Development and Application of In Vivo Protein Labeling Methods in Caenorhabditis Elegans

Abstract

The existence and preservation of life relies on the concerted orchestra of numerous biological processes of which proteins are the primary workhorse. In the highly complex intracellular environment, proteins must be synthesized, properly folded, carry out their specific functions, and be efficiently degraded when no longer required. Over time, we have learned even the most minute mutation in protein can have profound impacts on health and disease. Thus, developing methods to dissect protein dynamics and function are critical to understand the basic molecular processes that govern life. Protein pulse-chase is a versatile tool that can be used to study protein dynamics in cells and multicellular organisms. Original protein pulse-chase methods involved global, non-specific labeling of the entire proteome. While informative, spatial expression and dynamics across tissues are lost. The development of self-labeling enzyme tags such as HaloTag enable protein-specific pulse-chase. Pulse-chase experiments using these tags have been performed in various biological systems. However, their broader application is limited by significant technical and economic challenges. This work outlines the development and application of a HaloTag fluorescent pulse-chase method in the model organism, Caenorhabditis elegans. Strains expressing intestine-specific HaloTag histone reporter proteins were generated and could be efficiently pulse-labeled by soaking animals in the presence of HaloTag fluorescent ligand. Stability of labeled histone reporter protein could be feasibly monitored over time using confocal microscopy and was dependent on the animals’ nutritional state. HaloTag fluorescent labeling was further applied to investigate and characterize aberrant extranuclear divisions in the anterior intestine of C. elegans driven by the exogenous expression of a histone H3.3 protein. Collectively, this work serves as entrée to illustrate the potential of in vivo labeling methodologies in C. elegans. Technical advancements made in C. elegans will serve as a stepping-stone to address a variety of questions in metazoan biology. From pulse-labeling to detection, the method is highly adaptable to cater investigation into a wide variety of biological processes in vivo.