In vitro assays, semi-intact cells, intact cells: what's next for studies of membrane trafficking?
Authors: Louvard D. 
Published: April 2021

One of the last Nobel Prizes for Medicine and Physiology of this century was awarded to Gunther Blobel for his pioneering work on the mechanisms of secretion in eukaryotic and prokaryotic cells. This prize for molecular cell biology followed that awarded to George Palade, Christian DeDuve and Albert Claude in 1974 and demonstrated that the control of the subcellular localization of proteins is achieved by molecular machines that read ‘addresses’ that are embedded in the primary sequence of a protein. In the case of secretory proteins, these addresses are known as signal sequences. Sorting of proteins from their site of synthesis to their final destination is now known to be a key cellular process, which, if impaired, can give rise to lethal defects. Gunther Blobel and his group established an experimental approach that is now used by numerous researchers to study secretory pathways. The analysis of the secretory pathway by George Palade and his collaborators in the 1960s took advantage of advanced ultrastructural analysis and cell-fractionation procedures. Subsequently, in the 1970s, a number of investigators recognized that further studies at the molecular level were required. For this purpose, Gunther Blobel, together with Bernhard Dobberstein, set up an assay that was one of the first to reconstitute in vitro the translocation of a nascent polypeptide chain across vesicles derived from the rough endoplasmic reticulum. This in vitro assay led the researchers to establish the signal-peptide hypothesis, which was first proposed by David Sabatini and Gunther Blobel in 1971. The complete characterization of the translocation machinery itself, which is composed of ribonucleoprotein complexes and integral membrane proteins, was achieved after much effort and imaginative studies performed by several research groups from different countries. The in vitro assay not only was used by numerous investigators in the field but it also provided the conceptual framework necessary for unravelling of the molecular machinery involved in the different steps of the intracellular transport of proteins. At the beginning of the 1980s, James Rothman and his colleagues developed another type of in vitro assay, which was based upon the use of fractions derived from different membrane compartments and cytosolic extracts. The goal of these researchers was to reconstitute the specific fusion between a donor membrane and an acceptor membrane. In less than one decade, the assay was refined to the point where it became a very powerful tool for biochemical characterization of cellular machines that move proteins from one organelle to another. Membrane-fusion assays allow the identification not only of protein complexes but also of the energy requirements of the fusion events. Several groups who focus on the endocytic pathway have subsequently developed similar assays. While the biochemical experiments were in progress, Randy Scheckman and his colleagues launched a genetic approach to study secretory pathways, using yeast as a model system. Because of the high degree of conservation of this essential cellular function, gene product(s) identified in yeast have mammalian homologues that were characterised simultaneously by the biochemical approaches described above. Together, these strategies gave rise to a molecular description of the minimal protein machinery necessary for the numerous transport pathways in eukaryotic cells. As with all experimental methods designed by biologists, however, even the powerful reconstitution and genetic approaches had their limitations. Fortunately, another original strategy emerged, in which cells could be permeabilized by physical trauma or chemical treatments so that small holes appeared in the plasma membrane. This procedure empties cells of their cytoplasmic components but maintains their structural organization and allows investigators to reintroduce well-defined products such as activator(s) or inhibitor(s). The permeabilized-cell strategy has opened new possibilities for future studies, because one now has biochemical access to information contained in the structural organisation of cells - information that is lost in cell-extract and genetics-based studies. Most investigators in the field had faith in the specificity of the biochemical reactions of the transport processes and were less interested in the efficiency of the process in vivo. The importance of this lapse of attention to detail became evident when the reaction rates in the in vitro systems were compared with those during normal physiological membrane transport in vivo. Consequently, it was only after the discovery of molecular motors that reaction rates were studied seriously and the role of motor proteins was considered in experiments designed by groups interested in molecular analysis of membrane trafficking. However, one should note the interest of those who developed in vitro assays for membrane transport in studies of the roles of molecular motors in movement of intracellular cargo along microtubules or microfilaments. The myriad of molecular motors that move the various molecular cargos along cellular tracks in opposite directions is bewildering. Another challenge for the future, well illustrated by studies that take advantage of recombinant DNA technology, will be to describe the spatial and temporal plasticity of the protein that make up machines that participate in vesicular trafficking. In this respect, the use of chimeric proteins tagged with the green fluorescent protein(s) is already very significant but still only in its infancy. The systematic use of videomicroscopy recording, which allows subsequent quantitative and multiparametric analysis at the single-cell or multicellular level, has allowed development of new perspectives on membrane trafficking. Our knowledge of the numerous proteins capable of forming molecular machines will be facilitated by the complete description of the genomes of model organisms - whether or not we study them in a unicellular or a multicellular context. Looking ahead, more research should be done on membrane trafficking and its regulation by the cytoskeleton in different cell types. Although numerous studies have already been performed on polarized cells, such as epithelial cells and neurons, many differentiated cell types have not been investigated at all. Moreover studies of cell differentiation during development or during physiopathological processes are still scarce and, here too, more research is needed to characterise the molecular features of trafficking during these processes. The pioneering work by numerous investigators over the past thirty years indeed provides a solid basis for future investigation in this research area. Certainly, these illuminating studies have opened new avenues and point to exciting challenges for the next generation of molecular cell biologists, who will experience even more spectacular discoveries.