Unraveling division plane positioning and endocytic protein complexes in Arabidopsis thaliana

At the onset of mitosis, plant cells reorganize their cortical microtubule array into a narrow ring encircling the nucleus until late prophase. This transient cytoskeletal structure is termed the preprophase band (PPB) and is believed to establish the plasma membrane (PM) identity at the cortical division zone (CDZ) which persists until cytokinesis. The CDZ is determined by the presence or particular exclusion of several marker proteins with a cell plate guiding function. The expanding phragmoplast mediates the directional growth of the cell plate towards the CDZ, where the cell plate anchors to the parental plasma membrane at the end of cytokinesis. Therefore, the PPB defines the division plane early in mitosis and spatially controls plant cell division. However, the molecular mechanisms by which the identity of the CDZ is established and maintained as well as the role of the division zone markers in supporting cell plate guidance remains unknown. Although a variety of proteins have thus far been described to be involved in division plane positioning, information about their biochemical properties and interconnections is largely missing. The aim of this doctoral research was to increase our current understanding of division zone demarcation in plant cells and attempt to link the known players. Because the biological function of a protein is predominantly determined by protein interaction networks, a targeted interactomics approach based on the tandem affinity purification (TAP)-technology was set up starting from a set of bait proteins recognized to play a role in division zone demarcation. The TAP technology allows a high-throughput isolation of in vivo protein complexes under native conditions and requires no prior knowledge on the protein complexes in which the bait participates. Through the co-purification of interaction partners, a division zone interactome can be built which might ultimately enable us to correlate the known players, but will definitely identify novel candidate proteins involved in the process of division plane positioning. Based on subsequent fluorescence microscopy screening, those interactors which specifically localize to division plane figures such as the preprophase band, the PM at the cortical division zone or the expanding cell plate will be selected for further functional characterization using a combination of genetic and live-cell imaging analysis. This doctoral thesis can be subdivided in two major parts, each considering a specific functional branch present in the division zone interactome. Part one (chapters 1-3) provides a general overview of the obtained division zone interactome and describes novel players and protein complexes involved in division plane positioning and division zone demarcation. This targeted interactomics approach resulted amongst others in the isolation of a TON1/TRM/FASS PP2A phosphatase (TTP) complex, which was fully characterized in a collaborative effort with the research group of Prof. Dr. David Bouchez and Dr. Martine Pastuglia (INRA, Versailles). Our data tightly connected TON1, which shows homology to human centrosomal proteins, and the PP2A B’’ regulatory subunit FASS to a heterotrimeric PP2A holo-enzyme, which is targeted to the cortical cytoskeleton by the TRM protein family Scope 4 to exert its function. Moreover, we illustrated the necessity of FASS/PP2A phosphatase activity for PPB formation. Part two of this doctoral thesis (chapters 4-6) focusses on endocytosis at the plasma membrane and how endocytosis participates in cell plate anchoring at the CDZ. In light of this question, the cell biological function of the plant-specific multi-subunit complex comprising TPLATE, isolated through the targeted division zone interactomics approach described in part one, was functionally investigated. Besides the occurrence of two distinct recruitment pathways for the TPLATE complex and CLC2 during somatic cytokinesis, a cell plate recruitment pathway and a CDZ recruitment pathway during cell plate anchoring, we show that the TPLATE complex performs a more general function in clathrin-mediated endocytosis at the PM throughout plant development. The TPLATE complex could therefore provide a functional link between plasma membrane endocytosis and the maintenance of CDZ identity