MICROSCOPY RESEARCH AND TECHNIQUE 51:511–529 (2000)
The Beauty of the Yeast: Live Cell Microscopy at the Limits
of Optical Resolution
SEPP D. KOHLWEIN*
SFB Biomembrane Research Center, Department of Biochemistry, Technical University Graz, A-8010 Graz, Austria
KEY WORDS
green fluorescent protein; immunofluorescence; protein tagging; confocal and
video microscopy; Saccharomyces cerevisiae
ABSTRACT
The yeast Saccharomyces cerevisiae is a very powerful system for cell biological
research. Recent advances in electronic light microscopy together with the application of green
fluorescent protein and other in vivo staining techniques have allowed novel and exciting insights
into structural organization and dynamics of cells as small as yeast. Methods for staining yeast for
microscopic inspection and for introducing tags for localization studies of proteins in living or fixed
cells are summarized. Electronic light microscopy, video/deconvolution methods, and confocal laser
scanning microscopy as novel tools for structural analyses, and their practical applications in yeast,
are discussed. Microsc. Res. Tech. 51:511–529, 2000. © 2000 Wiley-Liss, Inc.
INTRODUCTION
The yeast Saccharomyces cerevisiae is an important
model system for cell biological research. It was the
first eukaryotic organism whose genome was completely sequenced (Goffeau et al., 1996). Since then,
yeast has become a paradigm of genomics research: the
development of DNA chip technology for the analysis of
the transcriptome (Brown and Botstein, 1999), genome-wide analyses of gene function [e.g., EUROFAN
European Function Analysis Network (Oliver, 1996)]
and protein localization (Ding et al., 2000; Ross-Macdonald et al., 1997, 1999), as well as biochemical
genomics (Carlson, 2000; Martzen et al., 1999). A comprehensive protein-protein interaction map by 2-hybrid
analysis was recently completed (Oliver, 2000; Uetz et
al., 2000). Multiple human disease genes have homologues in yeast (Ploger et al., 2000), and the high conservation of metabolic and regulatory mechanisms has
contributed to the wide use of yeast as a model system
for cell biological studies.
The analysis of subcellular structures in living or
fixed yeast cells using light microscopic techniques,
however, is a particular experimental challenge due to
the small size of the cells, between 5 and 10 ␮m. Nevertheless, microscopy methods—mainly based on fluorescence—are well established for yeast (Hašek and
Streiblová, 1996; Pringle et al., 1989, 1991; Streiblová
and Hašek, 1996). Since the size of yeast cells is only
about one order of magnitude above the wavelength of
visible light, resolution is rather limited and microscopic results are often not very satisfying. As a rule of
thumb, optical resolution is defined by 1/2 ␭, which
limits the ability to discriminate two fluorescent spots
to a distance of about 0.25 ␮m, using blue light for
excitation in conventional wide-field (“epi-”) fluorescence microscopy. Out-of-focus fluorescence, i.e., the
fluorescence emission of areas of the cell that are not in
the focal plane, blurs the image and results in a major
loss of resolution. The thick yeast cell wall is highly
diffractive and causes interference patterns, most obvious in transmission light microscopy. The movement
of unfixed cells and the internal dynamics of structures
©
2000 WILEY-LISS, INC.
in live cell observations, are further limitations. As
with all microscopic techniques, maximum resolution
in a particular application is determined by the size of
the object (magnification), wavelength and signal intensity (sensitivity of detection), and the duration of
microscopic observation (image acquisition time).
These “technical” limitations have to be considered in
the context of the main constraints imposed by the
living specimen, in particular, the potential interference of the staining method, illumination, and observation conditions with normal cellular physiology
(Fig. 1).
Many of these limitations can be partially overcome
by using specialized equipment and careful sample
preparation. Cell immobilization and embedding and
temperature-control techniques allow almost interference-free observation of yeast cells over extended periods of time. A major advantage of a small cell is the
excellent “statistics” of localization patterns: the homogeneity of a staining pattern and the significance of
structural features can be assessed by visual inspection
of a large population of cells, which is easy to achieve
with yeast. Advances in electronic light microscopy
such as video microscopy and confocal laser scanning
microscopy (Gustafsson et al., 1999; Pawley, 1995;
Shotton, 1993) have resulted in a major improvement
of yeast microscopic recordings by eliminating out-offocus blur and by increasing resolution and image contrast. As a consequence of these technological developments, yeast has also become a highly attractive system for structural and morphological studies and the
analysis of organelle dynamics and inheritance. A large
number of organelle-specific fluorescent dyes, indicators of physiological parameters (e.g., pH, Ca2⫹, mem-
Contract grant sponsor: Austrian Science Fund, FWF; Contract grant number:
F706; Contract grant sponsor: Oesterreichische Nationalbank; Contract grant
number: 7273; Contract grant sponsor: EU; Contract grant number: BIO4-CT972294; Contract grant sponsor: Austrian Ministry for Science and Transportation.
*Correspondence to: S.D. Kohlwein, SFB Biomembrane Research Center, Department of Biochemistry, Technical University Graz, Petersgasse 12, A-8010
Graz, Austria E-mail: kohlwein@ftugax.tu-graz.ac.at
Received 28 April 2000; accepted in revised form 20 July 2000
512
S.D. KOHLWEIN
Fig. 1. Limitations of electronic light microscopy. Signal intensity
and recording speed are main determinants of “resolution,” in the
context of live cell observations.
brane potential) and the application of green fluorescent protein (GFP) and its various spectral derivatives
as fluorescent tags for the analysis of protein localization in living cells provide novel insights and expand
the experimental potential of the yeast system into
multiple new dimensions.
METHODS AND TOOLS FOR YEAST
MICROSCOPY
Analyzing Protein Localization in Fixed and
Living Cells: Tagging of Proteins for Analyzing
Their Localization in Fixed Cells
Protein localization is a major application in yeast
cell microscopy. Traditionally, proteins are detected in
fixed and permeabilized cells by interaction with specific probes, i.e., fluorescently labeled antibodies or specific ligands. Actin can be detected with labeled phalloidin or phallacidin (heptapeptide mushroom toxins,
MW 800-850) after cell fixation (Hašek and Streiblová,
1996; Pringle et al., 1989; Streiblová and Hašek, 1996).
Biotin-containing proteins (e.g., acetyl-CoA carboxylase) are detectable using fluorescently labeled avidin
(an egg white glycoprotein) or streptavidin (bacterial
homolog), which strongly interact with biotin (Ivessa et
al., 1997).
Since the production of antibodies specific for individual proteins is a time-consuming process, and its
application is often limited by insufficient specificity,
alternative techniques have emerged, based on the introduction of heterologous tags, e.g., fragments of influenza virus hemagglutinin (HA-tag) or the myc protooncogene (myc-tag), into the coding sequences of the
proteins of interest. These tags are recognized by
highly specific and commercially available antibodies
that lack any noticeable background staining in yeast.
Their rather small size (10 –12 amino acids) makes
them unlikely to interfere with protein function, although this depends, of course, on the characteristics of
the tagged proteins and the specific sites of insertion of
the tags. In order to increase detection sensitivity, multiple tags can be introduced, which results in substan-
tial amplification of the immuno signal but also significantly increases the size and consequently the possible
structural alteration of the tagged protein. In any case,
testing of the tagged protein to ensure that it retains
normal function, e.g., by demonstrating rescue of the
relevant mutant phenotype, is an essential control that
is usually easily performed in yeast (see General Considerations for Creating and Analyzing Tagged Proteins). Alternative and independent methods for subcellular localization studies, e.g., cell fractionation,
have to be considered, to verify data obtained from
microscopic studies.
As with all immunodetection methods, this approach
for protein localization requires extensive preparation
of cells, which involves fixation, permeabilization, and
incubation with fluorescently labeled antibodies
(Hašek and Streiblová, 1996; Pringle et al., 1989,
1991). Since all these steps may introduce artifacts,
rigorous controls are necessary in order to correctly
interpret localization results obtained from immunofluorescence staining.
Alternative tagging methods for immunodetection of
proteins are designed to primarily detect proteins in
cellular extracts or for protein purification, e.g., the
IgG binding domain of S. aureus protein A [130 amino
acids; purification of the fusion by IgG Sepharose affinity chromatography (Puig et al., 1998)], HIS6 tag
(Ni⫹⫹ chelate chromatography), ␤-galactosidase (activity, immunodetection), glutathione-S-transferase (glutathione Sepharose affinity chromatography). However, many of the experimental strategies involved in
tagging with different tags are very similar, and even
allow multiple tags to be introduced for both live cell
analysis and immunodetection of proteins (see also
Methods for Introducing Tags; see Table 3).
Tagging With Green Fluorescent Protein:
Protein Localization in Living Cells
The introduction of Green Fluorescent Protein from
the jelly fish Aequorea victoria (Chalfie et al., 1994;
Prasher et al., 1992) has revolutionized the possibilities for protein localization studies and live cell microscopy (Misteli and Spector, 1997; Sullivan and Kay,
1999; Tsien, 1998) in a large number of species, including Saccharomyces cerevisiae (Ferrigno and Silver,
1999; Niedenthal et al., 1996; Shaw et al., 1997a),
Schizosaccharomyces pombe (Atkins and Izant, 1995;
Nabeshima et al., 1997; Sawin, 1999), Candida albicans (Morschhauser et al., 1998), and other fungi (Cormack, 1998; Du et al., 1999; Monosov et al., 1996;
Spellig et al., 1996; Suelmann et al., 1997). A large
number of spectral variants (Cormack et al., 1996; Cubitt et al., 1995; Haseloff, 1999; Yang et al., 1998), some
optimized for yeast codon usage (Cormack et al., 1997;
Kahana and Silver, 1996; Natarajan et al., 1998), are
available, and the number of broad application range
cloning vectors is increasing (see below) (Table 3).
GFP has a molecular weight of about 29 kDa and,
thus, represents a major structural alteration to the
tagged protein. Free GFP is a soluble enzyme that does
not display any interactions with membranes or other
cellular structures and resides in the cytosol in yeast.
Chromophore formation is independent of host factors
but requires oxygen, which limits its application to
aerobically growing yeast cells. The half life for chromophore formation is ⬃ 2 hours for the wild type GFP
YEAST LIGHT MICROSCOPY
TABLE 1. Common spectral variants of GFP
Common
name
GFP
EBFP1
BFP
ECFP1
EGFP1
S65T1
yEGFP1,2
EYFP1
GFP variant
␭ ex (nm)
␭ em (nm)
Wild type
F64L S65T Y66H Y145F
Y66H
K26R F64L S65T Y66W
N146I M153T V163A
N164H N212K
F64L S65T
S65T
S65G S72A1
S65G V68L S72A T203Y
396, 475
380
384
434
508
440
448
474
488
489
490
514
507
511
510
527
1
Suitable for applications at 37°C.
2
Yeast (Candida albicans) codon optimized (Cormack et al., 1997), in addition to
the two mutations affecting spectral characteristics.
and ⬃30 minutes for the improved spectral variants
(Tsien, 1998). Some evidence suggests that chromophore formation is delayed in the lumen of the endoplasmic reticulum, but no systematic studies are
available at present. Delay in chromophore formation
should be kept in mind when following pulse-chase
experiments for protein expression and translocation,
and may result in a lack of detectable signals of fusion
proteins that are subject to rapid turnover. The recently introduced RFP (Matz et al., 1999) has a half life
of chromophore formation of ⬃6 hours and may, thus,
require extensive modification prior to its successful
application in rapidly dividing yeast cells.
The ease of constructing cells expressing GFP-tagged
proteins has contributed to a wide range of fascinating
applications for analyzing protein localization and organelle structure and dynamics in living yeast (Table
2).
Use of GFP in Fixed-Cell Applications. In some
applications, it is desirable to observe GFP-tagged proteins in addition to other cellular components that require cell fixation. GFP fluorescence to some extent
resists fixation with 4% formaldehyde, paraformaldehyde, glutaraldehyde (0.25%; Rossanese et al., 1999),
and/or methanol (Brock et al., 1999). Some GFP-fusions, however, appear to be sensitive to methanol/
acetone fixation, whereas in other cases, formaldehyde/
paraformaldehyde treatment was found to be detrimental. Thus, the optimum fixation conditions may
depend on the particular GFP fusion protein and have
to be determined empirically for each case (Ferrigno
and Silver, 1999). As an alternative, GFP can be detected in fixed cells by immunoflourescence microscopy,
using highly specific anti-GFP antibodies.
Limitations of GFP. Besides having a major impact
on the structure of the tagged protein, and thus potentially affecting function, stability, and subcellular localization as discussed below, GFP attachment may
also lead to other problems, such as effects on transcriptional and translational efficiency (Sacchetti et al.,
2000) or even cytotoxicity (Liu et al., 1999). No reports
are as yet available to demonstrate such effects in
yeast.
GFP stability. Various GFP variants have been
tested for their stability in yeast cells. Whereas wildtype GFP has an estimated half life of ⬃7 hours in
yeast, some optimized mutant variants (F100S/M154T/
V164A) were shown to have an estimated half life of up
to 70 hours in S. cerevisiae (Natarajan et al., 1998).
513
However, upon fusion to other proteins, GFP stability
is highly variable and dependent mainly on the nature
of the tagged protein. Accumulation of GFP fluorescence in the yeast vacuole under some conditions (own
observations) may reflect a relatively high stability of
the compact GFP structure against protease attack in
this organelle.
Temperature effects on GFP. Chromophore formation
of the wild-type GFP is strongly temperature dependent, favoring expression at lower temperatures of 20 –
25°C. However, the temperature sensitivity of wild
type GFP was successfully used to observe post-synthetic localization of a nuclear protein in yeast, after a
shift to elevated temperature (Lim et al., 1995). Improved GFP variants (Kimata et al., 1997; Siemering et
al., 1996) impose no limitations for applications in the
physiological temperature range of yeast.
GFP Applications in Yeast Beyond Subcellular Localization Studies. The application of GFP in yeast is not
restricted to localization studies, as it can also serve as
a versatile tool for transcription analysis, employing
fluorescence-assisted cell sorting (FACS) (Atkins and
Izant, 1995; Bell et al., 1999; Niedenthal et al., 1996).
FACS analysis of a GFP-tagged marker has also been
used to monitor and quantitate chromosome and plasmid loss in yeast (Hegemann et al., 1999); quantitative
approaches of GFP detection also involve imaging of
cells (Patterson et al., 1998). In addition, GFP fusion
strategies have been employed successfully in mutant
screens to identify nuclear markers in fission yeast
(Sawin and Nurse, 1996) and as a marker in twohybrid screens to identify protein-protein interactions
in vivo (Mayer et al., 1999).
Alternative Protein Tagging Methods for Live
Cell Inspection
Although GFP and its spectral variants are the predominant protein tags currently used in live cell applications, alternative tagging techniques are emerging.
The introduction of a novel red-fluorescent protein
from the sea anemone Discosoma striata (Matz et al.,
1999) will undoubtedly lead to novel applications for
multiple protein detection, with significantly improved
spectral separation over conventional cyan, green, and
yellow fluorescent protein variants. Wildt and Deuschle (1999) recently introduced a novel protein tag,
based on the uroporphyrinogen III methyltransferase
(cobA) from Propionibacterium freudenreichii, that
may also prove useful in yeast transcription studies
and whole cell fluorescence applications. As with GFP,
cobA-mediated bright red fluorescence forms without
additional cofactors from the host cell, making it a
promising tool for expression analyses.
In another approach, Griffin at al. (1998) described
the construction of a recombinant tag that specifically
interacts with a membrane-permeant arsenic compound, 4⬘,5⬘-bis(1,3, 2-dithioarsolan-2-yl) fluorescein.
Recombinant proteins containing four cysteine residues at the x, x ⫹ 1, x ⫹ 4, and x ⫹ 5 positions of an
alpha helix can be fluorescently labeled in vivo with the
dye, which only fluoresces upon interaction with the
tetracysteine protein domain. The protein tag represents a much smaller alteration to the tagged protein
than does GFP. The feasibility of this approach in yeast
has not yet been demonstrated.
514
S.D. KOHLWEIN
TABLE 2. Applications of GFP fusions in yeasts: a selection1
Organelle
Cytosol
Mitochondria
Process
Localization
Targeting
Retention in mother cell
Nucleus
Nucleolus
Nucleolus, nucleus,
bud site
Endoplasmic reticulum
Fusion and fission
Sorting in zygotes
Localization, iron transporter
Nuclear/cytoplasmic
shuttling, stress
Transcription factors
Protein kinase A subunits
mRNA export
Nuclear protein
import/export
Ribosome export
Nuclear pore complex
dynamics
Nuclear movement during
meiosis
Mating
Dynamic changes in protein
localization
Membrane localization;
karmellae
Membrane localization; sterol
metabolism
Golgi
ER/exit to Golgi
Nuclear-vacuolar junction
Dynamics
COPII vesicles
Secretion; ER/Golgi transport
Proteasome
Ubiquitin hydrolase activity;
localization during meiosis
and mitosis
Autofluorescence ade1, ade2
mutants
Autophagy
Membrane; vacuole-vacuole,
vacuole-ER interaction
Vacuole
Late endosome
Endocytic
compartment
Plasma membrane
Cytosol-vacuole transport
Secretion, sorting, cargo
selection
Vacuole biogenesis
Signaling and membrane
traffic
Localization, recycling
Peroxisomes
PM quality control
Endocytosis
Endosome recycling
Biogenesis
Multi-vesicular bodies
Cytoskeleton
Metabolism
During life cycle and
sporulation
Pexophagy
Actin; dynamics
Actin filament assembly
Actin; role in polarized
growth
Actin-interacting proteins
S.p. cytology
mRNA localization
Protein fused to GFP
None (GFP alone)
COXIV-presequence-GFP cytochrome
oxidase subunit IV
CS1-presequence-GFP citrate
synthase
Dnm1p-GFP, Fts1p-GFP
Abf2p
Mft1p-GFP, Mft2p-GFP
Msn2p-GFP Msn4p-GFP
Organism
Reference
S.c.
S.c.
Figure 4
Figure 4
S.c.
Yang et al. (1999)
S.c.
S.c.
S.c.
S.c.
Sesaki and Jensen (1999)
Okamoto et al. (1998)
Li and Kaplan (1997)
Gorner et al. (1998)
S.c.
S.c.
Reiser et al. (1999)
Griffioen et al. (2000)
Lee et al. (1996)
Stade et al. (1997)
S.c.
S.c.
Hurt et al. (1999)
Bucci and Wente (1997)
GFP-Pol1p
S.p.
Hiraoka et al. (2000)
Ssf1-GFP
GFP-Glc7p protein phosphatase I
S.c.
S.c.
Hmg1p-GFP
S.c., S.p.
Kim and Hirsch (1998)
Bloecher and Tatchell
(2000)
Hampton et al. (1996);
Koning et al. (1996)
Hmg2p-GFP
Erg4p-GFP
S.c.
Zweytick et al. (2000a)
Are1p-GFP
Are2p-GFP
Sec12-GFP
Nvj1p-GFP
GFP-Sft2p
S.c.
Zweytick et al. (2000b)
S.c.
S.c.
S.c.
Sec13p-GFP, Sec23p-GFP, Sec24pGFP, Sec31p-GFP
GFP-Uch2p
Pad1p-GFP
S.c.
Rossanese et al. (1999)
Pan et al. (2000)
Wooding and Pelham
(1998)
Rossanese et al. (1999)
S.p.
Li et al. (2000); Wilkinson
et al. (1998)
—
S.c.
Figure 4
Apg7-GFP
Vac8p-GFP
S.c.
S.c.
preproLap1p-GFP (aminopeptidase I)
Ste2p-GFP alpha factor receptor,
GFP-Cps1p
Nhx1-GFP
GFP-FYVE RING finger domain
S.c.
S.c.
Kim et al. (1999)
Fleckenstein et al. (1998);
Pan and Goldfarb
(1998); Schneiter et al.
(2000)
Martinez et al. (1999)
Odorizzi et al. (1998)
S.c.
S.c.
Nass and Rao (1998)
Burd and Emr (1998)
Pho84p-GFP phosphate permease
Hxt2-GFP hexose transporter
Ste2p-GFP alpha factor receptor
Ste2p-GFP alpha factor receptor
GFP-Snc1p
GFP-SKL (pex targeting signal)
S.c.
Hog1p-GFP
Tpk1p-GFP Bcy1p-GFP
Npl3-GFP
Nuclear localization signal-GFP,
nuclear export signal-GFP Crm1pGFP
L25-GFP
GFP-Nup49p
GFP-PTS1
GFP-PTS2
GFP-AKL
GFP-SKL
P.p.
Petersson et al. (1999)
Kruckeberg et al. (1999)
Li et al. (1999)
Stefan and Blumer (1999)
Lewis et al. (2000)
Faber et al. (1998);
Monosov et al. (1996)
Kalish et al., (1996)
C.b.
S.c.
Nakagawa et al. (2000)
Gurvitz et al. (1998)
GFP-SKL
Abp1p-GFP, Sac6p-GFP (fimbrin)
P.p.
S.c.
Twf1p-GFP (twinfilin)
GFP-Cdc24p
S.c.
S.c.
Sakai et al. (1998)
Doyle and Botstein
(1996); Waddle et al.
(1996)
Goode et al. (1998)
Toenjes et al. (1999)
Rvs167p-GFP
Various
MS2-coat-protein-GFP (RNA-binding
protein)
S.c.
S.p.
S.c.
S.c.
S.c.
S.c.
P.p.
Balguerie et al. (1999)
Sawin (1999)
Beach et al. (1999);
Bertrand et al. (1998);
Gonzalez et al. (1999)
515
YEAST LIGHT MICROSCOPY
TABLE 2. (continued)
Organelle
Organism
Reference
Centromer behavior,
chromosome segregation
Process
GFP-Lacl-NLS; recipient strain
contains array of 256 LacO sites
S.c., S.p.
Microtubule dynamics
GFP-Tub1p
S.c., S.p.
Straight et al. (1996);
Maddox et al. (1999);
Straight et al. (1997)
Maddox et al. (1999);
Segal et al. (2000);
Shaw et al. (1997b)
Dyn1p-GFP
GFP-Kar9p
GFP-Tub1p
GFP-Duo1p, GFP-Dam1p
Cnm67p-GFP
S.c.
S.c.
S.c.
S.c.
Miller et al. (1999)
Maddox et al. (2000)
Hofmann et al. (1998)
Brachat et al. (1998)
Sad1p-GFP
S.p.
Nabeshima et al. (1998)
GFP-Cmd1p
S.c., S.p.
Moser et al. (1997)
Cyk1p-GFP
Myo1-GFP
Myo2p-GFP
CFP-Cd12p
CFP-Rng3
Cwp1-GFP
Cwp2-GFP
S.c.
Shannon and Li (1999)
S.p.
Bezanilla et al. (2000);
Chang, F. (2000); Wong
et al. (2000)
Ram et al. (1998)
Microtubule orientation
Spindle assembly
Mitotic spindle function
Spindle pole body; nuclear
migration and fusion
Spindle pole body during
mitosis
Spindle pole body;
calmodulin interaction
Actomyosin contractile ring
Myosin assembly
Cell wall
Localization
Protein fused to GFP
S.c.
1
S.c., Saccharomyces cerevisiae; S.p., Schizosaccharomyces pombe; P.p., Pichia pastoris, C.b., Candida boidinii.
Methods for Introducing Protein Tags
GFP and other tags usually are cloned on episomal
vectors or introduced by recombination into the genome, using PCR-based approaches. A major consideration with all tagging systems relates to the possible
interference of the tag with the localization and function of the protein under study (see below). In many
proteins, subcellular localization signals (e.g., mitochondrial, ER, or peroxisomal targeting sequences; ER
retention signals) or sites for post-translational modification (e.g., for N-terminal myristoylation, C-terminal GPI-anchor, or isoprenoid attachment) limit the
possibilities for the otherwise convenient N- or C-terminal tag attachment. A detailed computational analysis of the protein under study (e.g., using PROSITE;
Hofmann et al., 1999) should thus precede all attempts
to create the fusion genes. Internal tagging requires a
more detailed knowledge about the protein, i.e., functional and “non-functional” regions, and still depends
on trial and error to identify possible sites for tag
insertion that do not interfere with protein function
and/or localization. Insertion of “linker-sequences” to
allow for a more flexible connection between the protein under study and GFP may prove useful (Prescott
et al., 1999). Random approaches using transposon insertion of immuno tags or GFP in vitro (Merkulov and
Boeke, 1998) or on a genome-wide scale in vivo are also
having significant success in creating tagged proteins
for microscopic inspection (Ding et al., 2000; Kumar et
al., 2000; Ross-Macdonald et al., 1997; Sawin and
Nurse, 1996).
Numerous plasmids for constructing N- or C-terminal fusions to GFP, single or multiple HA, myc, or other
immuno tags, or combinations of immuno and GFP
tags have been described in the literature and are
summarized in Table 3 (see also web sites listed in
Table 5). When on plasmids, fusion gene expression is
driven either from the endogenous promoter or from a
heterologous promoter, e.g., ADH1 (constitutive),
GAL1/10 [galactose inducible; absence of glucose
(⬍0.01 %)], MET25 (induction on methionine-free medium), or CUP1 (induction with copper); in the latter
cases, changes in growth conditions are required for
induction.
As an alternative to episomal fusion plasmids, PCRbased approaches to integrate GFP or immuno tags at
the N- or C-terminus of the protein under study have
attracted increased popularity due to the simplicity of
the method and the lack of cloning steps (Bähler et al.,
1998; Knop et al., 1999; Krawchuk and Wahls, 1999;
Lafontaine and Tollervey, 1996; Longtine et al., 1998;
Puig et al., 1998; Schneider et al., 1995). For C-terminal tagging, GFP cDNA or the tag sequence together
with a selectable marker are amplified by PCR, using
long hybrid primers that are more than 50 nt homologous to the integration site, just before the stop codon
of the gene, and several nucleotides downstream, respectively. The linear PCR product is transformed into
haploid or diploid yeast cells and transformants are
selected based on the marker. Correct integration
should result in a C-terminal in-frame fusion, driven
by the endogenous promoter (Fig. 2; see also websites
listed in Table 5).
When C-terminal tagging is not possible, heterologous promoters cloned upstream of the GFP template
are typically used in constructs for N-terminal chromosomal tagging, following a similar strategy as outlined
for C-terminal fusions. As an alternative to maintain
expression driven by the endogenous promoter, we
have recently developed a novel PCR-based method
that allows the construction of N-terminal GFP fusions
by integration of a loxP-kanMX-loxP-GFP cassette into
the chromosome, upstream of the gene of interest
(Prein et al., unpublished data). After transformation
and selection of geneticin-resistant cells, the selection
marker is excised between the loxP sites by induction
of Cre recombinase. Although this procedure separates
the promoter of the tagged gene from the start ATG of
516
S.D. KOHLWEIN
TABLE 3. Multipurpose yeast vectors/cloning strategies for protein tagging1
Tag
Application
Cloning system
Fusion
Promoter
Marker
Organism
Reference
HA
HA(3)
Immuno detection
Immuno detection
PCR/chrom.
Plasmid&PCR/chr.
C-/NC-/N-
nmt1
nmt1
kanR or ura4⫹
ura4⫹
S.p.
S.p.
HA(3)
Immuno detection
PCR/chrom.
C-/N-
GAL1
S.c.
HA(3)
HA(3)
Immuno detection
Immuno detection
mTn transposon
mTn transposon
Internal
Internal
end.
end.
kanR, TRP1 or
sp his5⫹ 2
yl URA3
URA3
(Krawchuk and Wahls, 1999)
(Bähler et al., 1998; Craven
et al., 1998; Forsburg and
Sherman, 1997)
(Longtine et al., 1998)
Y.l.
S.c.
HA(3)
HA(3)
HA(6)
␤-Gal/HA(3,4)
Immuno detection
Immuno detection
Immuno detection
Activity/localization
PCR/chrom.
PCR/chrom.
PCR/chrom.
mTn transposon
Internal
Internal
Internal
Internal
end.
end.
end.
end.
URA3
kanR or HIS3
kl TRP12
URA3
S.c.
S.c.
S.c.
S.c.
myc
myc
myc(3)
myc(3)
myc(9)
myc(13)
Immuno detection
Immuno detection
Immuno detection
Immuno detection
Immuno detection
Immuno detection
Plasmid
PCR/chrom.
PCR/chrom.
PCR/chrom.
PCR/chrom.
PCR/chrom.
C-/NC-/NInternal
Internal
Internal
C-/N-
nmt1
nmt1
end.
end.
end.
GAL1
S.p.
S.p.
S.c.
S.c.
S.c.
S.c.
myc(13)
myc/His6
ProtA
ProtA
Immuno detection
Immuno det./purif.
Immuno detection
Immuno detection
PCR/chrom.
Plasmid
PCR/chrom.
PCR/chrom.
C-/NC-/NInternal
C-
nmt1
nmt1
end.
end.
S.p.
S.p.
S.c.
S.c.
(Bähler et al., 1998)
(Craven et al., 1998)
(Knop et al., 1999)
(Puig et al., 1998)
ProtA
Immuno detection
Plasmid
C-
end.
S.c.
(Puig et al., 1998)
ProtA/TEV/HIS6
GST
Immuno detection
Affinity purif.
PCR/chrom.
Plasmid
Internal
C-/N-
end.
nmt1
ura4⫹
kanR or ura4⫹
URA3
kanR or HIS3
kl TRP12
kanR, TRP1 or
sp his5⫹ 2
kanR or ura4⫹
ura4⫹
kanR
kl URA3, TRP1,
or HIS32
kl URA3, TRP1,
or HIS32
kanR or HIS3
ura4⫹
(Neuveglise et al., 1998)
(Kumar et al., 2000; RossMacdonald et al., 1997,
1999)
(Schneider et al., 1995)
(Knop et al., 1999)
(Knop et al., 1999)
(Kumar et al., 2000; RossMacdonald et al., 1997,
1999)
(Craven et al., 1998)
(Krawchuk and Wahls, 1999)
(Schneider et al., 1995)
(Knop et al., 1999)
(Knop et al., 1999)
(Longtine et al., 1998)
S.c.
S.p.
GST
GST
Affinity purif.
Affinity purif.
PCR/chrom.
PCR/chrom.
C-/NC-/N-
nmt1
GAL1
S.p.
S.c.
GFP
GFP
Vital
Vital
Plasmid
Plasmid
C-/NC-/N-
MET25
MET25
kanR or ura4⫹
kanR, TRP1, or
sp his5⫹ 2
URA3
URA3 or HIS3
(Knop et al., 1999)
(Forsburg and Sherman,
1997)
(Krawchuk and Wahls, 1999)
(Longtine et al., 1998)
GFP
GST/TEV/HIS6
(E)GFP
(E)GFP
Vital
Immuno detection
Vital
Vital
mTn transposon
PCR/chrom.
Plasmid
PCR/chrom.
Internal
Internal
C-/NC-/N-
end.
end.
nmt1
nmt1
yl URA3
kanR
ura4⫹
kanR or ura4⫹
Y.l.
S.c.
S.p.
S.p.
EGFP
Vital
PCR/chrom.
C-/N-
GAL1
S.c.
EGFP
EGFP
Vital
Vital
Plasmid
PCR/chrom.
C-/N
C-
ADH1
end.
kanR, TRP1, or
sp his5⫹ 2
HIS3
HIS3 or kanR
S.c.
S.c.
EGFP
Vital
PCR/chrom.
N-
GAL1
HIS3
S.c.
EGFP
EGFP/HA(3)
BFP
Vital
Vital/immuno
Vital
PCR/chrom.
mTn transposon
PCR/chrom.
Internal
Internal
C-
end.
end.
end.
kanR
URA3
kanR
S.c.
S.c.
S.c.
YFP
Vital
PCR/chrom.
C-
end.
sp HIS32
S.c.
Various
Vital/immuno
Plasmid
C-/N-
S.c.
S.c.
S.p.
(Niedenthal et al., 1996)
http://www.mips.biochem.
mpg.de/proj/yeast/info/tools/
hegemann/gfp. html
(Neuveglise et al., 1998)
(Knop et al., 1999)
(Craven et al., 1998)
(Bähler et al., 1998;
Krawchuk and Wahls,
1999)
(Longtine et al., 1998)
(Wach et al., 1997)
(Brachat et al., 2000; Wach
et al., 1997)
(Brachat et al., 2000; Wach
et al., 1997)
(Knop et al., 1999)
(Ross-Macdonald et al., 1997)
http://depts.washington.edu/
⬃yeastrc/fm_home1.htm
http://depts.washington.edu/
⬃yeastrc/fm_home2.htm
http://pingu.salk. edu/
⬃forsburg/vectors.html
1
HA: influenza virus hemagglutinin epitope; myc: myc oncogene epitope (-EQKLISEEDL-); GST: glutathione-S-transferase; GFP: see Table 1; ␤-Gal: ␤-galactosidase;
ProtA: IgG-binding domain of Staphylococcus aureus protein A; TEV: TEV protease cleavage site; end.: endogenous promoter; kanR: G418 resistance marker
(kanMX); K.l.: Kluyveromyces lactis; S.c. Saccharomyces cerevisiae; S.p.: Schizosaccharomyces pombe; Y.l.: Yarrowia lipolytica.
2
sp, kl: heterologous selection markers.
the GFP coding sequence by 53 nt, we found normal
expression of tagged genes that are essential, or in
response to nutritional stimuli (Prein et al., unpublished data).
General Considerations for Creating and
Microscopically Inspecting Tagged Proteins
(see also: Methods and Limitations for
Microscopy of Live Yeast Cells; figure 3)
Possible Mislocalization of Tagged Proteins.
Functionality of a tagged protein in yeast can be assessed using null mutants that are complemented by
the plasmid-borne fusion protein, or if the endogenous
gene is replaced by the respective fusion construct at
the chromosomal level. Thus, if the tagged protein restores a mutant phenotype to wild-type, it is generally
considered “functional” and likely to be localized to the
correct cellular site. Controls, such as growth tests of cells
expressing the fusion protein on different media and under various temperature conditions, or drug sensitivities
compared to wild type strains provide a good estimate as
to the full or partial complementation, and thus functionality, of a fusion construct. Ultimately, functionality
has to be proven at the biochemical level, e.g., by de-
YEAST LIGHT MICROSCOPY
Fig. 2. PCR strategy for creating chromosomal C-terminal GFP
fusions (Wach et al., 1997).
termining enzyme properties, binding affinities, and the
like. The situation becomes even more problematic, if the
function of the protein is unknown and if there is no
phenotype associated (or known) with loss of function of
the tagged protein.
517
However, even if the fusion protein turns out to be
active based on biochemical, morphological, and/or
other criteria, the localization pattern of the tagged
protein may still be misleading: only a few percent of
correctly localized protein may fulfill the cellular function while the majority of the tagged protein, which
ultimately will be detected in the microscope, may be
mislocalized, resulting in an erroneous interpretation
of protein localization. This is a particular problem
when fusion proteins are expressed from episomal plasmids and/or under control of strong heterologous promoters. Another consideration is the fact that many
proteins change their subcellular localization as they
are synthesized, targeted, matured, and eventually
subjected to degradation. For example, a protein synthesized in the endoplasmic reticulum and traveling
through the secretory pathway to the cell periphery
alters its localization throughout its life. Depending on
the steady-state kinetics of the delivery between the
different organelles, the tagged protein will be observed in one or several different locations along the
secretory pathway, and may also be directed to the
vacuole. Thus, for exemple, even if the protein is detected mainly in the Golgi (or the vacuole), it may
actually be functional only in the plasma membrane
(see Fig. 4). Although there are only a few “negative”
reports indicating that tagging of proteins failed to
unveil their localizations (Brachat et al., 2000), it
seems clear that in a number of cases this approach is
not feasible due to loss of function, alteration of protein
localization by the tag (Kunze et al., 1999), and/or the
steady state-distribution within the cell. Unfortunately, due to their “negative” nature, these results
rarely make it to the published literature, so that only
with time will one be able to obtain a more realistic
picture of the versatility of tagging proteins with GFP
and other tags. Thus, even if the protein fusion reveils
a defined staining pattern, it is important to substantiate localization data obtained by microscopic analyses
by independent means, e.g., cell fractionation experiments.
However, the ease of construction of tagged proteins
and the potential of obtaining relevant information
about protein function very directly from microscopic
localization experiments outweighs the potential risks
of this approach, which, however, should be kept in
mind.
Episomal Constructs Vs. Chromosomally Integrated Constructs. Plasmids generally allow more
flexibility as to the cloning strategy (e.g., for creating
N- and C-terminal fusions). However, (over)expression
of fusion proteins from episomal constructs leads, in all
cases that we have analyzed, to a rather heterogeneous
signal distribution among the cell population (see Fig.
4). As a consequence, the interpretation of localization
data (brightly fluorescent cells vs. weakly fluorescent
cells) is ambiguous. Centromere-based CEN plasmids
represent a major improvement because of their lower
copy number; in addition, they are mitotically more
stable and thus allow short-term cultivation of cells
without maintenance of selection pressure, which provides more flexibility for analyzing localization patterns under different growth and nutritional conditions. Two to three consecutive rounds of cultivation
(each 8 –12 hours) further improve signal homogeneity.
518
S.D. KOHLWEIN
Expression from the normal promoter on a plasmid is
more comparable to that of the endogenous chromosomal gene, which, on the other hand, may be too low in
some cases to yield a detectable signal. Inducible promoters are useful for short-term induction and pulsechase experiments to monitor the dynamics of protein
localization and turnover in living cells. Such applications are limited by the strength of the promoter, the
half life of chromophore formation, and the stability of
the fusion protein. It should also be noted that strong
promoters do not always result in higher expression
levels, as other levels of control (e.g., protein turnover)
may strongly affect the amount of fusion protein in the
cell. In addition, a heterologous promoter obscures specific expression patterns (e.g., throughout the cell cycle
or growth phase) and may result in mislocalization of
the fusion protein due to overexpression.
In contrast to episomal plasmids, integration of a
plasmid or a linear DNA-fragment, usually in single
copy, into the chromosome provides a very stable and
homogeneously expressed fusion (see Fig. 4). Integrative plasmids with heterologous promoters combine the
advantages of having regulatable gene expression and
homogeneous staining at the expense of losing specific
expression patterns and potential problems resulting
from overexpression. As an attractive alternative to
cloning strategies, the PCR approaches for creating Cor N-terminal fusions by chromosomal integration are
not only very simple and fast, they also provide a stable
integrant that renders the cells independent of any
selection pressure to maintain the fusion. The GFP
signal is typically very homogeneous and, in the case of
C-terminal fusions or with N-terminal fusions after
marker excision (see above), usually reflects endogenous expression levels and patterns. The potentially
insufficient level of expression for microscopic detection
and the possibility of a non-functional or mislocalized
fusion due to the modification at the C- or N-termini
are limits to this approach.
Brachat et al. (2000) recently reported on a “success
rate” of about 25% in detecting discernible GFP localization pattern for 21 tagged yeast proteins, using both
C-terminal chromosomal integration and N-terminal
fusions on episomal, GAL1-driven plasmids. In our
studies in the framework of the EUROFAN (European
Function Analysis Network) project, the success rate
for detecting significant localization patterns for essential proteins tagged with GFP was ⬃50% (Jandrositz et
al., unpublished data).
Vital Dyes
Vital dyes have been used successfully for a number
of applications in yeast, especially to monitor mitochondrial structure and inheritance, vacuolar biogenesis, the endoplasmic reticulum (nuclear envelope), and
the endocytic process. In addition, DNA intercalating
fluorescent dyes are used for detection of nuclear and
mitochondrial DNA and calcofluor is used to detect
chitin, especially in bud scars. Table 4 summarizes
some common vital dyes and their applications in
yeast. For vital staining, the cells are usually preincubated with the dye, washed, and subjected to microscopic inspection. This procedure reduces background fluorescence of non-incorporated dye, but may
also lead to washout of the dye if the staining is reversible. In addition, during sample preparation, physiolog-
ical conditions change for the cells, which may lead to
morphological alterations, a problem that is most obvious with the vacuole. Using the hanging agar block
method (see Long-Term Observation of Yeast Cells) for
cell immobilization provides some advantages for live
cell staining.
Fluorescently labeled phospholipids [(M-C6-NBDPE) phosphatidylethanolamine] have been employed
successfully to follow lipid flip-flop and lipid internalization (Kean et al., 1997). However, using fluorescently labeled metabolites requires extensive controls
to verify the nature of the internalized compound. Impurities in commercial preparations may also lead to
erroneous interpretation of microscopic data (Preston
et al., 1987).
Methods and Limitations for Microscopy of
Live Yeast Cells (see also Table 5)
A major advantage of live cell observation usually is
the ease of sample preparation, which also drastically
reduces potential artifacts associated with cell fixation,
immobilization, and immunofluorescence staining.
Nevertheless, a number of precautions should be observed when preparing yeast for live cell observation.
Culture Conditions. Usually, the culture conditions are defined by the requirements of the strain or
transformant harboring the desired plasmid. Even under selective growth conditions, heterogeneity of the
GFP fusion protein expression can be a severe problem
that hampers the statistical analysis of the experiment. Especially when episomal, high-copy-number
plasmids are used, the variability of signal between
individual transformants is severe. Multiple consecutive rounds of cultivation (8 –12 hours each) usually
lead to a more homogeneous population of GFP-labeled
cells.
Preparation of Cells. Cells are usually enriched
from a culture by centrifugation. It should be noted,
however, that high centrifugal force may alter vacuolar
morphology as well as the cytoskeleton (Pringle et al.,
1989). Washing of cells, change of temperature, and
placement of cells under the coverslip (quickly leading
to anaerobic conditions) represent major alterations to
the physiological conditions for the cells and may result
in stress responses and alterations of localization patterns. A number of immobilization methods have been
described to keep cells in place and under “physiological” conditions during microscopic inspection. As with
fixed cells for immunofluorescence microscopy, live
cells can be attached to polylysine, polyethyleneimine,
or Concanavalin A-treated cover slips (see Short-Term
Observation). These immobilization methods usually
are restricted to short/medium-term observations (a
few minutes) due to the nutritional limitations under
the cover slip. For longer term cell observation, immobilization in agarose or on gelatine-coated slides is
recommended (see Long-Term Observation of Yeast
Cells).
Background Fluorescence. Wild-type yeast cells
normally have a very low background fluorescence
when excited with visible light. High electronic amplification in confocal and video microscopy results in
some weak background signal of mitochondrial fluorescence (Kohlwein, unpublished data). Cultivation and
observation of cells in YPD complete medium contributes to some significant green background fluores-
519
YEAST LIGHT MICROSCOPY
‘
TABLE 4. Vital dyes and applications in yeast
Organelle
Dye
Concentration
Filter set
References
Remarks
DASPMI 4-(4-(dimethylamino)styryl)-Nmethylpyridinium
iodide
10-N-nonyl acridine
orange
Rhodamine 123
10–50 ␮g/ml
␭ex 475 nm,
␭em 605 nm; FITC
McConnell et al. (1990)
Potential sensitive, very
specific, stable
10–50 ␮g/ml
␭ex 495 nm,
␭em 519 nm; FITC
␭ex 507 nm,
␭em 529 nm; FITC
Gallet et al. (1995)
Cardiolipin-specific
DiOC6
20–100 ␮g/1
OD600
␭ex 478 nm,
␭em 496 nm; FITC
Lipid
particles
Nile red
10 ␮g/ml
␭ex 552 nm,
␭em 636 nm; TRITC
Endoplasmic
reticulum
DiOC6
10 ␮g/1
OD600
␭ex 478 nm,
␭em 496 nm; FITC
Vacuoles
CDCFDA carboxydichlorofluorescein
diacetate
Carboxyfluorescein
diacetate
10–50 ␮M
␭ex 500 nm,
␭em 530 nm; FITC
10–50 ␮M
␭ex 450/490 nm,
␭em 520 nm; FITC
FM1-43
1–5 ␮g/ml
FM4-64
10–40 ␮M
Koning et al. (1996);
Lum and Wright
(1995)
Vida and Emr (1995);
Zheng et al. (1998)
Pyranine
5 mg/ml
cMAC blue
1–5 ␮g/ml
␭ex 510 nm,
␭em 626 nm; FITC,
TRITC
␭ex 515 nm,
␭em 640 nm; FITC,
TRITC
␭ex 400/450 nm,
␭em 515 nm; UV
␭ex 353 nm,
␭em 466 nm; UV
various
FITC, TRITC
Haugland (1996)
Pringle et al. (1989)
Figure 4
FITC
Weisman et al. (1987);
Roberts et al. (1991)
Lumen; affects vacuolar
morphology
␭ex 428 nm,
␭em 540 nm
Riezman (1985)
Lumen, reversible
␭ex 590 nm,
␭em 610 nm; TRITC
␭ex 340–360 nm,
␭em 400–440 nm;
UV
Depends on
fluorochrome
Millard et al. (1997)
Viability test
Moore (1990); Pringle
et al. (1989)
Stains bud scars
Pringle et al. (1989);
Chant and Pringle
(1995)
Pringle et al. (1989)
Cell surface
Mitochondria
Vacuolar staining kit
Pigment in ade1, ade2
mutants
Quinacrine
Lucifer yellow carbohydrazide, lucifer yellow
vinylsulphone
FUN-1
10–50 ␮g/ml
Endogenous;
growth
phasedependent
10 mM (2%
glucose;
20 min)
5 mg/ml
2–20 ␮M
Cell wall;
chitin
Calcofluor White
25–500 ␮g/
ml
Cell wall;
␣-mannan
Concanavalin Afluorochrome
conjugates
DAPI 4⬘,6⬘-diamidino-2phenylindole
0.1 mg/ml
DNA
Cytosolic pH
0.5–1 ␮g/ml
␭ex 340–365 nm,
␭em 450–488 nm;
UV
␭ex 352 nm,
␭em 461 nm; UV
Haugland (1996);
Skowronek et al.
(1990)
Burgess et al. (1994);
Koning et al. (1993)
Yang et al. (1996);
Lum and Wright
(1995)
Burgess et al. (1994);
Koning et al. (1993)
Preston et al. (1989);
Pringle et al. (1989);
Roberts et al. (1991)
Preston et al. (1989);
Pringle et al. (1989)
Pringle et al. (1989)
Staining is concentration
and cell mass
dependent
Rapid bleaching
Staining is concentration
and cell mass
dependent
Lumen, pH dependent;
nonfluorescent
precursor
Lumen, pH dependent;
nonfluorescent
precursor
Membrane
Membrane, endocytosis
marker
pH indicator
Haugland (1996)
Hoechst 33258
0.25 ␮g/ml
Propidium iodide
0.5–1 ␮g/ml
␭ex 510–520 nm,
␭em 610 nm; FITC,
TRITC
Acridine orange
0.5–1 ␮g/ml
␭ex 470–503 nm,
␭em 523–650 nm;
FITC
Pringle et al. (1989)
Fluorescein diacetate;
carboxyfluorescein
diacetate
12 ␮M
␭ex 475 nm,
␭em 517 nm; FITC;
fluoresces only after
hydrolysis of the
acetate residues
Breeuwer et al. (1995)
Pringle et al. (1989)
Growth in the presence
of DAPI may generate
petites
Poorly permeant;
treatment of cells with
methanol and acetone
for permeabilization
Poorly permeant;
treatment of cells with
methanol and acetone
for permeabilization
Poorly permeant;
treatment of cells with
methanol and acetone
for permeabilization
Ratio measurement
520
S.D. KOHLWEIN
TABLE 5. Yeast microscopy sites on the web1
General yeast site
http://genome-www.stanford.edu/Saccharomyces/VL-yeast.html
Yeast microscopy sites
http://depts.washington.edu/⬃yeastrc/fm_home3.htm
http://ygac.med.yale.edu/triples/triples.htm
http://www.cis.tu-graz.ac.at/bioc/YGMBG/1micro.htm
Yeast microscopy videos
http://cbweb.med.harvard.edu/research/li/Webpages/
pageLippy!science.html
http://dfciwww.dfci.harvard.edu/pellmanlab/pages/movies.htm
http://genome-www.stanford.edu/group/botlab/people/doyle.html
http://info.med.yale.edu/ophtha/thom/movies.html
http://util.ucsf.edu/sedat/marsh/interphase.html
http://util.ucsf.edu/sedat/marsh/mito.html
http://www.cgc.maricopa.edu/instruction/scimath/biology/
visualization/INVSEE/cellphases.html
http://www.cooperlab.wustl.edu/
http://www.cooperlab.wustl.edu/Data/TSKPolSec/TSKPolSec.html
http://www.stanford.edu/⬃stearns/carminati.html
http://www.unc.edu/depts/biology/bloomlab/gfp.htm
Specific applications/images
ASH1 mRNA localization
http://www.zmbh.uni-heidelberg.de/Jansen/Res.act.html
Stress
http://www.at.embnet.org/bmz/groups/ruis1.htm
Nucleoporin interaction/FRET
http://cancerbiology.dfci.harvard.edu/cancerbiology/ResLabs/
Silver/research.html
Sporulation
http://www.sunysb.edu/biochem/BIOCHEM/facultypages/
neiman/micro.html
Nuclear localization signal
http://www.cc.rochester.edu/college/BIO/faculty/Goldfarb.html
Nuclear DNA quantification using microscopy
http://mcbio.med.buffalo.edu/CMB/huberman/Quant_Flu_
Microscopy/Quant_Flu_Micro.html
Mitochondrial retention in the mother cell
http://www.current-biology.com/supmat/cub/bb9s50s.pdf
Immuno-staining protocols
http://genome-www.stanford.edu/group/botlab/protocols/MeOH
IF.html
http://genome-www.stanford.edu/group/botlab/protocols/non MeOH
IF.html
http://genome-www.stanford.edu/group/botlab/protocols/rho pha
calc.html
http://www.biology.ucsc.edu/people/kellogg/Immunofluorescence.
html
http://www.sacs.ucsf.edu/home/HerskowitzLab/protocols/ashif.html
Tagging and vectors
http://depts.washington.edu/⬃yeastrc/fm home1.htm
http://pingu.salk.edu/⬃forsburg/vectors.html
http://www.mips.biochem.mpg.de/proj/yeast/info/tools/hegemann/
gfp.html
http://ycmi.med.yale.edu/YGAC/3xHA GFP info p.html
http://www.mips.biochem.mpg.de/proj/yeast/info/tools/hegemann/
gfp.html
S. pombe handbook
http://www.bio.uva.nI/pombe/handbook/default.html#section4
Guide to yeast morphology
http://bioclab.bioc.unsw.edu.au/iwd/AGtYM2.gif
GFP discussion forum (internet news group)
bionet.proteins.fluorescent
1
These sites can be accessed through: http://www.cis.tu-graz.ac.at/bioc/YGMBG/
yeastmicroscopy.htm
cence, due to vitamins (i.e., riboflavin) present in the
yeast extract. Under these conditions, the vacuole may
also contain substantial background fluorescence. ade1
and ade2 mutants (e.g., in such popular strains as
W303) accumulate a strongly fluorescent pigment in
the vacuole: the intermediates of the adenine biosynthetic pathway, CAIR (phosphoribosylaminoimidazole
carboxylate) in ade1 mutants and AIR (phosphoribosylimidazole) in ade2 mutants, are subject to oxidation
and condensation reactions and, ultimately, end up in
the vacuole as glutathione conjugates (Chaudhuri et
al., 1997). This pigment fluoresces brightly orange-red
upon blue light excitation; its emission spectrum overlaps significantly with that of GFP and is readily detected using an FITC filter cube. The pigment accumulates as cells enter stationary phase, but it is also
detectable during logarithmic growth. Additional supplementation of YPD medium with 5 mM adenine partially suppresses pigment formation. Background signal introduced by the ade-pigments can be eliminated
using electronic signal detection, e.g., in two-channel
confocal microscopy. Online channel-subtraction usually allows complete color separation between GFP and
vacuolar pigment (see Fig. 4).
Dye Interference With Organelles and Phototoxicity. Another problem with the application of vital
dyes is the potential interference with organelle structure and morphology. Indicator dyes fluoresce upon
interaction with their environment in the cell and can
thus affect the properties of the cellular structures.
These influences are further exacerbated by the exposure to light, the cell preparation technique, and conditions during microscopy. The following rules should
be kept in mind. Light intensity and field of exposure
should be reduced as much as possible, by using neutral density filters (the intensity of a xenon or mercury
arc lamp cannot be attenuated). “Longer wavelength
dyes” should be used where possible, because damage
to the cells is less severe at higher excitation wavelengths. Application of 2-photon microscopy for UV
dyes, thus, also provides major advantages (see Part II,
2-Photon Microscopy). Reduction of light exposure
time, e.g., by illuminating the specimen only during
film/camera exposure and application of the lowest concentration of dye possible to obtain the signal are further measures to increase the possibility of success of
the experiment. The illuminated area on the slide can
be delimited by an iris and frequent changes of the field
of view to unexposed areas immediately provide good
information as to the bleaching or other photodamage
that occurs during exposure. For sensitive stains, it is
useful to adjust the focus on one part of the slide and
then take the images from a different, previously unexposed field. In this case and for time-lapse recordings, an automatic shutter system needs to be implemented to block off the excitation light between exposures. A major consideration is, of course, the
maintenance of conditions that allow the cells to remain viable during staining and microscopic observation.
Phototoxic effects are obvious during extended exposure of cells labeled with mitochondrial dyes, e.g.,
DASPMI, and result in increased background fluorescence in the cytosol and swelling of mitochondria. Preparation of cells is critical for vacuole staining as well:
glucose levels and the energy state drastically affect
vacuolar morphology and may lead to coalescence of
multiple vacuolar vesicles into larger vacuoles if cells
are suspended in water (Pringle et al., 1989). The vacuolar dye quinacrine is also known to induce vacuole
coalescence (Pringle et al., 1989). Nile red, which is
used for labeling of membranes and especially of lipid
particles, is subject to rapid bleaching. In addition,
metabolically compromised or old cells are labeled
more readily with nile red, giving rise to a rather het-
YEAST LIGHT MICROSCOPY
521
Fig. 3. Immobilization of yeast cells. Cells
(0.5 ␮l of suspension) are covered with a thin
sheet of agarose on the cover slip. The cover
slip is mounted on a spacer device to allow for
sufficient airspace. Cells can be maintained
in a viable state for several hours during
microscopy, and incubated with vital dyes
(⬃1 ␮l of a 15⫻ concentrate). Prior to microscopy, cells can be adjusted to different temperatures and growth conditions. The spacer
device (plastic, aluminum) is assembled on a
regular microscopy slide using double-sided
sticky tape that also fixes the coverslip.
erogeneous labeling pattern (Kohlwein, unpublished
data).
Bleaching and Photodamage. Most chromophores are bleached upon interaction with light,
which results in a drastic reduction in quantum yield
and loss of fluorescence. To reduce bleaching, exposure
of cells (and chromophores) to the light source should
be kept to a minimum, especially in time-lapse recordings. Whereas in most applications bleaching represents a serious limitation, it can also be used advantageously to investigate the dynamics of recovery from
bleaching (White and Stelzer, 1999), as recently elegantly demonstrated in a study on yeast microtubule
dynamics (Maddox, et al., 2000).
Protocol 1: Cell Immobilization
Short-Term Observation. Coverslips are coated
with solutions of 1% poly-L-lysine or polyethyleneimine
or 0.1% Concanavalin A for 10 minutes; the solution is
aspirated off, and the slide is washed 5 times with
distilled water and air dried. Two microliters of cell
suspension are applied and allowed to sediment for
10 seconds. Unattached cells are rinsed off. The coverslip is mounted directly on a glas slide, with the application of gentle pressure to ensure a monolayer of cells,
and blotting with tissue to remove excess liquid. Coverslips are attached to the slide with small strips of
sticky tape or sealed with Valap (vaseline, lanolin, and
wax, 1:1:1) to avoid drying of the preparation. Cells
should be inspected immediately (within 30 minutes)
to avoid potential morphological alterations due to
anaerobiosis or lack of nutrients. In a modified version,
two strips of Parafilm on either side of the coverslip are
used to create a small chamber for the cell preparation
that leaves some airspace between the slide and coverslip.
Long-Term Observation of Yeast Cells. Various
immobilization methods have been described, including embedding of cells in 25% gelatin (Bloom et al.,
1999; Salmon et al., 1998), using agarose in an airpermeant Sylgard chamber (Koning et al., 1993), or
embedding in low-melting agarose (Doyle and Botstein,
1996). In our laboratory we are successfully using a
simple and reliable setup, based on the “hanging agar
block” method (Robinow, 1975) as outlined in Figure
3. Cells (⬃0.5 ␮l of a concentrated suspension) are
placed on a coverslip and covered with a thin sheet of
0.8 % agarose (prepared in a casting device for polyacrylamide gels and cut into 5⫻5 mm squares). The
agarose can be kept at 4°C for several days, and depending on the experiment, is soaked in various growth
media prior to application. The immobilized cells are
incubated in a moist chamber at the desired temperature for various periods of time to allow adaptation of
cells prior to microscopy. The coverslips are mounted
on spacer devices, usually made of 2-mm plastic glued
to microscope slides, using double-sided sticky tape
(Fig. 3). The doubling rate of the cells is comparable to
that during growth in suspension, and the airspace
provides sufficient oxygen for long-term (⬎24 hours)
cultivation. Multiple cell preparations can be mounted
on a single slide, allowing rapid switching between
samples during an experiment. A temperature-controlled device, based on the same principle but made
with an aluminum spacer that is temperature controlled by heated or cooled liquid, has been applied
successfully to maintain cells under optimum growth
conditions or to induce metabolic blocks in temperature-sensitive mutants during microscopic observation.
This device can be used for long-term observation of
cells labeled with GFP, to follow “on-line” induction of
GFP-fusion protein expression, or to label cells using
vital dyes.
Protocol 2. Staining of Cells With Vital Dyes
Several dyes require pretreatment of cells or a specific pH for labeling, to allow efficient uptake. It is
usually necessary to wash cells after staining to reduce
background fluorescence. Using the “hanging agar
block” technique described above, removal of non-incorporated dye is not required in many applications. Vital
dyes are applied as concentrates (⬃1 ␮l of a 15⫻ stock
of the final concentration) to the agarose slice and
incubated for 5–30 minutes prior to microscopy.
Bleaching, which represents a major problem for some
vital dyes (e.g., Nile red in solution; see above), is
drastically reduced due to the high dye capacity of the
agarose and some exclusion of oxygen (although the
522
S.D. KOHLWEIN
cells grow aerobically without any reduction in growth
rate). Whereas fluorescence intensity is not usually
affected by agarose embedding, transmission light microscopy (differential interference contrast) may be
somewhat obscured, although the closer match in refraction indices between the cell wall and the mounting
medium usually reveals more of the cell interior.
PART II: YEAST LIGHT MICROSCOPY:
INSTRUMENTAL SETUP
Microscope Setup
Pringle et al. (1989) provide an excellent overview on
how to set up a conventional microscope for fluorescence microscopy of yeast. High resolution objectives
with 63⫻ or 100⫻ magnification and a numerical aperture (NA) of 1.3 or 1.4 are crucial for yeast microscopy. The intensity of the observed fluorescence is related to the NA (a NA 1.4 lens is about 30% brighter
than a NA 1.3 lens) and inversely related to the magnification of the objective lens (a 63⫻ lens is about two
times brighter than a 100⫻ lens). For transmitted-light
microscopy, differential interference contrast (DIC; Nomarski) optics provide major improvements in resolution by contrast enhancement. Using DIC, yeast vacuoles and highly refractile dense bodies (lipid particles,
storage granules) can easily be visualized (see below).
For fluorescence microscopy, xenon or mercury arc
lamps (50 –200 W) provide the full spectrum of visible
and UV light. Xenon arcs provide a more uniform spectrum over mercury arcs but are usually less intense.
Fluorescence filters usually come as pre-packaged
sets that consist of excitation filter, dichroic mirror,
and emission filter, and cover most applications. For
GFP (S65T, EGFP) fluorescence microscopy, filters for
fluorescein detection (520-nm-long-pass filter or 510 –
560-nm band-pass filter) are widely used; it should be
noted, however, that a significant portion of the GFP
fluorescence, which has an emission maximum at
510 nm, is not detected with these filters.
A major limitation in multi-color applications is the
potential spectral overlap between chromophores, leading to “cross-talk” between fluorescence channels. This
is a particular problem if the dye intensities differ
greatly. Thus, in double-labeling experiments, it is critical to determine and control this cross-talk in order to
get meaningful results. Ideally, one would switch to
chromophores with well-separated emission spectra to
avoid this problem. Alternatively, electronic recording
of fluorescence signals with video cameras or confocal
microscopes allows correction for cross-talk between
fluorescence channels by electronic subtraction. Confocal laser scanning systems provide the advantage of
individual defined laser lines that can be electronically
selected and attenuated, together with prism-based
spectral detectors with continuously adjustable emission filter ranges.
Electronic Light Microscopy: Confocal, Video/
Deconvolution, and Two-Photon
Excitation Microscopy
Confocal Laser Scanning Microscopy (CLSM).
Although the theoretical principles of this technology
were worked out in the 1950s (Minsky, 1988), practically useful confocal microscopes became available only
in the 1980s (Brakenhoff et al., 1989a; Paddock, 1999a;
Pawley, 1995; Petran et al., 1986; Shotton, 1993; Webb,
1999; White et al., 1987). However, since then CLSM
has increased in popularity and it provides major advantages for observing organelle dynamics in yeast, as
elegantly demonstrated in a pioneering work by Koning et al. (1993). Confocal scanning and 3D reconstruction of multiple optical sections, as well as quantitative
applications of fluorescence microscopy, have reached
an astonishing level of resolution and precision, in
studies of fungi (Spear et al., 1999) as well as other
organisms (Meth Enzymol 307 (1999), “Confocal microscopy”), and make confocal microscopy the method
of choice for cells as small as yeast.
In CLSM, the specimen is illuminated by a point
light source, realized by a laser; fluorescence emitted
along the path of the laser beam is collected through
the objective lens and projected to a detection pinhole
before it hits a photodetector that converts the available fluorescent light into a photocurrent (Paddock,
1999a; Pawley, 1995; Shotton, 1993; Webb, 1999). The
size of the pinhole determines the width of the plane
that is actually recorded by the detector, thus increasing contrast by eliminating out-of-focus fluorescence
that obscures images in wide-field fluorescence microscopy. By scanning the laser beam through the specimen, a 2-dimensional array of fluorescence signals
(“pixels”) is recorded and assembled into a digital image, which is also referred to as an “optical section.”
The consecutive scanning of different focal planes of
the specimen provides 3-dimensional information (see
Fig. 4), with a theoretical vertical resolution of ⬃230
nm that can be obtained with a 1.4 numerical aperture
objective lens. The horizontal resolution limit in the
image plane is ⬃140 nm and depending on the wavelength of the laser light (Wilson, 1990). For work with
yeast, objectives with maximal numerical aperture
(e.g., 63⫻/NA 1.4 or 100⫻/NA 1.4) are required to obtain the best resolution. Since this high NA can only be
achieved with oil immersion lenses, the aqueous environment for yeast cells in live cell applications leads to
light diffraction at the glass-water interface. The consequence is that the scanned focal plane is actually
different from the plane set by the microscopy stage.
This effect becomes more pronounced the deeper the
laser scans into the specimen, making it necessary to
apply correction factors to account for these differences
in refraction indices during 3D-reconstructions. This,
of course, holds true also for other 3D-microscopy techniques (see Video/Deconvolution Microscopy). Furthermore, light intensity is significantly reduced in lower
sections due to light scattering. However, as long as
yeast cells are mounted close to the glass surface of the
cover slip, these limitations, which take significant effect at a depth ⬎10 ␮m, can be tolerated.
Not only does CLSM provide spatial information
close to the theoretical limits of light microscopy, but
the application of multiple lasers and multi-channel or
spectral detectors allows simultaneous detection of
multiple chromophores with great precision (Paddock,
1999b). Acousto-optical tunable filters (AOTFs), on the
excitation side, to select and attenuate individual, or
multiple, laser lines, and novel spectral emission detectors that lack glass filters and are thus more sensitive, make confocal systems extremely flexible. Multiple laser lines can be combined and modulated at will
in terms of intensity, which proves very valuable for
simultaneous multispectral analyses using different
YEAST LIGHT MICROSCOPY
fluorescent dyes that are present in variable concentrations. Spectral detectors make the user independent of
glass filters and allow optimization of fluorescence
emission detection and color separation. Pan et al.
(2000) have recently described 3-color simultaneous
CLSM of yeast cells, labeled with Hoechst 33258 dye
for DNA (UV excitation/detection), GFP (488 nm excitation, 500 –550 nm detection), and the red fluorescent
vacuolar membrane dye, FM4-64 (488 nm excitation,
560 nm longpass detection; Vida and Emr, 1995) in a
multi-laser CLSM system. Although these analyses require a highly advanced confocal system, the quality of
information that can be extracted justifies the investment in high-resolution microscopy technology for
yeast cell biology.
The signal obtained from the confocal microscope
directly reflects the fluorescence intensity in the focal
plane, so that no further computational manipulations
of the image information are a priori required, thus
providing “online” spatial information. Since the pioneering work of Brakenhoff to observe yeast mitochondria with CLSM (Brakenhoff et al., 1988, 1989a,b),
major technical innovations have become available,
and further improvements in resolution such as 4␲
confocal microscopy (Nagorni and Hell, 1998) are likely
to be implemented in future generations of confocal
microscopes, allowing them to reach the 100 nm range
of resolution in all three dimensions.
Limitations of Confocal Microscopy
As with all fluorescence techniques, the potential
interference of the light source with cells and dyes
imposes a major limitation. The intense laser beam
may result in rapid bleaching. However, this can be
reduced by the experimental setup and method of cell
immobilization (see Methods for Microscopy of Live
Yeast Cells), and the illumination time in each spot of
the specimen is rather short. The signal-to-noise ratio
is lower than with deconvolution microscopy (see below), as is the recording speed (in the range of seconds
per frame). The setup is rather costly, particularly if
multiple lasers are to be used for multi-spectral applications.
Video/Deconvolution Microscopy
Video microscopy is based on a conventional widefield fluorescence microscope equipped with a video or
cooled charge-coupled device (CCD) camera system for
image acquisition. Excellent results have been obtained using this technology especially for analyzing
cytoskeleton structure and dynamics in living yeast
(Bloom et al., 1999; Carminati and Stearns, 1997, 1999;
Doyle and Botstein, 1996; Maddox et al., 1999; Shaw et
al., 1997b; Waddle et al., 1996). High-resolution video
and digital-enhanced differential interference contrast
(DIC) light microscopy are further improvements to
video microscopy (Salmon et al., 1998). The DIC (Nomarski) method visualizes differences in refractive indices of subcellular structures in transmission light
microscopy; contrast can be further enhanced by acquiring images with a slow-scan cooled CCD camera
and subsequent digital image processing (“digitallyenhanced” DE-DIC). This method also allows combining DIC (transmission) imaging with the observation of
fluorescent vital dyes or GFP, using the same recording
device. The advantage of video-enhanced DIC (VE-
523
DIC) is that images are recorded at video rates of up to
30 frames/second. Both techniques require a more sophisticated microscope setup, such as a light scrambler
for uniform illumination, automated filter wheels for
fluorescence/multimode microscopy, and digital image
processing.
In video microscopy, out-of-focus fluorescence is a
serious limitation to resolution, and larger fluorescent
structures in the cell will appear blurred. In video/
deconvolution microscopy, the out-of-focus fluorescence
recorded in each image is eliminated by computation.
In wide-field microscopy, each fluorescent spot in a
specimen is recorded in a distorted form (Point Spread
Function, PSF) by the objective lens, which contributes
to the out-of-focus signal and loss of resolution. This
PSF can be determined experimentally using defined
fluorescent beads (Scalettar et al., 1996) or computer
simulated. In deconvolution microscopy, the inverse
PSF is applied to multiple optical sections, to reconstruct an undistorted image. The microscope setup is
rather simple and comparably inexpensive and is usually based on a conventional fluorescence microscope
with a motorized stage and a xenon or mercury arc
lamp that provides the full spectrum of light. The digital high-resolution cameras are rather fast, allowing
sampling of images in the msec range, which may be
crucial for the analysis of highly dynamic processes.
The method is more sensitive than confocal microscopy,
due to a better signal-to-noise ratio. The major limitations of the method are the quality of the deconvolution
software, which ultimately determines what is considered as a “real” signal or becomes eliminated as background, and the offline computing time required to
reconstruct the images. The results of the microscopic
observations are thus not immediately evident, and a
more direct online manipulation/interaction with the
specimen is lost. There may be some ambiguity in
selecting the “right” parameters for the PSF to get
“nice” images. Multispectral applications are a particular problem due to spherical aberration (Scalettar et
al., 1996). Although the number of yeast applications
using this technology is increasing, a direct comparison
of resolution between confocal and deconvolution systems is not yet available.
2-Photon Microscopy (Denk et al.,
1995; Piston, 1999)
Although, to my knowledge, no yeast applications of
this technology have been described yet in the literature, this exciting aspect of the confocal principle is
also likely to find applications in yeast. In 2-photon
microscopy, the specimen is illuminated in a confocal
microscope setup with high-intensity pulsed laser light
in the red/far-red wavelength range. If the photon density in the focal spot of the objective lens becomes high
enough, two photons may arrive nearly at the same
time at the chromophore, and together they excite dyes
that usually require excitation by high energy UV or
short wave-length blue light. Since fluorescence is only
emitted from the focal spot, no detection pinhole is
necessary for “confocal” signal registration. Despite the
high intensity of the excitation light, this approach is
much less harmful to living specimens than is UV
excitation; it minimizes both photodamage and photobleaching, which are otherwise limiting factors in live
cell microscopy. A further advantage is the deeper pen-
524
S.D. KOHLWEIN
Fig. 4. Examples of confocal laser scanning microscopy of yeast
and illustration of potential detection problems. Microscope setup:
Leica TCS 4D, Ar/Kr laser; 100⫻/1.4 NA objective; AOTF (acousto
optical tunable filter for laser line selection and attenuation). GFPoptimized filter (488 nm excitation; 500 nm dichroic mirror; 500 –550
nm detection filter). FITC detection filter (488 nm excitation; 510 nm
dichroic mirror; 515 nm long-pass detection filter). ade2-pigment detection filter (488 nm excitation; 500 dichroic mirror; 560 nm longpass detection filter). Bars ⫽ 10 ␮m. I: GFP expressed under MET25
control from an episomal plasmid (A, GFP-fluorescence; B, DIC) and
from a chromosomal C-terminal fusion (C, GFP fluorescence; D, DIC).
GFP is present in the cytosol and absent from vacuoles (black areas).
Note the heterogeneity of the fluorescence signal in different cells, due
to variable expression levels of plasmid-borne GFP (A); arrowheads
indicate lack of GFP expression in some cells, despite growth on
selective medium. Expression of the chromosomally integrated construct is very homogeneous (C). II: Fluorescence crosstalk between
GFP targeted to the endoplasmic reticulum and the red vacuolar
pigment in ade2 mutant cells (log phase). C, F: DIC. A: The FITC
filter set detects both GFP and substantial red fluorescence of the
vacuolar pigment (large arrowheads), at 488 nm excitation. B: The
ade2-pigment detection filter shows both red pigment ⫹ some GFP
fluorescence (small arrowheads). D: Online electronic subtraction of
the red signal (⬎560 nm) from the GFP signal (500 –550 nm) results
in a “clean” GFP signal. In contrast, there is still some GFP background in the “red” channel (E: small arrowheads). Depending on the
age of the cells, the vacuolar pigment can become very prominent (E,
large arrowheads). III: Aberrant localization of a plasma membrane
protein as a function of overexpression and induction time. The PMGFP marker was expressed under control of the MET25 promoter
(Niedenthal et al., 1996; Hegemann et al., personal communication)
for 2 h (A, B) and overnight (C, D). In A, note the vesicles (Golgi?
small arrowheads), which may indicate some delay en route to the
plasma membrane. Long-term overexpression leads to a dramatic
accumulation of GFP fluorescence in the vacuole (C and D). (Figure
continued on next page.)
YEAST LIGHT MICROSCOPY
525
Figure 4. (Continued.) IV: 3D scan (serial optical sections) of cells
expressing COXIV⬘-GFP; this construct (provided by Rob Jensen)
contains the mitochondrial targeting sequence of cytochrome oxidase
subunit IV fused to GFP, under control of the constitutive ADH1
promoter. Note the reticular structure of the mitochondrial network,
localized close to the periphery of the cell. Distance between sections ⫽ 2 ␮m (A–C) and 0.5 ␮m (C–D). V: Sporulation of a heterozy-
gous ERG6/ERG6-GFP strain demonstrates spore-autonomous expression of Erg6p-GFP, which is present only in 2 of 4 spores (A, large
arrowheads). Erg6p localizes to the surface of lipid particles that are
readily visible in DIC microscopy (DIC; C). Vacuolar pigment is
present in all 4 ade2 spores (B) (preparation courtesy Edith Oberhofer). Note the resolution power in DIC.
etration (⬎100 ␮m) into the specimen without significant loss of resolution, which, however, is not relevant
for yeast.
Perspectives
The yeast system has without doubt revolutionized
cell biological reserach. Digital high-resolution light
microscopy has expanded its great potential also to an
experimental system for morphological and dynamic
analyses. Further improvements in microscope technology, vital dyes, and protein-tagging methods can be
expected in the coming years. The combination of protein localization data on a genome-wide basis (e.g.,
TRIPLES database; Ding et al., 2000; Kumar et al.,
2000; Habeler et al., YPL.db (Yeast Protein Location
database) http://ypl.tu-graz.ac.at) with biochemical
pathways will further contribute to a better understanding of cellular organization and regulation. Computer reconstruction and animation of microscopic data
(Mohler, 1999) are also fascinating new tools with
which to further reveil the beauty of the yeast.
Specialized Microscopy Techniques
The number of highly sophisticated microscopy techniques is increasing, and these are likely to continue
the Renaissance of light microscopy in cell biological
research during the next decade. For example, FLIM
(fluorescence lifetime imaging microscopy) discriminates among various fluorophores with similar spectral
characteristics based on their fluorescence lifetimes.
This approach allows, for instance, imaging of multiple
fluorophores with a single excitation wavelength and a
single broad-range emission filter. In combination with
various GFP variants, FLIM opens novel dimensions
through which protein processes can be imaged simultaneously in a cell (Pepperkok et al., 1999). In addition,
FRET (fluorescence resonance energy transfer) microscopy was recently introduced to study protein-protein
interactions in living yeast (Sagot et al., 1999). In
FRET, a primary fluorophore is excited that may transfer its absorbed energy in a radiationless process to a
second chromophore upon close interaction. In an
elegent approach using FRET between enhanced cyan
and yellow fluorescent proteins (ECFP, EYFP), dynamic interactions between nuclear receptors and
nucleoporin and overlapping pathways of importin and
exportin traffic through the nuclear pore complex were
recently reveiled in yeast (Damelin and Silver, 2000).
ACKNOWLEDGMENTS
I thank the members of my lab for helpful suggestions, and especially Anita Jandrositz for providing
some artwork, Jiři Hašek and Trisha Davis for critically reading the manuscript and helpful suggestions,
and the reviewers of this article for invaluable comments and suggestions. Many thanks to Günther
Daum for his patience during preparation of the manuscript. I gratefully acknowledge support for the work in
our laboratory that comes from the Austrian Science
Fund, FWF (project F706), the Oesterreichische Nationalbank (project 7273), EU-grant BIO4-CT97-2294),
526
S.D. KOHLWEIN
and the Austrian Ministry for Science and Transportation (Project “Essential Genes,” supplement; Biocomputing).
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