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Methods Mol Biol. 2018;1840:137-161. doi: 10.1007/978-1-4939-8691-0_12.

Functional Analysis of the Yeast LINC Complex Using Fluctuation Spectroscopy and Super-Resolution Imaging.

Author information

1
Stowers Institute for Medical Research, Kansas City, MO, USA.
2
Stowers Institute for Medical Research, Kansas City, MO, USA. slj@stowers.org.
3
Department of Molecular and Integrative Physiology, University of Kansas Medical Center, Kansas City, KS, USA. slj@stowers.org.

Abstract

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.

KEYWORDS:

Fluctuation spectroscopy; Fluorescence cross-correlation spectroscopy; Fluorescence resonance energy transfer; Line scanning; Mps2/Kms1/Kms2; Mps3/Sad1; Single particle averaging; Super-resolution microscopy

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