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.

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.

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.

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.

The Phragmoplast-Orienting Kinesin-12 Class Proteins Translate the Positional Information of the Preprophase Band to Establish the Cortical Division Zone in Arabidopsis thaliana.

The preprophase band (PPB) is a faithful but transient predictor of the division plane in somatic cell divisions. Throughout mitosis the PPBs positional information is preserved by factors that continuously mark the division plane at the cell cortex, the cortical division zone, by their distinct spatio-temporal localization patterns. However, the mechanism maintaining these identity factors at the plasma membrane after PPB disassembly remains obscure. The pair of kinesin-12 class proteins PHRAGMOPLAST ORIENTING KINESIN1 (POK1) and POK2 are key players in division plane maintenance. Here, we show that POK1 is continuously present at the cell cortex, providing a spatial reference for the site formerly occupied by the PPB. Fluorescence recovery after photobleaching analysis combined with microtubule destabilization revealed dynamic microtubule-dependent recruitment of POK1 to the PPB during prophase, while POK1 retention at the cortical division zone in the absence of cortical microtubules appeared static. POK function is strictly required to maintain the division plane identity factor TANGLED (TAN) after PPB disassembly, although POK1 and TAN recruitment to the PPB occur independently during prophase. Together, our data suggest that POKs represent fundamental early anchoring components of the cortical division zone, translating and preserving the positional information of the PPB by maintaining downstream identity markers.

Nitrosative stress triggers microtubule reorganization in Arabidopsis thaliana.

Microtubules (MTs) are essential components of the cytoskeleton and fulfil multiple cellular functions in developmental processes, readily responding to intrinsic and external cues. Nitric oxide signalling is well established in plants, and the MT cytoskeleton is one of its potential targets. To mimic low level nitrosative stress, growth medium was supplemented with 3-nitro-L-tyrosine (NO2-Tyr), a nitrated form of the amino acid tyrosine, and concentration-dependent changes in root growth rate and a reduction in cell division frequencies in Arabidopsis thaliana were observed. In addition, it is reported that exposure to low NO2-Tyr concentrations was not detrimental to plant health and caused subtle and reversible defects. In contrast, growth defects caused by high NO2-Tyr concentrations could not be reversed. Live cell imaging of an MT reporter line revealed that treatment with a low concentration of NO2-Tyr correlated with disorganized cortical MT arrays and associated non-polar cell expansion in the elongation zone. NO2-Tyr treatment antagonized the effects of taxol and oryzalin, further supporting the association of NO2-Tyr with MTs. Furthermore, oblique division plane orientations were observed which were probably induced prior to cytokinesis.

Potential roles for Kinesins at the cortical division site.

Spatial control of cytokinesis is critical for cell and plant morphology. The plane of cell division is established at G2/M transition and is initially demarcated at the cortex of the cell by the cytoskeletal preprophase band (PPB) and subsequently throughout mitosis by the cortical division zone (CDZ). Few kinesins, belonging to different classes of the superfamily, either display a distinct spatio-temporal localization at the PPB and CDZ, or genetic evidence proposes a specific function there. Protein phosphorylation and degradation are likely directing the cell cycle-dependent localization and activity of some of these kinesins, as indicated by mutation of respective conserved motifs. Furthermore, kinesins are required for continuous recruitment of CDZ identity markers to the CDZ. This review summarizes the limited current knowledge of kinesins potentially involved in the steps required for correctly oriented division planes, considering localization patterns and genetic evidence, and discussing kinesin function in context with interaction partners and cell cycle regulation.