The visualisation of cytoskeletal proteins and their dynamics in live cells has led to invaluable insights into cytoskeleton-driven processes as various as cell migration, chromosome segregation during division, cytokinesis, and phagocytosis, as well as the establishment of cell-substrate or cell-cell adhesion. The cytoskeleton of eukaryotic cells can be grossly divided into three filamentous systems: actin filaments, microtubules, and intermediate filaments.
This article focuses on revealing the dynamics of the actin cytoskeleton and the microtubule system, as well as some proteins associating with them in interphase cells. It compares classical approaches with more recent advancements in this enormously growing field.
More specifically, this article discusses visualisation of the cytoskeleton using purified components chemically modified with fluorescent dyes and compares this method with ectopic expression of cytoskeletal genes fused to fluorescent protein tags such as green fluorescent protein (GFP). While the efficient introduction of fluorescently labelled cytoskeletal proteins is usually achieved by microinjection, which requires additional equipment and experimental effort, the expression of genes tagged to fluorescent proteins is obtained by transfection of the respective fusion constructs. We will show that both methods can lead to equally satisfying results, but as the transfection of nucleic acids seems to be continuously improved and developed further, the latter method may be used more frequently for most common tissue culture cells. However, because studies on cytoskeleton dynamics are sometimes performed on cell types that have so far not been reported to mediate the expression of ectopic genes, such as epidermal fish keratocytes, protein microinjection and subsequent analysis are still performed. In addition, we briefly mention the microinjection of specific antibodies to monitor the dynamics of cytoskeletal components.
Finally, this article gives examples of how to approach the visualisation of two distinct cytoskeletal components in the same living cell using two-colour fluorescence video microscopy.
II. MATERIALS AND INSTRUMENTATION
Inverted microscope (e.g., Axiovert 135TV, Carl Zeiss Jena GmbH) equipped for epifluorescence and phase-contrast microscopy with 40x/1.3NA and 100x/1.4NA oil immersion objectives, 1.6 and 2.5 optovar intermediate magnification, electronic shutters (e.g., Uniblitz Electronic 35-mm shutter including driver Model VMMD-1, BFI Optilas) to allow for computer-controlled opening of the light paths, filter wheel (e.g., LUDL Electronic Products LTD, SN: 102691 and driver SN: 1029595) to enable two-colour epifluorescence in combination with appropriate dichroic beam splitters and emission filters (Omega Optical Inc. or Chroma Technology Corp.), and tungsten lamps (Osram, HLX64625, FCR 12V, 100W) for both phase contrast and fluorescence light paths. Tungsten lamps are not as bright as mercury lamps, but the latter cause photodamage and bleaching with higher probability.
2. Data Acquisition
Preferably by a back-illuminated, cooled chargecoupled- device camera (e.g., Princeton Research Instruments TKB 1000 x 800, SN:J019820; Controller SN:J0198609) driven, for instance, by IPLab (Scanalytics Inc.) or Metamorph software (Universal Imaging Corporation). Dependent on cell types and/ or processes studied, this "epifluorescence setting" can be extended to the various options of confocal microscopy (see contributions on confocal imaging within this volume).
Commercial microinjection capillaries (Femtotips I; Cat. No.: 5242952.008, Eppendorf AG) or, alternatively, self-made needles requiring a "needle puller" (e.g., Model PN-30, Narishige International LTD), mechanical (Cat. No.: 520137, Leica Microsystems), oilpressure- driven (e.g., Model M0188NE, SN:99069, Narishige International LTD), or electronic (e.g., Model 5171, Eppendorf AG) micromanipulators used in combination with an air pressure device (e.g., Transjector 5246, Eppendorf AG); flexible microloaders for loading microinjection capillaries (Cat. No.: 5242956.003, Eppendorf AG).
Cooled high-speed benchtop centrifuges (e.g., Biofuge fresco, Heraeus) to pellet protein aggregates prior to injection.
Open (e.g., Series 20 chamber platform, Model PH4, Warner Instruments used with heater controller model TC-324B, SN:1176) or, alternatively, closed heating chambers (e.g., Model FCS2, Bioptechs Inc.).
Cells growing at room temperature are observed on coverslips mounted to the bottom of plastic dishes harbouring a central hole or in comparable commercial devices.
Round glass coverslips, 15 or 40mm in diameter, cleaned in 6/4 ethanol/HCl (37%), washed extensively with H2O, dried, and sterilised by exposure to ultraviolet light or in a dry heat sterilizer at 220°C (ethanol: Sigma, Cat. No.: E-7023; HCl: Sigma, Cat. No.: H-7020).
General equipment and plastic-ware (e.g., from Becton Dickinson Biosciences or Greiner Bio-One GmbH) for molecular biology techniques and tissue culture.
B. Cells and Media
Mouse melanoma cells B16-F1 (American Type Culture Collection: CRL-6323) and goldfish fin fibroblasts (CAR, American Type Culture Collection: CCL-71).
All reagents are from Invitrogen Corp. unless stated otherwise. For B16-F1: Dulbecco's modified Eagle's medium (Cat. No.: 41965-039) supplemented with 10% fetal calf serum (FCS, PAA Laboratories, Clone, Cat. No.: All-041), 2mM glutamine (Cat. No.: 25030-024), 1% antibiotics (Cat. No.: 15070-063). For CAR: basal Eagle's medium with Hank's balanced salt solution (Cat. No.: 21370-028), 15% serum (Hyclone, Cat. No.: SH30070.03), 1mM glutamine, 1mM nonessential amino acids (Cat. No.: 11140-035), and 1% antibiotics. Upon confluence, cells are detached using trypsin (Cat. No.: 25300-054) and seeded according to standard protocols.
3. B16 Medium for Microscope
Ham's F12 HEPES-buffered medium (Sigma, Cat. No.: N8641) including complete supplements of the regular growth medium (see point 2).
Laminin (Sigma, Cat. No.: L2020) or fibronectin (Roche, Cat. No.: 1051407).
With Superfect transfection reagent (Qiagen GmbH, Cat. No.: 301305) or FuGENE6 (Roche, Cat. No.: 1 814 443).
C. Proteins and Constructs
All chemicals are from Sigma-Aldrich.
A. Visualisation Using a Fluorescently Conjugated Protein
B. Visualisation Using a GFP-Tagged Protein (upon Transient Expression)
IV. GENERAL COMMENTS ON FLUORESCENT TAGGING
In order to obtain reliable data on the dynamics of any given cytoskeletal protein, it is important to consider potential interference of the fluorescent tag with proper subcellular targeting of the protein or with general protein function. It is therefore essential to make sure that the fluorescent analogue of a given protein incorporates identically to the endogenous protein, which can be tested for by cross-staining with appropriate antibodies. Chemical coupling of a purified protein to a conventional fluorescent tag such as fluoresceine or rhodamine can be advantageous over a fluorescent protein tag (such as GFP) due to the much lower molecular size of the former. However, certain coupling buffers (dependent on coupling chemistry) may affect proper protein folding and therefore lead to irreversible damage to a given protein avoiding its proper incorporation into the cytoskeleton. In general, monovalent dyes are advantageous over bi- or tetravalent dyes because the latter can cause irreversible protein multimerisation due to the coupling reaction (which can be tested for by SDS-PAGE).
For the fusion of cDNAs to fluorescent protein tags, the most common of which currently is the enhanced green fluorescent protein (EGFP, Clontech), also known as GFPmutl (Cormack et al., 1996), we recommend always comparing the fusion of the given cDNA to both the N and the C terminus of the tag because it is difficult to predict whether the fusion to the protein tag on either side may interfere with protein function. For instance, in order to generate reliable fusion proteins for the visualisation of the actin nucleating complex Arp2/3 (Higgs and Pollard, 2001), we have so far generated N- and C-terminal fusions to each of the seven components and tested their incorporation in cells, with varying results. Some of the constructs showed poor incorporation or even interfered with lamellipodia protrusion (p34-EGFP). Good results were obtained with small molecular weight components of the complex (p16 and p21), although both Nand C-terminally tagged Arp3 also proved useful (Stradal et al., 2001). Figure 2 shows the dynamics of p16B (ARPC5B), a novel isoform of p16 (Millard et al., 2003) fused to the C terminus of EGFP in B16-F1 cells. In addition, spacers between EGFP and the gene of interest can be crucial for incorporation of the fusion constructs (Geese et al., 2000).
As an alternative possibility to follow the dynamics of cytoskeletal proteins, it is worth mentioning purification, fluorescent coupling, and microinjection of monoclonal antibodies directed against a cytoskeletal protein, which has been used in the past for zyxin (Rottner et al., 2001). This requires that the epitopes are freely accessible in the native protein and that injected antibodies do not interfere with proper protein positioning and function, prerequisites that can again only be evaluated experimentally.
V. CHOICE OF COMBINATIONS FOR DUAL LABELING
As with conventional fluorescent dyes, fluorescent protein tags today also come in different colours. As opposed to multiple labellings of fixed samples (see article by Prast et al.), for live cell imaging as described here, only dual-labelling experiments have proven practical so far, probably due to the lack of sufficiently bright dyes excitable by UV excitation. We provide examples here for imaging EGFP-tagged proteins together with injected proteins tagged to rhodamine derivatives or to Cy3 (see Fig. 3). An important alternative is the use of the red fluorescent protein from Discosoma sp. (dsRED), exemplified here by a dsREDzyxin probe combined with EGFP-tubulin (Fig. 4). However, in contrast to EGFP, dsRED and even the improved variant termed dsRED2 (Clontech) are known to form a tetramer when fluorescent (Baird et al., 2000). Unfortunately, this feature can interfere with the function of certain cytoskeletal proteins. For instance, expression of a dsRED-calmodulin fusion protein was reported to cause severe aggregation and therefore mislocalisation of the tagged protein (Mizuno et al., 2001).
As an alternative, mutations in EGFP have led to spectrally separable variants, ECFP (cyan) (Heim and Tsien, 1996) and EYFP (yellow) (both Clontech), which were used successfully for dual-labelling experiments of cytoskeletal proteins in the past (Geese et al., 2002). However, the excitation and emission maxima of both dyes are relatively close, making it more difficult to separate them clearly. In addition, ECFP is significantly dimmer than EYFP, hence the simultaneous imaging of complex cytoskeletal structures may prove more difficult using these variants as compared to the red/green combinations given earlier.
Exciting more recent developments include far red fluorescent proteins, which appear separable not only from EGFP, but also from ECFP and EYFP. One was obtained from mutating a nonfluorescent chromoprotein from Heteractis crispa, termed HcRED-2A in (Gurskaya et al., 2001), and is available as HcRED-1 (Clontech). The protein is a dimer and can now also be obtained as a "monomeric" tandem variant (evrogen.com).
In addition, Tsien and colleagues have developed a true monomeric red fluorescent protein (termed mRFP1) with excitation and emission peaks at 584 and 607, respectively, by introducing multiple mutations into dsRED (Campbell et al., 2002). Comparisons of HcRED-1, its tandem variant, and mRFP1 as fusion proteins with actin and other cytoskeletal components revealed that mRFP1-at least in fusion with the components tested-was superior to the former red variants, mainly due to the lack of aggregation (not shown). Hence, although about 25% of the brightness of the original dsRED, the mRFP1 probe in combination with EGFP is expected to prove very useful for live cell dual imaging.
We thank Drs. Mario Gimona and Fedor Severin for kindly providing purified smooth muscle proteins and Cy3-tagged tubulin, respectively, Dr. R. Y. Tsien for mRFP1 cDNA, Drs. Anna S. Akhmanova, Marcus Geese, and Anna Huttenlocher for expression constructs, and Petra Hagendorff for excellent technical assistance.
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