Jeanne Ahlberg

Autophagy, heterophagy, microautophagy and crinophagy as the means for intracellular degradation.

SummaryIt is generally accepted that the lysosomal compartment plays an important role in the degradation of cellular components.In this communication we discuss various experimental models which have been used to study mechanisms of intralysosomal degradation and also discuss the evidence obtained in support of the following proposals:1.The autophagosomes can be isolated into high purity and are the subcellular locus of induced protein degradation.2.Different membrane components such as proteins and lipids are degraded at different rates inside the lysosomes. Intralysosomal hydrolysis is not the rate limiting step in degradation.3.Lysosomes take up soluble material in vitro by invagination and pinching off of their membranes (microautophagy).4.Secretory vesicles can degrade their secretory contents by fusing with the lysosomes.

Comparison of different autophagic vacuoles with regard to ultrastructure, enzymatic composition, and degradation capacity—Formation of crinosomes

The number of rat liver autophagic vacuoles (AVs) was increased by separate injection of three different inhibitors--vinblastine, leupeptin, and chloroquine--of lysosomal protein degradation. The different mechanisms of action of the agents correlated to the ultrastructure of the AVs. Accumulation of the base chloroquine with ensuing influx of water into AVs caused a significant swelling. The leupeptin-induced AVs were processed into residual-body-like structures within a few hours of exposure in line with the presence of a leupeptinase in liver tissue. Vinblastine was the most efficient agent in increasing the occurrence of AVs. The effect of vinblastine lasted for the entire study period (36 hr) with continuous formation of nascent AVs. In addition, vinblastine caused the appearance of a subpopulation of AVs laden with VLDL particles. The term crinosomes was suggested for these hybrid organelles, since they seemed to evolve by fusion between secretory granules and lysosomes. In addition to sequestered cell organelles, the AVs harbored cytosolic enzyme activities (LDH and aldolase). Leupeptin was the only agent that caused a decrease in cathepsin B and L activities. Similarly, leupeptin impeded protein breakdown in isolated AVs, whereas vinblastine and chloroquine evoked an increase. In vivo, chloroquine and vinblastine block protein degradation. The reason for this discrepancy is probably that during in vivo exposure the substrate (cytoplasmic proteins) is built up in the AVs because degradation is retarded. Upon isolation of the AVs the inhibitor block is released, and proteolysis proceeds at enhanced rates over control due to excess of substrates. Leupeptin, on the other hand, caused a substantial inhibition of thiol proteinases; this block remained in the isolated AVs. Accordingly, leupeptin-induced AVs displayed decreased protein degradation following shorter exposure times. Later, when leupeptin was metabolized, catch-up proteolysis was noted. The differing mechanisms of action of the inhibitors were also apparent as regards lipid contents and lipolysis. Whereas chloroquine and vinblastine increased the amounts of cholesterol and triglycerides parallel to proteins, leupeptin had no such effect. Lipolysis proceeded at normal rate following leupeptin administration, which was not the case after vinblastine and chloroquine exposure. Leupeptin has no effect on acid lipases; therefore lipids do not accumulate in AVs of hepatocytes that are exposed to leupeptin.

In vitro uptake of particles by lysosomes

Abstract Isolated rat liver lysosomes were incubated with Percoll particles in vitro at 25 and 37 °C. On morphological examination the incubated lysosomes contain vesicles some of which enclose Percoll particles, indicating that invagination of the lysosomal membrane occurs in vitro by means of microautophagy. Vesiculation occurs by formation of flaplike processes or cuplike invaginations. At later time points of incubation Percoll particles can be seen free in the lysosomal matrix indicating rupture or digestion of the vesicular membrane. The uptake of isotopically pre-labelled Percoll particles increases with incubation time and temperature. It is concluded that lysosomes show microautophagic activity in vitro and that this may be a mechanism for degradation of soluble cytoplasmic proteins also in vivo.

Degradation of short- and long-lived proteins in perfused liver and in isolated autophagic vacuoles—Lysosomes

The lysosomal contribution to breakdown of prelabeled short- and long-lived cell proteins in the perfused rat liver was monitored in two ways. In the first, either leupeptin or chloroquine was injected into rats as well as added to the perfusate. The extent of suppression of proteolysis compared to that of controls was equated with the lysosomal share of protein breakdown. Both compounds inhibited degradation of short-lived proteins by some 30% and long-lived proteins by some 60%. In the second approach, lysosomal-autophagic vacuolar (LAV) fractions were isolated from livers pretreated and perfused as above. The LAV fractions generated different amounts of degradation products during subsequent incubations. They were, in decreasing order of magnitude, that of the LAV fraction isolated from livers perfused with chloroquine, without inhibitor, and with leupeptin. The LAV fraction from control livers was considered to yield the most reliable estimate of the lysosomal share of proteolysis as recorded during liver perfusions. It was 54% for short-lived proteins and 75% for long-lived proteins. These figures are higher than those obtained in the perfusion experiments in the first approach. It is therefore suggested that quantitative assessment concerning the lysosomal share of overall proteolysis may be underestimated when based on inhibition experiments performed on intact cells. The ultrastructure of hepatocytes and LAV fractions was also investigated. Leupeptin-treated hepatocytes displayed more frequent and larger AVs than those of control livers. Cell vacuolation was an additional characteristic of chloroquine-exposed hepatocytes. The LAV fractions isolated from control and leupeptin-treated livers mirrored the observations made in liver tissue. The constituents of the LAV fraction isolated from chloroquine-treated tissue were, however, less swollen than in situ, probably due to the extraction of water during isolation.

Isolation of two lysosomal populations from iron-overloaded rat liver with different iron concentration and proteolytic activity

SummaryIron overload results in an accumulation of electron-dense iron-containing particles (IPs) such as ferritin and hemosiderin within the lysosomes of rat liver cells. In order to evaluate the effect or iron overload on lysosomal function, efforts were made to isolate lysosomes with different iron contents by means of ultracentrifugation in Percoll and Metrizamide gradients.Lysosomes isolated on the Percoll gradient were characterized ultrastructurally by a uniform matrix consisting mainly of IPs and these lysosomes contained a high iron concentration and showed a very low proteolytic activity. They may, therefore, constitute, or be equated, with a special type of residual bodies. They were also fragile, as judged by their significant release of enzymes during incubation in vitro.Lysosomes isolated in the Metrizamide gradient contained remnants of sequestered organelles and some IPs. These organelles displayed a somewhat impeded proteolytic activity compared with control lysosomes, as well as preserved membrane stability during incubation in vitro. We suggest that these may be precursors of the heavily iron-laden lysosomes recovered in the Percoll gradient.Our findings demonstrate that different populations of lysosomes exist in iron-overloaded rat liver cells, which show specific characteristics with regard to ultrastructural appearance, iron content and proteolytic activity. Differing iron contents is the most likely reason for their diverging densities and membrane integrities, whereas the difference in proteolytic activity could be a result of varying amounts of degradable substrate.

Characterization of the proteolytic compartment in rat hepatocyte nodules

SummaryPersistant liver nodules (hepatocyte nodules, neoplastic nodules) were produced in rat liver by intermittent feeding with 2-acetylaminofluorene. Dense bodies (secondary lysosomes) were purified and characterized from the nodules. The purity of the dense body fraction was 90%. The levels of various lysosomal enzyme activities were lower in these dense bodies in comparison with dense bodies from control liver. Similarly, protein degradation was 50% lower in dense bodies from liver nodules than in control liver. The number of autophagic vacuoles (AVs) in the nodular tissue increased considerably after 3 h vinblastine treatment. We have taken advantage of this expansion in an effort to isolate these organelles from liver nodules. Autophagic vacuoles have been isolated recently from liver and kidney but not from putatively premalignant liver nodules. Fraction purity of AVs from liver nodules was 95%. As with dense bodies, AVs from nodular tissue displayed lower activities of proteinases and lower rates of protein degradation when compared with their counterparts from normal liver tissue.Accordingly, the lower rate of overall protein degradation in liver nodules can be ascribed to a decrease in lysosomal activity. A diminished autophagic sequestration capacity is the most plausible explanation for the decreased rate of proteolysis in cells. This could conceivably give these nodular cells a growth advantage and assist in their selective outgrowth as well as in their transformation from neoplastic into true cancer cells.

Proteolysis in isolated autophagic vacuoles from rat liver. Effect of pH and of proteolytic inhibitors.

Abstract Autophagic vacuoles (AV) were purified from livers of rats which were pretreated with vinblastine (VBL) to increase the occurrence of AV. To measure proteolysis in the isolated AV rats were labelled with [ 14 C]leucine 2 or 16 h before sacrifice. The integrity of the AV was studied by measuring the leakage of hydrolytic enzymes during incubation at various pHs. VBL causes an increase in the degradation rate of liver homogenate and isolated AV. This increase was moderate if proteolysis was measured at neutral pH, whereas adjustment to acidic pH enhanced the rate of autodegradation in the AV several-fold. This indicates that the VBL-induced AV have acquired hydrolytic enzymes either by fusion with lysosomes or possibly by the sequestering endoplasmic reticulum (ER) membranes forming the limiting membranes of the AV. The internal pH is not optimal for degradation in vitro of sequestered proteins, indicating insufficient acidification of the isolated AV. Lysosomotropic inhibitors, like chloroquine and propylamine, but not asparagine, impede proteolysis in isolated AV, but not more than 40%.

Rapid isolation of rat liver secondary lysosomes—Autophagic vacuoles—Following chloroquine administration☆

A technique is presented for the rapid isolation of secondary lysosomes--autophagic vacuoles (AVs)--from rat liver by a one-step centrifugation in a discontinuous Metrizamide gradient. To this end chloroquine was injected into rats in order to increase the number of AVs, a prerequisite for their isolation, since they are rare in control liver tissue. Fraction purity was some 85-90% as judged from morphological analyses. To assess the proteolytic ability of the AVs they were isolated from livers of rats injected with [14C]leucine. Proteolysis increased significantly peaking after 60 min of chloroquine exposure, by far exceeding values for lysosomes isolated from control livers. This is in contrast to AVs (secondary lysosomes) obtained after leupeptin treatment which display lowered proteolysis as compared with control. After longer exposure times to chloroquine, proteolysis gradually returned to basal level. It is surmised that the augmented proteolysis in the isolated AVs is due to increased contents of substrate. So far, the chloroquine-induced AVs (secondary lysosomes) seem to be the best candidates for further analyses of proteolytic events in these organelles.

Effects on in vivo and in vitro administration of vinblastine on the perfused rat liver—Identification of crinosomes

Livers of nonstarved rats were perfused for up to 4 hr in a recirculating system. Bile production, transaminases, and the lactate/pyruvate ratio remained at normal values. The ultrastructure of the hepatocytes was also well preserved even after the 4-hr perfusion. When vinblastine was given either in vivo or in vitro by addition to the perfusion fluid, it caused a conspicuous expansion of the autophagic-lysosomal compartment. Initially, nascent autophagic vacuoles developed, followed by the appearance of more mature ones and finally an increase in dense bodies was observed. In addition, administration of vinblastine in vivo gave rise to an increased occurrence of a subpopulation of lysosomes laden with VLDL-like particles. The term crinosomes seems appropriate for these lysosomal vesicles, since they apparently evolve by means of fusion between retained secretory granules and preexisting lysosomes (dense bodies). Addition of vinblastine of the perfusion fluid decreased the rate of proteolysis whether four times the serum concentration of amino acids were added or not. However, when vinblastine was given in vivo, proteolysis as measured in the perfusate decreased during the initial 3 hr of VBL treatment, whereas by longer times of pretreatment protein degradation exceeded the control value, constituting an example of catch-up proteolysis. Autophagic vacuoles isolated after short exposure to vinblastine in vivo exhibited high rates of protein degradation when incubated at acid pH. Insufficient proton pumping rather than lack of hydrolytic enzymes seems to be the most plausible explanation for this prompt pH effect.

Isolation and characterization of autophagic vacuoles from rat kidney cortex

SummaryThe number of autophagic vacuoles in the proximal tubule cells of the rat kidney increased considerably after 3 h of vinblastine treatment. This increase was paralleled by stimulated proteolysis in an homogenate prepared from the cortex. We have taken advantage of this expansion in autophagic vacuoles in an effort to isolate these organelles from rat kidney cortex on a discontinuous Metrizamide gradient. Autophagic vacuoles have recently been purified from liver but not from other tissues.The purity of the isolated fraction was 95% of which 55% consisted of typical intact autophagic vacuoles containing sequestered organelles and 45% of other types of secondary lysosome. On plane section many of these displayed one or several intramatrical vesicles or flap like processes forming apparant vesicles at the pole of the organelles, which occasionally contained pinocytosed membranous material. These lysosomes were designated microautophagic vacuoles. It is suggested that the microautophagic vacuoles could be the morphological expression of uptake into lysosomes of small portions of cytosol. The isolated autophagic vacuole fraction was enriched in lysosomal enzymes (acid phosphatase and cathepsin D activities) and displayed high proteolytic rates, especially at acid pH.