Roux-Osovitz Michelle M, Foltz Kathy R, Oulhen Nathalie, Wessel Gary
Department of Baccalaureate Biology, St. Petersburg College, Clearwater, FL, United States.
Department of Molecular, Cellular and Developmental Biology and Marine Science Institute, UC Santa Barbara, Santa Barbara, CA, United States.
Methods Cell Biol. 2019;151:283-304. doi: 10.1016/bs.mcb.2018.11.003. Epub 2019 Jan 2.
The exquisite synchronicity of sea urchin development provides a reliable model for studying maternal proteins in the haploid egg as well as those involved in egg activation, fertilization and early development. Sea urchin eggs are released by the millions, enabling the quantitative evaluation of maternally stored and newly synthesized proteins over a range of time (seconds to hours post fertilization). During this window of development exist many hallmark and unique biochemical interactions that can be investigated for the purpose of characterizing profiles of kinases and other signaling proteins, manipulated using pharmacology to test sufficiency and necessity, for identification of post translational modifications, and for capturing protein-protein interactions. Coupled with the fact that sea urchin eggs and embryos are transparent, this synchronicity also results in large populations of cells that can be evaluated for newly synthesized protein localization and identification through use of the Click-iT technology. We provide basic protocols for these approaches and direct readers to the appropriate literature for variations and examples.
海胆发育的精确同步性为研究单倍体卵中的母体蛋白以及参与卵激活、受精和早期发育的蛋白提供了一个可靠的模型。海胆会释放出数百万枚卵,这使得我们能够在一定时间范围内(受精后数秒至数小时)对母体储存的和新合成的蛋白进行定量评估。在这个发育窗口期存在许多标志性的独特生化相互作用,可用于研究激酶和其他信号蛋白的特征谱、通过药理学手段进行操纵以测试充分性和必要性、鉴定翻译后修饰以及捕捉蛋白质-蛋白质相互作用。再加上海胆卵和胚胎是透明的这一事实,这种同步性还产生了大量的细胞群体,通过使用Click-iT技术可以对这些细胞群体进行新合成蛋白的定位和鉴定评估。我们提供了这些方法的基本方案,并引导读者查阅适当的文献以获取变体和示例。