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  • Structural insights into actin filament recognition by commonly used cellular actin markers.

Structural insights into actin filament recognition by commonly used cellular actin markers.

The EMBO journal (2020-06-23)
Archana Kumari, Shubham Kesarwani, Manjunath G Javoor, Kutti R Vinothkumar, Minhajuddin Sirajuddin
ABSTRACT

Cellular studies of filamentous actin (F-actin) processes commonly utilize fluorescent versions of toxins, peptides, and proteins that bind actin. While the choice of these markers has been largely based on availability and ease, there is a severe dearth of structural data for an informed judgment in employing suitable F-actin markers for a particular requirement. Here, we describe the electron cryomicroscopy structures of phalloidin, lifeAct, and utrophin bound to F-actin, providing a comprehensive high-resolution structural comparison of widely used actin markers and their influence towards F-actin. Our results show that phalloidin binding does not induce specific conformational change and lifeAct specifically recognizes closed D-loop conformation, i.e., ADP-Pi or ADP states of F-actin. The structural models aided designing of minimal utrophin and a shorter lifeAct, which can be utilized as F-actin marker. Together, our study provides a structural perspective, where the binding sites of utrophin and lifeAct overlap with majority of actin-binding proteins and thus offering an invaluable resource for researchers in choosing appropriate actin markers and generating new marker variants.

MATERIALS
Product Number
Brand
Product Description

Roche
Anti-His6, from mouse IgG1
SAFC
McCoyā€²s 5A Medium, Modified, with L-glutamine, without sodium bicarbonate, powder, suitable for cell culture
Sigma-Aldrich
Protein Gā€² from Streptococcus sp., recombinant, expressed in E. coli