KINESIN-12E regulates metaphase spindle flux and helps control spindle size in Arabidopsis.

The bipolar mitotic spindle is a highly conserved structure among eukaryotes that mediates chromosome alignment and segregation. Spindle assembly and size control are facilitated by force-generating microtubule-dependent motor proteins known as kinesins. In animals, kinesin-12 cooperates with kinesin-5 to produce outward-directed forces necessary for spindle assembly. In plants, the relevant molecular mechanisms for spindle formation are poorly defined. While an Arabidopsis thaliana kinesin-5 ortholog has been identified, the kinesin-12 ortholog in plants remains elusive. In this study, we provide experimental evidence for the function of Arabidopsis KINESIN-12E in spindle assembly. In kinesin-12e mutants, a delay in spindle assembly is accompanied by the reduction of spindle size, demonstrating that KINESIN-12E contributes to mitotic spindle architecture. Kinesin-12E localization is mitosis-stage specific, beginning with its perinuclear accumulation during prophase. Upon nuclear envelope breakdown, KINESIN-12E decorates subpopulations of microtubules in the spindle and becomes progressively enriched in the spindle midzone. Furthermore, during cytokinesis, KINESIN-12E shares its localization at the phragmoplast midzone with several functionally diversified Arabidopsis KINESIN-12 members. Changes in the kinetochore and in prophase and metaphase spindle dynamics occur in the absence of KINESIN-12E, suggest it might play an evolutionarily conserved role during spindle formation similar to its spindle-localized animal kinesin-12 orthologs.

Cell Cycle Dynamics during Stomatal Development: Window of MUTE Action and Ramification of Its Loss-of-Function on an Uncommitted Precursor.

Plants develop in the absence of cell migration. As such, cell division and differentiation need to be coordinated for functional tissue formation. Cellular valves on the plant epidermis, stomata, are generated through a stereotypical sequence of cell division and differentiation events. In Arabidopsis, three master regulatory transcription factors, SPEECHLESS (SPCH), MUTE and FAMA, sequentially drive initiation, proliferation and differentiation of stomata. Among them, MUTE switches the cell cycle mode from proliferative asymmetric division to terminal symmetric division and orchestrates the execution of the single symmetric division event. However, it remains unclear to what extent MUTE regulates the expression of cell cycle genes through the symmetric division and whether MUTE accumulation itself is gated by the cell cycle. Here, we show that MUTE directly upregulates the expression of cell cycle components throughout the terminal cell cycle phases of a stomatal precursor, not only core cell cycle engines but also check-point regulators. Time-lapse live imaging using the multicolor Plant Cell Cycle Indicator revealed that MUTE accumulates up to the early G2 phase, whereas its successor and direct target, FAMA, accumulate at late G2 through terminal mitosis. In the absence of MUTE, meristemoids fail to differentiate and their G1 phase elongates as they reiterate asymmetric divisions. Together, our work provides the framework of cell cycle and master regulatory transcription factors to coordinate a single symmetric cell division and suggests a mechanism for the eventual cell cycle arrest of an uncommitted stem-cell-like precursor at the G1 phase.

Shouting out loud: signaling modules in the regulation of stomatal development.

Stomata are small pores on the surface of land plants that facilitate gas exchange for photosynthesis while minimizing water loss. The function of stomata is pivotal for plant growth and survival. Intensive research on the model plant Arabidopsis (Arabidopsis thaliana) has discovered key peptide signaling pathways, transcription factors, and polarity components that together drive proper stomatal development and patterning. In this review, we focus on recent findings that have revealed co-option of peptide-receptor kinase signaling modules-utilized for diverse developmental processes and immune response. We further discuss an emerging connection between extrinsic signaling and intrinsic polarity modules. These findings have further enlightened our understanding of this fascinating developmental process.

Dual localized kinesin-12 POK2 plays multiple roles during cell division and interacts with MAP65-3.

Kinesins are versatile nano-machines that utilize variable non-motor domains to tune specific motor microtubule encounters. During plant cytokinesis, the kinesin-12 orthologs, PHRAGMOPLAST ORIENTING KINESIN (POK)1 and POK2, are essential for rapid centrifugal expansion of the cytokinetic apparatus, the phragmoplast, toward a pre-selected cell plate fusion site at the cell cortex. Here, we report on the spatio-temporal localization pattern of POK2, mediated by distinct protein domains. Functional dissection of POK2 domains revealed the association of POK2 with the site of the future cell division plane and with the phragmoplast during cytokinesis. Accumulation of POK2 at the phragmoplast midzone depends on its functional POK2 motor domain and is fine-tuned by its carboxy-terminal region that also directs POK2 to the division site. Furthermore, POK2 likely stabilizes the phragmoplast midzone via interaction with the conserved microtubule-associated protein MAP65-3/PLEIADE, a well-established microtubule cross-linker. Collectively, our results suggest that dual localized POK2 plays multiple roles during plant cell division.

Phragmoplast Orienting Kinesin 2 Is a Weak Motor Switching between Processive and Diffusive Modes.

Plant development and morphology relies on the accurate insertion of new cell walls during cytokinesis. However, how a plant cell correctly orients a new wall is poorly understood. Two kinesin class-12 members, phragmoplast orienting kinesin 1 (POK1) and POK2, are involved in the process, but how these molecular machines work is not known. Here, we used in vivo and single-molecule in vitro measurements to determine how Arabidopsis thaliana POK2 motors function mechanically. We found that POK2 is a very weak, on average plus-end-directed, moderately fast kinesin. Interestingly, POK2 switches between processive and diffusive modes characterized by an exclusive-state mean-squared-displacement analysis. Our results support a model that POK motors push against peripheral microtubules of the phragmoplast for its guidance. This pushing model may mechanically explain the conspicuous narrowing of the division site. Together, our findings provide mechanical insight into how active motors accurately position new cell walls in plants.

Deceleration of the cell cycle underpins a switch from proliferative to terminal divisions in plant stomatal lineage.

Differentiation of specialized cell types requires precise cell-cycle control. Plant stomata are generated through asymmetric divisions of a stem-cell-like precursor followed by a single symmetric division that creates paired guard cells surrounding a pore. The stomatal-lineage-specific transcription factor MUTE terminates the asymmetric divisions and commits to differentiation. However, the role of cell-cycle machineries in this transition remains unknown. We discover that the symmetric division is slower than the asymmetric division in Arabidopsis. We identify a plant-specific cyclin-dependent kinase inhibitor, SIAMESE-RELATED4 (SMR4), as a MUTE-induced molecular brake that decelerates the cell cycle. SMR4 physically and functionally associates with CYCD3;1 and extends the G1 phase of asymmetric divisions. By contrast, SMR4 fails to interact with CYCD5;1, a MUTE-induced G1 cyclin, and permits the symmetric division. Our work unravels a molecular framework of the proliferation-to-differentiation switch within the stomatal lineage and suggests that a timely proliferative cell cycle is critical for stomatal-lineage identity.

Putative RopGAPs impact division plane selection and interact with kinesin-12 POK1.

Cell shape is defined by the surrounding cell walls in plants. Thus, spatial control over cell division planes and cell expansion polarity are essential to maintain cell morphology. In eukaryotes, cell polarity and expansion are controlled by Rho GTPase signalling, regulating cytoskeletal reorganization and vesicle trafficking(1). However, until now, Rho signalling was not implicated in mitotic events in plants. Here, we report a pair of putative Rho GTPase activating proteins (RhoGAPs) that interact with the mitosis-specific kinesin-12 POK1, a core component of the cortical division zone/site (CDZ/CDS) that is required for division plane maintenance in Arabidopsis(2-4). The designated pleckstrin homology GAPs (PHGAPs) are cytoplasmic and plasma membrane associated in interphase, but during mitosis they additionally localize to the CDZ/CDS in a POK-dependent manner. In contrast to pok1 pok2 mutants, phgap1 phgap2 double mutants show moderate cell wall positioning defects as a consequence of inaccurate positioning of the cortical division zone marker POK1. We conclude that loss of PHGAP function interferes with division plane selection in proliferative cell divisions.

Mechanisms of plant cell division.

Plant cells are confined by a network of cellulosic walls that imposes rigid control over the selection of division plane orientations, crucial for morphogenesis and genetically regulated. While in animal cells and yeast, the actin cytoskeleton is instrumental in the execution of cytokinesis, in plant cells the microtubule cytoskeleton is taking the lead in spatially controlling and executing cytokinesis by the formation of two unique, plant-specific arrays, the preprophase band (PPB) and the phragmoplast. The formation of microtubule arrays in plant cells is contingent on acentrosomal microtubule nucleation. At the onset of mitosis, the PPB defines the plane of cell division where the partitioning cell wall is later constructed by the cytokinetic phragmoplast, imposing a spatio-temporal relationship between the two processes. Current research progress in the field of plant cell division focuses on identifying and tying the links between early and late events in spatial control of cytokinesis and how microtubule array formation is regulated in plant cells.