The central portion of the midbody, a cytoplasmic bridge between nascent daughter cells at the end of cell division, has generally been thought to be retained by one of the daughter cells, but has, recently, also been shown to be released into the extracellular space. The significance of midbody-retention versus -release is unknown. Here we show, by quantitatively analysing midbody-fate in various cell lines under different growth conditions, that the extent of midbody-release is significantly greater in stem cells than cancer-derived cells. Induction of cell differentiation is accompanied by an increase in midbody-release. Knockdown of the endosomal sorting complex required for transport family members, Alix and tumour-suppressor gene 101, or of their interaction partner, centrosomal protein 55, impairs midbody-release, suggesting mechanistic similarities to abscission. Cells with such impaired midbody-release exhibit enhanced responsiveness to a differentiation stimulus. Taken together, midbody-release emerges as a characteristic feature of cells capable of differentiation.

Every cell produces thousands of distinct lipid species, but insight into how lipid chemical diversity contributes to biological signaling is lacking, particularly because of a scarcity of methods for quantitatively studying lipid function in living cells. Using the example of diacylglycerols, prominent second messengers, we here investigate whether lipid chemical diversity can provide a basis for cellular signal specification. We generated photo-caged lipid probes, which allow acute manipulation of distinct diacylglycerol species in the plasma membrane. Combining uncaging experiments with mathematical modeling, we were able to determine binding constants for diacylglycerol–protein interactions, and kinetic parameters for diacylglycerol transbilayer movement and turnover in quantitative live-cell experiments. Strikingly, we find that affinities and kinetics vary by orders of magnitude due to diacylglycerol side-chain composition. These differences are sufficient to explain differential recruitment of diacylglycerol binding proteins and, thus, differing downstream phosphorylation patterns. Our approach represents a generally applicable method for elucidating the biological function of single lipid species on subcellular scales in quantitative live-cell experiments.

Photoreleaseo fc aged compoundsi sa mong the most powerful experimentala pproaches for studying cellularf unctions on fast timescales. However, its full potential has yett ob ee xploited, as the number of caged small molecules available for cell biological studies has been limited by synthetic challenges. Addressing this problem, as traightforward, one-step procedure fore fficiently synthesizing caged compounds was developed.A n in situ generated benzylic coumarin triflate reagent was used to specifically functionalize carboxylate and phosphate moieties in the presence of free hydroxy groups, generating variousc aged lipid metabolites, including a numbero fG PCR ligands. By combining the photo-caged ligandsw ith the respective receptors, an easily implementable experimental platform for the optical control and analysis of GPCR-mediated signal transduction in living cells wasd eveloped. Ultimately,t he described synthetic strategy allows rapid generation of photo-caged small moleculesa nd thus greatlyf acilitates the analysis of their biologicalr oles in live cell microscopy assays.

Transport systems for K+ in Escherichia coli are not detectable in membrane vesicles, but vesicles will take up K+ (and Rb+) in the presence of valinomycin. It is generally believed that valinomycin acts as a lipid-soluble cation carrier and that it does not interact with or activate cation transport systems. This view is challenged by Bhattacharyya et al. (Proc. Natl. Acad. Sci. USA 68:1448-1492, 1971), who reported reduced uptake in vesicles from E. coli mutants with K+ transport defects. We reexamined this question with some of the same mutants and were unable to confirm a correlation of valinomycin-induced vesicle transport with transport properties in intact cells. We found great variability in transport activity of vesicles from these E. coli K-12 strains and believe such variability as well as possible contamination with intact cells accounts for the earlier report. Our data do not support the idea that valinomycin-mediated transport in vesicles is related to physiological K+ transport systems.

18Every cell produces thousands of distinct lipid species, but methodology for studying the biological 19 roles of individual lipids is insufficient. Using the example of diacylglycerols, prominent second 20 messengers, we here investigate whether lipid chemical diversity can provide a basis for cellular 21 signal specification. We developed novel photo-caged lipid probes, which allow acute manipulation 22 of distinct diacylglycerol species in the plasma membrane. Combining uncaging experiments with 23 mathematical modelling enabled the determination of binding constants for diacylglycerol-protein 24 interactions and kinetic parameters for diacylglycerol transbilayer movement and turnover in 25 quantitative live-cell experiments. Strikingly, we find that affinities and kinetics vary by orders of 26 magnitude due to diacylglycerol structural diversity. These differences are sufficient to explain 27 differential recruitment of diacylglycerol binding proteins and thus differing downstream 28 phosphorylation patterns. Our approach represents a generally applicable method for elucidating the 29 biological function of single lipid species on subcellular scales. 30 Intriguingly, a growing body of evidence suggests that changes in the levels of individual lipid species 43 rather than entire lipid classes determine cellular signalling outcome. For instance, early studies 44 reported that activation of individual cell surface receptors leads to the formation of molecularly 45 distinct patterns of diacylglycerol (DAG) species during signal transduction (13-15), suggesting that 46 crucial information could be encoded in the molecular spectrum of signalling lipids generated. 47Supporting this notion, drastically altered levels of distinct lipid species were correlated with cellular 48 processes, e.g. the increase of a phosphatidic acid ether lipid during cytokinesis (16) or the reciprocal 49 regulation of ceramide species during toll-like receptor signaling in innate immunity (17). DAGs 50 appear to be prime targets to study the importance of lipid heterogeneity in cell signalling, as they act 51 as second messengers at the plasma membrane and function in many cellular processes, including 52 insulin signalling, ion channel regulation and neurotransmitter release (18, 19). Many of these 53 processes involve effector proteins such as protein kinase C (PKC) isoforms, which are recruited to 54 cellular membranes by DAG binding to their C1 domains (20). Faithful process initiation thus 55 requires the activation of a subset of DAG effector proteins in the presence of others as observed 56 during the formation of the immunological synapse (21), but the molecular mechanisms of such 57 specific recruitment events are not well understood. Here, specificity could be provided by 58 differential activation of effectors by structurally distinct DAG species which recruit DAG binding 59 proteins due to differences in lipid-protein affinities, local lipid densities and lifetimes. Determining 60 these parameters requires quantitative experim...