MEMBRANE DIFFUSION MEASURED USING FCS IN POLARIZED S CEREVISIAE

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The cellular membrane not only serves as the interface and barrier between the extracellular environment and intracellular biochemical processes, but also it is a dynamic organelle where proteins are embedded, recruited and organized, in many cases free to diffuse around the cell surface creating a homogeneous protein concentration. During polarization this uniformity is broken and localization of some proteins changes. To explore the role of diffusion and how changes of a protein's diffusion rate may be involved in polarization, Fluorescence Correlation Spectroscopy (FCS) was utilized to measure membrane diffusion in the budding yeast, S. cerevisiae, during mating projection formation. Slow diffusion rates were determined for cytosolic and membrane bound (EGFPmem) EGFP which were consistent with previously reported values obtained using FRAP [1]. a-cells were exposed to varying concentrations of a-factor (0, 10, 100, 1000 nM) and localization of EGFPmem and the a-factor receptor (Ste2-EGFP) after the formation of a projection were observed. Localization of the Ste2-EGFP was highly polarized whereas localization of EGFPmem was only slightly weighted toward the projection. This may be the result of increased membrane order in the projection [2]. Diffusion rates of both Ste2-EGFP and EGFPmem were measured at the front and back of the polarized cell. Future work will incorporate these measured diffusion rates as well as FCS derived molecule numbers into mathematical models of yeast cell polarization.