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

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 Physcomitrella patens, 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.

Endogenous localizome identifies 43 mitotic kinesins in a plant cell.

Kinesins are microtubule (MT)-based motor proteins that have been identified in every eukaryotic species. Intriguingly, land plants have more than 60 kinesins in their genomes, many more than that in yeasts or animals. However, many of these have not yet been characterized, and their cellular functions are unknown. Here, by using endogenous tagging, we comprehensively determined the localization of 72 kinesins during mitosis in the moss Physcomitrella patens. We found that 43 kinesins are localized to mitotic structures such as kinetochores, spindle MTs, or phragmoplasts, which are MT-based structures formed during cytokinesis. Surprisingly, only one of them showed an identical localization pattern to the animal homolog, and many were enriched at unexpected sites. RNA interference and live-cell microscopy revealed postanaphase roles for kinesin-5 in spindle/phragmoplast organization, chromosome segregation, and cytokinesis, which have not been observed in animals. Our study thus provides a list of MT-based motor proteins associated with the cell division machinery in plants. Furthermore, our data challenge the current generalization of determining mitotic kinesin function based solely on studies using yeast and animal cells.

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

Proper positioning of the nucleus is critical for the functioning of various cells. Actin and myosin have been shown to be crucial for the localization of the nucleus in plant cells, whereas microtubule (MT)-based mechanisms are commonly utilized 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 center 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 center took place normally; however, before reaching the center, 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 down-regulation. 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 localization among plant, animal and fungal cells.

Identification of two novel endoplasmic reticulum body-specific integral membrane proteins.

The endoplasmic reticulum (ER) body, a large compartment specific to the Brassicales, accumulates ß-glucosidase and possibly plays a role in the defense against pathogens and herbivores. Although the ER body is a subdomain of the ER, it is unclear whether any ER body-specific membrane protein exists. In this study, we identified two integral membrane proteins of the ER body in Arabidopsis (Arabidopsis thaliana) and termed them MEMBRANE PROTEIN OF ENDOPLASMIC RETICULUM BODY1 (MEB1) and MEB2. In Arabidopsis, a basic helix-loop-helix transcription factor, NAI1, and an ER body component, NAI2, regulate ER body formation. The expression profiles of MEB1 and MEB2 are similar to those of NAI1, NAI2, and ER body ß-glucosidase PYK10 in Arabidopsis. The expression of MEB1 and MEB2 was reduced in the nai1 mutant, indicating that NAI1 regulates the expression of MEB1 and MEB2 genes. MEB1 and MEB2 proteins localize to the ER body membrane but not to the ER network, suggesting that these proteins are specifically recruited to the ER body membrane. MEB1 and MEB2 physically interacted with ER body component NAI2, and they were diffused throughout the ER network in the nai2 mutant, which has no ER body. Heterologous expression of MEB1 and MEB2 in yeast (Saccharomyces cerevisiae) suppresses iron and manganese toxicity, suggesting that MEB1 and MEB2 are metal transporters. These results indicate that the membrane of ER bodies has specific membrane proteins and suggest that the ER body is involved in defense against metal stress as well as pathogens and herbivores.

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

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-ß 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.

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

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 Physcomitrella patens 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.

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

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 Physcomitrella patens 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.

NAI2 is an endoplasmic reticulum body component that enables ER body formation in Arabidopsis thaliana.

Plants develop various endoplasmic reticulum (ER)-derived structures, each of which has specific functions. The ER body found in Arabidopsis thaliana is a spindle-shaped structure that specifically accumulates high levels of PYK10/BGLU23, a beta-glucosidase that bears an ER-retention signal. The molecular mechanisms underlying the formation of the ER body remain obscure. We isolated an ER body-deficient mutant in Arabidopsis seedlings that we termed nai2. The NAI2 gene (At3g15950) encodes a member of a unique protein family that is only found in the Brassicaceae. NAI2 localizes to the ER body, and a reduction in NAI2 gene expression elongates ER bodies and reduces their numbers. NAI2 deficiency does not affect PYK10 mRNA levels but reduces the level of PYK10 protein, which becomes uniformly diffused throughout the ER. NAI1, a transcription factor responsible for ER body formation, regulates NAI2 gene expression. These observations indicate that NAI2 is a key factor that enables ER body formation and the accumulation of PYK10 in ER bodies of Arabidopsis. Interestingly, ER body-like structures are also restricted to the Brassicales, including the Brassicaceae. NAI2 homologs may have evolved specifically in Brassicales for the purpose of producing ER body-like structures.