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Focal laminate segments in cytoplasmic processes of mouse myocardial fibroblasts.

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Focal Laminate Segments in Cytoplasmic Processes of
Mouse Myocardial Fibroblasts
M. S. F O R B E S AND N. S P E R E L A K I S
Department of Physiology, University of Virgznia School of Medrcine,
Charlottesudle, Virginia 22908
In mouse ventricular myocardium, we have found unusual
fibroblasts whose cellular processes in some regions are particularly flattened and which contain linearly-arranged, electron-opaque structures (“central laminae”). The morphology of these focal laminate segments of fibroblast
processes suggests that the intracellular laminae are adhesive entities which
hold the plasmalemmata above and below them in close parallel apposition for
short distances,
A complex system of interconnected extracellular compartments is present throughout the mammalian heart. This system has
been termed the “tissue space” (Melax and
Leeson, ’72) and comprises the perivascular
spaces, the subepicardial and subendocardial
spaces, and the interstitial spaces between the
cardiac muscle cells themselves. Within the
myocardial tissue space, the most populous
cells are fibroblasts (Melax and Leeson, ’72).
Aside from their constituting a nuisance in
the cuIture of heart muscle cells, myocardial
fibroblasts have enjoyed little attention. However, we have found in mouse heart a unique
structure, the “central lamina,” that occurs
within particularly thin processes of fibroblasts. The fibroblastic segments containing
such laminae are somewhat similar in configuration to flattened regions seen in various
cell types in vitro (Conley and Herman, ’73;
Franke et al., ’78). We conclude that such
complexes represent sites of intracellular
The animals investigated were mice of
either the ICR or C57BL strains. Each animal
was anesthetized with an intraperitoneal injection of pentobarbital and its thoracic cavit y was opened, exposing the beating heart.
Fixative solution was injected into the circulatory system through a butterfly infusion
needle inserted into the apex of the left ventricle, and left the system via an incision in
the right atrium. Fixative solutions used included: (1) 3% glutaraldehyde in aqueous 3%
ANAT. REC. (1979) 195: 575-586.
dextrose-3% dextran, with or without 50 mM
CaC1, added (modified from Rostgaard and
Behnke, ’65); (2) 2%paraformaldehyde and 2%
glutaraldehyde in 0.1 M sodium cacodylate
with 50 mM CaC12 (McNutt and Weinstein,
’70); or (3) 2% paraformaldehyde and 3% glutaraldehyde in Hank’s balanced salt solution
(Bois, ’73). After 5 minutes of perfusion fixation, the heart was removed, and portions
were fixed an additional 3 hours by immersion
in fixative solution. All tissues were postfixed
in 1%phosphate-buffered osmium tetroxide
(Millonig, ’621, stained en bloc for 30 minutes
in saturated aqueous uranyl acetate, dehydrated in alcohols, passed through propylene
oxide and embedded in Epon 812 resin. Thin
sections were cut with a diamond knife, affixed to copper mesh grids and stained sequentially with saturated uranyl acetate in 50%
acetone (2 min) and 0.4%alkaline lead citrate
(45 sec) (Venable and Coggeshall, ’65). The
sections were examined in either Philips EM200 or Zeiss EM 9A electron microscopes,
which instruments were calibrated periodically against a replica of a diffraction grating.
For critical measurements of structures, micrographs were taken a t high magnification,
and the microscopes then calibrated at that
magnification; photographs were prepared a t
10 x enlargement (approximately 400,000 X
total magnification), and t h e cytological
structures of interest were measured with vernier calipers. Some sections were analyzed by
tilting in a Siemens 101 electron microscope
equipped with a goniometric specimen stage.
Received Apr. 5, ”79.Accepted
June 13, ’79.
In the tissue space of mouse heart, fibroblasts can be readily distinguished from
neighboring cell types by their lack of a covering of basal lamina (figs. 1-3,8,9).Myocardial
fibroblasts are characterized by their extensive thin cytoplasmic processes, which follow
a meandering course through the tissue space
(fig. 1).The majority of fibroblast processes
within mouse heart are unremarkable in
terms of their ultrastructure; however, highly-structured fibroblast processes were found
in the ventricular myocardium of the six mice
used in this study (figs. 1-10).In each of the
more than 30 examples examined, a portion of
each process was particularly a t t e n u a t e ,
averaging approximately 40 nm in thickness
and ranging from 164-918 nm in length. Inspection of such segments usually revealed
the presence of a distinct intracellular electron-opaque line (“central lamina”), approximately 12.5 nm in width, positioned midway
between the profiles of the limiting plasma
membranes (figs. 1-10].
In no instance did the intracellular laminae
of myocardial fibroblasts extend beyond a
thickness of more than three consecutive thin
sections. We have in fact found series of
thin sections in which laminae appeared and
then disappeared, either leaving fibroblast
profiles which showed cytoplasmic continuity
throughout their lengths or revealing profiles
in which laminate areas appeared a t a deeper
level of the process. We conclude therefrom
that such structures are ribbon-like bodies,
probably no more than 200 nm in width
(fig. 3).
Under close scrutiny, bridging structures
were sometimes found to be present between
the central laminae and the inner surfaces of
the limiting cell membranes of some fibroblast processes (figs. 3-7, 8-10). The laminae
themselves were seen to be composed either
of material arranged into a confluent membrane-like array (figs. 1-4, 6, 8-10) or of single
rows of spheroidal or ellipsoidal subunits (figs.
5 , 7, 8 ) . These two configurations could be
present in alternate sections of the same lamina (figs. 4,5) or might appear in the same section a t different points along the length of the
lamina (fig. 8 ) . Tilting of fibroblast segments
did not result in the resolution of separate
subunits within examples of the confluent
form of lamina.
A rather close resemblance was seen be-
tween focal laminate processes of fibroblasts
and saccules of myocardial junctional sarcoplasmic reticulum (J-SR) (fig. 91, in that both
of these structures contain ordered internal
material: the fibroblast process possesses a
central lamina (fig. 101, the J-SR an intrasaccular collection of opaque “junctional granules” that often fall into a linear arrangement
(fig. 11).
Focal laminate segments of fibroblast processes in mouse heart closely resemble the
thin profiles found in cultured acoustic
Schwannoma cells (Conley and Herman, ’73)
and in the P t K z rat kangaroo kidney cell line
(Franke et al., ’78). The presumed adhesive
structures described within all these cells are
totally intracellular and therefore are associated with the inner faces of the plasma membranes. They therefore may be distinguished
from the “intracytoplasmic” junctions described in heart by Buja et al. (’741, since
those structures are produced by the folding
over of cells on themselves, which brings the
extracellular faces of specialized membranes,
such as those of the intercalated discs, to abut
one another.
The attenuated processes in Schwannoma
cells (Conley and Herman, ’731 bear somewhat
greater similarity to focal laminate segments
of fibroblasts in terms of their dimensions
(lengths of 400-600 nm and total estimated
thickness of 37 nm) than do the PtK2 segments, which may reach 2 p m in length
(Franke et al., ’781, and whose thickness appears to be less than 30 nm. The pronounced
central lamina of the fibroblast processes,
found in the mouse heart in vivo, is absent
from the flattened cell segments of cultured
cells, which instead exhibit a distinct septa1
appearance. This appearance is derived from
the presence of “thin linear ladder-like formations” (Conley and Herman, ’73) that span the
entire intracellular gap, thereby creating
structures which resemble the “septate desmosomes” formed intercellularly in a variety
of invertebrate and vertebrate tissues (e.g.,
Rose, ’71; Friend and Gilula, ’72; Sotelo and
Llinas, ’72; Noirot-Timothee and Noirot, ‘73;
Schwartz, ’73). Although a septate configuration usually is not obvious in focal laminate
segments of fibroblasts, the subunits appearing in some laminae (figs. 5,7) resemble those
within the intercellular spaces of rat adrenal
cell-to-cell contacts (Friend and Gilula, ’72).
Other intracytoplasmic complexes that
have been described include those formed by
interaction of an intracellular organelle with
the inner side of the plasma membrane or its
derivatives. Such complexes include: (a) the
subsarcolemmal saccules of sarcoplasmic reticulum (“peripheral” junctional SR) of cardiac muscle (Forbes and Sperelakis, ’77) and
skeletal muscle (Spray et al., ’74); (b) interior couplings between junctional SR and
transverse tubules in cardiac (Forbes and
Sperelakis, ’77) and skeletal muscle cells
(Kelly, ’69; Franzini-Armstrong, ’70); (c) the
subsurface cisterns of endoplasmic reticulum
in neurons (Rosenbluth, ’62; Le Beux, ’72);
and (d) the “imaged-desmosomes” of fetal
guinea pig myocardial cells, which appear to
be composites of desmosomes and SR (Forbes
and Sperelakis, ’75).
Intraorganellar junctions are found as
well: under a variety of conditions, the luminal surfaces of certain portions of skeletal
muscle SR collapse upon themselves to form
pentalaminar membrane appositions (“zippers”: Wallace and Sommer, ’75; Forbes and
Sperelakis, ’79). In mitochondria, linear and
septate structures have been discovered in the
intermembrane spaces (Saito e t al., ’74; Smith
and Klima, ’76).
The focal laminate segments of fibroblasts
superficially resemble extensive tight junctions (zonulae occludentes). However, close inspection of the segments reveals no fusion of
the inner lamellae of the limiting unit membranes. Rather, the central linear opacity
which characterizes focal laminate segments
is not related to membrane structure, but instead resembles the “central extracellular
lamina” found in the intercellular space between the plaques of desmosomes (Rayns et
al., ’69). The function of the fibroblast lamina
may be similar to that of the external leaflet
of the desmosome. That is, the “intracellular
lamina” may exert an adhesive action from
within the cell, thereby causing the characteristic close apposition of the membranes of
these segments. The irregularly-disposed
“bridges” (figs. 4, 6, 8) may be the means
whereby the narrowing effect is achieved; a
similar role is attributed t o the “side arms”
associated with the external lamina of desmosomes (Rayns et al., ’69). In focal laminate
segments, the bridges appear to emanate from
the central lamina, and usually are not oriented in register; hence, they do not extend
completely across the intracellular gap as do
the “rungs” within intracellular septate desmosomes (Conley and Herman, ’73).
A “central dense layer” within cardiac junctional SR (J-SR) has been described by Walker
e t al. (’70); the opaque structural material
there has also been assigned the term “junctional granules” by Sommer and Johnson
(‘68). It has been speculated that this intrasaccular material is the framework with
which Ca ++,Mg”-dependent ATPase molecules are associated (Forbes and Sperelakis,
’74). In addition, junctional granules may perform a mechanical function. A t the point of
transition between the tubules of “free” or
“network” SR and the saccules of J-SR there
is usually found a pronounced flattening of
the SR profile (Forbes and Sperelakis, ’77); a
similar change of configuration is found a t the
junction between non-laminate and laminate
segments of myocardial fibroblast processes
(fig. 9). Walker et al. (’70) detected projections from the SR central dense layer that are
similar to the bridges between the central
lamina and the innermost lamellae of the cell
membrane of the myocardial fibroblast (fig.
11). They argue that such connections “exert
a holding force,” acting thereby to narrow the
J-SR lumen. The degree of parallelism
achieved by J-SR limiting membranes is in
most instances surpassed by that of the focal
laminate segments, probably because of the
far more elaborate intraluminal architecture
of the latter structures.
The focal laminate fibroblast processes
present in vivo in mouse heart, together with
the intracellular septate desmosomes found in
cultured cells (Conley and Herman, ‘73;
Franke et al., ’78), form a category of structures that are unique examples of seeming
adhesions between the inner plasmalemmal
faces of the same cell. It appears that the focal
laminate segment is itself unique within this
category in two respects. First, it is present
within the intact organism; second, it contains an array of subunits distinctly aligned
parallel to the planar axis of the cell process,
rather than exhibiting a collection of vertically-oriented bodies.
It should be noted that focal laminate segments are present (as are the structural variations of their central laminae) after treatment of mouse heart with a variety of
aldehyde-based fixatives (MATERIALS AND
METHODS). Such modified fibroblasts, furthermore, are not limited in occurrence to mouse
heart; we have recently discovered similar
focal laminate segments in fibroblasts of c a t
atrial myocardium (Forbes, unpublished observations).
This research was supported in part by
American Heart Grant-in-Aid 78-753. We are
grateful to Dr. John Rash of t h e Department of Pharmacology and Experimental
Therapeutics of t h e University of Maryland
School of Medicine for the use of t h e Siemens
electron microscope.
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Schwannoma in uitro. J. Neurocytology, 2: 457-464.
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the junctional sarcoplasmic reticulum of lizard myocardial cells. J. Cell Biol., 60: 602-615.
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1 Left ventricular wall of mouse myocardium. A slender process of a fibroblast (FP)is
inserted in the interstice (“tissue space”) between two transversely-sectioned cardiac muscle cells (MC). A prominent feature of the fibroblast process is the presence
of a particularly narrow segment along part of its profile (between arrows). This attenuate segment, approximately 0.92 p m in length, is characterized by a n opaque
intracytoplasmic line (fig. 2), which gives the fibroblast process a t this level a
multilayered or laminated appearance. X 34,500.
2 Detail of t h e laminate fibroblast segment shown in figure 1. At all points along t h e
segment, the limiting membranes on either side of t h e centrally-located lamina
exhibit a close, parallel relationship to one another as well as to t h e lamina. Note
that basal laminar material is not present a t the outer surface of the fibroblast, although a distinct cell coat is associated with the adjacent muscle cells. x 94,000.
M. S. Forbes and N. Sperelakis
Left ventricular wall. A laminate segment of a fibroblast process is sandwiched between a cardiac muscle cell (MC) and another fibroblast (F). This laminar profile is
particularly short (0.185 Fm), and may be an end-on view of a lamina whose length
is far greater than its width. x 68,000.
Detail of the laminate segment shown in figure 3. The central lamina cannot be resolved a t any point into a unit membrane, although the trilaminar structure of the
cell membrane profiles (CM) is obvious in this section. At several points, projections
(arrows) extend between the lamina and the inner surface of the adjacent cell membrane. x 220,000.
5 Another section of the laminate segment shown in figures 3 and 4. At this level, the
lamina seems to be composed of a series of ellipsoidal bodies (asterisks), which gives
it a scalloped appearance. x 220,000.
Laminate fibroblast process found in 3 periarterial space in right ventricle. Numerous perpendicularly-oriented “bridges” are present (arrows) between the lamina and
the cell membranes. x 220.000.
7 Laminate fibroblast process in which the lamina does not appear a s a single continuous structure, but instead is composed of discrete bodies that in profile are ellipsoidal or circular. At several points (arrows), these subunits of the lamina appear to
be connected to t h e inner cell membrane surfaces. x 220,000,
8 The left-hand portion of this laminate segment of a fibroblast contains a distinct,
confluent lamina with cross-bridges (arrows); in contrast, the rightmost laminar region is made up of discrete, linearly-arranged particles (asterisks). x 153,500.
M. S. Forbes and N. Sperelakis
9 A laminate fibroblast segment, shown for the comparison of its morphology with
t h a t of a saccule of junctional sarcoplasmic reticulum (J-SR) located a t the periphery of t h e adjacent myocardial cell. Note the abrupt narrowing of the profile of t h e fibroblast a t the junction between its nonlaminate and laminate portions.
x 109,000.
10 Detail of the fibroblast in figure 11. I t s central lamina averages 11.4 nm in width,
and the total thickness of the process is ca. 45 nm. X 272,000.
11 A portion of the J-SR saccule shown in figure 11. The overall thickness of the saccule is somewhat less (ca. 29-37 nm) than t h a t of the laminate fibroblast segment
(cf. fig. lo), and its internal opaque contents (“junctional granules”) are less
highly-structured, although in some regions they can be resolved into linear structures (arrows) having a width of 7-8 nm. Some of these structures appear to contact the inner surface of the limiting membrane of the saccule ( p i n t s of contact
marked by the asterisk). The bodies (‘‘junctional processes”; arrowheads) t h a t lie
in t h e space between t h e J-SR and the inner face of t h e sarcolemma (SL) may be
evaginations of the J-SR membrane. X 272,000.
M. S. Forbes and N . Sperelakis
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cytoplasmic, focal, myocardial, mouse, processes, segmento, fibroblasts, laminated
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