The POK2 tail: a versatile connectivity hub

Abstract

Plants exist in a plethora of shapes and sizes. Plants achieve such diverse morphological feats through cell patterning, which is particularly challenging, as plant cells are rigidly bound to one another by their adjoining cell walls. Hence, the emergent morphology of the whole organism is determined at the level of cytokinesis, i.e. cell division. Consequently, cell division, particularly division plane positioning, is highly regulated in plants. Plant cells utilise chemical and mechanical signals to determine the division plane position and to mark this site with division plane marking proteins. To localise such proteins to the division site, a microtubule structure, known as the preprophase band, is constructed at the division plane and acts as a recruitment hub for division markers. One such division marker is the PHRAGMOPLAST ORIENTING KINESIN 2 (POK2). POK2 and its close homologue, POK1, are kinesins that reside at the division site to guide the microtubule-based cell division machinery, the phragmoplast, to the division site. pok1/pok2 double mutants display cell patterning defects due to incorrect positioning of the phragmoplast. Importantly, pok1/pok2 mutants do not exhibit cytokinetic defects, indicating that the POKs are important for spatial control of cytokinesis but not for function of cytokinesis itself. Reflective of its function, POK2 localises to the plasma membrane at the division site and also to the phragmoplast midzone. As a plus-end directed kinesin, POK2 is recruited to the phragmoplast midzone by way of the activity of its N-terminally located kinesin motor. Meanwhile, the C-terminal region of POK2, i.e. the POK2 tail is responsible for localisation to the division site. However, the mechanism of specific recruitment and retention at the division site was unclear. To uncover the mechanism of POK2 recruitment and residence at the division site, I purified the POK2 tail and performed in vitro characterisation of its intrinsic biochemical properties. In vitro experiments revealed that the POK2 tail is capable of direct binding to microtubules and anionic lipids. On microtubules, the POK2 tail is able to diffuse along the length of the microtubule. Microtubule binding assays with shorter POK2 tail constructs narrowed down the final ∼100 amino acid residues as important for the binding to microtubules and lipids. Additionally, reconstitution of previously reported in vivo interaction between the POK2 tail and the microtubule bundling protein, MAP65-3, revealed that MAP65-3 enhances POK2 tail microtubule binding. At the single molecule level, MAP65-3 and the POK2 tail are able to interact and co-diffuse at a slower rate than the POK2 tail diffusing alone. Interestingly, the final ∼100 amino acid residues also seem to be important for MAP65-3-based enhancement of POK2 tail microtubule binding. Overall, the ability of the POK2 tail to interact and bind several substrates insinuates a robust and possibly hierarchical accumulation at its in vivo sites. Specifically, the in vitro results indicate that POK2 could be capable of directly binding preprophase band microtubules and the plasma membrane at the division site where interaction with MAP65-3 could confer further spatial and temporal specificity. In conclusion, this thesis work has revealed an aspect of the POK2 tail’s biochemical capabilities, propounding the POK2 tail as a versatile interaction hub necessary for POK2’s function in division plane guidance.