The exocyst complex is essential for polarized secretion and growth in eukaryotic cells and has been extensively studied across kingdoms. Despite these studies, its mechanism of function and regulation are still not fully un- derstood. Without this understanding, it will not be possible to manipulate, and address diseases associated with defects and deregulation of this evolutionarily conserved complex. The long-term goal is to have a mechanistic understanding of the regulation of polarized exocytosis in eukaryotic cells. The overall objective of this application is to determine the dynamic composition and regulation of the exocyst in plant cells by using biochemical purifi- cation, protein-protein interaction assays, and in vivo analyses of localization and dynamics. The central hypoth- esis is that the regulation of exocyst in plants is dependent on subcellular localization and its association with membrane proteins and phosphoinositides, and not heavily dependent on subcomplex association and dissoci- ation. This hypothesis was formulated based on localization analysis of Sec6 in moss cells and from existing work in other plants. The rationale for the proposed research is that, with this new knowledge, it will be possible to elucidate critical facets of the regulation of polarized secretion, and how it has evolved since the divergence from the last eukaryotic common ancestor. The moss Physcomitrella patens, because of its genetic, cell biolog- ical and microscopy tools, offers a powerful and unique model system to investigate this hypothesis in plants. The hypothesis will be tested by the following two specific aims: 1) Isolate the exocyst complex from plant cells and determine the regulation of its structure by binding interactions; and 2) Determine the in vivo dynamics of the exocyst and establish computational simulations of its assembly and interaction dynamics. Under the first aim, an approach based on affinity purification techniques, proteomics, and in vitro interactions of purified com- ponents will be used. All these activities will be performed by teams of undergraduate students from Biology and Biochemistry majors. Under the second aim, endogenous loci of exocyst subunits will be tagged with fluorescent protein fusions and analyzed by high-resolution multi-color imaging, quantitative microscopy, and fluorescence recovery after photobleaching. To advance a mechanistic understanding of exocyst function and regulation, a computer simulation approach will be used based on the working hypothesis that diffusion, assembly dynamics, and localization all participate in the regulation of exocyst function. These experiments and analyses will be completed by teams of undergraduate students from Biology, Bioinformatics and Computational Biology, and Physics majors. The approach proposed is innovative, because it uses the model plant, P. patens, and a com- bination of microscopy, structural biochemistry, and simulations to make major steps forward in understanding how exocyst is regulated. The proposed research is significant, because it will provide evidence for the presence or absence of subcomplexes and the dynamic localization of the exocyst in plants cells. It will also provide a theoretical framework to interpret microscopy observations and derive realistic models of exocyst regulation.
The proposed research is relevant to public health because comparisons of the regulation and dynamics of the exocyst across evolutionarily distant systems are expected to result in the identification of valuable applications for the control of mammalian cell growth and proliferation. The project is relevant to NIH?s mission because it is aimed at understanding the principles and mechanisms of exocytosis across living organisms, by using Physcomitrella patens as a novel research model.
