Intramembrane particles and filipin labelling on the membranes of autophagic vacuoles and lysosomes in mouse liver

Abstract

Morphologically detectable protein (intramembrane particles) and cholesterol (filipin labelling) in the membranes of autophagic vacuoles and lysosomes were studied in mouse hepatocytes using thin-section and freeze-fracture electron microscopy. Both isolated autophagic vacuoles and lysosomes, and intact tissue blocks were used due to the facts (i) that lysosomes are difficult to recognize in freeze-fracture replicas of intact hepatocytes, and (i) that filipin penetration into the tissue blocks is unsatisfactory. Intramembrane particle density was low in the membranes of early autophagic vacuoles (defined as round-shaped vacuoles in which an inner membrane parallel with the outer limiting membrane was clearly visible). The lysosomal membranes contained considerably more intramembrane particles. Particle-rich lysosomes or other vesicles were observed to fuse with the early autophagic vacuoles. The membranes of nascent autophagic vacuoles with morphologically intact contents were usually not labelled by filipin, whereas the membranes of all other autophagic vacuoles and lysosomes were heavily labelled. The increased cholesterol in the membranes of slightly older autophagic vacuoles is presumably derived from cholesterol-rich lysosomes or other vesicles fusing with the vacuoles and from the degrading organelles inside the autophagic vacuoles.

Maturation of Autophagic Vacuoles in Mammalian Cells

“…We observed that the same applies to membrane cholesterol. Phagophores have no detectable cholesterol, but the cholesterol content increases during the maturation to early and late autophagic vacuoles 35 (Fig. 2).…”

“…Based on morphological, cytochemical and immunoelectron microscopy data, autophagosomes are proposed to originate from the membranes of the endoplasmic reticulum. 11 However, the structural and biochemical characteristics of these membranes are different from the endoplasmic reticulum, [35][36][37][38] and thus the phagophores or isolation membranes should probably be regarded as distinct organelles.…”

Guidelines for the use and interpretation of assays for monitoring autophagy

“…In quickly frozen and fractured cells the fracture runs preferentially along the hydrophobic plane of the membranes, allowing characterization of the limiting membranes of the different types of autophagic vacuoles and visualization of their limited protein intramembrane particles (IMPs, or integral membrane proteins). Several studies have been performed using this technique on yeast, 53 as well as on mammalian cells or tissue, first on mouse exocrine pancreas, 54 then on mouse and rat liver, 55,56 mouse seminal vesicle epithelium 24,51 or cancer cell lines (e.g., breast cancer MDA-MB-231) 57 to investigate the various phases of autophagosome maturation, and to reveal useful details about the origin and evolution of their limiting membranes. 2,8,[58][59][60] The phagophore and the limiting membranes of autophagosomes contain few, or no detectable, IMPs ( Fig.…”

“…3C). 8,24,[53][54][55][56]61,62 Autolysosomes are generally delimited by a single membrane because, in addition to the engulfed material, the inner membrane is degraded by the lytic enzymes. Similarly, the limiting membrane of autophagic bodies in yeast and plants is also quickly broken down under normal conditions.…”

Comigration of Two Autophagosome-Associated Dehydrogenases on Two-Dimensional Polyacrylamide Gels

“…2,9 Furthermore, freeze-fracture studies have shown that phagophores and autophagosomes differ from other membranebounded organelles in having delimiting membranes with exceptionally few of the intramembrane particles that correspond to transmembrane proteins. [10][11][12][13] However, although autophagosomes and phagophores may be deficient with respect to transmembrane proteins, recent studies have identified several proteins that appear to be peripherally associated with the membranes of these organelles. In mouse embryonic stem cells, the mammalian homologues of Atg5 and Atg8, two of the proteins required for autophagy in yeast, were found to bind to sequestering membranes that apparently correspond to phagophores.…”