Molecular Control of Division Site Establishment

Plant cells are surrounded by rigid cell walls which confine their shape and determine their location within tissues. Thus the position of a new cell wall formed during cytokinesis contributes to the shape of the cell and consequently to the overall morphology of the plant.

In plants the positional information for the future division site is specified early, at G2/M transition by the formation of the preprophase band. This transient cytoskeletal array forms at the cell cortex and demarcates the future division plane, but disassembles upon entry into mitosis. Subsequently, the division plane is identified by the absence of cytoskeletal proteins (actin depletion zone, KCA1 kinesin depletion zone) and the recruitment of specific cortical division site markers TANGLED and a component of the Ran signaling network RanGAP (Fig. 1, Müller et al., 2009). Previously, we have characterized a pair of closely related kinesins PHRAGMOPLAST ORIENTING KINESIN (POK) 1 and 2, which are implicated in the positioning of new cell walls after cell division (Müller et al., 2006).

Figure 1: Schematic representation of the cortical division site throughout mitosis.

Cortical division site (CDS) and associated proteins at different cell cycle stages are indicated in blue. Note that KCA1 remains absent from this site throughout mitosis.

pok1pok2 double mutants exhibit pronounced cell wall positioning defects as a consequence of misguided phragmoplasts during cytokinesis (Fig. 2). Genetic and in vitro evidence suggest that POK kinesins are required for the maintenance of TANGLED and RanGAP1 at the cortical division site from metaphase onward (Walker et al., 2007, Xu et al., 2008).

In our current work we focus on the functional characterization of POK kinesins and on the identification of novel regulators of division site establishment.

Functional characterization of POKs

To investigate POK function we generate translational fusions of full length POK and POK fragments with fluorescent protein variants to perform live cell imaging. In the context of cell cycle and cytoskeletal markers, these experiments will allow us to determine POK requirements at spatial and temporal resolution. Furthermore, we will determine the protein domains that confer sub-cellular specificity during different cell cycle stages.

Figure 2: Aberrant cell wall positioning in pok double mutants.

Cell walls of root meristems are visualized via Propidium Iodide staining. (A) In wild type root meristems, regular cell divisions result in an ordered pattern of cell walls. (B) In the pok double mutant new cell walls are inserted arbitrarily causing the irregular pattern.

Preliminary localization analysis of C-terminal POK fragments indicate that both kinesins require their C-terminal domain for localization at the cortical division site (Fig. 3). The localization pattern is reminiscent of TANGLED and RanGAP1 localization and we will perform co-localization and FRET analysis.

Figure 3: Localization of POK1-C terminal fragment. GFP-POK1C co-localizes with the PPB (A and B) and subsequently becomes delimited to the site of cell wall fusion during cytokinesis (C and D).

Novel regulators of division site establishment

In yeast two hybrid screens we have identified regulatory components of ROP signaling as interaction partners of POK-C terminal fragments. ROP signaling has recently been implicated in the regulation of actin and microtubule cytoskeleton organization (Yang, 2008). Thus, we pursue a reverse genetics approach for these novel ROP regulatory proteins to elucidate their developmental relevance and specific role in division plane establishment.

Alternatively, we use a sensitized mutant screen to identify enhancers of pok1 single mutants, which are indistinguishable from wild type plants. We have selected several mutants for further analysis. One candidate exhibits an enhanced pok double mutant phenotype and was confirmed to be a novel pok1pok2 allele combination. The EMS induced mutation caused a premature Stop codon in POK2, abolishing most of the coiled coil interaction domains. We continue with our screen and we have initiated mapping of promising candidates.

Phylogenic analysis showed that a third POK-like kinesin clusters with POK1 and POK2. It is specifically expressed during mitosis (Menges et al., 2003). A T-DNA allele for POK-like is lacking full length transcript and we will determine the localization pattern of POK-like.

Mitosis Movie

Movie: Microtubule arrays during mitosis in the Arabidopsis root meristem. The cytoskeleton is a major player in the execution of cell division. Even before a cell begins to divide, the plane of division is highlighted by a plant specific cytoskeletal array, the preprophase band. The predictive band disassembles upon entry into mitosis. Nevertheless, later in mitosis the cytokinetic apparatus or phragmopast directs the formation of the separating cell plate in the plane of cell division as forcast by the preprophase band. Microtubules are visualized with the GFP-MAP4 reporter gene (Marc et al., 1998, Plant Cell 10, 1927-1940).