Abstract
BackgroundEukaryotic cells strictly regulate the structure and assembly of their actin filament networks in response to various stimuli. The actin binding proteins that control filament assembly are therefore attractive targets for those who wish to reorganize actin filaments and reengineer the cytoskeleton. Unfortunately, the naturally occurring actin binding proteins include only a limited set of pointed-end cappers, or proteins that will block polymerization from the slow-growing end of actin filaments. Of the few that are known, most are part of large multimeric complexes that are challenging to manipulate.Methodology/Principal FindingsWe describe here the use of phage display mutagenesis to generate of a new class of binding protein that can be targeted to the pointed-end of actin. These proteins, called synthetic antigen binders (sABs), are based on an antibody-like scaffold where sequence diversity is introduced into the binding loops using a novel “reduced genetic code” phage display library. We describe effective strategies to select and screen for sABs that ensure the generated sABs bind to the pointed-end surface of actin exclusively.Conclusions/SignificanceFrom our set of pointed-end binders, we identify three sABs with particularly useful properties to systematically probe actin dynamics: one protein that caps the pointed end, a second that crosslinks actin filaments, and a third that severs actin filaments and promotes disassembly.
Highlights
The actin cytoskeleton found in all eukaryotes defines many of the essential mechanical properties of the cell
A reactive free cysteine residue at the barbed end of actin was biotinylated to allow actin to be immobilized on a bead surface during the phage display sorting
DNase I is known to bind to the pointed end of G-actin monomers but not well to F-actin filaments
Summary
The actin cytoskeleton found in all eukaryotes defines many of the essential mechanical properties of the cell. The F-actin filament is polar, with distinct ends known as the barbed and pointed ends. These two ends maintain distinct polymerization and depolymerization rates, a property that requires the hydrolysis of bound ATP after polymerization [3]. Over a hundred distinct actin binding proteins (ABPs) modulate the properties of actin to establish filaments at precise locations, while preventing spontaneous assembly throughout the cell [4]. Examples of ABP function include the nucleation of filament formation in response to upstream signals, capping filaments to prevent elongation from the barbed end, depolymerization or severing of filaments, modulation of filament stiffness, bundling or crosslinking filaments into higher order assemblies, and sequestering actin monomers to block spontaneous nucleation. Of the few that are known, most are part of large multimeric complexes that are challenging to manipulate
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