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Autophagy in Tobacco BY-2 Cells Cultured under Sucrose Starvation Conditions: Isolation of the Autolysosome and its Characterization Regular Paper Chihiro Takatsuka1, Yuko Inoue3, Tomoya Higuchi2, Stefan Hillmer4, David G. Robinson4 and Yuji Moriyasu1,2,* 1 Graduate School of Science and Engineering, Saitama University, Saitama, Japan School of Science, Saitama University, Saitama, Japan 3 Graduate School of Food and Nutritional Sciences, University of Shizuoka, Shizuoka, Japan 4 Department of Plant Cell Biology, Centre for Organismal Studies, University of Heidelberg, Heidelberg, Germany *Corresponding author: E-mail, [email protected]; Fax, +81-48-858-3408. (Received June 3, 2011; Accepted October 7, 2011) 2 Tobacco culture cells carry out a large-scale degradation of intracellular proteins in order to survive under sucrose starvation conditions. We have previously suggested that this bulk degradation of cellular proteins is performed by autophagy, where autolysosomes formed de novo act as the major lytic compartments. The digestion process in autolysosomes can be retarded by addition of the cysteine protease inhibitor E-64c to the culture medium, resulting in the accumulation of autolysosomes. In the present study, we have investigated several properties of autolysosomes in tobacco cells. Electron microscopy showed that the autolysosomes contain osmiophilic particles, some of which resemble partially degraded mitochondria. It also revealed the presence of two kinds of autolysosome precursor structures; one resembled the isolation membrane and the other the autophagosome of mammalian cells. Immunofluorescence microscopy showed that autolysosomes contain acid phosphatase, in accordance with cytochemical enzyme analyses by light and electron microscopy in a previous study. Autolysosomes isolated by cell fractionation on Percoll gradients showed the localization of acid phosphatase, vacuolar H+-ATPase and cysteine protease. These results show that starvation-induced autophagy in tobacco cells follows a macroautophagic-type response similar to that described for other eukaryotes. However, our results indicate that, although the plant vacuole is often described as being equivalent to the lysosome of the animal cell, a new low pH lytic compartment—the autolysosome—also contributes to proteolytic degradation when tobacco cells are subjected to sucrose deprivation. Keywords: Autophagy Autolysosome/autophagosome Cysteine protease Endocytosis Tobacco BY-2 Vacuole. Abbreviations: Atg, autophagy-related protein; ConcA, concanamycin A; GFP, green fluorescent protein; 3-MA, 3-methyladenine. Introduction Autophagy involves the sequestration of portions of the cytoplasm including organelles in a membrane-bound compartment followed by the subsequent degradation of the contents in a lytic compartment. This process was first considered to be an induced response to nutrient depletion, and recent research on yeast and mammalian cells has put autophagy into a broader perspective as representing a major degradative pathway and as contributing to some of the regulation pathways in cellular homeostasis (Klionsky and Emr 2000). It is generally accepted that all eukaryotic cells have a lytic compartment: the lysosome in animal cells, the lysosome/ vacuole in yeast cells and the vacuole in plant cells. By macroautophagy, the cytoplasm to be degraded becomes trapped within a de novo formed, double-membrane compartment, the autophagosome. In mammalian cells, the autophagosome then develops into an autolysosome by acquiring hydrolytic enzymes and the vacuolar H+-ATPase in the boundary membrane. This transformation has been considered to be achieved by vesicle-mediated delivery of newly synthesized enzymes as well as through fusion to pre-existing primary lysosomes (Dunn 1994, Eskelinen 2005). In contrast, in yeast, an autophagosome does not differentiate into a lytic organelle. Instead, it fuses directly with the vacuole and releases an inner membrane-bound structure, termed an autophagic body, into the vacuolar lumen (Klionsky and Ohsumi 1999). The autophagic body is subsequently degraded by vacuolar hydrolases (Teter et al. 2001). Microautophagy is a process in which parts of the cytoplasm are invaginated by the membranes of lysosomes/vacuoles themselves and then degraded. Such a process has been observed with the lysosome of mammalian hepatocytes (Ahlberg and Glaumann 1985) and with the vacuole of the methylotrophic yeast Pichia pastoris (Tuttle and Dunn 1995, Sakai et al. 1998). Plant Cell Physiol. 52(12): 2074–2087 (2011) doi:10.1093/pcp/pcr137, available online at www.pcp.oxfordjournals.org ! The Author 2011. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: [email protected] 2074 Plant Cell Physiol. 52(12): 2074–2087 (2011) doi:10.1093/pcp/pcr137 ! The Author 2011. Autophagy in tobacco cells Autophagic processes that seem to be relevant to developmental processes have been described at the ultrastructural level in plants on a number of occasions. These include autophagy relating to the biogenesis of lytic vacuoles in vegetative tissues, protein storage vacuoles in developing seed tissues and the transformation of protein storage vacuoles to lytic vacuoles during seed germination (Robinson and Hinz 1997, Herman and Larkins 1999, Marty 1999, Moriyasu and Hillmer 2000, Moriyasu and Klionsky 2004, Bassham 2007). However, none of these cases fits into the classical molds of macro- and microautophagy as given above for mammalian and yeast cells. On the other hand, it has been thought that suspensioncultured cells perform a form of macroautophagy when placed under conditions of carbon deprivation (Aubert et al. 1996, Moriyasu and Ohsumi 1996, Rose et al. 2006). However, the actual lytic compartments in this cell system have not been characterized (Bassham et al. 2006). Sycamore cells start to form autophagic ‘vacuoles’ approximately 14 h after transfer into sucrose-free medium. The majority of these structures were surrounded by two membranes, and some singlemembrane vesicles with similar contents were observed inside the vacuole (Aubert et al. 1996). A small number of autophagic ‘vacuoles’ were bounded by a single membrane with partially degraded contents. By inhibiting cysteine protease activity, which increases dramatically upon sucrose deprivation, the autophagic process is slowed down in tobacco BY-2 cells, and, as a result, numerous autophagic ‘vacuoles’ 1–6 mm in diameter accumulated in the perinuclear cytoplasm (Moriyasu and Ohsumi 1996). Because the cytoplasmic debris was still recognizable in these autophagic ‘vacuoles’ they were termed autolysosomes by the authors. The accumulation and thus the formation of these autolysosomes were blocked by the autophagy inhibitor 3-methyladenine (3-MA) (Takatsuka et al. 2004). In Arabidopsis cells cultured in a sucrose-free medium, the formation of three types of autophagic ‘vacuoles’, which correspond to autophagosomes to autolysosomes, has been described (Rose et al. 2006). Taken together, these studies have shown that macroautophagy accompanying the formation of autophagosomes and/or autolysosomes occurs in plant cells cultured in nutrient-starved media. In yeast (Saccharomyces cerevisiae, P. pastoris and Hansenula polymorpha), 35 autophagy-related proteins (Atgs) have been identified (Klionsky et al. 2003). Seventeen of these proteins, which are evolutionarily conserved, play an essential role in autophagosome formation (Hanaoka et al. 2002, Inoue and Klionsky 2010). One of these proteins, the ubiquitin-like protein Atg8, localizes to the membranes of autophagosomes and is involved in the elongation of autophagosomal membranes in yeast cells. The formation of autophagosomes can therefore be monitored using Atg8. Thus, in Arabidopsis and tobacco cells, autophagosomes have been visualized by the expression of Atg8 fused to fluoroproteins such as green fluorescent protein (GFP) and yellow fluorescent protein (YFP) (Yoshimoto et al. 2004, Contento et al. 2005, Thompson et al. 2005, Toyooka et al. 2006). When transgenic plants were treated with concanamycin A (ConcA), the structures that derive from autophagosomes, which appear to correspond to the autophagic bodies in yeast cells, are seen in the central vacuole (Yoshimoto et al. 2004, Thompson et al. 2005, Xiong et al. 2007). This result has been taken as evidence to show that autophagosomes fuse directly with the central vacuole as occurs in yeast cells (Kirisako et al. 1999). Furthermore, it has recently been reported that ribulose bisphosphate-containing bodies, which are formed by the evagination of chloroplasts into the cytoplasm during senescence, are transported to the central vacuoles by macroautophagy (Ishida et al. 2008). In this paper, we establish that starvation-induced macroautophagy in plant cells is qualitatively different from that in other eukaryotic cells. We show that while autophagosomes originate as double-membrane-bound structures, the digestion of ingested cytoplasmic contents occurs to a great extent before they fuse with the central vacuole. In agreement with this are the observations that vacuolar H+-ATPase is detectable in the membrane of the autolysosome, and that hydrolytic enzymes are present in its lumen. Results The characterization of autolysosomes by light and electron microscopy Tobacco cells accumulate autolysosomes in a sucrose-free culture medium containing a cysteine protease inhibitor (Moriyasu and Ohsumi, 1996). The accumulation of autolysosomes was recognized in almost all cells cultured in sucrose-free medium containing E-64c using a light microscope equipped with Nomarski optics (Fig. 1A, arrow), whereas such an accumulation was not seen in cells cultured without the inhibitor (Fig. 1B). In sections stained with toluidine blue, we found small (>6 mm in diameter) vacuoles containing densely stained particles in cells treated with E-64c (Fig. 1C, arrow). Particles stained with toluidine blue were also seen in cells cultured without E-64c, but the frequency was lower (Fig. 1D). Thus autolysosomes in tobacco cells had the appearance of small vacuoles containing toluidine blue-stained particles. Electron microscopy revealed that an autolysosome has a few electron-dense particles (Fig. 1E, arrow). These electron-dense particles seemed to be the degradation intermediates of parts of the cytoplasm that had been enclosed in autolysosomes. Indeed, some of the particles were occasionally less degraded and partially retained the morphology of double-membrane-bound organelles such as mitochondria or plastids (Fig. 1F, arrowhead; Supplementary Fig. S1). Fusion between an autolysosome and the central vacuole was often observed, suggesting that some autolysosomes fuse with the vacuolar membrane to release electron-dense contents into the central vacuole (Fig. 1E). Similar electron-dense particles were occasionally observed in the central vacuole (Fig. 1E, arrowhead). Toyooka et al. (2006) also analyzed autophagosomes labeled with a YFP–Atg8 fusion protein in tobacco Plant Cell Physiol. 52(12): 2074–2087 (2011) doi:10.1093/pcp/pcr137 ! The Author 2011. 2075 C. Takatsuka et al Fig. 1 Light and electron micrographs of tobacco cells cultured under sucrose starvation conditions in the presence of the cysteine protease inhibitor E-64c. Tobacco cells were cultured in a sucrose-free culture medium for 24 h (A–G) or 10 h (H) in the presence (A, C, E, F, G and H) or absence (B and D) of the cysteine protease inhibitor E-64c. (A and B) Living cells were observed under a light microscope with Nomarski optics. (C–H) Cells were fixed, embedded in Spurr’s resin, sectioned and observed by light microscopy (C and D) or by electron microscopy (E–H). Arrows indicate autolysosomes in A, C and E. Arrowheads indicate partially degraded cytoplasm in the central vacuole in E, partially degraded organelles such as mitochondria in an autolysosome in F, a putative isolation membrane in G and a putative autophagosome in H. n, nucleus; v, the central vacuole; m, mitochondrion. Bar: 20 mm in A and B; 10 mm in C and D; 2 mm in E; and 500 nm in F–H. 2076 Plant Cell Physiol. 52(12): 2074–2087 (2011) doi:10.1093/pcp/pcr137 ! The Author 2011. Autophagy in tobacco cells cells under nitrogen starvation by time-lapse video microscopy, and observed two distinct pathways in which autophagosomes are delivered to the central vacuole. One is a direct delivery of autophagosomes to the central vacuole, and the other involves the engulfment of autophagosomes into small vacuolar structures and subsequent fusion of small vacuoles with the central vacuole. The small vacuolar structures seem to be autolysosomes, and the latter pathway may correspond to the pathway we have observed using E-64c in the present study. However, it should be noted that we observed almost all the particles in autolysosomes and only a few in the central vacuole, suggesting that the latter pathway is operating dominantly and that degradation is already well underway before the autolysosomes fuse with the central vacuole. In cells cultured under sucrose starvation conditions without E-64c, we occasionally found a structure resembling an autolysosome (a small vacuole containing a few electrondense particles) (Supplementary Fig. S2). This suggests that autolysosomes, which have been recognized in the presence of E-64c, are real intermediate structures emerging during the autophagic process in tobacco cells. In addition to autolysosomes, we found two types of structures that are also probably involved in autophagy (Fig. 1G, H; arrowhead). One resembles the isolation membrane and the other the autophagosome of mammalian cells. Both structures were distinguishable from autolysosomes in that two or more membranes were present at their boundary and the cytoplasm enclosed showed no signs of degradation. Similar structures have also been observed by Toyooka et al. (2006). These morphological observations suggest that the autophagy that occurs in tobacco cells cultured under sucrose starvation proceeds in a manner more similar to mammalian than yeast macroautophagy. Immunofluorescence study of autolysosomes In a previous study, we showed the localization of acid phosphatase, a marker enzyme of mammalian lysosomes, in autolysosomes of tobacco cells by enzyme cytochemistry (Moriyasu and Ohsumi 1996). In the present study, we performed immunofluorescence microscopy on protoplasts that had accumulated autolysosomes, using an affinity-purified antibody against acid phosphatase from potato (Fig. 2). The autolysosomes stained positively, whereas control cells not accumulating autolysosomes did not show such clear staining (Fig. 2A vs. B). This result provides confirmatory evidence that autolysosomes contain acid phosphatase. Curiously, although the central vacuole contains acid phosphatase, it remained unstained. We assume that this is because the concentration of proteins including acid phosphatase in the central vacuole is too low to be recognized by immunofluorescence. Isolation of autolysosomes Using acid phosphatase as a marker, we attempted to isolate the autolysosomes of tobacco cells. The cell walls were first digested to make protoplasts, which were then gently ruptured and fractionated by isopycnic centrifugation through a Percoll gradient. All fractions were then analyzed for acid phosphatase activity. Fig. 3A shows the profile of the activity from control cells, which were 4-day-old cells immediately after transfer to a sucrose-free medium. The acid phosphatase activity collected exclusively in a sharp peak around fraction No. 21. A similar profile was obtained in the fractionation of the cells that had been put under sucrose starvation conditions for 24 h without E-64c (Fig. 3C, filled circle), suggesting that starvation per se does not alter the intracellular localization of acid phosphatase. In contrast, the curve of acid phosphatase activity altered very significantly in the centrifugation tube prepared from the 24 h starved cells treated with E-64c; another peak (Fig. 3B, arrow) showed up close to fraction No. 5 near the bottom, in addition to a peak at around fraction No. 21 (Fig. 3B, filled circle). This suggested that this peak contained autolysosomes. Thus, the density of autolysosomes was estimated to be 1.08–1.11 mg ml–1 using density marker beads (Pharmacia Biotech). Cyt c oxidase, a marker for mitochondria, and catalase, a marker for microbodies, were distributed differently from each other. However, a portion of Cyt c oxidase and almost all of the catalase activity co-equilibrated in the same peak fractions as acid phosphatase (Fig. 3B, C). We interpret these data as showing that partially disrupted products of the three organelles, the central vacuoles, mitochondria and microbodies, collected in these fractions. The distributions of Cyt c oxidase and catalase did not change significantly when the cells with an accumulation of autolysosomes were fractionated (Fig. 3B vs. C). Next we analyzed polypeptides in the fractions (Fig. 4). Acid phosphatase activity in each fraction was measured again in order to localize the fractions containing autolysosomes (Fig. 4A, arrow). Silver staining of SDS gels showed that the fractions of autolysosomes (fraction Nos. 4–8) had many obscure polypeptide bands on a relatively high background (Fig. 4B, left). In contrast, such staining was not seen in the corresponding fractions from the control cells (Fig. 4B, right). Thus, some of these polypeptides may represent proteins of cytoplasmic origin that were enclosed and partially degraded in autolysosomes. Western blot analysis showed that the fractions of autolysosomes contained acid phosphatase (Fig. 4C), which is consistent with the results obtained by immunostaining (Fig. 2) and enzyme cytochemistry (Moriyasu and Ohsumi 1996). The autolysosomal fractions also contained the A and B subunits of vacuolar H+-ATPase (Fig. 4C). This suggests the presence of a functional H+-ATPase on the membanes of autolysosomes, which is consistent with their having an acidic lumen (Moriyasu and Ohsumi 1996). We cannot exclude, however, the possibility that they originated from the cytoplasm enclosed in autolysosomes. We also tried to examine the existence of H+-pyrophosphatase in the autolysosomal fractions, but did not obtain a conclusive result because H+-pyrophosphatase was detected throughout the gradient. Plant Cell Physiol. 52(12): 2074–2087 (2011) doi:10.1093/pcp/pcr137 ! The Author 2011. 2077 C. Takatsuka et al Fig. 2 Immunostaining of tobacco protoplasts for acid phosphatase. Protoplasts were prepared from cells cultured under sucrose starvation for 1 d in the presence (A and C) or absence (B and D) of E-64c. Protoplasts were chemically fixed and bound with anti-potato acid phosphatase antibody and rhodamine-conjugated secondary antibody. (A and B) Fluorescence images. (C and D) Nomarski images of A and B, respectively. Significant staining was not observed when the first antibody was omitted. Arrows, autolysosomes. n, nucleus. Bar, 10 mm. Autolysosomes have cysteine protease activity E-64c is an inhibitor that binds covalently to papain-type cysteine proteases. Thus cysteine protease activity is not expected to be recovered in fractions prepared from cells treated with E-64c. However, leupeptin, which is also able to cause autolysosomes to accumulate (Moriyasu and Ohsumi 1996), inhibits cysteine protease non-covalently. Thus we expected that if we isolated autolysosomes from cells treated with leupeptin, we would be able to remove the inhibitor and detect protease activity. First we examined whether or not we really could restore protease activity in the homogenate prepared from cells treated with leupeptin (Fig. 5). In order to detect protease activity, we performed activity staining of protease in native gels containing a standard substrate, gelatin. In the gels, one main band was detected (Fig. 5, arrowhead). We have previously reported that the protease activity that can be measured using casein as a substrate increased in response to sucrose starvation (Moriyasu and Ohsumi 1996). Consistent with this result, gelatin-degrading activity increased in response to sucrose starvation (Fig. 5; 0 d vs. 1 d, MeOH and Water). When the homogenate was prepared from cells that had 2078 been incubated with E-64c, no protease activity was detected (Fig. 5; 1 d, E-64c). In contrast, two or more activity bands were detected in the homogenate prepared from the cells treated with leupeptin, although the band pattern was different from the controls, presumably due to the interaction of protease and leupeptin and their dissociation during electrophoresis (Fig. 5; 1 d, Leu; marked with stars). To confirm the localization of proteases in isolated autolysosomes, we treated the cells with leupeptin instead of E-64c. From the profile of acid phosphatase activity, the fractions of autolysosomes at around fraction No. 6 were identified (Fig. 6A; left, arrow). In these fractions, we were able to detect protease activity, consisting of two or more bands on a native gel (Fig. 6B; left, marked with stars). These bands were barely detectable when the gels were treated with E-64c after electrophoresis (data not shown), suggesting that these activities are due to cysteine protease. In contrast, neither protease activity nor acid phosphatase activity was seen in the corresponding fractions from control cells (Fig. 6A, B; right). These results support the notion that cysteine proteases are located in autolysosomes and degrade the enclosed cytoplasm, Plant Cell Physiol. 52(12): 2074–2087 (2011) doi:10.1093/pcp/pcr137 ! The Author 2011. Autophagy in tobacco cells and that the inhibition of the proteases by E-64c or leupeptin leads to the accumulation of the degradation intermediates within autolysosomes. The autophagy inhibitor 3-MA releases the E-64c inhibition of endocytosis. Fig. 3 Fractionation of tobacco cells cultured under sucrose starvation conditions for 1 d in the presence or absence of the cysteine protease inhibitor E-64c. Tobacco cells were homogenized and fractionated by density equilibrium centrifugation on a Percoll gradient. Acid phosphatase (filled circle), catalase (open circle) and Cyt c oxidase (triangle) activities in each fraction were measured. Fraction numbers are in the order from the bottom to the top of centrifuge tubes. (A) Immediately after starvation treatment. (B) After 1 d of culture in the presence of E-64c. The arrow indicates the peak of the autolysosome fractions. (C) After 1 d of culture in the absence of E-64c. E-64c blocks the endocytotic pathway from the plasma membrane to the membrane of the central vacuole (Yano et al. 2004). To confirm this result, we stained the plasma membrane by incubating the cells with FM4-64 at 0 C and, after washing out unbound dye, the cells were placed in a fresh culture medium without the dye at 26 C. Immediately after the start of culture, the plasma membrane was seen to be heavily stained, together with a punctate staining in the cell cortex (Fig. 7A). These punctate structures (Fig. 7A, arrowhead) were assumed to be endosomes. FM4-64 fluorescence thereafter moved from the plasma membrane to the central vacuole in cells cultured in a sucrose-free medium for 24 h (Fig. 7B). In contrast, the majority of the fluorescence associated with autolysosomes rather than the vacuolar membrane when the cells were starved for 24 h in the presence of E-64c (Fig. 7C, arrowhead; see also Fig. 1A in Yano et al. 2004). This result suggests that some of the membrane for the formation of autolysosomes flows from the endocytotic pathway leading to the central vacuole. 3-MA is an inhibitor of autophagy (Gordon and Seglen 1982, Seglen and Gordon 1982, Takatsuka et al. 2004), but does not perturb the movement of FM4-64 from the plasma membrane to the vacuolar membrane (Fig. 7D). We next treated the cells simultaneously with E-64c and 3-MA. In this situation, autolysosomes did not accumulate (Takatsuka et al. 2004) and FM4-64 moved normally from the plasma membrane to the vacuolar membrane (Fig. 7E). Thus, the inhibition of autophagy by 3-MA abolishes the inhibition of FM4-64 endocytosis by E-64c. Yamada et al. (2005) have claimed that the structures accumulating in the presence of E-64d (an esterified form of E-64c) are endosomes, and proposed that cysteine protease inhibitors such as E-64c or E-64d inhibit the proteases that are involved in the fusion of endosomes with the central vacuole, causing the accumulation of endosomes, which are called autolysosomes in the present study. It is therefore possible that 3-MA treatment simply reverses the E-64c inhibition of protease activity, and thus prevents the accumulation of ‘autolysosomes/endosomes’. However, measurements of cellular protease activities do not support this (Fig. 8). First, protease activity measured in cell homogenates at an acidic pH using casein as a substrate increased >2-fold in 24 h (Fig. 8A). This was reduced to approximately 20% by the addition of E-64c to the culture medium. Inclusion of 3-MA did not perturb the inhibition of the protease activity by E-64c. Secondly, as shown in Fig. 5, one major band depicting gelatin-degrading activity in native gels increased in response to sucrose starvation (Fig. 8B). This band was not visible in gels prepared from starved cells which were exposed to E-64c, and the same was observed Plant Cell Physiol. 52(12): 2074–2087 (2011) doi:10.1093/pcp/pcr137 ! The Author 2011. 2079 C. Takatsuka et al Fig. 4 Western blot analysis of vacuolar proteins in each fraction from tobacco cells cultured under different conditions. Tobacco cells were cultured under sucrose starvation conditions with (left) or without (right) E-64c for 1 d and were then fractionated on a Percoll gradient. (A) Acid phosphatase activity in each fraction. Fraction numbers are in the order from the bottom to the top of centrifuge tubes. The arrow indicates the peak of the autolysosome fractions. (B) Proteins in each fraction were separated by SDS–PAGE and stained with silver. The lane numbers correspond to the fraction numbers in A. (C) The presence of acid phosphatase (APase), and vacuolar H+-ATPase subunits A (ATPaseA) and B (ATPaseB) was analyzed by Western blotting. The lane numbers correspond to the fraction numbers in A. when 3-MA was included. These results show that 3-MA has nothing to do with the protease levels in cells. Together, the data strongly suggest that 3-MA releases the inhibition of fusion between ‘autolysosomes/endosomes’ and the central vacuole without perturbing the E-64c inhibition of protease activity, and therefore question the notion that some protease sensitive to E-64c is required for the fusion between ‘autolysosomes/ endosomes’ and the central vacuole. Discussion The exact pathway of macroautophagy is not fully understood in plant cells. In mammalian cells, an autophagosome fuses with 2080 a lysosome thereby converting it into an autolysosome. Similarly in yeast cells, an autophagosome fuses with the vacuole which, since it contains assorted hydrolytic enzymes, is equivalent to the lysosome. In contrast, in plant cells, it remains unclear whether an autophagosome fuses directly with the central vacuole (also a lysosome equivalent) or whether the autophagosome is transformed into an autolysosome prior to fusion with the central vacuole. The crucial question is therefore: do autophagosomes become enriched in acid hydrolases? In previous studies, we showed that special membrane compartments accumulated in tobacco cells cultured under sucrose starvation conditions in the presence of a cysteine protease inhibitor (Moriyasu and Ohsumi 1996, Inoue and Plant Cell Physiol. 52(12): 2074–2087 (2011) doi:10.1093/pcp/pcr137 ! The Author 2011. Autophagy in tobacco cells Fig. 5 Staining of protease activity in native gels prepared from tobacco cell homogenates cultured under different conditions. Tobacco cells cultured under sucrose starvation conditions for 1 d in the presence of E-64c or leupeptin were homogenized, and proteins were separated on non-denaturing polyacrylamide gels containing gelatin as a protease substrate. After electrophoresis, gels were kept in 50 mM acetate-Na (pH 5.0) containing 2.8 mM 2-mercaptoethanol at 37 C overnight and then stained with Coomassie brilliant blue. 0 d, immediately after treatment of sucrose starvation; MeOH, cells cultured for 1 d in the presence of 1% (v/v) methanol as a solvent control of E-64c addition; Water, cells cultured for 1 d with the addition of water as a solvent control of leupeptin addition; E-64c, cells cultured for 1 d in the presence of 10 mM E-64c; Leu, cells cultured for 1 d in the presence of 10 mM leupeptin. The arrowhead indicates the main band of protease; the stars mark several activity bands in the Leu lane. Moriyasu 2006). These compartments were acidic and contained acid phosphatase, as was shown by vital staining with quinacrine and by cytochemical enzyme analysis with b-glycerophosphate as a substrate. Moreover, the compartments contained electron-dense materials, which assumed the appearance of partially degraded parts of the cytoplasm. Interestingly, when mammalian cells executing autophagy are treated with protease inhibitors such as leupeptin, autolysosomes accumulate in them (Kominami et al. 1983). On the other hand, when yeast cells under nitrogen starvation conditions are treated with the protease inhibitor phenylmethylsulfonyl fluoride, autophagic bodies, i.e. membrane-bound parts of the cytoplasm, accumulate in the vacuole (Takeshige et al. 1992). Thus, by analogy to mammalian rather than yeast cells, it is reasonable to suppose that the compartments accumulated by the inhibition of proteolysis in tobacco cells are autolysosomes. In the present study, we now show by electron microscopy that the autolysosomes induced in tobacco cells contained partially degraded organelles such as mitochondria or plastids (Fig. 1F), confirming that autophagosomes acquire degradative properties. We have furthermore identified the precursory structures of the autolysosomes, which appear to be enclosing a part of the cytoplasm (Fig. 1G) or just have enclosed it (Fig. 1H). Similar structures have been observed in sycamore, Arabidopsis and tobacco cells cultured under nutrient starvation conditions (Aubert et al. 1996, Marty 1999, Rose et al. 2006, Toyooka et al. 2006). We have successfully isolated cell fractions enriched in autolysosomes and biochemically characterized them. Our results suggest that autolysosomes have a cysteine protease(s) in their lumen and most probably an H+-ATPase activity in their membranes. Thus it would seem that autolysosomes in tobacco cells are able to create and maintain the acidic environment required for the action of hydrolytic enzymes. This indicates that autophagosomes acquires hydrolytic enzymes and proton pump complexes before they coalesce with the central vacuole. Accordingly, in the autophagy that is performed by tobacco cells, it is autolysosomes which play a major role in the degradation of the enclosed cytoplasm. A similar conclusion was reached by Rose et al. (2006) when working on cultured Arabidopsis cells. The vacuole therefore acts as a recipient for partially degraded material and completes the degradation. On the other hand, experiments with ConcA support the notion that the central vacuole acts as the main lytic compartment. When nitrogen-starved Arabidopsis hypocotyl and root cells are treated with ConcA, autophagic bodies accumulate in the lumen of the central vacuole. These are cytoplasmic droplets (with organelles) surrounded by a single boundary membrane (Yoshimoto et al. 2004). ConcA is a well-known inhibitor of vacuolar H+-ATPase and prevents the acidification of the vacuole interior, implying that autophagic bodies become visible because they cannot be degraded. However, in mammalian cells, the effects of ConcA and bafilomycin, another inhibitor of vacuolar H+-ATPase, seem to be complicated (Klionsky et al. 2008), and in plants it is known that ConcA also acts at the level of the Golgi apparatus by preventing the formation and release of the trans-Golgi network (Robinson et al. 2004, Dettmer et al. 2005, Dettmer et al. 2006, Viotti et al., 2010). Thus, although ConcA treatment does enable an easier detection of autophagy in plant cells, it obviously distorts the normal autophagic process in plant cells. How an autophagosome acquires the hydrolytic enzymes necessary for the degradation of enclosed cytoplasm and transforms into an autolysosome in tobacco BY-2 cells remains unclear. It is possible that autophagosomes in tobacco cells are different from genuine autophagosomes in that they have hydrolytic enzymes right from the start of their formation. According to Marty (1978), small vacuoles in root meristematic cells develop from tubular structures which contain acid phosphatase. These tubules surround a part of the cytoplasm to form a double-membrane compartment, a kind of autophagosome, which subsequently becomes a vacuole after digestion of the inner membrane and enclosed cytoplasm. Plant Cell Physiol. 52(12): 2074–2087 (2011) doi:10.1093/pcp/pcr137 ! The Author 2011. 2081 C. Takatsuka et al Fig. 6 Staining of protease activity in native gels from subcellular fractions obtained from tobacco cells cultured under different conditions. Tobacco cells were cultured under sucrose starvation conditions for 1 d in the presence (left) or absence (right) of leupeptin. (A) Acid phosphatase activity in each fraction. The arrow indicates the peak of the autolysosome fractions. (B) Staining of protease activity on native gels containing gelatin. The lane numbers correspond to the fraction number in A. The stars mark the activity bands of protease. Another possibility is that autophagosomes acquire hydrolytic enzymes secondarily by an as yet unknown interaction with the central vacuole. When the vacuolar membranes of BY-2 cells are labeled with the fluorescent dye FM4-64, the dye moves to autolysosomes upon shifting the cells to starvation conditions in the presence of E-64c (Yano et al. 2004). Conversely, the staining intensity of FM4-64 on the vacuolar membranes decreases. Similarly, a lumenal GFP-tagged vacuolar protein also moves to autolysosomes, when autolysosomes are formed (Yano et al. 2004). These results suggest that there is a contribution of the central vacuole to the formation of autolysosomes, and explain how they acquire hydrolytic enzymes from the central vacuole. Interestingly, in the absence of E-64c, FM4-64 remains on the vacuolar membrane during starvation-induced autophagy (Yano et al. 2004). Taken together, it would seem that parts of the vacuolar membrane and lumen join in the formation of autolysosomes, and autolysosomes once formed return to the central vacuole in a normal autophagic pathway in the absence of E-64c. It would appear that the autophagic pathway merges with the endocytotic pathway in tobacco cells as well as in mammalian cells, since blockage of the autophagic pathway by E-64c 2082 leads to blockage of the endocytotic pathway (Liou et al. 1997, Yano et al. 2004). However, it has been proposed that endosomes accumulate in the presence of cysteine protease inhibitors because they inhibit the fusion of endosomes with the central vacuole (Yamada et al. 2005). In contradiction to this, we now show that the autophagy inhibitor 3-MA overrules the E-64c block and allows the normal endocytotic pathway to recover. Thus, the apparent inhibition of endocytosis by E-64c is due to the inhibition of the autophagic pathway by this inhibitor. We think that the accumulation of undegraded materials in autolysosomes makes autolysosomes larger and hinders their fusion with the central vacuole. Whether or not the same autophagic pathway as described here for tobacco BY-2 cells occurs in other plant cells is not yet known. We have observed young and mature root cells treated with E-64 d, a more membrane-permeable analog of E-64c, in barley and Arabidopsis (Moriyasu et al. 2003, Inoue et al. 2006). In these cells, partially degraded materials derived from the cytoplasm appeared to accumulate in large vacuoles and small vacuoles such as autolysosomes. The detailed observation of autolysosomes and the central vacuole is now in progress in our laboratory. Plant Cell Physiol. 52(12): 2074–2087 (2011) doi:10.1093/pcp/pcr137 ! The Author 2011. Autophagy in tobacco cells Materials and Methods Plant material Tobacco culture cells (BY-2) were cultured in a 300 ml Ehrenmeyer flask at 26 ± 1 C with rotation of 110 r.p.m. The culture was maintained by transferring 1.5 ml of cell suspension at the stationary phase to 80 ml of fresh culture medium once a week. The culture medium consisted of water, 4.3 g l–1 Murashige and Skoog salt mixture (M7150, Sigma), 3% (w/v) sucrose, 0.2 mg l–1 2,4-D, 2 mg l–1 glycine, 100 mg l–1 myo-inositol, 0.5 mg l–1 nicotinic acid, 0.5 mg l–1 pyridoxineHCl and 0.1 mg l–1 thiamine-HCl. A sucrose-free culture medium was prepared by omitting sucrose from the culture medium and was used for putting cells under carbon starvation conditions. The pH of these culture media was adjusted to between 5.8 and 6.0 with fresh KOH. Sucrose starvation Four-day-old cells, which were at the logarithmic phase, were collected by centrifugation at 500g for 5 min. The cells were suspended in the original volume of the sucrose-free culture medium and precipitated again by centrifugation. They were suspended again in the same volume of fresh sucrose-free culture medium and kept at 26 ± 1 C with rotation of 110 r.p.m. When the cells were treated with E-64c (a gift from Taisho Pharmaceutical Company), one-hundredth of the culture medium of 1 mM E-64c dissolved in methanol was added to the cell culture except in experiments for cell fractionation. Thus the final concentration of E-64c was 10 mM. As a control of solvent effects, the same volume of methanol alone was added. Thus the final concentration of methanol was 1% (v/v). Microscopy In order to observe whole living cells and immunostained protoplasts, a light microscope (OptiPhoto, Nikon) that is fitted with Nomarski interference optics and an epifluorescence apparatus was used. Photographs were taken through a 20 or 40 objective lens and a 5 projection lens using color film (Fujicolor ASA400, Fuji). To observe cells stained with FM4-64, confocal laser microscopes (MRC-1024, Bio-Rad, or FV1000-D, Olympus) were used. For sectioning, the cells were embedded in Spurr’s resin. They were fixed with 2% (w/v) glutaraldehyde and 1% (w/v) formaldehyde in 0.1 M sodium cacodylate-HCl (pH 6.9) at room temperature for 1 h and at 4 C overnight, and post-fixed with Fig. 7 The effect of the autophagy inhibitor 3-MA on the endocytosis of FM4-64. Plasma membranes of tobacco cells were stained with FM4-64, and endocytosis was observed. (A) Immediately after staining with FM4-64. (B) After 1 d in the absence of E-64c and 3-MA. (C) After 1 d in the presence of E-64c alone. (D) After 1 d in the presence of 3-MA alone. (E) After 1 d in the presence of both E-64c and 3-MA. Arrowheads indicate putative endosomes in A and the accumulation of autolysosomes/endosomes in C. n, nucleus. Bar, 50 mm. Plant Cell Physiol. 52(12): 2074–2087 (2011) doi:10.1093/pcp/pcr137 ! The Author 2011. 2083 C. Takatsuka et al Immunofluorescence staining for acid phosphatase Fig. 8 The effect of the autophagy inhibitor 3-MA on intracellular protease activities in tobacco cells. After tobacco cells were cultured in a sucrose-free medium for 1 d in the presence or absence of E-64c and 3-MA, the cells were homogenized. (A) Protease activity in the homogenate was measured using casein as a substrate. Values relative to the value at 0 d are shown. 0 d, immediately after culture in a sucrose-free medium; 1 d, 1 d after culture in the absence of E-64c and 3-MA; 1 d+E64, 1 d after culture in the presence of E-64c alone; 1 d+3MA+E64, 1 d after culture in the presence of both 3-MA and E-64c. (B) Activity staining of protease on a native polyacrylamide gel. + and – mean the presence and absence of the respective inhibitors. 1% (w/v) osmium tetroxide at room temperature for 2 h. After they were immersed in 1% (w/v) uranyl acetate at room temperature for 2 h, the cells were dehydrated with a series of ethanol/water mixtures and propylene oxide. They were then embedded in the resin. Sections of 1 mm thick were made from the blocks and stained with toluidine blue. They were observed by light microsocpy (OptiPhoto, Nikon). Sections of approximately 70 nm thick were made from the blocks and stained with uranyl acetate and lead nitrate. They were observed by electron microscopy (H-7000, Hitachi). 2084 To make protoplasts, cells were collected by centrifugation and kept at 28 C for 60 min in 10 mM MES-Na buffer (pH 5.5) containing 1% (w/v) Cellulase Onozuka RS (Yakult Pharmaceutical Industry Co., Ltd.), 0.1% (w/v) Pectolyase Y-23 (Seishin Pharmaceutical Co., Ltd.) and 0.4 M sorbitol, and protoplasts were collected by centrifugation at 500g for 5 min, washed with 0.4 M sorbitol, and fixed at room temperature for 1 h with 50 mM phosphate-Na (pH 7.0) containing 3.7% (w/v) formaldehyde, 5 mM EGTA, 3% (w/v) sorbitol and 0.02% (w/v) sodium azide. After the fixative was exchanged with a fresh one, protoplasts were stored at 4 C until the next step. In the next step, the protoplasts were washed with 50 mM phosphate-Na buffer (pH 7.0) containing 5 mM EGTA, 3% (w/v) sorbitol and 0.02% (w/v) NaN3 for 5 min three times and for 1 h once. They were then treated with 0.5% (w/v) Triton X-100 for 5 min at room temperature. After they were washed with the same buffer for 5 min three times, the protoplasts were kept for 30 min in blocking solution {phosphate-buffered saline [P-3813, Sigma; 10 mM phosphate buffer (pH 7.4), 138 mM NaCl, 2.7 mM KCl], 0.25% (w/v) bovine serum albumin (BSA), 0.25% (w/v) gelatin and 0.05% (w/v) NP-40}. The blocking solution was removed and the protoplasts were incubated overnight at 4 C in the blocking solution containing antibody against acid phosphatase. The protoplasts were washed with the blocking solution for 5 min three times and then incubated with the blocking solution containing 1/400 diluted anti-rabbit IgG–tetramethylrhodamine isothiocyanate conjugate (T-6778, Sigma) for 1 h at room temperature. After the protoplasts were washed with the blocking solution three times each for 5 min, they were observed by epifluorescence microscopy. Antibody against acid phosphatase used in this staining was affinity-purified using nitrocellulose paper blotted with acid phosphatase from potato according to the method by Pringle et al. (1991). Briefly, acid phosphatase from potato (P-1146, Sigma) was separated by SDS–PAGE and transferred electrophoretically onto nitrocellulose paper. The membrane strip with the major band of potato acid phosphatase (approximately 45 kDa) was cut out and used as an affinity purification matrix. Cell fractionation Four-day-old cells in 80 ml of culture medium were precipitated by centrifugation at 500g for 5 min. Supernatant was removed and the cells were suspended in 80 ml of sucrose-free culture medium. Cells were precipitated again and the supernatant was removed. Cells were then suspended in 80 mk of fresh sucrose-free culture medium. After 80 ml of 10 mM E-64c or methanol (as a solvent control) was added, the suspensions were cultured at 26 C with rotation of 110 r.p.m. Immediately or 24 h after sucrose starvation, cells were collected by centrifugation at 500g for 5 min. To prepare protoplasts, cells were Plant Cell Physiol. 52(12): 2074–2087 (2011) doi:10.1093/pcp/pcr137 ! The Author 2011. Autophagy in tobacco cells washed once with 0.4 M sorbitol and kept in 80 ml of 10 mM MES-Na buffer (pH 5.5) containing 1% (w/v) Cellulase Onozuka RS, 0.1% (w/v) Pectolyase Y-23 and 0.4 M sorbitol at 26 C for 1 h with rotation. The resulting protoplasts were collected by centrifugation at 500g for 10 min, and the supernatant was discarded. Protoplasts were suspended in 0.4 M sorbitol and pelleted by centrifugation again. They were then disrupted in 10 ml of 0.4 M sorbitol containing 50 mM HEPES-K (pH 7.5), 1 mM EDTA, 10 mM leupeptin (Peptide Institute), 10 mM pepstatin A (Peptide Institute) and 1 mM 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride by passing through a needle. Organelles that were released from the cells were pelleted by centrifugation at 17,000g (JA-20 rotar, Beckman) for 5 min. The pellet was suspended in 5 ml of 0.4 M sorbitol containing 10 mM HEPES-K (pH 7.5) and 1 mM EDTA and then filtered through nylon mesh (mesh size: 10 mm). The filtrate was put on 60 ml of 0.4 M sorbitol containing 30% (v/v) Percoll (Amerciam-Pharmacia), 10 mM HEPES-K (pH 7.5) and 1 mM EDTA, and then centrifuged at 50,000g for 1 h (RP45T rotar, Hitachi) to carry out density gradient and organelle separation simultaneously. After centrifugation, the content of a centrifuge tube was divided into about 30 fractions (each 2 ml). Acid phosphatase activity in each fraction was measured in a mixture of 500 ml of 0.1 M acetate-Na (pH 5.0), 380 ml of water, 100 ml of each fraction and 20 ml of 50 mM p-nitrophosphate. The reaction was performed at 37 C for 20 min, and then stopped by the addition of 1.6 ml of 1 M Na2CO3. After Percoll in the reaction mixture was precipitated by centrifugation at 1,000 r.p.m. for 5 min, the A405 of the supernatant was measured. To measure Cyt c oxidase activity, a marker of mitochondria, in each fraction, 100 ml of each fraction was mixed with 100 ml of 4 mg ml–1 digitonin. The mixture (50 ml) was mixed with 900 ml of Cyt c substrate solution consisting of 26.4 mg of Cyt c and 28 mg of Na2S2O3 dissolved in 40 ml of 100 mM phosphate-K (pH 7.0) in a cuvette, and then the decrease in the A240 at 37 C was monitored. To measure catalase activity, a marker of microbodies, in each fraction, 1 ml of 10 mM H2O2 in 50 mM phosphate-K (pH 7.0) was mixed with 50 ml of each fraction in a cuvette, and then the decrease in the A240 was monitored at 37 C. (w/v) skim milk, 150 mM NaCl and 0.05% (w/v) Tween-20 at 4 C overnight. Anti-acid phosphatase (potato) antibody (10 mg ml–1, Polyscience) was used at 1 : 3,000 dilution without further purification; anti-A and B subunits of vacuolar H+-ATPase antibody and anti-H+-pyrophosphatase antibody (generous gifts from Dr. Maeshima, Nagoya University) were at 1 : 2,500 dilution. The secondary antibody used was anti-rabbit IgG antibody conjugated with alkaline phosphatase (S3731, Promega). The binding of the primary and secondary antibodies to membranes was done in 10 mM Tris–HCl (pH 8.0) containing 150 mM NaCl and 0.05% Tween-20. Nitroblue tetrazolium and 5-bromo-4chloro-3-indolyl phosphate were used to detect the activity. Activity staining of gelatin-degrading activity on a polyacrylamide gel A polyacrylamide gel [7.5% (w/v)] containing 0.04% (w/v) gelatin was made by polymerizing the mixture of acrylamide and methylene-bis-acrylamide together with gelatin. Cell homogenate, prepared in the same way as for measuring casein-degrading activity, was mixed with one-fifth volume of 20% (w/v) sucrose and 0.01% (w/v) bromophenol blue and run on the gel at 4 C according to Davis (1964). After electrophoresis, the gel was washed three times with 20 mM MES-Na (pH 5.5) containing 2.8 mM 2-mercaptoethanol, and incubated in the same buffer at 37 C overnight. The gel was thereafter stained with Coomassie Brilliant Blue, destained and photographed. Endocytosis of FM4-64 Four-day-old culture (5 ml) was centrifuged at 500 r.p.m. for 5 min and the sedimented cells were suspended in 5 ml of sucrose-free culture medium. The suspension was centrifuged again and the sedimented cells were resuspended in 5 ml of sucrose-free culture medium. To 1 ml of cell suspension in the sucrose-free culture medium, 10 ml of 1 mM E-64c or methanol alone and 0.5 ml of 0.1 M 3-MA or water alone were added. Immediately, 100 ml of the suspension was put into a polypropylene tube (12 75 mm) and cooled down to 0 C. To this tube, 1 ml of 10 mM FM4-64 (Molecular Probes) solution in dimethylsulfoxide (DMSO) was added, and the culture was kept at 0 C for 20 min. FM4-64 was washed off and the culture was returned to 26 C; this was defined as t = 0. SDS–PAGE and western blotting An aliquot of each fraction was mixed with the same volume of sample preparation buffer (Tris–SDS–bME, Enprotech), and then heated at 100 C for 5 min. Proteins were separated on 10% polyacrylamide gels by SDS–PAGE according to Laemmli (1970). After electrophoresis, gels were stained with silver. Proteins in another gel were electrotransferred onto a nitrocellulose membrane in a solution consisting of 25 mM Tris, 192 mM glycine and 20% methanol. After transfer, membranes were immersed in 10 mM Tris–HCl (pH 8.0) containing 5% Measurement of casein-degrading activity The cells were homogenized and casein-degrading activity was measured in almost the same way as reported previously (Moriyasu and Inoue 2008). Cells in 3 ml of culture were collected by centrifugation at 500g for 5 min. After the cells were washed with 3 ml of sucrose-free culture medium, they were homogenized at 4 C with 100 mM MES-Na (pH 5.5) containing 28 mM 2-mercaptoethanol using a mortar-driven Teflon homogenizer. The homogenate was centrifuged at 15,000 r.p.m. for Plant Cell Physiol. 52(12): 2074–2087 (2011) doi:10.1093/pcp/pcr137 ! The Author 2011. 2085 C. Takatsuka et al 10 min. 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