Formation and destabilization of actin filaments with tetramethylrhodamine-modified actin |
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| Authors: | Kudryashov Dmitry S Phillips Martin Reisler Emil |
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| Affiliation: | Department of Chemistry and Biochemistry, and the Molecular Biology Institute, University of California, Los Angeles, California, USA. dkudryas@ucla.edu |
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| Abstract: | Actin labeling at Cys(374) with tethramethylrhodamine derivatives (TMR-actin) has been widely used for direct observation of the in vitro filaments growth, branching, and treadmilling, as well as for the in vivo visualization of actin cytoskeleton. The advantage of TMR-actin is that it does not lock actin in filaments (as rhodamine-phalloidin does), possibly allowing for its use in investigating the dynamic assembly behavior of actin polymers. Although it is established that TMR-actin alone is polymerization incompetent, the impact of its copolymerization with unlabeled actin on filament structure and dynamics has not been tested yet. In this study, we show that TMR-actin perturbs the filaments structure when copolymerized with unlabeled actin; the resulting filaments are more fragile and shorter than the control filaments. Due to the increased severing of copolymer filaments, TMR-actin accelerates the polymerization of unlabeled actin in solution also at mole ratios lower than those used in most fluorescence microscopy experiments. The destabilizing and severing effect of TMR-actin is countered by filament stabilizing factors, phalloidin, S1, and tropomyosin. These results point to an analogy between the effects of TMR-actin and severing proteins on F-actin, and imply that TMR-actin may be inappropriate for investigations of actin filaments dynamics. |
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| Keywords: | ANP n-(4-azido-2-nitrophenyl) putrescine BeFx berillium fluoride DTT dithiothreitol EGTA ethyleneglycol bis(aminoethylether) tetraacetic acid S1 myosin subfragment 1 SDS-PAGE sodium dodecylsulphate polyacrylamide gel electrophoresis TMR tetramethylrhodamine maleimide/iodoacetamide |
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