Research Overview
Changes in cell shape and adhesion are two fundamental hallmarks of the differentiation of eucaryotic cells. To better understand the mechanistic basis of these processes we are studying their roles in the life cycle of the budding yeast, Saccharomyces cerevisiae.
Three main projects are presently underway in the laboratory:
- Analyses of a novel paxillin homolog and Rho GTPase signaling modulator, Pxl1p;
- Studies of the functions, traffic, and inhibition of fungal adhesins;
- Functional genomics of fungal control of lipid and membrane homeostasis.
Analyses of a Novel Paxillin Homolog and Rho GTPase Signaling Modulator, Pxl1p
The scaffolding protein paxillin and its homologs play key roles at sites of polarized cell growth in vertebrate cells, such as focal contacts. Paxillin family homologs are present in the predicted sequences of a growing number of invertebrate species, including budding yeast. Due to its central role in cell signaling and polarized cell growth in vertebrate cells, we have begun studying how the homologous protein encoded by the PXL1 (Paxillin-like protein 1) gene functions in yeast cells.
Our studies to date show that, like vertebrate paxillin, the Pxl1 protein (Pxl1p) is found specifically at sites of polarized cell growth. Several lines of evidence show that yeast cells lacking Pxl1p have altered signaling through the Rho1 cell integrity pathway. Rho1p functions normally to help coordinate polarized cell growth at the levels of the organization of the actin cytoskeleton and assembly of glucan in the fungal cell wall. The cell wall is a major target of some classes of antifungal drugs and therefore better understanding of its regulation may aid in further antifungal drug development. We are presently using a variety of genetic and biochemical approaches to examine the mechanisms controlling the localization of Pxl1p to polarized growth sites and to determine what other proteins it interacts with at such sites.
Studies of the Functions, Traffic, and Inhibition of Fungal Adhesins
The ability of fungal cells to colonize different substrates and persist in diverse environments frequently involves regulated changes in their ability to bind substrates and/or one another; such changes are mediated cell surface adhesion proteins known as adhesins. We are dissecting the functions of model adhesins in the simple and easily manipulated system of budding yeast. In a complementary approach, we are applying methodologies for the directed identification of lead compounds with high specificity to functional domains of budding yeast adhesion proteins and major determinants of adhesion in the human pathogenic yeast Candida albicans. Such compounds are a means of developing novel antifungal drugs that disrupt fungal adhesion interactions. Drugs of this kind may have both stand-alone prophylactic value and combinatorial synergy with other antifungal drugs presently employed in immune-compromised patients suffering from systemic fungal infections.
The genes encoding adhesion proteins often undergo dramatic regulation in response to different life cycle transitions and in different fungal species. Two species of interest to us, and in which a large number of bona fide and candidate adhesion proteins are already known, are budding yeast, S. cerevisiae and the human commensal pathogen, Candida albicans. To be successful, pathogenic cells must initially adhere tightly to host tissues (a heterotypic adhesion interaction) and also, to a lesser extent, one another (a homotypic adhesion interaction). The adhesion proteins identified to date in these two fungal systems share a number of common domains of protein sequence similarity and undergo common types of post-translational modifications during their biogenesis that can be important to their to their function and localization at the fungal cell surface. Presently, the significance of these modifications and domains, as well as the specific function of a number of the proteins that contain them remain to be understood. Similarly, the cellular processes that regulate the disposition of these proteins in the fungal cell wall and that presumably therefore may represent virulence factors, are also incompletely understood. Our aim is to learn more about this important class of fungal proteins through a variety of functional studies, including developing methods for rapidly generating inhibitors of specific fungal adhesion proteins.
Functional Genomics of Fungal Control of Lipid and Membrane Homeostasis
The synthesis, maintenance and regulated dynamics of a wide number of lipids and membranes are crucial to the organization and function of all eucaryotic cells. Despite their fundamental roles, their challenging chemistry and complex natures have inhibited broad investigation of this fundamentally important area of fungal cell biology. In collaboration with W. Thomas Starmer in our department, we have undertaken a systematic functional genomics study in S. cerevisiae of genes that may mediate resistance to plant natural products that govern the generation of wild species of yeasts in the genus Pichia. As these natural products are membrane disturbing saponins, we expect these studies to aid in our further understanding of the cellular components that play roles in fungal lipid and membrane homeostasis.
Wild yeasts of the genus Pichia occur on decaying tissues of different species of cacti found in the Sonoran Desert and throughout the world. The presence of plant natural products, specifically triterpene glycosides, occurring at high levels naturally in these plant tissues creates unique challenges for such fungi to utilize these valuable niches in such ecosystems. Triterpene glycosides are a stunningly diverse chemical scaffold that nature has repeatedly utilized to create a broad array pharmacologically active chemicals found in a very large number of plant species, including birch bark, licorice, black cohosh and many others. We have found that different commonly used wild type strains of budding yeast differ markedly and specifically in their response to triterpene glycosides found in certain Sonoran Desert cacti.
A collection of “knock-out” strains corresponding to most of the approximately 6,200 genes of the budding yeast genome is being screened for genes whose inactivation leads to sensitivity or resistance to triterpene glycosides. As the identity of the predicted gene knocked out in each of these several thousand strains is already known, we can rapidly identify a functional network of genes involved in cellular responses to the presence of the triterpene glycoside. We have already identified known genes by this screen whose functions are associated with lipid and membrane regulation. As might be expected for such a fundamental but relatively understudied area of fungal cell biology, we have also identified a wide variety of previously uncharacterized genes encoding proteins whose cellular roles remain to be understood. We are assigning such genes the name LMH for Lipid and Membrane Homeostasis.
Based on current estimates, we expect to identify roughly 800 genes in the yeast genome whose activities affect lipids and membranes either directly or indirectly. While this number appears relatively high, about a third of all yeast genes encode proteins that may be predicted to reside in membranes. A sizeable number of additional proteins are also peripherally associated with membranes. Still other proteins play known roles in the synthesis of lipids, membrane components and their precursors. For example, synthesis of the predominant membrane sterol, ergosterol, begins with acetyl CoA and requires 19 enzymatic steps; branches of this pathway are also required to produce dolichol and geranylgeranyl groups which function in post-translational modification of proteins. Hence, the large number of genes uncovered by this screen may, in part, be expected considering the ubiquitous nature of lipid and membrane function in eucaryotic, and particularly fungal, cells. Ergosterol is the direct or indirect target of most classes of commonly employed antifungal drugs, making information on these genes of fundamental biomedical relevance. Efforts are presently underway through additional secondary screens and bioinformatic methods to classify functional subgroups of the LMH genes and further elucidate the cellular functions of their gene products.