MEMBRANE DIFFUSION MEASURED USING FCS IN POLARIZED S CEREVISIAE

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 细胞膜不仅是细胞外环境和细胞内生化过程之间的界面和屏障，而且是一个动态的细胞器，蛋白质在其中嵌入、招募和组织，在许多情况下可以自由扩散到细胞表面，产生均匀的蛋白质浓度。在极化过程中，这种一致性被打破，一些蛋白质的定位发生变化。为了探索扩散的作用以及蛋白质扩散速率的变化如何参与极化，利用荧光相关光谱(FCS)测量了酿酒酵母(S.cerevisiae)交配投影形成过程中的膜扩散。细胞液和膜结合(EGFPmem)的EGFP的缓慢扩散速率被测定，这与以前用FRAP[1]得到的值一致。将A细胞暴露于不同浓度的α-因子(0、10、100、1000 nM)中，观察突起形成后EGFP膜和α-因子受体(Ste2-EGFP)的定位。Ste2-EGFP的定位是高度极化的，而EGFPmem的定位仅略有偏向投影。这可能是由于突起中膜的有序性增加[2]。在极化细胞的前后测量了Ste2-EGFP和EGFPmem的扩散速率。 未来的工作将把这些测量的扩散速率以及FCS得出的分子数纳入酵母细胞极化的数学模型中。

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.