Abstract
Self-assembly of complex structures is commonplace in biology but often poorly understood. In the case of the actin cytoskeleton, a great deal is known about the components that include higher order structures, such as lamellar meshes, filopodial bundles, and stress fibers. Each of these cytoskeletal structures contains actin filaments and cross-linking proteins, but the role of cross-linking proteins in the initial steps of structure formation has not been clearly elucidated. We employ an optical trapping assay to investigate the behaviors of two actin cross-linking proteins, fascin and alpha-actinin, during the first steps of structure assembly. Here, we show that these proteins have distinct binding characteristics that cause them to recognize and cross-link filaments that are arranged with specific geometries. alpha-Actinin is a promiscuous cross-linker, linking filaments over all angles. It retains this flexibility after cross-links are formed, maintaining a connection even when the link is rotated. Conversely, fascin is extremely selective, only cross-linking filaments in a parallel orientation. Surprisingly, bundles formed by either protein are extremely stable, persisting for over 0.5 h in a continuous wash. However, using fluorescence recovery after photobleaching and fluorescence decay experiments, we find that the stable fascin population can be rapidly competed away by free fascin. We present a simple avidity model for this cross-link dissociation behavior. Together, these results place constraints on how cytoskeletal structures assemble, organize, and disassemble in vivo.
Highlights
A great deal of work has been done examining how cytoskeletal proteins are regulated and how they are sorted within the cell
Filopodial nucleation is thought to occur by formation of the filopodial tip complex bringing together actin filament barbed ends, allowing local elongation leading to filopodial growth [17]
We set out to determine whether ␣-actinin and fascin could bind filaments in any orientation presented or if they would only form cross-links when presented with filaments already arranged in a specific orientation
Summary
Protein Purification—Actin was purified using an established protocol [42]. Actin was polymerized at a concentration of 10 M monomer in assay buffer (AB: 25 mM imidazole, pH 7.5, 25 mM KCl, 1 mM EGTA, 4 mM MgCl2, and 10 mM dithiothreitol) in the presence of 2 mM ATP using 90% dark (unlabeled) actin and 10% biotinylated actin and stabilized with tetramethylrhodamine (TMR)3-phalloidin. Reservoir one was loaded with 4 l of neutravidin-coated beads in 1 ml of observation buffer (AB plus 0.86 mg/ml glucose oxidase, 0.14 mg/ml catalase, 9 mg/ml glucose). Reservoir two was loaded with 1 l of 10 M F-actin diluted in 1 ml of observation buffer. Reservoir four contained a final concentration of 1 M cross-linking protein in observation buffer for angular dependence assays and 0.1 M cross-linking protein when making bundles for unzipping experiments. The bead and actin structures were moved into the cross-linking protein lane. The chamber was washed with an additional observation buffer wash to remove any free fascin from solution. Cross-linking protein washes were performed using 3 M protein in observation buffer
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