Recovery of the actin cytoskeleton during cellular repair is independent of protein synthesis

M. Hosford, R. Sandoval, B. A. Molitoris

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Abstract

Several laboratories have shown that renal proximal tubule cellular G-actin decreases rapidly during ischemia in vivo and ATP depletion in vitro. The purpose of the present studies was to determine the effect of cellular ATP recovery on G-actin levels, its intracellular distribution and whether or not protein synthesis was required for normalization. Confluent LLCPK1 cells were ATP depleted using substate depleted media containing 0.1 μM Antimycin A for 60 minutes. Cellular G-actin was followed immunoflourescently using a G-actin specific monoclonal Ab and quantified biochemically using the ethidium bromide DNase reaction. Protein synthesis was inhibited with cycloheximide (10μg/ml) Under physiologic conditions G-actin was found in small discrete well dispursed punctate areas throughout the cell, and accounted for 40-50% of total cellular actin. With ATP depletion G-actin levels decreased in a time-dependent fashion to 20% of total cellular actin, and accumulated in large aggregates throughout the cytoplasm. This was accompanied by a corresponding increase in cellular F-actin. There was no overlap between the G-actin and F-actin (rhodamine phalloidin staining) aggregates within the cell. With ATP repletion cellular G actin increased to physiologic levels over 1 hour and the large intracellular accumulations resolved. Cycloheximide was without effect on G- or F-actin concentrations and intracellular distribution during ATP depletion or repletion. These data indicate the cell can rapidly and quantitatively reutilize intracellular actin to achieve pysiologic F and G concentrations. They imply alterations in both F- and G-actin, induced by ATP depletion, result in a metabolically stable cytoplasmic pool of reusable G- and F-actin.

Original languageEnglish (US)
Pages (from-to)169A
JournalJournal of Investigative Medicine
Volume44
Issue number1
StatePublished - Jan 1 1996

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ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)

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