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Cell division, the physical separation of cellular content, is a fundamental process for all living organisms. As a result of the rigidity of the cellulosic plant cell wall, and contrary to the acto-myosin contractile ring, which constricts the cell surface inwards during cytokinesis in animal cells, vascular plant cells cope with their spatial demands by an inwards-to-outwards directed division mode. The latter is accomplished after the orchestrated and successive assembly of distinct cytoskeletal arrays. Prior to mitosis, a ring of cortical microtubules, the preprophase band (PPB) emerges and prescribes the future division site where the developing cell plate will fuse with the parental cell wall. The PPB disassembles at the end of prophase and simultaneously the nuclear envelope breaks down (NEB). Although the PPB is absent following prophase, the positional information, provided by the PPB, is preserved through mitosis and cytokinesis by a set of highly dynamic components, occupying a zone beneath the PPB established at the cell cortex, named cortical division zone (CDZ). As prophase proceeds a bipolar prospindle is assembled by the reorganization/nucleation of perinuclear microtubules at the surface of the nuclear envelope. After NEB, bundles of microtubules that connect to the kinetochores are formed leading to the generation of a bipolar barrel shaped spindle, which mediates the alignment of the chromosome pairs at the equatorial plane in metaphase. After chromosome segregation in anaphase, the assembly of a newly synthesized cell plate is driven by the rapid centrifugal expansion of a plant specific bipolar cytoskeletal apparatus called phragmoplast. Fusion of the cell plate with the parental cell wall at the CDZ results in the generation of two identical daughter cells at the end of cytokinesis. Although several aspects of this complicated process have been characterized over the years, yet, little is known about the exact molecular interplay of proteins during plant cell division. Of particular importance during cell division are several proteins that belong to the kinesin-12 class. This class of motor proteins consists of six members in Arabidopsis (Kinesin-12A-F). Kinesin-12A/B accumulate at the phragmoplast midzone and together are necessary for proper cell plate formation in the male gametophyte. An additional pair of functionally redundant kinesin-12 members, the PHRAGMOPLAST ORIENTING KINESIN (POK) 1 and 2 (Kinesin-12D/C) preserve the spatial information of the CDZ throughout cytokinesis. Simultaneous mutations in POK1 and POK2 cause tilting of phragmoplasts and random cell plate insertions, compromising the usual co-alignment of PPB, phragmoplast and cell plate fusion site. While residing exclusively at the PPB and CDZ, POK1 act as scaffold that retains TAN1 and RanGAP1 from metaphase on at the CDZ supporting its crucial role in CDZ establishment/ maintenance. Little is known regarding the existence of additional proteins that mediate the latter and the dependency on POKs for their function and localization. In the present thesis the identification of two closely related putative Rho-GTPase Activating proteins, namely PH-GAP1 and 2, which are necessary for proper division plane selection during cell division was reported (Stöckle et al., 2016). Briefly, taking advantage of different interaction assays, we were able to show that PHGAPs interact with kinesin-12 POK1 and in addition interact with small ROP GTPases in vivo. The GUS reporter gene expression system revealed a similar expression pattern for both genes including the root and leaf meristematic tissue, young leaf epidermal cell as well as the leaf-vascular tissue indicating that they share of a common biological function. Moreover, using fusion proteins and live cell imaging approaches we investigated the intracellular localization pattern of both PHGAP1 and PHGAP2. Interestingly, although PHGAPs decorate the plasma membrane of all cells, in dividing cells both proteins were restricted to the CDZ in a POK-dependent manner from metaphase onward. However, contrary to pok1 pok2 double mutants, simultaneous loss of PHGAP1 and PHGAP2 results only in mild cell wall positioning defects reflecting and inaccurate positioning of POK1. Collectively, our data shed some light on POK-dependent and small GTPase cell polarity signalling-based mechanisms that control plant cell division in a POK1/2 dependent manner. The aim of the second project of this thesis was to further characterize the function of POK2. It was found that POK2 exhibits a role in mediating moderate phragmoplast expansion, which explained the reduced phragmoplast expansion rate, monitored in cytokinetic root cells of pok1 pok2 double mutants (Herrmann et al., 2018). To investigate the latter, we analysed the spatio-temporal localization pattern of a functional POK2 GFP-fusion protein and found that it displays a dual localization pattern throughout cell division, as it is present at the CDZ from prophase onward and at the phragmoplast midzone during cytokinesis. Further dissection of different protein domains revealed that the accumulation of POK2 at the phragmoplast midzone depends on its functional motor-domain, whereas its carboxy-terminal domain is sufficient for CDZ targeting. Intriguingly, the conserved microtubule associated protein MAP65-3/PLE interacts with POK2. Furthermore in collaboration we analysed the mechanistic properties of POK2 motor domain demonstrating that it acts as a weak plus end directed motor in vitro as well as in vivo (Chugh et al., 2018). The last project of my thesis focused on the investigation of a plant kinesin-12 with potentially conserved function during spindle assembly, considering that the molecular mechanism of the latter is still poorly understood in plants. For this, aiming to identify the closest relative of kinesin-12 metazoan orthologue HsKIf15 and KLP2, we performed phylogenetic tree analysis based on full length protein sequence of different kinesin-12 homologues in selected plant species. Among those we decided to functionally characterize the closest relative of POK1 and POK2, the Kinesin-12E, which we renamed into PHRAGMOPLAST ORIENTING KINESIN-LIKE (POK-like). Live cell imaging of a functional POK-like GFP-fusion protein uncovered a cell cycle specific and microtubule dependent localization pattern. Using a set of protein deletions, we were able to show that the timely recruitment of POK-like is accomplished by distinct protein domains. POK-like in interphase cells displays a cytoplasmic accumulation whereas it is recruited to the nuclear envelope in prophase and upon NEB localizes to overlapping antiparallel kinetochore microtubule bundles. Finally, POK-like is specifically targeted to the phragmoplast midzone during cytokinesis. As determined by immuno-labeling experiments, dividing meristematic root cells of pok-like mutants display significantly reduced spindle length and prolonged duration of the prometaphase/metaphase stage in plants. Furthermore, co-localization analysis with the kinetochore marker RFP-HTR12 combined with the microtubule depolymerizing agent oryzalin recorded an additional accumulation GFP-POK-like close to chromosome-associated structures. In addition, we confirmed RanGAP1 as a novel interaction partner of POK-like in planta. Finally, we determined that motor domain activity of POK-like is a prerequisite for its midzone targeting of bipolar microtubule arrays. Collectively, the data presented in this thesis highlight the crucial role of kinesin-12 class motor proteins during plant cell division. We characterized new interactions partners for both POK1 and POK2 and identified a plant kinesin-12 specific function for POK2 during phragmoplast establishment. Furthermore, we investigated the yet uncharacterized protein POK-like, suggesting a functionally conserved activity of kinesin-12 members during spindle assembly throughout the eukaryotic kingdom. |
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