Mechanistic biology in the next quarter century.

Biologists study the transfer of information at many levels. Biochemists have traditionally investigated the structure and function of individual components of the cell to understand the mechanisms underlying biological transformations, e.g., enzymatic modifications of small molecules and macromolecules, the transformation of genetic information into the sequence of an RNA or a protein, and the transduction of signals through molecular interactions. Cell biologists have investigated the organization of cells, the molecular mechanisms of cellular activities, the regulation of cellular processes by cues from the extracellular environment, and the assembly of cells into tissues. Recent advances in microscopy have facilitated an effective convergence of biochemistry and cell biology—we can visualize molecules and cells, and individual molecules within cells, in real time. Thus, we can study spatial organization and dynamic changes in organization on various distance and time scales—enzymatic reactions, protein associations, signal relays, organelle function, cellular translocations, and many other molecular processes. It is no surprise that spatial organization matters, but we can now follow it directly—from the in vitro behavior of single molecular assemblies to changes in gene expression at any one time within a single cell. Stephen C. Harrison Joan S. Brugge This theme is likely to become increasingly important during the next two decades of research and discovery in molecular and cellular biology for two reasons: first, because the technological advances that have enabled single-molecule detection and visualization of macromolecular complexes within living organisms are still at early stages of development; and second, because visualizing molecular activity will be part of solving many frontier problems in biology and medicine. The following list is a rough outline of some of these problems. 1) The molecular mechanisms of membrane dynamics and remodeling and of cytoskeletal dynamics, which together underlie much of the spatial organization and compartmentalization in a cell, will be analyzed in real time, at high spatial resolution, and with multiple, component-specific optical tags, leading to molecular descriptions of organelle assembly, disassembly, and reorganization. 2) The spatial organization of signaling will be studied with increasing resolution (in both time and space) and at increasingly complex levels of molecular interaction, ultimately connecting with systems approaches to the same questions. 3) Control of gene expression (and of epigenetic inheritance)—the most profound output of cellular signal processing—will be analyzed in individual cells and at large numbers of independently monitored genetic loci within those cells. 4) Integration of cellular processes will be investigated by recording the sequence of events as reported by optical probes, to understand how perturbations of one cellular complex, machine, or signaling pathway affects others. 5) It will be possible to analyze cellular heterogeneity in real time, to understand cause–effect relationships in terms of the probabilities with which specific events succeed others. (Standard biochemical/signaling cell lysate analyses and “omics” analyses necessarily record population averages, masking important mechanistic information.) 6) At the level of tissues and organs, it will be possible to move from molecular descriptions of spatial and temporal organization in cells to mechanistic analyses of pattern formation in development, paracrine effects, cell movement, cell–matrix and cell–cell interactions, and the influence of these properties on both morphogenesis and disease pathology. 7) Imaging of metabolic events is likely to become feasible, with development of suitable probes and reporters. The potential of this new biochemical and structural cell biology for twenty-first century therapeutics—molecular, cellular, and genetic—is evident. Just as mechanistic enzymology has changed the directions of drug development during the past 25–30 years, so does mechanistic cell biology promise to redirect it during the next 25 years. But even more generally, because the spatial organization of a cell is just as critical (or more so) for its properties and interactions as its genetic, epigenetic, or physiological “state,” a realization of the goals just outlined will be essential for a rational program to link systems' analysis to translational application. Examples of frontier technologies that will drive the continuing fusion of cell biology and biochemistry as disciplines are in vivo imaging, in vitro single-molecule analysis, in silico simulation, and mass spectrometry. 1) Imaging, both of individual living cells and of larger-scale cellular organizations and tissues, will be driven by new microscopy technologies (e.g., superresolution methods), by novel computational methods in image analysis, and (perhaps most crucially) by development of new optical probes. Realizing the potential of these technical advances for exciting biological discovery will require close interaction between computer scientists and experimental biologists, to optimize procedures for data acquisition and data analysis. 2) Single-molecule methods will become a dominant approach to in vitro biochemistry, allowing far more definitive analysis of molecular mechanism than possible with ensemble experiments. Structural biology will shift in this direction as well, as “single-particle” methods in electron cryomicroscopy become more powerful. 3) Computational simulations of protein folding, protein assembly, and complex molecular processes in cells are likely to become increasingly realistic and increasingly useful for predicting and interpreting molecular behavior. 4) Large-scale mass spectrometry will define total changes in the expression of cellular components (e.g., protein, nucleic acid, metabolite), their modification, and their interactions, revealing the breadth and nature of changes that define and regulate cellular states. In addition, development of cell culture models that better recapitulate in vivo tissue biology will improve the relevance of data derived from cell culture analyses. The advent of single-molecule biochemistry and live organism imaging means that we can anticipate studying the biochemistry and structure of single molecules and molecular assemblies in living cells and integrating in vitro understanding of reactions and reorganizations into pictures of the in vivo spatial and temporal coordination of such processes. Although genetic analysis in vivo and reconstitution approaches in vitro will remain crucial, especially as single-molecule experiments become more powerful, the development of more sensitive and more flexibly used optical probes will allow the cell itself to become a principal “test tube,” permitting a direct experimental link between studies of molecular activity and genetic or regulatory modifications.