Investigations of plant cell division would greatly benefit from a fast, inducible system. Therefore, we aimed to establish a mitotic model by transiently expressing D-type cyclins in tobacco leaf cells.Two different D-type cyclins, CYCD3;1 and CYCD4;2 from Arabidopsis thaliana, were expressed by agrobacterial infiltration in the cells of expanded leaves in tobacco (Nicotiana benthamiana). Leaf pavement cells were examined after cyclin expression while target and reference (histone or tubulin) proteins were marked by fluorescent protein-tagging.Ectopic expression of the D-type cyclin induced pavement cells to re-enter cell division by establishing mitotic microtubule arrays. The induced leaf cells expressed M phase-specific genes of Arabidopsis encoding the mitotic kinase AtAurora 1, the microtubule-associated proteins AtEDE1 and AtMAP65-4, and the vesicle fusion protein AtKNOLLE by recognizing their genomic elements. Their distinct localizations at spindle poles (AtAurora1), spindle microtubules (AtEDE1), phragmoplast microtubules (AtMAP65-4) and the cell plate (AtKNOLLE) were indistinguishable from those in their native Arabidopsis cells. The dividing cells also revealed two rice (Oryza sativa) microtubule-associated proteins in the phragmoplast and uncovered a novel spindle-associated microtubule motor protein.Hence, this cell division-enabled leaf system predicts hypothesized cell cycle-dependent functions of heterologous genes by reporting the dynamics of encoded proteins.

Contents SummaryI.IntroductionII.MT arrays in plant cellsIII.γ‐Tubulin and MT nucleationIV.MT nucleation sites or flexible MTOCs in plant cellsV.MT‐dependent MT nucleationVI.Generating new MTs for spindle assemblyVII.Generation of MTs for phragmoplast expansion during cytokinesisVIII.MT generation for the cortical MT arrayIX.MT nucleation: looking forward AcknowledgementsReferences Summary Cytoskeletal microtubules (MTs) have a multitude of functions including intracellular distribution of molecules and organelles, cell morphogenesis, as well as segregation of the genetic material and separation of the cytoplasm during cell division among eukaryotic organisms. In response to internal and external cues, eukaryotic cells remodel their MT network in a regulated manner in order to assemble physiologically important arrays for cell growth, cell proliferation, or for cells to cope with biotic or abiotic stresses. Nucleation of new MTs is a critical step for MT remodeling. Although many key factors contributing to MT nucleation and organization are well conserved in different kingdoms, the centrosome, representing the most prominent microtubule organizing centers (MTOCs), disappeared during plant evolution as angiosperms lack the structure. Instead, flexible MTOCs may emerge on the plasma membrane, the nuclear envelope, and even organelles depending on types of cells and organisms and/or physiological conditions. MT‐dependent MT nucleation is particularly noticeable in plant cells because it accounts for the primary source of MT generation for assembling spindle, phragmoplast, and cortical arrays when the γ‐tubulin ring complex is anchored and activated by the augmin complex. It is intriguing what proteins are associated with plant‐specific MTOCs and how plant cells activate or inactivate MT nucleation activities in spatiotemporally regulated manners.

The evolutionarily conserved WD40 protein budding uninhibited by benzimidazole 3 (BUB3) is known for its function in spindle assembly checkpoint control. In the model plant Arabidopsis thaliana, nearly identical BUB3;1 and BUB3;2 proteins decorated the phragmoplast midline through interaction with the microtubule-associated protein MAP65-3 during cytokinesis. BUB3;1 and BUB3;2 interacted with the carboxy-terminal segment of MAP65-3 (but not MAP65-1), which harbours its microtubule-binding domain for its post-mitotic localization. Reciprocally, BUB3;1 and BUB3;2 also regulated MAP65-3 localization in the phragmoplast by enhancing its microtubule association. In the bub3;1 bub3;2 double mutant, MAP65-3 localization was often dissipated away from the phragmoplast midline and abolished upon treatment of low doses of the cytokinesis inhibitory drug caffeine that were tolerated by the control plant. The phragmoplast microtubule array exhibited uncoordinated expansion pattern in the double mutant cells as the phragmoplast edge reached the parental plasma membrane at different times in different areas. Upon caffeine treatment, phragmoplast expansion was halted as if the microtubule array was frozen. As a result, cytokinesis was abolished due to failed cell plate assembly. Our findings have uncovered a novel function of the plant BUB3 in MAP65-3-dependent microtubule reorganization during cytokinesis.

Olfactory ensheathing cells (OECs) are a special type of glial cells that have characteristics of both astrocytes and Schwann cells. Evidence suggests that the regenerative capacity of OECs is induced by soluble, secreted factors that influence their microenvironment. These factors may regulate OECs self-renewal and/or induce their capacity to augment spinal cord regeneration. Profiling of plasma membrane and extracellular matrix through a high-throughput expression proteomics approach was undertaken to identify plasma membrane and extracellular matrix proteins of OECs under serum-free conditions. 1D-shotgun proteomics followed with gene ontology (GO) analysis was used to screen proteins from primary culture rat OECs. Four hundred and seventy nonredundant plasma membrane proteins and 168 extracellular matrix proteins were identified, the majority of which were never before reported to be produced by OECs. Furthermore, plasma membrane and extracellular proteins were classified based on their protein-protein interaction predicted by STRING quantitatively integrates interaction data. The proteomic profiling of the OECs plasma membrane proteins and their connection with the secretome in serum-free culture conditions provides new insights into the nature of their in vivo microenvironmental niche. Proteomic analysis for the discovery of clinical biomarkers of OECs mechanism warrants further study. Key words: Olfactory ensheathing cells; Neural repair; 1D-shotgun proteomics; Plasma membrane; Extracellular matrix INTRODUCTIONare aided by glia unique to the olfactory system, the olfactory ensheathing cells (OECs) (45). For instance, OECs have been transplanted into sites In the mammalian adult central nervous system (CNS), recovery from injury is generally poor, and of spinal cord injury and other CNS lesions, with the anticipation that OECs may recreate the plastic environspontaneous nerve regeneration occurs only in restricted regions of the CNS. The damage to nerve fiber pathways ment of the olfactory system elsewhere (12,21). Ao et al.'s research shows that the effectiveness of OECs inresults in a devastating loss of function, due to the disconnection of nerve fibers from their targets (22,34,59). duced neural differentiation of neural stem cells (NSCs) and their culture medium concentrations of total protein An exception to this rule is found in the olfactory system, in which olfactory receptor neurons (ORNs) unsecretion was positively correlated. OEC conditioned medium (OCM) can induce NSCs to differentiate into dergo natural and injury-induced turnover and functional neurogenesis throughout life (25). To accommodate sucneurons, but the same protein concentration of conditioned medium of astrocytes mainly induced differentiacessful adult axon targeting, newly generated ORNs from the peripheral nervous system (PNS) that extend tion to glial cells of NSCs (2). The results of other research groups also showed that the OCM can promote axons to synapse in the olfactory bulb (OB) of the CNS and secondary fluorescent an...

In animals and fungi, cytoplasmic dynein is a processive minus-end-directed motor that plays dominant roles in various intracellular processes. In contrast, land plants lack cytoplasmic dynein but contain many minus-end-directed kinesin-14s. No plant kinesin-14 is known to produce processive motility as a homodimer. OsKCH2 is a plant-specific kinesin-14 with an N-terminal actin-binding domain and a central motor domain flanked by two predicted coiled-coils (CC1 and CC2). Here, we show that OsKCH2 specifically decorates preprophase band microtubules in vivo and transports actin filaments along microtubules in vitro. Importantly, OsKCH2 exhibits processive minus-end-directed motility on single microtubules as individual homodimers. We find that CC1, but not CC2, forms the coiled-coil to enable OsKCH2 dimerization. Instead, our results reveal that removing CC2 renders OsKCH2 a nonprocessive motor. Collectively, these results show that land plants have evolved unconventional kinesin-14 homodimers with inherent minus-end-directed processivity that may function to compensate for the loss of cytoplasmic dynein.