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
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
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,
Cooled high-speed benchtop centrifuges (e.g.,
, 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
Round glass coverslips, 15 or 40mm in diameter,
cleaned in 6/4 ethanol/HCl (37%), washed extensively
O, 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
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.:
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
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
C. Proteins and Constructs
- TAMRA-vinculin: From turkey gizzard, coupled
to carboxytetramethylrhodamine (5'-TAMRA) succinimidyl
ester (Molecular Probes, Cat. No.: C-2211) as
described (Rottner et al., 1999) and upon addition of
2mg sucrose/mg protein stored in 30-µl aliquots at
1 mg/ml at -70°C
- TAMRA-α-actinin: From turkey gizzard, kindly
provided by M. Gimona (Salzburg) and fluorescently
modified and stored as described for vinculin
- Cy3-tubulin: As described (Hyman, 1991); for a
protocol of tubulin labelling (by John Peloquin) we
- EGFP-tubulin: Kindly provided by M. Geese
(Braunschweig), comprises murine β3-tubulin fused
into EGFP-C2 (Clontech, Cat. No.: 6083-1)
- EGFP-EBI: As described (Stepanova et al., 2003)
- EGFP-p16-B: p16B-cDNA, also known as
ARPC5B (Millard et al., 2003), was amplified from a
human EST clone (Acc. No.: 12652556, RZPD cloneID
IRALp962P167) and subcloned into EGFP-C1
(Clontech; Cat. No.: 6084-1)
- Zyxin-EGFP: As described (Rottner et al., 2001)
- Zyxin-dsRED: As described (Bhatt et al., 2002)
All chemicals are from Sigma-Aldrich.
- Phosphate-buffered saline (PBS) working solution:
140 mM NaCl (Cat. No.: S-7653), 2.7 mM KCl (Cat. No.:
P-1338), 10mM Na2HPO4 (Cat. No.: S-7907), 1.8mM KH2PO4 (Cat. No.: P-0662), pH 7.4
- Urea: 2M in H2O (Cat. No.: U-0631)
- Laminin coating buffer: 50mM Tris (Cat. No.: T-
1503) adjusted with HCl to pH 7.5, 150mM NaCl
- Microinjection buffer (for vinculin and α-actinin):
2 mM Tris-acetate (acetic acid: Cat. No.: A-0808), 50 mM KCl (Cat. No.: P-1338), 0.1mM dithioerythritol (DTE,
Cat. No.: D-8255), pH 7.0. Note that microinjection
buffers vary significantly depending on the cytoskeletal
protein injected [e.g., buffers for microinjecting
actin generally lack KCl (Wang, 1984), whereas buffers
for injecting myosin II require KCl (up to 450mM) in
order to avoid polymerisation of the respective component
in the needle (Verkhovsky and Borisy, 1993)].
- Microinjection buffer (for tubulin): 80mM PIPES
(Cat. No.: P-6757) adjusted with KOH (Cat. No.: P-
6310) to pH 6.8, 1 mM MgCl2 (Cat. No.: M-2670), 1 mM EGTA (Cat. No.: E-1644) (Fig. 1).
|FIGURE 1 Examples for specific incorporation of fluorescently tagged, microinjected proteins: CAR fish
fibroblasts microinjected with TAMRA-vinculin (A) or TAMRA-α-actinin (B). Note the specific recruitment
of both probes to focal adhesions and the additional periodic incorporation of α-actinin into stress fibres.
(C) EGFP-β-tubulin (kind gift of M. Geese, Braunschweig) expression (green) in a CAR cell additionally microinjected
with Cy3-tubulin (kind gift of F. Severin, Dresden; red). Note that both probes reveal an identical
microtubule pattern (merge) and can therefore be used equally to study microtubule dynamics.
A. Visualisation Using a Fluorescently
B. Visualisation Using a GFP-Tagged Protein
(upon Transient Expression)
- Various different fibroblast cell lines survive
microinjections better when grown on fibronectin. For
coating of coverslips, dissolve fibronectin at a concentration
of 1 mg/ml in 2M urea and store at 4°C.
- Dilute fibronectin stock 1:20 to 50µg/ml with
PBS and coat sterile coverslips for 1 h at room temperature
(150µl for 15-mm-diameter coverslips). Before
seeding the cells, wash thoroughly with PBS to remove
urea (two to three times).
- Upon washing, seed the cells onto coverslips in
a petri dish and let them attach and spread for 12-
72h in an incubator.
- Thaw an aliquot of fluorescently coupled protein.
If not in appropriate buffer, dialyse the sample into
microinjection buffer using Slide-A-Lyzer (e.g., Pierce,
10 kDa cutoff Cat. No.: 66415) or a similar device for
the dialysis of small volumes and concentrate using
microcon concentrators (Amicon, YM-10, Cat. No.:
42407) (for proteins such as vinculin or α-actinin, concentrations
of 0.5-1mg/ml are ideal for injections).
- Spin the probe for microinjection in a cooled centrifuge
at maximum speed (approximately 10,000g) for
at least 30min before loading the needle in order to
remove protein aggregates that may clog up the needle
- Mount cells onto the microscope in a chamber
freely accessible to the microinjection needle.
- Upon loading of the needle from the back using
a flexible pipette tip, carefully inspect the needle tip for
the absence of air bubbles. If required, remove them by
gently hitting the needle shaft, but proceed rapidly in
order to avoid drying of the tip.
- Apply pressure (20-100 hPa) to the needle holder,
attach needle, and rapidly move needle into the
medium. (The "background pressure" applied before
entering the medium is required to avoid sucking up
of the medium due to capillary force of the needle tip.)
- Bring the needle close to the cells.
- Before injections, check the needle flow by
- In case of flow, gently inject the cells, manually
or using a so-called half-automatic system (Eppendorf
AG, see earlier discussion). For manual injections,
carefully approach the membrane and remove immediately
upon needle flow into the cell.
- Upon injection of a sufficient number of cells,
allow the cells to recover and the cytoskeletal proteins
to incorporate (e.g., 30-60min). For actin, full incorporation
into the cytoskeleton may take up to 2h. In
contrast, vinculin targets to focal adhesions within
- After incorporation of the injected protein,
darken the room and search for interesting cells using
epifluorescence and preferably a 40x oil immersion
lens. In case no fluorescent cells can be found by eye,
it will be difficult to pick up a satisfying signal using
a back-illuminated CCD camera.
- After selection of an interesting cell, switch to
higher magnification (100x) with or without optovar
intermediate magnification and test for the minimal
exposure time required to get a good signal/noise
ratio. (For important details on fluorescence imaging
and image processing, see articles by Anderson and by
- In order to record a stack of fluorescent images,
set the appropriate number of images to be taken and the time period in between individual frames
(dependent on experiment). In order to avoid photodamage,
illuminate cells with strong fluorescent light
as rarely as possible. Avoid focusing in between frames
using epifluorescence; focus using transmitted light,
for example, in phase contrast, because for transmitted
optics, much lower lamp intensities are required.
IV. GENERAL COMMENTS ON
- Purify the expression vector for your GFP-tagged
open reading frame of interest using standard DNA
purification columns (e.g., Qiagen-tip 500); dilute the construct in H2O in concentrations of not less than
- Seed the cells into 3.5-cm-diameter dishes
(Falcon, Cat. No.: 35.3001) and allow them to attach
and spread for 12-24h.
- Transfect cells in 3.5-cm dishes 12-24 h according
to standard protocols, e.g., using FuGENE (Roche; 3 µl
FuGENE/µg DNA) or Superfect transfection reagent
(Qiagen; 6µl Superfect/µg DNA). In our hands, 1-2 µg
of DNA is sufficient per 3.5-cm-diameter dish.
- Detach transfected cells using trypsin and seed
onto coverslips. For studying the dynamics of GFPtagged
proteins during the actin-based motility of B16-
F1 melanoma cells, we coat coverslips with 25µg/ml
laminin in laminin-coating buffer (1 h at room temperature);
laminin stock (1mg/ml) is stored in small
aliquots at -20°C.
- Upon spreading (for B16-F1 cells, wait 3-5h),
mount coverslips on the microscope using an appropriate
open or closed chamber system, including a
heating device if required. Compensate for lack of CO2 by using HEPES-buffered growth media if needed.
Changing to HEPES-buffered media may require some
adaption time (dependent on cell type).
- Darken the room and search for transfected cells
with your eyes with epifluorescence preferably using
a 40x/1.3NA oil immersion lens.
- Upon identification of an interesting cell, proceed
as in steps 13 and 14 in Section III,A.
|FIGURE 2 Arp2/3 dynamics in motile B16-F1 melanoma
cell as revealed by transient expression of the
GFP-tagged novel isoform p16B (ARPC5B). (A) Note
that EGFP-p16B incorporated into the lamellipodial
actin meshwork at the cell periphery and into highly
dynamic surface ruffles, also known as actin flowers or
clouds, as expected. (B) The time-lapse sequence from
the region boxed in A (right panel: phase contrast)
reveals reorganisation of the Arp2/3 complex during
advancement of the cell periphery. Time is in minutes
and seconds. Note the relatively constant lamellipodium
width (double-headed arrow in B) during forward
movement and the virtual exclusion of this Arp subunit
from protruding filopodia (arrows).
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
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.
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.
|FIGURE 3 Examples of simultaneous visualisation of two distinct cytoskeletal components in the same
cell. (A) Dynamics of microtubule plus ends in fish fibroblast CAR. Microinjected Cy3-tubulin (red) combined
with ectopically expressed EGFP-EB1 (kind gift of A. S. Akhmanova, Rotterdam; green). Top image,
overview. Bottom images, zoom into the boxed region in the overview, subsequent frames, time shown in
minutes and seconds. Note that EB1 localises to the tips of growing microtubules (e.g., arrowheads in frames
0'00" and 0'40"), but is absent from pausing or shrinking microtubule ends (e.g., arrowhead in frame 1'20").
(B) EGFP-zyxin expressing CAR cell microinjected with TAMRA-α-actinin: As shown in Fig. 2B, the α-actinin
probe (top panel and red in the merged image at the bottom) mainly incorporates along stress fibers (arrowheads)
and in focal adhesions (arrows). Zyxin (middle panel and green at the bottom), thought to be recruited
to the cytoskeleton via α-actinin binding (Reinhard et at., 1999), shows a similar, although not identical distribution.
The merged image (bottom) reveals that zyxin (green) is enriched in focal adhesions, whereas α-actinin
(red) targets more prominently to stress fibres, indicating that the subcellular positioning of zyxin is
more complex than simple α-actinin-mediated recruitment.
|FIGURE 4 Simultaneous visualisation of two cytoskeletal
using two distinct fluorescent protein tags.
EGFP-β-tubulin (pseudocoloured red)
and dsRED-zyxin (kind gift
of A. Huttenlocher, Madison;
green) expressed ectopically in a CAR
fish fibroblast. The
combination reveals the formation and microtubule
targeting of focal adhesions on this slowly protruding cell
edge. Subsequent frames, time shown in minutes and
newly formed adhesions (frame 3'45") and
with them (arrowheads).
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.
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
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
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