Using Single Molecule Imaging to Explore Intracellular Heterogeneity

TL;DR

This paper discusses how single-molecule imaging techniques can be used to quantitatively analyze intracellular molecular condensates, enhancing understanding of their roles in cell signaling and organization.

Contribution

It introduces methods for applying single-molecule imaging to quantify and distinguish molecular behaviors within and outside condensates in living cells.

Findings

Single-molecule imaging can differentiate behaviors inside and outside condensates.

Techniques vary in effectiveness depending on temporal and spatial scales.

Combining imaging methods improves identification of condensate-associated molecular behaviors.

Abstract

Despite more than 100 years of study, it is unclear if the movement of proteins inside the cell is best described as a mosh pit or an exquisitely choreographed dance. Recent studies suggest the latter. Local interactions induce molecular condensates such as liquid-liquid phase separations (LLPSs) or non-liquid, functionally significant molecular aggregates, including synaptic densities, nucleoli, and Amyloid fibrils. Molecular condensates trigger intracellular signaling and drive processes ranging from gene expression to cell division. However, the descriptions of condensates tend to be qualitative and correlative. Here, we indicate how single-molecule imaging and analyses can be applied to quantify condensates. We discuss the pros and cons of different techniques for measuring differences between transient molecular behaviors inside and outside condensates. Finally, we offer suggestions for how imaging and analyses from different time and space regimes can be combined to identify molecular behaviors indicative of condensates within the dynamic high-density intracellular environment.