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Our goal is to understand the processes that generate compartments of the secretory pathway, including ER exit sites (ERES; also known as transitional ER or tER sites) and the cisternae of the Golgi apparatus. Self-organization models provide the conceptual framework. Specifically, we postulate that ERES are generated together with early Golgi cisternae by an integrated self-organization pathway, and that early cisternae progressively mature into late cisternae.
For exploring these ideas, our main experimental system is a pair of budding yeasts. In Saccharomyces cerevisiae, Golgi cisternae are dispersed throughout the cytoplasm and the ER contains multiple small ERES, whereas in Pichia pastoris, ordered Golgi stacks are located next to large, stable ERES. These two yeasts have complementary advantages for testing specific hypotheses about the secretory pathway. We use a combination of genetics, molecular biology, 4D confocal microscopy, and electron tomography. This work is revealing evolutionarily conserved principles of cellular organization.
A second project in the lab involves optimizing fluorescent proteins, including the red fluorescent protein DsRed. Wild-type DsRed matures very slowly. We overcame this problem by using directed evolution to create the first rapidly maturing DsRed variants, one of which is marketed commercially as DsRed-Express. More recent work yielded a noncytotoxic variant called DsRed-Express2, as well as a far-red variant called E2-Crimson. These engineering efforts inspired a basic research project in which we clarified the pathway of DsRed chromophore formation. Current efforts are focused on creating improved monomeric green and red fluorescent proteins.
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