High-resolution fractionation of signaling endosomes containing different receptors.
Traffic. 2009 Jul; 10(7):938-50
This article presents a two-dimensional fractionation method for the separation of physically distinct endosome populations isolated from cells. The method uses both density and size for fractionation, providing the necessary resolution to separate endosomes that would largely overlap on conventional cell fractionation gradients. The result is a unique window into a stunning heterogeneity of endosomes engaged in different signaling and sorting events.
The authors concentrated on primary endocytic vesicles containing ligand-activated receptors that had been newly internalized from the PC12 cell plasma membrane. Surprisingly, three classes of activated receptors, including receptor tyrosine kinases, a G-protein-coupled receptor and a TNF superfamily receptor were found in physically separable endocytic organelles. This unexpected degree of heterogeneity means that primary endocytic vesicles contain distinct molecular components and regulatory molecules, traffic differently, and may mature along distinct pathways toward very different fates. Importantly, the new method potentially provides the opportunity to characterize subpopulations of any type of organelle, including those not resolvable by light microscopy. Furthermore, since immunoblotting is used to assay the fractions, an unlimited number of endogenous marker proteins may be assessed relative to the cargo of interest. In addition, protein isoforms or post-translational modifications that are not distinguishable by microscopy may be assigned to organelle subpopulations using this method.
Hay J: F1000Prime Recommendation of [McCaffrey G et al., Traffic 2009, 10(7):938-50]. In F1000Prime, 15 Apr 2009; DOI: 10.3410/f.1159026.619306. F1000Prime.com/1159026#eval619306
F1000Prime Recommendations, Dissents and Comments for [McCaffrey G et al., Traffic 2009, 10(7):938-50]. In F1000Prime, 12 Dec 2013; F1000Prime.com/1159026
Nature. 2013 Mar 28; 495(7442):534-8
J Cell Biol. 2006 Mar 27; 172(7):1045-56
Receptor endocytosis is regulated by ligand binding, and receptors may signal after endocytosis in signaling endosomes. We hypothesized that signaling endosomes containing different types of receptors may be distinct from one another and have different physical characteristics. To test this hypothesis, we developed a high-resolution organelle fractionation method based on mass and density, optimized to resolve endosomes from other organelles. Three different types of receptors undergoing ligand-induced endocytosis were localized predominately in endosomes that were resolved from one another using this method. Endosomes containing activated receptor tyrosine kinases (RTKs), TrkA and EGFR, were similar to one another. Endosomes containing p75(NTR) (in the tumor necrosis receptor superfamily) and PAC1 (a G-protein-coupled receptor) were distinct from each other and from RTK endosomes. Receptor-specific endosomes may direct the intracellular location and duration of signal transduction pathways to dictate response to signals and determine cell fate.
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