Chapter 9 - New and Old Reagents for Fluorescent Protein Tagging of Microtubules in Fission Yeast: Experimental and Critical Evaluation
Introduction
The green fluorescent protein (GFP) of the jellyfish Aequorea victoria was purified in the early 1960s (Shimomura et al., 1962). After the gene encoding GFP was cloned (Prasher et al., 1992), GFP was used as a marker for gene expression (Chalfie et al., 1994) and was quickly adopted as a protein tag by cell biologists, including fission yeast researchers (Nabeshima et al., 1995, Sawin and Nurse, 1996). In this chapter, we discuss and evaluate reagents currently available to image GFP-labeled microtubules in fission yeast, with particular reference to time-lapse applications. We also introduce new tagging cassettes for several novel red fluorescent proteins (RFPs) as well as new RFP-tubulin strains that can be added to the set of tools available for in vivo imaging of microtubules and microtubule-associated proteins (MAPs). We will not address basic fission yeast tagging and growth protocols as these subjects are well-covered elsewhere (e.g., Bähler et al., 1998, Moreno et al., 1991, Sato et al., 2009).
GFP is a 26.9 kD protein consisting of an 11-stranded β-barrel surrounding a coaxial α-helix, with the chromophore, a cyclic derivative of the tripeptide sequence serine-dehydrotyrosine-glycine contained within the α-helix (Cody et al., 1993, Örmo et al., 1996). GFP has become the mainstay of in vivo imaging for several reasons, including its bright and photostable fluorescence, low phototoxicity upon prolonged illumination of the fluorophore, and (in most cases) relatively minimal impact on the function of proteins to which it is fused. GFP has been subject to multiple rounds of mutagenesis to improve its spectral characteristics and folding efficiency. The first major improvement in spectral characteristics of GFP was a single point mutation in the chromophore (S65T), which generated GFP with a fluorescence signal six-fold brighter than the original GFP (Heim et al., 1995, Patterson et al., 1997). This mutation also shifted the excitation maximum from 396 to 488 nm, making the fluorophore much more amenable to imaging with standard fluorescein filters. Most of the mutations affecting the spectral properties of GFP are contained within the central α-helix and the contacting β-strands, with mutations affecting folding more widely distributed through the protein (Shaner et al., 2007). Versions of GFP with blue, cyan, and yellow fluorescence are available, and on-going studies continue to further improve the brightness, photostability, and brightness of these and other variants.
Microtubules are highly dynamic, intracellular polymers composed of dimers of α and β tubulin (Mandelkow and Mandelkow, 1985, Nogales et al., 1998, Nogales et al., 1999). The fission yeast Schizosaccharomyces pombe contains a single β-tubulin isoform encoded by nda3+ (Hiraoka et al., 1984) and two isoforms of α-tubulin, Nda2 and Atb2 (Toda et al., 1984). The nda2+ gene is essential, and its level of expression is tightly regulated by the total cellular α-tubulin concentration; the atb2+ gene is nonessential and is constitutively expressed (Adachi et al., 1986). Microtubules are organized into relatively simple arrays in S. pombe, making it an attractive model for studying microtubule dynamics (Sawin and Tran, 2006). Fission yeast cells contain three to five bundles of antiparallel microtubules, which align with the long axis of the cell (Drummond and Cross, 2000, Marks et al., 1986, Tran et al., 2001). Two or three independently regulated microtubules are present within each bundle (Hoog et al., 2007, Sagolla et al., 2003). Microtubules are nucleated from specific sites in the cell called microtubule organizing centers and, once nucleated, rapidly become bundled at their slow-growing, minus ends by the microtubule bundling protein Ase1 (Piel and Tran, 2009). The rapidly growing, plus ends exhibit behavior known as “dynamic instability,” in which individual microtubules stochastically switch between periods of growth and shrinkage (Mitchison and Kirschner, 1984). Regulation of microtubule dynamics is complex and involves many factors, including the local concentration of tubulin dimers, cell cycle position, and the concerted activity of a host of MAPs. Much attention has focused on the group of proteins associated with the microtubules plus ends, the +TIPs (reviewed by Akhmanova and Steinmetz, 2008) including EB1, CLIP170, and the fission yeast protein Tea1.
The dynamic growth pattern of microtubules allows cells to respond to constantly changing cellular requirements by remodeling microtubule arrays. The most significant alteration in microtubule organization occurs at cell division, when the cytoplasmic microtubules depolymerize and an intranuclear mitotic spindle is formed. In fission yeast the spindle is nucleated from the nucleoplasmic face of the spindle pole bodies (SPB) as cells enter prophase. It remains at constant length while the chromosome become bioriented on the metaphase plate, and then rapidly elongates once the cells enter anaphase (Mallavarapu et al., 1999, Nabeshima et al., 1998, Tatebe et al., 2001). While cells are in prometaphase, the SPB also nucleates highly dynamic short nuclear microtubules in addition to kinetochore microtubules (Sagolla et al., 2003, Zimmerman et al., 2004). Once the cell initiates spindle elongation in anaphase, astral microtubules are nucleated from the cytoplasmic side of the SPB. As the cell completes anaphase, microtubule organization changes again and a postanaphase array (PAA) of microtubules is nucleated from a novel equatorial microtubule organizing centre located on the division plane (Hagan, 1998).
Section snippets
Which GFP-Tubulin Should I Use?
This is what everyone reading this article really wants to know. However, before discussing in detail what versions of GFP-tubulin may be most appropriate for physiological imaging, it is important to consider some general criteria for what makes a “good” GFP-tubulin, as these have been satisfied by different GFP-tubulin expression systems with varying degrees of success. These criteria apply equally to tubulin fused to RFPs (discussed further below):
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GFP-tubulin expression should not strongly
Searching for the “GFP” of RFPs
It is becoming increasingly important to be able to image microtubules with other proteins of interest, using different fluorescent tags. While labeling microtubules with GFP has become commonplace, it has been much more difficult to identify a red fluorescent protein that is the equal of GFP in terms of brightness, photostability/phototoxicity, chromophore maturation time, and effects on protein function. The tetrameric dsRed protein from the mushroom coral Discosoma striata was subjected to
Generation and Evaluation of New RFPs in Fission Yeast
Armed with several of the new RFPs we undertook a critical study to evaluate their properties in fission yeast and determine which might be most suitable for general use and for microtubules in particular. We previously generated tagging cassettes for tdTomato and mCherry (Snaith et al., 2005). As part of the present study we created additional tagging cassettes for mKate, mOrange2, and TagRFP-T (Table I), as their properties suggested that they may be the most useful for in vivo time-lapse
The Hunt for Red Tubulin
In light of our experience in tagging a variety of cytoskeletal proteins with the different RFPs, we wanted to investigate which RFP would be best-suited to tagging tubulin. Plasmids expressing nmt1:mRFP1-Atb2 (Yamashita et al., 2005), nmt1:mCherry-Atb2 (Terenna et al., 2008), or nmt81:mCherry-Atb2 (Grallert et al., 2006, Hauf et al., 2007) have all been described, and these have been used primarily to image mitotic or meiotic spindles. However, episomal expression of RFP-tubulin is subject to
Successful Fluorescent Imaging of Fission Yeast Microtubules and Associated Proteins
Several factors contribute to high-quality fluorescence imaging of fission yeast. Some of these are hardware-related (e.g., the quality of the imaging system) and may be expensive to optimize. However, there are many simple, less expensive ways to maximize image quality.
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Emission filters should be matched to fluorophores to ensure that the maximum signal emitted by one’s protein of interest is collected. Widely available fluorescein filters match the spectral properties of GFP(S65T) very well
Acknowledgments
We thank D. Kelly for help with image processing, C. Bicho and E. Lynch for help with strain construction, and F. Chang and Y. Watanabe for strains. K.E.S. is a Wellcome Trust Senior Research Fellow in Basic Biomedical Sciences. This work was supported by a grant from the Wellcome Trust.
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