The lysosomotropic agent monodansylcadaverine also acts as a solvent polarity probe.

The autofluorescent substance monodansylcadaverine has recently been reported as a specific in vivo marker for autophagic vacuoles. However, the mechanism for this specific labeling remained unclear. Our results reveal that the common model of ion trapping in acidic compartments cannot completely account for the observed autophagic vacuole staining. Because autophagic vacuoles are characterized by myelin-like membrane inclusions, we tested whether this lipid-rich environment is responsible for the staining properties of monodansylcadaverine. In in vitro experiments using either liposomes or solvents of different polarity, monodansylcadaverine showed an increased relative fluorescence intensity in a hydrophobic environment as well as a Stokes shift dependent on the solvent polarity. To test the effect of autophagic vacuoles or autophagic vacuole lipids on monodansylcadaverine fluorescence, we isolated autophagic vacuoles and purified autophagic vacuole lipids depleted of proteins. Entire autophagic vacuoles and autophagic vacuole lipids had the same effect on monodansylcadaverine fluorescence properties, suggesting lipids as the responsible component. Our results suggest that the in vivo fluorescence properties of monodansylcadaverine do not depend exclusively on accumulation in acidic compartments by ion trapping but also on an effective interaction of this molecule with autophagic vacuole membrane lipids. (J Histochem Cytochem 48:251-258, 2000)     

Vital staining of autophagic vacuoles in differentiating cells of Dictyostelium discoideum

Abstract. Vital staining of differentiating slime mold cells of Dictyostelium discoideum was studied, with reference to autophagic vacuoles they contain. By microscopically comparing within individual cells neutral-red staining granules with acid phosphatase positive granules, we identified vitally stained granules as autophagic vacuoles. At the early stages of differentiation, although the majority of cells were strongly acid phosphatase positive and there was little difference in the number of autophagic vacuoles between prestalk and prespore cells, only the former (about 25% of the total) were strongly vitally stained. It was thus concluded that autophagic vacuoles of prestalk cells are intensely stained with neutral-red while those of prespore cells are only weakly stained. Strong vital staining of prestalk cells was bleached by lysosomotropic agents such as NH₄Cl, methylamine, and chloroquine which are known to increase intra-lysosomal pH. This suggests that autophagic vacuoles of prestalk cells are strongly stained because of their acidity.     

Autophagy Promotes Microglia Activation Through Beclin-1-Atg5 Pathway in Intracerebral Hemorrhage

Previous study demonstrates that intracerebral hemorrhage (ICH) promotes microglia activation and inflammation. However, the exact mechanism of microglia activation induced by ICH is not clear. In this experiment, microglia autophagy was examined using electron microscopy, conversion of light chain 3(LC3), and monodansylcadaverine (MDC) staining to detect autophagic vacuoles. We found that ICH induced microglia autophagy and activation. The suppression of autophagy using either pharmacologic inhibitors (3-methyladenine, bafilomycin A1) or RNA interference in essential autophagy genes (BECN1 and ATG5) decreased the microglia activation and inflammation in ICH. Moreover, autophagy inhibitors reduced brain damage in ICH. In conclusion, these data indicate that ICH contributes to microglia autophagic activation through BECN1 and ATG5 and provide the therapeutical strategy for ICH.     

The protective role of metformin in autophagic status in peripheral blood mononuclear cells of type 2 diabetic patients

Autophagy plays an important role in the pathophysiology of type 2 diabetes (T2D). Metformin is the most common antidiabetic drug. The main objective of this study was to explore the molecular mechanism of metformin in starvation‐induced autophagy in peripheral blood mononuclear cells (PBMCs) of type 2 diabetic patients. PBMCs were isolated from 10 diabetic patients and 7 non‐diabetic healthy volunteers. The autophagic puncta and markers were measured with the help of monodansylcadaverine staining and western blot. Additionally, transmission electron microscopy was also performed. No significant changes were observed in the initial autophagy marker protein levels in PBMCs of T2D after metformin treatment though diabetic PBMCs showed a high level of phospho‐mammalian target of rapamycin, p62 and reduced expression of phospho‐AMP‐activated protein kinase and lysosomal membrane‐associated protein 2, indicating a defect in autophagy. Also, induction of autophagy by tunicamycin resulted in apoptosis in diabetic PBMCs as observed by caspase‐3 cleavage and reduced expression of Bcl2. Inhibition of autophagy by bafilomycin rendered consistent expression of p62 indicating a defect in the final process of autophagy. Further, electron microscopic studies also confirmed massive vacuole overload and a sign of apoptotic cell death in PBMCs of diabetic patients, whereas metformin treatment reduced the number of autophagic vacuoles perhaps by lysosomal fusion. Thus, our results indicate that defective autophagy in T2D is associated with the fusion process of lysosomes which could be overcome by metformin.     

Intracellular distribution of acridine derivatives in platelets and their suitability for cytoplasmic pH measurements

The site and mechanism of accumulation of acridine derivatives into platelets and their isolated organelles were investigated. In addition, their suitability as indicators of cytoplasmic pH was analysed. Direct microscopic observation showed that quinacrine and 9-aminoacridine are concentrated inside organelles in platelets. Using fractionation studies, the acridine derivatives were found to accumulate particularly in dense and α-granules. Uptake into these organelles is driven by a pH differential across their membrane (acidic inside). Because of their cellular distribution, acridine derivatives were found to be poor indicators of cytoplasmic pH. In contrast, a poorly permeant dicarboxylated fluorescein derivative, generated in situ by cytosolic enzymes, is shown to be a more reliable probe of intracellular pH. The results are compared with previous reports of the use of 9-aminoacridine as a cytoplasmic pH probe in platelets and of quinacrine as a selective dense-granule marker.     

Autophagy, cathepsin L transport, and acidification in cultured rat fibroblasts.

The mechanisms of enzyme delivery to and acidification of early autophagic vacuoles in cultured fibroblasts were elucidated by cryoimmunoelectron microscopic methods. The cation-independent mannose-6-phosphate receptor (MPR) was used as a marker of the pre-lysosomal compartment, and cathepsin L and an acidotropic amine (3-(2,4-dinitroanilino)-3'-amino-N-methyl-dipropylamine (DAMP), a cytochemical probe for low-pH organelles) as markers of both pre-lysosomal and lysosomal compartments. In addition, cationized ferritin was used as an endocytic marker. In ultrastructural double labeling experiments, the bulk of all the antigens was found in vesicles containing tightly packed membrane material. These vesicles also contained small amounts of endocytosed ferritin and probably correspond to the MPR-enriched pre-lysosomal compartment. Some immunolabeling was also visible in the trans-Golgi network. In addition, cathepsin L, DAMP, and large amounts of ferritin were found in smaller vesicles which can be classified as mature lysosomes. Early autophagic vacuoles were defined as vesicles containing recognizable cytoplasm. MPR, cathepsin L, and DAMP, but not ferritin, were detected in the early vacuoles. Inhibition of the acidification in the early vacuoles by monensin did not prevent the delivery of MPR and cathepsin L. The presence of MPR in the vacuoles suggests that cathepsin L is not delivered to early autophagic vacuoles solely by fusion with mature, MPR-deficient lysosomes. Furthermore, although lysosomes were loaded with endocytosed ferritin, it was not detected in autophagic vacuoles. Either the trans-Golgi network or the MPR-enriched pre-lysosomes may be the main source of enzymes and acidification machinery for the autophagic vacuoles in fibroblasts.(ABSTRACT TRUNCATED AT 250 WORDS)     

Studies on the mechanisms of autophagy: maturation of the autophagic vacuole

Data presented in the accompanying paper suggests nascent autophagic vacuoles are formed from RER (Dunn, W. A. 1990. J. Cell Biol. 110:1923-1933). In the present report, the maturation of newly formed or nascent autophagic vacuoles into degradative vacuoles was examined using morphological and biochemical methods combined with immunological probes. Within 15 min of formation, autophagic vacuoles acquired acid hydrolases and lysosomal membrane proteins, thus becoming degradative vacuoles. A previously undescribed type of autophagic vacuole was also identified having characteristics of both nascent and degradative vacuoles, but was different from lysosomes. This intermediate compartment contained only small amounts of cathepsin L in comparison to lysosomes and was bound by a double membrane, typical of nascent vacuoles. However, unlike nascent vacuoles vet comparable to degradative vacuoles, these vacuoles were acidic and contained the lysosomal membrane protein, lgp120, at the outer limiting membrane. The results were consistent with the stepwise acquisition of lysosomal membrane proteins and hydrolases. The presence of mannose-6-phosphate receptor in autophagic vacuoles suggested a possible role of this receptor in the delivery of newly synthesized hydrolases from the Golgi apparatus. However, tunicamycin had no significant effect on the amount of mature acid hydrolases present in a preparation of autophagic vacuoles isolated from a metrizamide gradient. Combined, the results suggested nascent autophagic vacuoles mature into degradative vacuoles in a stepwise fashion: (a) acquisition of lysosomal membrane proteins by fusing with a vesicle deficient in hydrolytic enzymes (e.g., prelysosome); (b) vacuole acidification; and (c) acquisition of hydrolases by fusing with preexisting lysosomes or Golgi apparatus-derived vesicles.     

The Dynamics of Autophagy Visualised in Live Cells: from Autophagosome Formation to Fusion with Endo/lysosomes

Autophagy has been implicated in a range of disorders and hence is of major interest. However, imaging autophagy in real time has been hampered by lack of suitable markers. We have compared the potential of monodansylcadaverine, widely used as an autophagosomal marker, and the Atg8 homologue LC3, to follow autophagy by fluorescence microscopy whilst labelling late endosomes and lysosomes simultaneously using EGFP-CD63. Monodansylcadaverine labelled only acidic CD63-positive compartments in response to a range of autophagic inducers in various live or post-fixed cells, staining being identical in atg5+/+ and atg5-/- MEFs in which autophagosome formation is disabled. Monodansylcadaverine staining was essentially indistinguishable from that of LysoTracker Red, LAMP1 or LAMP2. In contrast, 60-90% of EGFP-LC3-positive punctate organelles did not colocalise with LAMP1/LAMP2/CD63 and were monodansylcadaverine-negative while EGFP-LC3 puncta that did colocalise with LAMP1/LAMP2/CD63 were also monodansylcadaverine-positive. Hence monodansylcadaverine is no different from other markers of acidic compartments and it cannot be used to follow autophagosome formation. In contrast, fusion of mRFP-LC3-labelled autophagosomes with EGFP-CD63-positive endosomes and lysosomes and sequestration of dsRed-labelled mitochondria by EGFP-LC3- and EGFP-CD63-positive compartments could be visualised in real time. Moreover, transition of EGFP-LC3-I (45 kDa) to EGFP-LC3-II (43 kDa) - traced by immunoblotting and verified by [3H]ethanolamine labelling - revealed novel insights into the dynamics of autophagosome homeostasis, including the rapid activation of autophagy by the apoptotic inducer staurosporine prior to apoptosis proper. Use of fluorescent LC3 and a counterfluorescent endosomal/lysosomal protein clearly allows the entire autophagic process to be followed by live cell imaging with high fidelity.     

A method to measure cardiac autophagic flux in vivo

Autophagy, a highly conserved cellular mechanism wherein various cellular components are broken down and recycled through lysosomes, has been implicated in the development of heart failure. However, tools to measure autophagic flux in vivo have been limited. Here, we tested whether monodansylcadaverine (MDC) and the lysosomotropic drug chloroquine could be used to measure autophagic flux in both in vitro and in vivo model systems. Using HL-1 cardiac-derived myocytes transfected with GFP-tagged LC3 to track changes in autophagosome formation, autophagy was stimulated by mTOR inhibitor rapamycin. Administration of chloroquine to inhibit lysosomal activity enhanced the rapamycin-induced increase in the number of cells with numerous GFP-LC3-positive autophagosomes. The chloroquine-induced increase of autophagosomes occurred in a dose-dependent manner between 1 microM and 8 microM, and reached a maximum 2 hour after treatment. Chloroquine also enhanced the accumulation of autophagosomes in cells stimulated with hydrogen peroxide, while it attenuated that induced by Bafilomycin A1, an inhibitor of V-ATPase that interferes with fusion of autophagosomes with lysosomes. The accumulation of autophagosomes was inhibited by 3-methyladenine, which is known to inhibit the early phase of the autophagic process. Using transgenic mice expressing 3 mCherry-LC3 exposed to rapamycin for 4 hr, we observed an increase in mCherry-LC3-labeled autophagosomes in myocardium, which was further increased by concurrent administration of chloroquine, thus allowing determination of flux as a more precise measure of autophagic activity in vivo. MDC injected 1 hr before sacrifice colocalized with mCherry-LC3 puncta, validating its use as a marker of autophagosomes. This study describes a method to measure autophagic flux in vivo even in non-transgenic animals, using MDC and chloroquine.     

The dynamics of autophagy visualized in live cells: from autophagosome formation to fusion with endo/lysosomes

Autophagy has been implicated in a range of disorders and hence is of major interest. However, imaging autophagy in real time has been hampered by lack of suitable markers. We have compared the potential of monodansylcadaverine, widely used as an autophagosomal marker, and the Atg8 homologue LC3, to follow autophagy by fluorescence microscopy whilst labelling late endosomes and lysosomes simultaneously using EGFP-CD63. Monodansylcadaverine labelled only acidic CD63-positive compartments in response to a range of autophagic inducers in various live or post-fixed cells, staining being identical in atg5(+/+) and atg5(-/-) MEFs in which autophagosome formation is disabled. Monodansylcadaverine staining was essentially indistinguishable from that of LysoTracker Red, LAMP-1 or LAMP-2. In contrast, 60-90% of EGFP-LC3-positive punctate organelles did not colocalise with LAMP-1/LAMP-2/CD63 and were monodansylcadaverine-negative while EGFP-LC3 puncta that did colocalise with LAMP-1/LAMP-2/CD63 were also monodansylcadaverine-positive. Hence monodansylcadaverine is no different from other markers of acidic compartments and it cannot be used to follow autophagosome formation. In contrast, fusion of mRFP-LC3-labelled autophagosomes with EGFP-CD63-positive endosomes and lysosomes and sequestration of dsRed-labelled mitochondria by EGFP-LC3- and EGFP-CD63-positive compartments could be visualized in real time. Moreover, transition of EGFP-LC3-I (45 kDa) to EGFP-LC3-II (43 kDa)-traced by immunoblotting and verified by [(3)H]ethanolamine labelling-revealed novel insights into the dynamics of autophagosome homeostasis, including the rapid activation of autophagy by the apoptotic inducer staurosporine prior to apoptosis proper. Use of fluorescent LC3 and a counter-fluorescent endosomal/lysosomal protein clearly allows the entire autophagic process to be followed by live cell imaging with high fidelity.     

Minocycline inhibits the growth of glioma by inducing autophagy

Minocycline has been shown to alleviate several neurological disorders. Unexpectedly, we found that minocycline had opposite effects on glioma cells: minocycline induced nonapoptotic cell death in glioma cells. The glioma cell death was associated with the presence of autophagic vacuoles in the cytoplasm. Minocycline induced autophagy was confirmed by acridine orange, monodansylcadaverine (MDC) stainings of vesicle formation and the conversion of microtubule-associated proteins light chain 3 (LC3-I) to LC3-II. Pretreatment with autophagy inhibitor 3-methyladenine (3-MA) suppressed the induction of acidic vesicular organelles and the accumulation of LC3-II to the autophagosome membrane in glioma cells treated with minocycline. Despite the pretreatment of 3-MA, minocycline induced cell death which could result from the activation of caspase-3. Minocycline effectively inhibited tumor growth and induced autophagy in the xenograft tumor model of C6 glioma cells. These results suggest that minocycline may kill glioma cells by inducing autophagic cell death. When autophagy was inhibited, minocycline still induced cell death through the activation of caspase-3. Thus, minocycline is a promising agent in the treatment of malignant gliomas.     

4th International Symposium on Autophagy: Exploiting the Frontiers of Autophagy Research

Minocycline has been shown to alleviate several neurological disorders. Unexpectedly, we found that minocycline had opposite effects on glioma cells: minocycline induced nonapoptotic cell death in glioma cells. The glioma cell death was associated with the presence of autophagic vacuoles in the cytoplasm. Minocycline induced autophagy was confirmed by acridine orange, monodansylcadaverine (MDC) stainings of vesicle formation and the conversion of microtubule-associated proteins light chain 3 (LC3-I) to LC3-II. Pretreatment with autophagy inhibitor 3-methyladenine (3-MA) suppressed the induction of acidic vesicular organelles and the accumulation of LC3-II to the autophagosome membrane in glioma cells treated with minocycline. Despite the pretreatment of 3-MA, minocycline induced cell death which could result from the activation of caspase-3. Minocycline effectively inhibited tumor growth and induced autophagy in the xenograft tumor model of C6 glioma cells. These results suggest that minocycline may kill glioma cells by inducing autophagic cell death. When autophagy was inhibited, minocycline still induced cell death through the activation of caspase-3. Thus, minocycline is a promising agent in the treatment of malignant gliomas.     

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 localization of cytoplasmic phosphatases in preimplantation mouse embryos.

The appearance and localization of the cytoplasmic phosphatases [acid phosphatase (AcPase) as a marker of lysosomes, TPPase as a marker of the Golgi apparatus, and NDPase (IDPase) as enzymatic marker of the endoplasmic reticulum (ER)] were cytochemically studied on the ultrastructural level in secondary oocytes and in preimplantation mouse embryos. The detectable AcPase activity, located on the inner surface of the membrane delimiting some cytoplasmic vacuoles (lysosomes and autophagic vacuoles), appears at the eight-cell stage and grows pregressively stronger up to the blastocyst stage. Golgi-associated reaction for TPPase was detectable in oocytes, dropped in one-cell embryos and became negative in the two-cell embryos. The reaction for TPPase and IDPase was present in plasma membranes of oocytes and early embryos and appeared in the delimiting membrane of some cytoplasmic vesicles in eight-cell embryos. Some activity of IDPase was found in small segments of the ER at the morula and blastocyst stage. The observed results suggest that the lysosomes are the first organelles in early embryos showing activity of the marker enzymes of the phosphatase type, while the activity of other marker enzymes is mainly concentrated in the plasma membrane of blastomeres. It cannot be excluded, however, that positive reaction for TPPase and IDPase in the plasma membrane results from nonspecific action of other phosphatases.     

Autophagy-related vacuoles in mouse gallbladder epithelium

The mouse gallbladder epithelial cells contain very heterogeneous vacuolar population. In an attempt to classify these vacuoles we identified NADPase and TPPase activity as well as the location of HRP which is used as the endocytotic marker. The results of the present study show that the vacuoles can be classified into three categories: (1) the vacuoles predominantly containing loose membrane coils related to the nascent autophagic vacuoles, (2) vacuoles containing densely packed membranes and exhibiting a positive HRP reaction, indicating the convergence of endocytotic and autophagic pathway, and (3) vacuoles composed of degraded membrane structures and containing the reaction product of NADPase activity, showing that the fusion of the lysosomes with the autophagosome-endosome took place. The highly developed cis, medial and trans Golgi compartments reflect the biosynthetic and endocytotic activity of the gallbladder epithelium.     

How Yersinia pestis becomes a foreign obstruction in the digestive system of the macrophage

Yersinia pestis, a facultative intracellular bacterial pathogen, survives and replicates within macrophage phagosomes. Macrophages can use an autophagic pathway known as xenophagy to destroy pathogens in an acidic autolysosome or autophagolysosome. Yersinia-containing vacuoles (YCVs) in macrophages can acquire LC3, a marker of autophagic membranes. However, YCVs fail to acidify, which likely prevents their maturation to the autophagolysosome stage. We suggest that this process bypasses the cell’s attempt to use xenophagy to destroy the pathogen. It remains to be determined how Y. pestis blocks YCV acidification. Although autophagy is not required for Y. pestis survival in macrophages, it is possible that sequestration of autophagic membrane in YCVs allows the pathogen to induce cell death in the macrophage.     

The role of neutral lipid nanospheres in Plasmodium falciparum haem crystallization

The intraerythrocytic malaria parasite constructs an intracellular haem crystal, called haemozoin, within an acidic digestive vacuole where haemoglobin is degraded. Haem crystallization is the target of the widely used antimalarial quinoline drugs. The intracellular mechanism of molecular initiation of haem crystallization, whether by proteins, polar membrane lipids or by neutral lipids, has not been fully substantiated. In the present study, we show neutral lipid predominant nanospheres, which envelop haemozoin inside Plasmodium falciparum digestive vacuoles. Subcellular fractionation of parasite-derived haemozoin through a dense 1.7 M sucrose cushion identifies monoacylglycerol and diacylglycerol neutral lipids as well as some polar lipids in close association with the purified haemozoin. Global MS lipidomics detects monopalmitic glycerol and monostearic glycerol, but not mono-oleic glycerol, closely associated with haemozoin. The complex neutral lipid mixture rapidly initiates haem crystallization, with reversible pH-dependent quinoline inhibition associated with quinoline entry into the neutral lipid microenvironment. Neutral lipid nanospheres both enable haem crystallization in the presence of high globin concentrations and protect haem from H2O2 degradation. Conceptually, the present study shifts the intracellular microenvironment of haem crystallization and quinoline inhibition from a polar aqueous location to a non-polar neutral lipid nanosphere able to exclude water for efficient haem crystallization.     

Membrane markers of endoplasmic reticulum preserved in autophagic vacuolar membranes isolated from leupeptin-administered rat liver

We isolated membranes from leupeptin-induced autophagic vacuoles and compared them with lysosomal membranes purified from dextran-administered rats. In protein composition, autophagic vacuole membranes prepared from long term-starved (36 h) rats bear marked resemblance to lysosomal membranes, whereas vacuole membranes prepared from short term-starved (12 h) animals differ significantly from lysosomal membranes. Immunoblotting analyses showed that only autophagic vacuole membranes from short term-starved rats possess endoplasmic reticulum markers such as cytochrome P450 and NADPH-cytochrome c reductase. None of the membranes contain sialyltransferase, a Golgi membrane marker. In experiments in which rats were starved after feeding to induce autophagy, the appearance of the endoplasmic reticulum markers occurred during 6-12 h of starvation, concomitantly with increases in vacuolar proteins and sequestered cytosolic aldolase. The endoplasmic reticulum membrane markers and sequestered aldolase declined gradually after 20-36 h of starvation, suggesting that prolonged starvation causes no further increase in the formation of autophagic vacuoles but an increase in the population of matured autophagic vacuoles. Thus, the prominent markers of endoplasmic reticulum from which autophagosomes originate are well preserved in autophagic vacuole membranes, and retention of these markers is highly dependent on the formation and subsequent maturation process of autophagic vacuoles.     

Induction of autophagy causes dramatic changes in the subcellular distribution of GFP-Rab24

Rab GTPases comprises a large family of proteins, with more than 50 gene products localized in distinct subcellular compartments. Rab24 is a member of this family whose function is not presently known. In order to elucidate the role of this protein we have generated a GFP-tagged Rab24 and studied the distribution of this chimera by fluorescence microscopy. GFP-Rab24 showed a perinuclear reticular localization that often encircled the nucleus. This reticular pattern partially overlapped with ER markers, cis-Golgi, and the ER-Golgi intermediate compartment. Surprisingly, when GFP-Rab24-transfected cells were starved to induce autophagy the distribution of the protein changed dramatically. GFP-Rab24 localized in large dots, cup-shaped structures and ring-shaped vesicles. Some of these vesicles were labeled with monodansylcadaverine , a specific autophagosome marker. In the presence of vinblastine, an agent that induces the formation of very large autophagic vesicles, GFP-Rab24 accumulated in the large vacuoles that were also labeled by monodansylcadaverine. Furthermore, Rab24 colocalized with LC3, a mammalian homolog of the yeast protein Apg8/Aut7, an essential gene for autophagy. This is the first report indicating that Rab24 localizes on autophagosomes, suggesting that this Rab protein is involved in the autophagic pathway.