As live imaging plays an increasingly critical role in cell biology research, the desire to label and track individual protein molecules in vivo has been growing. To address this, in this protocol we describe steps for sparse labeling using two different HaloTag ligand dyes in C. elegans. This labeling approach is simple, is non-invasive, and preserves the view of the bulk protein population. We further describe how to carry out single-particle tracking experiments and extract information about particle diffusion behavior. For complete details on the use and execution of this protocol, please refer to Chang and Dickinson (2022).1.

The heat shock protein 90 (Hsp90) family of chaperones are well-known, highly important components of the cellular systems which regulate protein homeostasis. Essential in eukaryotes, Hsp90s is also found in prokaryotes, including archaea. Hsp90 is a dimeric protein, with each monomer consisting of three separate structural domains, and undergoes large conformational changes as part of its functional cycle. This cycle is driven by interactions with nucleotides, cochaperone proteins, client proteins and allosteric effects enacted by these and by posttranslational modifications. All of these influence the rate and degree of the opening and closing of the dimer as well as the relative domain orientations and its overall rigidity. Optical tweezers, which can access many of these functionally important conformational changes, therefore provide a unique tool for the study of this large and complex molecular chaperone. Here, we provide protocols for the design and implementation of different Hsp90 constructs and optical tweezers experiments for addressing the many open questions about the function of this important molecular chaperone.

BAX activation techniques are crucial to studying the intrinsic pathway of apoptosis- thousands of pro-apoptotic signals converge on BAX activation. Current methodologies are predominantly limited to membrane permeabilization studies, which assess endpoint functionality of oligomeric BAX, but overlook early activation steps of cytosolic BAX. Here we detail FLAMBE: a fluorescence polarization ligand assay for monitoring BAX early-activation in solution. We also describe a dual-metric parameterization strategy for distillation of kinetic data and comparative analyses when studying candidate ligands. For complete details on the use and execution of this protocol, please refer to Gelles et al. (2022).