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 the 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.
Highlights
The network of actin filaments in eukaryotic cells is involved in processes ranging from intracellular trafficking to cell movement, cell division, and shape control [1]
Yeast viability upon Lifeact–maltose-binding protein (MBP) overexpression under the galactose promoter was analyzed by a drop test: 5-fold serial dilutions of cell suspensions were prepared from overnight agar cultures by normalizing OD600 measurements, spotted onto agar plates, and incubated for 2 to 3 days at 30 ̊C
Prior studies have indicated a molecular basis for stabilization of actin filaments by phalloidin [7,8,9]
Summary
The network of actin filaments in eukaryotic cells is involved in processes ranging from intracellular trafficking to cell movement, cell division, and shape control [1]. Numerous actin-visualizing compounds were developed to enable this These include small molecules, labeled toxins, recombinant tags, as well as actin-binding proteins and peptides (see [2] for a detailed review). Using these molecules to study actin in vivo often alters the properties of actin filaments to such an extent that normal homeostasis of the cytoskeleton is impaired. Since these side effects cannot be avoided, it is important to know their molecular basis in order to be able to adequately interpret the experimental data
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