Johnny Silva Cancer Research

The Amphisome

Amphisomes are defined as organelles formed by the fusion of autophagosomes with endosomes or heterophagosomes. Amphisomes can thus be classified as either autophagic or heterophagic/endocytic organelles. The existence of these hybrid organelles was first indicated by biochemical experiments that used electroinjected, radiolabelled lactose to investigate the autophagic-lysosomal pathway. Cytoplasmic lactose was found to be taken up into autophagosomes by autophagy, but was degraded by the lysosomal a-galactosidase upon delivery of autophagocytosed material to the lysosome.49 However, high concentrations of aspar-agine inhibited the autophagic-lysosomal flux, causing lactose to accumulate in prelysosomal autophagic organelles. The surprising discovery was that this prelysosomal lactose could be degraded by endocytosed a-galactosidase, suggesting that it resided in a vacuolar compartment accessible to both autophagic and endocytic inputs, i.e., an amphisome.7,73 Subsequent studies showed that amphisome accumulation could also be induced by leupeptin treatment, apparently due to the relative fusion incompetence of degradation-inhibited lysosomes filled up with autophagocytosed cytoplasm.8 A large fraction of the "autolysosomes" isolated after leupeptin treatment63 would thus actually be amphisomes. The amphisomes could be physically separated from lysosomes on density gradients, and eventually purified by a modification8 of the procedure used for autophagosome preparation,27 where lysosomes are selectively removed by substrate-induced osmotic disruption. 5

Amphisomes can be recognized ultrastructurally as autophagic organelles containing both autophagocytosed cytoplasm and endocytosed markers (e.g., asialoglycoprotein-gold conjugates, Fig. 5A-E), but lacking lysosomal markers.6 They have an easily recognizable, but often slightly denatured, cytoplasmic contents, and are usually delimited by a single membrane, which can sometimes be partially or completely double, depending on the extent of autophagosome-endosome fusion and on the plane of section.8 The amphisomes are often complex, clearly being the result of multiple fusion events involving several autophagosomes as

Figure 6. Lysosomes. Rat hepatocytes, incubated for 2 h at 37°C under autophagic conditions, were processed for conventional electron microscopy (A), or used to prepare purified lysosomes8 subjected to freeze-fracture electron microscopy (B).4

well as several endosomes (Fig. 5B). In freeze-fracture images, the fusion between particle-rich endosomes and smooth autophagosomes is well visualized (Fig. 5F,G).4 Eventually, the endosomal intramembrane particles (transmembrane proteins) diffuse out and distribute evenly over the merged membrane of the amphisome, giving the amphisomes an average particle density of about 90 particles/mm2, i.e., 4-5x higher than the average autophagosome (about 20 particles/mm2), but still considerably lower than the average lysosome (Fig. 6B; about 2000 particles/mm2).4

Amphisomes have been shown to have an acidic interior,74 suggesting that the observed denaturation of contents may be due to acidification by a proton pump, brought in by the endosomal fusion partner. Most of the autophagic organelles of intermediate morphology (contents denatured, but not degraded) described and classified as AVi/d, are thus likely to be amphisomes.9,60,75 The presence of endocytic markers cleanly distinguishes amphisomes from autophagosomes, but the distinction versus lysosomes can be more problematic, because the endosomal fusion partner may furnish the amphisome with a variable portifolio of lysosomal enzymes.76 For example, acid phosphatase was found to be present in the majority of pancreatic amphisomes (type I AVs), whereas cathepsins B and D, the lysosomal membrane glycoprotein Lgp120 and the cation-independent mannose 6-phosphate receptor were found exclusively in lysosomes (type II AVs). 5 In rat hepatocytes, cathepsin D and Lgp120 were similarly confined to lysosomes,6,60 and would appear to be suitable as lysosomal markers.

Is amphisome formation an obligatory step in the autophagic-lysosomal pathway, required to make prelysosomal autophagic organelles competent for fusion with lysosomes? In yeast cells, the ATPase Vps4, required for endosomal translocation and multivesicular body formation (see chapter 14), was found to be necessary for autophagy as well.77 Mutations in its mammalian homologue, Skd1, prevented amphisome formation and caused an accumulation of autophagosomes in HeLa cells.78 Starvation-induced protein degradation was strongly suppressed under these conditions, suggesting that the autophagic-lysosomal flux proceeded predominantly through amphisome formation. However, the possibility should also be considered that Skd1 might be directly involved in autophagosomal translocation, thereby preventing autophagosome-lysosome fusion independent of its effect on endosomes (see chapter 15).

In isolated rat hepatocytes, an ultrastructural study of vacuole fusion profiles indicated that about 80% of the autophagosomal fusion events were with endosomes, and that 20% represented direct autophagosome-lysosome fusion.60 The amphisomal pathway would thus seem to be predominant, but not obligatory. Apparently, both early and late endosomes can fuse with autophagosomes to form amphisomes.8 Although one report observed autophagosome-lysosome fusion only, this was probably due to a very low rate of marker en-docytosis in the cell line studied, with negligible endocytic flux into either amphisomes or lysosomes.79 In hepatocytes, there was rapid delivery of endocytosed material both to amphisomes and directly to lysosomes.80 The endocytic-lysosomal pathway was operative even if autophagy had been blocked by 3-methyladenine,81 i.e., amphisome formation is not an obligatory step in this pathway either.80 In vivo, hepatic endocytosis was found to proceed mainly along the direct pathway to the lysosome, with little amphisome formation.82 Convergence of the endocytic and autophagic pathways at the amphisome level would thus seem to represent a condition-dependent, probably regulated event.

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