Whether in cytoplasm, nucleoplasm, membranes, or phase-separated compartments, proteins exist and function in complex cellular environments. Stark differences between the physico-chemical characteristics of these crowded native environments and the ideal dilute solutions in which proteins and their biochemistry are traditionally studied, pose at least two fundamental challenges for understanding protein activity and functionality in vivo. In the first place, extrapolation of conformational dynamics, or reaction rates between environments of vastly different macromolecular composition and density, is generally not straightforward. Second, the capacity of proteins to co-evolve with the biological context in which they function enables their activity to be fine-tuned for the physico-chemical properties of their native environment. So not only is it hard to predict how a protein-protein reaction studied in an ideal solution would proceed in, say, a phase-separated compartment, but some biologically important activities might not even be observed in the dilute limit. This underscores the importance of interrogating proteins under conditions that faithfully mimic those of their native cellular milieu, if not directly in cells. As a first step in this direction, our paper in the April 10 issue of the Biophysical Journal examines the self-assembly reaction of the cytoskeletal protein actin in the crowded environment of phase-separated polyelectrolyte complex coacervate droplets.
Our cover image shows actin filaments and filament bundles localized to the periphery of coacervate droplets. Monomeric actin proteins partition passively into the coacervate phase, which forms from the associative phase separation of positively charged polylysine and negatively charged polyglutamic acid from the surrounding solution. By increasing the local actin concentration within the coacervate phase relative to solution, partitioning accelerates the assembly of actin filaments.
The cover image highlights our observation that, once formed, actin filaments localize primarily to the coacervate periphery. This localization came as a surprise to us, since the protein bovine serum albumin (BSA), which is very similar to an actin monomer in terms of shape, size, and net charge, localizes uniformly throughout the coacervate interior. We find that filament bending, macromolecular crowding, and interfacial attraction all contribute to the peripheral localization of actin filaments.
The image is composed of maximum intensity projections of fluorescently labeled actin filaments from the upper or lower hemispheres of individual coacervates, obtained from confocal z-sections. The raw images were acquired from multiple fields of view of a single experimental sample. The fluorescence intensities of individual projections were false-colored using Fiji (Is Just ImageJ), and the projections were arranged in a spiral from smallest to largest using Adobe Illustrator. The diameter of the largest coacervate is 35 µm.
This work resulted from the close collaboration of three experimental research groups from physics, biochemistry, and molecular engineering, and was enabled by the NSF-funded Materials Research Science and Engineering Center at the University of Chicago.
- Patrick M. McCall, Samanvaya Srivastava, Sarah L. Perry, David R. Kovar, Margaret L. Gardel, and Matthew V. Tirrell