Figure 7. Active Secretion of DNA and Histones through an Amphisome-Dependent Mechanism.

(A) DKO-1 cells stained for DAPI, and endogenous CD63 and CD9, and imaged with 3D SIM. (Top) Z-stack projections. (Bottom) Enlarged inserts. See also Figure S7A.

(B) Z-slice image from (A), and greyscale enlarged insert.

(C) Size and structure of CD63-positive multivesicular endosomes (MVEs) by 3D SIM. (Top) Two types of CD63-positive MVE structure. Cropped images of DKO-1 cells stained for CD63. (Bottom) Data are mean ± SD. n = 57, data from five independent experiments.

(D) Cropped images of DKO-1 cells localized for CD63 and double-stranded DNA (dsDNA). (Left) Greyscale CD63 and dsDNA, and colorized merge. Yellow arrows in dsDNA channel indicate dsDNA peak intensity in line scans. Line scans were performed at yellow dotted lines in colorized merge. (Right) Line scans indicate drop of CD63 signal at dsDNA peak intensity. For line scan graphs, individual channels were normalized to display on graph. Scale bars, 250 nm.

(E) Cropped images of DKO-1 cells localized for CD63 and Histone H3 by 3D SIM. (Left) Greyscale CD63 and Histone H3, and colorized merge. Displayed are sequential image slices (125 nm) through the Z-stack. (Right) Enlarged image of the CD63-positive MVE with yellow line indicating longest axis. Scale bar, 200 nm.

(F) Cropped images of DKO-1 cells localized for CD63 and p62 by 3D SIM. (Left) Greyscale CD63 and p62, and colorized merge. Displayed are sequential image slices (125 nm) through the Z-stack. (Right) Enlarged image of the CD63-positive MVE with yellow line indicating longest axis. Scale bar, 200 nm.

(G) CD63-and LC3B-positive amphisomes in DKO-1 cells. Cells were stained for endogenous CD63 and LC3B. Greyscale CD63 and LC3B, colorized merge, and enlarged inserts. Yellow arrows in LC3B channel and enlarged inserts indicate LC3B localized to CD63-positive compartments. Scale bars for enlarged inserts, 200 nm.

(H) Localization of dsDNA in CD63-, LC3B-positive amphisomes in DKO-1 cells. (Left) Cropped images of cells localized for endogenous CD63, LC3B and dsDNA and imaged by three-color 3D SIM. X-Y axis “top-down view” of greyscale CD63, LC3B and dsDNA, and colorized merge. (Right) X-Z axis “side view”. Yellow arrow indicates CD63-positive compartment, and blue arrow indicates LC3B-positive compartments, both co-localized at the plasma membrane. Scale bars, 200 nm.

(I) Detection LC3B-PE positive EVs. (Left) Immunoblots of DKO-1 whole cell lysates, large EVs (P15) and crude small EVs (P120). (Right) Immunoblots of whole cell lysates and crude small EVs (P120) isolated from matched tissue/interstitial fluid of a colorectal cancer (CRC), adjacent normal and lymph node metastasis. N, normal; T, tumor; LM, lymph node metastasis.

(J) Gradient (6–30%) density fractionation of crude DKO-1 sEVs (P120).

(K) DIC of CD81-, CD63-and CD9-positive exosomes. Immunoblots of crude sEV pellet (P120) and bead-captured exosomes from DKO-1 (top) and Gli36 cells (bottom).

(L) Model of amphisome-dependent, exosome-independent secretion. For autophagy, cytosolic LC3 is lipidated by conjugation with phosphatidylethanolamine to form LC3-PE.

1) Nuclear membranes can bleb in a process dependent on LC3B and the nuclear lamina protein Lamin B1, causing the appearance of cytoplasmic chromatin fragments. 2) During autophagy, cytoplasmic components are sequestered as a phagophore begins to engulf material. 3) Continued expansion of autophagic membranes requires LC3-PE and results in formation of the double-membrane autophagosome. 4) As early endosomes develop to late endosomes, the pH decreases and continued invagination of limiting membranes generates intraluminal vesicles (ILVs). A fully developed CD63-positive multivesicular endosome (MVE) contains numerous ILVs. 5) Fusion of the autophagosome with a MVE causes degradation of the inner autophagosome membrane generating an amphisome, a single-membrane hybrid compartment. 6) The amphisome fuses with a lysosomal compartment to form the autolysosome followed by degradation of cargo, or alternatively, 7) the amphisome fuses with the plasma membrane causing extracellular release of dsDNA and histones, and separately, the ILVs as exosomes.

See also Figure S7.