There are a variety of fluorescent and biotinylated phalloidins that label F-actin. These conjugates are water-soluble, allowing for convenient labeling, identifying, and quantitating of F-actin in tissue sections, cell cultures, and cell-free experiments. The binding properties of phalloidin conjugates do not change appreciably with actin from different species, including plants and animals. In addition, they exhibit negligible non-specific staining, resulting in a clear and distinct contrast between stained and unstained areas.
Phalloidin overview
Phalloidin is a bicyclic peptide that belongs to a family of toxins isolated from the deadly Amanita phalloides “death cap” mushroom and is commonly used in imaging applications to selectively label F-actin in fixed cells, permeabilized cells, and cell-free experiments. Phalloidin conjugates 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 non-muscle cells; they reportedly do not bind to monomeric G-actin.
Fluorescent phalloidin conjugates can be used with other actin-binding proteins such as myosin, troponin, and DNase I. Phalloidin-stained actin filaments are still functional and able to contract. Fluorescent phalloidins can also be used to quantitate the amount of F-actin in cells.
Selection guide for phalloidin conjugates
Alexa Fluor and Alexa Fluor Plus phalloidin conjugates
Fluorescent Alexa Fluor and Alexa Fluor Plus dye conjugates of phalloidin are widely used F-actin stains, with color choices across the full spectral range (Figures 1–4). These phalloidin conjugates provide researchers with fluorescent probes that are designed to be superior in brightness and photostability to all other spectrally similar conjugates tested.
Alexa Fluor Plus Phalloidin conjugates have 3–5 times more signal sensitivity and brightness compared to Alexa Fluor conjugates. This extra brightness is especially useful when conducting challenging F-actin imaging, such as with SIM, or STORM, or when reliable staining is needed of stress fibers.
Figure 1. Bovine pulmonary artery endothelial cells visualized using and Alexa Fluor phalloidin conjugates. Nuclei and F-actin were stained, respectively, with DAPI and Alexa Fluor 680 phalloidin (top left), SYTOX Green dye and Alexa Fluor 568 phalloidin (top right), 7-AAD and Alexa Fluor 488 phalloidin (bottom left), and TO-PRO 3 dye and Alexa Fluor 350 phalloidin (bottom right).
Figure 2. U2OS cells labeled with Alexa Fluor Plus 647 Phalloidin. U2OS cells labeled using NucBlue Live ReadyProbes Reagent, CellLight Talin-GFP BacMam 2.0, CellLight Mitochondria-RFP BacMam 2.0, and Alexa Fluor Plus 647 Phalloidin show multiplexing capability and staining specificity. Cells were mounted in ProLong Glass Antifade Mountant. Images were generated using an EVOS M7000 Imaging System with an Olympus 60X Apochromat Oil objective using DAPI, GFP, RFP, and Cy5 EVOS light cubes.
Figure 3. HeLa cells labeled with Alexa Fluor Plus 750 Phalloidin. HeLa cells labeled using NucBlue Live ReadyProbes Reagent, CellLight Talin-GFP BacMam 2.0, CellLight Mitochondria-RFP BacMam 2.0, and Alexa Fluor Plus 750 Phalloidin show multiplexing capability and staining specificity. Cells were mounted in ProLong Glass Antifade Mountant. Images were generated using an EVOS FL Auto 2 Imaging System with an Olympus 60X Apochromat Oil objective using DAPI, GFP, RFP, and Cy7 EVOS light cubes.
Figure 4. HeLa cells labeled with Alexa Fluor Plus 405 Phalloidin. HeLa cells labeled using Alexa Fluor Plus 405 Phalloidin, CellLight Talin-GFP BacMam 2.0, CellLight Mitochondria-RFP BacMam 2.0, and DRAQ5 show multiplexing capability and staining specificity. Cells were mounted in ProLong Glass Antifade Mountant. Images were generated using an EVOS FL Auto 2 Imaging System with an Olympus 60X Apochromat Oil objective using DAPI, GFP, RFP, and Cy5 EVOS light cubes.
Other phalloidin conjugates
There are other phalloidin conjugates available for staining actin filaments. These conjugates have very little non-specific staining which allows for high-contrast discrimination of actin. Fluorescein and Oregon Green 488 are conjugated to green fluorescent dyes, while BODIPY 558/568 is conjugated to a bright, red fluorescent dye. These phalloidins allow for multiplexing with other fluorescent stains such as fluorescent proteins and Alexa Fluor conjugates including secondary antibodies. Rhodamine Phalloidin is conjugated to the red-orange fluorescent dye, tetramethylrhodamine (TRITC) (Figure 5). Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594, and Texas Red-X (Figure 6) phalloidins are available replacements for rhodamine phalloidin since their emission spectra are better separated from green-fluorescent dyes.
Unlabeled phalloidin is also offered for use as a control in blocking F-actin staining by labeled phalloidins and for promoting actin polymerization.
Figure 5. Muntjac skin fibroblast labeled with rhodamine phalloidin. The actin cytoskeleton of a fixed and permeabilized muntjac skin fibroblast was labeled with rhodamine phalloidin; the nucleus was stained with DAPI. The sample was mounted and imaged in SlowFade Gold antifade reagent.
Figure 6. UV-irradiated muntjac skin fibroblasts labeled with Texas Red-X phalloidin. UV-irradiated muntjac skin fibroblasts showing mitochondrial damage. Fixed, permeabilized cells were stained with Texas Red-X phalloidin to visualize F-actin in the cytoskeleton. Endogenous biotin in the mitochondria was detected with Alexa Fluor 488 streptavidin. Nuclei were counterstained with DAPI.
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