Synthetic control of actin nucleation in vivo

肌动蛋白成核因子的数量和结构多样性的不断增加给结构-功能研究带来了巨大的挑战。许多影响细胞内肌动蛋白聚合的因素，如亚细胞蛋白分布或生物膜的存在，在生化测定中不能容易地再现。此外，许多肌动蛋白调节剂如profilin、胸腺素、CAP或cofilin影响体内聚合动力学。然而，到目前为止，还缺乏一个细胞系统，其中不同的成核剂可以在可比的生理条件下进行表征。因此，我们建议使用芽殖酵母S。酿酒酵母作为一个最小的生理系统，在体内定量测试肌动蛋白成核剂。我们将首先建立一个强大的测定和控制的细胞环境，以监测肌动蛋白成核在皮层的芽殖酵母细胞。使用这个系统，我们将初步定量表征的三个内源性酵母成核剂和他们的监管机构。然后，我们将扩大我们的分析，系统地表征和比较所有类型的成核剂从各种来源，以及修改和合成肌动蛋白成核剂。在技术上，我们将结合联合收割机强大的酵母遗传学和分子生物学，以精确地操纵肌动蛋白成核剂的表达和定位（自上而下的合成生物学），与活细胞全内反射荧光显微镜（TIRFM）和2D反卷积，以高时空分辨率监测肌动蛋白成核。我们的研究旨在为系统描述肌动蛋白成核剂提供参考。

The ever growing number and structural variety of actin nucleation factors poses a huge challenge to structure-function studies. Many factors influencing actin polymerization in cells, such as subcellular protein distribution or presence of biological membranes cannot be easily reproduced in biochemical assays. In addition, a host of actin regulators such as profilin, thymosin, CAP or cofilin influence polymerization kinetics in vivo. However, a cellular system where different nucleators can be characterized under comparable physiological conditions has so far been lacking. We therefore propose to use the budding yeast S. cerevisiae as a minimal physiological system to quantitatively test actin nucleators in vivo. We will first establish a robust assay and controlled cellular environment to monitor actin nucleation at the cortex of budding yeast cells. Using this system we will initially quantitatively characterize the three endogenous yeast nucleators and their regulators. We will then expand our analysis to systematically characterize and compare all types of nucleators from various sources, as well as modified and synthetic actin nucleators. Technically we will combine powerful yeast genetics and molecular biology to precisely manipulate expression and localization of actin nucleators (top down synthetic biology), with live cell Total Internal Reflection Fluorescence Microscopy (TIRFM) and 2D deconvolution to monitor actin nucleation at high temporal and spatial resolution. With our study we aim to provide a reference for the systematic description of actin nucleators.