Investigating the role of phosphoinositides during plant cytokinesis – INTERPLAY

Cytokinesis is the final step of cell division, partitioning the cytoplasm between the daughter cells. In animal cells, this event is driven by cortical contraction that squeezes the cell into two and requires coordination between cytoskeleton and plasma membrane. Higher plants do not constrict the plasma membrane (PM) during division, but instead, vesicles are targeted to the center of the division plane and partition the cell through cytoskeleton-driven expansion of the cell plate. While cytokinesis is executed in radically different manners in animals and plants, they both rely on the dynamic interplay between cytoskeleton and PM to precisely deliver molecular components to the future site of cell division. It is recently emerging that, in non-plant organisms, strict regulation of the levels and distribution of phosphoinositides (PIPs), which are minor components of the membrane lipids, is required for successful cytokinesis. In particular, localized PI4P/PI(4,5)P2 act as a signaling hub by promoting proper actin cytoskeleton organization and thereby directing membrane trafficking to the division plane. Yet, the role of PI4P/PI(4,5)P2 during cytokinesis is largely overlooked.
We recently identified strong cytokinesis defects in the sac9 mutant. AtSAC9 is an atypical plant PIP-phosphatase that regulates the balance between PI4P/PI(4,5)P2. Our preliminary data show that while AtSAC9 localizes at the cell plate during plant cell division, PI(4,5)P2 is excluded from it until the late telophase when the cell plate joins the mother cell wall. We also observed that in the absence of AtSAC9, PI(4,5)P2 accumulates ectopically in endosomes while it localizes exclusively at the PM in the wild-type. We found that ectopic accumulation of PI(4,5)P2 in sac9 mutant leads to an increase in F-actin around endosomes, as reported for the loss of function of the PI(4,5)P2 5-phosphatase HsOCRL in animal cells. Both HsOCRL and AtSAC9 have PIP-phosphatase domains but are very different in terms of protein domain organization and phylogenetic origin. To our knowledge, AtSAC9 is the first molecular component shown to regulate PI(4,5)P2 subcellular targeting in plants.
In the INTERPLAY project, we propose to investigate the role of phosphoinositides during plant cytokinesis. In the first work package, we will determine biochemically the enzymatic activity of AtSAC9 in vitro (Task1a) and evaluate in planta the substrate of AtSAC9 (Task1b). We then propose to analyze AtSAC9 subcellular localization during cell division (Task2a) and the dynamic of the cytoskeleton in the absence of AtSAC9 in dividing cells by in vivo confocal microscopy (Task2b). In particular, cell division will be monitored in pericycle cells, in order to dissect the phenotype of sac9 during lateral root initiation (Task2c). We will next evaluate the impact on cell division progression of chemical-induced perturbation of PI4P/PI(4,5)P2 using microfluidic technology on Arabidopsis PIP-reporter lines (Task2d). While the study of AtSAC9 will give us a first molecular handle on how PIP might control cell division in plants, we will tackle this question in a second work package with higher spatial and temporal resolution by developing an optogenetic approach for the first time in plants. We will first build tools to selectively remove (or produce) PI4P/PI(4,5)P2 during each step of cell division in synchronized cell culture (Task3a) and follow the progression of cell division by live imaging after light-induced recruitment of PIP-metabolizing enzymes (Task3b). We will next assess the effect of PI4P/PI(4,5)P2 deficit (or accumulation) on the establishment and maintenance of the division plane in targeted zones such as the cell equator or poles (Task4). Results gained from this proposal will provide a better understanding of the signaling mechanisms coupling PIP metabolism and cell division, the latest being fundamental in regulating organismal growth.