Autophagic Compartments Research Articles

Binding preference of p62 towards LC3-ll during dopaminergic neurotoxin-induced impairment of autophagic flux

Accumulating evidence has revealed that autophagy may be beneficial for treatment of neurodegenerative diseases through removal of abnormal protein aggregates. However, the critical autophagic events during neurodegeneration remain to be elucidated. Here, we investigated whether prototypic autophagic events occur in the MN9D dopaminergic neuronal cell line upon exposure to N-methyl-4-phenylpyridinium (MPP+), a well-known dopaminergic neurotoxin. MPP+ treatment induced both morphological and biochemical characteristics of autophagy, such as accumulation of autophagic vacuoles and LC3-II form and decreased p62 levels. Further investigation revealed that these phenomena were largely the consequences of blocked autophagic flux. Following MPP+ treatment, levels of LC3-II formed and p62 dramatically increased in the Triton X-100-insoluble fraction. Levels of ubiquitinated proteins also increased in this fraction. Further colocalization analyses revealed that the punctated spots positive for both p62 and LC3 were more intense following MPP+ treatment, suggesting drug-induced enrichment of these two proteins in the insoluble fraction. Intriguingly, reciprocal immunoprecipitation analysis revealed that p62 mainly precipitated with LC3-II form following MPP+ treatment. Transient transfection of the mutant form of Atg4B, Atg4BC74A, which inhibits LC3 processing, dramatically decreased binding between p62 and LC3-II form. Taken together, our results indicate that p62 can be efficiently localized to autophagic compartments via preferential binding with LC3-II form. This colocalization may assist in removal of detergent-insoluble forms of damaged cellular proteins during dopaminergic neurotoxin-induced impairment of autophagic flux.

Cellular machinery to fuse antimicrobial autophagosome with lysosome

Autophagy is an intracellular bulk degradation/recycling system that turns over cellular constituents and also functions to degrade intracellular foreign microbial invaders by a process termed xenophagy (antimicrobial autophagy). We previously showed that intracellular group A Streptococcus (GAS) organisms are captured by xenophagosomes, then degraded following fusion with lysosomes. Very recently, we analyzed the molecular mechanism underlying xenophagosome/lysosome fusion and found that endocytic soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) are involved. Knockdown of the combinational SNARE proteins Vti1b and VAMP8 with siRNAs disturbed autophagic fusion with lysosomes, and cellular bactericidal efficiency was significantly diminished. Furthermore, knockdown of those SNAREs inhibited the fusion of canonical autophagosomes with lysosomes. In addition, important findings showed that Vti1b is derived from autophagic compartments, whereas VAMP8 originates from lysosomes. Together, these results strongly suggest that SNARE proteins Vti1b and VAMP8 mediate the fusion of antimicrobial and canonical autophagosomes with lysosomes, an essential event for autophagic degradation.

Devil inside: does plant programmed cell death involve the endomembrane system?

Eukaryotic cells have to constantly cope with environmental cues and integrate developmental signals. Cell survival or death is the only possible outcome. In the field of animal biology, tremendous efforts have been put into the understanding of mechanisms underlying cell fate decision. Distinct organelles have been proven to sense a broad range of stimuli and, if necessary, engage cell death signalling pathway(s). Over the years, forward and reverse genetic screens have uncovered numerous regulators of programmed cell death (PCD) in plants. However, to date, molecular networks are far from being deciphered and, apart from the autophagic compartment, no organelles have been assigned a clear role in the regulation of cellular suicide. The endomembrane system (ES) seems, nevertheless, to harbour a significant number of cell death mediators. In this review, the involvement of this system in the control of plant PCD is discussed in-depth, as well as compared and contrasted with what is known in animal and yeast systems.

Autophagic Compartments Gain Access to the MHC Class II Compartments in Thymic Epithelium

The presentation of self-peptides in the context of MHC molecules by thymic epithelial cells (TECs) is essential for T cell repertoire selection in the thymus. However, the underlying mechanisms of this process have not been fully elucidated. To address whether autophagy, a catabolic process involving the degradation of a cell's components through the lysosomal machinery, intersects the MHC class II-restricted Ag presentation pathway in TECs, we investigated the colocalization of LC3, a peculiar autophagy marker molecule, with MHC class II compartments in in vitro-established TEC lines by immunofluorescence microscopy and Western blotting analyses. We found that in both cortical and medullary TEC lines, LC3 was colocalized with the H2-DM-positive lysosomal compartments, in which MHC class II plus class II-associated invariant chain peptides complexes are formed. Furthermore, our analysis of thymic cryosections from 1-day-old mice revealed that LC3 colocalizes with the H2-DM-positive compartments in TECs. These results strongly suggest that the cytoplasmic self-Ags gain access to the H2-DM-positive compartments via the autophagic process in the thymus.

Autophagy is involved in starvation response and cell death in Blastocystis

Previous studies have demonstrated that colony forms of Blastocystis undergo cell death with numerous membrane-bound vesicles containing organelles located within the central vacuole, resembling morphological features of autophagy. In this study, we investigated whether Blastocystis underwent autophagy upon amino acid starvation and rapamycin treatment. Concurrently, we provide new insight into a possible function of the central vacuole. The use of the autophagy marker monodansylcadaverine, and the autophagy inhibitors 3-methyladenine and wortmannin, showed the existence of autophagy in amino-acid-starved and rapamycin-treated Blastocystis. Confocal microscopy and transmission electron microscopy studies also showed morphological changes that were suggestive of autophagy. The unusually large size of the autophagic compartments within the parasite central vacuole was found to be unique in Blastocystis. In addition, autophagy was found to be triggered when cells were exposed to the cytotoxic antibody mAb 1D5, and autophagy was intensified in the presence of the caspase inhibitor zVAD.fmk. Taken together, our results suggest that the core machinery for autophagy is conserved in Blastocystis, and that it plays an important role in the starvation response and cell death of the parasite.

Varicella-Zoster Virus Infection Induces Autophagy in both Cultured Cells and Human Skin Vesicles

When grown in cultured cells, varicella-zoster virus (VZV) forms many aberrant light particles and produces low titers. Various studies have explored the reasons for such a phenotype and have pointed to impaired expression of specific late genes and at lysosomal targeting of egressing virions as possible causes. In the studies presented here, we report that the autophagic degradation pathway was induced at late time points after VZV infection of cultured cells, as documented by immunoblot analysis of the cellular proteins LC3B and p62/SQSTM1, along with electron microscopy analysis, which demonstrated the presence of both early autophagosomes and late autophagic compartments. Autophagy was induced in infected cells even in the presence of phosphonoacetic acid, an inhibitor of viral late gene expression, thus suggesting that accumulation of immediate-early and early viral gene products might be the major stimulus for its induction. We also showed that the autophagic response was not dependent on a specific cell substrate, virus strain, or type of inoculum. Finally, using immunofluorescence imaging, we demonstrated autophagosome-specific staining in human zoster vesicles but not in normal skin. Thus, our results document that this innate immune response pathway is a component of the VZV infectious cycle in both cultured cells and the human skin vesicle, the final site of virion formation in the infected human host.

Selective vulnerability of dorsal root ganglia neurons in experimental rabies after peripheral inoculation of CVS-11 in adult mice

The involvement of dorsal root ganglia was studied in an in vivo model of experimental rabies virus infection using the challenge virus standard (CVS-11) strain. Dorsal root ganglia neurons infected with CVS in vitro show prolonged survival and few morphological changes, and are commonly used to study the infection. It has been established that after peripheral inoculation of mice with CVS the brain and spinal cord show relatively few neurodegenerative changes, but detailed studies of pathological changes in dorsal root ganglia have not previously been performed in this in vivo experimental model. In this study, adult ICR mice were inoculated in the right hindlimb footpad with CVS. Spinal cords and dorsal root ganglia were evaluated at serial time points for histopathological and ultrastructural changes and for biochemical markers of cell death. Light microscopy showed multifocal mononuclear inflammatory cell infiltrates in the sensory ganglia and a spectrum of degenerative neuronal changes. Ultrastructural changes in gangliocytes included features characteristic of the axotomy response, the appearance of numerous autophagic compartments, and aggregation of intermediate filaments, while the neurons retained relatively intact mitochondria and plasma membranes. Later in the process, there were more extensive degenerative neuronal changes without typical features of either apoptosis or necrosis. The degree of degenerative neuronal changes in gangliocytes contrasts with observations in CNS neurons in experimental rabies. Hence, gangliocytes exhibit selective vulnerability in this animal model. This contrasts markedly with the fact that they are, unlike CNS neurons, highly permissive to CVS infection in vitro. Further study is needed to determine mechanisms for this selective vulnerability, which will give us a better understanding of the pathogenesis of rabies.

Induction of Autophagy Promotes Fusion of Multivesicular Bodies with Autophagic Vacuoles in K562 Cells

Morphological and biochemical studies have shown that autophagosomes fuse with endosomes forming the so-called amphisomes, a prelysosomal hybrid organelle. In the present report, we have analyzed this process in K562 cells, an erythroleukemic cell line that generates multivesicular bodies (MVBs) and releases the internal vesicles known as exosomes into the extracellular medium. We have previously shown that in K562 cells, Rab11 decorates MVBs. Therefore, to study at the molecular level the interaction of MVBs with the autophagic pathway, we have examined by confocal microscopy the fate of MVBs in cells overexpressing green fluorescent protein (GFP)-Rab11 and the autophagosomal protein red fluorescent protein-light chain 3 (LC3). Autophagy inducers such as starvation or rapamycin caused an enlargement of the vacuoles decorated with GFP-Rab11 and a remarkable colocalization with LC3. This convergence was abrogated by a Rab11 dominant negative mutant, indicating that a functional Rab11 is involved in the interaction between MVBs and the autophagic pathway. Interestingly, we presented evidence that autophagy induction caused calcium accumulation in autophagic compartments. Furthermore, the convergence between the endosomal and the autophagic pathways was attenuated by the Ca2+ chelator acetoxymethyl ester (AM) of the calcium chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), indicating that fusion of MVBs with the autophagosome compartment is a calcium-dependent event. In addition, autophagy induction or overexpression of LC3 inhibited exosome release, suggesting that under conditions that stimulates autophagy, MVBs are directed to the autophagic pathway with consequent inhibition in exosome release.

Dissection of the Autophagosome Maturation Process by a Novel Reporter Protein, Tandem Fluorescent-Tagged LC3

During the process of autophagy, autophagosomes undergo a maturation process consisting of multiple fusions with endosomes and lysosomes, which provide an acidic environment and digestive function to the interior of the autophagosome. Here we found that a fusion protein of monomeric Red-fluorescence protein and LC3, the most widely used marker for autophagosomes, exhibits a quite different localization pattern from that of GFP-LC3. GFP-LC3 loses fluorescence due to lysosomal acidic and degradative conditions but mRFP-LC3 does not, indicating that the latter can label the autophagic compartments both before and after fusion with lysosomes. Taking advantage of this property, we devised a novel method for dissecting the maturation process of autophagosomes. mRFP-GFP tandem fluorescent-tagged LC3 (tfLC3) showed a GFP and mRFP signal before the fusion with lysosomes, and exhibited only the mRFP signal subsequently. Using this method, we provided evidence that overexpression of a dominant negative form of Rab7 prevented the fusion of autophagosomes with lysosomes, suggesting that Rab7 is involved in this step. This method will be of general utility for analysis of the autophagosome maturation process.

Role for Gingipains inPorphyromonas gingivalisTraffic to Phagolysosomes and Survival in Human Aortic Endothelial Cells

Gingipains are cysteine proteinases that are responsible for the virulence of Porphyromonas gingivalis. Recent studies have shown that P. gingivalis is trapped within autophagic compartments of infected cells, where it promotes survival. In this study we investigated the role of gingipains in the intracellular trafficking and survival of this bacterium in human aortic endothelial cells and any possible involvement of these enzymes in the autophagic pathway. Although autophagic events were enhanced by infection with either wild-type (WT) P. gingivalis strains (ATCC 33277, 381, and W83) or an ATCC 33277 mutant lacking gingipains (KDP136), we have found that more than 90% of intracellular WT and KDP136 colocalized with cathepsin B, a lysosome marker, and only a few of the internalized cells colocalized with LC3, an autophagosome marker, during the 0.5- to 4-h postinfection period. This was further substantiated by immunogold electron microscopic analyses, thus implying that P. gingivalis evades the autophagic pathway and instead directly traffics to the endocytic pathway to lysosomes. At the late stages after infection, WT strains in phagolysosomes retained their double-membrane structures. KDP136 in these compartments, however, lost its double-membrane structures, representing a characteristic feature of its vulnerability to rupture. Together with the ultrastructural observations, we found that the number of intracellular viable WT cells decreased more slowly than that of KDP136 cells, thus suggesting that gingipains contribute to bacterial survival, but not to trafficking, within the infected cells.

Lysosomes and autophagy in cell death control

Lysosomal hydrolases participate in the digestion of endocytosed and autophagocytosed material inside the lysosomal/autolysosomal compartment in acute cell death when released into the cytosol and in cancer progression following their release into the extracellular space. Lysosomal alterations are common in cancer cells. The increased expression and altered trafficking of lysosomal enzymes participates in tissue invasion, angiogenesis and sensitization to the lysosomal death pathway. But lysosomal heat-shock protein 70 locally prevents lysosomal-membrane permeabilization. Similarly, alterations in the autophagic compartment are linked to carcinogenesis and resistance to chemotherapy. Targeting these pathways might constitute a novel approach to cancer therapy.

Cellular autophagic capacity changes during azaserine-induced tumour progression in the rat pancreas. Up-regulation in all premalignant stages and down-regulation with loss of cycloheximide sensitivity of segregation along with malignant transformation.

The knowledge of alterations in regulation of autophagy during tumorigenesis may also help our understanding of its normal control. We established an experimental system and reported recently that autophagic capacity, measured as the cell's capability of increasing segregation (formation of autophagosomes) and subsequent degradation of cytoplasmic quanta were highly increased in premalignant nodule cells 6 months after initiation by azaserine in the rat pancreas in vivo. In the present study, we followed changes of these autophagic functions throughout the tumour progression. We carried out electron-microscopic morphometrical analysis of the expansion of autophagic vacuole compartment and subcompartments induced by vinblastine (an in vivo segregation enhancer), as well as their regression upon segregation-inhibitor cycloheximide post-treatment. Premalignant tumour samples were taken at month 5, month 8 (nodules), month 10 and month 15 (adenomas) after initiation. In all these stages, a highly increased and varying autophagic capacity was found compared with the host tissue. The basal (non-stimulated) autophagic compartment was measurable only at month 5 and month 15, and its regression upon cycloheximide was consistent with increased basal autophagic activity. Compared with the host tissue, autophagic capacity profoundly decreased in the differentiated and anaplastic adenocarcinomas at month 20, when, surprisingly, cycloheximide was unable to inhibit segregation. Our conclusion is that down-regulation of the cycloheximide sensitive segregation and a partly compensatory up-regulation of an alternative pathway of segregation might occur along with malignant transformation.

The convergent point of the endocytic and autophagic pathways in leydig cells.

Endocytic tracers and marker enzyme of lysosomes were used in the present study to analyze the processes of autophagocytosis and endocytosis, and the convergent point of these two pathways in Leydig cells. The endocytic and autophagic compartments can be easily identified in Leydig cells, which makes easier to define the stages of two pathways than was possible before. The evidences indicated that the late endosomes (dense MVBs) deliver their endocytosed gold tracers together with lysosomal enzymes to the early autophagosomes and they are the convergent point of the two pathways. During this convergent process, the early autophagosomes transform into late autophagosomes and the late endosomes transform into mature lysosomes.

Dynamics of vinblastine-induced autophagocytosis in murine pancreatic acinar cells: Influence of cycloheximide post-treatments

Accumulation of autophagic vacuoles (AVs) was monitored by electron microscopic morphometry in murine pancreatic acinar cells during the 5-hr period after a single injection of vinblastine (VBL). The expansion of the autophagic compartment (AC) occurred in two waves. AVs accumulated in the first 90 min and regressed in the next hour, but thereafter AC expanded again, and 5 hr following the VBL injection, as much as 5.3% of the cytoplasmic volume was found sequestered into the AC. The high rates of accumulation of AVs indicated that VBL stimulated AV formation ( segregation) during both expansion phases. To have a deeper insight into the dynamics of the process segregational inhibitor cycloheximide (CHI) was given 1 and 3 hr after VBL and the subsequent regression of the AC and its subcompartments (i.e., early, advanced, and late AVs) were measured during the next 90 min. We found that regression of AVs was fast in the first expansion and slowed down in the second expansion phase during which only early AVs regressed. CHI proved to be a fast and effective inhibitor of autophagic segregation, whether it was given before, simultaneously, or after the VBL injection. The aforementioned results argue for a dual mode of action of VBL (i.e., a prompt stimulation of segregation and a delayed retardation of AV maturation). The two effects of the alkaloid prevail differently along the time course. A further analysis of the behavior of the AC subcompartments showed that CHI perhaps inhibits segregational step(s) occurring prior to the actual formation of the autolysosomes.

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.

Regression of autophagic vacuoles in house pancreatic cells: A morphometric study of the effect of methylamine and chloroquine followed by cycloheximide treatment

Treatment of mice with methylamine and chloroquine for two hours markedly increased the volume fraction of autophagic vacuoles in the pancreatic acinar cells of the mouse. The autophagic vacuoles disappeared, from the cells within 20 min after the administration of a suppressor of autophagic sequestration cycloheximide to animals pretreated with 0,25 mg b.w. dose of methylamine. In contrast, no regression or very slow decay was seen in cells of mice pretreated with higher doses of methylamine (0,50-0,70 mg/g b.w.) and chloroquine (0,08 mg/g b.w.). Our data show that the two drugs retard the disintegration of autophagic vacuole content. It is concluded, that accumulation of autophagic vacuoles due to their slow turnover is an important mechanism of the expansion of autophagic vacuole compartment under the effect of methylamine and chloroquine.

Regression of autophagic vacuoles in pancreatic acinar, seminal vesicle epithelial, and liver parenchymal cells: A comparative morphometric study of the effect of vinblastine and leupeptin followed by cycloheximide treatment

Treatment of mice with both leupeptin (0.06 mg/g body wt) and vinblastine (0.05 mg/g body wt) for 2 h caused a many-fold enlargement of the autophagic-lysosomal compartment of pancreatic acinar, seminal vesicle epithelial, and liver parenchymal cells. In all three types of cells a predominance of large, dense bodies was seen after leupeptin treatment and that of typical autophagic vacuoles were seen after vinblastine treatment. An exponential decrease of the volume fraction of autophagic vacuoles was observed in leupeptin-treated cells after the administration of cycloheximide (0.2 mg/g body wt). The half-life of autophagic vacuoles estimated from the decay curve was 5.3, 5.7, and 6.6 min for pancreatic, seminal vesicle, and liver cells, respectively. Our data suggest that sequestered cytoplasmic material rapidly enters the lysosomes in leupeptin-treated cells and accumulates in this compartment. In contrast, no regression of the autophagic vacuole compartment of pancreatic and seminal vesicle cells was observed after the administration of cycloheximide to animals pretreated with vinblastine, and only a slight decrease was seen in liver cells. These observations show that the lifetime of autophagic vacuoles is prolonged by vinblastine resulting in their accumulation in the cells. However, our measurements also lend support to the view that in addition to the accumulatory effect on undegraded cytoplasmic material, stimulation of sequestration may play a role in the enlargement of the autophagic lysosomal compartment after treatment with leupeptin as well as with vinblastine in all three types of cells investigated.

Morphometric evaluation of the turnover of autophagic vacuoles after treatment with Triton X-100 and vinblastine in murine pancreatic acinar and seminal vesicle epithelial cells.

Large numbers of autophagic vacuoles were found in murine pancreatic acinar and seminal vesicle epithelial cells following the administration of Triton X-100 or vinblastine for 4 h. The autophagic vacuoles disappeared rapidly from the cells after the administration of cycloheximide to animals pretreated with Triton X-100. The decay in seminal vesicle cells appeared to follow first-order kinetics with an estimated t1/2 of 8.7 min. The regression in pancreatic cells was equally rapid and less than half the initial volume of autophagic vacuoles was found at the 12th min after cycloheximide injection. This time, the decay curve appeared to be linear rather than exponential. Our data, together with the work of others, support the view that the average half-life of autophagic vacuoles is a fairly constant parameter kept within the range of 6-9 min in various types of mouse and rat cell when the late steps of autophagocytosis (i.e. the fusion of autophagosomes and lysosomes and the degradation within lysosomes) are not affected. The regression of autophagic vacuoles was slow in mice pretreated with vinblastine (t1/2 of about 27-30 min) suggesting that this drug slows down the turnover of autophagic vacuoles. Morphometric evaluation of the regression of the autophagic vacuole compartment after cycloheximide treatment can be used as a tool to distinguish between treatments which elevate the amount of autophagic vacuoles within the cells by increasing the rate of sequestration from those which expand the autophagic vacuole compartment by causing accumulation of autophagic vacuoles as a result of blockade of the late steps of the autophagic process.

Participation of lysosomes in basal proteolysis in perfused rat liver: Discrepancy between leupeptin-induced lysosomal enlargement and inhibition of proteolysis

Livers of non-starved rats were perfused cyclically for 2 h in the presence of either 4x the normal concentration of amino acids (known to suppress proteolysis to basal level), or the same medium together with leupeptin (an inhibitor of cathepsin B, H and L). Stereologic analysis revealed that the drug elicited a linear increase in the fractional cytoplasmic volume of the hepatocytic autophagic vacuolar compartment amounting to 1.34%/h. Measurements of proteolysis under the same experimental conditions showed that addition of leupeptin to the perfusate reduced proteolysis from 1.74%/h to 1.34%/h, i.e. an inhibition of 21.6% was observed. Thus, although proteolysis was only little inhibited by leupeptin, cytoplasm was sequestered at a rate that almost fully accounted for overall protein breakdown during basal state. The reasons for this discrepancy, such as subtotal inhibition of proteinases, increase of lysosomal contents and compensatory increased nonlysosomal degradation are discussed.

Regression of autophagic vacuoles in seminal vesicle cells following cycloheximide treatment

A 0.25% fraction of cytoplasm was present in the autophagic vacuole compartment of the seminal vesicle cells of fasted and estrogen-treated mice. Autophagic vacuoles regressed rapidly after administration of 0.2 mg/g body weight cycloheximide to the animals and the decay appeared to follow first-order kinetics (average half-life 5.8 min, rate constant of regression 0.120 min −1). Our observations suggest that the drug suppressed the formation of autophagosomes. The fraction of cytoplasm removed by autophagy from the cells was estimated to be about 1.8%/h under steady-state conditions.