DivFUSE

In plants, patterning by cell division relies on division plane establishment, on the biogenesis of a membrane compartment called the cell plate, and on cell plate insertion at the division site. It is generally accepted that a landmark is laid down at the plasma membrane before prophase, and that the leading edge of the expanding cell plate is guided towards this cue at the end of cytokinesis. The nature of this positional cue remains elusive and the processes of cell plate guidance and insertion are poorly understood. Lipids are increasingly recognized as important membrane landmarks, but whether specific lipids mark the division site has yet to be investigated. Interestingly, inhibiting the synthesis of phosphoinositides, sterols or very-long chain fatty acids, an essential component of sphingolipids, results in cytokinesis defects. Preliminary data indicate that PI(4,5)P2 phosphoinositides and its phosphatase SAC9 are involved in defining the spatial site of cell plate insertion. PI(4,5)P2 distribution appears to be driven by homeostatic lipid-lipid interactions. Indeed, we found that the acyl-chain length of sphingolipids is involved in regulating the level of PI(4,5)P2 and that sphingolipids localize at the cell plate. In animals, specific combinations of phosphoinositides and Rab GTPases are responsible for maintaining and co-ordinating intersecting trafficking pathways in the cell. Our preliminary data suggest that in plants, Rab-E GTPases might play a role in cell plate insertion. Furthermore, we propose that Rab-E GTPases are regulated by the TRAPPII tethering complex, a putative Guanine nucleotide Exchange Factor. TRAPPII plays a pivotal role in the spatiotemporal control of plant cytokinesis. In this project, we will address how lipids and key trafficking proteins mediate cell plate insertion by deploying a unique combination of genetic, cell biological and biochemical tools. These include lipid biosensors, lipid click-chemistry, inducible depletion of lipids by optogenetics, super-resolution microscopy, targeted immuno-isolation of membrane complexes and cutting-edge proteomics and lipidomics. Our project is structured around three main work packages, each of which involves all partners. These address the following questions: i) how are lipids sorted at the expanding cell plate or division site? ii) does lipid sorting require a TRAPPII-Rab-E GTPase module? iii) conversely, do lipid landmarks regulate TRAPPII/Rab-E GTPase function? Altogether, our project proposes to gather internationally recognized and unique expertise in cutting-edge plant cell biology and lipid membrane biology and biochemistry. The outcome of this project will break new ground in tackling the question of how plants achieve patterning by cell division, a crucial process for plant growth and development. Additionally, the production of new tools and genetic resources within the frame of this project will be useful for the entire cell biology community.