RESUMO
Phosphoinositides are a family of membrane lipids essential for many biological and pathological processes. Due to the existence of multiple phosphoinositide regioisomers and their low intracellular concentrations, profiling these lipids and linking a specific acyl variant to a change in biological state have been difficult. To enable the comprehensive analysis of phosphoinositide phosphorylation status and acyl chain identity, we develop PRMC-MS (Phosphoinositide Regioisomer Measurement by Chiral column chromatography and Mass Spectrometry). Using this method, we reveal a severe skewing in acyl chains in phosphoinositides in Pten-deficient prostate cancer tissues, extracellular mobilization of phosphoinositides upon expression of oncogenic PIK3CA, and a unique profile for exosomal phosphoinositides. Thus, our approach allows characterizing the dynamics of phosphoinositide acyl variants in intracellular and extracellular milieus.
Assuntos
Classe I de Fosfatidilinositol 3-Quinases/genética , Metaboloma , PTEN Fosfo-Hidrolase/genética , Fosfatidilinositóis/metabolismo , Neoplasias da Próstata/metabolismo , Animais , Cromatografia de Afinidade , Classe I de Fosfatidilinositol 3-Quinases/metabolismo , Fator de Crescimento Epidérmico/farmacologia , Exossomos/química , Exossomos/metabolismo , Expressão Gênica , Células HEK293 , Células HeLa , Humanos , Masculino , Espectrometria de Massas , Camundongos , Células PC-3 , PTEN Fosfo-Hidrolase/deficiência , Fosfatidilinositóis/química , Fosfatidilinositóis/classificação , Fosfatidilinositóis/isolamento & purificação , Próstata/química , Próstata/efeitos dos fármacos , Próstata/patologia , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Pirimidinas/farmacologia , Quinazolinas/farmacologia , EstereoisomerismoRESUMO
Cardiolipin (CL) localizes to curved membranes such as cristae in mitochondria as well as cell poles and division sites in rod-shaped bacteria. CL is believed to stabilize the membrane curvature by localizing to sites of negative curvature. However, this hypothesis has not been tested because of a lack of appropriate tools to distinguish CL inside and outside lipid bilayers. In this study, we provided the first evidence that CL localized to regions of negative curvature in Escherichia coli using the novel CL probe erylysin A-EGFP (EryA-EGFP). Staining in E.coli illustrated that CL localized to the inner leaflets at cell poles and the outer leaflets at division sites. Furthermore, we revealed that EryA-EGFP inhibited cytokinesis. We propose that cytokinesis completes after CL in the outer leaflets transfers to the inner leaflets at division sites by inspecting the mechanism of inhibition of cytokinesis. Moreover, the cytoskeletal protein RodZ was abnormally distributed when cytokinesis was inhibited by EryA-EGFP, suggesting that RodZ participates in cytokinesis. In summary, we revealed the detailed distribution of CL and proposed a new model of cytokinesis.