The cytoskeleton is an essential component of a cell's structure and one of the easiest to label with fluorescent reagents. This section describes Molecular Probes labeling reagents for both monomeric actin (G-actin) and filamentous actin (F-actin); reagents for staining tubulin and other cytoskeletal proteins are described in Probes for Tubulin and Other Cytoskeletal Proteins—Section 11.2.
Alexa Fluor Actin and Unlabeled Actin
Fluorescently labeled actin (Figure 11.1.1) is an important tool for investigating the structural dynamics of the cytoskeleton. We offer highly purified actin from rabbit muscle (A12375), as well as fluorescent actin conjugates labeled with four of our brightest and most photostable dyes. The green-fluorescent Alexa Fluor 488 actin conjugate (A12373) has excitation and emission maxima similar to fluorescein actin, but it is brighter and more photostable, and its spectra are much less pH dependent. The red-orange–fluorescent Alexa Fluor 568 (A12374, ), red-fluorescent Alexa Fluor 594 (A34050) and far-red–fluorescent Alexa Fluor 647 (A34051) actin conjugates are more fluorescent than the spectrally similar Lissamine rhodamine B, Texas Red and Cy5 conjugates, respectively.
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. 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.
Figure 11.1.1 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 (T6027) has been used to covalently attach the dye to a specific cysteine residue (Cys 374). Image provided by Roberto Dominguez, Boston Biomedical Research Institute, Watertown, Massachusetts. Reprinted with permission from Science (2001) 293:708. Copyright 2001 American Association for the Advancement of Science.
GFP- and RFP-Labeled Actin
The requirement for intracellular delivery of Alexa Fluor dye–labeled actin conjugates by microinjection typically limits their applications for live cell imaging to experiments involving no more than a few (<10) cells. For applications such as high-content screening (HCS) assays requiring larger sample sizes, GFP–actin fusions are well-established probes for imaging cytoskeletal structure and dynamics. CellLight Actin-GFP (C10506, C10582), CellLight Actin-RFP (C10502, C10583; Figure 11.1.2) expression vectors (CellLight reagents and their targeting sequences—Table 11.1) generate autofluorescent proteins fused to the N-terminus of human β-actin and incorporate all the generic advantages of BacMam delivery technology (BacMam Gene Delivery and Expression Technology—Note 11.1). In particular, the viral dose can be readily adjusted to modulate expression levels if GFP- or RFP-dependent perturbation of cellular structural or functional properties is a concern.
Figure 11.1.2 HeLa cell labeled with CellLight Actin-RFP (C10502, C10583) and CellLight MAP4-GFP (C10598) reagents and with Hoechst 33342 nucleic acid stain.
CellLight Null Control Reagent
The CellLight Null (control) reagent (C10615), a suspension of baculovirus particles lacking mammalian genetic elements, is designed for use in parallel with our CellLight targeted fluorescent protein reagents (CellLight reagents and their targeting sequences—Table 11.1). For example, microarray expression analysis on cells treated with the CellLight Null (control) reagent can be used to assess down-regulation or up-regulation of host cell genes elicited by baculovirus infection.
We prepare a number of fluorescent and biotinylated derivatives of phalloidin and phallacidin for selectively labeling F-actin. Phallotoxins are bicyclic peptides isolated from the deadly Amanita phalloides mushroom (www.grzyby.pl/gatunki/Amanita_phalloides.htm). They can be used interchangeably in most applications and bind competitively to the same sites on F-actin. Spectral characteristics of Molecular Probes actin-selective probes—Table 11.2 lists the available phallotoxin derivatives, along with their spectral properties.
A detailed staining protocol (Phallotoxins) is included with each phallotoxin derivative, and extensive product bibliographies are available at www.invitrogen.com. 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 (P3457) 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. F-actin in live neurons can be efficiently labeled using cationic liposomes containing fluorescent phallotoxins, such as BODIPY FL phallacidin (B607). 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. An extensive study on visualizing the actin cytoskeleton with various fluorescent probes in cell preparations, as well as in live cells, has been published.
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. Phallotoxins have further advantages over antibodies for actin labeling, in that 1) their binding properties do not change appreciably with actin from different 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. 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.
Alexa Fluor Phalloidins
We have taken advantage of the outstanding fluorescence characteristics of our Alexa Fluor dyes (Alexa Fluor Dyes Spanning the Visible and Infrared Spectrum—Section 1.3) to create a series of Alexa Fluor dye–labeled phalloidins (, , , ), 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 (). 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 is available at the Journal of Cell Biology web site (www.jcb.org/cgi/content/full/150/2/361/DC1).
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 (Fluorescein, Oregon Green and Rhodamine Green Dyes—Section 1.5) to prepare Oregon Green 488 phalloidin (O7466, ) and the slightly longer-wavelength Oregon Green 514 phalloidin (O7465). 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. As shown in Figure 11.1.3, Oregon Green 514 phalloidin is more photostable than fluorescein phalloidin, making it easier to visualize and photograph.
Figure 11.1.3 Photostability comparison for Oregon Green 514 phalloidin (O7465) and fluorescein phalloidin (F432). 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 5 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.
BODIPY phallotoxin conjugates (B607, B3475, B12382; , ) have some important advantages over the conventional NBD, fluorescein and rhodamine phallotoxins. BODIPY dyes are more photostable than these traditional fluorophores and have narrower emission bandwidths (Fluorescein, Oregon Green and Rhodamine Green Dyes—Section 1.5), making them especially useful for double- and triple-labeling experiments. BODIPY FL phallacidin (B607), which reportedly gives a signal superior to that of fluorescein phalloidin, has been used for quantitating F-actin and determining its distribution in cells.
The BODIPY FL and BODIPY 558/568 phallotoxins (B607, B3475) exhibit excitation and emission spectra similar to those of fluorescein and rhodamine B, respectively, and can be used with standard optical filter sets. BODIPY 650/665 phalloidin (B12382) is the longest-wavelength BODIPY phallotoxin conjugate available, increasing the options for multicolor analysis. BODIPY 650/665 phalloidin, Alexa Fluor 647 phalloidin (A22287) and Alexa Fluor 660 phalloidin (A22285) are among the few probes available that can be excited by the 647 nm spectral line of the Ar-Kr laser.
Rhodamine Phalloidin and Other Red-Fluorescent Phalloidins
Rhodamine phalloidin (R415, ) has been the standard for red-fluorescent phallotoxins. Rhodamine phalloidin is excited efficiently by the mercury-arc lamp in most fluorescence microscopes. However, our Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594 and Texas Red-X phalloidins (A22283, A12380, A12381, T7471; , ) 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.
Other Labeled Phallotoxins
The original yellow-green–fluorescent NBD phallacidin (N354) and green-fluorescent fluorescein phalloidin (F432) remain in use despite their relatively poor photostability (Figure 11.1.3). Photostability of fluorescein phalloidin and some other fluorescent phallotoxins can be considerably improved () by mounting the stained samples with our ProLong Antifade Kit or ProLong Gold antifade reagent (P7481, P36930, P36934; Fluorescence Microscopy Accessories and Reference Standards—Section 23.1). We recommend the Alexa Fluor 488, Oregon Green 488, Oregon Green 514 and BODIPY FL phallotoxins as the preferred green-fluorescent actin stains. Alexa Fluor 350 phalloidin (A22281) is the only blue-fluorescent phallotoxin conjugate currently available for staining actin.
Biotin-XX phalloidin (B7474) also permits detection of F-actin by electron microscopy and light microscopy techniques. This biotin conjugate can be visualized with fluorophore- or enzyme-labeled avidin and streptavidin (Avidin, Streptavidin, NeutrAvidin and CaptAvidin Biotin-Binding Proteins and Affinity Matrices—Section 7.6) or with tyramide signal amplification (TSA) technology (TSA and Other Peroxidase-Based Signal Amplification Techniques—Section 6.2). Biotin-XX phalloidin, in conjunction with streptavidin or CaptAvidin agarose (S951, C21386; Avidin, Streptavidin, NeutrAvidin and CaptAvidin Biotin-Binding Proteins and Affinity Matrices—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.
Bovine pancreatic deoxyribonuclease (DNase I, ~31,000 daltons) binds much more strongly to monomeric G-actin than to filamentous F-actin, with binding constants of 5 × 108 M-1 and 1.2 × 104 M-1, respectively. Because of this strong, selective binding to G-actin, fluorescent DNase I conjugates have proven very useful for detecting and quantitating the proportion of unpolymerized actin in a cell. We 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 (C962, Secondary Immunoreagents—Section 7.2). We found that the monoclonal antibody, which binds to both G-actin and F-actin, colocalized with the DNase I and phalloidin conjugates, suggesting that these three probes recognize unique binding sites on the actin molecule. Researchers can choose DNase I conjugates labeled with either the green-fluorescent Alexa Fluor 488 (D12371) or red-fluorescent Alexa Fluor 594 (D12372) dyes, depending on their multicolor application and their detection instrumentation (Spectral characteristics of Molecular Probes actin-selective probes—Table 11.2).
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 and to study the disruption of microfilament organization in live nonmuscle cells. Rhodamine phalloidin (R415) 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. A mouse fibroblast labeled with both Texas Red DNase I and Oregon Green 488 phalloidin (O7466) permitted visualization of the G-actin and the complex network of F-actin throughout the cytoplasm, as well as at the cell periphery (). The influence of cytochalasins on actin structure in monocytes has been quantitated by flow cytometry using Texas Red DNase I and BODIPY FL phallacidin (B607) to stain the G-actin and F-actin pools, respectively. Fluorescent DNase I has also been used as a model system to study the interactions of nucleotides, cations and cytochalasin D with monomeric actin.
Assays for Quantitating F-Actin and G-Actin Polymerization
Quantitative assays for F-actin have employed fluorescein phalloidin, rhodamine phalloidin, BODIPY FL phallacidin and NBD phallacidin. An F-actin assay based on fluorescein phalloidin was used to demonstrate the loss of F-actin from cells during apoptosis. The addition of propidium iodide (P1304MP, P3566, P21493; Nucleic Acid Stains—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. 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 (A12379) and ethidium homodimer-1 (E1169, Nucleic Acid Stains—Section 8.1).
The fluorescence of actin monomers labeled with pyrene iodoacetamide (P29) 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.
Jasplakinolide: A Cell-Permeant F-Actin Probe
We offer jasplakinolide (J7473, ), a macrocyclic peptide isolated from the marine sponge Jaspis johnstoni. Jasplakinolide is a potent inducer of actin polymerization in vitro by stimulating actin filament nucleation and competes with phalloidin for actin binding (Kd = 15 nM). 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, 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.
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, alter the shape of cells and inhibit receptor-mediated endocytosis. Latrunculin A (L12370, ) binds to monomeric G-actin in a 1:1 ratio at submicromolar concentrations (Howard Petty, Wayne State University, personal communication) and is frequently used to establish the effects of F-actin disassembly on particular physiological functions such as ion transport and protein localization. The activity of latrunculin B (L22290) mimics that of latrunculin A in most applications.
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 proteins. We have used the change in fluorescence of rhodamine phalloidin (R415) to determine the dissociation constant of various phallotoxins. 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. 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.
|A12379||~1320||F,L||MeOH, H2O||494||78,000||517||pH 7||1, 2, 3|
|A12380||~1590||F,L||MeOH, H2O||578||88,000||600||pH 7||1, 2, 3|
|A12381||~1620||F,L||MeOH, H2O||593||92,000||617||pH 7||1, 2, 3|
|A22281||~1100||F,L||MeOH, H2O||346||17,000||446||pH 7||1, 2, 3|
|A22282||~1350||F,L||MeOH, H2O||528||81,000||555||pH 7||1, 2, 3|
|A22283||~1800||F,L||MeOH, H2O||554||112,000||570||pH 7||1, 2, 3|
|A22284||~1900||F,L||MeOH, H2O||621||159,000||639||MeOH||1, 2, 3, 4|
|A22285||~1650||F,L||MeOH, H2O||668||132,000||697||MeOH||1, 2, 3, 4|
|A22286||~1850||F,L||MeOH, H2O||684||183,000||707||MeOH||1, 2, 3, 4|
|A22287||~1950||F,L||MeOH, H2O||650||275,000||672||MeOH||1, 2, 3, 4|
|A34054||~1800||F,L||MeOH, H2O||622||145,000||640||MeOH||1, 2, 3, 4|
|A34055||~1900||F,L||MeOH, H2O||555||155,000||572||MeOH||1, 2, 3|
|B607||~1160||F,L||MeOH, H2O||505||83,000||512||MeOH||1, 2, 3|
|B3475||~1115||F,L||MeOH, H2O||558||85,000||569||MeOH||1, 2, 3|
|B7474||~1300||F||MeOH, H2O||<300||none||1, 2|
|B12382||~1200||F,L||MeOH||647||102,000||661||MeOH||1, 3, 5|
|F432||~1175||F,L||MeOH, H2O||496||84,000||516||pH 8||1, 2, 3|
|N354||~1040||F,L||MeOH, H2O||465||24,000||536||MeOH||1, 2, 3|
|O7465||~1280||F,L||MeOH, H2O||511||85,000||528||pH 9||1, 2, 3|
|O7466||~1180||F,L||MeOH, H2O||496||86,000||520||pH 9||1, 2, 3|
|P29||385.20||F,D,L||DMF, DMSO||339||26,000||384||MeOH||6, 7|
|P3457||~790||F||MeOH, H2O||<300||see Notes||2, 8|
|R415||~1250||F,L||MeOH, H2O||542||85,000||565||MeOH||1, 2, 3, 9|
|T7471||~1490||F,L||MeOH, H2O||583||95,000||603||MeOH||1, 2, 3, 9|