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- Title
Structure of the Lifeact–F-actin complex.
- Authors
Belyy, Alexander; Merino, Felipe; Sitsel, Oleg; Raunser, Stefan
- Abstract
Lifeact is a short actin-binding peptide that is used to visualize filamentous actin (F-actin) structures in live eukaryotic cells using fluorescence microscopy. However, this popular probe has been shown to alter cellular morphology by affecting the structure of the cytoskeleton. The molecular basis for such artefacts is poorly understood. Here, we determined the high-resolution structure of the Lifeact–F-actin complex using electron cryo-microscopy (cryo-EM). The structure reveals that Lifeact interacts with a hydrophobic binding pocket on F-actin and stretches over 2 adjacent actin subunits, stabilizing the DNase I-binding loop (D-loop) of actin in the closed conformation. Interestingly, the hydrophobic binding site is also used by actin-binding proteins, such as cofilin and myosin and actin-binding toxins, such as a hypervariable region of TccC3 (TccC3HVR) from Photorhabdus luminescens and ExoY from Pseudomonas aeruginosa. In vitro binding assays and activity measurements demonstrate that Lifeact indeed competes with these proteins, providing an explanation for the altering effects of Lifeact on cell morphology in vivo. Finally, we demonstrate that the affinity of Lifeact to F-actin can be increased by introducing mutations into the peptide, laying the foundation for designing improved actin probes for live cell imaging. Lifeact is a short actin-binding peptide that is widely used to visualize filamentous actin structures in live cell fluorescence microscopy. This cryo-EM study reveals how Lifeact competes with actin-binding proteins such as cofilin and myosin, providing an explanation for how Lifeact alters cell morphology, while structure-guided site directed mutagenesis demonstrates that the affinity of Lifeact can be modulated.
- Subjects
PHOTORHABDUS luminescens; CELL morphology; ACTIN; BINDING site assay; CELL imaging; HYPERVARIABLE regions; MYOSIN; MICROFILAMENT proteins
- Publication
PLoS Biology, 2020, Vol 18, Issue 11, p1
- ISSN
1544-9173
- Publication type
Article
- DOI
10.1371/journal.pbio.3000925