High‐Resolution Fractionation of Signaling Endosomes Containing Different Receptors

Abstract

contributed equally to this work.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. Receptorspecific endosomes may direct the intracellular location and duration of signal transduction pathways to dictate response to signals and determine cell fate.

Assembly of Arenavirus Envelope Glycoprotein GPC in Detergent-Soluble Membrane Microdomains

“…Membrane fractionation. Vero cells infected with Candid#1 virus were solubilized in cold PBS lysis buffer containing 1 mM EDTA, 1 mM EGTA, and 1% TX-100 (44). Cells were incubated on ice for 1 h with occasional mixing to ensure complete segregation of soluble and insoluble membranes.…”

“…The detergent-insoluble membranes were resuspended in cold buffer B (38 mM each of the potassium salts of aspartic acid, glutamic acid, and gluconic acid, 20 mM morpholinepropanesulfonic acid [MOPS; pH 7.1], 10 mM potassium bicarbonate, 0.5 mM magnesium carbonate, 1 mM EDTA, 1 mM EGTA) also containing protease, phosphatase inhibitors, and glutathione (44) and were adjusted to 1% TX-100. This suspension was treated with Benzonase for 30 min on ice, adjusted to 49% OptiPrep (Sigma) using a 60% OptiPrep stock solution, and subjected to flotation equilibrium density gradient centrifugation (44). Five hundred microliters was placed in a polyallomer ultracentrifuge tube and overlaid with a gradient of 48 to 15% OptiPrep in cold buffer B (without TX-100).…”

“…Samples were centrifuged to equilibrium using an SW55 Ti rotor (100,000 ϫ g for 18 h at 4°C), and fractions of ϳ200 l each were manually collected from the bottom of the tube. The refractive indices of the fractions were determined and used to calculate the respective densities (44). Proteins were concentrated by overnight precipitation in 10% cold trichloroacetic acid and were collected by high-speed centrifugation.…”

“…These pellets were washed in cold acetone, air dried, solubilized in 50 l of 7 M urea sample buffer (7 M urea, 125 mM Tris HCl [pH 6.95], 2% SDS, 0.1 mM dithiothreitol, 1 mM EDTA, 0.1% bromophenol blue), and heated to 55°C for 15 min. Samples were resolved by SDS-PAGE (10% Bis-Tris gel) and transferred to nitrocellulose membranes as previously described (44). GPC was detected using the G2-directed MAb F106G3.…”

“…(Bottom) Caveolin-containing lipid rafts were detected in parallel gradients by Western blot analysis. Buoyant densities calculated from the refractive indices of fractions (44) are plotted along the horizontal axes to standardize individual gradients.…”