RNA localisation during the development of hyphae in the human pathogen Candida albicans

The fungus Candida albicans is a major human pathogen. It is the causative agent of vaginitis in women. In addition, it can cause life-threatening blood stream infections in vulnerable patients such as the new born and certain intensive care patients, especially those undergoing cancer chemotherapy, immunosuppressant therapy or catheterisation. A striking feature of its biology is its ability to grow as a unicellular yeast or in filamentous forms called hyphae and pseudohyphae. Hyphae are chains of long thin cells that grow exclusively from the hyphal tip. This ability to switch growth forms is important for virulence. It is thought that the hyphae can penetrate epidermal barriers while the yeast can circulate in the blood stream to establish lethal secondary infections in the kidneys and heart valves. Hyphal growth is restricted to their tip; our research focuses on the molecular mechanisms responsible for this highly polarised form of growth. If we understood these mechanisms, it might be possible to develop better anti-fungal drugs by finding ways to block them. However, this is a long term goal that is unlikely to be realised as a direct result of this research. So far, we have shown that a special structure called a Spitzenkörper is located at the tip. It is thought that membrane-bound secretory vesicles, which contain the raw materials for the new cell wall and membranes, are transported to the Spitzenkörper where they accumulate before being moving on to the cell surface. Thus we first need to understand the mechanisms responsible for the aggregation of secretory vesicles in the Spitzenkörper. Our research has focussed on a protein called Sec2p, which is associated with secretory vesicles and plays a key role in their movement to sites of polarised growth. This proposal is prompted by the consideration that as a yeast cell switches to hyphal growth the nucleus remains in the mother yeast cell which can be a considerable distance from the hyphal tip where many proteins need to aggregate to facilitate polarised growth. We wondered whether the mRNA molecules, which encode the information for the synthesis of new proteins, may be specifically transported from the nucleus, where they are transcribed from DNA, to the hyphal tip where they are translated into new proteins. To test this idea we carried out a preliminary experiment designed to detect a direct physical interaction between Sec2p and mRNA molecules, and found that this was indeed the case. We now wish to identify the individual mRNA molecules attached to Sec2p and resolve such questions as: 1) What is the role of the proteins encoded by these mRNA molecules in hyphal growth? 2) Is it important that mRNAs encoding these proteins are transported to the hyphal tip? 3) Do the mRNAs interact directly with Sec2p or is the interaction indirect / that is, do the mRNAs interact with another protein that is part of a molecular complex that includes Sec2p? 4) Are the mRNAs transported on the outside of secretory vesicles? 5) What is the nature of the molecular motors that transport the mRNA molecules and the nature of the tracks along which the motors move? The proposed research exploits the latest genome-based technologies as well as state-of-the-art microscopy. Moreover, this is a collaborative proposal between Professor Peter Sudbery an expert in Candida albicans cell biology and molecular genetics and Dr Stuart Wilson a world leader in the study of mRNA movement within cells.

Polarised growth of C. albicans hyphae depends on multiprotein structures such as the exocyst and polarisome, and the Spitzenkörper, an apical body rich in secretory vesicles and ribosomes. During the outgrowth of a hyphal germ from a yeast cell the nucleus becomes separated by a considerable distance from the hyphal tip where proteins act to drive polarised growth. Thus, in addition to mechanisms of protein localisation, mRNA encoding these proteins may also be actively transported from the nucleus to the tip. We tested this hypothesis by determining whether mRNAs co-immune precipitate with Sec2p, a secretory vesicle-associated protein and found that this was indeed the case. In order to identify the mRNAs that associate with Sec2p we will use RIP-CHIP and validate hits using RT-PCR. The cellular localisation of selected validated transcripts will be investigated by in situ hybridisation experiments and the role of the encoded protein in hyphal morphogenesis investigated by constructing gene deletions. We shall delete the 3' UTR of validated transcripts to determine whether it plays a role in localisation and if it does, we shall further construct N-terminal YFP fusions to determine the importance of RNA localisation in protein localisation. To determine whether the mRNAs are attached to secretory vesicles we will fractionate Sec2p into membrane-associated and cytosolic fractions and determine which fraction contains mRNA. We shall determine whether the interaction of the mRNA with Sec2p is direct or indirect using an mRNP capture assay. If the interaction appears indirect we will use the mRNP capture assay on proteins known to associate with Sec2p. Finally we will investigate whether RNA localisation depends on actin filaments or microtubules using specific actin and microtubular inhibitors, and by examining the involvement of She2/She3 required for actin cable-based mRNA localisation in S. cerevisiae and the microtubular associated motors dynein and kinesin.