Macromolecular crowding dramatically affects cellular processes such as protein folding and assembly, regulation of metabolic pathways, and condensation of DNA. Despite increased attention, we still lack a definition for how crowded a heterogeneous environment is at the molecular scale and how this manifests in basic physical phenomena like diffusion. Here, we show by means of fluorescence correlation spectroscopy and computer simulations that crowding manifests itself through the emergence of anomalous subdiffusion of cytoplasmic macromolecules. In other words, the mean square displacement of a protein will grow less than linear in time and the degree of this anomality depends on the size and conformation of the traced particle and on the total protein concentration of the solution. We therefore propose that the anomality of the diffusion can be used as a quantifiable measure for the crowdedness of the cytoplasm at the molecular scale.

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.

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. INTRODUCTIONFormation of buds to generate intracellular transport vesicles from membranes such as Golgi cisternae involves both coat binding and local lipid conversion (for reviews and theoretical models, see Kirchhausen, 2000;Shemesh et al., 2003;Weiss and Nilsson, 2003;Bethune et al., 2006). For COPI vesicles, formation of buds is initiated by the small GTPase ARF1 (ADP-ribosylation factor 1) which, in its GTPconferred conformation, drives coatomer recruitment from the cytosol to both Golgi and pre-Golgi membranes (Palmer et al., 1993). Indeed, ARF1 and coatomer are sufficient for both bud and vesicle formation as evidenced in in vitro experiments using liposomes forming coated vesicles in a controlled manner (Spang et al., 1998). Addition of ARF-GAP1, a GTPase-activating protein for ARF1, then yielded uncoated vesicles of the expected size of ϳ50-60 nm in diameter (Reinhard et al., 2003).The situation in biological membranes is likely more refined involving additional as well as alternative components to promote or prevent vesicle formation such that Golgi function is maintained. Here, both ARF1 and ARFGAP1 have been implicated in vesicle formation through direct or indirect modulation of lipid synthesis such that bud formation and membrane fission are promoted. For example, ARF1 stimulates the production of phosphatidic acid (PA) from phosphatidylcholine (PC; Brown et al., 1993;Cockcroft et al., 1994) through the activation of phospholipase D (PLD) in a nucleotide (GTP)-specific manner Houle et al., 1995;Ktistakis et al., 1995). Such ARF1-mediated PLD stimulation results in an increased vesicle production (Ktistakis et al., 1996;Chen et al., 1997). This ability of ARF1 to stimulate lipid formation in the Golgi apparatus offers a possibility to mechanistically link lipid conversion with coat recruitment. PA may also be converted to diacylglycerol (DAG) and the ratio between DAG and PC seems to influence protein transport through the Golgi apparatus in yeast (Rivas et al., 1999). PA can also be synthesized from lysophosphatidic acid (LPA) by a LPA acyltransferase-dependent pathway through...

Assembly of the coat protein I (COPI) vesicle coat is controlled by the small GTPase ADP ribosylation factor 1 (ARF1) and its GTPase-activating protein, ARFGAP1. Here, we investigate the diffusional behaviours of coatomer, the main component of the coat, and also those of ARF1 and ARFGAP1. Using fluorescence-correlation spectroscopy, we found that most ARF1 and ARFGAP1 molecules are highly mobile in the cytosol (diffusion constant D ≈ 15 μm 2 s -1 ), whereas coatomer diffuses 5-10 times more slowly than expected (D ≈ 1 μm 2 s -1 ). This slow diffusion causes diffusion-limited binding kinetics to Golgi membranes, which, in FRAP (fluorescence recovery after photobleaching) experiments, translates into a twofold slower binding rate. The addition of aluminium fluoride locks coatomer onto Golgi membranes and also decreases the binding kinetics of both ARF1 and ARFGAP1, suggesting that these proteins function in concert to mediate sorting and vesicle formation.

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