single-molecule-measurement-protein-interaction-dynamics-within

Single-Molecule Measurement of Protein Interaction Dynamics Within Biomolecular Condensates

Biomolecular condensates formed via liquid-liquid phase separation (LLPS) have been considered critical in cellular organization and an increasing number ...

California Institute of Technology

Fluorescence microscopy techniques have been widely adopted in biology for their ability to visualize the structure and dynamics of a wide range of cellular and subcellular processes. The specificity and sensitivity that these techniques afford have made them primary tools in the characterization of protein localizations within cells. Many of the fluorescence microscopy techniques require cells to be fixed via chemical or alternative methods before being imaged. However, some fixation methods have been found to induce the redistribution of particular proteins in the cell, resulting in artifacts in the characterization of protein localizations and functions under physiological conditions. Here, we review the ability of commonly used cell fixation methods to faithfully preserve the localizations of proteins that bind to chromatin, undergo liquid-liquid phase separation (LLPS), and are involved in the formation of various membrane-bound organelles. We also review the mechanisms underlying various fixation artifacts and discuss potential alternative fixation methods to minimize the artifacts while investigating different proteins and cellular structures. Overall, fixed-cell fluorescence microscopy is a very powerful tool in biomedical research; however, each experiment demands the careful selection of an appropriate fixation method to avoid potential artifacts and may benefit from live-cell imaging validation.

Visualizing Protein Localizations in Fixed Cells: Caveats and the Underlying Mechanisms.

Transcription factors (TFs) activate enhancers to drive cell-specific gene programs in response to signals, but our understanding of enhancer assembly during signaling events is incomplete. Here, we show that androgen receptor (AR) forms condensates through multivalent interactions mediated by its N-terminal intrinsically disordered region (IDR) to orchestrate enhancer assembly in response to androgen signaling. AR IDR can be substituted by IDRs from selective proteins for AR condensation capacity and its function on enhancers. Expansion of the poly(Q) track within AR IDR results in a higher AR condensation propensity as measured by multiple methods, including live-cell single-molecule microscopy. Either weakening or strengthening AR condensation propensity impairs its heterotypic multivalent interactions with other enhancer components and diminishes its transcriptional activity. Our work reveals the requirement of an optimal level of AR condensation in mediating enhancer assembly and suggests that alteration of the fine-tuned multivalent IDR-IDR interactions might underlie AR-related human pathologies.

Shawn R. Yoshida

Division of Chemistry and Chemical Engineering,
Division of Biology and Biological Engineering

California Institute of Technology

Single-Molecule Measurement of Protein Interaction Dynamics Within Biomolecular Condensates

Shawn R. Yoshida

Division of Chemistry and Chemical Engineering,Division of Biology and Biological Engineering

California Institute of Technology

Single-Molecule Measurement of Protein Interaction Dynamics Within Biomolecular Condensates

Division of Chemistry and Chemical Engineering,
Division of Biology and Biological Engineering