Toyoko Ishikawa

Immunocytochemical study of the surrounding envelope of autophagic vacuoles in cultured rat hepatocytes

By the use of electron immunoperoxidase cytochemistry at the ultrastructural level, the relationship of the surrounding sac of the autophagic vacuoles to the different cytomembranes was studied. When the endoplasmic reticulum was completely stained for microsomal carboxyesterase E1, the enzyme was not found to be labeled in the developed envelopes forming autophagic vacuoles. The autophagic envelope at the formative stages was also devoid of albumin which intensely stained Golgi cisternae. However, although it was rare, the endoplasmic reticulum showed an electron-lucent region like an early autophagic envelope in its cisternae which was lacking in carboxyesterase E1. In addition, deeply curving swelled cisternae where carboxyesterase E1 was found at the edges were occasionally encountered. These observations suggest that the segregating membranes arise from an endoplasmic reticulum and the structural characteristics of the endoplasmic membranes change at very early stages of formation of autophagic vacuoles. Acid phosphatase, a lysosomal marker enzyme, began to be localized on sections of the double membranes of newly created autophagic vacuoles. The enzyme spread all along the limiting membranes of the autophagic vacuoles, while, at the same time, the double membranes were converted into a single membrane. A lysosomal membrane glycoprotein (LGP107) was also localized on the surrounding envelope of autophagic vacuoles in a fashion similar to that of acid phosphatase. Lysosomal hydrolases seem to play some role in the conversion of double limiting membranes into a single limiting membrane.

Ultrastructural studies on autolysosomes in rat hepatocytes after leupeptin treatment

We have studied the morphological alterations of the lysosomal compartment in rat hepatocytes following intraperitoneal administration of leupeptin, using electron microscopy and cytochemical techniques. At 30 min after the injection, autophagic vacuoles (autophagosomes and autolysosomes), containing cytoplasmic organelles, increased in number in the vicinity of bile canaliculi and also near the Golgi apparatus. At 1 h, most of the autophagic vacuoles were autolysosomes, single membrane-limited bodies positive for acid phosphatase activity. Development of the autolysosomes was accompanied by the reciprocal disappearance of pre-existing secondary lysosomes. From 1 to 8 h, the autolysosomes varied to a great extent in both size and shape as a result of coalescence. Segregated organelles within the autolysosomes were gradually degraded into electron-lucent unidentifiable debris. At later, residual bodies were abundant in the cytoplasm, and occasionally, their contents were discharged into the space of Disse. From 9 to 12 h, the autolysosomes decreased in the volume and number and secondary lysosomes of normal shape and size appeared. The autolysosomes seem to persist for long periods because of a retarded degradation of sequestered materials in leupeptin-treated hepatocytes.

Two lysosomal membrane proteins, LGP85 and LGP107, are delivered to late endosomes/lysosomes through different intracellular routes after exiting from the trans-Golgi network

Lysosomal membrane proteins are delivered from their synthesis site, the endoplasmic reticulum (ER) to late endosomes/lysosomes through the Golgi complex. It has been proposed that after leaving the Golgi they are transported either directly or indirectly (via the cell surface) to late endosomes/lysosomes. In the present study, we examined the transport routes taken by two structurally different lysosomal membrane proteins, LGP85 and LGP107, in rat 3Y1-B cells. Here we show that newly synthesized LGP85 and LGP107 are delivered to late endosomes/lysosomes via a direct route without passing through the cell surface. Interestingly, although LGP107 is delivered from the Golgi to early endosomes containing internalized horseradish peroxidase-conjugated transferrin (HRP-Tfn) en route to lysosomes, LGP85 does not pass through the HRP-Tfn-positive early endosomes. These results suggest, therefore, that LGP85 and LGP107 are sorted into distinct transport vesicles at the post-Golgi, presumably the trans-Golgi network (TGN), after which LGP85 is delivered directly to late endosomes/lysosomes, but significant fractions of LGP107 are targeted to early endosomes before transport to late endosomes/lysosomes. This study provides the first evidence that after exiting from the Golgi, LGP85 and LGP107 are targeted to late endosomes/lysosomes via a different pathway.

Accumulation of autolysosomes after receptor-mediated introduction of Ep459-asialofetuin conjugate into lysosomes in rat hepatocytes.

Administration of Ep459-asialofetuin conjugate (Ep459-AF) and pepstatin-asialofetuin conjugate (Ps-AF) to rats effectively inhibited lysosomal BANA hydrolase and cathepsin D in the liver, respectively, at a very low dose. Ep459-AF treatment also led to an accumulation of autolysosomes in rat liver. There was a close correlation between the accumulation of autolysosomes and the inhibition of BANA hydrolase activity. However, as opposed to the inhibition of thiol proteases, the inhibition of cathespin D did not cause accumulation of autolysosomes in the rat liver. These results suggest that autophagy in rat hepatocytes is a common occurrence under normal physiological conditions and that thiol proteases are digestive enzymes essential for the autolysomes.

Transport of acid phosphatase to lysosomes does not involve passage through the cell surface

In foregoing studies, we reported that LGP107, a major lysosomal membrane glycoprotein in the rat liver, distributes in and circulates continuously throughout the endocytic membrane system (endosomes, lysosomes and plasma membrane), in hepatocytes (1,2). In the present study we examined whether acid phosphatase (APase), an enzyme that is transported to lysosomes as a transmembrane protein, passes through the cell surface during intracellular transport, because transport of newly synthesized APase to lysosomes involves the passage of endosomes containing a ligand which is internalized via receptors on the cell surface and is finally dispatched to lysosomes for degradation (3). When localization of APase in rat hepatocytes was investigated by immunoelectron microscopy, APase was found to be localized in lysosomes and endosomes, but not in coated pits on the cell surface, which are positive for LGP107, and from which antibodies for LGP107 are internalized. Further, unlike LGP107, newly synthesized APase was not detected in plasma membranes isolated from livers of rats given [35S]methionine, and when cultured hepatocytes were exposed to 125I-labeled anti APase IgG at 37 degrees C, there was no transfer of the antibody to lysosomes even after 24 h incubation. Therefore, these results indicate that intracellular movement of APase does not involve cell surface passage in rat hepatocytes, and clearly differs from the recent report that human APase is transported to lysosomes via the cell surface in BHK cells transfected with its cDNA (4).

Requirement of phosphatidylglycerol for flagellation of Escherichia coli.

We report that phosphatidylglycerol is required for flagellation of Escherichia coli. Cells carrying the pgsA3 mutation did not form swarm rings in semisolid agar. P1 transduction experiments revealed that the potential for phosphatidylglycerol synthesis and for the formation of swarm rings was co‐transducible. The pgsA3 mutant transformed with the wild type pgsA + gene cloned into the R‐plasmid vector had the potential for both phosphatidylglycerol synthesis and cell motility. Electronmicroscopic and SDS‐PAGE analyses showed that the pgsA3 mutation causes the lack of flagellation.

Isolation of Golgi fractions from colchicine-treated rat liver. I. Morphological and enzymic characterization.

1. Three Golgi fractions, GF-1, GF-2 and GF-3, were isolated from the livers of rats pretreated with colchicine, which gave better yields of the fractions than ethanol treatment of rats. 2. Electron microscopic observation showed that GF-1 was composed mainly of secretory vesicles, GF-3 consisted predominantly of small tubules and flattened cisternae, and GF-2 was an intermediate fraction composed of secretory vesicles and cisternal elements. 3. Among these three fractions the highest activity of galactosyl transferase, marker enzyme of the Golgi complex, was found in GF-3 and the lowest activity was in GF-1, although a different distribution of the enzymes was observed in fractions obtained from ethanol-treated rat liver. 4. Enzymatic characterization of these fractions showed that no significant contamination with other subcellular components occurred in GF-1 and GF-2.