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• W2396001259 abstract "An active fluorescent derivative of the actin- binding mushroom toxin phallacidin has been synthesized. Convenient methods were developed to stain actin cytoskeletal structures in living and fixed cultured animal cells and actively streaming algal cells. Actin binding specificity was demon- strated by competitive binding experiments and comparative staining of well-known structures. Large populations of living animal cells in culture were readily stained by using a relatively mild lysolecithin permeabilization procedure facilitated by the small molecular size of the label. Actin in animal cells was stained in stress fibers, ruffles, the cellular geodome, and in diffuse appearing distributions apparently associated with the plasma membrane. Staining of actin cables in algae with ni- trobenzoxadiazole (NBD)phallacidin did not inhibit cytoplas- mic streaming. NBD-phallacidin provides a convenient actin- specific fluorescent label for cellular cytoskeletal structures with promise for use in studies of actin dynamics in living sys- tems. To pographical fluorescence microscopy images of the major features of the cellular cytoskeleton have advanced our knowledge of cytoskeletal structures during the last few years. Labeling of fixed cells by indirect immunofluorescence with antiactin antibodies (1, 2) or by fluorescent heavy meromyosin (3) has been used to study microfilaments and their relationship with other cytoskeletal components. More recently, microin- jection of fluorescent actin has been used to study these struc- tures in living cells (4-6). Here we report the development and application of an al- ternative fluorescent marker for cellular F-actin. It is applicable to living cells and thereby offers potential for observing the dynamics of cellular processes. We first outline the synthesis of the fluorescent derivative of phallacidin. Next we demon- strate its specificity and applicability by three illustrative studies. In the first, images of the actin cytoskeleton of fixed tissue culture fibroblasts provide familiar structures for con- formation of specificity and quality of staining. Second, pre- liminary observations of living animal cells in culture confirm the practicability of incorporating the actin marker into living functioning cells by simple permeabilization procedures and suggest some of the possibilities for observing the dynamics of cytoskeletal processes. Third, we present an application in which the actin filaments associated with the rotational cyto- plasmic streaming in the alga Chara australis are observed without inhibition of streaming in perfused living cells. We sought a fluorescent marker for actin that could be in- troduced conveniently into large populations of living cells," @default.
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• W2396001259 date "2016-01-01" @default.
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• W2396001259 title "Fluorescence staining of the actin cytoskeleton in living cells with 7-nitrobenz-2-oxa- 1 ,3-diazole-phallacidin (phallacidin/phalloidin/immunofluorescence/cytoplasmic streaming/Chara australis)" @default.
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