RESUMO
ATP production requires the establishment of an electrochemical proton gradient across the inner mitochondrial membrane. Mitochondrial uncouplers dissipate this proton gradient and disrupt numerous cellular processes, including vesicular trafficking, mainly through energy depletion. Here we show that Endosidin9 (ES9), a novel mitochondrial uncoupler, is a potent inhibitor of clathrin-mediated endocytosis (CME) in different systems and that ES9 induces inhibition of CME not because of its effect on cellular ATP, but rather due to its protonophore activity that leads to cytoplasm acidification. We show that the known tyrosine kinase inhibitor tyrphostinA23, which is routinely used to block CME, displays similar properties, thus questioning its use as a specific inhibitor of cargo recognition by the AP-2 adaptor complex via tyrosine motif-based endocytosis signals. Furthermore, we show that cytoplasm acidification dramatically affects the dynamics and recruitment of clathrin and associated adaptors, and leads to reduction of phosphatidylinositol 4,5-biphosphate from the plasma membrane.
Assuntos
Ácidos/metabolismo , Clatrina/metabolismo , Endocitose/efeitos dos fármacos , Mitocôndrias/metabolismo , Desacopladores/farmacologia , Trifosfato de Adenosina/deficiência , Trifosfato de Adenosina/metabolismo , Arabidopsis/efeitos dos fármacos , Arabidopsis/metabolismo , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Metabolismo Energético/efeitos dos fármacos , Células HeLa , Humanos , Mitocôndrias/efeitos dos fármacos , Organelas/efeitos dos fármacos , Organelas/metabolismo , Transporte Proteico/efeitos dos fármacos , Quinolonas/química , Quinolonas/farmacologiaRESUMO
Endomembrane trafficking relies on the coordination of a highly complex, dynamic network of intracellular vesicles. Understanding the network will require a dissection of cargo and vesicle dynamics at the cellular level in vivo. This is also a key to establishing a link between vesicular networks and their functional roles in development. We used a high-content intracellular screen to discover small molecules targeting endomembrane trafficking in vivo in a complex eukaryote, Arabidopsis thaliana. Tens of thousands of molecules were prescreened and a selected subset was interrogated against a panel of plasma membrane (PM) and other endomembrane compartment markers to identify molecules that altered vesicle trafficking. The extensive image dataset was transformed by a flexible algorithm into a marker-by-phenotype-by-treatment time matrix and revealed groups of molecules that induced similar subcellular fingerprints (clusters). This matrix provides a platform for a systems view of trafficking. Molecules from distinct clusters presented avenues and enabled an entry point to dissect recycling at the PM, vacuolar sorting, and cell-plate maturation. Bioactivity in human cells indicated the value of the approach to identifying small molecules that are active in diverse organisms for biology and drug discovery.