New and old reagents for fluorescent protein tagging of microtubules in fission yeast; experimental and critical evaluation.
The green fluorescent protein (GFP) has become a mainstay of in vivo imaging in many experimental systems. In this chapter, we first discuss and evaluate reagents currently available to image GFP-labeled microtubules in the fission yeast Schizosaccharomyces pombe, with particular reference to time-lapse applications. We then describe recent progress in the development of robust monomeric and tandem dimer red fluorescent proteins (RFPs), including mCherry, TagRFP-T, mOrange2, mKate, and tdTomato, and we present data assessing their suitability as tags in S. pombe. As part of this analysis, we introduce new PCR tagging cassettes for several RFPs, new pDUAL-based plasmids for RFP-tagging, and new RFP-tubulin strains. These reagents should improve and extend the study of microtubules and microtubule-associated proteins in S. pombe.
Snaith
Hilary A
HA
Wellcome Trust Centre for Cell Biology, School of Biological Sciences, University of Edinburgh, Edinburgh EH93JR, United Kingdom.
Anders
Andreas
A
Samejima
Itaru
I
Sawin
Kenneth E
KE
D019411:Clinical Laboratory Techniques; D005456:Fluorescent Dyes / Q000494:pharmacology*; D007202:Indicators and Reagents / Q000494:pharmacology; D008869:Microtubule-Associated Proteins / Q000032:analysis / Q000378:metabolism; D008870:Microtubules / Q000737:chemistry / Q000378:metabolism*; D008954:Models, Biological; D012568:Schizosaccharomyces / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D029702:Schizosaccharomyces pombe Proteins / Q000032:analysis / Q000378:metabolism; D013194:Staining and Labeling / Q000379:methods*
D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D008869:Microtubule-Associated Proteins; D029702:Schizosaccharomyces pombe Proteins