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The association of hemidesmosome-like plaque and dense coating with the pinocytic uptake of a heterologous fibrillar protein (amyloid) by macrophages
© 1970 by Academic Press, Inc. J. ULTRASTRUCTURE RESEARCH
33, 587-597 (1970)
587
The Association of Hemidesmosome-like Plaque and Dense Coating with the Pinocytic Uptake of a Heterologous Fibrillar Protein (Amyloid) by Macrophages 1 TSURANOBU SHIRAHAMAAND ALAN S. COHEN
The Arthritis and Connective Tissue Disease Section of the Evans Department of Clinical Research, University Hospital, and the Department of Medicine, Boston University School of Medicine, Boston University Medical Center, Boston, Massachusetts 02118 Received April 27, 1970 Mouse peritoneal macrophages incubated in vitro with isolated human amyloid fibrils not only phagocytized the fibrils, but also demonstrated a hemidesmosome-like structure on the contact surface with the amyloid. The extracellular amyloid fibrils attached closely to the cell surface at the hemidesmosome-like structure, and seemed to be attracted to the small plasmalemmal pit which often formed at that site. Occasionally the pit was deep and had a narrow stalk or neck. The hemidesmosome-like plaque associated with a deep plasmalemmal pit was usually less electron dense than that with the plasma membrane with a shallow indentation or none, and spike-like structures appeared to be present in it. In the cytoplasm near the surface abutting the amyloid, coated vesicles were frequently observed, some containing amyloid-like fibrillar material. From these findings we have proposed a sequential series of events leading to the formation of the coated vesicles from the hemidesmosome-like structures, which then participate in the uptake of amyloid fibrils. A number of electron microscopic studies of the pinocytic vesicles (2, 3, 13, 19, 20, 24) as well as of hemidesmosomes (lO, 12, 13, 16) have clearly defined their ultrastructure and provided information leading to an understanding of their biological significance. Macrophages from a variety of sources have also been studied extensively by electron microscopy (4, 6-9, 11, 22, 23, 32), and coated vesicles have been described as a common cytoplasmic element in the macrophages (2, 6, 11) as in other cells. Although much circumstantial evidence is available, the sequence of formation and the function of the coated vesicles has not been clearly understood. In our studies on the phagocytosis of amyloid, mouse peritoneal macrophages incubated with isolated human amyloid fibrils not only phagocytized the fibrils (31) 1 Grants in support of these investigations have been received from the United States Public Health Service, National Institutes of Arthritis and Metabolic Diseases, Grants Nos. AM-04599 and T1-AM5385, from the Arthritis Foundation, and the Massachusetts Chapter of the Arthritis Foundation.
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but also on occasion demonstrated a peculiar hemidesmosome-like structure on the contact surface with the extracellular amyloid. Detailed observations have strongly suggested the participation of the hemidesmosome-like device in the "pinocytic" uptake of the extracellular substance (amyloid fibrils) and the formation of the coated vesicles. This communication reports ultrastructural aspects of the pinocytosis associated with the hemidesmosome-like plaque and sequential formation of the coated vesicles.
MATERIALS AND METHODS The present materials and methods were comparable to those published in detail elsewhere (31). For the present analysis, however, several hundred new electron micrographs were added to the data previously used. In brief, with the intention of stimulating macrophages (6, 8) 0.5 ml of 10 % casein solution in 0.05 N sodium hydroxide was injected intraperitioneally into each of several C3H mice. Three or four days after the injection, the peritoneal exudate cells were washed out with 5 ml of Hank's balanced salt solution, and then spun down at 500 g for 5 minutes in a Servall refrigerated centrifuge. Amyloid fibrils were isolated from human amyloidotic spleens by a sucrose gradient centrifugation method (5, 28), lyophilized, and stored at -10°C prior to use. The cells obtained from mice (mainly macrophages) were then suspended in tissue culture medium 199 containing 20 % calf serum with untreated or Congo red stained amyloid, at a concentration of approximately 2 × 106 cells with 0.5 mg amyloid (dry weight) per 1 ml of the medium. They were incubated at 37 ° for 0, 10, 20, 30, 60, 120 or 360 minutes while covered by air containing 5 % carbon dioxide. As controls, the cells were incubated alone or with latex particles in the same fashion. After incubation, the samples were collected by centrifugation at 500 g for 5 minutes, fixed with 2 % osmium tetroxide in phosphate buffer (21), dehydrated in graded ethanols, and embedded in Epon (18). Sections were cut on an LKB Ultrotome, stained with uranyl acetate (34) and lead citrate (26), and examined in a Siemens Elmiskop I at initial magnifications of 1 000-80 000 ×. RESULTS General cytological details of the macrophage structure (whether before or after incubation with or without amyloid) were comparable to those previously reported by m a n y investigators (4, 6-9, 11, 22, 23, 32). The macrophages incubated with amyloid gave evidence that they phagocytized the amyloid fibrils. Attachment, infolding, and ingestion could be sequentially observed. The amyloid-containing phagocytic vacuoles were more frequent in samples of the Congo red stained amyloid, and in specimens incubated longer (31). By low power electron microscopy, portions of the plasma membrane of the macrophages were on occasion observed to be thicker and have greater electron
HEMIDESMOSOME-L1KE PLAQUE AND COATED VESICLES
589
Fro. 1. After 120 minutes of incubation, accumulations of dense material (arrows) are seen in the peripheral cytoplasm close to the plasma membrane on the surface contacting the extracellular amyloid fibrils (Am). These densities are observed at fairly regular intervals, and measure 300500 ~ in width (including the plasma membrane) and 2 000-8 000 ~, in length. Coated vesicles (arrow heads) are frequent in the peripheral cytoplasm near these plasmalemmal thickenings. The contact between two macrophages (Mac) is very close (open arrow), x 20 000. FIG. 2. As noted in Fig. 1. Extracellular amyloid fibrils (Am) closely approximate the plasma membrane at the sites of the membrane thickening. A portion of the plasma membrane associated with the hemidesmosome-like plaque forms a deep pit (open arrow). × 20 000.
density o n the contact surface with amyloid. Closer observation of the micrographs of m o d e r a t e m a g n i f i c a t i o n revealed that the local increased thickness a n d density of the m e m b r a n e was due to a n intracytoplasmic a c c u m u l a t i o n of dense material close to the p l a s m a m e m b r a n e . A t the involved portions, the dense area measured 300-500 N i n thickness (including the p l a s m a m e m b r a n e ) a n d was 2 000-8 000 A in length. The p l a s m a m e m b r a n e often showed a shallow or deep i n d e n t a t i o n at the site
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(Figs. 1 a n d 2). The areas of m e m b r a n e thickening were on occasion f o u n d at a l m o s t r e g u l a r intervals (Figs. 1 a n d 2). I n the extracellular space, a m y l o i d fibrils were generally thin ( a b o u t 100 A thick), rigid, n o n b r a n c h i n g , of i n d e t e r m i n a t e length, a n d a r r a n g e d in r a n d o m array. A t the site of this specific m e m b r a n e structure, the a m y l o i d fibrils were often s o m e w h a t c o n d e n s e d in their d i s t r i b u t i o n a n d closer to the p l a s m a l e m m a (Fig. 2). W h e n the p l a s m a l e m m a l i n d e n t a t i o n was very deep, a g r o u p of a m y l o i d fibrils seemed to be p u l l e d into the pit a n d to be a r r a n g e d in p a r a l l e l to each o t h e r at the neck of the pit (Figs. 2-4). O n occasion the neck of the pit was very n a r r o w a n d a l m o s t p i n c h e d off (Figs. 2-4, 10, a n d 11). T h e c o a t e d vesicles were o b s e r v e d in the c y t o p l a s m n e a r the site of the m e m b r a n e t h i c k e n i n g m o r e frequently t h a n elsewhere in the c y t o p l a s m . T h e y were a b o u t 7 0 0 2 500 A in diameter, a n d their c y t o p l a s m i c surface was c o a t e d b y a 200-400 A t h i c k c o r o n a of dense m a t e r i a l (Figs. 1-3, 5, 7, a n d 13). Some of t h e m a p p e a r e d to a t t a c h to the p l a s m a m e m b r a n e (Fig. 11). I n these c o a t e d vesicles, fibrillar structures of d i a m e t e r c o m p a r a b l e with the a m y l o i d fibrils were occasionally n o t e d (Figs. 5 a n d 13). A n a l y s i s of high p o w e r electron m i c r o g r a p h s of these structures led to the following findings. T h e specific m e m b r a n e structure associated with dense p l a q u e a n d the vesicles c o a t e d b y dense c o r o n a are p r o b a b l y of the same derivation. T h e p l a s m a
FIG. 3. Higher magnification of a portion of the area shown in Fig. 2. The plasma membrane forms a deep pit (at the right upper corner) at the site associated in part with the dense plaque (D). Amyloid fibrils are primarily in the extracellular space (Am) and also can be followed within the plasmalemmal pit. At the narrow opening of the pit, amyloid fibrils are almost in parallel with one another. Coated (CV) and uncoated vesicles (V) also are seen. The corona of the coated vesicle in the deeper cytoplasm (at the right bottom comer) clearly shows "spikes." Scattered free ribosomes, smooth endoplasmie reticulum (SER) and cytoplasmic filaments are also seen. × 60 000. FIG. 4. A deep plasmalemmal pit with a relatively long, narrow neck is associated with the dense plaque (D) at its base. Extraeellular amyloid fibrils (Am) are arranged in parallel array at the opening of the pit and can be followed to its bottom. This finding gives the impression that the amyloid fibrils fixed at the hemidesmosome-like area have been "pulled" into the plasmalemmal pit as it has formed. R: free ribosomes, RER: rough endoplasmic reticulum, x 60 000. FI6. 5. A coated vesicle in the peripheral cytoplasm contains fibrillar material whose dimensions are comparable to those of the amyloid fibrils. Am: extracellular amyloid fibrils, R: free ribosomes. x 60 000. FIG. 6. High power electron micrograph showing a portion of the border between a macrophage (Mac) and amyloid (Am), after 120 minute incubation. A hemidesmosome-like structure (D) is observed in part in cross section. Dense homogeneous material accumulates on the cytoplasmic side of the plasma membrane and forms a thinner band close to the plasma membrane, an electron lucent space (arrows), and a thicker band apart from the plasma membrane. This structure is distinct from the plasma membrane cut in cross (C) and oblique (O) sections. The portion surrounded by dotted lines is seen at higher magnification in the inset, x 160 000. Inset: Higher magnification of a hemidesmosome-like structure. The plasma membrane is distinct and consists of the outer dense layer (a), the intermediate electron lucent space (b), and the inner dense layer (c). A narrow band of the dense homogeneous material (30-60/~ thick) (e) is seen close to the inner dense layer of the plasma membrane (c) without appreciable space (d). Separated by an electron lucent space (50-150 A wide) (f), the additional dense material forms a thicker band (150200 ~ thick) (g). Thin dense strands are seen to bridge the outer thin and the inner thick bands (arrows). × 500 000.
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FIG. 7. High power electron micrograph of a coated vesicle in the peripheral cytoplasm. The limiting membrane is 70-90 A thick and shows features of the unit membrane. The dense coating is 200300 A in thickness. Although the coating has a comparable structure with the dense hemidesmosomelike plaque, the strands perpendicular to the limiting membrane (or the bridges between the outer thin and the inner thick bands) are prominent while the outer thin and the inner thick bands are barely seen. The perpendicular strands (arrows) are arranged at fairly regular intervals, x 200 000.
Flas. 8-13. These electron micrographs show a possible sequence of transition from the hemidesmosome-like structure to the coated vesicle. PM, plasma membrane; AM, extracellular amyloid fibrils. x 140 000. FIG. 8. A hemidesmosome-like plaque associated with a portion of the plasma membrane with shallow indentation. F~G. 9. A small, moderately deep indentation of the plasma membrane associated with the dense plaque. Fro. 10. A deep plasmalemmal pit and a shallow indentation with the dense plaque adjoin one another. The opening of the deep pit is very narrow and the opposing membranes at its neck seen to be about to fuse. FIG. 11. A slightly indented plasma membrane and a vesiclecoated by dense homogeneous material are connected by a membranous structure at one point. This micrograph again suggests that a newly formed coated vesicle is pinched off. FIG. 12. A coated vesicle in the peripheral cytoplasm is connected with the plasma membrane by a long membranous structure surrounded by dense homogeneous material (arrow). FIG. 13. A coated vesicle in the peripheral cytoplasm with no apparent connection with the plasma membrane. In the dense corona of the vesicle, spike-like structures are prominent. In the coated vesicle fibrillar structures comparable to the amyloid fibrils are indicated (arrows).
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SHIRAHAMA AND COHEN
membrane of the area or the limiting membrane of the vesicle is about 70-90 A thick and consists of outer and inner dense layers separated by an intermediate electron lucent space. Close to the inner layer of the membrane (with no appreciable space), dense homogeneous material forms a very thin layer (30-60 A in thickness) (this will be referred as "the outer thin band"). Separated from the outer thin band by an electron lucent layer (50-150 A wide), the dense material forms an another thicker band of about 150-200 A thickness ("the inner thick band"). Thin (50-100 A) dense strands bridge the outer thin and the inner thick bands. The strands usually stand perpendicularly to the plasma or the limiting membrane and are often arranged at fairly regular (150-250 A) intervals (Figs. 6 and 7). In the plaques where the plasma membrane showed no or only a shallow indentation, the outer thin and the inner thick bands were very to moderately dense and more predominant than the perpendicular strands (Figs. 6, 8, and 9). At the deep plasmalemmal pit, density of the bands seemed slightly decreased (Figs. 3 and 10). In the corona of the coated vesicles, the bands were often less electron dense and thinner, so that the perpendicular strands were relatively prominent and often showed the features that could be termed "spikes" (2) (Figs. 3, 7, and 13). The transition between the membrane structure associated with the plaque and the coated vesicle has been suggested by (a) flask-shaped plasmalemmal pit connected to the outer space with a relatively long, narrow opening (Figs. 3, 4, and 10), (b) apparent fusion of the apposing plasmalemma at the neck of the pit (Fig. 11), and (c) coated vesicles seemingly connected to the plasma membrane by a strand of the dense material (Fig. 12). The above findings were found primarily in the samples incubated longer than 30 minutes. With this longer incubation, macrophages often came into close contact with each other, a finding rarely observed with shorter incubation times. On occasion the structure of a tight junction or desmosome was suggestive at the contact sites though we could not obtain the typical micrograph of one in the present study. The presently described membrane structure associated with the dense plaque was not observed on the surface of the macrophages with no contact with amyloid or in controls. DISCUSSION Peripheral cytoplasmic accumulation of dense material in close relation to extracellular microfibrils has been described on many occasions. Porter (25) has observed indistinctness of portions of the plasma membrane associated with cytoplasmic dense accumulations in fibroblasts in intimate relation to extracellular collagen microfibrils. In amyloidotic tissues, the intimate structural relationship of a group of well oriented amyloid fibrils, indistinct plasma membrane and peripheral cytoplasmic
HEMIDESMOSOME-LIKE PLAQUE AND COATED VESICLES
595
accumulation of dense material has also been found in a variety of cells, and has been interpreted as the possible site of amyloid formation (29, 30, 33). A similar structural combination has been observed where the lymphatic anchoring filaments approximate the lymphatic endothelium (17, 30). Leak and Burke have stated that some of these dense areas resembled the hemidesmosomes of the basal epithelial layer of the epidermis (17). Since the ultimate significance of these comparable, but poorly understood morphologic observations is not known, it is difficult to define interrelations or precisely to relate the present observation to them. While many similarities exist among those structures, the present structure differs from the previous ones in several ways; i.e., the experimental conditions, the distinctness of the plasma membrane, the uniformity in the size of the structure, the apparent relation to the coated vesicles. The plasma membrane thickening herein described resembles the hemidesmosome (10, 12, 13, 16) in many respects; i.e., the localization, the form and the size of the dense plaque, their uniformity and fairly regular arrangement along the plasma membrane, the distinctness of the plasma membrane. However, the following differences do exist: (a) the present structure has been found in macrophages, while typical hemidesmosomes have been reported only in epithelial cells, (b) the classical hemidesmosomes have faced the basement membrane while this structure has occurred on the cell surface in contact with amyloid and without a basement membrane, and (c) associated cytoplasmic filaments have not been seen in our material, but are usually found with the hemidesmosomes. Under the present experimental conditions it is an unlikely possibility that synthesis of amyloid by macrophages plays a major role, and the present results are most likely interpreted as follows. The hemidesmosome is widely accepted as the attachment device to the extracellular elements, and the hemidesmosome-like structure of the present study presumably has a similar function. The macrophage facing the extracellular amyloid mass forms the hemidesmosome-like plaque and attaches to the amyloid fibrils at the site. As the plasmalemmal indentation occurs locally, a group of amyloid fibrils attaching to it are pulled into the pit and separated from the main amyloid mass. The plasmalemmal pit eventually forms a coated vesicle after the plasma membrane fuses at the neck of the pit. In the sequence of the formation of the coated vesicle and perhaps after the coated vesicle is formed, the dense material forming the outer thin and the inner thick bands of the plaque or the corona becomes less and less apparent. A few observations somewhat comparable to the present study have been previously published. Berry and Friend (1) have observed that hemidesmosome-coutaining areas of the cell membrane invaginate and appear to pinch off and migrate centrally when rat liver parenchymal cells have been isolated. They also have found plaques similar to the hemidesmosomes but less dense on vacuoles deep within the cells and on
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multivesicular bodies, and have interpreted them as the probable precursors of the hemidesmosome plaques. Similar observations have also been made by Gona (15) in the resorbing tadpole tail fin where "free" hemidesmosomes have formed vesicles containing globular layer material. In the usual process of formation or discharge of the coated vesicles, the plasma membrane structure associated with a dense plaque such as described in the present study has not been observed. However, observations similar to the present study have been reported on the oocytes in the process of taking up the yolk protein (13, 27). The short spine-like projections coating the pinocytic vesicles in the cytoplasm have also attached to the portions of the plasma membrane where the condensation of yolk protein has been observed on the external surface of the cell membrane. Friend and Farquhar have also reported similar observations during protein (exogenous peroxidase) absorption in the rat vas deferens (14).
Finally, we have reported here the formation of hemidesmosome-like plaque in macrophages in contact with a heterologous fibrillar protein (amyloid) in vitro. This structure has more points of resemblance to the hemidesmosome than to any other known structure, and can be interpreted as an attachment device for the extracellular amyloid fibrils. The site has become indented and eventually formed a coated vesicle. This process has probably subserved the uptake of the amyloid fibrils or a substance closely related to them. If one accepts the above findings and interpretation, our observations may provide additional information leading to a better understanding of the modes of formation of and the nature of the hemidesmosomes and the coated vesicles, as well as the nature of amyloid.
REFERENCES 1. BERRY, M. N. and FRIEND, D. S., J. Cell Biol. 43, 506 (1969). 2. BRUNS, R. R. and PALADE,G. E., J. Cell Biol. 37, 244 (1968). 3. - ibid. 37, 277 (1968). 4. CARR, I., Z. Zellforsch. Mikrosk. Anat. 80, 534 (1967). 5. COHEN,A. S., Lab. Invest. 15, 66 (1966). 6. COHN, Z. A., Advan. Immunol. 9, 163 (1968). 7. COHN, Z. A., FEDORKO, M. E. and HIRSCH, J. G., J. Exp. Med. 123, 757 (1966). 8. COHN, Z. A., HmSCH, J. G. and FEDORKO, M. E., J. Exp. Med. 123, 747 (1966). 9. DEPETRIS,S., KARLSBAD,G. and PERMS, B., J. Ultrastruct. Res. 7, 39 (1962). 10. DOUaLAS, W. H. J., RrPLEY, R. C. and ELLIS, R. A., J. CellBiol. 44, 211 (1970). 11. DUMONT,A., J. Ultrastruet. Res. 29, 191 (1969). 12. FARQUHAR, M. G. and PALADE,G. E., J. Cell Biol. 26, 263 (1965). 13. FAWCETT, D. W., The Cell. Its Organelles and Inclusions. Saunders, Philadelphia, Pensylvania, 1966. 14. FRtEND, D. S. and FARQUHAR, M. G., J. Cell Biol. 35, 357 (1967).
HEMIDESMOSOME-LIKEPLAQUE AND COATED VESICLES 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34.
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GONA, A. G., Y. Ultrastruct. Res. 30, 103 (1970). KELLY, D. E., J. Cell Biol. 28, 51 (1966). LEAK, L. V. and BURKE, J. F., Y. Cell Biol. 36, 129 (1968). L~JrT, J. H., J. Biophys. Biochern. Cytol. 9, 409 (1961). LUFT, J. H., The Inflammatory Process, p. 121. Academic Press, New York, 1965. MAJNO, G., Handbook of Physiology, Section 2, p. 2293. American Physiological Society, Washington, D.C., 1965. MILLONIG, G., J. Appl. Phys. 32, 1637 (1961). NORTH, R. J., J. Ultrastruet. Res. 16, 83 (1966), -ibid. 16, 96 (1966). PALADE,G. E. and BRUNS, R. R., J. Cell Biol. 37, 633 (1968). PORTER, K. R., Biophys. J. 4. Suppl., 167 (1964). REYNOLDS,E. S., J. Cell Biol. 17, 208 (1963). ROTH, T. F. and PORTER, K. R., J. Cell Biol. 20, 313 (1964). SHmAHAMA,T. and COHEN, A. S., J. Cell Biol. 33, 679 (1967). -Amer. J. Pathol. 51, 869 (1967). - - - - Exp. Mol. Pathol. 11, 300 (1969). SHIRAHAMA,T., COHEN,A. S. and RODGERS,0. G., submitted for publication. SUTTON, J. S. and WEISS, L., aT. Cell Biol. 28, 303 (1966). TRUMP, B. F. and BENDITT, E. P., Lab. Invest. 11, 753 (1962). WATSON, M. L., aT. Biophys. Biochem. Cytol. 4, 475 (1958).
33, 587-597 (1970)
587
The Association of Hemidesmosome-like Plaque and Dense Coating with the Pinocytic Uptake of a Heterologous Fibrillar Protein (Amyloid) by Macrophages 1 TSURANOBU SHIRAHAMAAND ALAN S. COHEN
The Arthritis and Connective Tissue Disease Section of the Evans Department of Clinical Research, University Hospital, and the Department of Medicine, Boston University School of Medicine, Boston University Medical Center, Boston, Massachusetts 02118 Received April 27, 1970 Mouse peritoneal macrophages incubated in vitro with isolated human amyloid fibrils not only phagocytized the fibrils, but also demonstrated a hemidesmosome-like structure on the contact surface with the amyloid. The extracellular amyloid fibrils attached closely to the cell surface at the hemidesmosome-like structure, and seemed to be attracted to the small plasmalemmal pit which often formed at that site. Occasionally the pit was deep and had a narrow stalk or neck. The hemidesmosome-like plaque associated with a deep plasmalemmal pit was usually less electron dense than that with the plasma membrane with a shallow indentation or none, and spike-like structures appeared to be present in it. In the cytoplasm near the surface abutting the amyloid, coated vesicles were frequently observed, some containing amyloid-like fibrillar material. From these findings we have proposed a sequential series of events leading to the formation of the coated vesicles from the hemidesmosome-like structures, which then participate in the uptake of amyloid fibrils. A number of electron microscopic studies of the pinocytic vesicles (2, 3, 13, 19, 20, 24) as well as of hemidesmosomes (lO, 12, 13, 16) have clearly defined their ultrastructure and provided information leading to an understanding of their biological significance. Macrophages from a variety of sources have also been studied extensively by electron microscopy (4, 6-9, 11, 22, 23, 32), and coated vesicles have been described as a common cytoplasmic element in the macrophages (2, 6, 11) as in other cells. Although much circumstantial evidence is available, the sequence of formation and the function of the coated vesicles has not been clearly understood. In our studies on the phagocytosis of amyloid, mouse peritoneal macrophages incubated with isolated human amyloid fibrils not only phagocytized the fibrils (31) 1 Grants in support of these investigations have been received from the United States Public Health Service, National Institutes of Arthritis and Metabolic Diseases, Grants Nos. AM-04599 and T1-AM5385, from the Arthritis Foundation, and the Massachusetts Chapter of the Arthritis Foundation.
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SHIRAHAMA AND COHEN
but also on occasion demonstrated a peculiar hemidesmosome-like structure on the contact surface with the extracellular amyloid. Detailed observations have strongly suggested the participation of the hemidesmosome-like device in the "pinocytic" uptake of the extracellular substance (amyloid fibrils) and the formation of the coated vesicles. This communication reports ultrastructural aspects of the pinocytosis associated with the hemidesmosome-like plaque and sequential formation of the coated vesicles.
MATERIALS AND METHODS The present materials and methods were comparable to those published in detail elsewhere (31). For the present analysis, however, several hundred new electron micrographs were added to the data previously used. In brief, with the intention of stimulating macrophages (6, 8) 0.5 ml of 10 % casein solution in 0.05 N sodium hydroxide was injected intraperitioneally into each of several C3H mice. Three or four days after the injection, the peritoneal exudate cells were washed out with 5 ml of Hank's balanced salt solution, and then spun down at 500 g for 5 minutes in a Servall refrigerated centrifuge. Amyloid fibrils were isolated from human amyloidotic spleens by a sucrose gradient centrifugation method (5, 28), lyophilized, and stored at -10°C prior to use. The cells obtained from mice (mainly macrophages) were then suspended in tissue culture medium 199 containing 20 % calf serum with untreated or Congo red stained amyloid, at a concentration of approximately 2 × 106 cells with 0.5 mg amyloid (dry weight) per 1 ml of the medium. They were incubated at 37 ° for 0, 10, 20, 30, 60, 120 or 360 minutes while covered by air containing 5 % carbon dioxide. As controls, the cells were incubated alone or with latex particles in the same fashion. After incubation, the samples were collected by centrifugation at 500 g for 5 minutes, fixed with 2 % osmium tetroxide in phosphate buffer (21), dehydrated in graded ethanols, and embedded in Epon (18). Sections were cut on an LKB Ultrotome, stained with uranyl acetate (34) and lead citrate (26), and examined in a Siemens Elmiskop I at initial magnifications of 1 000-80 000 ×. RESULTS General cytological details of the macrophage structure (whether before or after incubation with or without amyloid) were comparable to those previously reported by m a n y investigators (4, 6-9, 11, 22, 23, 32). The macrophages incubated with amyloid gave evidence that they phagocytized the amyloid fibrils. Attachment, infolding, and ingestion could be sequentially observed. The amyloid-containing phagocytic vacuoles were more frequent in samples of the Congo red stained amyloid, and in specimens incubated longer (31). By low power electron microscopy, portions of the plasma membrane of the macrophages were on occasion observed to be thicker and have greater electron
HEMIDESMOSOME-L1KE PLAQUE AND COATED VESICLES
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Fro. 1. After 120 minutes of incubation, accumulations of dense material (arrows) are seen in the peripheral cytoplasm close to the plasma membrane on the surface contacting the extracellular amyloid fibrils (Am). These densities are observed at fairly regular intervals, and measure 300500 ~ in width (including the plasma membrane) and 2 000-8 000 ~, in length. Coated vesicles (arrow heads) are frequent in the peripheral cytoplasm near these plasmalemmal thickenings. The contact between two macrophages (Mac) is very close (open arrow), x 20 000. FIG. 2. As noted in Fig. 1. Extracellular amyloid fibrils (Am) closely approximate the plasma membrane at the sites of the membrane thickening. A portion of the plasma membrane associated with the hemidesmosome-like plaque forms a deep pit (open arrow). × 20 000.
density o n the contact surface with amyloid. Closer observation of the micrographs of m o d e r a t e m a g n i f i c a t i o n revealed that the local increased thickness a n d density of the m e m b r a n e was due to a n intracytoplasmic a c c u m u l a t i o n of dense material close to the p l a s m a m e m b r a n e . A t the involved portions, the dense area measured 300-500 N i n thickness (including the p l a s m a m e m b r a n e ) a n d was 2 000-8 000 A in length. The p l a s m a m e m b r a n e often showed a shallow or deep i n d e n t a t i o n at the site
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(Figs. 1 a n d 2). The areas of m e m b r a n e thickening were on occasion f o u n d at a l m o s t r e g u l a r intervals (Figs. 1 a n d 2). I n the extracellular space, a m y l o i d fibrils were generally thin ( a b o u t 100 A thick), rigid, n o n b r a n c h i n g , of i n d e t e r m i n a t e length, a n d a r r a n g e d in r a n d o m array. A t the site of this specific m e m b r a n e structure, the a m y l o i d fibrils were often s o m e w h a t c o n d e n s e d in their d i s t r i b u t i o n a n d closer to the p l a s m a l e m m a (Fig. 2). W h e n the p l a s m a l e m m a l i n d e n t a t i o n was very deep, a g r o u p of a m y l o i d fibrils seemed to be p u l l e d into the pit a n d to be a r r a n g e d in p a r a l l e l to each o t h e r at the neck of the pit (Figs. 2-4). O n occasion the neck of the pit was very n a r r o w a n d a l m o s t p i n c h e d off (Figs. 2-4, 10, a n d 11). T h e c o a t e d vesicles were o b s e r v e d in the c y t o p l a s m n e a r the site of the m e m b r a n e t h i c k e n i n g m o r e frequently t h a n elsewhere in the c y t o p l a s m . T h e y were a b o u t 7 0 0 2 500 A in diameter, a n d their c y t o p l a s m i c surface was c o a t e d b y a 200-400 A t h i c k c o r o n a of dense m a t e r i a l (Figs. 1-3, 5, 7, a n d 13). Some of t h e m a p p e a r e d to a t t a c h to the p l a s m a m e m b r a n e (Fig. 11). I n these c o a t e d vesicles, fibrillar structures of d i a m e t e r c o m p a r a b l e with the a m y l o i d fibrils were occasionally n o t e d (Figs. 5 a n d 13). A n a l y s i s of high p o w e r electron m i c r o g r a p h s of these structures led to the following findings. T h e specific m e m b r a n e structure associated with dense p l a q u e a n d the vesicles c o a t e d b y dense c o r o n a are p r o b a b l y of the same derivation. T h e p l a s m a
FIG. 3. Higher magnification of a portion of the area shown in Fig. 2. The plasma membrane forms a deep pit (at the right upper corner) at the site associated in part with the dense plaque (D). Amyloid fibrils are primarily in the extracellular space (Am) and also can be followed within the plasmalemmal pit. At the narrow opening of the pit, amyloid fibrils are almost in parallel with one another. Coated (CV) and uncoated vesicles (V) also are seen. The corona of the coated vesicle in the deeper cytoplasm (at the right bottom comer) clearly shows "spikes." Scattered free ribosomes, smooth endoplasmie reticulum (SER) and cytoplasmic filaments are also seen. × 60 000. FIG. 4. A deep plasmalemmal pit with a relatively long, narrow neck is associated with the dense plaque (D) at its base. Extraeellular amyloid fibrils (Am) are arranged in parallel array at the opening of the pit and can be followed to its bottom. This finding gives the impression that the amyloid fibrils fixed at the hemidesmosome-like area have been "pulled" into the plasmalemmal pit as it has formed. R: free ribosomes, RER: rough endoplasmic reticulum, x 60 000. FI6. 5. A coated vesicle in the peripheral cytoplasm contains fibrillar material whose dimensions are comparable to those of the amyloid fibrils. Am: extracellular amyloid fibrils, R: free ribosomes. x 60 000. FIG. 6. High power electron micrograph showing a portion of the border between a macrophage (Mac) and amyloid (Am), after 120 minute incubation. A hemidesmosome-like structure (D) is observed in part in cross section. Dense homogeneous material accumulates on the cytoplasmic side of the plasma membrane and forms a thinner band close to the plasma membrane, an electron lucent space (arrows), and a thicker band apart from the plasma membrane. This structure is distinct from the plasma membrane cut in cross (C) and oblique (O) sections. The portion surrounded by dotted lines is seen at higher magnification in the inset, x 160 000. Inset: Higher magnification of a hemidesmosome-like structure. The plasma membrane is distinct and consists of the outer dense layer (a), the intermediate electron lucent space (b), and the inner dense layer (c). A narrow band of the dense homogeneous material (30-60/~ thick) (e) is seen close to the inner dense layer of the plasma membrane (c) without appreciable space (d). Separated by an electron lucent space (50-150 A wide) (f), the additional dense material forms a thicker band (150200 ~ thick) (g). Thin dense strands are seen to bridge the outer thin and the inner thick bands (arrows). × 500 000.
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FIG. 7. High power electron micrograph of a coated vesicle in the peripheral cytoplasm. The limiting membrane is 70-90 A thick and shows features of the unit membrane. The dense coating is 200300 A in thickness. Although the coating has a comparable structure with the dense hemidesmosomelike plaque, the strands perpendicular to the limiting membrane (or the bridges between the outer thin and the inner thick bands) are prominent while the outer thin and the inner thick bands are barely seen. The perpendicular strands (arrows) are arranged at fairly regular intervals, x 200 000.
Flas. 8-13. These electron micrographs show a possible sequence of transition from the hemidesmosome-like structure to the coated vesicle. PM, plasma membrane; AM, extracellular amyloid fibrils. x 140 000. FIG. 8. A hemidesmosome-like plaque associated with a portion of the plasma membrane with shallow indentation. F~G. 9. A small, moderately deep indentation of the plasma membrane associated with the dense plaque. Fro. 10. A deep plasmalemmal pit and a shallow indentation with the dense plaque adjoin one another. The opening of the deep pit is very narrow and the opposing membranes at its neck seen to be about to fuse. FIG. 11. A slightly indented plasma membrane and a vesiclecoated by dense homogeneous material are connected by a membranous structure at one point. This micrograph again suggests that a newly formed coated vesicle is pinched off. FIG. 12. A coated vesicle in the peripheral cytoplasm is connected with the plasma membrane by a long membranous structure surrounded by dense homogeneous material (arrow). FIG. 13. A coated vesicle in the peripheral cytoplasm with no apparent connection with the plasma membrane. In the dense corona of the vesicle, spike-like structures are prominent. In the coated vesicle fibrillar structures comparable to the amyloid fibrils are indicated (arrows).
w~
J~
m4
J~
m~
J~
594
SHIRAHAMA AND COHEN
membrane of the area or the limiting membrane of the vesicle is about 70-90 A thick and consists of outer and inner dense layers separated by an intermediate electron lucent space. Close to the inner layer of the membrane (with no appreciable space), dense homogeneous material forms a very thin layer (30-60 A in thickness) (this will be referred as "the outer thin band"). Separated from the outer thin band by an electron lucent layer (50-150 A wide), the dense material forms an another thicker band of about 150-200 A thickness ("the inner thick band"). Thin (50-100 A) dense strands bridge the outer thin and the inner thick bands. The strands usually stand perpendicularly to the plasma or the limiting membrane and are often arranged at fairly regular (150-250 A) intervals (Figs. 6 and 7). In the plaques where the plasma membrane showed no or only a shallow indentation, the outer thin and the inner thick bands were very to moderately dense and more predominant than the perpendicular strands (Figs. 6, 8, and 9). At the deep plasmalemmal pit, density of the bands seemed slightly decreased (Figs. 3 and 10). In the corona of the coated vesicles, the bands were often less electron dense and thinner, so that the perpendicular strands were relatively prominent and often showed the features that could be termed "spikes" (2) (Figs. 3, 7, and 13). The transition between the membrane structure associated with the plaque and the coated vesicle has been suggested by (a) flask-shaped plasmalemmal pit connected to the outer space with a relatively long, narrow opening (Figs. 3, 4, and 10), (b) apparent fusion of the apposing plasmalemma at the neck of the pit (Fig. 11), and (c) coated vesicles seemingly connected to the plasma membrane by a strand of the dense material (Fig. 12). The above findings were found primarily in the samples incubated longer than 30 minutes. With this longer incubation, macrophages often came into close contact with each other, a finding rarely observed with shorter incubation times. On occasion the structure of a tight junction or desmosome was suggestive at the contact sites though we could not obtain the typical micrograph of one in the present study. The presently described membrane structure associated with the dense plaque was not observed on the surface of the macrophages with no contact with amyloid or in controls. DISCUSSION Peripheral cytoplasmic accumulation of dense material in close relation to extracellular microfibrils has been described on many occasions. Porter (25) has observed indistinctness of portions of the plasma membrane associated with cytoplasmic dense accumulations in fibroblasts in intimate relation to extracellular collagen microfibrils. In amyloidotic tissues, the intimate structural relationship of a group of well oriented amyloid fibrils, indistinct plasma membrane and peripheral cytoplasmic
HEMIDESMOSOME-LIKE PLAQUE AND COATED VESICLES
595
accumulation of dense material has also been found in a variety of cells, and has been interpreted as the possible site of amyloid formation (29, 30, 33). A similar structural combination has been observed where the lymphatic anchoring filaments approximate the lymphatic endothelium (17, 30). Leak and Burke have stated that some of these dense areas resembled the hemidesmosomes of the basal epithelial layer of the epidermis (17). Since the ultimate significance of these comparable, but poorly understood morphologic observations is not known, it is difficult to define interrelations or precisely to relate the present observation to them. While many similarities exist among those structures, the present structure differs from the previous ones in several ways; i.e., the experimental conditions, the distinctness of the plasma membrane, the uniformity in the size of the structure, the apparent relation to the coated vesicles. The plasma membrane thickening herein described resembles the hemidesmosome (10, 12, 13, 16) in many respects; i.e., the localization, the form and the size of the dense plaque, their uniformity and fairly regular arrangement along the plasma membrane, the distinctness of the plasma membrane. However, the following differences do exist: (a) the present structure has been found in macrophages, while typical hemidesmosomes have been reported only in epithelial cells, (b) the classical hemidesmosomes have faced the basement membrane while this structure has occurred on the cell surface in contact with amyloid and without a basement membrane, and (c) associated cytoplasmic filaments have not been seen in our material, but are usually found with the hemidesmosomes. Under the present experimental conditions it is an unlikely possibility that synthesis of amyloid by macrophages plays a major role, and the present results are most likely interpreted as follows. The hemidesmosome is widely accepted as the attachment device to the extracellular elements, and the hemidesmosome-like structure of the present study presumably has a similar function. The macrophage facing the extracellular amyloid mass forms the hemidesmosome-like plaque and attaches to the amyloid fibrils at the site. As the plasmalemmal indentation occurs locally, a group of amyloid fibrils attaching to it are pulled into the pit and separated from the main amyloid mass. The plasmalemmal pit eventually forms a coated vesicle after the plasma membrane fuses at the neck of the pit. In the sequence of the formation of the coated vesicle and perhaps after the coated vesicle is formed, the dense material forming the outer thin and the inner thick bands of the plaque or the corona becomes less and less apparent. A few observations somewhat comparable to the present study have been previously published. Berry and Friend (1) have observed that hemidesmosome-coutaining areas of the cell membrane invaginate and appear to pinch off and migrate centrally when rat liver parenchymal cells have been isolated. They also have found plaques similar to the hemidesmosomes but less dense on vacuoles deep within the cells and on
596
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multivesicular bodies, and have interpreted them as the probable precursors of the hemidesmosome plaques. Similar observations have also been made by Gona (15) in the resorbing tadpole tail fin where "free" hemidesmosomes have formed vesicles containing globular layer material. In the usual process of formation or discharge of the coated vesicles, the plasma membrane structure associated with a dense plaque such as described in the present study has not been observed. However, observations similar to the present study have been reported on the oocytes in the process of taking up the yolk protein (13, 27). The short spine-like projections coating the pinocytic vesicles in the cytoplasm have also attached to the portions of the plasma membrane where the condensation of yolk protein has been observed on the external surface of the cell membrane. Friend and Farquhar have also reported similar observations during protein (exogenous peroxidase) absorption in the rat vas deferens (14).
Finally, we have reported here the formation of hemidesmosome-like plaque in macrophages in contact with a heterologous fibrillar protein (amyloid) in vitro. This structure has more points of resemblance to the hemidesmosome than to any other known structure, and can be interpreted as an attachment device for the extracellular amyloid fibrils. The site has become indented and eventually formed a coated vesicle. This process has probably subserved the uptake of the amyloid fibrils or a substance closely related to them. If one accepts the above findings and interpretation, our observations may provide additional information leading to a better understanding of the modes of formation of and the nature of the hemidesmosomes and the coated vesicles, as well as the nature of amyloid.
REFERENCES 1. BERRY, M. N. and FRIEND, D. S., J. Cell Biol. 43, 506 (1969). 2. BRUNS, R. R. and PALADE,G. E., J. Cell Biol. 37, 244 (1968). 3. - ibid. 37, 277 (1968). 4. CARR, I., Z. Zellforsch. Mikrosk. Anat. 80, 534 (1967). 5. COHEN,A. S., Lab. Invest. 15, 66 (1966). 6. COHN, Z. A., Advan. Immunol. 9, 163 (1968). 7. COHN, Z. A., FEDORKO, M. E. and HIRSCH, J. G., J. Exp. Med. 123, 757 (1966). 8. COHN, Z. A., HmSCH, J. G. and FEDORKO, M. E., J. Exp. Med. 123, 747 (1966). 9. DEPETRIS,S., KARLSBAD,G. and PERMS, B., J. Ultrastruct. Res. 7, 39 (1962). 10. DOUaLAS, W. H. J., RrPLEY, R. C. and ELLIS, R. A., J. CellBiol. 44, 211 (1970). 11. DUMONT,A., J. Ultrastruet. Res. 29, 191 (1969). 12. FARQUHAR, M. G. and PALADE,G. E., J. Cell Biol. 26, 263 (1965). 13. FAWCETT, D. W., The Cell. Its Organelles and Inclusions. Saunders, Philadelphia, Pensylvania, 1966. 14. FRtEND, D. S. and FARQUHAR, M. G., J. Cell Biol. 35, 357 (1967).
HEMIDESMOSOME-LIKEPLAQUE AND COATED VESICLES 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34.
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GONA, A. G., Y. Ultrastruct. Res. 30, 103 (1970). KELLY, D. E., J. Cell Biol. 28, 51 (1966). LEAK, L. V. and BURKE, J. F., Y. Cell Biol. 36, 129 (1968). L~JrT, J. H., J. Biophys. Biochern. Cytol. 9, 409 (1961). LUFT, J. H., The Inflammatory Process, p. 121. Academic Press, New York, 1965. MAJNO, G., Handbook of Physiology, Section 2, p. 2293. American Physiological Society, Washington, D.C., 1965. MILLONIG, G., J. Appl. Phys. 32, 1637 (1961). NORTH, R. J., J. Ultrastruet. Res. 16, 83 (1966), -ibid. 16, 96 (1966). PALADE,G. E. and BRUNS, R. R., J. Cell Biol. 37, 633 (1968). PORTER, K. R., Biophys. J. 4. Suppl., 167 (1964). REYNOLDS,E. S., J. Cell Biol. 17, 208 (1963). ROTH, T. F. and PORTER, K. R., J. Cell Biol. 20, 313 (1964). SHmAHAMA,T. and COHEN, A. S., J. Cell Biol. 33, 679 (1967). -Amer. J. Pathol. 51, 869 (1967). - - - - Exp. Mol. Pathol. 11, 300 (1969). SHIRAHAMA,T., COHEN,A. S. and RODGERS,0. G., submitted for publication. SUTTON, J. S. and WEISS, L., aT. Cell Biol. 28, 303 (1966). TRUMP, B. F. and BENDITT, E. P., Lab. Invest. 11, 753 (1962). WATSON, M. L., aT. Biophys. Biochem. Cytol. 4, 475 (1958).