J. Cell Sci. 18, S4S-S5I (i97S)
Printed in Great Britain
545
A NEW OCCLUDENS-LIKE JUNCTION
LINKING ENDOTHELIAL CELLS OF SMALL
CAPILLARIES (PROBABLY VENULES) OF
RAT JEJUNUM
L. A. STAEHELIN
Department of Molecular, Cellular and Developmental Biology,
University of Colorado, Boulder, Colorado 80302, U.S.A.
SUMMARY
Freeze-cleave replicas of small capillaries of rat jejunum have revealed the presence of a new
type of junction linking endothelial cells. This new junction resembles tight junctions (zonulae
occludentes) in that the adjacent plasma membranes are held closely together along lines of
attachment organized in the form of a loose, but frequently discontinuous network. In contrast
to tight junctions, the A-face ridges possess a very low profile, and only at low shadowing angles
can a repeating, particulate substructure occasionally be resolved. The shallow B-face furrows
lack any particulate components. Images of cross-fractured focal points of attachment suggest
that the external leaflets of adjacent membranes are closely apposed but not actually fused, as
is the case with zonulae occludentes. It appears that this new type of endothelial junction is
characteristic of small venules. Thus we propose that it be termed small venule endothelial
junction.
INTRODUCTION
Although the morphology of endothelial cell junctions has been extensively investigated in thin sections and with the help of tracers (Karnovsky, 1967, 1970; Bruns &
Palade, 1968 a, b; Cotran & Karnovsky, 1968; Clementi & Palade, 1969; Simionescu,
Simionescu & Palade, 1973), no clear consensus has yet been reached as to whether
they represent tight junctions {zonulae occludentes, Farquhar& Palade, 1963; Staehelin,
1973 a), maculae occludentes (discontinuous areas of obliteration of the intercellular
space, Karnovsky, 1967), or even a different kind of junction not recognized so far.
Knowledge of the fine structure of these junctions is important for understanding
endothelial cell function, since it has been proposed that small discontinuities in these
junctions (slits) could correspond to the small pores that physiologists and morphologists have proposed to account for certain permeability characteristics of capillary
walls (Renkin, 1964; Karnovsky, 1967).
High-resolution thin section micrographs of endothelial cell junctions cut precisely
at right angles to the junctional membrane segments usually exhibit focal points of
membrane attachment or membrane fusion (Farquhar & Palade, 1963; Karnovsky,
1967; Bruns & Palade, 1968a; Clementi & Palade, 1969). In some instances the focal
points of fusion are indistinguishable from those of zonulae occludentes found between
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L. A. Staehelin
epithelial cells, while in others the membranes appear separated by a gap up to 4 nm
wide and which can be penetrated by electron-opaque tracers (Karnovsky, 1967).
Freeze-cleaved tight junctions appear in face views either as band-like meshworks
of interconnected ridges (on A-fracture faces), or as complementary meshworks of
furrows on B-faces (Friend &Gilula, 1972; Staehelin, 1973 a). The ridges and furrows
correspond to the sealing elements of these junctions (for a review see Staehelin, 1974)
and as demonstrated by Claude & Goodenough (1973), the number of sealing elements
in the apical-basal direction is related to the tightness of the junction. This characteristic appearance makes tight junctions prominent structures in freeze-cleave replicas
and allows an easy distinction between tight junctions and gap junctions. Thus a
freeze-cleave study of endothelial cell junctions should permit one to determine
unambiguously whether they possess the same supramolecular architecture as zonulae
occludentes or whether they have a different and unique fine structure.
To date over ten papers dealing with the freeze-cleave morphology of endothelial
cells have been published (Friederici, 1968, 1969; Leak, 1968, 1970, 1971; Nickel &
Grieshaber, 1969; Weinstein & McNutt, 1970; Wisse, 1970; Maul, 1971; Dempsey,
Bullivant & Watkins, 1973; Smith, Ryan & Smith, 1973; Simionescu, Simionescu &
Palade, 1974a). Nearly all of these reports have focused on the size, form and distribution of plasmalemmal infoldings, vesicles and fenestrae, and in only two instances
(Weinstein & McNutt, 1970; Leak, 1971) have the junctions connecting endothelial
cells received more than passing attention. The micrographs of junctional regions
published by these latter authors reveal structures resembling tight junctions, but the
limited magnification and resolution of their pictures precludes an unambiguous
interpretation of the fine structure of the junctional elements. This lack of information
on freeze-cleaved endothelial junctions can be attributed to the fact that such images
are only rarely observed, because the geometry of the endothelial cells favours cleavage
planes along the extensive plasma membrane sheets bordering the tissue and lumenal
cell surfaces.
In the present study, which is part of a larger investigation of the freeze-cleave
morphology of tissues of the digestive tract (Mukherjee & Staehelin, 1971; Staehelin,
1972, 1973 a), we demonstrate that the junctions linking endothelial cells of small
capillaries (less than 20 /tm in diameter) of the jejunum have a structure distinct from
that of normal zonulae occludentes.
MATERIALS AND METHODS
Jejunal tissues of adult albino rats were processed for freeze-etching as already described
by Staehelin (1973 a). Only tissues prefixed with glutaraldehyde were examined. Arrows in the
lower right corners of micrographs indicate the direction of shadowing.
RESULTS
The thin-section image of endothelial cell junctions of intestinal capillaries has been
described in great detail by Clementi & Palade (1969). Our own thin-section micrographs have confirmed but not extended those findings. Similarly, we have been able
New type of endothelial junction
547
to confirm the freeze-cleave morphology of the ribbon-like parajunctional region
as reported by Weinstein & McNutt (1970), Leak (1971) and Simionescu et al.
(1974 a).
Cross-fractured endothelial cell junctions of small diameter intestinal capillaries
(Figs. 1 and 2) appear very similar to corresponding thin section images in that they
exhibit focal points of attachment of the adjacent membranes. Careful examination of
such points of attachment (Fig. 2) suggests that the external leaflets of the adjacent
membranes are closely apposed, but not actually fused, as is the case for tight junctions.
Where these occludens-like junctions are revealed in face views (Figs. 3-6) simple
meshworks of low profile ridges on A-faces, and corresponding shallow meshworks of
grooves on B-faces are seen. The distribution of ridges and furrows corresponds to
the distribution of attachment lines of adjacent membranes. As seen in Figs. 3 and 5
the branching frequency of the attachment lines is rather low and many individual
lines do not even seem to link up with any of their neighbours, or only to one other
line at one end. Fig. 3 demonstrates, furthermore, that the meshwork of lines of
attachment is discontinuous. Sizeable gaps appear to separate different segments of
the meshwork.
At higher magnifications the low profile A-face ridges exhibit a faint substructure
in the form of very small, irregularly shaped particles which frequently possess a
periodicity of approximately 12 nm (Figs. 4-6). In some instances (Fig. 6) the particles
take on the form of short, thin ridges oriented at right angles to the main axis of the
attachment lines. The faint grooves usually appear devoid of particles and other
substructures (Fig. 4).
DISCUSSION
The present study has shown that the endothelial cells of small capillaries (less than
20 /tm in diameter) of the jejunum are linked by occludens-like junctions possessing
a freeze-cleave morphology different from epithelial tight junctions of the same tissue
(Staehelin, 1973 a). As shown in Fig. 2, the adjacent membranes of these endothelial
junctions become closely apposed along lines of attachment, but, in contrast to zonulae
occludentes, no fusion of the outer membrane leaflets nor interruption of these leaflets
by bridging particles is seen (compare Fig. 2 withfig.8 of Staehelin, 1973 a). Thus, the
freeze-cleave image of cross-fractured occludens-like junctions parallels the appearance of certain types of endothelial cell junctions in high-resolution thin sections,
whose membranes remain separated by a narrow gap (~4 nm wide) even at the focal
points of membrane attachment (Karnovsky, 1967; Clementi & Palade, 1969). Such
junctions would be expected to be leakier than zonulae occludentes, especially since
they also seem to have larger discontinuities in the lines of attachment (Fig. 3).
The lack of large and closely spaced membrane connecting elements in the endothelial occludens-like junctions is also reflected in the low profile of the freeze-cleaved
lines of attachment seen in face views of the junctional membrane regions. Although
tight junctions of different tissues show a certain variability in their freeze-fracturing
behaviour, as evidenced by the distribution of their bridging particles on A- and
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L. A. Staehelin
B-fracture faces (Friend & Gilula, 1972; Staehelin, 1973 a), in no instance would it
appear too difficult to distinguish a tight junction from the type of endothelial cell
junction demonstrated in this paper, providing the replicas are of good quality (for
criteria of good quality replicas see Staehelin, 19736). The A-face particles of the
occludens-like junctions of the endothelium are in most instances so small that they
show up clearly only at low shadowing angles (compare Figs. 5 and 6). This relatively
small size of the particles compared to their average spacing, frequently gives the
A-face views of the attachment lines a stitched appearance. In contrast, the particles
and ridges of zonulae occludentes have a size and height that makes them readily discernible at virtually all shadowing angles. Furthermore, the meshworks of ridges and
grooves of tight junctions are more regularly interconnected than the occludens-like
junctions of endothelial cells. They also lack discontinuities of the type shown in
Fi
g- 3During the final stages of preparation of this manuscript Simionescu, Simionescu &
Palade (19746) reported on differences between the endothelial junctions of arterioles,
and their capillaries, as well as the emerging venules in sequential segments of the
microvasculature of rat mesentery and omentum as visualized by freeze-cleave microscopy. Their micrographs revealed that in arterioles, the endothelium has continuous
and elaborate zonulae occludentes. The adjacent capillary endothelium which follows
the arterioles is also provided with normal tight junctions, the strands and grooves of
which exhibit a more staggered arrangement than in the arterioles. The endothelial
cells of immediately post capillary venules (pericytic venules) have 'loose' tight
junctions with discontinuous strands that give rise to low profile ridges and grooves
that resemble those illustrated in this article. Since the same authors found corresponding junctional structures on muscular venules as well, it appears that the tight
junction-like endothelial cell junction described in this article is characteristic of small
venules. Thus, after consulting with the above mentioned authors, we propose that
the new type of junction be termed small venule endothelial junction.
I gratefully acknowledge the technical assistance of Stephanie Krah. Thanks are also due to
Barbara Hull for reading the manuscript. This research was supported by the National Institutes
of General Medical Sciences under Grant GM18639.
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Fig. i. Cross-fractured junction linking endothelial cells of a small capillary in rat
jejunum. Numerous vesicular structures appear associated with the endothelial
plasma membrane bordering the tissue. The 2 cross-fractured junctional membranes
in the lower half of the micrograph depict a focal point of membrane attachment
(arrow), which is shown at higher magnification in Fig. 2. x 50000.
Fig. 2. Higher magnification of the junctional membrane region (arrow) shown in
Fig. 1. The bilayer structure of the 2 membranes is clearly visible. At the focal point
of membrane attachment the 2 membranes come close together, but, in contrast to
true tight junctions, the external membrane leaflets appear neither fused nor interrupted by bridging particles, x 100000.
Fig. 3. Face view of the junctional region between 2 endothelial cells of a small capillary
(probably a small venule). On the A-fracture face (a) the lines of attachment of adjacent
membranes appear as low profile ridges (r) with a faint substructure illustrated in
greater detail in Figs. 4 and 6. Notice the small amount of cross-linking between the
ridges and the presence of discontinuities in the network of ridges. A small ridge
segment completely separated from the main network is indicated by a small arrow.
Faint furrows (/) devoid of any particles and corresponding in their distribution to
the A-face ridges may be recognized on the B-fracture faces (b). The capillary
lumen is seen in the top third of the micrograph, x 36000.
New type of endothelial junction
Fig. 4. Higher magnification of the junctional membrane region on the left side of
Fig. 3. Some small, irregularly shaped particles can be detected on the crest of the
low profile ridges. The B-face (b) grooves lack any particles. The inset demonstrates
regularly spaced particles (small arrowheads) associated with the 2 ridges shown in
the upper left corner of Fig. 4. a, A-face;/, furrows, x 80000; inset, x 160000.
Fig. 5. Micrograph of a junctional region between a flat and a tubular endothelial cell
process. The faint A-face (a) ridges exhibit very little branching, and due to the
relatively high shadowing angle, the substructure of the ridges is only barely detectable
(compare with Fig. 6). x 80000.
Fig. 6. Higher magnification of the junctional region on the right side of Fig. 4. Due
to the low shadowing angle over most of the depicted membrane area, the substructure
of the low profile A-face (a) ridges appears very distinct. In particular, the elongated
form and the regular spacing (small arrowheads) of many of the small ridge particles
can be recognized, x 110000.