N-Rh-PE is sorted to discrete domains of the T cell plasma membrane. (A–C) Jurkat cells pulse labeled with N-Rh-PE at 4°C for 1 h were washed, returned to growth medium, and examined by fluorescence microscopy at 0 (A), 1 (B), and 24 (C) h after labeling. (D) 24 h after labeling, exosomes were collected, bound to coverglass, and examined by fluorescence microscopy. (E–L) Unpermeabilized Jurkat cells that had been pulse labeled with N-Rh-PE and incubated at 37°C overnight were fixed and processed for fluorescence microscopy using antibodies specific for CD81 (E–H) or CD63 (I–L). (M–P) Unpermeabilized Jurkat cells that had been pulse labeled with N-F-PE and incubated at 37°C overnight were fixed and processed for fluorescence microscopy using antibodies specific for CD63. (Q–T) Unpermeabilized Jurkat cells were pulse labeled with N-Rh-PE for 1 h, incubated for 16 h at 37°C, pulse labeled with N-F-PE for 1 h, incubated for an additional 16 h at 37°C, and processed for fluorescence microscopy. (U and V) Exosomes were collected from the second 16-h incubation, bound to coverglass, and examined by fluorescence microscopy. (U) N-Rh-PE–containing exosomes; (V) N-F-PE–containing exosomes. N-F-PE–containing exosomes are marked by white circles. (W–Z) Unpermeabilized Jurkat T cells that had been pulse labeled with N-Rh-PE and incubated at 37°C overnight were fixed and processed for fluorescence microscopy using antibodies specific for Lamp1. (A′–D′) Unpermeabilized Jurkat T cells that had been pulse labeled with N-Rh-PE were fixed, stained with antibodies specific for CD63, and examined by fluorescence microscopy. Primary T cells that had been pulse labeled with N-Rh-PE were also examined by fluorescence microscopy and found to possess single (E′ and F′) or multiple (G′ and H′) domains enriched for this lipid. Bars, 10 μm.

Class E VPS proteins at discrete domains of the Jurkat T cell plasma membrane. (A–D) Unpermeabilized Jurkat T cells expressing AIP1/VPS31-YFP were processed for fluorescence microscopy using antibodies specific for CD81. (A) AIP1/VPS31-YFP. (E–H) Unpermeabilized Jurkat T cells expressing AIP1/VPS31-DsRed were processed for fluorescence microscopy using antibodies specific for CD63. (E) AIP1/VPS31-DsRed. (I–S) Unpermeabilized Jurkat T cells (I–P) or K562 cells (Q–S) expressing VPS4B/K180Q-DsRed were processed for fluorescence microscopy using antibodies specific for CD63. (I, M, and Q) VPS4B/K180Q-DsRed. (T–W) N-Rh-PE–labeled Jurkat T cells were fixed, permeabilized, and processed for fluorescence microscopy using antibodies specific for TSG101. Bars (I–P and T–W), 10 μm; (Q–S) 5 μm.

HIV Gag is sorted to endosome-like domains of T cells and K562 cells. (A–H) Unpermeabilized Jurkat cells expressing HIV Gag-GFP were processed for fluorescence microscopy using antibodies specific for CD81 (A–D) or CD63 (E–H). (I–L) Unpermeabilized Jurkat cells pulse labeled with N-Rh-PE and expressing HIV Gag-GFP were processed for fluorescence microscopy. (M–P) Unpermeabilized Jurkat T cells expressing AIP1/VPS31-DsRed and HIV Gag-GFP were processed for fluorescence microscopy. (Q–S) K562 cells expressing VPS4BK180Q-DsRed and HIV Gag-YFP were processed for fluorescence microscopy. (Q) VPS4B-K180Q-DsRED. (T–W) Unpermeabilized, N-Rh-PE–labeled Jurkat T cells expressing HIV Gag-GFP were processed for fluorescence microscopy. Bars, 10 μm.

HIV virions bud from the plasma membrane in association with CD81. 293T cells expressing a proviral clone of HIV were processed for cryoimmunoelectron microscopy using antibodies specific for CD81 and gold-conjugated secondary antibodies. Arrows denote position of gold particles. Bars, 100 nm.

Jurkat T cells sort exosomal proteins to discrete domains of the plasma membrane. (A and B) Sucrose density gradient analysis of exosomes from Jurkat T cells (A) and K562 cells (B). Densities of the different fractions are listed at the top in grams/milliliters. Note the significant enrichment of CD81 and CD63 in exosome fractions relative to the levels of the proteins in the whole cell lysate (wcl; CD81 could not be detected in the whole cell lysate sample). The same amount of whole cell lysate and gradient fractions were used for all three blots. (C–J) Jurkat T cells were processed for fluorescence microscopy using antibodies specific for CD81 (C–F) or CD63 (G–J) under permeabilizing (C, D, G, and H) or nonpermeabilizing (E, F, I, and J) conditions. Shown are fluorescence (C, E, G, and I) and phase-contrast (D, F, H, and J) images of representative cells. (K–N) Unpermeabilized Jurkat T cells were fixed and processed for fluorescence microscopy using antibodies specific for CD81 (K) and CD63 (L). (M) Merge of K and L. (N) Phase-contrast image of the cell. Bars, 10 μm.

Domain dynamics. (A–D) Jurkat T cells (A and B) or K562 cells (C and D) were pulse labeled with N-Rh-PE for 1 h, incubated overnight, and examined by live cell video microscopy. (E–J) Jurkat T cells were pulse labeled with N-Rh-PE for 1 h, incubated for 16 h, incubated with 15 mM β-cyclodextrin for 30 min, washed, and incubated for various lengths of time before examination by fluorescence microscopy. (E and F) Mock-treated cells examined at t = 0 after treatment. (G and H) β-cyclodextrin–treated cells examined at t = 0 after treatment. (I and J) Methyl-β-cyclodextrin–treated cells examined at t = 8 h after treatment. (K and L) The monocyte cell line U937 was labeled with N-Rh-PE for 1 h, grown overnight, fixed, and processed for immunofluorescence microscopy using FITC-conjugated antibodies specific for CD43. Shown are two representative cells in which the N-Rh-PE (red), surface CD43 (green), and phase-contrast images have been merged. Bars, 10 μm.

Exosomes appear to bud from discrete domains of the Jurkat T cell plasma membrane. (A and B) A presumptive outward budding intermediate (nascent exosome) at the plasma membrane of a K562 cell (B) or Jurkat T cells (A, left) adjacent to either another outward budding vesicle or a completely budded vesicle (A, right). (C and D) HRP/DAB detection of CD63 at the Jurkat T cell surface. (C) A cluster of three CD63-positive regions (arrowheads). (D) A higher magnification image of the boxed region, possibly with a presumptive outward budding intermediate. (E–I). Immunogold labeling of Jurkat T cell cryosections. (E) Most of the Jurkat T cell was devoid of label. (F) When CD81 (arrows) could be detected, it was typically clustered. (G) CD81 (arrowheads) decorated presumptive outward vesicle budding intermediates. (H) A higher magnification view of the budding intermediate (boxed area) from G. Arrows denote the positions of immunogold labeling for CD81. (I) CD81 (arrowheads) can be detected in domains that span micrometers of the plasma membrane. (J and K) Immunogold labeling (arrowheads) of Jurkat T cells for N-Rh-PE (6 nm gold) and CD81 (18 nm gold). (J) CD81 and N-Rh-PE colocalized at presumptive outward budding intermediates. (K) N-Rh-PE was enriched at discrete domains of the plasma membrane. Bars (A, B, D–H, J, and K), 100 nm; (C and I) 1 μm.

HIV Gag buds from endosome-like domains of Jurkat T cells and K562 cells. (A) Jurkat T cells processed for transmission electron microscopy of cryosections possess nascent HIV Gag buds at their plasma membrane. (B and C) Jurkat T cells processed for transmission electron microscopy of cryosections using antibodies specific for CD81 and gold-conjugated secondary antibodies. Arrows highlight immunogold labeling for surface CD81. (D–J) K562 cells expressing HIV Gag were fixed, surface labeled with monoclonal antibodies specific for CD63 and gold-conjugated secondary antibodies, and processed for electron microscopy. Boxed area in D is shown at a higher magnification in E. Arrows and arrowheads highlight immunogold labeling for surface CD63. Bars, 100 nm.

HIV Gag is secreted from cells in exosomes. (A and B) Exosomes were collected from Jurkat cells pulse labeled with N-Rh-PE and expressing HIV Gag-GFP and were examined by fluorescence microscopy. Circles mark secreted vesicles that contain HIV Gag-GFP. (C and D) Thin-section electron micrographs of exosomes collected from Jurkat cells expressing HIV Gag. Arrows denote the position of exosomes that contain the electron-dense HIV Gag core. (E and F) Immunoblot analysis of whole cell lysates (wcl) and sucrose density gradient fractions from conditioned media prepared from HIV Gag–expressing (E) Jurkat cells and (F) K562 cells. Bars (A and B), 20 μm; (C) 500 nm; (D) 100 nm.