Whole-body cellular mapping in mouse using standard IgG antibodies.

Whole-body imaging techniques play a vital role in exploring the interplay of physiological systems in maintaining health and driving disease. We introduce wildDISCO, a new approach for whole-body immunolabeling, optical clearing and imaging in mice, circumventing the need for transgenic reporter animals or nanobody labeling and so overcoming existing technical limitations. We identified heptakis(2,6-di-O-methyl)-ß-cyclodextrin as a potent enhancer of cholesterol extraction and membrane permeabilization, enabling deep, homogeneous penetration of standard antibodies without aggregation. WildDISCO facilitates imaging of peripheral nervous systems, lymphatic vessels and immune cells in whole mice at cellular resolution by labeling diverse endogenous proteins. Additionally, we examined rare proliferating cells and the effects of biological perturbations, as demonstrated in germ-free mice. We applied wildDISCO to map tertiary lymphoid structures in the context of breast cancer, considering both primary tumor and metastases throughout the mouse body. An atlas of high-resolution images showcasing mouse nervous, lymphatic and vascular systems is accessible at http://discotechnologies.org/wildDISCO/atlas/index.php .

Commuting between Golgi cisternae--mind the GAP!

Intracellular transport has remained central to cell biology now for more than 40 years. Despite this, we still lack an overall mechanistic framework that describes transport in different parts of the cell. In the secretory pathway, basic questions, such as how biosynthetic cargo traverses the pathway, are still debated. Historically, emphasis was first put on interpreting function from morphology at the ultrastructural level revealing membrane structures such as the transitional ER, vesicular carriers, vesicular tubular clusters, Golgi cisternae, Golgi stacks and the Golgi ribbon. This emphasis on morphology later switched to biochemistry and yeast genetics yielding many of the key molecular players and their associated functions that we know today. More recently, microscopy studies of living cells incorporating biophysics and system analysis has proven useful and is often used to readdress earlier findings, sometimes with surprising outcomes.

Toward understanding and exploiting tumor heterogeneity.

The extent of tumor heterogeneity is an emerging theme that researchers are only beginning to understand. How genetic and epigenetic heterogeneity affects tumor evolution and clinical progression is unknown. The precise nature of the environmental factors that influence this heterogeneity is also yet to be characterized. Nature Medicine, Nature Biotechnology and the Volkswagen Foundation organized a meeting focused on identifying the obstacles that need to be overcome to advance translational research in and tumor heterogeneity. Once these key questions were established, the attendees devised potential solutions. Their ideas are presented here.

Novel scoring function for modeling structures of oligomers of transmembrane alpha-helices.

Specific non-covalent interactions between transmembrane (TM) alpha-helices are important in a variety of biological processes. Experimental and computational studies have shown that van der Waals interactions play an important role in the tight packing between TM alpha-helices, although polar interactions can also be important in some instances. Based on the assumption that van der Waals interaction alone is sufficient for a meso-scale (residue-scale) description of the interaction between TM alpha-helices, we have designed a novel residue-scale scoring function for modeling structures of oligomers of TM alpha-helices. We first calculated atomistic van der Waals interaction energies between two amino acids, X and Y, of a pair of parallel alpha-helices, glycine-X-glycine and glycine-Y-glycine and compiled them according to three variables, the distance between the two C(alpha) atoms and the rotational angles of X and Y about their helical axes. Upon averaging over the rotational angles, we obtained one-dimensional interaction energy profiles that are functions of the distance between C(alpha) atoms only. Each of the interaction energy profiles was fitted with a generic fitting function of the distance between C(alpha) atoms, yielding analytical scoring functions for all possible amino acid pairs. For glycophorin A, neu/erbB-2, and phospholamban, lowest-energy conformations obtained through exhaustive scanning of the entire conformational space using the scoring functions were compatible with available experimental data.

Early stages of Golgi vesicle and tubule formation require diacylglycerol.

We have investigated the role for diacylglycerol (DAG) in membrane bud formation in the Golgi apparatus. Addition of propranolol to specifically inhibit phosphatidate phosphohydrolase (PAP), an enzyme responsible for converting phosphatidic acid into DAG, effectively prevents formation of membrane buds. The effect of PAP inhibition on Golgi membranes is rapid and occurs within 3 min. Removal of the PAP inhibitor then results in a rapid burst of buds, vesicles, and tubules that peaks within 2 min. The inability to form buds in the presence of propranolol does not appear to be correlated with a loss of ARFGAP1 from Golgi membranes, as knockdown of ARFGAP1 by RNA interference has little or no effect on actual bud formation. Rather, knockdown of ARFGAP1 results in an increase in membrane buds and a decrease of vesicles and tubules suggesting it functions in the late stages of scission. How DAG promotes bud formation is discussed.

Cisternal maturation and vesicle transport: join the band wagon! (Review).

'No cellular organelle has been the subject of as many, as long-lasting or as diverse polemics as the Golgi apparatus'. This statement was made by Whaley almost 30 years ago in the book The Golgi Apparatus and still holds true today, perhaps more then ever. Why is this? How come something as mundane as a series of intracellular membrane bound structures continues to fascinate and captivate a large section of the cell biology community? One simple reason (putting polemics aside) is that the secretory pathway appears deceptively simple. Once probed, however, it has a persistent habit of developing into an enigma. Is one then not closer than 30 years ago? In a sense yes, in that one has more components and a better understanding of inherent membrane dynamics, but it is still not known how newly synthesized proteins and lipids make their way from the ER to the plasma membrane. Is it by vesicles, cisternal carriers or transient tubular connections? It has also been learned that newly synthesized proteins are segregated away from the resident components throughout the pathway, but not how. Do coat proteins hold the key? It is understood that the cytoskeleton is important, but not really why. It is known that each Golgi stack is a fully functional unit, but not why stacks are connected laterally into a large ribbon (the Golgi apparatus). This review focuses on how proteins make their way through the pathway, a basic question that remains to be answered.