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An ordered complex of filaments surrounding the lipid droplets in developing adipose cells.

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An 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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