In intact (control) cells, MT bundles that are associated with kinesin-ARKs were frequently observed around the moving nucleus. In contrast, such MT bundles were not identified after kinesin-ARK downregulation. An in vitro MT-gliding assay showed that kinesin-ARK is a plus-end-directed motor protein. These results indicate that MTs and the MT-based motor drive nuclear migration in the moss cells, thus showing a conservation of the mechanism underlying nuclear localisation among plant, animal, and fungal cells.This work was supported by the Next Generation grant (Japan Society for Promotion of Science; JSPS), James A. and Faith Miller Memorial Fund, and Human Frontier Science Program (to G.G.). T.M. is a recipient of a pre-doctoral fellowship of the JSPS.2016-01-1

Augmin : a protein complex required for centrosome-independent microtubule generation within the spindle

© 2008 Goshima et al. This article is distributed under the terms of a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license). The definitive version was published in Journal of Cell Biology 181 (2008): 421-429, doi:10.1083/jcb.200711053.Since the discovery of γ-tubulin, attention has focused on its involvement as a microtubule nucleator at the centrosome. However, mislocalization of {gamma}-tubulin away from the centrosome does not inhibit mitotic spindle formation in Drosophila melanogaster, suggesting that a critical function for γ-tubulin might reside elsewhere. A previous RNA interference (RNAi) screen identified five genes (Dgt2–6) required for localizing γ-tubulin to spindle microtubules. We show that the Dgt proteins interact, forming a stable complex. We find that spindle microtubule generation is substantially reduced after knockdown of each Dgt protein by RNAi. Thus, the Dgt complex that we name "augmin" functions to increase microtubule number. Reduced spindle microtubule generation after augmin RNAi, particularly in the absence of functional centrosomes, has dramatic consequences on mitotic spindle formation and function, leading to reduced kinetochore fiber formation, chromosome misalignment, and spindle bipolarity defects. We also identify a functional human homologue of Dgt6. Our results suggest that an important mitotic function for γ-tubulin may lie within the spindle, where augmin and γ-tubulin function cooperatively to amplify the number of microtubules.This work is supported by the Special Coordination Funds for Promoting Science and Technology (MEXT, Japan), the Global COE Program “Advanced Systems-Biology: Designing the Biological Function” (MEXT), and the Uehara Memorial Foundation

Shortening of microtubule overlap regions defines membrane delivery sites during plant cytokinesis

© The Author(s), 2016. This is the author's version of the work and is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Current Biology 27 (2017): 514-520, doi:10.1016/j.cub.2016.12.043.Different from animal cells that divide by constriction of the cortex inwards, cells of land plants divide by initiating a new cell wall segment from their centre. For this, a disk-shaped, membrane-enclosed precursor termed the cell plate is formed that radially expands towards the parental cell wall. The synthesis of the plate starts with the fusion of vesicles into a tubulo-vesicular network. Vesicles are putatively delivered to the division plane by transport along microtubules of the bipolar phragmoplast network that guides plate assembly. How vesicle immobilisation and fusion are then locally triggered is unclear. In general, a framework for how the cytoskeleton spatially defines cell plate formation is lacking. Here we show that membranous material for cell plate formation initially accumulates along regions of microtubule overlap in the phragmoplast of the moss Physcomitrella patens. Kinesin-4 mediated shortening of these overlaps at the onset of cytokinesis proved to be required to spatially confine membrane accumulation. Without shortening, the wider cell plate membrane depositions evolved into cell walls that were thick and irregularly shaped. Phragmoplast assembly thus provides a regular lattice of short overlaps on which a new cell wall segment can be scaffolded. Since similar patterns of overlaps form in central spindles of animal cells, involving the activity of orthologous proteins, we anticipate that our results will help uncover universal features underlying membrane-cytoskeleton coordination during cytokinesis.The work has been financially supported by HFSP grant RGP0026/2011 to MEJ and GG.2018-01-2

Reconstitution of dynamic microtubules with Drosophila XMAP215, EB1, and Sentin

© The Author(s), 2012. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Journal of Cell Biology 199 (2012): 849-862, doi:10.1083/jcb.201206101.Dynamic microtubules (MTs) are essential for various intracellular events, such as mitosis. In Drosophila melanogaster S2 cells, three MT tip-localizing proteins, Msps/XMAP215, EB1, and Sentin (an EB1 cargo protein), have been identified as being critical for accelerating MT growth and promoting catastrophe events, thus resulting in the formation of dynamic MTs. However, the molecular activity of each protein and the basis of the modulation of MT dynamics by these three factors are unknown. In this paper, we showed in vitro that XMAP215msps had a potent growth-promoting activity at a wide range of tubulin concentrations, whereas Sentin, when recruited by EB1 to the growing MT tip, accelerated growth and also increased catastrophe frequency. When all three factors were combined, the growth rate was synergistically enhanced, and rescue events were observed most frequently, but frequent catastrophes restrained the lengthening of the MTs. We propose that MT dynamics are promoted by the independent as well as the cooperative action of XMAP215msps polymerase and the EB1–Sentin duo.This work was supported by a Next Generation grant (Japan Society for the Promotion of Science), the Inoue Foundation, and the Human Frontier Science Program (to G. Goshima). W. Li was supported by the Global Centers of Excellence program, the Leading Graduate School program, and the State Scholarship Study Abroad Program of the Chinese Scholarship Council.2013-05-2