Introduction to the Minireview Series on Modern Technologies for In-cell Biochemistry

Abstract

The last decade has seen enormous progress in the exploration and understanding of the behavior of molecules in their natural cellular environments at increasingly high spatial and temporal resolution. Advances in microscopy and the development of new fluorescent reagents as well as genetic editing techniques have enabled quantitative analysis of protein interactions, intracellular trafficking, metabolic changes, and signaling. Modern biochemistry now faces new and exciting challenges. Can traditionally "in vitro" experiments, e.g. analysis of protein folding and conformational transitions, be done in cells? Can the structure and behavior of endogenous and/or nontagged recombinant proteins be analyzed and altered within the cell or in cellular compartments? How can molecules and their actions be studied mechanistically in tissues and organs? Is personalized cellular biochemistry a reality? This thematic series summarizes recent studies that illustrate some first steps toward successfully answering these modern biochemical questions. The first minireview focuses on utilization of three-dimensional primary enteroids and organoids for mechanistic studies of intestinal biology with molecular resolution. The second minireview describes application of single chain antibodies (nanobodies) for monitoring and regulating protein dynamics in vitro and in cells. The third minireview highlights advances in using NMR spectroscopy for analysis of protein folding and assembly in cells.The ultimate goal of biochemistry is to understand how molecules work in a complex cellular environment at atomic level. Although we are still far away from this goal, great progress has been made toward characterization of metabolic identities of various cells and the behavior of many essential molecules in diverse intracellular contexts. Chemically induced dimerization of recombinant proteins has enabled dissection of signaling events in distinct intracellular locations with fine temporal resolution (1). Advances with cell sorting, single cell RNA sequencing, and computational methods revealed a rich diversity of cell identities and the existence of previously unknown cell subtypes (2, 3). Single molecule tracking, correlation spectroscopy, and time-resolved fluorescence energy transfer protein dynamics and interactions in cells in unprecedented detail and allow high-throughput screening for new protein modulators (4, 5). This thematic series aims to add to the excitement by highlighting recent methodological advances in three areas that differ markedly with respect to their scale and specific experimental goals but that, together, bring us closer to the ultimate goal of learning about cell function at the atomic level.In the first minireview of this thematic series (6), Zachos et al. (6) focus on the advantages of primary human enteroids and organoids for studies of intestinal physiology and pathobiology. In recent years, it has become increasingly clear that studies directed toward the understanding and treatment of human disorders require exp...

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