RESUMEN
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.
Asunto(s)
Diglicéridos/química , Lípidos/química , Proteínas/metabolismo , Adenosina Trifosfato/metabolismo , Técnicas Biosensibles , Membrana Celular/metabolismo , Membrana Celular/efectos de la radiación , Supervivencia Celular , Isoenzimas/metabolismo , Cinética , Luz , Modelos Biológicos , Proteína Quinasa C/metabolismo , Transducción de SeñalRESUMEN
Photorelease of caged compounds is among the most powerful experimental approaches for studying cellular functions on fast timescales. However, its full potential has yet to be exploited, as the number of caged small molecules available for cell biological studies has been limited by synthetic challenges. Addressing this problem, a straightforward, one-step procedure for efficiently synthesizing caged compounds was developed. An in situ generated benzylic coumarin triflate reagent was used to specifically functionalize carboxylate and phosphate moieties in the presence of free hydroxy groups, generating various caged lipid metabolites, including a number of GPCR ligands. By combining the photo-caged ligands with the respective receptors, an easily implementable experimental platform for the optical control and analysis of GPCR-mediated signal transduction in living cells was developed. Ultimately, the described synthetic strategy allows rapid generation of photo-caged small molecules and thus greatly facilitates the analysis of their biological roles in live cell microscopy assays.
Asunto(s)
Cumarinas/química , Lípidos/síntesis química , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal/efectos de los fármacos , Humanos , Ligandos , Lípidos/química , Receptores Acoplados a Proteínas G/química , Transducción de Señal/fisiologíaRESUMEN
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.