Biophysical Society Thematic Meeting| Lima 2019

Revisiting the Central Dogma of Molecular Biology at the Single-Molecule Level

Friday Speaker Abstracts

TOXICITY AMPLIFICATION MECHANISM OF ACTIN CROSSLINKING TOXIN REVEALED BY SINGLE-MOLECULE IMAGING Elena Kudryashova 1 ; David Heisler 1,2 ; Blake Williams 1 ; Kyle Shafer 1 ; Alyssa Harker 3 ; David Kovar 3 ; Margot Quinlan 4 ; Dimitrios Vavylonis 5 ; Dmitri Kudryashov 1 ; 1 The Ohio State University, Department of Chemistry and Biochemistry, Columbus, OH, USA 2 University of Texas, Department of Microbiology, Dallas, TX, USA 3 University of Chicago, Department of Biochemistry and Molecular Biology , Chicago, IL, USA 4 University of California, Los Angeles, Department of Chemistry and Biochemistry, Los Angeles, CA, USA 5 Lehigh University, Department of Physics, Bethlehem, PA, USA Efficiency of actin-targeting toxins is hampered by an overwhelming abundance of the target: cytoskeletal actin is among the most abundant proteins in eukaryotic cells. Consequently, toxins employ sophisticated mechanisms of toxicity amplification. One such mechanism is demonstrated by the actin crosslinking domain (ACD)-containing toxins of Vibrio cholerae, Vibrio vulnificus, and Aeromonas hydrophila, which catalyze the formation of covalently crosslinked actin oligomers with actin subunits connected by side-chain amide bonds. Since ACD-produced oligomers are non-polymerizable, crosslinking bulk amounts of actin eventually leads to failure of its functions and cell rounding; however, this mechanism requires high doses of toxin to be effective. Conversely, our data imply that ACD-conferred cytotoxic effects are evident when only 2-6% actin is crosslinked, suggesting that low doses of actin oligomers are highly toxic. We discovered that ACD toxicity is amplified via a “gain-of-function” mechanism whereby ACD-crosslinked actin oligomers act as potent secondary toxins that directly inhibit proteins involved in nucleation, elongation, severing, and branching of actin filaments. Affected actin-regulatory proteins possess multiple G-actin-binding domains either organized in tandem in a single polypeptide or through oligomerization of several polypeptides and, therefore, serve as a multivalent platform for high-affinity interaction with actin oligomers. Single-molecule TIRFM and bulk actin polymerization assays revealed that actin oligomers bind with abnormally high affinity and potently inhibit formins, Ena/VASP, Spire, and NPFs of the Arp2/3 complex. In live cells, single-molecule speckle (SiMS) microscopy corroborated these findings and revealed potent inhibition and halted dynamics of these proteins in lamellipodia leading to massive disarray of the cytoskeleton upon low-dose ACD treatment. This study redefines ACD as an indirect, universal inhibitor of tandem-organized G-actin-binding proteins that overcomes the abundancy of actin by redirecting the toxicity cascade towards less abundant targets whose inhibition by actin oligomers leads to disorganization of actin cytoskeleton disabling normal cellular functions (published in Science-2015 and Current Biology-2018).

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