Use of the avidin-biotin complex for the localization of actin and myosin with fluorescence microscopy.

Abstract

In the course of studies on the ultrastructural localization and functions of contractile proteins in nonmuscle cells, we have found it useful to adapt the avidin-biotin complex technique introduced by Heitzmann and Richards (7) to the specific labeling of actin and myosin components in these cells. These authors modified cell surfaces by covalent attachment of biotinyl residues, and then visualized these residues in electron microscopy by staining with a ferritin-avidin conjugate, thereby making use of the extraordinarily high binding affinity of biotin for avidin (5). We have used this general procedure to develop two specific fluorescence staining procedures, one for intracellular actin, the other for myosin. The actin procedure involves the successive reaction with biotin-labeled heavy meromyosin (HMM) followed by fluorescein-labeled avidin, which in our hands has given results superior to those obtained with the use of direct fluorescein-labeled HMM as described by Sanger (11). The myosin labeling procedure involves the successive reaction with biotin-labeled antimyosin antibody followed by fluorescein-labeled avidin, which provides an alternative and comparably effective staining procedure to the usual indirect immunofluorescence technique. MATERIALS AND METHODS Muscle ProteinsActin was purified from acetone powders of rabbit back muscle by the method of Spudich and Watt (14). HMM was prepared from rabbit skeletal muscle myosin (15) by a limited tryptic digestion as described by Lowey et al. (10). These proteins were stored in 50% glycerol at -20~ Rabbit myofibrils were prepared by the procedure of Zak et ai. (18). Biotinyl-N-Hydroxysuccinimide Ester ( BOSu)This reagent was synthesized by the method of Heitzmann and Richards (7), and was stored over dessicant at 4~ It was found to be stable for at least 12 mo. Radioactive BOSu (6.4 • 104 cpm/~mol) was prepared by adding [~4C]biotin (Amersham/Searle Corp., Arlington Heights, I11.) to the biotin used for the synthesis. Biotin-Labeled HMM4 mg of fibrous actin and 4 mg of HMM were dialyzed separately overnight against 0.15 M KC1 in 0.01 M potassium phosphate buffer, pH 7.5 (PBK). The two protein solutions were combined, so as to protect the actin-binding site of HMM during the chemical modification (11). The volume was adjusted to 7.5 ml with PBK, and this solution was brought to room temperature. The pH of the protein solution was then raised to 8.8 by the addition of 2.5 ml of a buffer containing 0.2 M NaHCO.~ and 0.15 M KCI, pH 8.8. The reaction was initiated by adding 5 ~1 (preparation A) or 8/~1 (preparation B) of a freshly prepared solution of BOSu (12 mg/ml) in dimethylformamide. The solution was mixed gently and allowed to react for 5 rain with periodic gentle stirring. The reaction was then quenched by the addition of 50 pl of 1 M NH4CI (pH 6). The modified acto-HMM was pelleted by centrifugation for 1 h at 180,000 g. This pellet was recovered, suspended in 2 ml of PBK containing sodium pyrophosphate (0.01 M) and MgCI~ (0.002 M) (to dissociate the ac...

Transmembrane interactions and the mechanism of capping of surface receptors by their specific ligands.

“…After such surface labeling, the cells were lightly fixed with formaldehyde, infused with 1.2 M sucrose, frozen, and sectioned in the frozen state to a thickness of about 1 gm. The thawed sections were then stained either for actin, by using a rhodamine fluorescence method based on heavy meromyosin binding (6), or for myosin, by using a rhodamine indirect immunofluorescence procedure that did not interfere with the antibodies used for surface labeling. The stained sections were then examined in a Zeiss photomicroscope with a X63 oil-immersion lens and an epi-illuminator, with appropriate filter combinations.…”

Intracellular distributions of mechanochemical proteins in cultured fibroblasts

“…Actin was localized in these cells using the reagents biotinconjugated heavy meromyosin and fluorescein-conjugated avidin as described (5).…”

Antibody-induced linkages of plasma membrane proteins to intracellular actomyosin-containing filaments in cultured fibroblasts.

“…Biotinyl-heavy meromyosin and fluorescein isothiocyanate-conjugated avidin (Fl-avidin) used to stain intracellular actin were prepared as described (11 In all cases, after fixation the cells were rinsed and treated with 0.1 M glycine in phosphate-buffered saline and stained indirectly with two successive layers of rhodamine-conjugated antibodies. The use of two layers of fluorescent antibodies was necessary to amplify the signal arising from these membrane proteins, which were relatively sparsely present on the surface.…”