Unruh Jay R, Slaughter Brian D, Jaspersen Sue L
Stowers Institute for Medical Research, Kansas City, MO, USA.
Department of Molecular and Integrative Physiology, University of Kansas Medical Center, Kansas City, KS, USA.
Methods Mol Biol. 2018;1840:137-161. doi: 10.1007/978-1-4939-8691-0_12.
The Saccharomyces cerevisiae and Schizosaccharomyces pombe genomes encode a single SUN domain-containing protein, Mps3 and Sad1, respectively. Both localize to the yeast centrosome (known as the spindle pole body, SPB) and are essential for bipolar spindle formation. In addition, Mps3 and Sad1 play roles in chromosome organization in both mitotic and meiotic cells that are independent of their SPB function. To dissect the function of Mps3 at the nuclear envelope (NE) and SPB, we employed cell imaging methods such as scanning fluorescence cross-correlation spectroscopy (SFCCS) and single particle averaging with structured illumination microscopy (SPA-SIM) to determine the strength, nature, and location of protein-protein interactions in vivo. We describe how these same techniques can also be used in fission yeast to analyze Sad1, providing evidence of their applicability to other NE proteins and systems.
酿酒酵母和粟酒裂殖酵母的基因组分别编码一种含SUN结构域的蛋白，即Mps3和Sad1。二者均定位于酵母中心体（即纺锤体极体，SPB），并且对于双极纺锤体的形成至关重要。此外，Mps3和Sad1在有丝分裂和减数分裂细胞的染色体组织中发挥作用，且这些作用独立于它们的SPB功能。为了剖析Mps3在核膜（NE）和SPB处的功能，我们采用了细胞成像方法，如扫描荧光互相关光谱法（SFCCS）和结构光照显微镜单颗粒平均法（SPA-SIM），以确定体内蛋白质-蛋白质相互作用的强度、性质和位置。我们描述了如何将这些相同的技术也用于裂殖酵母中分析Sad1，从而证明它们适用于其他NE蛋白和系统。
