In single-molecule colocalization (“CoSMoS”) experiments, “the most challenging aspects of the technique are often not the preparation of the molecules or making the microscope observations, but in analyzing the resulting data. Data analysis has two primary challenges. First, one is studying inherently stochastic processes (thermally driven reactions of single molecules), so that analysis is inherently statistical. Second, the number of photons that can be emitted by a single fluorophore is limited by photobleaching, so images often have low signal-to-noise ratios, making discrimination of real signals from noise a challenge.”  In this article, Larry Friedman provides a step-by-step description the the procedures he has developed to reliably and efficiently analyze CoSMoS data.  While the examples discussed in the paper focus on analysis of transcription mechanisms, the methods should be applicable to CoSMoS studies on a broad range of biological systems.

Resources: Computer software that implements methods described in this article is available here.

“The mechanisms by which cells destabilize and rapidly disassemble filamentous actin networks have remained elusive; however, Coronin, Cofilin and AIP1 have been implicated in this process.”   Silvia Jansen from Bruce Goode’s lab and co-authors showed in this study “using multi-wavelength single-molecule fluorescence imaging…that mammalian Cor1B, Cof1 and AIP1 work in concert through a temporally ordered pathway to induce highly efficient severing and disassembly of actin filaments. Cor1B binds to filaments first, and dramatically accelerates the subsequent binding of Cof1, leading to heavily decorated, stabilized filaments. Cof1 in turn recruits AIP1, which rapidly triggers severing and remains bound to the newly generated barbed ends. New growth at barbed ends generated by severing was blocked specifically in the presence of all three proteins. This activity enabled us to reconstitute and directly visualize single actin filaments being rapidly polymerized by formins at their barbed ends while simultanteously being stochastically severed and capped along their lengths, and disassembled from their pointed ends.”

In this study, a collaboration between the Gelles lab and Stephen P. Bell’s lab at MIT, Simina Ticau and co-authors studied the mechanism of “licensing”, the initial process in setting up DNA replication in eukaryotic cells. “Licensing is loading of the ring-shaped Mcm2-7 helicase around DNA origins of replication. During loading, Cdc6, Cdt1, and the origin-recognition complex (ORC) assemble two heterohexameric Mcm2-7 complexes into a head-to-head double hexamer that facilitates bidirectional replication initiation. Using multi-wavelength single-molecule fluorescence to monitor the events of helicase loading, we demonstrate that double-hexamer formation is the result of sequential loading of individual Mcm2-7 complexes. Loading of each Mcm2-7 molecule involves the ordered association and dissociation of distinct Cdc6 and Cdt1 proteins. In contrast, one ORC molecule directs loading of both helicases in each double hexamer. Based on single-molecule FRET, arrival of the second Mcm2-7 results in rapid double-hexamer formation that anticipates Cdc6 and Cdt1 release, suggesting that Mcm-Mcm interactions recruit the second helicase. Our findings reveal the complex protein dynamics that coordinate helicase loading and indicate that distinct mechanisms load the oppositely oriented helicases that are central to bidirectional replication initiation.”

This article was featured in a Cell Preview “Single-Molecule Visualization of MCM2-7 DNA Loading: Seeing Is Believing” by Gheorghe Chisto and Johannes C. Walter. Cell 161, 429-430 (2015) 10.1016/j.cell.2015.04.006.  It was also discussed in
“Single Molecule Enzymology Finds its Stride” by Jeffrey Perkel. BioTechniques 59:183–187 (2015) 10.2144/000114337.

This new Methods in Enzymology chapter by Ben Smith, Jeff Gelles, and Bruce Goode “describe[s] techniques for acquisition and analysis of single-molecule data, applied to the novel challenges of studying the filament assembly and disassembly activities of actin-associated proteins in vitro. We discuss the advantages of single-molecule analysis in directly visualizing the order of molecular events, measuring the kinetic rates of filament binding and dissociation, and studying the coordination among multiple factors. The methods described… complement traditional biochemical approaches in elucidating actin-regulatory mechanisms in reconstituted filamentous networks.”