Cofilin, actin and their complex observed in vivo using fluorescence resonance energy transfer.
Actin is the principal component of microfilaments. Its assembly/disassembly is essential for cell motility, cytokinesis, and a range of other functions. Recent evidence suggests that actin is present in the nucleus where it may be involved in the regulation of gene expression and that cofilin binds actin and can translocate into the nucleus during times of stress. In this report, we combine fluorescence resonance energy transfer and confocal microscopy to analyze the interactions of cofilin and G-actin within the nucleus and cytoplasm. By measuring the rate of photobleaching of fluorescein-labeled actin in the presence and absence of Cy5-labeled cofilin, we determined that almost all G-actin in the nucleus is bound to cofilin, whereas approximately (1/2) is bound in the cytoplasm. Using fluorescence resonance energy transfer imaging techniques we observed that a significant proportion of fluorescein-labeled cofilin in both the nucleus and cytoplasm binds added tetramethylrhodamine-labeled G-actin. Our data suggest there is significantly more cofilin-G-actin complex and less free cofilin in the nucleus than in the cytoplasm.
Chhabra
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Muscle Research Unit, School of Medical Sciences, Institute for Biomedical Research, University of Sydney, Sydney, New South Wales, Australia. dchhabra@anatomy.usyd.edu.au
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D000199:Actins / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D000818:Animals; D002467:Cell Nucleus / Q000378:metabolism; D002522:Chlorocebus aethiops; D051340:Cofilin 2 / Q000737:chemistry / Q000502:physiology*; D003593:Cytoplasm / Q000378:metabolism; D003850:Deoxyribonuclease I / Q000737:chemistry / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism; D016650:Fluorescein-5-isothiocyanate / Q000494:pharmacology; D005452:Fluoresceins / Q000494:pharmacology; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D007150:Immunohistochemistry; D007700:Kinetics; D018613:Microscopy, Confocal; D008954:Models, Biological; D018482:Muscle, Skeletal / Q000378:metabolism; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry; D013997:Time Factors; D014709:Vero Cells
D000199:Actins; D051340:Cofilin 2; D005452:Fluoresceins; D011994:Recombinant Proteins; C015296:5-iodoacetamidofluorescein; D003850:Deoxyribonuclease I; D016650:Fluorescein-5-isothiocyanate