Abstract
Cofilin/ADF proteins are actin-remodeling proteins, essential for actin disassembly in various cellular processes, including cell division, intracellular transport, and motility. Cofilins bind actin filaments cooperatively and sever them preferentially at boundaries between bare and cofilin-decorated (cofilactin) segments. The cooperative binding to actin has been proposed to originate from conformational changes that propagate allosterically from clusters of bound cofilin to bare actin segments. Estimates of the lengths over which these cooperative conformational changes propagate vary dramatically, ranging from 2 to >100 subunits. Here, we present a general, structure-based method for detecting from cryo-EM micrographs small variations in filament geometry (i.e. twist) with single-subunit precision. How these variations correlate with regulatory protein occupancy reveals how far allosteric, conformational changes propagate along filaments. We used this method to determine the effects of cofilin on the actin filament twist. Our results indicate that cofilin-induced changes in filament twist propagate only 1-2 subunits from the boundary into the bare actin segment, independently of the boundary polarity (i.e. irrespective of whether or not the bare actin segment flanks the pointed or barbed-end side of the boundary) and the pyrene fluorophore labeling of actin. These observations indicate that the filament twist changes abruptly at boundaries between bare and cofilin-decorated segments, thereby constraining mechanistic models of cooperative actin filament interactions and severing by cofilin. The methods presented here extend the capability of cryo-EM to analyze biologically relevant deviations from helical symmetry in actin as well as other classes of linear polymers.
Highlights
Cofilin/ADF proteins are actin-remodeling proteins, essential for actin disassembly in various cellular processes, including cell division, intracellular transport, and motility
Procedure for deriving filament geometry and cofilin occupancy from electron micrographs We acquired a set of 500 micrographs of unlabeled actin filaments and 197 micrographs of pyrene-labeled actin filaments, both partially decorated with cofilin at a binding density (v) of ϳ0.5 cofilin per actin subunit
We limit our analysis here to boundaries defined by contiguous clusters of bound cofilin flanked by bare regions and were distinguishable by eye; smaller clusters are vulnerable to corruption from background noise levels inherent to cryo-EM
Summary
Procedure for deriving filament geometry and cofilin occupancy from electron micrographs We acquired a set of 500 micrographs of unlabeled actin filaments and 197 micrographs of pyrene-labeled actin filaments, both partially decorated with cofilin at a binding density (v) of ϳ0.5 cofilin per actin subunit. We performed a focused 3D classification by subtracting all but the central subunit from image segments using a masked reference volume (see under “Experimental procedures”) This procedure yielded bare and cofilin-decorated classes (Fig. 1A), with approximately half of the subunits assigned to each class. The cumulative twist difference displays an abrupt transition at boundaries between bare and cofilin-decorated segments within an individual filament (Fig. 2, A and B) Averaging this quantity over many boundaries reduces the noise and reveals that this structural transition occurs abruptly. The twist change at a given boundary appears to spread bidirectionally into the bare and cofilin-decorated segments, as determined from the best fit of the average twist angle (Fig. 2, E and F), i.e. the midpoint of the “twist transition” lies between the bare and cofilin-decorated filament segments, extending ϳ1 subunit to each side of the boundary (Fig. 2, E and F)
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