The role of plant Rab GTPases in cytokinesis

Cell division is fundamental to life, but how does it happen? Considering that cell division is common to all living cells, it is perhaps surprising to learn that animal and plant cells divide quite differently. In animals the cell is constricted by an internal ring of protein and when the cell membranes meet in the centre and two daughter cells pinch off. But in plant cells, which are surrounded by a rigid cell wall and are under great internal pressure, a new cell wall is laid down within a specialised internal compartment, the cell plate, that grows rapidly across the centre of the cell. The cell plate forms a disc that partitions the cell and when it fuses with the cell membrane, 2 separate daughter cells are produced. How does all this happen? Work on this project aims to identify some of the molecules that are involved in orchestrating these events and to explain how they work. It seems that despite the differences in mechanics of cell division in plants and animals, some of the same molecules may be involved. If so, is this evidence of an ancient shared ancestral mechanism, or a case of evolution arriving independently at similar solutions? This project involves the use of transgenic plants to visualise the behaviour of these molecules in living plant cells and to perturb their activities to learn about their importance in cell division. It is an example of GMOs helping us to understand and appreciate fundamental processes in living organisms.

Rab GTPases co-ordinate many aspects of membrane traffic in eukaryotic cells. In higher plants, some branches of the Rab family tree have become greatly expanded suggesting that these Rab proteins may act in plant-specific transport processes. One such is the plant Rab-A branch. We have tagged various members of this family with YFP and have found that some Rab-A proteins relocate from punctate cytoplasmic structures to the cell plate during cytokinesis. The assembly of the cell plate in plants is a highly specialised and possibly unique membrane trafficking process. Only a few components of this trafficking pathway have been identified. We hypothesise that the Rab-A GTPases that locate to the cell plate play a role in co-ordinating membrane traffic to or from the cell plate. In this project we wish to: 1. Use anti-GFP antibodies to precisely localise Rab-A proteins using electron microscopy of interphase and dividing cells. 2. Raise anti-peptide antisera (commercially) to localise the native proteins similarly. 3. Use inducible expression of siRNAs or dominant mutants to ask whether the Rab-A proteins perform an essential role in cytokinesis. If no cytokinesis defects are observed we will ask whether these transgenic lines exhibit increased sensitivity to inhibitors that act on membrane traffic or cell plate biogenesis. We will also perform crosses to mutants with mild cytokinesis defects to ask whether the phenotype is enhanced after induction. This will involve the use of inducible gene expression technology developed in my laboratory. 4. To use a yeast two-hybrid screen of a cDNA library made from rapidly dividing root cells to look for interactors of 2 Rab-A proteins that locate to the cell plate. We will establish whether these interactors bind specifically to particular members of the Rab-A family in the yeast two hybrid assay. 5. We will use anti-peptide antisera or tagged versions of the interactors to determine whether they colocalise with their Rab-binding partners during interphase and/or cytokinesis.