Visualizing a protein decision complex in actin filament length control

Seen at the Gelles Lab Little Engine Shop blog this week, commentary on a new paper in Nature Communications, published in collaboration with the Goode Lab and researchers from New England Biolabs.

“Single-molecule visualization of a formin-capping protein ‘decision complex’ at the actin filament barbed end”

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Regulation of actin filament length is a central process by which eukaryotic cells control the shape, architecture, and dynamics of their actin networks. This regulation plays a fundamental role in cell motility, morphogenesis, and a host of processes specific to particular cell types. This paper by recently graduated [Biophysics and Structural Biology] Ph.D. student Jeffrey Bombardier and collaborators resolves the long-standing mystery of how formins and capping protein work in concert and antagonistically to control actin filament length. Bombardier used the CoSMoS multi-wavelength single-molecule fluorescence microscopy technique to to discover and characterize a novel tripartite complex formed by a formin, capping protein, and the actin filament barbed end. Quantitative analysis of the kinetic mechanism showed that this complex is the essential intermediate and decision point in converting a growing formin-bound filament into a static capping protein-bound filament, and the reverse. Interestingly, the authors show that “mDia1 displaced from the barbed end by CP can randomly slide along the filament and later return to the barbed end to re-form the complex.” The results define the essential features of the molecular mechanism of filament length regulation by formin and capping protein; this mechanism predicts several new ways by which cells are likely to couple upstream regulatory inputs to filament length control.

Single-molecule visualization of a formin-capping protein ‘decision complex’ at the actin filament barbed end
Jeffrey P. Bombardier, Julian A. Eskin, Richa Jaiswal, Ivan R. Corrêa, Jr., Ming-Qun Xu, Bruce L. Goode, and Jeff Gelles
Nature Communications  6:8707 (2015)

The capping protein expression plasmid described in this article is available from Addgene.

Readers interested in this subject should also see a related article by Shekhar et al published simultaneously in the same journal.  We are grateful to the authors of that article for coordinating submission so that the two articles were published together.

A facilitated diffusion confusion dissolution

To utilize the information contained within a cell’s genes, the enzyme RNA polymerase must find the beginning of each gene (the promoter).  Finding the beginning is a prodigious task:  RNAP must start at a particular base pair of DNA, but the cell contains millions of base pairs to choose from.  It has been proposed that gene-finding challenge is aided by a process termed ‘facilitated diffusion’ (FD).  In FD, RNA polymerase first binds to a random position on DNA and then slides along the DNA like a bead on a string until it encounters the target DNA sequence.

In a recently published study in PNAS (1), biophysicists Larry Friedman and Jeffrey Mumm worked with Prof. Jeff Gelles in the Brandeis Biochemistry department to test key predictions of the FD model.  They used a novel light microscope that Friedman and colleagues invented and built at Brandeis, a microscope that can directly observe the binding of an individual RNA polymerase to a single DNA.  The scientists studied the σ⁵⁴ RNA polymerase holoenzyme, an RNA polymerase found in most species of bacteria.  Surprisingly, none of the three predictions of the FD model that the experiments tested were found to be valid, demonstrating that target finding by the polymerase is not accelerated by sliding along DNA.  Friedman and colleagues instead propose that RNA polymerases are present in such large numbers that they can diffuse through the cell and efficiently bind to their target sites directly.  The absence of FD may explain how other proteins can bind to positions on the DNA that flank gene start sites and yet not interfere with RNA polymerase finding the gene.

Is this the end of the story? Not likely, given previous publications suggesting FD plays a role for some other DNA binding proteins. Using single-molecule techniques like those developed in the Gelles lab, scientists in next few years should give us a better idea if FD is very rare or very common. [editor: as a chemical engineer, I’m sad to see FD not have a role — it seemed like such a nice theory…]

Friedman LJ, Mumm JP, Gelles J. RNA polymerase approaches its promoter without long-range sliding along DNA.  Proc Natl Acad Sci U S A. 2013 May 29. [Epub ahead of print]