11.1 Probes for Actin
The cytoskeleton is an essential component of a cell’s structure and one of the easiest
to label with fluorescent reagents. Section 11.1 describes our probes for both monomeric
actin (G-actin) and filamentous actin (F-actin); tubulin, tubulin conjugates and reagents
for tubulin and other cytoskeletal proteins are described in Section 11.2.
Unlabeled and Fluorescent Actin
Fluorescently labeled actin (Figure 11.1) is an important tool for investigating cytoskeleton dynamics in vivo.1–5 Molecular Probes offers highly purified actin from rabbit
muscle (A-12375), as well as fluorescent actin conjugates labeled with two of our brightest and most photostable dyes. The green-fluorescent Alexa Fluor 488 actin conjugate
(A-12373) has excitation and emission maxima similar to fluorescein actin (Figure 7.54),
but it is brighter and more photostable, and its spectra are much less pH dependent. The
red-orange–fluorescent Alexa Fluor 568 actin conjugate (A-12374, Figure 11.2) is more
fluorescent than the spectrally similar Lissamine rhodamine B conjugate.
Both of our fluorescent actin conjugates are prepared by reacting amine residues of
polymerized F-actin with the succinimidyl ester of the appropriate dye using a modification of the method described by Alberts and co-workers.2 After labeling, the conjugates
are subjected to depolymerization and subsequent polymerization to ensure that the actin
conjugates are able to assemble properly. The labeled actin that polymerizes is then
separated from remaining monomeric actin by centrifugation, depolymerized and packaged in monomeric form.
Phallotoxins for F-Actin
Figure 11.1 Ribbon diagram of the structure of uncomplexed actin in the ADP state. The four subdomains are represented in different colors, and ADP
is bound at the center where the four subdomains
meet. Four Ca2+ ions bound to the actin monomer
are represented as gold spheres. In this structure,
tetramethylrhodamine-5-maleimide (T-6027) has
been used to covalently attach the dye to a specific
cysteine residue (Cys 374). Figure provided by
Roberto Dominguez, Boston Biomedical Research
Institute, Watertown, Massachusetts. Reprinted
with permission from Science 293, 708 (2001).
Copyright 2001 American Association for the Advancement of Science.
Molecular Probes prepares numerous fluorescent and biotinylated derivatives of phalloidin and phallacidin for selectively labeling F-actin (Figure 11.3, Figure 11.4). Phallotoxins are bicyclic peptides isolated from the deadly Amanita phalloides mushroom.
They can be used interchangeably in most applications and bind competitively to the
same sites on F-actin. Table 11.1 lists the available phallotoxin derivatives, along with
their spectral properties. A detailed staining protocol is included with each phallotoxin
derivative and extensive bibliographies are available on our Web site. One vial of the
fluorescent phallotoxin contains sufficient reagent for staining ~300 microscope slide
preparations; one vial of biotin-XX phalloidin, which must be used at a higher concentration, contains sufficient reagent for ~50 microscope slide preparations. We also offer
unlabeled phalloidin (P-3457) for blocking F-actin staining by labeled phallotoxins and
for promoting actin polymerization.
Properties of Phallotoxin Derivatives
The fluorescent and biotinylated phallotoxin derivatives stain F-actin selectively at
nanomolar concentrations and are readily water soluble, thus providing convenient labels
for identifying and quantitating actin in tissue sections, cell cultures or cell-free preparations.6–10 F-actin in live neurons can be efficiently labeled using cationic liposomes containing fluorescent phallotoxins, such as BODIPY FL phallacidin 11 (B-607). This procedure permits the labeling of entire cell cultures with minimum disruption. Because
fluorescent phalloidin conjugates are not permeant to most live cells, they can be used to
detect cells that have compromised membranes. However, it has been reported that unlabeled phalloidin, and potentially dye-labeled phalloidins, can penetrate the membranes of
certain hypoxic cells.12 An extensive study on visualizing the actin cytoskeleton with
various fluorescent probes in cell preparations as well as live cells has been published.6
Labeled phallotoxins have similar affinity for both large and small filaments and bind
in a stoichiometric ratio of about one phallotoxin per actin subunit in both muscle and
nonmuscle cells; they reportedly do not bind to monomeric G-actin, unlike some antibodies against actin.8,13 Phallotoxins have further advantages over antibodies for actin labeling, in that 1) their binding properties do not change appreciably with actin from different
Figure 11.2 Chick embryo fibroblasts injected with
the Alexa Fluor 568 conjugate of actin from rabbit
muscle (A-12374). The cells were then fixed and
permeabilized, and the filamentous actin was
stained with coumarin phallacidin (C-606). The
double-exposure image was acquired using longpass filter sets appropriate for rhodamine and
DAPI. Image contributed by Heiti Paves, Laboratory
of Molecular Genetics, National Institute of Chemical Physics and Biophysics, Estonia.
Section 11.1
455
Table 11.1 Spectral characteristics of our F-actin–selective probes.
Cat #
Figure 11.3 Actin filaments of chick heart fibroblasts stained with rhodamine phalloidin (R-415).
The subcompartments in the cytoskeleton are
readily apparent and labeled as follows: sf, stress
fiber; lam, lamellipodium; fil/ms, filipodium/microspike; am, actin meshwork; arc, dorsal arc. Figure
reprinted from “Visualizing the Actin Cytoskeleton.” J. Small et al. Microscopy Research & Technique 47, 3–17 (1999). Reprinted by permission of
Wiley-Liss, Inc., a subsidiary of John Wiley &
Sons, Inc., and J. Victor Small.
Labeling actin in fixed cell preparations
with our fluorescent phalloidin conjugates is one of the easiest and most
reliable techniques in cell biology.
Alexa Fluor 488 phalloidin (A-12379)
is highly recommended as the best
green-fluorescent F-actin stain.
Ex/Em *
Approximate MW
A-22281
Alexa Fluor 350 phalloidin
Actin-Selective Probe
346/442
1100
C-606
Coumarin phallacidin
355/443
1100
N-354
NBD phallacidin
465/536
1040
A-12379
Alexa Fluor 488 phalloidin
495/518
1320
F-432
Fluorescein phalloidin
496/516 †
1175
O-7466
Oregon Green 488 phalloidin
496/520 †
1180
B-607
BODIPY FL phallacidin
505/512
1125
O-7465
Oregon Green 514 phalloidin
511/528 †
1281
E-7463
Eosin phalloidin
524/544
1500
A-22282
Alexa Fluor 532 phalloidin
531/554
1350
R-415
Rhodamine phalloidin
554/573 †
1250
A-22283
Alexa Fluor 546 phalloidin
556/573
1800
B-3475
BODIPY 558/568 phalloidin
558/569
1115
A-12380
Alexa Fluor 568 phalloidin
578/600
1590
A-12381
Alexa Fluor 594 phalloidin
580/609
1620
B-7464
BODIPY TR-X phallacidin
589/617
1400
T-7471
Texas Red-X phalloidin
591/608 †
1490
A-22284
Alexa Fluor 633 phalloidin
632/647
1900
A-22287
Alexa Fluor 647 phalloidin
650/668
1950
B-12382
BODIPY 650/665 phalloidin
647/661
1200
A-22285
Alexa Fluor 660 phalloidin
663/690
1750
A-22286
Alexa Fluor 680 phalloidin
679/702
1850
B-7474
Biotin-XX phalloidin
NA
1300
P-3457
Phalloidin
NA
790
* Excitation (Ex) and emission (Em) maxima, in nm. Spectra of phallotoxins are either in aqueous buffer,
pH 7–9 (denoted †) or in methanol. NA = Not applicable.
Figure 11.4 Fixed, permeabilized bovine pulmonary artery endothelial cells
were labeled with Texas Red-X phalloidin (T-7471), which stains F-actin, and
counterstained with DAPI (D-1306, D-3571, D-21490). The panels show the
unprocessed image (left panel), after deconvolution (middle panel) and after
456
deconvolution and 3-D reconstruction (right panel). The image was deconvolved using Huygens software (Scientific Volume Imaging, www.svi.nl). 3-D
reconstruction was performed using Imaris software (Bitplane AG).
Chapter 11 — Probes for Cytoskeletal Proteins
www.probes.com
species, including plants and animals; and 2) their nonspecific staining is negligible; thus,
the contrast between stained and unstained areas is high.
Phallotoxins shift actin’s monomer/polymer equilibrium toward the polymer, lowering
the critical concentration for polymerization as much as 30-fold.14,15 Furthermore, depolymerization of F-actin by cytochalasins, potassium iodide and elevated temperatures is
inhibited by phallotoxin binding. Because the phallotoxin derivatives are relatively small,
with approximate diameters of 12–15 Å and molecular weights below 2000 daltons, a
wide variety of actin-binding proteins — including myosin, tropomyosin, troponin and
DNase I — can still bind to actin after treatment with fluorescent phallotoxins. Even
more significantly, phallotoxin-labeled actin filaments retain certain functional characteristics; labeled glycerinated muscle fibers still contract, and labeled actin filaments still
move on solid-phase myosin substrates.16–18
Alexa Fluor Phalloidins
We have taken advantage of the outstanding characteristics of our Alexa Fluor dyes
(Section 1.3) to create a series of 10 different Alexa Fluor dye–labeled phalloidins (Figure 7.96, Figure 11.5, Figure 11.6, Figure 24.20), which are now the preferred F-actin
stains for most applications across the full spectral range. The Alexa Fluor phalloidin
conjugates provide researchers with fluorescent probes that are superior in brightness and
photostability to all other spectrally similar conjugates tested (Section 1.3, Figure 1.10).
Spectra of the 11 Alexa Fluor dyes are given in Figure 1.14, Figure 1.21 and Figure 1.30.
For improved fluorescence detection of F-actin in fixed and permeabilized cells, we
encourage researchers to try these fluorescent phalloidins in their actin-labeling protocols. A series of videos showing Alexa Fluor 488 phalloidin–stained actin 19 is available
at the Journal of Cell Biology Web site (www.jcb.org/cgi/content/full/150/2/361/DC1).
Figure 11.5 Actin filaments of the turbellarian flatworm Archimonotresis sp. stained with Alexa
Fluor 488 phalloidin (A-12379) to reveal a meshwork of longitudinal, circular and diagonal muscles. The large, bright ring with muscle fibers radiating outward is the muscular pharynx, and the
small, bright ring at the posterior is part of the reproductive system. This epifluorescence image
was contributed by Matthew D. Hooge and Seth
Tyler, Department of Biological Sciences, University of Maine, Orono, Maine.
Oregon Green Phalloidins
Green-fluorescent actin stains are popular reagents for labeling F-actin in fixed and
permeabilized cells. Unfortunately, the green-fluorescent fluorescein phalloidin and NBD
phallacidin photobleach rapidly, making their photography difficult. We have used two of
our Oregon Green dyes (Section 1.5) to prepare Oregon Green 488 phalloidin (O-7466)
and the slightly longer-wavelength Oregon Green 514 phalloidin (O-7465, Figure 11.7).
The excitation and emission spectra of the Oregon Green 488 dye are virtually superimposable on those of fluorescein, and both the Oregon Green 488 and Oregon Green 514
dyes may be viewed with standard fluorescein optical filter sets (Table 24.6). As shown
in Figure 11.8, Oregon Green 514 phalloidin is more photostable than fluorescein phalloidin, making it easier to visualize and photograph (Figure 1.57).
BODIPY Phallotoxins
BODIPY phallotoxin conjugates (B-607, B-3475, B-7464, B-12382; Figure 8.109,
Figure 11.9, Figure 11.10) have some important advantages over the conventional NBD,
fluorescein and rhodamine phallotoxins. The BODIPY FL, BODIPY 558/568 and
BODIPY TR-X fluorophores exhibit excitation and emission spectra similar to those of
fluorescein, rhodamine B and Texas Red dyes, respectively, and can be used with standard optical filter sets (Table 24.8).
BODIPY 650/665 phalloidin (B-12382) is the longest-wavelength BODIPY phallotoxin conjugate available, increasing the options for multicolor analysis. BODIPY 650/
665 phalloidin, Alexa Fluor 647 phalloidin (A-22287) and Alexa Fluor 660 phalloidin
(A-22285) are among the few probes available that can be excited by the 647 nm spectral
line of the Ar–Kr laser used in many confocal laser-scanning microscopes.
Furthermore, BODIPY dyes are more photostable than these traditional fluorophores 20
and have narrower emission bandwidths (Figure 1.39), making them especially useful for
double- and triple-labeling experiments. BODIPY FL phallacidin (B-607), which reportedly gives a signal superior to that of fluorescein phalloidin,21 has been used for quantitating F-actin and determining its distribution in cells.22,23
Figure 11.6 Subcellular structures in fixed and
permeabilized bovine pulmonary arterial endothelial cells visualized with several fluorescent dyes.
Filamentous actin (F-actin) was identified with
Alexa Fluor 633 phalloidin (A-22284), which is
pseudocolored magenta. Lipophilic regions of the
cell, including intracellular membranes, were
stained with green-fluorescent DiOC6(3) (D-273).
Finally, nuclei were counterstained with blue-fluorescent DAPI (D-1306, D-3571, L-12490). The image was acquired using filters appropriate for fluorescein and DAPI and a special filter (courtesy of
Omega Optical) for the Alexa Fluor 633 dye, consisting of a narrow band exciter (630DF10), dichroic (640DRLP) and emitter (660DF10).
Rhodamine Phalloidin and Other Red-Fluorescent Phalloidins
Rhodamine phalloidin (R-415, Figure 11.3) has been the standard for red-fluorescent
phallotoxins, with more than 1300 citations in our bibliography database. Rhodamine
phalloidin is excited efficiently by the mercury-arc lamp in most fluorescence microscopes.
Section 11.1
457
However, our Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594 and Texas Red-X phalloidins 24 (A-22283, A-12380, A-12381, T-7471; Figure 7.73, Figure 11.11, Figure 11.12)
will be welcome replacements for rhodamine phalloidin in many multicolor applications
because their emission spectra are better separated from those of the green-fluorescent
Alexa Fluor 488, Oregon Green and fluorescein dyes. Moreover, the Alexa Fluor 568 and
Texas Red-X conjugates can be excited by the 568 nm spectral line of the Ar–Kr laser used
in several confocal laser-scanning microscopes, whereas the tetramethylrhodamine dye
used to prepare rhodamine phalloidin is poorly excited by this laser.
Figure 11.7 Simultaneous visualization of F- and
G-actin in a bovine pulmonary artery endothelial
cell (BPAEC) using F-actin–specific Oregon Green
488 phalloidin (O-7466) and G-actin–specific Texas
Red deoxyribonuclease I (D-972). The G-actin appears as diffuse red fluorescence that is more intense in the nuclear region where the cell thickness
is greater and stress fibers are less dense. The image was obtained by taking multiple exposures
through bandpass optical filter sets appropriate for
fluorescein and Texas Red.
Other Labeled Phallotoxins, Including Eosin Phalloidin
The original yellow-green–fluorescent NBD phallacidin (N-354) and green-fluorescent fluorescein phalloidin (F-432) remain in use despite their relatively poor photostability (Figure 11.8). Photostability of fluorescein phalloidin and some other fluorescent
phallotoxins can be considerably improved (Figure 24.22) by mounting the stained samples with our Prolong antifade reagent (in Kit P-7481, Section 24.1). We recommend the
Alexa Fluor 488 (Figure 11.7), Oregon Green 488, Oregon Green 514 and BODIPY FL
phallotoxins as the preferred green-fluorescent actin stains. Alexa Fluor 350 phalloidin
(A-22281) and coumarin phallacidin (C-606, Figure 11.2) are the only blue-fluorescent
phallotoxin conjugates currently available for staining actin.26
We have also prepared eosin phalloidin (E-7463), which may be useful for correlated
fluorescence and electron microscopy studies (see Fluorescent Probes for Photoconversion of Diaminobenzidine Reagents in Section 1.5). Deerinck and colleagues have reported that eosin-mediated photooxidation of diaminobenzidine followed by treatment with
osmium tetroxide yields an insoluble, electron-dense DAB oxidation product that can be
visualized by either light or electron microscopy, allowing 3-D reconstructions at the
electron microscopy level.24,27 Biotin-XX phalloidin (B-7474) also permits detection of
F-actin by electron microscopy and light microscopy techniques.28 This biotin conjugate
can be visualized with fluorophore- or enzyme-labeled avidin and streptavidin (Section
7.6), with tyramide signal-amplification (TSA) technology (Section 6.2), with our novel
ELF signal-amplification technology (Figure 6.24), or potentially with NANOGOLD or
Alexa Fluor FluoroNanogold streptavidin (Section 7.6). Biotin-XX phalloidin, in conjunction with streptavidin or Captavidin agarose (S-951, C-21386; Section 7.6), can be
used to precipitate F-actin from the cytosolic anti-phosphotyrosine–reactive fraction in
macrophages stimulated with colony-stimulating factor-1.29
DNase I Conjugates for Staining G-Actin
Figure 11.8 Photostability comparison for Oregon
Green 514 phalloidin (O-7465) and fluorescein
phalloidin (F-432). CRE BAG 2 fibroblasts were
fixed with formaldehyde, permeabilized with acetone and then stained with the fluorescent phallotoxins. Samples were continuously illuminated and
images were acquired every five seconds using a
Star 1 CCD camera (Photometrics); the average
fluorescence intensity in the field of view was calculated with Image-1 software (Universal Imaging
Corp.) and expressed as a fraction of the initial intensity. Three data sets, representing different
fields of view, were averaged for each labeled phalloidin to obtain the plotted time courses.
458
Bovine pancreatic deoxyribonuclease (DNase I, ~31,000 daltons) binds to monomeric
G-actin with an affinity of about 5 × 108 M-1.30–34 Like unlabeled DNase I, our fluorescent DNase I conjugates (Table 11.2) selectively label G-actin and have proven very
useful for detecting and quantitating the proportion of unpolymerized actin in a cell.
Molecular Probes’ scientists have triple-labeled endothelial cells with fluorescein DNase I,
BODIPY 581/591 phalloidin and a monoclonal anti-actin antibody detected with a Cascade Blue dye–labeled secondary antibody 35 (C-962, Section 7.3, Table 7.3). They found
that the monoclonal antibody, which binds to both G-actin and F-actin, co-localized with
the DNase I and phalloidin conjugates, suggesting that these three probes recognize
unique binding sites on the actin molecule. Researchers can choose fluorescein (D-970),
Alexa Fluor 488 (D-12371), Oregon Green 488 (D-7497), Alexa Fluor 594 (D-12372) or
Texas Red (D-972) DNase I conjugates (Table 11.2), depending on their multicolor
application and their detection instrumentation.
Fluorescein DNase I and the Alexa Fluor 488 and Alexa Fluor 594 DNase I conjugates
have been used in combination with fluorescently labeled phallotoxins to simultaneously
visualize G-actin pools and filamentous F-actin 35–40 and to study the disruption of microfilament organization in live nonmuscle cells.41 Rhodamine phalloidin (R-415) has
been used in conjunction with Oregon Green 488 DNase I to determine the F-actin:
G-actin ratio in Dictyostelium using confocal laser-scanning microscopy.42 A mouse
fibroblast labeled with both Texas Red DNase I and Oregon Green 488 phalloidin
(O-7466) permitted visualization of G-actin and the complex network of F-actin throughout the cytoplasm, as well as at the cell periphery (Figure 11.7). The influence of cytocha-
Chapter 11 — Probes for Cytoskeletal Proteins
www.probes.com
lasins on actin structure in monocytes has been quantitated by flow cytometry using Texas
Red DNase I and BODIPY FL phallacidin (B-607) to stain the G-actin and F-actin pools,
respectively.43 Fluorescent DNase I has also been used as a model system to study the
interactions of nucleotides, cations and cytochalasin D with monomeric actin.44
Probes and Assays for Actin Quantitation and Polymerization
Assays for Quantitating F-Actin and G-Actin Polymerization
Quantitative assays for F-actin have employed fluorescein phalloidin,45,46 rhodamine
phalloidin,47 BODIPY FL phallacidin 23 and NBD phallacidin.48 An F-actin assay based
on fluorescein phalloidin was used to demonstrate the loss of F-actin from cells during
apoptosis.49 The addition of propidium iodide (P-1304, P-3566; FluoroPure Grade,
P-21493; Section 8.1) to the cell suspensions enabled these researchers to estimate the
cell-cycle distributions of both the apoptotic and nonapoptotic cell populations. The
change in F-actin content in proliferating adherent cells has been quantitated using the
ratio of rhodamine phalloidin fluorescence to ethidium bromide fluorescence.50 The
spectral separation of the signals in this assay may be improved by using a green-fluorescent stain for F-actin and a high-affinity red-fluorescent nucleic acid stain, such as the
combination of Alexa Fluor 488 phalloidin (A-12379) and ethidium homodimer-1
(E-1169, Section 8.1).
The fluorescence of actin monomers labeled with pyrene iodoacetamide (P-29) has
been demonstrated to change upon polymerization, making this probe an excellent tool
for following the kinetics of actin polymerization and the effects of actin-binding proteins
on polymerization.51–53
Figure 11.9 FluoCells prepared slide #1 (F-14780),
consisting of bovine pulmonary artery endothelial
cells incubated with MitoTracker Red CMXRos
(M-7512) to label the mitochondria. After fixation
and permeabilization, the cells were stained with
BODIPY FL phallacidin (B-607) to label the filamentous actin (F-actin) and finally counterstained
with DAPI (D-1306, D-3571, D-21490) to label the
nucleus. The multiple-exposure image was acquired using bandpass filters appropriate for Texas
Red dye, fluorescein and DAPI.
Jasplakinolide — A Cell-Permeant F-Actin Probe
Molecular Probes offers jasplakinolide (J-7473, Figure 11.13), a macrocyclic peptide
isolated from the marine sponge Jaspis johnstoni.54–56 Jasplakinolide is a potent inducer
of actin polymerization in vitro by stimulating actin filament nucleation 57,58 and competes with phalloidin for actin binding (Kd = 15 nM).59 Moreover, unlike other known
actin stabilizers such as phalloidins and virotoxins, jasplakinolide appears to be somewhat cell permeant and therefore can potentially be used to manipulate actin polymerization in live cells. This peptide, which also exhibits fungicidal, insecticidal and antiproliferative activity,55,60–62 is particularly useful for investigating cell processes mediated by
actin polymerization and depolymerization, including cell adhesion, locomotion, endocytosis and vesicle sorting and release. Jasplakinolide has been reported to enhance apoptosis induced by cytokine deprivation.63
Latrunculin A and Latrunculin B — Cell-Permeant Actin Antagonists
Latrunculins are powerful disruptors of microfilament organization. Isolated from a
Red Sea sponge, these G-actin binding compounds inhibit fertilization and early embryological development,64 alter the shape of cells 65,66 and inhibit receptor-mediated endocytosis.67 Latrunculin A (L-12370, Figure 11.14) binds to monomeric G-actin in a 1:1 ratio
at submicromolar concentrations 63,65,66,68,69 and is frequently used to establish the effects
of F-actin disassembly on particular physiological functions such as ion transport 70 and
protein localization.71 The activity of latrunculin B (L-22290) mimics that of latrunculin
A in most applications.65,67,72–74
Table 11.2 Spectral characteristics of our G-actin–selective probes.
Cat #
Actin-Selective Probe
Ex/Em *
D-970
DNase I, fluorescein conjugate
494/517
D-12371
DNase I, Alexa Fluor 488 conjugate
495/519
D-7497
DNase I, Oregon Green 488 conjugate
496/516
D-12372
DNase I, Alexa Fluor 594 conjugate
590/617
D-972
DNase I, Texas Red conjugate
597/618
Figure 11.10 Actin labeled with BODIPY FL phallacidin (B-607) and vinculin, a cytoskeletal focal adhesion protein, tagged with a monoclonal anti-vinculin antibody that was subsequently probed with
Texas Red goat anti–mouse IgG antibody (T-862).
The large triangular cell is a fibroblast containing
green actin stress fibers terminating in red focal
adhesions. The neighboring polygonal cell, a rat
neonatal cardiomyocyte, contains green striated
actin in the myofibrils terminating in the focal adhesions. The close apposition of the two stains results in a yellowish-orange color. Image contributed by Mark B. Snuggs and W. Barry VanWinkle,
University of Texas, Houston.
* Excitation/emission maxima, in nm. Spectra of the DNase I conjugates are in aqueous buffer, pH 7–8.
Section 11.1
459
Fluorescent Cytochalasins
Our fluorescent cytochalasin derivatives promise to be useful probes for live-cell
staining of actin filaments. Cytochalasins are a group of natural compounds that bind to
actin and alter its polymerization. Activities reported for cytochalasin D, which binds to
the barbed (faster-growing) end of actin with high affinity (Kd ~50 nM),75 include capping the barbed end of actin, cleaving actin filaments and increasing the rate of actin
assembly. Cytochalasin B, which binds elsewhere on actin, has been shown to increase
the rate of actin assembly and is not believed to have a capping activity. We have prepared the green-fluorescent BODIPY FL and orange-fluorescent BODIPY TMR derivatives of cytochalasin D (C-12377, C-12378) and the green-fluorescent BODIPY FL
derivative of cytochalasin B (C-12376). BODIPY TMR cytochalasin D has been shown
to colocalize with Oregon Green phalloidin in NIH 3T3 fibroblasts. Migrating human
neutrophils appear to show fluorescent cytochalasin D staining approximately 1–2 µm
inside the leading edge.68
Figure 11.11 A section of mouse intestine stained
with a combination of fluorescent stains. Fibronectin, an extracellular matrix adhesion molecule, was
labeled using a chicken primary antibody against
fibronectin and visualized using green-fluorescent
Alexa Fluor 488 goat anti–chicken IgG antibody
(A-11039). The filamentous actin (F-actin) prevalent in the brush border was stained with red-fluorescent Alexa Fluor 568 phalloidin (A-12380). Finally, the nuclei were stained with DAPI (D-1306,
D-3571, D-21490).
Cofilin — An F-Actin Depolymerizing Factor
Molecular Probes offers high-purity, recombinant chicken muscle cofilin (C-22280),
isolated from Escherichia coli. Cofilin, along with the related actin–depolymerizing
factor (ADF), promotes the depolymerization of actin filaments in vivo, a process that
is required for a variety of cellular responses, including cytokinesis, chemotaxis and
formation of lamellipodia.76–79 This low molecular weight protein (~18,800 daltons) is
ubiquitous in tissues of eukaryotes and particularly abundant in embryonic tissue and
in developing and degenerating muscle. At pH <7.0, cofilin complexes with F-actin at a
stoichiometry of 1:1 with the actin subunits; its name is derived from this cofilamentous
structure. At pH >7.0, cofilin causes an increase in the G-actin pool and, in muscle,
favors dissociation from the pointed (minus) ends of actin filaments. Cofilin binding to Factin results in a loss of the phalloidin binding site and is also competitive with tropomyosin binding. The activity of cofilin in vivo is regulated by the phosphorylation of cofilin
by LIM kinase at a single serine residue in the N-terminal region. Phosphorylated cofilin
does not bind to either G-actin or F-actin. LIM kinase is, in turn, regulated by Rho, a
small GTPase of the Ras family.80,81
Molecular Probes’ cofilin preparation has an estimated purity of >99% by SDS-polyacrylamide gel electrophoresis, and its actin-binding activity is confirmed by its comigration with G-actin in native gel electrophoresis. The binding constant for our cofilin
to the ATP-form of G-actin is ~0.2 µM.
Assays for Actin-Binding Proteins
Enhancement of the fluorescence of certain phallotoxins upon binding to F-actin can
be a useful tool for following the kinetics and extent of binding of specific actin-binding
Figure 11.12 Confocal micrograph of the cytoskeleton of a mixed population of granule neurons and
glial cells. The F-actin was stained with red-fluorescent Texas Red-X phalloidin (T-7471). The microtubules were detected with a mouse monoclonal
anti–β-tubulin primary antibody and subsequently
visualized with the green-fluorescent Alexa Fluor
488 goat anti–mouse IgG antibody (A-11001). Image contributed by Jonathan Zmuda, Immunomatrix, Inc.
Figure 11.13 J-7473 jasplakinolide.
460
Chapter 11 — Probes for Cytoskeletal Proteins
Figure 11.14 L-12370 latrunculin A.
www.probes.com
proteins. We have used the change in fluorescence of rhodamine
phalloidin (R-415) to determine the dissociation constant of
various phallotoxins 82. The enhancement of rhodamine phalloidin’s fluorescence upon actin binding has also been used to measure the kinetics and extent of gelsolin severing of actin filaments.83 In this study, the ion indicator mag-fura-5 (M-3103,
Section 20.2) was employed to determine the dependence of this
severing on divalent ion concentrations. The affinity and rate
constants for rhodamine phalloidin binding to actin are not affected by saturation of actin with either myosin subfragment-1 or
tropomyosin, indicating that these two actin-binding proteins do
not bind to the same sites as the phalloidin.11
In Section 11.2 are described our probes for tubulin and other
cytoskeletal proteins, including the following probes for actinbinding proteins:
• Recombinant Endostatin protein (E-23377), which binds to
tropomyosin, an actin-binding protein
• Fluorescent phosphoinositides and related probes, which bind
to actin-binding proteins, including cofilin I, through pleckstrin homology (PH) domains and other binding motifs
• An antibody to the actin-binding protein, synapsin I (A-6442)
References
1. Cell Struct Funct 22, 59 (1997); 2. Development
103, 675 (1988); 3. J Cell Biol 102, 1074 (1986);
4. J Cell Biol 101, 597 (1985); 5. Proc Natl Acad
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(1994).
Data Table — 11.1 Probes for Actin
Cat #
A-12379
A-12380
A-12381
A-22281
A-22282
A-22283
A-22284
A-22285
A-22286
A-22287
B-607
B-3475
B-7464
B-7474
B-12382
C-606
C-12376
C-12377
C-12378
E-7463
F-432
J-7473
L-12370
L-22290
N-354
O-7465
MW
~1320
~1590
~1620
~1100
~1350
~1800
~1900
~1650
~1850
~1950
~1160
~1115
~1400
~1300
~1200
~1100
753.69
781.70
887.83
~1500
~1175
709.68
421.55
395.51
~1040
~1280
Storage
F,L
F,L
F,L
F,L
F,L
F,L
F,L
F,L
F,L
F,L
F,L
F,L
F,L
F
F,L
F,L
F,D,L
F,D,L
F,D,L
F,L
F,L
F,D
F,D
F,D
F,L
F,L
Soluble
MeOH, H2O
MeOH, H2O
MeOH, H2O
MeOH, H2O
MeOH, H2O
MeOH, H2O
MeOH, H2O
MeOH, H2O
MeOH, H2O
MeOH, H2O
MeOH, H2O
MeOH, H2O
MeOH
MeOH, H2O
MeOH
MeOH, H2O
DMSO
DMSO
DMSO
MeOH, H2O
MeOH, H2O
MeOH
DMSO
DMSO
MeOH, H2O
MeOH, H2O
Abs
494
578
593
346
528
554
621
668
684
650
505
558
589
<300
647
355
503
504
545
524
496
278
<300
<300
465
511
EC
78,000
88,000
92,000
17,000
81,000
112,000
159,000
132,000
183,000
275,000
83,000
85,000
62,000
102,000
16,000
80,000
80,000
56,000
100,000
84,000
8,000
24,000
85,000
Em
517
600
617
446
555
570
639
697
707
672
512
569
617
none
661
443
510
511
571
544
516
none
none
none
536
528
Solvent
pH 7
pH 7
pH 7
pH 7
pH 7
pH 7
MeOH
MeOH
MeOH
MeOH
MeOH
MeOH
MeOH
MeOH
MeOH
MeOH
MeOH
MeOH
MeOH
pH 8
MeOH
MeOH
pH 9
Notes
1, 2, 3
1, 2, 3
1, 2, 3
1, 2, 3
1, 2, 3
1, 2, 3
1, 2, 3, 4
1, 2, 3, 4
1, 2, 3, 4
1, 2, 3, 4
1, 2, 3
1, 2, 3
1, 3, 5
1, 2
1, 3, 5
1, 2, 3
1, 2, 3
1, 2, 3
1, 2, 3
1, 2, 3
Section 11.1
461
Data Table — 11.1 Probes for Actin — continued
Cat #
O-7466
P-29
P-3457
R-415
T-7471
MW
~1180
385.20
~790
~1250
~1490
Storage
F,L
F,D,L
F
F,L
F,L
Soluble
MeOH, H2O
DMF, DMSO
MeOH, H2O
MeOH, H2O
MeOH, H2O
Abs
496
339
<300
542
583
EC
86,000
26,000
85,000
95,000
Em
520
384
see Notes
565
603
Solvent
pH 9
MeOH
MeOH
MeOH
Notes
1, 2, 3
6, 7
2, 8
1, 2, 3, 9
1, 2, 3, 9
For definitions of the contents of this data table, see “How to Use This Book” on page viii.
Notes
1. Phallotoxin conjugates have approximately one label per peptide.
2. Although this phallotoxin is water soluble, storage in water is not recommended, particularly in dilute solution.
3. The value of EC listed for this phallotoxin conjugate is for the labeling dye in free solution. Use of this value for the conjugate assumes a 1:1 dye:peptide labeling ratio and no change
of EC due to dye–peptide interactions.
4. In aqueous solutions (pH 7.0), Abs/Em = 625/645 nm for A-22284, 661/689 nm for A-22285, 677/699 nm for A-22286 and 649/666 nm for A-22287.
5. B-7464 and B-12382 are not directly soluble in H2O. Aqueous dispersions can be prepared by dilution of a stock solution in MeOH.
6. Spectral data of the 2-mercaptoethanol adduct.
7. Iodoacetamides in solution undergo rapid photodecomposition to unreactive products. Minimize exposure to light prior to reaction.
8. This bicyclic peptide is very weakly fluorescent in aqueous solution (Em ~380 nm) (Biochim Biophys Acta 760, 411 (1983)).
9. In aqueous solutions (pH 7.0), Abs/Em = 554/573 nm for R-415 and 591/608 nm for T-7471.
Product List — 11.1 Probes for Actin
Cat #
Product Name
A-12375
A-12373
A-12374
A-22281
A-12379
A-22282
A-22283
A-12380
A-12381
A-22284
A-22287
A-22285
A-22286
B-7474
B-607
B-3475
B-12382
B-7464
C-22280
C-606
C-12376
C-12377
C-12378
D-12371
D-12372
D-970
D-7497
D-972
E-7463
F-432
J-7473
L-12370
L-22290
N-354
O-7466
O-7465
P-3457
P-29
R-415
T-7471
actin from rabbit muscle ......................................................................................................................................................................................
actin from rabbit muscle, Alexa Fluor® 488 conjugate *in solution* ....................................................................................................................
actin from rabbit muscle, Alexa Fluor® 568 conjugate *in solution* ....................................................................................................................
Alexa Fluor® 350 phalloidin ..................................................................................................................................................................................
Alexa Fluor® 488 phalloidin ..................................................................................................................................................................................
Alexa Fluor® 532 phalloidin ..................................................................................................................................................................................
Alexa Fluor® 546 phalloidin ..................................................................................................................................................................................
Alexa Fluor® 568 phalloidin ..................................................................................................................................................................................
Alexa Fluor® 594 phalloidin ..................................................................................................................................................................................
Alexa Fluor® 633 phalloidin ..................................................................................................................................................................................
Alexa Fluor® 647 phalloidin ..................................................................................................................................................................................
Alexa Fluor® 660 phalloidin ..................................................................................................................................................................................
Alexa Fluor® 680 phalloidin ..................................................................................................................................................................................
biotin-XX phalloidin ..............................................................................................................................................................................................
BODIPY® FL phallacidin ........................................................................................................................................................................................
BODIPY® 558/568 phalloidin ................................................................................................................................................................................
BODIPY® 650/665 phalloidin ................................................................................................................................................................................
BODIPY® TR-X phallacidin ...................................................................................................................................................................................
cofilin, chicken muscle, recombinant from Escherichia coli .................................................................................................................................
coumarin phallacidin ............................................................................................................................................................................................
cytochalasin B, BODIPY® FL conjugate ................................................................................................................................................................
cytochalasin D, BODIPY® FL conjugate ................................................................................................................................................................
cytochalasin D, BODIPY® TMR conjugate ............................................................................................................................................................
deoxyribonuclease I, Alexa Fluor® 488 conjugate .................................................................................................................................................
deoxyribonuclease I, Alexa Fluor® 594 conjugate .................................................................................................................................................
deoxyribonuclease I, fluorescein conjugate ..........................................................................................................................................................
deoxyribonuclease I, Oregon Green® 488 conjugate ............................................................................................................................................
deoxyribonuclease I, Texas Red® conjugate .........................................................................................................................................................
eosin phalloidin ....................................................................................................................................................................................................
fluorescein phalloidin ...........................................................................................................................................................................................
jasplakinolide ........................................................................................................................................................................................................
latrunculin A .........................................................................................................................................................................................................
latrunculin B .........................................................................................................................................................................................................
N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)phallacidin (NBD phallacidin) .....................................................................................................................
Oregon Green® 488 phalloidin ..............................................................................................................................................................................
Oregon Green® 514 phalloidin ..............................................................................................................................................................................
phalloidin ..............................................................................................................................................................................................................
N-(1-pyrene)iodoacetamide ..................................................................................................................................................................................
rhodamine phalloidin ............................................................................................................................................................................................
Texas Red®-X phalloidin .......................................................................................................................................................................................
462
Unit Size
Chapter 11 — Probes for Cytoskeletal Proteins
1 mg
200 µg
200 µg
300 U
300 U
300 U
300 U
300 U
300 U
300 U
300 U
300 U
300 U
50 U
300 U
300 U
300 U
300 U
50 µg
300 U
100 µg
100 µg
100 µg
5 mg
5 mg
5 mg
5 mg
5 mg
300 U
300 U
100 µg
100 µg
100 µg
300 U
300 U
300 U
1 mg
100 mg
300 U
300 U
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