Publications by authors named "Gohta Goshima"

68 Publications

Plant stem cell research is uncovering the secrets of longevity and persistent growth.

Plant J 2021 Apr 25;106(2):326-335. Epub 2021 Mar 25.

Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, 464-8601, Japan.

Plant stem cells have several extraordinary features: they are generated de novo during development and regeneration, maintain their pluripotency, and produce another stem cell niche in an orderly manner. This enables plants to survive for an extended period and to continuously make new organs, representing a clear difference in their developmental program from animals. To uncover regulatory principles governing plant stem cell characteristics, our research project 'Principles of pluripotent stem cells underlying plant vitality' was launched in 2017, supported by a Grant-in-Aid for Scientific Research on Innovative Areas from the Japanese government. Through a collaboration involving 28 research groups, we aim to identify key factors that trigger epigenetic reprogramming and global changes in gene networks, and thereby contribute to stem cell generation. Pluripotent stem cells in the shoot apical meristem are controlled by cytokinin and auxin, which also play a crucial role in terminating stem cell activity in the floral meristem; therefore, we are focusing on biosynthesis, metabolism, transport, perception, and signaling of these hormones. Besides, we are uncovering the mechanisms of asymmetric cell division and of stem cell death and replenishment under DNA stress, which will illuminate plant-specific features in preserving stemness. Our technology support groups expand single-cell omics to describe stem cell behavior in a spatiotemporal context, and provide correlative light and electron microscopic technology to enable live imaging of cell and subcellular dynamics at high spatiotemporal resolution. In this perspective, we discuss future directions of our ongoing projects and related research fields.
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http://dx.doi.org/10.1111/tpj.15184DOI Listing
April 2021

Ran-GTP Is Non-essential to Activate NuMA for Mitotic Spindle-Pole Focusing but Dynamically Polarizes HURP Near Chromosomes.

Curr Biol 2021 Jan 12;31(1):115-127.e3. Epub 2020 Nov 12.

Division of Biological Science, Graduate School of Science, Nagoya University, Chikusa-ku, Nagoya 464-8602, Japan; Precursory Research for Embryonic Science and Technology (PRESTO) Program, Japan Science and Technology Agency, 4-1-8 Honcho Kawaguchi, Saitama 332-0012, Japan; Okinawa Institute of Science and Technology Graduate University, 1919-1 Tancha, Onna-son, Kunigami-gun, Okinawa 904-0495, Japan. Electronic address:

Spindle assembly is spatially regulated by a chromosome-derived Ran- GTP gradient. Previous work proposed that Ran-GTP activates spindle assembly factors (SAFs) around chromosomes by dissociating inhibitory importins from SAFs. However, it is unclear whether the Ran-GTP gradient equivalently activates SAFs that localize at distinct spindle regions. In addition, Ran's dual functions in interphase nucleocytoplasmic transport and mitotic spindle assembly have made it difficult to assess its mitotic roles in somatic cells. Here, using auxin-inducible degron technology in human cells, we developed acute mitotic depletion assays to dissect Ran's mitotic roles systematically and separately from its interphase function. In contrast to the prevailing model, we found that the Ran pathway is not essential for spindle assembly activities that occur at sites spatially separated from chromosomes, including activating NuMA for spindle-pole focusing or for targeting TPX2. On the other hand, Ran-GTP is required to localize HURP and HSET specifically at chromosome-proximal regions to set proper spindle length during prometaphase. We demonstrated that Ran-GTP and importin-β coordinately promote HURP's dynamic microtubule binding-dissociation cycle, which maintains HURP near chromosomes during metaphase. Together, we propose that the Ran pathway acts on spindle assembly independently of its interphase functions in mitotic human cells but does not equivalently regulate all Ran-regulated SAFs. Ran-dependent spindle assembly is likely coupled with additional parallel pathways that activate SAFs distantly located from the chromosomes.
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http://dx.doi.org/10.1016/j.cub.2020.09.091DOI Listing
January 2021

Rho of Plants GTPases and Cytoskeletal Elements Control Nuclear Positioning and Asymmetric Cell Division during Physcomitrella patens Branching.

Curr Biol 2020 07 28;30(14):2860-2868.e3. Epub 2020 May 28.

Division of Biological Science, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8602, Japan; Sugashima Marine Biological Laboratory, Graduate School of Science, Nagoya University, Sugashima-cho, Toba 517-0004, Japan.

Branching morphogenesis is a widely used mechanism for development [1, 2]. In plants, it is initiated by the emergence of a new growth axis, which is of particular importance for plants to explore space and access resources [1]. Branches can emerge either from a single cell or from a group of cells [3-5]. In both cases, the mother cells that initiate branching must undergo dynamic morphological changes and/or adopt oriented asymmetric cell divisions (ACDs) to establish the new growth direction. However, the underlying mechanisms are not fully understood. Here, using the bryophyte moss Physcomitrella patens as a model, we show that side-branch formation in P. patens protonemata requires coordinated polarized cell expansion, directional nuclear migration, and orientated ACD. By combining pharmacological experiments, long-term time-lapse imaging, and genetic analyses, we demonstrate that Rho of plants (ROP) GTPases and actin are essential for cell polarization and local cell expansion (bulging). The growing bulge acts as a prerequisite signal to guide long-distance microtubule (MT)-dependent nuclear migration, which determines the asymmetric positioning of the division plane. MTs play an essential role in nuclear migration but are less involved in bulge formation. Hence, cell polarity and cytoskeletal elements act cooperatively to modulate cell morphology and nuclear positioning during branch initiation. We propose that polarity-triggered nuclear positioning and ACD comprise a fundamental mechanism for increasing multicellularity and tissue complexity during plant morphogenesis.
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http://dx.doi.org/10.1016/j.cub.2020.05.022DOI Listing
July 2020

Kinesin-13 and Kinesin-8 Function during Cell Growth and Division in the Moss .

Plant Cell 2020 03 9;32(3):683-702. Epub 2020 Jan 9.

Division of Biological Science, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8602, Japan

Kinesin-13 and Kinesin-8 are well-known microtubule (MT) depolymerases that regulate MT length and chromosome movement in animal mitosis. While much is unknown about plant Kinesin-8, Arabidopsis () and rice () Kinesin-13 have been shown to depolymerize MTs in vitro. However, the mitotic function of both kinesins has yet to be determined in plants. Here, we generated complete null mutants of and in moss (). Both kinesins were found to be nonessential for viability, but the knockout (KO) line had increased mitotic duration and reduced spindle length, whereas the KO line did not display obvious mitotic defects. Surprisingly, spindle MT poleward flux, which is mediated by Kinesin-13 in animals, was retained in the absence of Kinesin-13. MT depolymerase activity was not detectable for either kinesin in vitro, while MT catastrophe-inducing activity (Kinesin-13) or MT gliding activity (Kinesin-8) was observed. Interestingly, both KO lines showed waviness in their protonema filaments, which correlated with positional instability of the MT foci in their tip cells. Taken together, the results suggest that plant Kinesin-13 and Kinesin-8 have diverged in both mitotic function and molecular activity, acquiring roles in regulating MT foci positioning for directed tip growth.
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http://dx.doi.org/10.1105/tpc.19.00521DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7054034PMC
March 2020

A versatile microfluidic device for highly inclined thin illumination microscopy in the moss Physcomitrella patens.

Sci Rep 2019 10 23;9(1):15182. Epub 2019 Oct 23.

Division of Biological Science, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Aichi, 464-8602, Japan.

High-resolution microscopy is a valuable tool for studying cellular processes, such as signalling, membrane trafficking, or cytoskeleton remodelling. Several techniques of inclined illumination microscopy allow imaging at a near single molecular level; however, the application of these methods to plant cells is limited, owing to thick cell walls as well as the necessity to excise a part of the tissue for sample preparation. In this study, we utilised a simple, easy-to-use microfluidic device for highly inclined and laminated optical sheet (HILO) microscopy using a model plant Physcomitrella patens. We demonstrated that the shallow microfluidic device can be used for long-term culture of living cells and enables high-resolution HILO imaging of microtubules without perturbing their dynamics. In addition, our microdevice allows the supply and robust washout of compounds during HILO microscopy imaging, for example, to perform a microtubule regrowth assay. Furthermore, we tested long-term (48 h) HILO imaging using a microdevice and visualised the developmental changes in the microtubule dynamics during tissue regeneration. These novel applications of the microfluidic device provide a valuable resource for studying molecular dynamics in living plant cells.
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http://dx.doi.org/10.1038/s41598-019-51624-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6811556PMC
October 2019

Moss Kinesin-14 KCBP Accelerates Chromatid Motility in Anaphase.

Cell Struct Funct 2019 ;44(2):95-104

Division of Biological Science, Graduate School of Science, Nagoya University.

KCBP is a microtubule (MT) minus-end-directed kinesin widely conserved in plants. It was shown in Arabidopsis that KCBP controls trichome cell shape by orchestrating MT and actin cytoskeletons using its tail and motor domains. In contrast, the KCBP knockout (KO) line in the moss Physcomitrella patens showed a defect in nuclear and organelle positioning in apical stem cells. Moss KCBP is postulated to transport the nucleus and chloroplast via direct binding to their membranes, since it binds to and transports liposomes composed of phospholipids in vitro. However, domains required for cargo transport in vivo have not been mapped. Here, we performed a structure-function analysis of moss KCBP. We found that the FERM domain in the tail region, which is known to bind to lipids as well as other proteins, is essential for both nuclear and chloroplast positioning, whereas the proximal MyTH4 domain plays a supporting role in chloroplast transport. After anaphase but prior to nuclear envelope re-formation, KCBP accumulates on the chromosomes, in particular at the centromeric region in a FERM-dependent manner. In the KCBP KO line, the rate of poleward chromosome movement in anaphase was reduced and lagging chromosomes occasionally appeared. These results suggest that KCBP binds to non-membranous naked chromosomes via an unidentified protein(s) for their transport. Finally, the liverwort orthologue of KCBP rescued the chromosome/chloroplast mis-positioning of the moss KCBP KO line, suggesting that the cargo transport function is conserved at least in bryophytes.Key words: kinesin, mitosis, chromosome segregation, kinetochore, dynein.
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http://dx.doi.org/10.1247/csf.19015DOI Listing
April 2020

Identification of 15 New Bypassable Essential Genes of Fission Yeast.

Cell Struct Funct 2019 Sep 31;44(2):113-119. Epub 2019 Aug 31.

Division of Biological Science, Graduate School of Science, Nagoya University.

Every organism has a different set of genes essential for its viability. This indicates that an organism can become tolerant to the loss of an essential gene under certain circumstances during evolution, via the manifestation of 'masked' alternative mechanisms. In our quest to systematically uncover masked mechanisms in eukaryotic cells, we developed an extragenic suppressor screening method using haploid spores deleted of an essential gene in the fission yeast Schizosaccharomyces pombe. We screened for the 'bypass' suppressors of lethality of 92 randomly selected genes that are essential for viability in standard laboratory culture conditions. Remarkably, extragenic mutations bypassed the essentiality of as many as 20 genes (22%), 15 of which have not been previously reported. Half of the bypass-suppressible genes were involved in mitochondria function; we also identified multiple genes regulating RNA processing. 18 suppressible genes were conserved in the budding yeast Saccharomyces cerevisiae, but 13 of them were non-essential in that species. These trends suggest that essentiality bypass is not a rare event and that each organism may be endowed with secondary or backup mechanisms that can substitute for primary mechanisms in various biological processes. Furthermore, the robustness of our simple spore-based methodology paves the way for genome-scale screening.Key words: Schizosaccharomyces pombe, extragenic suppressor screening, bypass of essentiality (BOE), cut7 (kinesin-5), hul5 (E3 ubiquitin ligase).
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http://dx.doi.org/10.1247/csf.19025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6877344PMC
September 2019

Transient cotransformation of CRISPR/Cas9 and oligonucleotide templates enables efficient editing of target loci in Physcomitrella patens.

Plant Biotechnol J 2020 03 9;18(3):599-601. Epub 2019 Sep 9.

Division of Biological Science, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Japan.

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http://dx.doi.org/10.1111/pbi.13238DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7004911PMC
March 2020

Editorial overview: Cell division - from molecules to tissues.

Curr Opin Cell Biol 2019 10 26;60:iii-v. Epub 2019 Jul 26.

PSL, UMR 3215 U934 Department of Genetics and Developmental Biology, Institut Curie, Paris, France. Electronic address:

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http://dx.doi.org/10.1016/j.ceb.2019.06.006DOI Listing
October 2019

Kinetochore protein depletion underlies cytokinesis failure and somatic polyploidization in the moss .

Elife 2019 03 5;8. Epub 2019 Mar 5.

Division of Biological Science, Graduate School of Science, Nagoya University, Nagoya, Japan.

Lagging chromosome is a hallmark of aneuploidy arising from errors in the kinetochore-spindle attachment in animal cells. However, kinetochore components and cellular phenotypes associated with kinetochore dysfunction are much less explored in plants. Here, we carried out a comprehensive characterization of conserved kinetochore components in the moss and uncovered a distinct scenario in plant cells regarding both the localization and cellular impact of the kinetochore proteins. Most surprisingly, knock-down of several kinetochore proteins led to polyploidy, not aneuploidy, through cytokinesis failure in >90% of the cells that exhibited lagging chromosomes for several minutes or longer. The resultant cells, containing two or more nuclei, proceeded to the next cell cycle and eventually developed into polyploid plants. As lagging chromosomes have been observed in various plant species in the wild, our observation raised a possibility that they could be one of the natural pathways to polyploidy in plants.
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http://dx.doi.org/10.7554/eLife.43652DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6433463PMC
March 2019

kinesin-8 stabilizes the kinetochore-microtubule interaction.

J Cell Biol 2019 02 11;218(2):474-488. Epub 2018 Dec 11.

Division of Biological Science, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Japan

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 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.
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http://dx.doi.org/10.1083/jcb.201807077DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6363442PMC
February 2019

Microtubule nucleation and organization without centrosomes.

Curr Opin Plant Biol 2018 12 2;46:1-7. Epub 2018 Jul 2.

Division of Biological Science, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8602, Japan. Electronic address:

Centrosomes play various critical roles in animal cells such as microtubule nucleation and stabilization, mitotic spindle morphogenesis, and spindle orientation. Land plants have lost centrosomes and yet must execute many of these functions. Recent studies have revealed the crucial roles played by morphologically distinct cytoplasmic microtubule-organizing centers (MTOCs) in initiating spindle bipolarity and maintaining spindle orientation robustness. These MTOCs resemble centrosomes in many aspects, implying an evolutionary divergence of MT-organizing structures in plants. However, their functions rely on conserved nucleation and amplification mechanisms, indicating a similarity in MT network establishment between animals and plants. Moreover, recent characterization of a plant-specific MT minus-end tracking protein suggests that plants have developed functionally equivalent modules to stabilize and organize MTs at minus ends. These findings support the theory that plants overcome centrosome loss by utilizing modified but substantially conserved mechanisms to organize MT networks.
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http://dx.doi.org/10.1016/j.pbi.2018.06.004DOI Listing
December 2018

The KCH Kinesin Drives Nuclear Transport and Cytoskeletal Coalescence to Promote Tip Cell Growth in .

Plant Cell 2018 07 7;30(7):1496-1510. Epub 2018 Jun 7.

Division of Biological Science, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8602, Japan

Long-distance transport along microtubules (MTs) is critical for intracellular organization. In animals, antagonistic motor proteins kinesin (plus end directed) and dynein (minus end directed) drive cargo transport. In land plants, however, the identity of motors responsible for transport is poorly understood, as genes encoding cytoplasmic dynein are absent in plant genomes. How other functions of dynein are brought about in plants also remains unknown. Here, we show that a subclass of the kinesin-14 family, KCH (kinesin with calponin homology domain), which can also bind actin, drives MT minus end-directed nuclear transport in the moss When all four genes were deleted, the nucleus was not maintained in the cell center but was translocated to the apical end of protonemal cells. In the knockout (KO) line, apical cell tip growth was also severely suppressed. KCH was localized to MTs, including at the MT focal point near the tip of protonemal cells, where MT plus ends coalesced with actin filaments. MT focus was not stably maintained in KO lines, whereas actin destabilization also disrupted the MT focus in wild-type lines despite KCH remaining on unfocused MTs. KCH had distinct functions in nuclear transport and tip growth, as a truncated KCH construct restored nuclear transport activity, but not tip growth retardation of the KO line. Thus, our study identified KCH as a long-distance retrograde transporter as well as a MT cross-linker, reminiscent of the versatile animal dynein.
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http://dx.doi.org/10.1105/tpc.18.00038DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6096588PMC
July 2018

SPIRAL2 Stabilises Endoplasmic Microtubule Minus Ends in the Moss Physcomitrella patens.

Cell Struct Funct 2018 03 15;43(1):53-60. Epub 2018 Mar 15.

Division of Biological Science, Graduate School of Science, Nagoya University.

Stabilisation of minus ends of microtubules (MTs) is critical for organising MT networks in land plant cells, in which all MTs are nucleated independent of centrosomes. Recently, Arabidopsis SPIRAL2 (SPR2) protein was shown to localise to plus and minus ends of cortical MTs, and increase stability of both ends. Here, we report molecular and functional characterisation of SPR2 of the basal land plant, the moss Physcomitrella patens. In protonemal cells of P. patens, where non-cortical, endoplasmic MT network is organised, we observed SPR2 at minus ends, but not plus ends, of endoplasmic MTs and likely also of phragmoplast MTs. Minus end decoration was reconstituted in vitro using purified SPR2, suggesting that moss SPR2 is a minus end-specific binding protein (-TIP). We generated a loss-of-function mutant of SPR2, in which frameshift-causing deletions/insertions were introduced into all four paralogous SPR2 genes by means of CRISPR/Cas9. Protonemal cells of the mutant showed instability of endoplasmic MT minus ends. These results indicate that moss SPR2 is a MT minus end stabilising factor.Key words: acentrosomal microtubule network, microtubule minus end, P. patens, CAMSAP/Nezha/Patronin.
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http://dx.doi.org/10.1247/csf.18001DOI Listing
March 2018

Cytoplasmic MTOCs control spindle orientation for asymmetric cell division in plants.

Proc Natl Acad Sci U S A 2017 10 2;114(42):E8847-E8854. Epub 2017 Oct 2.

Division of Biological Science, Graduate School of Science, Nagoya University, Chikusa-ku, Nagoya 464-8602, Japan;

Proper orientation of the cell division axis is critical for asymmetric cell divisions that underpin cell differentiation. In animals, centrosomes are the dominant microtubule organizing centers (MTOC) and play a pivotal role in axis determination by orienting the mitotic spindle. In land plants that lack centrosomes, a critical role of a microtubular ring structure, the preprophase band (PPB), has been observed in this process; the PPB is required for orienting (before prophase) and guiding (in telophase) the mitotic apparatus. However, plants must possess additional mechanisms to control the division axis, as certain cell types or mutants do not form PPBs. Here, using live imaging of the gametophore of the moss , we identified acentrosomal MTOCs, which we termed "gametosomes," appearing de novo and transiently in the prophase cytoplasm independent of PPB formation. We show that gametosomes are dispensable for spindle formation but required for metaphase spindle orientation. In some cells, gametosomes appeared reminiscent of the bipolar MT "polar cap" structure that forms transiently around the prophase nucleus in angiosperms. Specific disruption of the polar caps in tobacco cells misoriented the metaphase spindles and frequently altered the final division plane, indicating that they are functionally analogous to the gametosomes. These results suggest a broad use of transient MTOC structures as the spindle orientation machinery in plants, compensating for the evolutionary loss of centrosomes, to secure the initial orientation of the spindle in a spatial window that allows subsequent fine-tuning of the division plane axis by the guidance machinery.
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http://dx.doi.org/10.1073/pnas.1713925114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5651782PMC
October 2017

Human microcephaly ASPM protein is a spindle pole-focusing factor that functions redundantly with CDK5RAP2.

J Cell Sci 2017 Nov 7;130(21):3676-3684. Epub 2017 Sep 7.

Division of Biological Science, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8602, Japan

Nonsense mutations in the gene have been most frequently identified among familial microcephaly patients. Depletion of the orthologue () causes spindle pole unfocusing during mitosis in multiple cell types. However, it remains unknown whether human ASPM has a similar function. Here, by performing CRISPR-based gene knockout (KO) and RNA interference combined with auxin-inducible degron, we show that ASPM functions in spindle pole organisation during mitotic metaphase redundantly with another microcephaly protein, CDK5RAP2 (also called CEP215), in human tissue culture cells. Deletion of the gene alone did not affect spindle morphology or mitotic progression. However, when the pericentriolar material protein CDK5RAP2 was depleted in KO cells, spindle poles were unfocused during prometaphase, and anaphase onset was significantly delayed. The phenotypic analysis of CDK5RAP2-depleted cells suggested that the pole-focusing function of CDK5RAP2 is independent of its known function to localise the kinesin-14 motor HSET (also known as KIFC1) or activate the γ-tubulin complex. Finally, a hypomorphic mutation identified in ASPM microcephaly patients similarly caused spindle pole unfocusing in the absence of CDK5RAP2, suggesting a possible link between spindle pole disorganisation and microcephaly.
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http://dx.doi.org/10.1242/jcs.203703DOI Listing
November 2017

14-3-3 regulation of Ncd reveals a new mechanism for targeting proteins to the spindle in oocytes.

J Cell Biol 2017 10 31;216(10):3029-3039. Epub 2017 Aug 31.

Wellcome Centre for Cell Biology, School of Biological Sciences, University of Edinburgh, Edinburgh, Scotland, UK

The meiotic spindle is formed without centrosomes in a large volume of oocytes. Local activation of crucial spindle proteins around chromosomes is important for formation and maintenance of a bipolar spindle in oocytes. We found that phosphodocking 14-3-3 proteins stabilize spindle bipolarity in oocytes. A critical 14-3-3 target is the minus end-directed motor Ncd (human HSET; kinesin-14), which has well-documented roles in stabilizing a bipolar spindle in oocytes. Phospho docking by 14-3-3 inhibits the microtubule binding activity of the nonmotor Ncd tail. Further phosphorylation by Aurora B kinase can release Ncd from this inhibitory effect of 14-3-3. As Aurora B localizes to chromosomes and spindles, 14-3-3 facilitates specific association of Ncd with spindle microtubules by preventing Ncd from binding to nonspindle microtubules in oocytes. Therefore, 14-3-3 translates a spatial cue provided by Aurora B to target Ncd selectively to the spindle within the large volume of oocytes.
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http://dx.doi.org/10.1083/jcb.201704120DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5626551PMC
October 2017

Multiple kinesin-14 family members drive microtubule minus end-directed transport in plant cells.

J Cell Biol 2017 06 25;216(6):1705-1714. Epub 2017 Apr 25.

Division of Biological Science, Graduate School of Science, Nagoya University, Nagoya 464-8602, Japan

Minus end-directed cargo transport along microtubules (MTs) is exclusively driven by the molecular motor dynein in a wide variety of cell types. Interestingly, during evolution, plants have lost the genes encoding dynein; the MT motors that compensate for dynein function are unknown. Here, we show that two members of the kinesin-14 family drive minus end-directed transport in plants. Gene knockout analyses of the moss revealed that the plant-specific class VI kinesin-14, KCBP, is required for minus end-directed transport of the nucleus and chloroplasts. Purified KCBP directly bound to acidic phospholipids and unidirectionally transported phospholipid liposomes along MTs in vitro. Thus, minus end-directed transport of membranous cargoes might be driven by their direct interaction with this motor protein. Newly nucleated cytoplasmic MTs represent another known cargo exhibiting minus end-directed motility, and we identified the conserved class I kinesin-14 (ATK) as the motor involved. These results suggest that kinesin-14 motors were duplicated and developed as alternative MT-based minus end-directed transporters in land plants.
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http://dx.doi.org/10.1083/jcb.201610065DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5461021PMC
June 2017

Shortening of Microtubule Overlap Regions Defines Membrane Delivery Sites during Plant Cytokinesis.

Curr Biol 2017 Feb 26;27(4):514-520. Epub 2017 Jan 26.

Laboratory of Cell Biology, Wageningen University, Droevendaalsesteeg 1, 6708 PB Wageningen, the Netherlands; Marine Biological Laboratory, 7 MBL Street, Woods Hole, MA 02543, USA. Electronic address:

Different from animal cells that divide by constriction of the cortex inward, cells of land plants divide by initiating a new cell-wall segment from their center. For this, a disk-shaped, membrane-enclosed precursor termed the cell plate is formed that radially expands toward the parental cell wall [1-3]. The synthesis of the plate starts with the fusion of vesicles into a tubulo-vesicular network [4-6]. Vesicles are putatively delivered to the division plane by transport along microtubules of the bipolar phragmoplast network that guides plate assembly [7-9]. How vesicle immobilization 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 [10, 11], we anticipate that our results will help uncover universal features underlying membrane-cytoskeleton coordination during cytokinesis.
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http://dx.doi.org/10.1016/j.cub.2016.12.043DOI Listing
February 2017

Mitotic Spindle Assembly in Land Plants: Molecules and Mechanisms.

Biology (Basel) 2017 Jan 25;6(1). Epub 2017 Jan 25.

Graduate School of Science, Division of Biological Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8602, Japan.

In textbooks, the mitotic spindles of plants are often described separately from those of animals. How do they differ at the molecular and mechanistic levels? In this chapter, we first outline the process of mitotic spindle assembly in animals and land plants. We next discuss the conservation of spindle assembly factors based on database searches. Searches of >100 animal spindle assembly factors showed that the genes involved in this process are well conserved in plants, with the exception of two major missing elements: centrosomal components and subunits/regulators of the cytoplasmic dynein complex. We then describe the spindle and phragmoplast assembly mechanisms based on the data obtained from robust gene loss-of-function analyses using RNA interference (RNAi) or mutant plants. Finally, we discuss future research prospects of plant spindles.
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http://dx.doi.org/10.3390/biology6010006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5371999PMC
January 2017

Five factors can reconstitute all three phases of microtubule polymerization dynamics.

J Cell Biol 2016 Nov 31;215(3):357-368. Epub 2016 Oct 31.

Division of Biological Science, Graduate School of Science, Nagoya University, Nagoya 464-8602, Japan

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, XMAP215, Sentin, kinesin-13, and CLASP). When singly mixed with tubulin, CLASP strongly inhibited MT catastrophe and reduced the growth rate. However, in the presence of the other four factors, CLASP acted as an inducer of pausing. The mitotic kinase Plk1 modulated the activity of CLASP and kinesin-13 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.
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http://dx.doi.org/10.1083/jcb.201604118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5100292PMC
November 2016

Live Cell Microscopy-Based RNAi Screening in the Moss Physcomitrella patens.

Methods Mol Biol 2016 ;1470:225-46

Division of Biological Science, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, 464-8602, Japan.

RNA interference (RNAi) is a powerful technique enabling the identification of the genes involved in a certain cellular process. Here, we discuss protocols for microscopy-based RNAi screening in protonemal cells of the moss Physcomitrella patens, an emerging model system for plant cell biology. Our method is characterized by the use of conditional (inducible) RNAi vectors, transgenic moss lines in which the RNAi vector is integrated, and time-lapse fluorescent microscopy. This method allows for effective and efficient screening of >100 genes involved in various cellular processes such as mitotic cell division, organelle distribution, or cell growth.
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http://dx.doi.org/10.1007/978-1-4939-6337-9_18DOI Listing
December 2017

Imaging Mitosis in the Moss Physcomitrella patens.

Methods Mol Biol 2016 ;1413:263-82

Division of Biological Science, Graduate School of Science, Nagoya University, Room 227, Building A, Furo-cho, Chikusa-ku, Nagoya, 464-8602, Japan.

At first glance, mitosis in plants looks quite different from that in animals. In fact, terrestrial plants have lost the centrosome during evolution, and the mitotic spindle is assembled independently of a strong microtubule organizing center. The phragmoplast is a plant-specific mitotic apparatus formed after anaphase, which expands centrifugally towards the cell cortex. However, the extent to which plant mitosis differs from that of animals at the level of the protein repertoire is uncertain, largely because of the difficulty in the identification and in vivo characterization of mitotic genes of plants. Here, we discuss protocols for mitosis imaging that can be combined with endogenous green fluorescent protein (GFP) tagging or conditional RNA interference (RNAi) in the moss Physcomitrella patens, which is an emergent model plant for cell and developmental biology. This system has potential for use in the high-throughput study of mitosis and other intracellular processes, as is being done with various animal cell lines.
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http://dx.doi.org/10.1007/978-1-4939-3542-0_17DOI Listing
December 2017

Intra-spindle Microtubule Assembly Regulates Clustering of Microtubule-Organizing Centers during Early Mouse Development.

Cell Rep 2016 Apr 24;15(1):54-60. Epub 2016 Mar 24.

Division of Biological Science, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8602, Japan. Electronic address:

Errors during cell division in oocytes and early embryos are linked to birth defects in mammals. Bipolar spindle assembly in early mouse embryos is unique in that three or more acentriolar microtubule-organizing centers (MTOCs) are initially formed and are then clustered into two spindle poles. Using a knockout mouse and live imaging of spindles in embryos, we demonstrate that MTOC clustering during the blastocyst stage requires augmin, a critical complex for MT-dependent MT nucleation within the spindle. Functional analyses in cultured cells with artificially increased numbers of centrosomes indicate that the lack of intra-spindle MT nucleation, but not loss of augmin per se or overall reduction of spindle MTs, is the cause of clustering failure. These data suggest that onset of mitosis with three or more MTOCs is turned into a typical bipolar division through augmin-dependent intra-spindle MT assembly.
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http://dx.doi.org/10.1016/j.celrep.2016.02.087DOI Listing
April 2016

Augmin shapes the anaphase spindle for efficient cytokinetic furrow ingression and abscission.

Mol Biol Cell 2016 Mar 13;27(5):812-27. Epub 2016 Jan 13.

Division of Biological Science, Graduate School of Science, Nagoya University, Nagoya 464-8602, Japan.

During anaphase, distinct populations of microtubules (MTs) form by either centrosome-dependent or augmin-dependent nucleation. It remains largely unknown whether these different MT populations contribute distinct functions to cytokinesis. Here we show that augmin-dependent MTs are required for the progression of both furrow ingression and abscission. Augmin depletion reduced the accumulation of anillin, a contractile ring regulator at the cell equator, yet centrosomal MTs were sufficient to mediate RhoA activation at the furrow. This defect in contractile ring organization, combined with incomplete spindle pole separation during anaphase, led to impaired furrow ingression. During the late stages of cytokinesis, astral MTs formed bundles in the intercellular bridge, but these failed to assemble a focused midbody structure and did not establish tight linkage to the plasma membrane, resulting in furrow regression. Thus augmin-dependent acentrosomal MTs and centrosomal MTs contribute to nonredundant targeting mechanisms of different cytokinesis factors, which are required for the formation of a functional contractile ring and midbody.
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http://dx.doi.org/10.1091/mbc.E15-02-0101DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4803307PMC
March 2016

The microtubule catastrophe promoter Sentin delays stable kinetochore-microtubule attachment in oocytes.

J Cell Biol 2015 Dec 14;211(6):1113-20. Epub 2015 Dec 14.

Wellcome Trust Centre for Cell Biology, School of Biological Sciences, The University of Edinburgh, Edinburgh EH9 3BF, Scotland, UK

The critical step in meiosis is to attach homologous chromosomes to the opposite poles. In mouse oocytes, stable microtubule end-on attachments to kinetochores are not established until hours after spindle assembly, and phosphorylation of kinetochore proteins by Aurora B/C is responsible for the delay. Here we demonstrated that microtubule ends are actively prevented from stable attachment to kinetochores until well after spindle formation in Drosophila melanogaster oocytes. We identified the microtubule catastrophe-promoting complex Sentin-EB1 as a major factor responsible for this delay. Without this activity, microtubule ends precociously form robust attachments to kinetochores in oocytes, leading to a high proportion of homologous kinetochores stably attached to the same pole. Therefore, regulation of microtubule ends provides an alternative novel mechanism to delay stable kinetochore-microtubule attachment in oocytes.
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http://dx.doi.org/10.1083/jcb.201507006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4687879PMC
December 2015

Microcephaly protein Asp focuses the minus ends of spindle microtubules at the pole and within the spindle.

J Cell Biol 2015 Dec;211(5):999-1009

Division of Biological Science, Graduate School of Science, Nagoya University, Chikusa-ku, Nagoya 464-8602, Japan

Depletion of Drosophila melanogaster Asp, an orthologue of microcephaly protein ASPM, causes spindle pole unfocusing during mitosis. However, it remains unclear how Asp contributes to pole focusing, a process that also requires the kinesin-14 motor Ncd. We show that Asp localizes to the minus ends of spindle microtubule (MT) bundles and focuses them to make the pole independent of Ncd. We identified a critical domain in Asp exhibiting MT cross-linking activity in vitro. Asp was also localized to, and focuses the minus ends of, intraspindle MTs that were nucleated in an augmin-dependent manner and translocated toward the poles by spindle MT flux. Ncd, in contrast, functioned as a global spindle coalescence factor not limited to MT ends. We propose a revised molecular model for spindle pole focusing in which Asp at the minus ends cross-links MTs at the pole and within the spindle. Additionally, this study provides new insight into the dynamics of intraspindle MTs by using Asp as a minus end marker.
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http://dx.doi.org/10.1083/jcb.201507001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4674282PMC
December 2015

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

Nat Plants 2015 Jul;1(7)

Marine Biological Laboratory (MBL), Woods Hole, Massachusetts 02543, USA ; Division of Biological Science, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8602, Japan.

The molecular motors kinesin and dynein drive bidirectional motility along microtubules (MTs) in most eukaryotic cells. Land plants, however, are a notable exception, because they contain a large number of kinesins but lack cytoplasmic dynein, the foremost processive retrograde transporter. It remains unclear how plants achieve retrograde cargo transport without dynein. Here, we have analysed the motility of the six members of minus-end-directed kinesin-14 motors in the moss 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 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, observations of green fluorescent protein-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.
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http://dx.doi.org/10.1038/NPLANTS.2015.87DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4548964PMC
July 2015

NACK kinesin is required for metaphase chromosome alignment and cytokinesis in the moss Physcomitrella patens.

Cell Struct Funct 2015 ;40(1):31-41

Division of Biological Science, Graduate School of Science, Nagoya University.

The NACK kinesins (NACK1, NACK2 in tobacco and AtNACK1/HINKEL, AtNACK2/STUD/TETRASPORE in Arabidopsis), members of a plant-specific kinesin-7 family, are required for cytokinesis. Previous studies using tobacco and Arabidopsis cells showed that NACK1 and AtNACK1 at the phragmoplast midzone activate the MAP kinase cascade during the late M phase, which is critical for the cell plate formation. However, the loss-of-function phenotype has not been investigated in details in living cells and the molecular activity of this kinesin remains to be determined. Here, we report the mitotic roles and activity of the NACK kinesins in the moss Physcomitrella patens. When we simultaneously knocked down three PpNACKs by RNA-interference (RNAi) in protonemal cells, we observed a cytokinesis failure following a defect in phragmoplast expansion. In addition, misaligned chromosomes were frequently detected in the pre-anaphase spindle and the anaphase onset was significantly delayed, indicating that PpNACK also plays a role in pre-anaphase. Consistent with the appearance of early and late mitotic phenotypes, endogenous PpNACK was localised to the interpolar microtubule (MT) overlap from prometaphase through telophase. In vitro MT gliding assay and single motor motility assay showed that PpNACK-b is a processive, plus-end-directed motor, suggesting that PpNACK is capable of transporting cargoes along the spindle/phragmoplast MT. Our study using Physcomitrella patens demonstrated that PpNACK is an active motor protein and identified unexpected and conserved roles of PpNACK during the mitosis of P. patens.
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http://dx.doi.org/10.1247/csf.14016DOI Listing
November 2015

Cytoplasmic nucleation and atypical branching nucleation generate endoplasmic microtubules in Physcomitrella patens.

Plant Cell 2015 Jan 23;27(1):228-42. Epub 2015 Jan 23.

Division of Biological Science, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8602, Japan Marine Biological Laboratory, Woods Hole, Massachusetts 02543

The mechanism underlying microtubule (MT) generation in plants has been primarily studied using the cortical MT array, in which fixed-angled branching nucleation and katanin-dependent MT severing predominate. However, little is known about MT generation in the endoplasm. Here, we explored the mechanism of endoplasmic MT generation in protonemal cells of Physcomitrella patens. We developed an assay that utilizes flow cell and oblique illumination fluorescence microscopy, which allowed visualization and quantification of individual MT dynamics. MT severing was infrequently observed, and disruption of katanin did not severely affect MT generation. Branching nucleation was observed, but it showed markedly variable branch angles and was occasionally accompanied by the transport of nucleated MTs. Cytoplasmic nucleation at seemingly random locations was most frequently observed and predominated when depolymerized MTs were regrown. The MT nucleator γ-tubulin was detected at the majority of the nucleation sites, at which a single MT was generated in random directions. When γ-tubulin was knocked down, MT generation was significantly delayed in the regrowth assay. However, nucleation occurred at a normal frequency in steady state, suggesting the presence of a γ-tubulin-independent backup mechanism. Thus, endoplasmic MTs in this cell type are generated in a less ordered manner, showing a broader spectrum of nucleation mechanisms in plants.
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http://dx.doi.org/10.1105/tpc.114.134817DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4330588PMC
January 2015