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PMID: 15298916 Published · ppublish English Evaluation Study Journal Article Research Support, U.S. Gov't, Non-P.H.S. Research Support, U.S. Gov't, P.H.S.

Formation and destabilization of actin filaments with tetramethylrhodamine-modified actin.

Biophysical journal ·Vol. 87 ·No. 2 ·2004-08-00 ·Pages 1136-45

Kudryashov DS, Phillips M, Reisler E

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.

MeSH Terms
Actin Cytoskeleton/chemistry,ultrastructure Actins/chemistry Animals Artifacts Dimerization Microscopy, Fluorescence/methods Molecular Motor Proteins/chemistry Muscle, Skeletal/chemistry Protein Conformation Protein Denaturation Rabbits Rhodamines/chemistry Staining and Labeling/methods
Chemicals
Actins Molecular Motor Proteins Rhodamines tetramethylrhodamine
Authors & Affiliations
3 authors, click to expand affiliations / ORCID
Kudryashov Dmitry S
Department of Chemistry and Biochemistry, and the Molecular Biology Institute, University of California, Los Angeles, California, USA. dkudryas@ucla.edu
Phillips Martin
Reisler Emil
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Article Info
Journal
Biophysical journal
Abbr.
Biophys J
ISSN
0006-3495
DOI
10.1529/biophysj.104.042242
Email
dkudryas@ucla.edu
Published
2004-08-00
Pages
1136-45
Language
English
Region
United States
NLM ID
0370626
PMCID
PMC1304452
Subset
IM
Grants
NIAMS NIH HHS · R01 AR022031 · United States
NIAMS NIH HHS · AR 22031 · United States
External Links
PubMed source DOI: 10.1529/biophysj.104.042242 PMC Full Text View journal details
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