The autophagic pathway is actively modulated by phase II Coxiella burnetii to efficiently replicate in the host cell

The etiologic agent of Q fever Coxiella burnetii, is an intracellular obligate parasite that develops large vacuoles with phagolysosomal characteristics, containing multiple replicating bacteria. We have previously shown that Phase II C. burnetii replicative vacuoles generated after 24-48 h post infection are decorated with the autophagic protein LC3. The aim of the present study was to examine, at earlier stages of infection, the distribution and roles of the small GTPases Rab5 and Rab7, markers of early and late endosomes respectively, as well as of the protein LC3 on C. burnetii trafficking. Our results indicate that: (i) Coxiella phagosomes (Cph) acquire the two Rab proteins sequentially during infection; (ii) overexpression of a dominant negative mutant form of Rab5, but not of Rab7, impaired Coxiella entry, whereas both Rab5 and Rab7 dominant negative mutants inhibited vacuole formation; (iii) Cph colocalized with the protein LC3 as early as 5 min after infection; acquisition of this protein appeared to be a bacterially driven process, because it was inhibited by the bacteriostatic antibiotic chloramphenicol and (iv) C. burnetii delayed the arrival of the typical lysosomal protease cathepsin D to the Cph, which delay is further increased by starvation-induced autophagy. Based on our results we propose that C. burnetii transits through the normal endo/phagocytic pathway but actively interacts with autophagosomes at early times after infection. This intersection with the autophagic pathway delays fusion with the lysosomal compartment possibly favouring the intracellular differentiation and survival of the bacteria.     

Autophagosomes: a fast-food joint for unexpected guests

Coxiella burnetii is a Gram-negative obligate intracellular bacterium that infects a wide range of hosts including humans, causing Q fever, a disease characterized by high fever and flu-like symptoms. After its internalization the Coxiella-containing phagosomes interact with intracellular compartments and generate a large replicative vacuole that displays certain characteristics of a phagolysosome. We have shown that this bacterially-customized replicative vacuole also has the hallmarks of an autophagosomal compartment. Furthermore, in a recent publication we have reported that induction of autophagy is beneficial for the replication and survival of Coxiella. Different morphological forms of this bacterium have been described during its developmental cycle. Here we present additional data and discuss a model indicating that induction of autophagy favors the differentiation of the Coxiella small cell variants to the metabolically active large cells variants. We postulate that nutrient acquisition, likely by fusion with the nutrient-rich autophagic vacuoles, triggers the development of the large cell variants which actively multiply in the host cell.     

Beclin 1 modulates the anti-apoptotic activity of Bcl-2: Insights from a pathogen infection system

Coxiella burnetii is an obligate intracellular bacterium that generates large vacuoles in which this pathogen replicates and survives. We have previously demonstrated that C. burnetii interacts with the autophagic pathway as a strategy for its survival and replication. Coxiella displays an anti-apoptotic activity to maintain host cell viability, leading to a persistent infection. Our recent study reveals that Beclin 1 is recruited to the Coxiella-membrane vacuole favoring its development and bacterial replication. In contrast, the anti-apoptotic protein Bcl-2 alters the normal development of the Coxiella-replicative compartment. In addition, our results indicate that C. burnetii infection modulates autophagy and apoptotic pathways via Beclin 1-Bcl-2 interplay to establish a successful infection in the host cell. Of note, this pathogen-host cell model has allowed uncovering a novel function of Beclin 1 as a regulator of the anti-apoptotic activity of Bcl-2. We have also established that a proper interplay between Beclin 1 and Bcl-2 is required for both autophagy and apoptosis modulation.     

Acidification modulates the traffic of Trypanosoma cruzi trypomastigotes in Vero cells harbouring Coxiella burnetii vacuoles

We studied the fate of different Trypanosoma cruzi trypomastigote forms after they invade Vero cells persistently colonised with Coxiella burnetii. When the invasion step was examined we found that persistent C. burnetii infection per se reduced only tissue-culture trypomastigote invasion, whereas raising vacuolar pH with Bafilomycin A1 and related drugs, increased invasion of both metacyclic and tissue-culture trypomastigotes when compared with control Vero cells. Kinetic studies of trypomastigote transfer indicated that metacyclic trypomastigotes parasitophorous vacuoles are more efficiently fused to C. burnetii vacuoles. The higher tissue-culture trypomastigote hemolysin and transialidase activities appear to facilitate their faster escape from the parasitophorous vacuole. Sialic acid deficient Lec-2 cells facilitate the escape of both forms. Endosomal-lysosomal sequential labelling with EEA1, LAMP-1, and Rab7 of the parasitophorous vacuoles formed during the entry of each infective form revealed that the phagosome maturation processes are also distinct. Measurements of C. burnetii vacuolar pH disclosed a marked preference for trypomastigote fusion with more acidic rickettsia vacuoles. Our results thus suggest that intravacuolar pH modulates the traffic of trypomastigote parasitophorous vacuoles in these doubly infected cells.     

Survival of Trypanosoma cruzi metacyclic trypomastigotes within Coxiella burnetii vacuoles: differentiation and replication within an acidic milieu

Coxiella burnetii, the etiological agent of Q fever, is an obligate intracellular bacterium that resides within acidified vacuoles with secondary lysosomal characteristics. Infective stages of Trypanosoma cruzi, the causative agent of Chagas' disease, actively invade a wide variety of cells, a process followed by lysosomal recruitment. Recently, we have investigated and characterized early events that occur in Vero cells persistently colonized with C. burnetii when doubly infected with T. cruzi trypomastigote forms. Kinetic studies of trypomastigote transfer indicated that parasitophorous vacuoles (PV) of metacyclic trypomastigotes are rapidly and efficiently fused to C. burnetii vacuoles. Based on these observations we have investigated the behavior of metacyclic trypomastigotes within C. burnetii vacuoles beyond 12 h of co-infection inside Vero cells. Using indirect immunofluorescence with MAb against different developmental stages, it was possible to follow the T. cruzi differentiation process within C. burnetii vacuoles after up to 96 h post-invasion. We observed that metacyclic trypomastigotes began to differentiate after 12 h of infection, and 24 h later amastigotes were the prevailing forms within C. burnetii vacuoles. T. cruzi amastigote replication within C. burnetii vacuoles was confirmed using video and time-lapse confocal microscopy and around 36 h of co-infection, cytokinesis took about 70 min to occur. After 72 h, we observed that amastigote forms seemed to escape from C. burnetii vacuoles. Labeling of amastigotes within C. burnetii vacuoles using a polyclonal antibody to C9 complement protein suggested that TcTOX (T. cruzi hemolysin) could play a role in parasite escape from C. burnetii. We concluded that T. cruzi has an outstanding adaptation capability and can survive within a hostile milieu such as C. burnetii vacuoles.     

Coxiella burnetii survival in THP-1 monocytes involves the impairment of phagosome maturation: IFN-gamma mediates its restoration and bacterial killing

The subversion of microbicidal functions of macrophages by intracellular pathogens is critical for their survival and pathogenicity. The replication of Coxiella burnetii, the agent of Q fever, in acidic phagolysosomes of nonphagocytic cells has been considered as a paradigm of intracellular life of bacteria. We show in this study that C. burnetii survival in THP-1 monocytes was not related to phagosomal pH because bacterial vacuoles were acidic independently of C. burnetii virulence. In contrast, virulent C. burnetii escapes killing in resting THP-1 cells by preventing phagosome maturation. Indeed, C. burnetii vacuoles did not fuse with lysosomes because they were devoid of cathepsin D, and did not accumulate lysosomal trackers; the acquisition of markers of late endosomes and late endosomes-early lysosomes was conserved. In contrast, avirulent variants of C. burnetii were eliminated by monocytes and their vacuoles accumulated late endosomal and lysosomal markers. The fate of virulent C. burnetii in THP-1 monocytes depends on cell activation. Monocyte activation by IFN-gamma restored C. burnetii killing and phagosome maturation as assessed by colocalization of C. burnetii with active cathepsin D. In addition, when IFN-gamma was added before cell infection, it was able to stimulate C. burnetii killing but it also induced vacuolar alkalinization. These findings suggest that IFN-gamma mediates C. burnetii killing via two distinct mechanisms, phagosome maturation, and phagosome alkalinization. Thus, the tuning of vacuole biogenesis is likely a key part of C. burnetii survival and the pathophysiology of Q fever.     

Maturation of the Coxiella burnetii parasitophorous vacuole requires bacterial protein synthesis but not replication

This study examined whether protein synthesis and replication are required for maturation and fusogenicity of the lysosomal-like, large and spacious parasitophorous vacuole (PV) of Coxiella burnetii, an obligate intracellular bacterium. Large and spacious PV with multiple non-replicating C. burnetii were observed by phase microscopy in Vero cells infected at a multiplicity of infection of ten and treated with a bacteriostatic concentration of nalidixic acid or carbenicillin, antimicrobics that inhibit DNA and cell wall biosynthesis respectively. Conversely, large and spacious PV were not observed in cells treated with a bacteriostatic concentration of the protein synthesis inhibitor chloramphenicol. Rather, fluorescence microscopy of individual cells revealed multiple, acidic PV harbouring a single organism tightly bounded by a LAMP-1 positive vacuolar membrane. These vacuoles homotypically fused to form a large and spacious PV upon removal of the drug. Chloramphenicol also inhibited trafficking of latex beads to large and spacious PV and caused mature PV to collapse. Collectively, these results demonstrate that C. burnetii protein synthesis, but not replication, is required for fusion between nascent C. burnetii PV and latex bead phagosomes, and also for formation and maintenance of large and spacious, replicative PV. However, transit of nascent PV through the endocytic pathway to ultimately acquire lysosomal markers appears to occur irrespective of Coxiella protein synthesis.     

Lounging in a lysosome: the intracellular lifestyle of Coxiella burnetii

Most intracellular parasites employ sophisticated mechanisms to direct biogenesis of a vacuolar replicative niche that circumvents default maturation through the endolysosomal cascade. However, this is not the case of the Q fever bacterium, Coxiella burnetii. This hardy, obligate intracellular pathogen has evolved to not only survive, but to thrive, in the harshest of intracellular compartments: the phagolysosome. Following internalization, the nascent Coxiella phagosome ultimately develops into a large and spacious parasitophorous vacuole (PV) that acquires lysosomal characteristics such as acidic pH, acid hydrolases and cationic peptides, defences designed to rid the host of intruders. However, transit of Coxiella to this environment is initially stalled, a process that is apparently modulated by interactions with the autophagic pathway. Coxiella actively participates in biogenesis of its PV by synthesizing proteins that mediate phagosome stalling, autophagic interactions, and development and maintenance of the mature vacuole. Among the potential mechanisms mediating these processes is deployment of a type IV secretion system to deliver effector proteins to the host cytosol. Here we summarize our current understanding of the cellular events that occur during parasitism of host cells by Coxiella.     

Coxiella burnetii express type IV secretion system proteins that function similarly to components of the Legionella pneumophila Dot/Icm system

Coxiella burnetii is an obligate intracellular pathogen that replicates in large endocytic vacuoles. Genomic sequence data indicate that 21 genes encoding products that are similar to components of the Legionella pneumophila Dot/Icm type IV secretion system are located on a contiguous 35 kb region of the Coxiella chromosome. It was found that several dot/icm genes were expressed by Coxiella during host cell infection and that dot/icm gene expression preceded the formation of large replicative vacuoles. To determine whether these genes encode a functional type IV secretion system, we have amplified the Coxiella dotB, icmQ, icmS and icmW genes and produced the encoded proteins in Legionella mutants in which the native copy of each gene had been deleted. The Coxiella dotB, icmS and icmW products restored dot/icm-dependent growth of Legionella mutants in eukaryotic host cells. The Coxiella IcmQ protein and the Legionella IcmR protein did not interact, which could explain why the Coxiella icmQ gene was unable to restore growth to a Legionella icmQ mutant. Thus, Coxiella encodes functional components of a type IV secretion system expressed in vivo that is mechanistically related to the Legionella Dot/Icm apparatus. These studies suggest that a dot/icm-related secretion system could play an important role in creating the specialized vacuole that supports Coxiella replication.     

Construction of chimeric phagosomes that shelter Mycobacterium avium and Coxiella burnetii (phase II) in doubly infected mouse macrophages: an ultrastructural study.

Dual infection of cells may divert pathogens to intracellular compartments different from those occupied in mono-infected cells. In the present studies, mouse bone marrow in vitro-derived macrophages were first infected with virulent Mycobacterium avium, which are normally singly lodged within tight phagosomes. These phagosomes do not mature; they undergo homotypic fusion with early endosomes and do not fuse with lysosomes. Seven days later, the cultures were superinfected with phase II (non-virulent) Coxiella burnetii, organisms sheltered in lysosome- (or prelysosome)-like, multi-occupancy phagosomes. The latter can attain large size and engage in efficient homo- and heterotypic fusion with other phagosomes. Cultures were fixed for transmission electron microscopy 6, 12, 24, and 48 h later. Other M. avium-infected cultures were superinfected with amastigotes of the trypanosomatid flagellate Leishmania amazonensis, which are also sheltered in lysosome- (or prelysosome)-like multi-occupancy vacuoles, and fixed at the same time periods. Chimeric phagosomes containing both M. avium and C. burnetii, were found already at 6 h and the proportion of M. avium that colocalized with C. burnetii in the same phagosomes reached over 90% after 48 h. In such phagosomes, both organisms were ultrastructurally well preserved. In contrast, colocalization of M. avium and L. amazonensis was rarely found. Speculative scenarios that could underlie the formation of chimeric phagosomes could involve delayed maturation of C. burnetii-containing phagosomes in presence of M. avium, which would allow for fusion of C. burnetii- and M. avium-containing phagosomes; the production, by C. burnetii, of molecules that upregulate the fusion of M. avium-containing phagosomes with those that contain C. burnetii; and the secretion of factors that could favour the survival of M. avium within chimeric vacuoles.     

Infection of Vero cells with Coxiella burnetii phase II: relative intracellular bacterial load and distribution estimated by confocal laser scanning microscopy and morphometry

Coxiella burnetii, the agent of Q fever in man and of coxiellosis in other species, is an intracellular pathogen not yet grown axenically. Confocal laser fluorescence microscopy and morphometry were used to measure relative C. burnetii phase II loads and their intracellular distribution in aldehyde fixed and DAPI stained Vero cell monolayers. The fluorescence of single horizontal optical sections provided useful information on relative loads of bacteria in cells and vacuoles. The relative density of the bacteria in the vacuoles was inferred from ratios of fluorescence to vacuolar section areas. Relative bacterial loads, bacterial densities and section areas of large vacuoles increased exponentially between days 2 and 4 of the infection of gamma-irradiated host cells, stabilized between days 4 and 6, and decreased thereafter. Estimated minimum doubling times were higher for the overall complement of the intracellular organisms (about 12 h) than for bacteria that were confined to larger vacuoles (about 10 h).     

In exocrine pancreas, the basolateral endocytic pathway converges with the autophagic pathway immediately after the early endosome

Intracisternal granules (ICGs) are insoluble aggregates of pancreatic digestive enzymes and proenzymes that develop within the lumen of the rough endoplasmic reticulum of exocrine pancreatic cells, especially in guinea pigs. These ICGs are eliminated by autophagy. By morphological criteria, we identified three distinct and sequential classes of autophagic compartments, which we refer to as phagophores, Type I autophagic vacuoles, and Type II autophagic vacuoles. Lobules of guinea pig pancreas were incubated in media containing HRP for periods of 5-120 min to determine the relationship between the endocytic and autophagic pathways. Incubations with HRP of 15 min or less labeled early endosomes at the cell periphery that were not involved in autophagy of ICGs, but after these short incubations none of the autophagic compartments were HRP positive. After 30-min incubation with HRP, early endosomes at the cell periphery, late endosomes in the pericentriolar region, and, in addition, Type I autophagic vacuoles containing ICGs were all labeled by the tracer. Type II autophagic vacuoles were not labeled after 30-min incubation with HRP but were labeled after incubations of 60-120 min. Phagophores did not receive HRP even after 120 min incubations. We concluded that the autophagic and endocytic pathways converge immediately after the early endosome level and that Type I autophagic vacuoles precede Type II autophagic vacuoles on the endocytic pathway. We studied the distribution of acid phosphatase, lysosomal proteases and cation-independent-mannose-6-phosphate receptor (CI-M6PR) in the three classes of autophagic compartments by histochemical and immunocytochemical methods. Phagophores, the earliest autophagic compartment, contained none of these markers. Type I autophagic vacuoles contained acid phosphatase but, at most, only very low levels of cathepsin D and CI-M6PR. Type II autophagic vacuoles, by contrast, are enriched for acid phosphatase, cathepsin D, and other lysosomal enzymes, and they are also enriched for CI-M6PR. Moreover, soluble fragments of bovine CI-M6PR conjugated to colloidal gold particles heavily labeled Type II but not Type I autophagic vacuoles, and this labeling was specifically blocked by mannose-6-phosphate. This indicates that the lysosomal enzymes present in Type II autophagic vacuoles carry mannose-6-phosphate monoester residues. Using 3-C2, 4-dinitroanilino-3'-amino-N-methyldipropylamine (DAMP), we showed that Type II autophagic vacuoles are acidic. We interpret these findings as indicating that Type II autophagic vacuoles are a prelysosomal compartment in which the already combined endocytic and autophagic pathways meet the delivery pathway of lysosomal enzymes.     

Synthesis of ribonucleotides and their participation in ribonucleic acid synthesis by Coxiella burnetii.

Synthesis of ribonucleic acid (RNA) by the deoxyribonucleic acid-dependent RNA polymerase of Coxiella burnetii required adenosine, uridine, guanosine, and cytidine 5'-triphosphates. Cell-free preparations of this obligate intracellular procaryotic parasite had competence to phosphorylate ribonucleoside mono- and diphosphates in the presence of exogenous adenosine and guanosine 5'-triphosphates to the corresponding di- and triphosphates. C. burnetii contained about 2 nmol of adenosine 5'-triphosphate per mg of protein, which could serve as a approximately P donor for in vivo synthesis of nucleoside triphosphates. The latter were then used as substrates in the synthesis of RNA in a coordinated metabolic system with C. burnetii RNA polymerase. It is suggested that during infection the rickettsiae might obtain the nucleotides necessary for RNA synthesis from the vacuoles in which C. burnetii proliferates.     

Mouse resident peritoneal macrophages partially control in vitro infection with Coxiella burnetii phase II

Coxiella burnetii, the agent of Q fever in man and of coxiellosis in other species, is a small, dimorphic, obligate intracellular bacterium, sheltered within large, acidified, and hydrolase-rich phagosomes. Although several primary and established cell lines, macrophage-like cells, and primary macrophages from other species have been infected with C. burnetii, the infection of mouse primary macrophages has not been sufficiently characterized. In this report quantification of DAPI (4', 6-diamino-2-phenylindole) fluorescence images acquired by confocal microscopy, and transmission electron microscopy were used to compare the infection of three mouse-derived cells, L929 fibroblasts, J774 macrophage-like cells, and resident peritoneal macrophages, with a phase II clone of C. burnetii known to be non-virulent for mammals. Infected peritoneal phagocytes differed from L929 or J774 cells in that: (a) large vacuoles took longer to appear (3-5 d instead of 2), and were only found in a subset (20-30%) of macrophages, as opposed to in more than 70% of the other cells; (b) total and vacuole-associated relative bacterial loads in L929 and J774 cells were several-fold higher than in peritoneal macrophages; (c) estimated doubling times of the bacteria were about 68 h in the primary macrophages, 18 h in J774 and 22 h in L929 cells. Thus, mouse resident peritoneal macrophages control both the formation of the large vacuoles and the intracellular proliferation of C. burnetii phase II.     

Comparative sensitivity of four different cell lines for the isolation of Coxiella burnetii

Coxiella burnetii is an obligate intracellular bacterium that causes the disease Q-fever. This is usually diagnosed by serology (immunofluorescence assay) and/or PCR detection of C.?burnetii DNA. However, neither of these methods can determine the viability of the bacterium. Four different cell lines were compared for their ability to amplify very low numbers of viable C.?burnetii. Two different isolates of C.?burnetii were used. For the Henzerling isolate, DH82 (dog macrophage) cells were the most sensitive with an ID (50) (dose required to infect 50% of cell cultures) of 14.6 bacterial copies. For the Arandale isolate, Vero (monkey epithelial) cells were the most sensitive with an ID (50) of less than one bacterium in a 100-μL inoculum. The Vero cell line appeared highly useful as vacuoles could be seen microscopically in unstained infected cells. The findings of this study favour the use of Vero and DH82 tissue culture cell lines for isolation and growth of C.?burnetii in vitro. The other cell lines, XTC-2 and L929, were less suitable.     

Fluorescence properties and staining behavior of monodansylpentane, a structural homologue of the lysosomotropic agent monodansylcadaverine.

We have recently shown that monodansylcadaverine labels autophagic vacuoles. Analysis of the mechanism underlying the labeling revealed that monodansylcadaverine acts as a lysosomotropic agent, being concentrated into acidic compartments by an ion-trapping mechanism, and as a solvent polarity probe, increasing its relative fluorescence intensity by interacting with membrane lipids that are highly concentrated in the autophagic vacuoles. In this study, we synthesized three structurally related derivatives of monodansylcadaverine, replacing the primary amino group of monodansylcadaverine with a neutral (dansylamylamine; MDH), a polar (dansylaminopentanol; MDOH), or an acidic group (dansylaminovaleric acid; MDA), to replace the lysosomotropic character of the marker. Whereas MDH showed a specific staining of autophagic vacuoles, the polar and acidic derivatives did not show any staining. We further demonstrate that the MDH staining of autophagic vacuoles is independent on the acidic pH and thus on an ion-trapping mechanism, but it still shows the same preferences for autophagic membrane lipids as monodansylcadaverine. We propose that MDH can specifically interact with lamellar bodies of the autophagic type as a solvent polarity probe. Therefore, dansylated aminopentane can be used as a specific marker for autophagic vacuoles in vivo and in fixed cells.(J Histochem Cytochem 49:177-185, 2001)     

Analysis of whole cell lysate from the intercellular bacterium Coxiella burnetii using two gel-based protein separation techniques

Coxiella burnetii, the causative agent of Q fever, is an obligate intracellular gamma-proteobacterium, which replicates within large phagolysosome-like compartments formed in the host cell. The global protein profile of intracellular C. burnetii strain Nine Mile phase II was analyzed by two gel-based approaches coupled to MALDI-TOF MS. Colloidal Coomassie brilliant blue-stained 2-DE gels at the pH range 3-10 resolved over 600 protein spots and 125 spots in doubled-SDS-PAGE gels. Mass spectra obtained for each trypsin-digested protein-spot were compared to the C. burnetii genome database, and a total number of 185 different C. burnetii proteins were identified by both techniques. 2-DE in combination with MALDI-TOF MS, as a high-throughput method, allowed the identification of 172 proteins. On the other hand, the application of doubled-SDS-PAGE allowed the identification of 38 proteins, with some of them being very alkaline and membrane proteins not identified in the 2-DE approach. Most identified proteins were predicted to be involved in metabolism and biosynthesis. Several identified proteins are speculated to have a distinct and vital role in the pathogenesis and survival of C. burnetii within the harsh phagolysosomal environment.     

Disturbed cholesterol traffic but normal proteolytic function in LAMP-1/LAMP-2 double-deficient fibroblasts

Mice double deficient in LAMP-1 and -2 were generated. The embryos died between embryonic days 14.5 and 16.5. An accumulation of autophagic vacuoles was detected in many tissues including endothelial cells and Schwann cells. Fibroblast cell lines derived from the double-deficient embryos accumulated autophagic vacuoles and the autophagy protein LC3II after amino acid starvation. Lysosomal vesicles were larger and more peripherally distributed and showed a lower specific density in Percoll gradients in double deficient when compared with control cells. Lysosomal enzyme activities, cathepsin D processing and mannose-6-phosphate receptor expression levels were not affected by the deficiency of both LAMPs. Surprisingly, LAMP-1 and -2 deficiencies did not affect long-lived protein degradation rates, including proteolysis due to chaperone-mediated autophagy. The LAMP-1/2 double-deficient cells and, to a lesser extent, LAMP-2 single-deficient cells showed an accumulation of unesterified cholesterol in endo/lysosomal, rab7, and NPC1 positive compartments as well as reduced amounts of lipid droplets. The cholesterol accumulation in LAMP-1/2 double-deficient cells could be rescued by overexpression of murine LAMP-2a, but not by LAMP-1, highlighting the more prominent role of LAMP-2. Taken together these findings indicate partially overlapping functions for LAMP-1 and -2 in lysosome biogenesis, autophagy, and cholesterol homeostasis.     

Combined effects of fasting and vinblastine treatment on serum insulin level, the size of autophagic-lysosomal compartment, protein content and lysosomal enzyme activities of liver and exocrine pancreatic cells of the mouse

1. The volume fraction of autophagic vacuoles in liver parenchymal and exocrine pancreatic cells was smallest and the serum insulin level highest in the 24 hr prestarved mouse immediately after 3 hr feeding period. 2. The size of the autophagic vacuole and lysosome (dense body) compartments increased in both types of cells during 2-72 hr fasting parallel with decreasing serum insulin levels. 3. The protein content of the cells decreased and the DNA-based activity of acid phosphatase showed little change throughout fasting. The activity of cathepsin D increased during days 2 and 3 of food deprivation. 4. Vinblastine (50 mg/kg body wt) applied for the last 2 hr of different periods (2, 12, 24, 48 and 72 hr) of fasting decreased serum insulin level and increased the fractional cytoplasmic volume of autophagic vacuoles and dense bodies. This increase was smaller when the drug was applied shortly after feeding and much larger after prolonged fasting. The increase was more pronounced in the pancreatic than in the liver cells. 5. Our data show that the effect of vinblastine on the size of the autophagic-lysosomal compartment depends on the feeding status of the animals.     

Development of an autophagic system in differentiating cells of the cellular slime moldDictyostelium discoideum

Summary Changes in an autophagic system during differentiation of cells ofDictyostelium discoideum, NC-4 were studied under light and electron microscopes, and it was demonstrated cytochemically that acid phosphatase was almost exclusively localized in food and autophagic vacuoles. Autophagic vacuoles first appeared during formation of loose aggregates, coupled with the defecation of food vacuoles. Autophagic vacuoles seem to originate from flat sacs which segregate parts of the cytoplasm. No acid phosphatase was detected in the vacuoles when first formed, but activity appeared later probably due to fusion with Golgi-like vesicles. When starved cells were not allowed to aggregate due to a low cell density, they formed no autophagic vacuoles but retained many food vacuoles. This indicates that the formation of autophagic vacuoles is not simply due to starvation, but to cell interaction mediated by cell contact. Autophagic vacuoles containing acid phosphatase rapidly increased in number in all cells in the early stage of aggregation. After papillae formed, however, they selectively decreased in the prespore cells, but developed further and grew larger in the prestalk cells.