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Author:
Date:
2021-04-05
[Abstract] The Target of Rapamycin kinase Complex I (TORC1) is the master regulator of cell growth and metabolism in eukaryotes. In the presence of pro-growth hormones and abundant nutrients, TORC1 is active and drives protein, lipid, and nucleotide synthesis by phosphorylating a wide range of proteins. In contrast, when nitrogen and/or glucose levels fall, TORC1 is inhibited, causing the cell to switch from anabolic to catabolic metabolism, and eventually enter a quiescent state. In the budding yeast Saccharomyces cerevisiae, TORC1 inhibition triggers the movement of TORC1 from its position around the vacuole to a single focus/body on the edge of the vacuolar membrane. This relocalization depends on the activity of numerous key TORC1 regulators and thus analysis of TORC1 localization can be used to ...
[摘要] [摘要]雷帕霉素激酶复合体I（TORC1）的靶标是真核生物中细胞生长和代谢的主要调节剂。在促生长激素和丰富营养素的存在下，TORC1活跃并通过磷酸化多种蛋白质来驱动蛋白质，脂质和核苷酸合成。相反，当氮和/或葡萄糖水平下降时，TORC1被抑制，导致细胞从合成代谢转换为分解代谢，并最终进入静止状态。在发芽酵母中酿酒酵母 抑制TORC1会触发TORC1从其在液泡周围的位置移动到液泡膜边缘的单个焦点/物体。这种重新定位取决于许多关键的TORC1调节子的活动，因此可以使用TORC1定位分析来跟踪通过TORC1途径发出的信号。在这里，我们提供了使用荧光显微镜测量TORC1（特别是Kog1-YFP）重新定位/信号传递的详细协议。重点放在确保以下方面的程序上：（1）尽管葡萄糖和氮饥饿期间它们相对较低的荧光和自发荧光灶的积累，但仍正确地识别（并计数）TORC1抗体；（2）在每次实验开始时，使细胞保持对数生长期，以便正确监测TORC1体形成的动力学；（3）使用适当的荧光标签来避免检查错误定位的TORC1。
[背景]雷帕霉素的靶激酶复合物I（TORC1 ）是真核生物的细胞生长控制网络中的中央轮毂（Loewith和Hall，2011;冈萨雷斯和Hall，2017; Liu和萨巴蒂尼，2020 ...
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Author:
Date:
2020-05-05
[Abstract] The Ras homologous protein (Rho) GTPase subfamily, including RhoA, RhoB, and RhoC are small molecules (~21 kDa) that act as molecular switches in a wide range of signaling pathways to orchestrate biological processes associated with both physiological and tumorigenic cellular states. The Rho GTPases are crucial regulators of actin cytoskeleton rearrangements and FA dynamics and are required for effective cell migration and invasion, as well as cell cycle progression and apoptosis. The Rho GTPases activity is regulated by conformational switching between GTP-bound (active) and GDP-bound (inactive) states. This GTP/GDP cycling is tightly controlled by the guanine nucleotide exchange factors (GEFs), which function as activators by catalyzing the exchange of GDP for GTP and by the ...
[摘要] [摘要] 包括RhoA，RhoB和RhoC在内的Ras同源蛋白（Rho）GTPase亚家族是小分子（〜21 kDa ），在广泛的信号传导途径中充当分子开关，以协调与生理和致瘤细胞状态相关的生物学过程。该Rho GTP酶是至关重要的调节肌动蛋白细胞骨架重排和FA动态和所需要的有效细胞迁移和侵袭，以及细胞周期进程和凋亡，其Rho GTP酶的活动是监管下的构象之间切换GTP结合（有源）和GDP -- GTP / GDP循环受鸟嘌呤核苷酸交换因子（GEF）严格控制，鸟嘌呤核苷酸交换因子（GEF）通过催化GTP的GDP交换和GTPase激活蛋白（GAP）来激活，从而实现水解作用。导致Rho GTPase失活的GTP 的详细信息。在此，我们描述了执行RhoB G-LISA活化测定以检测体外GTP负载的RhoB的水平的详细协议。这是第一个专门用于测量RhoB活化的比色测定。该方法是通过改编RhoA G-LISA激活检测试剂盒（Cytoskeleton，Inc.）而开发的，可在不到3 小时的时间内精确测量RhoB活性，这种快速方法可广泛用于评估GTP负载的RhoB 的水平在任何种类的细胞模型中，要了解RhoB激活在生理过程，疾病，致癌转化中的作用或在高通量筛选中发现药物的作用。
[背景 ] 尽管Rho GTP酶的RhoA，RhoB的，和RhoC占有率超过85 ...
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