Molecular Recognition in Biological Systems and Bioinformatics

Genetics; microscopy; signal transduction; genomics; cell polarity

Specificity of Cdc42-, PAK-, and MAPK-Pathways
A recent surprising finding is that signal transduction pathways share components. How does the activation of a pathway containing general factors induce a specific response? Indeed, inappropriate cross-talk between signaling pathways is a trigger for cancer. In yeast, the FG pathway, which is required for filamentous growth, is composed chiefly of generic factors that function in multiple MAPK pathways. Few specific FG pathway components have been identified. Using genomic approaches, I discovered the presumptive receptor for the FG pathway, called Msb2. I am interested in understanding the mechanism whereby Msb2, which is a signaling mucin receptor communicates to Cdc42 and PAK to trigger activation of the FG pathway. Proteomic approaches (co-IP, MASS SPEC, 2-hybrid) will be used to identify protein-protein interactions at the head of the FG pathway. Moreover, characterizing the mucin receptor will ultimately result in elucidation of the ligand that triggers the FG pathway. Ultimately, these studies will lead to the design of drugs to receptors that induce filamentous growth in fungal pathogens.

Mammalian Mucins and Polarity
I discovered that loss of the mucin domain of Msb2 can trigger hyperactivation of the protein. This surprising result suggests that mucin-domain loss may trigger signal pathway activation and possibly cancer in mammalian cells. This result has broad implications in drug design and tumor diagnosis, and therefore I am beginning to extend my findings to mammalian systems. For example, do the mucin domains in metazoan signaling mucins inhibit pathway activity? Is there a correlation between mucin-domain deletion and metastasis in human tumors? I am also interested in the possibility that mammalian mucins play a role in cell polarity not previously appreciated. Using live-cell imaging of tissue culture cells I hope to watch the effects of mammalian mucin activation on the actin cytoskeleton in the cell.

New Connections to MAPK Pathways
I am interested in identifying new connections to the Msb2-Cdc42/PAK-MAPK pathway that controls filamentous growth in yeast. We are currently performing a variety of traditional and high-throughput genetic screens and we anticipate the identification of a number of factors, including scaffolding proteins, negative regulators, and modulators of Cdc42 activity. The most provocative connection I have found links the cell cycle at M phase/Start to MAPK activation and is governed by the mitotic exit network and cytokinesis.

Cell-cell Adhesion, Cellular Invasion, and Biofilm Formation
Many interesting questions surround the FG pathway that I am beginning to explore. For example, FG occurs on a solid surface but not in liquid culture - how does this discrimination occur? Perhaps the receptor for the FG pathway plays a role in detecting this environment. Another avenue of investigation is the outputs of the FG pathway, which include cell-cell adhesion, invasion of cells into substrate, and cell migration (microbial biofilm formation). Understanding these processes has general applications to cancer (i.e. cellular invasion and adhesion) and to fungal pathogenesis. Bioinformatics approaches comparing genomes of budding yeast to true filamentous fungi will be used to identify conserved mechanisms that contribute to FG across species.

Zeiss APOTOME microscope

Saccharomyces cerevisiae, budding yeast

• MAP kinases
• Mucins
• cell-surface proteins
• Cdc42
• GTPases
• receptors

Basic biomedical research, causes underlying heart disease, cancer, human genetics, signal specificity

• yeast genetics
• protein biochemistry
• molecular biology
• two-hybrid approaches
• microscopy

Yeast genetics tools, two-hybrid constructs

Two-hybrid yeast strains, filamentous yeast strains

http://wwwbiology.nsm.buffalo.edu/cullen_web/index.html