Lysosomal movements during heterophagy and autophagy: With special reference to nematolysosome and wrapping lysosome

JOURNAL OF ELECTRON MICROSCOPY TECHNIQUE 12:lOl-131 (1989)

Lysosomal Movements During Heterophagy and Autophagy: With Special Reference to Nematolysosome and Wrapping Lysosome MASAHIRO SAKAI, NOBUKAZU ARAKI, AND KAZUO OGAWA Department of Amtomy, Faculty of Medicine, Kyoto University, Kyoto 606, Japan

KEY WORDS

ABSTRACT Recent studies on lysosomal movements during heterophagy and autophagy performed in our laboratory for the past several years were reviewed; methods for the investigation of lysosomes and the cytoskeleton in these studies mainly involved electron microscopic cytochemistry.

Lysosomal movements during heterophagy were observed in cultured rat alveolar macrophages taking up horseradish peroxidase (HRP) and rat peroxidase-antiperoxidase (PAP) by f hid-phase pinocytosis and adsorptive pinocytosis, respectively. A characteristic lysosomal change which was induced by the pinocytosis was the appearance of long, threadlike lysosomes (nematolysosomes) in the cytoplasm. The effects of actin filament destabilizer and antimicrotubular drug on lysosomal changes revealed that the appearance of nematolysosomes was dependent on the presence of both actin filaments and microtubules. The close morphological relationship between lysosomes and cytoskeletal elements, such as actin filaments and microtubules in the alveolar macrophages, supports the participation of the cytoskeletal system in the regulatory mechanism of lysosomal movements.

In the study of the lysosomal wrapping mechanism (LWM), which is one type of lysosomal movement that occurs during autophagy, it was found that the occurrence of LWM was dependent on energy-namely, the supply of ATP-and on the presence of actin filaments. However, deconstruction of microtubules induced or favored the occurrence of LWM. It is conceivable that the LWM is also related to the cytoskeletal system.

We conclude that intracellular dynamics of lysosomes during heterophagj and autophagy are largely a consequence of complicated modulation by the cytoskeletal system.

Lysosome, Heterophagy, Autophagy, Cytoskeleton, Cytochemistry

INTRODUCTION Lysosomes, which work as an important intracellu-

lar digestive system, contain a large number and variety of hydrolytic enzymes. Since these enzymes have the capacity to digest most macromolecules com- posing a living body, such as proteins, nucleic acids, lipids, and carbohydrates, an ingenious regulatory mechanism must exist in order to fulfill main lysoso- ma1 functions systematically. This intracellular digestive system associated with lysosomes can be classified, on the basis of materials taken into lysosomes, into two categories: heterophagy or the digestion of exogenous materials, and autophagy for the catabolism of endogenous substances in the cell. However, it seems difficult to separate these two processes from each other clearly, because they can be usually detected in some cells at the same time (Kindberg et al., 1987; Piao et al., 19831, suggesting that they are correlated and play some important parts in the whole intracellular metabolism.

Numerous investigations about the relationship between lysosomes and either heterophagy or autophagy have been performed so far as a means of studying the intracellular metabolism morphologically, but little attention has been paid to the details of the regulatory

mechanism of the intracellular movement of lysosomes with regard to their function.

Heterophagy is easily observed in some cells which have high phagocytic activity such as macrophages and leukocytes; exogenous materials taken into the cell by phagocytosis or pinocytosis are degraded in phagoly- sosomes which are formed by the fusion between lysosomes and phagosomes or pinosomes. Meanwhile, lysosomes actively move through the cytoplasm and reveal polymorphic behavior in their shape and size.

It is currently agreed that cytoskeletal elements such as microtubules and actin filaments play an important role in completing the process of heterophagy (Alison et al., 1971; Silverstein et al., 1977) as well as in retaining both cellular external structure and distribution of organelles in the cell (Ball et al., 1982; Collot et al., 1984; Matteoni and Kreis, 1987). In the first part of this review, we focus on the translocation and transformation of lysosomes during heteroph-

Received May 26, 1988 accepted in revised form July 8, 1988. Address reprint requests ta Masahiro Sakai, Department of Anatomy, Faculty

of Medicine, Kyoto University, Konoecho, Yoshida, Sakyo-ku, Kyota 606, Japan.

0 1989 ALAN R. LISS, INC

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Fig. 1. Conventional electron micrograph of control rat alveolar macrophage. Many lysosomes polymorphic in shape and size are seen in the cytoplasm. x 11,000.

agy and examine how cytoskeletal elements behave when lysosomes reveal dynamic changes in their shape by detecting the effects of cytoskeleton-affecting drugs. Moreover, the direct interaction of actin with lysosomes isolated from alveolar macrophages is investigated in order to corroborate their relationship in vitro.

Autophagy is defined as the intracellular activity by which the cell's own components are sequestered within membrane-bound vacuoles and degraded by lysosomal enzymes. Although a large number of investigations have been carried out so far concerning autophagy on various materials under various conditions morphologically and biochemically (see reviews; Glaumann et al., 1981; Hirsimaki et al., 1983; Seglen, 19871, the exact mechanism responsible for the regulation of autophagy remains to be clarified. In the second half of this paper, we will delineate the generally established concept of autophagy and some evidence related to the regulatory mechanism of autophagy, particularly focusing on the lysosomal wrapping mechanism (LWM), which is one theory we have proposed (Ogawa, 1981) to interpret how autophagic vacuoles form, in terms of the energy supply and the participation of the cytoskeleton in the autophagic process.

LYSOSOMAL MOVEMENT DURING HETEROPHAGY

Nematolysosome Induced by Pinocytosis In alveolar macrophages, lysosomes have an extraor-

dinary polymorphic structure in terms of shape, size, and internal structure (Fig. 1). The polymorphic struc-

ture of lysosomes seems to result from saltatory movements during heterophagy and/or autophagy. Recently we have observed saltatory lysosomal movements ac- companied by a characteristic transformation in alveolar macrophages, which were stimulated by fluid- phase pinocytosis or adsorptive pinocytosis, by the following methods (Araki and Ogawa, 1987b).

First, horseradish peroxidase (HRP) was employed as a fluid-phase pinocytic marker. Primary cultured alveolar macrophages harvested from lungs of Wistar rats were incubated with HRP at a dose of 1 mg/ml in the culture medium (Medium 199 containing 10% newborn calf serum) at 37" C for 20 min and then reincubated in the culture medium without HRP for 0-48 hr. In the second method, rat peroxidase- antiperoxidase immune complex (PAP) was employed as an adsorptive pinocytic marker. The cultured alveolar macrophages were exposed to PAP at a concentration of 0.25 mgiml in the culture medium or in phosphate-buffered saline (PBS) at 4" C for 30 min and then reincubated in the culture medium without PAP at 37" C for 0-120 min. These cells were fixed with 1% glutaraldehyde in cacodylate buffer, pH 7.4, containing 5% sucrose at room temperature (above 25" C) for 20 min. After rinsing with the buffered solution, peroxidase activities of HRP and PAP were detected by the method of Graham and Karnovsky (1966). For the cytochemical identification of lysosomes, acid phosphatase (AcPase) activity was detected by the lead-salt method of Gomori (1952). These specimens were dehydrated with graded ethanol series and embedded in Spurr's resin. The blocks of Spurr's resin were cut into

NEMATOLYSOSOME AND WRAPPING LYSOSOME 103

ultrathin sections and into 1-pm semithin sections. Ultrathin sections and 1-pm semithin sections were observed under electron microscopes (JEM lOOCX, JEM 1200EX, JEM 2000ES) at accelerating voltages to 100 kV or 200 kV.

HRP was taken up into various-size pinosomes in the macrophages by fluid-phase pinocytosis a few minutes after incubation with HRP at 37” C. Subsequently, many threadlike structures appeared in the cytoplasm and fused to pinosomes, so that the macrophages ac- quired many threadlike structures entirely filled with HRP (Fig. 2). Since the threadlike structures were cytochemically demonstrable for AcPase activity (Fig. 31, we termed the threadlike structures “nematolysosomes.” The nematolysosomes had varying diameters (0.1-0.5 pm) and were several pm in maximal length. The volume of lysosomal compartments filled with HRP was increased; in contrast, the pinosomes containing HRP decreased with a period of incubation in the HRP-free medium at 37” C for 20-60 min. This finding implies that nematolysosomes moving through the cytoplasm contribute to the digestion of exogenous solute materials which are pinocytozed into pinosomes as a heterophagic process.

The nematolysosomes could be induced in the cytoplasm of alveolar macrophages by adsorptive pinocytosis of PAP as well as fluid-phase pinocytosis of HRP. PAP was bound to the Fc receptors all along the plasma membranes of macrophages at 4” C (Fig. 4). When the cells were warmed to 37”C, the receptor-mediated pinocytosis started, and PAP was incorporated into pinosomes. Subsequent to the fusion of the pinosomes to lysosomes, a remarkable increment of nematolysosomes in the cytoplasm was observed (Figs. 5 , 6). The amount of nematolysosomes in the cells tended to increase with the incubation period for 20-60 min and then plateaued.

Recent studies by Swanson et al. (1987a,b) revealed that pinocytosis of the fluorescent dye lucifer yellow labeled elongated tubular organelles which could be identified aslysosomes by AFPase cytochemistry and immunof luorescence localization of catepsin L in thioglycollate-elicited mouse peritoneal macrophages and the macrophagelike cell line 5774.2. The tubular lysosomes mentioned in their reports must be identified as the same organelles as nemetolysosomes. Since the threadlike organelle is not a tubule, we recommend the use of the appropriate term nematolysosome. Similar elongated lysosomes have been observed in some types of cells: cultured smooth muscle cells and myocardial cells (Robinson et al., 1986; 19871, striated muscle cells (Okada et al., 19861, and pinealocytes (Krstic, 1985).

In exocrine acinar cells of pancreas, parotid gland, and lacrimal gland, basal elongated lysosomes not showing AcPase activity but showing trimetaphosphatase activity were observed by Oliver (1980, 1983). Beaudoin et al. (1984,1985) found “snake-like tubules” which were morphologically similar to basal elongated lysosomes in the exocrine pancreas. These lysosomes were also related to pinocytosis (Oliver, 1982) and distinguished from GERL (Golgi-associated ER from which Lysosomes form) by their differences in the cytochemical localization of enzyme activities (Beaudoin

et al., 1984,1985; Oliver, 1980,1983). In earlier studies on lysosomes in macrophages, similar lysosomal elements to nematolysosomes were regarded as a part of GERL (Essner and Haimes, 1977; Novikoff et al., 1981), because the extended definition of GERL could be applied to them with some morphological and cytochemical hallmarks: a delimiting membrane of thick variety and subjacent “halo” and demonstrable acid hydrolase. However, GERL is a term used to describe a hydrolase- rich structure located near the trans side of the Golgi saccule (Novikoff, 1964, 1976). From this viewpoint, nematolysosomes should not be categorized as GERL, because nematolysosomes were distributed not only near the trans side of Golgi apparatus but also throughout the cytoplasm in macrophages. It is not known whether nematolysosomes in macrophages and snake- like tubules or “basal elongated lysosomes” in exocrine acinar cells have the same character in their functions. But it appeared that both of their shapes of lysosomes resulted from their active dynamics of intracellular lysosomal movements related to heterophagy, especially pinocytosis, but not phagocytosis. It can be interpreted that the threadlike shape of lysosomes results from quick directional movements against the resistance of viscous cytoplasm.

Nematolysosomes and Cytoskeleton: Effects of Cytoskeleton-affecting Drugs on Lysosomal Movements

In general, the cytoskeletal system organized by microtubules, actin filaments, and intermediate filaments plays a role of supporting and moving of cells and cell components. It is thought that motile organelles including secretory granules might be driven through the cytoplasm by actin filaments and microtubules (Carr, 1972; Couchman and Rees, 1982; Fowler and Pollard, 1982; Freed and Lebowitz, 1970; Kachar et al., 1987; Vale, 1987; Vale et al., 1987). Therefore, it could be supposed that lysosomal translocation and transformation such as caused by nematolysosomes also might be affectd by the cytoskeletal system.

It is well known that actin filaments and microtubules are involved in the process of pinocytosis and phagocytosis. It is difficult to clarify the direct role of such cytoskeletal elements in lysosomal movements, because the lysosomal movement is a phenomenon subsequent to endocytosis. Phagocytosis taking up solid materials is inhibited by actin filament destabilizer or by an antimicrotubular drug, and fluid-phase pinocytosis is also partially inhibited by them. How- ever, it is said that adsorptive pinocytosis is little affected by the cytoskeletal elements (Allison, 1973; Allison et al., 1971; Silverstein et al., 1977; Wills et al., 1972). Therefore, in order to elucidate whether the cytoskeletal system directly participates in the regulation of the intracellular lysosomal movements during heterophagy, we have examined the effects of actin filament destabilizers and antimicrotubular drugs on the lysosomal movements, including nematolysosomes induced by adsorptive pinocytosis of PAP (Araki and Ogawa, 1987b).

The method which we used is as follows: cytochalasin B, cytochalasin D (actin filament destabilizers), and

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Fig. 2. Alveolar macrophages incubated with HRP (1 mgiml in Medium 199) for 30 min at 37°C. The cells were tested for peroxidase activity of HFtP by the DAB reaction. HRP is incorporated in pinosomes by fluid-phase pinocytosis. It can be noticed that numerous threadlike structures filled with reaction products of HRP were elongated in the cytoplasm. x 15,000.

Fig. 3. Cytochemical demonstration of acid phosphatase (AcPase) activity in alveolar macrophage incubated with HRP for 20 min at 37°C. The threadlike structures induced by fluid-phase pinocytosis of HRP demonstrate AcPase activity, which is a marker enzyme of lysosome. x 13,000.


Fig. 4. A surface portion of alveolar macrophage exposed to PAP (0.25 mgiml in PBS) at 4°C for 30 min. The cell was tested for peroxidase activity of PAP by the DAB reaction. PAP is localized all

nocodazole (an antimicrotubular drug) were dissolved in dimethyl sulfoxide (DMSO) at a concentration of 4 mM as stock solutions. The stock solutions of these drugs were diluted in the culture medium to a final concentration of 20 pM. Colchicine (an antimicrotubular drug) was directly dissolved in the culture medium at a concentration of 20 pM. The alveolar macrophages were preincubated in the medium containing the drugs for 30 min at 37" C before exposure to PAP at 4" C. The pretreated cells were exposed to PAP at 4" C and reincubated in the PAP-free medium at 37" C in the presence of the drugs. The influence of the drugs on pinocytic process and on subsequent lysosomal movements was examined by electron microscopy, compar- ing with the case in which the drugs were absent.

In the presence of 20 pM cytochalasin B or D, PAP was bound to Fc receptors all along the plasma membrane of the cells pretreated with the drugs. Then, 20 min after incubation at 37" C in the PAP-free medium, PAP was pinocytozed into the cells, so many PAP- positive pinosomes were seen in the cytoplasm (Fig. 7). This implied that the destruction of actin filaments scarcely altered adsorptive pinocytic activity at a morphological level. However, a t this time, nematolysosomes filled with PAP were not increased in the cytoplasm by the pinocytosis (Fig. 7). Cytochemical demonstration of AcPase also revealed that there was no increase of nematolysosomes in the cells which were in the presence of cytochalasins (Fig. 8). The PAP- positive pinosomes remained in the cytoplasm for a longer time than in the case in which the drugs were

along the surface of the plasma membrane. The localization of PAP indicates that PAP is bound to Fc receptor on the plasma membrane. x 28,000.

absent (Fig. 9). It was suggested that cytochalasins suppressed lysosomal movement and the formation of nematolysosomes and that this resulted in inhibited conveyance of PAP to lysosomes from pinosomes. From these findings, it follows that actin filaments are involved in intracellular lysosomal movements including the formation of nematolysosomes.

In the presence of antimicrotubular drugs, 20 pM colchicine or nocodazole, the binding of PAP to Fc receptor on the plasma membrane did not change. And these antimicrotubular drugs also showed little inhib- itory effect on the adsorptive pinocytosis of PAP. Many PAP-positive pinosomes were observed in the cytoplasm after incubation at 37" C; however, no nematolysosome was observed in the cytoplasm of the cells treated with antimicrotubular drugs (Figs. 10-12). The antimicrotubular drugs led to the disappearance of intracytoplasmic microtubules and nematolysosomes. This result strongly suggested that microtubules were required for the maintenance of nematolysosomes.

In addition, these antimicrotubular drugs led to another lysosomal transformation which could be clearly distinguished from nematolysosomes. When the cells were reincubated in the PAP-free medium at 37" C after exposure to PAP at 4" C in the presence of antimicrotubular drugs, though the nematolysosomes were absent, some lysosomes transformed into wrapping lysosomes, which segregated a part of cytoplasm, usually including some small lysosomes (Figs. 11, 12). PAP was not accumulated in the wrapping lysosomes (Fig. 11). The transformation of wrapping lysosomes

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Fig. 5. Alveolar macrophage reincubated in PAP-free medium for 20 min at 37°C after exposure to PAP at 4°C. The cell was tested for PAP. Threadlike lysosomes (nematolysosomes) filled with reaction products of PAP are observed in the cytoplasm. x 30,000.

Fig. 6. Alveolar macrophage reincubated in PAP-free medium for 30 min at 37°C after exposure to PAP at 4°C. The cell was tested for AcPase and observed under 200 kV TEM as a semithin section. Long nematolysosomes showing AcPase activity are evident in the cytoplasm. x 9,000.


Fig. 7. In the presence of 20 p M cytochalasin B, alveolar macrophages were reincubated in PAP-free medium at 37°C for 20 min after exposure to PAP. The cell was tested for PAP. PAP is free from the cell

surface and incorporated into pinosomes (PI by adsorptive pinocytosis. However, no appearance of nematolysosomes is noticed. x 11,000.

was caused by only antimicrotubular drug treatment without pinocytosis. These results suggest that the transformation of wrapping lysosomes is independent of pinocytosis and not related to heterophagy. The transformation of a lysosome wrapping other small lysosornes was mentioned as lysosomophagy (Thyberg et al., 1982a,b), i.e., a selective process of autophagy by LWM. In the presence of both actin filament destabilizer and antimicrotubular drug, the appearance of wrapping lysosomes tended to decrease. Lysosomal wrapping mechanism during autophagy will be described in detail later.

Our findings from electron microscopy on alveolar macrophages are summarized in Table 1. As shown in Table 1, two individual lysosomal transformations, i.e., nematolysosomes and wrapping lysosomes, were classified by using a pinocytic marker and cytoskeleton- affecting drugs. In other words, it follows that lysoso- ma1 movements could be controlled experimentally.

Much attention has been paid to the role of cytoskel- eta1 elements in the cell function by many cell biolo- gists. Actin filament destabilizers and antimicrotubular drugs have been widely used as tools for investigation of the role of actin filaments and microtubules in multiple cell functions such as cell locomotion, cell division or cytokinesis, endocytosis, cytoplasmic streaming, and secretion of cellular materials. Recently, it has become clear that intracellular orga-

nelle movement can also be caused by the motile machinery composed of actin filaments and microtubules. It is likely that each organelle movement is characterized by motile machinery which may be dif- ferentiated among the kinds of organelles and cell types. Kachar et al. (1987) have described at least two classes of motility machinery for moving cellular organelles. The first class, which is characteristically microtubule-based and supports directional movements, can be directly visualized in cultured epithelial cells, in squid giant axon, and in protozoans. The second class of motility mechanism characteristically utilizes actin filaments, promotes undirectional movements, and can be directly visualized in characean algae cells. Our investigation using actin filament destabilizers and antimicrotubular drugs suggested that the motility mechanism which regulated intracellular lysosomal movements in macrophages utilized both actin filaments and microtubules. The lysosomal movements in macrophages seem to be regulated by the complicated mechanism of both first and second classes. The translocation and transformation of nematolysosomes in macrophages could be regarded as directional movement and require the association of microtubules and actin filaments. The transformation of wrapping lysosomes is undirectional movement and does not require the association of microtubules, but does require that of actin filaments.

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Fig. 8. AcPase activity in the semithin section of alveolar macrophages reincubated in PAP-free medium a t 37°C for 20 min after exposure to PAP in the presence of 20pM cytochalasin D. Demonstra- tion of AcPase shows that lysosomes are roughly spherical in shape and there is no increment of nematolysosomes. x 12,000.

Fig. 9. Alveolar macrophages reincubated in PAP-free medium at 37°C for 60 min after exposure to PAP in the presence of 20 p M cytochalasin D. The cell was tested for PAP. The pinosomes containing PAP still remain in the cytoplasm. But nematolysosomes are scarcely seen in the cytoplasm. x 11,000,


Fig. 10. Alveolar macrophages reincubated in PAP-free medium at 37" C for 20 min after exposure t o PAP in the presence of 20 p M nocodazole. The cell was tested for PAP. PAP is incorporated into

Videofluorescence microscopic study by Matteoni and Kreis (1987) revealed that translocation and clustering of endosomes and lysosomes in normal rat kidney (NRK) cells were dependent on microtubules, because translocation of endosomes and lysosomes occurs along linear tracks by discontinuous saltations, but this movement ceases when the interphase microtubules are depolymerized by the treatment of the cells with nocodazole. This is in common with the movement of nematolysosomes in macrophages. However, in NRK cells, the movement remained unaffected when the cells were treated with cytochalasin D (Matteoni and Kreis, 1987). This discrepancy may be owing to differences in the kinds of lysosomal movement or cell types, because the lysosomes in macrophages behave with more complicated movements with various morphological transformations than those in NRK cells, so macrophages are specialized phagocytes and the most motile cells, which contain a lot of actin filaments in their cytoplasm. The lysosomal movements in macrophages need the complicated mechanism for their regulation in order to respond to their functions of heterophagy and autophagy.

In video-enhanced fluorescence microscopy, the col- lapse of intermediate filaments in NRK cells by microinjection with anti-vimentin left the lysosomal movement unchanged (Matteoni and Kreis, 1987). Collot et al. (1984) also suggested that intermediate filaments had no active role in lysosomal movements in cultured

pinosomes (PI. However, no appearance of nematolysosomes is observed. Wrapping lysosomes which are not filled with PAP are seen (arrows). x 12,000.

fibloblasts. However, possible association of intermediate (10 nm) filaments with lysosomes was assured by some investigators (Phaire-Washington et al., 1980). Further investigation by electron microscopy will be required for assurance of the involvement of intermediate filaments in the lysosomal movements.

In Situ Morphological Relationship of Cytoskeletal Elements With Lysosomes

Although the investigation using cytoskeleton- affecting drugs suggested the participation of cytoskeletal elements such as actin filaments and microtubules in the regulatory mechanism of intracellular lysosomal movements, i t does not provide direct evidence for the interaction of cytoskeletal elements with lysosomes, because the cytoskeleton-affecting drugs have some effect on not only cytoskeletal elements but also on many other cell metabolisms, which have not all been clarified (Treves et al., 1987; Zigmond and Hirsch, 1972). Therefore, in order to support the results obtained by using cytoskeleton-affecting drugs, we examined the morphological relationship between cytoskeletal elements and lysosomes in alveolar macrophages in in situ and in vitro interaction of actin filaments with isolated lysosomes by the following three methods (Araki and Ogawa, 1987~):

In situ observation in ultrathin section. For in situ observation in ultrathin sections of alveolar macrophages, conventionally the cells were fixed with

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Fig. 11. Conventional electron micrograph of wrapping lysosomes during lysosomophagg. The cell was reincubated in PAP-free medium at 37°C for 20 min after exposure to PAP in the presence of 20 KM nocodazole. The wrapping lysosomes encompass segments of cytoplasm including several small lysosomes. x 40,000.

Fig. 12. AcPase activity in the semithin section of the alveolar macrophage reincubated in PAP-free medium a t 37°C for 20 min af'Eer exposure to PAP in the presence of 20 pM nocodazole. Nematolyso- somes are not present, but wrapping lysosomes showing AcPase are noticed (arrows). x 18,000.


TABLE 1. Summary of the data on lysosomcd movements by PAP-pinocytosis and effect of cytoskeletul-affecting drugs'

Wrapping Nematolysosomes lysosomes

Control f-+ f--

PAP-pinocytosis - + + + Actin filament destabilizer

PAP-pinocytosis 2 i Antimicrotubular drugs

PAP-pinocytosis -

Actin filament destabilizer + antimicrotubular drug PAP-pinocytosis ~

+ +

+ 'Frequency of appeamnces: -, not present; 2 , rare; +, present; + +, frequently present; + + + , very frequently present.

2% glutaraldehyde in 0.1 M cacodylate buffer containing 5% sucrose, pH 7.4, for 10 min at room temperature. Also, a mixture containing 1 or 2% glutaraldehyde, 1% saponin, and 0.4% tannic acid in 0.1 M cacodylate buffer, pH 7.4, was employed for fixation with the special aim of visualizing cytoskeletal elements which are embedded in cytosol. Then these specimens were processed for routine electron microscopy and observed under a JEM 1OOCX or 1200EX.

In Situ Three-Dimensional Observation in Crit- ical Point-dried Cells. For the three-dimensional observation in situ, critical-point-dried whole-mount cells were prepared by the method reported previously (Araki and Ogawa, 1986,1987a). In brief, the alveolar macrophages harvested from rat lungs were immedi- ately cultured on Formvar-coated grids. The adhering cells on Formvar-coated grids were extracted with 0.1% Triton X or saponin in PHEM buffer composed of 60 mM PIPES, 25 mM HEPES, 10 mM EGTA, and 2 mM MgClz at pH 6.8 (Schliwa and Blerkom, 1981), for 1 min at room temperature. After the extraction, some specimens were incubated with heavy meromyosin (HMM) at a concentration of 1 mg proteidml in PHEM buffer for 10 min in order to identify actin filaments (Ishikawa et al., 1969). The cells were rinsed with the PHEM buffer and fixed with a mixture of 1% glutaraldehyde and 0.2% tannic acid in PHEM buffer for 3 min at room temperature. After fixation, the cells were rinsed well with the buffer and distilled water and postfixed with 1% Os04 for 5 min. Subsequently, the cells were dehydrated briefly in ethanol series, substi- tuted by isoamyl acetate, and critical-point dried with liquid COZ in a critical-point dryer (Hitachi, HCP-2). The cells whole-mounted on Formvar-coated grids were observed under a high-voltage electron microscope (Hitachi H-l250M, National Institute for Physiological Science, Okazaki) operating at 1,000 kV. Some of the whole-mount cells were cleaved with a freon gas spray by the dry-blowing method (Araki and Ogawa, 1986, 1987a) and observed under a JEM 1OOCX electron microscope operating at 100 kV.

Negative-stained Electron Microscopy of In Vi- tro Interaction of Actin Filaments With Lysos- omes. Crude lysosomal fraction was prepared from alveolar macrophages by means of differential centrif- ugation. The solution used to isolate lysosomes was 0.05 M Tris-buffered 0.25 M sucrose solution with

protease inhibitors, 5 pM leupeptin, 5 pM pepstatin, and 1 mM phenylmethylsulfonyl fluoride (PMSF). G- actin (from rabbit skeletal muscle, Sigma Chemical Co.) was resuspended at a concentration of 2 mg/ml in a depolymerization buffer composed of 2 mM Tris, 0.2 mM ATP, 0.5 mM mercaptoethanol, and 0.2 mM CaClz at pH 8.0 (Spudich and Watt, 1971). Various concentrations of isolated lysosomes (ca. 0.01-0.5 mg pro- teiniml) were mixed with G-actin solution to give a final actin concentration of 0.1-0.5 mg proteidml. For polymerizing to F-actin from G-actin, a high-concentration salt actin polymerization buffer with protease inhibitors was added to give final concentrations of 50 mM KC1, 20 mM HEPES, 1 mM ATP, 2 mM MgC12, and 5 mM EGTA at pH 6.8 (Mehrabian et al., 1984). The mixture of actin and isolated lysosomes was then incubated at 37" C for 30 min and applied, negative- stained by 1% uranyl acetate, on Formvar-coated grids for electron microscopy. In some specimens, F-actin was decorated with HMM on the Formvar-coated grid before negative staining in order to examine the polarity of the filaments.

By observation of ultrathin sections of macrophages, it was found that some microtubules run along the nematolysosomes (Fig. 13). In the cells fixed with a mixture containing 1% glutaraldehyde, 1% saponin, and 0.4% tannic acid, many filamentous elements were clearly visualized in the cytoplasm of control alveolar macrophages (Fig. 14) and pinocytosis-activated macrophages (Fig. 15). Some of them were located in the vicinity of lysosomes (Figs. 14, 15). The filamentous elements located near lysosomes had a diameter of 5-10 nm, so it was difficult to differentiate actin filaments and intermediate filaments in the ultrathin section. Presumably, both kinds of filaments might exist.

In the critical-point-dried cells, a well-organized cytoskeletal system was observed three dimensionally all over the cytoplasm of macrophages (Fig. 16), and most of the filamentous elements located in the vicinity of lysosomes were identified as actin filaments which could be decorated with HMM, although a few intermediate filaments not decorated with HMM were also recognized (Fig. 17).

In negative-stained electron microscopy of in vitro cell-free experimentation, F-actin filaments were found to be preferentially assembled onto lysosomal membranes when actin was mixed with isolated lysosomes. The actin filaments contacted lysosomal membranes along the side of filaments rather than at the end. The polarity of actin filaments was not uniform against the lysosomal membrane (Fig. 18).

Our morphological observation revealed that microtubules were closely associated with nematolysosomes; similar results have been obtained by immunof luorescence microscopy of antitubulin and anticatepsin L, showing close correspondence between the orientation of lysosomes and microtubules, and by electron microscopy of lysosomes labeled by pinocytosis of microper- oxidase and microtubules in macrophages (Swanson et al., 198713). These morphological findings suggest that the microtubules are required for the maintenance of the nematoform of lysosomes and guide the directional movement of nematolysosomes.

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Fig. 13. Morphological viewing of the association of microtubules (arrows) with nematolysosome. The cell was reincubated in PAP-free

medium at 37" C for 20 min and processed for conventional electron microscopy. x 50,000.

In other cell types, association of microtubules with cytoplasmic organelles such as mitochondria has been reported in many light and electron microscopic studies (Carr, 1972; Couchman and Rees, 1982; Stebbings and Hunt, 1987). It is likely that some motile types of cytoplasmic organelles including lysosomes were guided by microtubules which provide support for the movement of organelles in the cytoplasm. It was conceivable that cross-bridge structures, which might be responsible for mutual binding of microtubules and organelles moving along the microtubules, might be present a t the gap between them (Kachar et al., 1987). The cross-bridge structures may be translocators such as dyneinlike molecules (Forman et al., 1984) or kinesin (Vale, 1987; Vale et al., 1985), which are force- generating proteins involved in microtubule-based motility. Although we have not identified the cross-bridge structure between microtubules and lysosomes, there is a great possibility that lysosomal movements associated with microtubules are also mediated by such translocators.

In the matter of actin filaments and lysosomal movements, our findings in alveolar macrophages in in situ observation showed that some actin filaments were located in the vicinity of lysosomes. An earlier study by Moore et al. (1976) also demonstrated a morphologically close relationship of some filamentous elements to lysosomes in polymorphonuclear leukocytes. Most of the filamentous elements located in the vicinity of

lysosomes could be regarded as actin filaments by their diameter (5-8 nm) and HMM decoration, although a few intermediate (10 nm) filaments were also present. Our findings in in vitro experimentation, which demonstrated that F-actin filaments were attached to isolated lysosomes in the cell-free condition, suggest that actin filaments located in the vicinity of lysosomes interact with the lysosomal membrane and may par- ticipate directly in the regulation of lysosomal movements in alveolar macrophages. These morphological observations of the relationship between actin filaments and lysosomes support the findings in the experimentation using actin filament destabilizers.

In nonmuscle cells, the cell motility-such as cell locomotion, endocytosis, and intracytoplasmic movements-accounted for the gel-sol transformation of actin molecules (Stossel and Hartwig, 1976; Stossel et al., 1981, 1985). It is generally considered that the gel-sol transformation of actin is mediated by actin- associated proteins. Several kinds of actin-associated proteins such as gelsolin (Yin and Stossel, 1980; Yin et al., 19811, a-actinin (Bennett et al., 19841, actin- binding protein (Hartwig and Stossel, 19751, and actin- bundling protein (Pacaud, 1986; Pacaud and Harri- cane, 1987) were isolated from the cytoplasm of macrophages and characterized. Some of these actin- associated proteins were considered to be involved in the gel-sol transformation of actin in the cytoplasm of macrophages (Hartwig and Stossel, 1975; Pacaud,


Fig. 14. Control alveolar macrophage fixed with 1% glutaral- Fig. 15. Association of filamentous elements with BSA-gold- labeled nematolysosomes (N). The cells were incubated in the medium containing BSA-gold at 37°C for 40 min and fixed with 1% glutaraldehyde, 1% saponin, and 0.4% tannic acid. It is difficult to distinguish actin filaments from intermediate filaments by their diameter. x 70.000.

dehyde, 1% saponin, and 0.4% tannic acid. Many filamentous elements can be seen in the cytoplasm. It can be noticed that some of the filaments (arrows) are closely associated with lysosomes (L). X 45,000.

114 M. SAKAI ET AL.

Fig. 16. High-voltage (1,000 kV) electron micrograph of whole- The basal potion of control alveolar macrophage prepared mount preparation of control alveolar macrophages. The three- by the dry-blowing method. The cell was incubated with HMM to dimensional association of filamentous elements with electron-dense identify actin filaments. Most of filamentous elements associated with granules, which are presumably lysosomes, can be observed. lysosome (L) show arrowhead structures with HMM decoration. x 35,000. x 37,000.

Fig. 17.


Fig. 18. Negative-stained electron microscopy of the interaction a d i n filaments with isolated lysosomes in vitro. The actin filaments are decorated with HMM, which indicates the polarity of the fila-

ments. It is found that actin filaments can be attached to lysosomal membrane along the side of filaments, and the polarity of the filaments (arrowheads) is not uniform. x 40,000.

1986; Pacaud and Harricane, 1987; Stossel and Hartwig, 1976; Stossel et al., 1981, 1985; Yin and Stossel, 1980; Yin et al., 1980). We speculate that some actin- associated protein which promotes mutual interaction of actin filaments with lysosomal membranes mhy be present between lysosome and actin, and among lactin filaments, although we have not been able to obberve the structure of an actin-associated protein by elebtron microscopy. Moore et al. (1976) observed cross-lidking short filaments between lysosomes and actin filaments in polymorphonuclear leukocytes. Furthermore, a biochemical study by Mehrabian et al. (1984) revealed that increase of the viscosity could be measured by using a falling ball viscometer when the isolated lysosomal membranes of liver were combined with actin. These results also strongly suggest the existence of actin-associated protein on the lysosomal membranes.

The interaction of actin with actin-associated proteins was reported to be sensitive to intracellular calcium ions, which are a second messenger on many cell functions (Bennett et al., 1984; Pacaud, 1986; Pacaud and Harricane, 1987; Stossel et al., 1985; Yin and Stossel, 1980; Yin et al., 1980). Since the intracellular concentration of calcium ion was connected with the ligand-receptor interaction and endocytosis (Young et al., 19841, the change of calcium ion concentration at

micromolar level might play a role as a signal for regulation of the lysosomal movement following endocytosis during heterophagy. The association of calmodulin, which is a known mediator of calcium action in many cell functions, with lysosomes in macrophages was observed by immunof luorescence microscopy, and it suggested a role for calcium ions in lysosomal function (Nielsen et al., 1987). The location of calmodulin on lysosomes may be also involved in the regulation of lysosomal function. From these findings, it can be speculated that lysosomal movements may be regulated by a complicated mechanism which consists of cytoskeletal elements (microtubules, actin filaments), cytoskeleton-associated protein, calcium ion calmodulin, etc., t o be translocated and transformed variously in response to the lysosomal function.

LYSOSOMAL MOVEMENT DURING AUTOPHAGY

General Aspects Autophagy is one of the important processes for

intracellular metabolism of many endogenous substances through the lysosomal compartment, although the existence of non-autophagic or nonlysosomal path- ways for protein degradation with cytosolic or membrane bound enzymes has been generally accepted in

116 M. SAKAI ET AL.

various cell types (see reviews; Ciechanover, 1987; Dice, 1987; Pontremoli and Melloni, 1986).

The representative structure of the autophagic process, designated autophagic vacuoles (AVs), constitutes membrane-bound organelles containing cytoplasmic components structurally intact or in various stages of disintegration. The term AVs is generally used in ultrastructural studies when it is not necessary or is impossible to define the maturation stage of the digestive vacuoles. In contradistinction, vacuoles, which have newly sequestered contents and do not yet contain hydrolases within their matrices, are called autophagosomes (De Duve and Wattiaux, 1966). Autophago- somes are subsequently fused with pre-existing primary or secondary lysosomes, resulting in the formation of autophagolysosomes or autolysosomes, and their contents become digested by lysosomal enzymes. As degradation proceeds, products of low molecular weight diffuse out of the autophagolysosome into cytosol, but larger degradation products or indigestible materials will accumulate in lysosomes, giving them the characteristic appearance of residual bodies or lipofuscin-containing granules (Brunk and Ericsson, 1972; Collins et al., 1980).

However, the number of morphologically detectable autophagosomes, even of autophagolysosomes, is usually small under physiological cell conditions, although it varies among different cell types. This may reflect the extremely short half-life of the AVs (8-10 min) compared with other cell organelles (days) (Mortimore and Schworer, 1977; Pfeifer, 1978). Therefore, many morphological investigations of autophagy have been performed under autophagy-induced conditions by various methods. The methods to induce autophagy morphologically seem to be classified in two divisions, depending on whether they can act on either the first (i.e., AV formation stage) or second step (i.e., fusion and degradation stage) of the autophagic process. That is to say, autophagy can be induced first by increasing intracellular substances destined for sequestration, which is observed during not only starvation (Ohshita et al., 1986; Pfeifer, 19731, cell injury (Odessey, 19871, and remoding of organs (Ericsson, 1969a,b) but also under the administration of some hormones such as glucagon (Deter, 1971; Schworer and Mortimore, 1979), and second, by interfering in the fusion of autophagosomes and lysosomes or by impairing the lysosomal enzyme activities within the AVs, which are caused by the administration of microtubular inhibitors-e.g., vinblastine (Kovacs et al., 1982)-1yso- somotropic weak bases-e.g., chloroquine (Gray et al., 1981)-and inhibitors of lysosomal enzymes-e.g., leupeptin (Furuno et al., 1982).

It should nevertheless be mentioned that the exact meaning of the induction of autophagy is applicable to only the first method and not to the second one, because although the appearance of AVs in various stages definitely increases morphologically with the second method, autophagy is conversely inhibited biochemically, judging from the protein degradation rate in the cell.

On the contrary, inhibitors of autophagy, such as insulin (Pfeifer, 1978), amino acids (Schworer et al.,

19811, and cycloheximide (Kovacs, 19831, have been also used in combination with quantitative electron microscopy t o clarify the kinetics of cellular autophagy. As pointed out above, a half-life for AVs on the order of 8-10 min has been calculated from decay studies of AV volume fraction after the treatment with various inhibitors.

Although lysosomal digestion through the autophagic process correlates with degradation and release of proteins, nucleic acids, carbohydrates, and lipids, as can be measured in a biological assay system, most biochemical work on autophagy is related to the proteolysis in the liver from rats either as a perfused organ or as isolated cells (Grinde, 1985). Several reviews are available for detailed information about the relationship between autophagy and protein degradation (Kovacs and Rez, 1979; Marzella and Glaumann, 1987; Seglen, 1987). In addition to the assay method for proteolysis associated with autophagy, a recently de- vised autophagy bioassay method based on electropermealization as a means of making hepatocytes tran- siently permeable to radiolabelled sufars-e.g., (14C)sucrose (Gordon and Seglen, 19821, ( 4C)lactose (Hflyvik et al., 1986), and (3H)raffinose (Seglen et al., 1986)-has the potential possibility of clarifying the control mechanism of the autophagic process from several viewpoints-e.g., estimating the turnover rate of soluble cytosol materials or studying the existence of selective uptake by autophagy.

The Origin of the AV Membrane The initial step of the autophagic process is the

sequestration of the cell’s own cytoplasmic constituents by an enclosing membrane, resulting in the formation of an autophagosome which frequently consists of two membrane sheets (Fig. 19) a t a very early stage (Ar- stila and Trump, 1968; Hirsimaki et al., 1975). Exam- ination of the nature, the structure, and the origin of the segregating membrane of the AV has been a major object of considerable effort in morphological work on autophagy in the past and has proposed two possible sources of AV membrane material: 1) membrane formation of de novo synthesis (Ashford and Porter, 1962; Pfeifer, 1971) and 2) the utilization of preexisting cytoplasmic membrane.

The preexisting membrane theory generally seems now to be accepted by most investigators rather than the de novo formation theory, based on evidence that autophagy can be successfully induced even when protein synthesis is nearly perfectly blocked by puromycin (Kovacs and Rez, 19791, implying the indepen- dence of specific protein synthesis on AV membrane formation. However, the possible existence of de novo formation of the AV membrane is undeniable because the preexisting membrane theory does not seem to allow any clear-cut interpretation as to the exact origin of the AV membrane.

Based on a large number of investigations using techniques of thin section electron microscopy, enzyme cytochemistry, osmium impregnation, and cell frac- tionations, several precursors for the AV membrane have been proposed (Fig. 20): 1) rough-surfaced endoplasmic reticulum after degranulation (Helminen and


2 3 5 6 7 8 1

20 Fig. 19. The early stage of two autophagic vacuoles (autophaogo-

somes) containing the fragments of rER. Note some part of the AV membrane consists of two membrane sheets (arrows). AV, autophagic vacuole. x 40,000.

Fig. 20. Schematic drawing of several origins of AV membrane, such as 1) rough surfaced ER, 2) smooth-surfaced ER, 3) Golgi complex, 4) GERL, 5) plasma membrane, 6) vesicles or vacuoles, 7) lysosomes, and 8) de novo synthesis.

118 M. SAKAI ET AL.

Ericsson, 1971; Rhz et al., 1976), 2) smooth-surfaced endoplasmic reticulum (Arstila and Trump, 1969; Erics- son, 196913; Hirsimaki and Reunanen, 1980) 3) Golgi complex (Frank and Christensen, 1968; Locke and Sykes, 1975) 4) GERL (Novikoff et al., 1971; Paavola, 1971), 5) plasma membrane (Quatacker, 1971) 6) vesicles and vacuoles (Moe and Behnke, 19621, and 7) lysosomes (Hamberg et al., 1977; Mayahara and Ogawa, 1972; Thyberg et al., 1982a,b).

Up to now, most reports favor the ER and Golgi apparatus membrane as the origin of the autophagoso- ma1 membrane from evidence such as the direct conti- nuity of ER membrane with the outer bounding membrane of the AV, and the osmium-staining common property of the AV membrane with ER or Golgi apparatus membrane.

Nevertheless, it has been proposed, on the bases of freeze replica studies of autophagy (Hirsimaki and Hirsimaki, 1982; Rez and Meldolesi, 19801, that nei- ther ER nor Golgi apparatus membrane is suggestive of an immediate origin of the AV membrane with regard to the density of intramembrane particles. Fur- thermore, Reunanen et al. (1985) reported that the content of unsaturated fatty acid in AV membrane is higher than that of other cellular membranes by the imidazole-buffered osmium tetroxide impregnation method. These results suggest a possibility that a preexisting membrane which is destined to form the AV membrane must change its structure or nature during the formation of the AV membrane.

As a means of assessing this possibility, we recently performed an investigation to clarify whether there are any detectable changes in AV membrane or in the membrane which is just becoming AV membrane in normal or autophagy-induced rat or mouse liver cells from the viewpoint of distribution of electric charges on AV membranes (Sakai et al., 1987).

The livers from normal rats or mice were fixed by perfusion with a mixture of 2% paraformaldehyde and 0.25% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) for 10 min at room temperature. In some cases, autophagy was induced in hepatocytes by intraperito- neal injection of vinblastine (VBL; 50 mgikg body weight) in animals 2 hr prior to sacrificing. Fixed small tissue blocks (ca. 1 mm3) were immersed in 2.3 M sucrose solution and rapidly frozen with liquid nitro- gen. Frozen thin sections 90-100 nm thick were prepared by using techniques of cryomicrotomy. Sections recovered on Formvar-coated copper grids using the method of Tokuyasu (Tokuyasu, 1973) were thoroughly washed to remove sucrose with PBS (phosphate- buffered saline, pH 7.4) and incubated with cationized or anionized ferritin (50-100 pgiml PBS) for 30 min at room temperature. After washing with PBS several times again, sections were refixed with 2% glutaraldehyde in 0.1 M phosphate buffer for 10 min. Sections were then stained with 2% uranyl acetate, embedded in a mixture of 0.2% methylcellulose and 2% polyethylene glycol, and examined under JEM 1OOCX or 1200EX electron microscopy.

On sections incubated with cationized ferritin, ferritin particles were observed throughout the cytoplasm and on the outer surface of the plasma membrane.

However, lysosomes and AVs revealed a denser distribution than other cellular organelles. Particularly, the inner surface of the AV membrane showed a high binding affinity to cationized ferritin compared with other intracellular membranes (Fig. 21). On the contrary, anionized ferritin showed no distinct dense distribution either on lysosomes or AVs.

These results suggest that if the AV membrane were to originate from preexisting membranes, such membranes must somehow intensify their negative electric charges. Further efforts will be required to obtain a clear answer to the question of how negative charges are produced on the precursor membranes during AV membrane formation.

One-step or Two-step Autophagic Uptake

Since the lysosomal enzymes-e.g., acid phosphatase-cannot usually be detected enzyme cytochemically either on the boundary membrane of autophagosomes or in the segregated cytoplasmic portion, the transformation from nonenzyme-containing AVS to autolysosomes must occur by the fusion of preexisting primary or secondary lysosomes (Arstila and Trump, 1968; Pfeifer, 1976). Thus, the well-known two-step mechanism of performing the autophagic process is sometimes referred to as “classic autophagy” or “mac- roautophagy” to distinguish it from other mechanisms of autophagic uptake such as the so-called “LWM” (Mayahara and Ogawa, 1972; Ogawa, 1981), “microautophagy” (Ahlberg et al., 1982; Marzella et al., 1980), or “lysosomophagy” (Thyberg et al., 1982a,b). These latter notions represent a segregating structure with preexisting lysosomes forming protrusions or invagination from their membranes. Since this mechanism implies that lysosomal enzymes can be supplied directly from the cavity of the surrounding lysosomal membranes, it can be defined as a one-step uptake of cytoplasmic materials by lysosomes.

As shown in Figure 20 (No. 7, light line), microautophagy can be applied to the sequestration of mostly cytosolic small components (glycogen, ribosome, soluble protein, etc.) by an invagination of the surface membrane of a spherical lysosome, and lysosomophagy is defined as the special lysosomal activity in which excess lysosomes caused by treatment of macrophages with nocodazole (an antimicrotubular drug) are sequestrated by the protrusion or invagination of other lysosomes to regulate the number of lysosomes in the cell. However, in our proposed LWM it is possible to take up not only a small part of the cytosolic components in a similar manner to microautophagy but also cellular organelles including lysosomes which are usually thought to be catabolized through classic autophagy. We regard both microautophagy and lysosomophagy as types of the LWM. The LWM in detail and the regulatory factors of the LWM will be discussed in the next section.

LWM The LWM implies a process in which the cell’s own

cytoplasmic components are directly enwrapped by preexisting primary or secondary lysosomes which


Fig. 21. Cationized ferritin-labelled autophagic vacuole (AV) from a frozen thin section of mouse hepatocyte. Many ferritin particles are

observed mainly on the inner surface of the AV membrane (arrows). MT, mitochondrion. ~49,000.

change their structure from spherical to elongated or form tail- or armlike extensions or invaginations from their membrane, resulting in the direct formation of autophagosomes bound by two membranes containing hydrolases in the intermembrane space (Fig. 22). After sealing sequestered materials perfectly with their membrane, lysosomal enzymes can be directly ac- quired, possibly by the degeneration of their inner membrane (Fig. 23). Following complete digestion or catabolism of sequestered cytoplasmic materials, autophagolysosomes return to the original round lysosomes.

The LWM was initially proposed as the possible one-step pathway of the autophagic process during the uptake and translocation of exogenous HRP in mouse histiocytes (Mayahara, 1972; Mayahara and Ogawa, 1972) and has been developed in various tissues under various conditions as follows: 1) liver cells after administration of glucagon (Saito and Ogawa, 19741, or CPIB (ethyl-p-chlorophenoxy-isobutyrate) (Matsushita et al., 1982), and of cyclic AMP (Abe and Ogawa, 1980), 2) Yoshida ascites cells treated with cortisone acetate in viva and in vitro (Abe and Ogawa, 1976), 3) splenic macrophage from y-ray-irradiated rat (Piao et al., 1983), 4) mouse histiocytes after ovalbumin uptake (Sakai and Ogawa, 1982), 5) macrophages from rat thymus (Piao and Ogawa, 1985), and 6 ) pinealocytes of rats (KrstiC, 1985).

We set up the in vitro model system which could easily induce the LWM in mouse histiocytes after administration of 1% ovalbumin (OA) in order to

examine the regulatory condition with regard to energy dependency and the participation of the cytoskeleton in the LWM.

Histiocytes, which reside in the abdominal subcutaneous connective tissue of the mouse, usually reveal a spindle shape similar to that of fibroblasts without any stimulation. Lysosomes in histiocytes are in fairly large number and show mostly a spherical structure. Acid phosphatase (AcPase, one of the marker enzymes of lysosomes) activity was cytochemically demonstrated in these lysosomes by the method of Gomori (Gomori, 1952) in order to trace the lysosomal movements responding to various stimulation.

After OA subcutaneous injection (5-10 mid , most histiocytes a t the site of injection suddenly transformed their shape from spindle to spherical and various- shaped lysosomes showing intense AcPase activity appeared in the cytoplasm (Fig. 24). The following shapes of lysosomes could be observed during OA uptake: an elongated and curved lysosome (Fig. 25a), a horse’s-hoof-shaped lysosome (Fig. 25b), a curved dumb- bell-shaped lysosome (Fig. 25c), a ring-shaped lysosome containing less electron-dense cytoplasmic area fragments of ER or mitochondria within the segregated space (Fig. 25d), a trifold ring-shaped lysosome (Fig. 25e), and a spherical or oval-shaped lysosome having invagination or various sizes of vacuoles (Fig. 25b,f). These unique appearances of the lysosomal structure were ones of thin-sectional phases of the wrapping lysosomes, which was clarified by the observation of a series of thin sections (Ogawa et al., 1984).

120 M. SAKAI ET AL.

Fig. 22. Several stages of the lysosomal wrapping are observed in a histiocyte. AcPase reaction. Arrows show the beginning of a mapping pathway. An arrowhead indicates the partial enwrapping of some part of the cytoplasmic area, and complete wrapping is pointed out by double arrows. X27,OOO.

Fig. 23. A lysosome (lys) completely encloses some part of the cytoplasmic area. Note the inner lysosomal membrane partially degenerates (arrows) and probably lysosomal enzymes are supplied to the matrix of AV from there. x 37,000.


Fig. 24. Five minutes after ovalbumin (OA) injection in mouse histiocytes. AcPase reaction. A histiocyte which has changed its

structure from spindle to spherical has many wrapping lysosomes (arrows). X 30,000.

These irregularly shaped lysosomes ceased to be prominent more than 30 min after OA injection by forming large round lysosomes in which could be detected intense AcPase activity and probably containing both exogenous (OA) and endogenous (cytoplasmic components) materials within their matrices (Fig. 26). Although it is not known why the LWM can be induced during OA uptake in histiocytes, we took advantage of the fairly rapid cycles of the LWM in the histiocyte system to investigate the control factors of the LWM.

Energy Dependency of the LWM It is generally accepted from early in vitro studies

that metabolic energy from ATP is necessary for autophagy to complete its process (Shelbure et al., 1973) and that autophagic protein degradation is also an energy- dependent process (Hopgood et al., 1977). However, as these previous studies do not supply any information about the steps of the autophagic pathway that require ATP, the investigation of the ATP dependency on the lysosomal movements associated with the LWM was performed by using inhibitors of ATP synthesis on the histiocyte system.

Pretreatment of histiocytes with either an inhibitor of glycolysis (2-deoxyglucose) or oxidative phosphorylation (sodium azide or 2-4 dinitrophenol) failed to prevent the occurrence of the LWM induced following OA injection, implying that the LWM has a low affinity for ATP levels in the cell if the ATP supply is partially

cut off by either inhibitor pretreatment. That is to say, many wrapping lysosomes showing intense AcPase activity were observed in histiocytes (Fig. 27). How- ever, when ATP supply was nearly completely blocked by the pretreatment of histiocytes with both inhibitors together, the LMW could no longer be induced following OA injection. Interestingly, the number of lysosomes showing intense AcPase activity decreased, and most spherical lysosomes showed very weak or no activity (Fig. 28). It was also observed that no nematolysosomes appeared at all in histiocytes pretreated with both inhibitors together. These results indicated that lysosomal movements associated with the LWM (the formation of autophagolysosome in this case) and with nematolysosomes formation require ATP as an energy source.

Plomp et al. (1987) recently reported, by using a (14C)sucrose loading method, by electropermealization that ATP is required at least for two steps in the autophagic pathway: the sequestration step and the operation of the lysosomal proton pump. Their results correspond well with ours with respect to the ATP- dependent sequestration step (i.e., autophagosomes formation) in the autophagic pathway, although it is not known whether loaded ('4C)sucrose in cytoplasm could be autophagocytosed through the LWM. It is very interesting that ATP depletion could suppress the autophagic process by impairing the function of the lysosomal membrane proton pump, which works to

122 M. SAKAI ET AL.

Fig. 25. Several irregularly shaped lysosomes being involved in the LWM. AcPase reaction. See the text for explanation. a, x 52,000; b, x 30,000; c, x 47,000; d, x 46,000; e, x 56,000; f, x 43,000.


Fig. 26. Thirty minutes after OA injection in histiocyte. AcPase Fig. 27. reaction. Large round lysosomes, which probably contain both endogenous and exogenous materials, are prominent instead of wrapping lysosomes. x 48,000. x 38,000.

Five minutes after OA injection which was done 30 min after initial injection with sodium azide (an inhibitor of oxidative phosphorylation). AcPase reaction. Wrapping lysosomes are observed.

124 M. SAKAI ET AL.

Fig. 28. Five minutes after OA injection which was done 30 min after injection with a mixture of 2-4-dinitrophenol and 2-deoxyglucose. AcPase reaction. Most lysosomes reveal round shapes and very weak or no AcPase activity (arrows). x 24,000.

Fig. 29. Five minutes after OA injection which was done 30 min after the initial cytochalasin B injection. AcPase reaction. Wrapping lysosomes are not observed. x 32,000.


Fig. 30. Wrapping lysosomes (lys) from 5 min aRer OA injection in saponin-treated histiocytes. The direct connection of the wrapping lysosomes and microfilaments can be detected (arrows). x 93,000.

maintain the intralysosomal acid pH, because in our experiment, lysosomes showing very weak or no AcP- ase activity in histiocytes under completely ATP- deficient conditions may reflect the increasing intralysosomal pH.

In addition to direct utilization of ATP by the lysosomal membrane proton pump, ATP may be used to support the lysosomal movement by other organelles such as cytoskeletal elements. As described earlier concerning the relationship between the cytoskeleton and lysosomal movement during heterophagy, microtubules or actin filaments are necessary mainly for directional or undirectional lysosomal movements, respectively. However, considered from the viewpoint of ATP utilization, myosin filaments which have ATPase activity are the most conceivable candidate for cooperating with actin to support the lysosomal movement. In nonmuscle cells it is nearly impossible to distinguish myosin filaments from other filaments by pure morphological observation, but the demonstration of Ca+ +

ATPase activity at the optimal condition for the detec- tion of myosin ATPase on adjacent cytoplasmic area to wrapping lysosomes (Ogawa et al., 1984) may support the notion that myosin filaments are involved in the lysosomal movement.

In conclusion, during the autophagic process ATP is

consumed as an energy source for, first, the sequestration step of both classic autophagy and the LWM, and, second, the degradation step, by maintaining optimal intralysosomal conditions for lysosomal enzymes.

Cytoskeleton and the LWM As already described earlier, cytoskeletal elements,

particularly microtubules and actin filaments, elabo- rately manipulate the intracellular lysosomal movements during heterophagy. It is a growing question whether the lysosomal movement related to the LWM is also regulated by cytoskeletal elements in the same manner as in the case of heterophagy. In order to answer the question, the effects of various cytoskeleton- affecting drugs on the LWM induced by OA in histiocytes were investigated.

Pretreatment of an actin filament destabilizer (cytochalasin B) or an actin filament stabilizer (phalloidin) could successfully inhibit the occurrence of wrapping lysosomes following OA injection in histiocytes (Fig. 29). This result was supported by the demonstration of a direct connection between actinlike filaments and wrapping lysosomes in saponin-treated histiocytes (Fig. 30). Taking into consideration the clear evidence for the ments in vivo and in vitro (Araki and Ogawa, 1987b1, we conclude that intact actin filaments are surely neces-

126 M. SAKAI ET AL.

Fig. 31. Five minutes after OA injection which was done 30 min after the initial injection of colchicine. AcPase reaction. Wrapping lysosomes are observed and some lysosomes are wrapped by other lysosomes (arrows). x 44,000.

Fig. 32. Five minutes after vinblastine injection only. AcPase reaction. Many wrapping lysosomes are observed, indicating that antimicrotubule drugs can induce the LWM by themselves. ~40,000.


Fig. 33. Thirty minutes after paraquat administration in histio- being wrapped by a lysosome. ~51,000. b: A mitochondrion is cytes. AcPase reaction. a: A damaged mitochondrion (an arrow) is completely wrapped by a ring-shaped lysosome. x 78,000.

sary for the intracellular lysosomal movements related to both wrapping lysosomes and nematolysosomes.

However) there seems to be a contrary opinion for actin filament dependency of the lysosomal movement in the case of microautophagy, based on the evidence that isolated intact lysosomes from liver were able to take up exogenously added protein by forming invagination and vesicles from their membranes in vitro (Ahlberg et al., 1982; Marzella et al., 1980). This discrepancy may come from the differences in cell types. As macrophages, including histiocytes, have well-developed cytoskeletal system to adapt themselves to their transformation or to their function of heterophagy and autophagy, the movement of intracellular organelles is most likely ruled by them in a very strict and complicated manner. It is likely that the cytoskeletal system in the cell, which has a fixed shape and low or no phaogocyotic activity, may act less on the movement of organelles. Another possible idea to explain the discrepancy is that the uptake of soluble or small-sized materials by the invagination of the lyso- soma1 membrane may be caused by the change of lysosomal membrane fluidity in in vivo condition, but that dynamic changes of lysosomal shape for wrapping of bulk material such as mitochondria) ER, and other lysosomes through the LWM in vivo is actin-filament- dependent process.

Pretreatment with so-called antimicrotubular drugs (colchicine or nocodazole) did not reveal any preventive

effects on the occurrence of the wrapping lysosome induced following OA injection in histiocytes (Fig. 31). On the contrary, these drugs induced the LWM by themselves without OA treatment (Fig. 32), coinciding with other evidence such as microautophagy induced by vinblastine in glial cells (Hamberg and Edman, 1983) and lysosomophagy by nocadazole in macrophages (Thyberg et al., 1982a,b). The characteristic appearance of lysosomes wrapping other small lysosomes was often observed in histiocytes treated with antimicrotubular drugs (Fig. 31).

The exact mechanism by which autophagy is induced by antimicrotubular drug treatments in various cells is not yet fully understood. It has been found that the LWM as well as classic autophagy could be induced by these drugs (Sakai and Ogawa, 1984; Hirsimaki et al, 1975). In the case of classic autophagy, the induction of autophagy (increase of AVs) can possibly be explained by the prevention of fusion between lysosomes and autophagosomes. This notion could not be applied in the case of the LWM for interpretation of the autophagy- inducing mechanism, because, as described above, the LWM is a one-step autophagic uptake and it is unnecessary to fuse preexisting lysosomes. Kovacs and Rez (1979) suggested another interesting theory for induction of classic autophagy by antimicrotubular drugs in which vinblastine exerts its autophagy-promoting effect through a breakdown of polyribosomes leading, to labilization of the ER with subsequent formation of

128 M. SAKAI ET AL.

(extrace? 1111 ar )

endocytosis

(extracell u l a r )

34

AUTOPHAGY

AL : amhilysosome AP : aolophagosme APL: autophagolysosome HP : heterophagosome HPL: heterophagolysosome LCY: large coated vesicle LWM: lysosomal wrapping mechanism MA : microautophagy NL : nmatolysosome PL : primary l y s o s m RB : residual body SCV: small coated vesicle

Fig. 34. A scheme showing changes of lysosomes during heterophagy and autophagy (modified from Ogawa, 1981).

sequestration membranes. This conceivable direct effect of vinblastine on membrane structure, which was supported from other evidence that vinblastine could induce membrane invagination in human erythrocytes (Ben-Bassat et al., 19721, may help explain the induction mechanism of the lysosomal wrapping by antimicrotubular drugs.

In addition to possible direct effects of antimicrotubular drugs on lysosomal membranes, it is tempting to speculate that since the lysosomal movements in macrophages are controlled by a complicated mechanism probably cooperating with microtubules and actin filaments, disorganization of microtubules in the cell may change the condition of the cytoplasmic area adjacent to lysosomes, where lysosomes easily reveal their wrapping transformation. Although this idea is far from being proved, the fact that under cytoskeleton- free conditions lysosomes isolated from liver without any treatment reveal very irregular shapes, similar to those observed in the case of microautophagy, may support this possibility indirectly.

In any case, antimicrotubular drugs may supply more appropriate conditions for wrapping transformation to lysosomes by affecting somehow both lysosomal membranes and their environment. However, the control of wrapping lysosomes by actin filaments seems to predominate over that by microtubules.

Other Aspects of the LWM

As described in the section The Origin of the AV Membrane, it has been generally accepted that protein synthesis is unnecessary for AV formation, particularly in the classic autophagic pathway. A similar result on the relationship between protein synthesis and the LWM was reported in an early study (Maya- hara, 1972). That is to say, the pretreatment of histiocytes with puromycin, an inhibitor of protein synthesis, revealed no preventive effects on the occurrence of the LWM following HRP injection. This result seems to be reasonable because as lysosomal wrapping is a fairly rapid phenomenon in the cell, newly synthesized protein may not be necessary to the occurrence of the LWM. However, it is worthwhile to reconfirm the relation of the lysosomal movement with protein synthesis by using an in vivo system in terms of not only protein synthesis and the LWM but also protein synthesis and nematolysosome formation.

Another interesting approach for the study of autophagy is to make a thorough search for the presence of the recognition mechanism. Although classic autophagy is believed to be a nonselective process (Glaumann et al., 1981), there is some evidence available suggesting the selectivity of the LWM, e.g., specific lysosomal uptake of 1) CPIB-induced peroxisome (Matsushita et


al., 1982),2) damaged mitochondria by paraquat (Fig. 33a,b) (Ogawa et al., 19841, and 3) excess lysosomes by nocadazole (Thyberg et al., 1982a,b).

Nevertheless, as there is currently no information about initiation factors of autophagy morphologically, e.g., structural modification, further experiments are necessary to answer the essential questions about autophagy by using newly developed methods such as microinjection or electroshock loading of labelled materials into the cell.

CONCLUSIONS In both heterophagic processes and autophagic pro-

cesses, lysosomes reveal dynamic changes in regard to their intracellular translocation and transformation (Fig. 34). Nematolysosomes and wrapping lysosomes could be regarded as typical function-related configu- rations representing heterophagy and autophagy respectively. In this review, we conclude that both the formation of nematolysosomes during heterophagy and the LWM during autophagy are a consequence of the modulation by the cytoskeletal system. In this sense, the cytoskeleton can be considered functionally the cytolocomotor system. Each of the intracellular lysosomal movements during heterophagy and autophagy have individual modes of regulatory mechanism complicated by the lysosome-cytoskeleton interaction. Thus, it can be said that the lysosome is not merely a sac containing hydrolase but a systematic organelle which has the ability to move with specific functional significance and recognize substances to be digested. This concept is only the beginning of studying the regulatory mechanism of the lysosomal function. These morphological observations may become an important clue to the understanding of hitherto-unknown lysoso- ma1 functions.

ACKNOWLEDGMENTS This research was supported by a Grant-in-Aid for

Special Project Research, No. 60127010, from the Min- istry of Education, Science and Culture, Japanese Government.

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Lysosomal movements during heterophagy and autophagy: With special reference to nematolysosome and wrapping lysosome