Phalloidin Actin labeling

Labeled phallotoxines (phalloidins) The bicyclic peptides isolated from Amanita phalloides mushroom bind selectively to F-actin in nanomolar concentrations. They have advantages over antibodies for actin labeling... 

Fixed embryos or tissues expressing GFP can also be labeled with antibodies to other proteins or with DNA stains or other compounds that bind specific cellular components, e.g., rhodamine-phalloidin to label F-actin (Wang and Hazelri 1994 Breitweiser et al. 1996 Edwards et al. 1997 Brand 1999). In our laboratory, we find that GFP s fluorescence is destroyed by extended exposure to methanol, so we omit steps that require methanol but others report that short exposures to methanol are acceptable (Brand 1999). The methods given below (Protocols 17.6-17.8) all use formaldehyde fixation, but glutaraldehyde fixation also preserves GFP s fluorescence (Ward 1998). 

Fig. 1 Lateral line neuromasts in zebrafish larvae, (a) Lateral view of live untreated 8 dpf zebrafish larvae (anterior to the left and dorsal up) stained with 5 pM 4-Di-2-Asp. (b) A neuromast from a fixed 5 dpf eleutheroembryo containing hair cells labeled for f-actin with phalloidin-Alexa 488. (c) Lateral view of a fixed 5 dpf larva containing lateral line neuromast stained with anti-acetylated alpha-tubulin, (d) Hair cells in a neuromast, with the stereocilia bundles in green (phalloidin-Alexa 488) and the kinocilia in red (alpha-tubulin, 1/1000)...

Remove blocking solution and add 100 iL of PBS+2% BSA with TO-PRO-3 (1 100) and phalloidin-Alexa Fluor 543 (1 100) to label cell nuclei and actin filaments, respectively. 

C2 toxin-induced depolymerization of actin filaments is visualized by fluorescence labeling of actin with FITC- or Rhodamine-phalloidin. 

The cytoskeleton is a cellular scaffold within the cytoplasm of the cell or within structures such as flagella, cilia, and lamellipodia. The cytoskeleton plays a crucial role in cellular integrity/structural support, cell division, cell motility, and intracellular transport. Staining of the cytoskeleton may be achieved by immunofluorescence in fixed cells however there are also dyes available, such as fluorescently labelled phalloidin, which directly labels actin filaments, as well as tubulin tracker which labels microtubules (Molecular Probes). Phalloidin is poorly permeable to living cells however, phalloidin derivatives with improved permeability properties are also available. Although both phalloidin derivatives and microtubule tracker can be used in live cells, it should be noted that both dyes are toxic as they inhibit cell division, and therefore limits their applications. 

Fig. 4. Actin cytoskeletal organization (labeled with phalloidin-FITC) of hFOB 1.19 grown for 48h on a) plastic support (A) - original magnification 16x b) sucrose film deposited on glass support SI (B) - original magnification 32x.

Fig. 12 Fluorescence micrographs of confluent MDCK II cell monolayers after the actin cytoskeleton has been stained by fluorescence-labeled phalloidin. a Control cells were not exposed to Cytochalasin D. b Cells were exposed to 5 xM Cytochalasin D for 100 min. The staining confirms that actin filaments have been degraded to small actin aggregates. The scale bar represents 25 xm. c Magnitude of the load impedance A 2l as a function of time when confluent MDCK II cell monolayers were exposed to 5 xM Cytochalasin D at the time indicated by the arrow. The value of Zi at the beginning of the experiment was set to zero...

A. Preparation of Rhodamine Phalloidin-Labeled Actin Filaments... 

FIGURE 2 Collage demonstrating the movement of rhodamine phalloidin-labeled actin filaments over a myosin-coated surface. Panels a-e are direct photographs of the video monitor at 30-sec intervals. Panel f shows a plot of the centroid position of selected actin filaments in the field, with arrows showing the direction of movement. The figure is taken from Collins et al. (1990). 

Two vol of 20 nM rhodamine phalloidin labeled actin in buffer B is used to wash the flow cell. 

FIGURE 3 Construction of the flow cell for the sliding actin in vitro motility assay. The paired irregular yellow strips represent the grease that adheres the coverslip (on top) to the slide. The coverslip slivers that are used as spacers are narrower than the top coverslip. The pink liquid represents a solution of rhodamine phalloidin-labeled actin that is being applied to the flow cell. 

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