The roles of C. elegans patched family genes in cell division

This project focuses on a cell biological process called cytokinesis. When a cell divides into 2 daughter cells, it first replicates its genetic material, so that each daughter cell inherits the same number of chromosomes and DNA content as its parent. Subsequently, the duplicated genetic material is evenly partitioned between what will become the 2 new daughter cells. During the terminal stages of cell division, the partitioning of the parental cell into daughter cells is completed when a constriction arises that physically separates the parental cell into two daughter cells - the process of cytokinesis. Thus, it is clear that understanding the mechanisms underlying cytokinesis is a fundamental problem in cell biology, and has consequences with regard to cell proliferation, developmental biology and cancer, as discussed below. We have identified a gene, named ptr-2 (for patched-related), which is required for cytokinesis in the model organism, the nematode C. elegans. C. elegans is a small transparent non-pathogenic worm, which is a widely used in biomedical studies because it possesses a number of experimental advantages, including the ability to grow large numbers at low cost. The lifecycle of the worm from egg to adult takes only 3 days, which makes it possible to analyse the effects of genetic mutation very rapidly. The worm was the first multicellular animal with a fully sequenced genome, so it is known that over 70% of genes associated with human disease are also present in the worm. However, the interpretation of gene function is often simplified in the worm because it only has ~1000 cells. Using a technique known as RNA mediated interference (RNAi), we have shown that the depletion or absence of ptr-2 causes cells of the early C. elegans embryo to have a cytokinesis defect. Cytokinesis defects are usually detected because a cell has multiple nuclei arising from the failure to partition the parental cell into daughter cells at each cell division. The involvement of ptr-2 in this process is of particular interest, because it is related to another gene, which encodes a protein named Patched (Ptch). In human cells, Ptch is a tumour suppressor, because the loss of Ptch activity causes both familial and sporadic carcinomas; mutations in Ptch are the leading cause of skin cancer. Defects in Ptch activity can also lead to developmental abnormalities. Hence an understanding of how the Ptch protein functions is important from both a cell biological standpoint and also from the perspective of human development and disease. To understand how ptr-2 affects cytokinesis, we are proposing to take a genetic approach by first analysing how the absence of ptr-2 affects cytokinesis. By determining what aspects of cell division are perturbed by the absence of ptr-2, we can then apply genetic logic to infer the normal role of ptr-2 in the cell. To make this analysis possible, we have obtained a genetic mutant that lacks the ptr-2 gene. Other clues that can help us to understand how ptr-2 affects cytokinesis and cell division will be obtained by determining where the PTR-2 protein is found in the cell. Protein localisation can be performed in either fixed cells using antibodies or in living cells using a fluorescent reporter, such as the green fluorescent protein from the jellyfish, fused to PTR-2. The PTR-2::GFP fusion can be detected in cells by shining light of the appropriate wavelength. This technique has the advantage that we can follow the behaviour of PTR-2::GFP by microscopic time-lapse recording during the course of an entire cell-division cycle. Once we know how PTR-2 behaves in the cell, we can then make and test predictions regarding the identity of other proteins that could interact with PTR-2. The outcome of this project is that we will expand our knowledge of the cell biology of cell division and also gain an improved understanding of the biochemical activities of Patched proteins.

Cytokinesis is the terminal step in cell division and is an integral component of cell proliferation and spatial patterning in development. In animal cells, cytokinesis is driven by a subcortical equatorial ring composed of filamentous actin and myosin II, which constricts like a purse-string to separate a parental cell into two daughter cells. Membrane trafficking is also required during cytokinesis to complete the final separation of daughter cells (abscission) after the contractile ring has completed ingression, and also to promote membrane addition during the expansion of the cleavage furrow. C. elegans is an excellent model for studying cell division in the context of a multicellular animal because of the large size of cells in the early embryo and because numerous resources and reagents are available for performing optical imaging analysis. In this application, we propose to explore the role of the C. elegans ptr-2 (for ptc-related) gene in cytokinesis; ptr-2 encodes a protein that is related to the Patched (Ptc) family of Hedgehog (Hh) receptors. The first indication that a Ptc protein could participate in cytokinesis was made when we showed that the C. elegans ptc-1 gene plays an essential role in germ line cytokinesis. We subsequently performed a screen of all ptc and ptr genes in the C. elegans genome by RNAi and identified a single candidate, ptr-2, which causes the early embryo to produce multinucleate cells, a hallmark of a cytokinesis defect. Given that members of the Ptc family of proteins participate in the trafficking of lipids or lipid modified proteins, we hypothesise that PTR-2 plays a role in the trafficking of vesicles or proteins required for cytokinesis. Hence, our study of PTR-2, which is now facilitated by the availability of a deletion mutant, should provide insights into the membrane trafficking events that occur during cytokinesis.