An ordered complex of filaments surrounding the lipid droplets in developing adipose cells.
код для вставкиСкачатьAn Ordered Complex of Filaments Surroundinq- the Lipid Droplets in Developing Adipose Cells ' EUNICE M. WOOD Department of Anatomy, HaTVaTd Medical School, Boston, Massachusetts ABSTRACT Examination of the lipid-cytoplasm interface of the weakly osmiophilic f a t droplets in developing adipose cells from chick bone marrow reveals that the lipid does not lie free in the cytoplasm, nor is it bounded by a membrane, but instead is encompassed by a highly ordered complex of small, 80A thick, filaments. In oblique sections, superimposition of the images of adjacent filaments results i n a more or less continuous line which resembles a membrane. Perhaps this view of the system of filaments, in sections where heavily osmicated f a t had retracted slightly from the interface, is responsible for reports that the lipid in adipose cells is enclosed by a membrane. It is suggested that a similar system of filaments may be visualized in adipose cells of other species after extraction of the strongly osmiophilic f a t which obscures details at the surface of the lipid droplets. Although the true functional significance of the filaments is not yet known, the morphological evidence alone suggests that they may provide support for the lipid A n interpretation of the geometry of the filaments, consistent with inclusions. the present evidence, is presented. Although there are several studies in the literature on the fine structure of white adipose cells (Sheldon, '64; Williamson, '64; Napolitano, '65), there is no agreement as to whether the lipid droplets are enclosed by a membrane or lie free and unsupported in the cytoplasm. The uncertainty is in part attributable to the intense osmiophilia of the lipid in the mammalian adipose cells studied. If a membrane were present it is questionable whether it could be resolved as a distinct structure at the periphery of a lipid droplet of matching density. The observations reported here on the adipose cells from the bone marrow of young chicks may help clarify the issue. The lipid in these cells is weakly osmiophilic and examination of the lipid-cytoplasm interface reveals that the fat is neither bounded by a membrane, nor is it unsupported, but instead is encompassed by a highly ordered complex of small filaments. ('65) formaldehyde-glutaraldehyde fixative. After a few minutes the plugs were cut into small blocks and fixation continued for a total of 10 to 30 minutes at room temperature. Other bone marrow was fixed 30 to 60 minutes in a modification of this fixative containing only half the recommended amount of glutaraldehyde. The tissues were rinsed briefly in cold, 0.1 M cacodylate buffer and postfixed in 1% cacodylate buffered osmium tetroxide for 1 to 2 hours at 4°C. Some tissue blocks were stained in 0.5% maleate buffered uranyl acetate for two hours prior to dehydration. Embedding was in Araldite. Thin sections, cut with a diamond knife on a Sorvall MT-1 microtome, were collected on uncoated grids, doubly stained with uranyl acetate and lead citrate (Venable and Coggeshall, '65), and examined in an RCA EMU3F electron microscope. OBSERVATIONS AND DISCUSSION MATERIALS AND METHODS Bone marrow was obtained from White Rock chicks 2 to 5 days old. The animals were killed by decapitation, the femurs quickly removed, split lengthwise, and the plugs of marrow placed in Karnovsky's ANAT. REC., 157: 437-448 The general cytological features of these developing adipose cells are illustrated in figure 1. The cytoplasm contains, in addi1 Supported by grant GM-406 from the Institute of General Medical Sciences, National Institutes of Health. 437 438 EUNICE M. WOOD tion to the weakly osmiophilic lipid droplets, numerous, small mitochondria, a well-developed Golgi complex, elements of both granular and agranular reticulum, and free ribosomes. The nucleus is distinguished by the presence of a dense, fibrous lamina against the inner membrane of the nuclear envelope (figs. 1-3), which is either interrupted or greatly thinned over the nuclear pores (figs. 1,2). This lamina appears to be identical to the one that Fawcett ('66a) described in the intestinal epithelial cells of Amphiuma. In favorable sections, the most striking feature of the developing adipose cells is the presence of numerous, tiny filaments at the interface of the lipid droplets with the surrounding cytoplasm (fig. 4). These are approximately 80 A thick and are quite uniformly distributed around the fat droplets at 200-300 A intervals. Higher magnification of the filaments (figs. 5-8) reveals a slightly beaded appearance, perhaps indicating a periodic substructure. In cross section (fig. 2), they appear as minute dots of uniform density. Regions where the filaments are sectioned obliquely are of particular interest (fig. 5 ) , for when viewed in this plane, superimposition of the images of adjacent filaments results in a more or less continuous dense line and gives the distinct impression of a membrane. It is possible that this view of the system of filaments, in sections where heavily osmicated fat had retracted slightly from the interface, is responsible for reports that the lipid in adipose cells is enclosed by a membrane. There appear to be two groups of filaments surrounding each droplet, one set inside the other, and the inner one intersecting the outer at nearly right angles (figs. 6, 8). Each set of filaments is visualized as a series of parallel rings which decrease in diameter from the equator to the poles. If this interpretation of their geometry is correct, each fat droplet would have four poles where the final ring of one set appears as a small circle while those of the other set course straight through. This particular configuration of filaments has not yet been encountered and this concept of their arrangement awaits the confirmation of serial sections. It is consistent, however, with the images examined to date. For example, in the lower portion of figure 7 are several filaments describing parallel arcs of circles with 2 or 3 straight filaments crossing at right angles. Such a region is probably very close to one of the postulated poles of the droplet. Also consistent with this interpretation is the fact that the filaments are very uniformly spaced in all planes of section, and they are never seen to converge on a point, as they would if arranged meridionally. In many sections, clusters of filaments are found in the cytoplasm adjacent to the fat droplets (figs. 1, 2). These filaments are randomly oriented and they appear to be similar in all respects to the tonofilaments present in epithelia, neuroglia, the interstitial cells of bird testes, and numerous other cell types (Fawcett, '66b). It is possible that in adipose cells these groups of irregularly arranged filaments provide new material for addition to the orderly complex around the fat droplets as the volume of lipid increases. If this is so, then the orthogonal system of Maments encompassing the droplets represents a special arrangement of structures that are common to many classes of cells. Questions concerning the origin of the filaments, the means by which they become so precisely arranged, and their fate during the coalescence of droplets, are beyond the scope of this brief report, and, indeed, are unanswerable at this time. Furthermore, in the absence of data on the appearance of the filaments after experimental treatment, their functional significance cannot be assessed. It seems likely, on the basis of the present morphological evidence, that the filaments provide support for the lipid inclusions, but the results of future research may well provide evidence for a less passive role. The weak osmiophilia of the lipid in adipose cells of chick bone marrow has favored the visualization of this orthogonal system of filaments, but inasmuch as they are apparent in this material even where none of the fat is preserved, it is suggested that they will be seen in adipose cells of other species after extraction of the strongly osmiophilic lipid which tends to obscure them. This prediction is not an unreasonable one, for the author has FILAMENTS SURROUNDING LIPID DROPLETS detected similar filaments in a few of the published micrographs of mammalian adipose cells. Although fat droplets are frequently bounded by a membrane in other cell types (e.g., liver, intestinal epithelium), it seems clear that in adipose cells the lipid is surrounded, not by a membrane, but by a highly ordered complex of filaments. ACKNOWLEDGMENT The author is grateful to Dr. Don W. Fawcett for his valuable advice and for his generous aid in the preparation of the manuscript. LITERATURE CITED Fawcett, D. W. 1966a On the occurrence of a fibrous lamina on the inner aspect of the 439 nuclear envelope in certain cells of vertebrates. Am. J. Anat., 119: 129-146. Fawcett, D. W. 19661, The Cell. Its Organelles and Inclusions. W. B. Saunders Co., Philadelphia. Karnovksy, M. J. 1965 A formaldehyde-glutaraldehyde fixative of high osmolality for use in electron microscopy. J. Cell Biol., 27: 137A. Napolitano, L. 1965 The fine structure of adipose tissues, p. 109-123. In: A. E. Renold and G. F. Cahill, Jr. (eds.), Handbook of Physiology; adipose tissue. American Physiological Society. Washington, D. C. Sheldon, H. 1964 The fine structure of the f a t cell, p. 41-68. In: K. Rodahl and B. Issekutz (eds.), Fat as a tissue. McGraw-HiU. New York. Venable, J. H., and R. E. Coggeshall 1965 A simplified lead citrate stain for use in electron microscopy. J. Cell Biol., 25: 407-408. Williamson, J. R. 1964 Adipose tissue: morphological changes associated with lipid metabolism. J. Cell Biol., 20: 57-74. Note added in proof: After this report was submitted for publication, a brief note by L. M. Luckenbill and A. S. Cohen appeared in the Journal of Cell Biology (31: 195-199, 3966). These authors described a similar array of filaments associated with the lipid droplets in avian subsynovial adipose cells. PLATE 1 EXPLANATION OF FIGURE 1 A portion of a developing adipose cell from chick bone marrow. Although this section does not favor clear visualization of the system of filaments at the lipid-cytoplasm interface, several groups of similar filaments are seen in the adjacent cytoplasm (indicated by arrows). Such irregularly arranged filaments, which appear to be identical to the tonofilaments seen in many different cell types, may provide new material for addition to the orderly complex around the fat droplets as the volume of lipid increases. Numerous, small mitochondria are present as well as a Golgi complex, short profiles of granular endoplasmic reticulum, and many smooth walled vesicles. Double walled structures, such as the one above the Golgi, are observed frequently in these cells, but nothing is known of their significance. A number of pores are evident in the nuclear envelope, and over each of them the dense, fibrous lamina is either thinned or absent. Magnification 34,000 X. 440 FILAMENTS SURROUNDING LIPID DROPLETS Eunice M. Wood PLATE 1 44 1 PLATE 2 EXPLANATION OF FIGURES Enlargements of selected areas from figure 1. 442 2 Several filaments, cut in cross section, are apparent at the interface of the lipid with the cytoplasm, and in addition, a large number of similar, randomly orientcd filaments are present farther out in the cytoplasm. It is evident that these structures are solid filaments. and not tubules. Magnification 85,000 X. 3 In some areas where the filaments are not preserved, it is possible to compare the naked rim of the fat droplet with a membrane, in this instance the outer member of the nuclear envelope (see at arrows). Such a comparison makes it obvious that no membrane exists around the lipid itself. However, when the filaments are well preserved, and sectioned as in figure 5, they can easily be confused with a membrane. Magnification 85,000 X. FILAMENTS SURROUNDING LIPID DROPLETS PLATE 2 Eunice M. Wood 443 PLATE 3 EXPLANATION OF FIGURE 4 444 A portion of inultilocular adipose cell from chick bone marrow. In this favorably sectioned cell, the filaments around ihe fat droplets are clearly visible. The regular, 200-300A spacing of the filaments is evident in many regions. Since these droplets were distorted froin their spherical form during specimen preparation, it is not clear whether the disruption of the filaments between the two droplets at the right of the figure represents the beginning of coalescence, or is merely a n artifact. Magnification 32,000 X . FILAMENTS SURROUNDING LIPID DROPLETS Eunice M. Wood PLATE: 3 445 PLATE 4 EXPLANATION OF FIGURES Enlargements of selected areas from the cell showii in figure 4. At this magnification the filaments show a slightly beaded appearance which may indicate a periodic substructure. 5 When the filaments are sectioned obliquely, the superimposition of adjacent images results in a continuous line which resembles a membrane (arrowj. Compare with figure 3. Magnification 87,000 X . 446 6 and 8 The concept that each fat droplet is surrounded by two groups of filaments, one set inside the other, and the two intersecting each other at nearly right angles, is based o n images such as these (see at arrows). Magnification 87,000 X . 7 If each set of filaments is a series of parallel rings which decrease in diamcter from the equator to the poles, then the lipid droplet would have four poles where the final ring of one set appears as a small circle while those of the other set pass straight through. The configuration of filainents in this figure (arrow) is interpreted as being close to one of these postulated poles. Magnification 87,000 X. FILAMENTS SURROUNDING LIPID DROPLETS Eunice M. Wood PLATE 4
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