Confinement of unliganded EGFR by tetraspanin nanodomains gates EGFR ligand binding and signaling.

The epidermal growth factor receptor (EGFR) is a central regulator of cell physiology. EGFR is activated by ligand binding, triggering receptor dimerization, activation of kinase activity, and intracellular signaling. EGFR is transiently confined within various plasma membrane nanodomains, yet how this may contribute to regulation of EGFR ligand binding is poorly understood. To resolve how EGFR nanoscale compartmentalization gates ligand binding, we developed single-particle tracking methods to track the mobility of ligand-bound and total EGFR, in combination with modeling of EGFR ligand binding. In comparison to unliganded EGFR, ligand-bound EGFR is more confined and distinctly regulated by clathrin and tetraspanin nanodomains. Ligand binding to unliganded EGFR occurs preferentially in tetraspanin nanodomains, and disruption of tetraspanin nanodomains impairs EGFR ligand binding and alters the conformation of the receptor's ectodomain. We thus reveal a mechanism by which EGFR confinement within tetraspanin nanodomains regulates receptor signaling at the level of ligand binding.

Analysis of conditional colocalization relationships and hierarchies in three-color microscopy images.

Colocalization analysis of multicolor microscopy images is a cornerstone approach in cell biology. It provides information on the localization of molecules within subcellular compartments and allows the interrogation of known molecular interactions in their cellular context. However, almost all colocalization analyses are designed for two-color images, limiting the type of information that they reveal. Here, we describe an approach, termed "conditional colocalization analysis," for analyzing the colocalization relationships between three molecular entities in three-color microscopy images. Going beyond the question of whether colocalization is present or not, it addresses the question of whether the colocalization between two entities is influenced, positively or negatively, by their colocalization with a third entity. We benchmark the approach and showcase its application to investigate receptor-downstream adaptor colocalization relationships in the context of functionally relevant plasma membrane locations. The software for conditional colocalization analysis is available at https://github.com/kjaqaman/conditionalColoc.

Divergent Effects of Cyclophilin-D Inhibition on the Female Rat Heart: Acute Versus Chronic Post-Myocardial Infarction.

BACKGROUND/AIMS: The mitochondrial permeability transition pore opening plays a critical role in the pathogenesis of myocardial infarction. Inhibition of cyclophilin-D (CyP-D), a key regulator of the mitochondrial permeability transition pore, has been shown to exert cardioprotective effects against ischemia-reperfusion injury on various animal models, mostly in males. However, failure of recent clinical trials requires a detailed elucidation of the cardioprotective efficacy of CyP-D inhibition. The aim of this study was to examine whether cardioprotective effects of sanglifehrin A, a potent inhibitor of CyP-D, on post-infarcted hearts depends on reperfusion. METHODS: Acute or chronic myocardial infarction was induced by coronary artery ligation with/without subsequent reperfusion for 2 and 28 days in female Sprague-Dawley rats. Cardiac function was estimated by echocardiography. Oxygen consumption rates, ROS production, permeability transition pore opening, protein carbonylation and respiratory supercomplexes were analyzed in isolated cardiac mitochondria. RESULTS: Sanglifehrin A significantly improved cardiac function of reperfused hearts at 2 days but failed to protect after 28 days. No protection was observed in non-reperfused post-infarcted hearts. The respiratory control index of mitochondria was significantly reduced in reperfused infarcted hearts at 2-days with no effect at 28-days post-infarction on reperfused and non-reperfused hearts. Likewise, only a minor increase in reactive oxygen species production was observed at 2-days in non-reperfused post-infarcted hearts. CONCLUSION: This study demonstrates that CyP-D inhibition exerts cardioprotective effects in reperfused but not in non-reperfused infarcted hearts of female rats, and the effects are observed only during acute post-infarction injury.