Identifying Intermolecular Interactions in Single-Molecule Localization Microscopy.

Intermolecular interactions underlie all cellular functions, yet visualizing these interactions at the single-molecule level remains challenging. Single-molecule localization microscopy (SMLM) offers a potential solution. Given a nanoscale map of two putative interaction partners, it should be possible to assign molecules either to the class of coupled pairs or to the class of non-coupled bystanders. Here, we developed a probabilistic algorithm that allows accurate determination of both the absolute number and the proportion of molecules that form coupled pairs. The algorithm calculates interaction probabilities for all possible pairs of localized molecules, selects the most likely interaction set, and corrects for any spurious colocalizations. Benchmarking this approach across a set of simulated molecular localization maps with varying densities (up to ∼ 50 molecules µm - 2 ) and localization precisions (5 to 50 nm) showed typical errors in the identification of correct pairs of only a few percent. At molecular densities of ∼ 5-10 molecules µm - 2 and localization precisions of 20-30 nm, which are typical parameters for SMLM imaging, the recall was ∼ 90%. The algorithm was effective at differentiating between non-interacting and coupled molecules both in simulations and experiments. Finally, it correctly inferred the number of coupled pairs over time in a simulated reaction-diffusion system, enabling determination of the underlying rate constants. The proposed approach promises to enable direct visualization and quantification of intermolecular interactions using SMLM.

Activation of the Notch Signaling Pathway In Vivo Elicits Changes in CSL Nuclear Dynamics.

A key feature of Notch signaling is that it directs immediate changes in transcription via the DNA-binding factor CSL, switching it from repression to activation. How Notch generates both a sensitive and accurate response-in the absence of any amplification step-remains to be elucidated. To address this question, we developed real-time analysis of CSL dynamics including single-molecule tracking in vivo. In Notch-OFF nuclei, a small proportion of CSL molecules transiently binds DNA, while in Notch-ON conditions CSL recruitment increases dramatically at target loci, where complexes have longer dwell times conferred by the Notch co-activator Mastermind. Surprisingly, recruitment of CSL-related corepressors also increases in Notch-ON conditions, revealing that Notch induces cooperative or "assisted" loading by promoting local increase in chromatin accessibility. Thus, in vivo Notch activity triggers changes in CSL dwell times and chromatin accessibility, which we propose confer sensitivity to small input changes and facilitate timely shut-down.