MEMBRANE DIFFUSION MEASURED USING FCS IN POLARIZED S CEREVISIAE

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目及 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 细胞膜不仅作为细胞外环境和细胞内生化过程之间的界面和屏障，而且它是一个动态的细胞器，其中蛋白质被嵌入，募集和组织，在许多情况下自由扩散到细胞表面周围，产生均匀的蛋白质浓度。 在极化过程中，这种均匀性被打破，一些蛋白质的定位发生了变化。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）测量了出芽酵母S.酿酒酵母，在交配突起形成期间。测定了胞质和膜结合（EGFP）EGFP的慢扩散速率，这与先前报道的使用FRAP获得的值一致[1]。将α-细胞暴露于不同浓度的α-因子（0、10、100、1000 nM），并观察形成突起后EGFP和α-因子受体（Ste 2-EGFP）的定位。 Ste 2-EGFP的定位是高度极化的，而EGFP的定位仅略微向投影加权。这可能是投影中膜有序度增加的结果[2]。在极化细胞的前部和后部测量Ste 2-EGFP和EGFP的扩散速率。 未来的工作将把这些测得的扩散速率以及FCS衍生的分子数纳入酵母细胞极化的数学模型。

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.