Methods Actin labeling

Actin was prepared from rabbit skeletal muscle by the method of Spudich and Watt [19] and was further pinified with gel-filtration over Sephadex G-150 gel [20]. Monomeric actin, labeled with the fluorophore tetramethylrhodamine maleimide, was a generous gift of Dr Ewa Prochniewcz at the Department of Biochemistry, University of Minnesota. It was concluded from a preliminary experiment that Spudich-Watt actin contained minor factorfs) which greatly accelerated the polymerization of actin at 0.5 mM Ca and at 8°C upon the addition of Ca ions, 100 pM Spudich-Watt actin rapidly polymerized, whereas the purified actin did not. 

Simon, J.R., and Taylor, D.L. (1988) Preparation of a fluorescent analog Acetamidofluoresceinyl labeled dictyostelium discoideum a-actin. In Methods in Enzymology, (R.B. Vallee, ed.), Vol. 134, p. 47. Academic Press, San Diego. 

Figure 3. Critical concentration behavior of actin self-assembly. For the top diagram depicting the macroscopic critical concentration curve, one determines the total amount of polymerized actin by methods that measure the sum of addition and release processes occurring at both ends. Examples of such methods are sedimentation, light scattering, fluorescence assays with pyrene-labeled actin, and viscosity measurements. Forthe bottom curves, the polymerization behavior is typically determined by fluorescence assays conducted under conditions where one of the ends is blocked by the presence of molecules such as gelsolin (a barbed-end capping protein) or spectrin-band 4.1 -actin (a complex prepared from erythrocyte membranes, such that only barbed-end growth occurs). Note further that the barbed end (or (+)-end) has a lower critical concentration than the pointed end (or (-)-end). This differential stabilization requires the occurrence of ATP hydrolysis to supply the free energy that drives subunit addition to the (+)-end at the expense of the subunit loss from the (-)-end.

VPA was obtained from Sigma (St. Louis, MO). Antibodies and cDNAs for various acyl-CoA dehydrogenases (SCAD, MCAD, LCAD and IVD) were gifts from Prof Kay Tanaka, Yale imiversity school of medicine. cDNAs for rat ornithine transcar-bamylase (OTC) and P-actin were previously synthesized in our laboratory using cDNA synthesis kit (Clontech, CA). For use as probes, cDNAs were radiolabeled with [a P]dCTP (3,000 mCi/mmol, Amersham Corp.) using a random primer DNA labeling method. ... 

Effects of intracellular C3. To confirm that active C3 was introduced into cells by the method, and to quantify the effect, cells that had been osmotically shocked after endocytosing C3 were later lysed in the presence of C3 and [ P]NAD. This measured the amount of p21.bot remaining to be labeled and, hence, the amount modified in situ. Six hrs after the introduction of C3 the available p21.bot was considerably decreased (Table 2). As expected, the available actin was imaffected. 

Actins are pyrene-labeled based on the method of Kouyama and Mihashi (Kouyama and Mihashi, 1981). 

Recently, a new optical microscopic technique has been developed to investigate the conformation of a specified actin monomer in F-actin (36). An actin molecule was labeled with two kinds of small fluorescent molecules at different amino acid residues, and their distance was measured by the method of fluorescence energy transfer. The result has shown that the distance is not kept constant, but changes with time, as in Fig. 7. The change happens discontinuously and reversibly, indicating that actin monomer repeats transitions between two differ-... 

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). 

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