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Traversing filaments in desmosomal and hemidesmosomal attachmentsFreeze-fracture approaches toward their characterization.

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THE ANATOMICAL RECORD 1991-14 (1981)
Traversing Filaments in Desmosomal and
Hemidesmosomal Attach ments : Freeze-Fractu re
Approaches Toward Thei r Characterization
DOUGLAS E. KELLY AND AILEEN M. KUDA
Department of Anatomy, Uniuersity of Southern California School of Medicine,
Los Angeles, California90033
ABSTRACT
Desmosomes and hemidesmosomes from larval newt epidermis
were examined by freeze-fracture methods incorporating low osm olality fixation,
short duration glycerination, complementary replica comparison, stereo imaging,
and dark shadow printing. These procedures provide new evidence regarding the
structure of "traversing" filaments as mediators of attachment between intermediate filaments and the cell membranes of desmosomes and hemidesmosomes. A
detailed analysis of intramembranous particles and other structure in these attachments has also been possible. The relationship of this evidence to models of
desmosomal structure suggested by other authors is discussed.
Desmosomes are now generally regarded a s
focal areas of firm intercellular membraneto-membrane adhesion a t which a complex
network of cytoskeletal intermediate filaments
(or tonofilaments) is anchored (reviewed by
McNutt and Weinstein, '73; Staehelin, '74).
Hemidesmosomes display outwardly similar
structure but serve to attach cells to basal
laminae and subjacent connective tissue strata
(Kelly, '66; Shienvold and Kelly, '76). Little
structural or biochemical information exists
either a s to the nature of the intercellular
adhesive vehicle or the mode of intermediate
filament attachment to cell membranes. However, a number of possibilities have recently
been proposed, largely as a result of studies
using freeze-fracture.
It is doubtful that intermediate filaments
adhere directly to the cell membrane in desmosomal regions inasmuch as transmission electron micrographs of sectioned material frequently show them to converge toward, then
arch away from, the dense proteinaceous cytoplasmic plaque that characterizes each side of a
typical desmosome (Fawcett, '61;Kelly, '66).In
some epithelia the intermediate filaments run
a course parallel to the plaque, but at a distance
of 20-40 nm from the cell membrane. In no
instance have intermediate filaments been
demonstrated to terminate a t a desmosome or
insert directly into the desmosomal membrane.
High resolution images often suggest a finely
filamentous network surrounding the plaque
and spanning the interval between desmo-
0003-276X/81/1991-0001$02.60
0 1981 ALAN R.LISS, INC.
somal membrane on the one side and intermediate filaments on the other. On the basis of
freeze-fracture images, McNutt and Weinstein
('73)first postulated that this network serves as
the anchoring vehicle between intermediate
filaments and the desmosomal membrane. We
have introduced the term "traversing filaments" to denote fine filamentous profiles
emanating from the intermediate filament region and extending onto the E-face of fractured
desmosomal membranes (Kelly and Shienvold,
'76). However, such images have been suspect
for their similarity to nonspecific stress lines
caused by plastic deformation.
Freeze-fractured desmosomal membranes
display aggregated intramembranous particles
that are slightly larger and more irregular
than the typical 10 nm intramembranous particles. These have been reported to cling predominantly to the E-fracture face in the desmosomes of unfixed non-keratinized epithelia
(McNutt and Weinstein, '73; Kelly and Shienvold, '76), to remain with the P-fracture face in
fixed non-keratinizing epithelia (Kelly and
Shienvold, '76; Ishimura et al., '79)) or to be
dispersed equally between the two faces
(Breathnach et al., '72, '76; Staehelin, '74;
Shimono and Clementi, '77). In keratinizing
epithelial cells, the particles are found on both
faces regardless of whether glutaraldehyde fixation has beenused (Breathnach et al., '73; '76).
~
Received June 13, 1980; accepted July 10, 1980.
2
DOUGLAS E. KELLY AND AILEEN M. KUDA
Some workers have suggested that a relationship exists between the fine cytoplasmic
traversing filaments and the intramembranous particles (McNutt and Weinstein, ’73;
Kelly and Shienvold, ’76; Leloup et al., ’791,but
Staehelin and Hull (‘78) diagrammed a scheme
in which a network of small caliber filaments
(termed “linkers”) extend from the desmosomal
plaque of one cell, directly through its membrane, across the intercellular gap, through the
membrane of a neighboring cell, and into its
plaque. In this scheme, intermediate filaments
are in turn anchored to the plaques by another
set of filaments, the plaque being a region of
interconnection between the two sets of small
caliber filaments. In contrast, Leloup et al. (’79)
have proposed that “protofilaments unravel
from tonofilaments, course through the plaque,
pierce their respective desmosomalmembranes
and end by anchoring in the midline of the
intercellular space, joining there with similar
units from the neighboring cell.” Hence, while
the various investigators are agreed on the existence of some form of traversing filaments,
and that these likely connect directly or indirectly t o intermediate filaments, there is no
consensus as to whether they terminate in the
plaque, within the membrane, or within the
intercellular space. An accurate interpretation
of the nature of particles and filaments of such
minute dimensions is obscured by imaging
phase granularity, replica granularity, replica
plastic deformity, and intentional or unintentional sublimation of the specimen prior to
replication.
In our prior investigations (Kelly and Shienvold, ’76), we were disturbed that images of
traversing filaments seen extending between
intermediate filaments and exposed desmosoma1 membrane E-face were far more obvious
and numerous in unfixed tissue than when glutaraldehyde fixation was employed. We have
therefore sought technical modifications that
might clarify the reality and extent of these
fine filamentous components. This report
summarizes results obtained by the use of abbreviated glycerination in conjunction with
glutaraldehyde fixation a t an osmolality lower
than has been used with prior methods. Stereo
imaging in which tilt axis and replica shadow
azimuth are coordinated, complementary replica comparison, and dark shadow photographic printing have also been used to advantage. We believe that these procedures allow
added assurance as to the reality of traversing
filaments and provide additional information
about their behavior during the freeze-fracture
replication process. The resultant observations
offer new insight on the mode of termination of
these filaments within desmosomal membranes, and also clarify the architecture of
hemidesmosomes.
MATERIALS AND METHODS
Larval west coast newts, Taricha torosa,
were maintained in the laboratory after hatching. As noted in our prior studies, the epidermis
of these animals provides highly ordered and
distinct desmosomes and hemidesmosomes.
Animals of 23-50 mm (approximate lengths)
were anesthetized with tricaine (ethyl maminobenzoate methanesulfonate) and tail
pieces were excised. The tissues used for the
images in this report were fixed a t room temperature for 30minutes in 1.Wo glutaraldehyde
with 0.1 M cacodylate buffer (pH 7.4) and 5mM
CaC1, (310mOsm), embedded in 3.0%agar, and
sliced on a Sorvall TC-2 tissue chopper.The 100
pm slices were freed of agar and glycerinated a t
room temperature for limited periods ranging
from 1 to 2.5 hr (total time) in 10% and 20%
glycerol in cacodylate butYer. After sandwiching between two gold discs the tissues were
quickly frozen in liquid Freon 22 cooled to liquid nitrogen temperature, then fractured a t
-110°C in a Balzers BAF 301 instrument
equipped with a double replica device. Platinum-carbon replication of the fractured surfaces was performed with minimal etching and
a t a 45“ angle. Additional carbon reinforcement was applied from 90”in two layers as the
tissue was allowed to warm to -30°C. Replicas were processed through methanol,
household bleach, distilled water, 1.0% acetic
acid, and several water rinses before being
picked up for study on 75 or 100 mesh parlodion-coated grids.
Replicas were examined with a JEOL-1OOC
microscope equipped with a goniometer stage;
stereo pairs were obtained after adjustment of
the goniometer stage to achieve parallel
alignment of tilt axis and the shadow azimuth
of the specimen. This maximizes the stereoscopic effect of the 12-14” angle of tilt (see
Observations-Technical). Final dark shadow
photographic prints were made with an intermediate positive transparency, which was a direct contact exposure of the original EM negative.
NOTE: Stereo-pair micrographs are mounted for viewing
with a standard cartographer‘s stereo-viewer or
using a wall-eyed viewing technique. Cross-eyed
viewing will provide a confused “false-stereo”
image.
DESMOSOMAL TRAVERSING FILAMENTS
3
Fig. 1. Two freeze-fracture stereo-electron micrograph
pairs of a desmosome depicting conventional light shadow
imaging (top pair) and dark shadow imaging (bottom pair).
Note that finer details of the desmosome such as 1&15 nm
tonofilaments (TO),6-8 nm traversing filaments VR), and
intramembranous particles of the P-face (P) are revealed
with more distinct elevation when the dark shadow imaging
is employed. The rest of the replica-stereo pairs in this report
have been reprodud by the dark shadow imaging technique. Note example of a traversing filament, whi+ extends
from the cytoplasm onto the desmosomal E-face (arrow). (El,
E-face; (IS),intercellular space. x 58,600. The accompanying
micrographof a sectioned desmosomedisplays major components and the approximate fracture (dotted line) achieved in
the replica.
4
DOUGLAS E. KELLY AND AILEEN M. KUDA
OBSERVATIONS
Technical considerations
The use of relatively low OSmOlalitY fixation
and shortened duration of glycerol treatment
has resulted in better quality freeze-fracture
replicas of lama1 amphibian desmosomes and
hem~desmosomesthan we have achieved with
Previous procedures. In particular, fine filaments, which heretofore were only suggested
in unfixed preparations and not seen with claritY in fixed material, are now revealed distinctly and with consistency.
Analysis of fine structural features in replicas of desmosomes and hemidesmosomes has
been aided by the coordinated use of several
refined techniques. First, stereo-imaging is
helpful in establishing a n accurate appreciation of the full three-dimensional relief contained in freeze-fracture replicas. Analysis Of
single images is reasonably accurate with regard to the two lateral dimensions but Often
provides a n underestimation of depth. Therefore, the illustrations in this article are portrayed in stereo-pairs. They are arranged for
viewing with a StereO-VieWer; crosseyed Viewing will require reversal of the images
presented, left for right, in order to avoid confusing false stereo-imaging. Wall-eyed viewing
can be used in lieu of a stereo-viewer and without reversing the images.
Second, we have found that fine details are
more distinctly presented when a "dark
shadow" print is used for final analysis. Two
identical stereo-images (made from the same
negatives) are shown in Figure 1. The
stereo-pair has been prepared by the conventional light shadow technique, and the
lower pair illustrates dark shadow processing
through the use of an intermediate tramparencY. Both Pairs Provide adequate stereo-imaging if oriented as presented here, SO that the
dark sides of fine raised details face downward
in each illustration. However, close comparison
of the two images will show a n enhanced distinctness and appreciation for elevation of finer
details (e.g. filaments and particles) in the
lower, dark shadow stereo-pair. Because of this
advantage, all subsequent figures in this report
are prints processed by the dark shadow technique. In such images, light areas in the replica
represent regions of heavy platinum deposition, and dark areas are regions protected from
such deposition (shadowed in a freer sense of
the word). With the exception of the top pair in
Figure 1, all figures are presented with the
direction (azimuth) of metal deposition from
the top. This is opposite to the conventional
orientation used for light shadow images.
Third, a n aspect commonly overlooked in the
use of stereo-pairs with freeze-fracture replicas
involves coordination of metal deposition direction and tilt =is. Much of the threedimensional quality of a single conventional micrograph of a freeze-fracture replica derives from
shadow direction, with the attendant necessity
of orienting the micrograph SO that the direction of metal deposition is from the bottom of a
figure. Inverting this relationship reverses
perceived depth and confuses true relief, unless
a reversed, or dark, shadow image isutilized. In
any stereo-pair, three-dimensional information is provided by the angular differencein the
optic
with
each member ofthe pair
has been obtained and viewed. This is achieved
tiit alongan axisthat necesthrough specimen
sarily lies vertical in the &sewer's field of vision. Rotation of that axis gradually reduces,
and at 90" cancels, the three-dimensional effect. Metal deposition direction and tilt axis
azimuth can be potentially reinforcing or antagonistic in presentation of depth. If maximal
reinforcement is toresult, then the metal &Position azimuth of areplica must be arranged to
coincide with the tilt axis used in stereo electron microscopy. All of the illustrations in this
report,as well as many others we have studied,
have been coordinated carefully to provide this
reinforcement.
Fourth, we report here the first published
complementary
replica images of desmosomes
of comand hemidesmosomes. comparison
plementary replicas provides some additional
insight into the arrangement of finer components of these adhesive structures. When complementary replicas have been achieved and
the same d e ~ ~ s o m e&midesmosame 10cated on each replica, we have taken stereo
pairs of both members using coordinated tilt
and &adow
in order to provide the advantages of all the above refinementsin a complementary pair comparison. Several examples
are included in this report ( ~ i2 ,~3 and
~ .8).
Biological findings
In previous studies (e.g. Breathnach et al.,
'72, '73, '76; McNutt and Weinstein, '73; Kelly
and Shienvold, '76; Caputo and Peluchetti, '77;
Staehelin and Hull, '78; Brown and Ilic, '79) it
has been noted that the intramembranous particles characteristic of desmosomes are about
10-15 nm in diameter, rather closely packed,
but of irregular shape and orientation. When
DESMOSOMAL TRAVERSING FILAMENTS
5
Fig. 2. Stereo-pairsof complementaryreplicas (topand bottom) in which split membranes of three desmosomal territories
are visible (encircledby dotted lines). Note the difference in the caliber of tonofilaments (TO)and traversing filaments (TR),
both of which appear as elevations on both sides of the replica. Typical desmosomal intramembranous particles are Seen on
P-faces (P)of each desmosomal territory, but the corresponding desmosomal E-face is relatively featureless in these replicas.
One desmosomal territory discloses traversingfilamentsextendingfrom the cytoplasm onto the surfaceof the E-face (arrows).
x 64,000
fixation is avoided, the particles of l q a l newt
epidermal desmosomes appear to remain adherent to the E-fracture face, but infixedpreparations they are found predominately on the Pfracture face (Kelly and Shienvold, '76). These
relations and sizes are confirmed in the present
observations of differently fixed epidermis
(Figs. 1, 2, 3 and 51, but in addition 8-10 nm
thread- or ridge-like profiles are also a part of
the P-face desmosomal territory, wedged
among the aggregated particles. In most fixed
preparations, the corresponding E-face appears
smooth and devoid of particulation. However,
in some of the present replicas, we have noted
6
DOUGLAS E. KELLY AND AILEEN M. KUDA
Fig. 3. Stereo-pairsof complementaryreplicas (topand bottom) of the split membranes of a desmosomedepicted at higher
magnification.The desmosomal P-face(P) displaysclosely aggregated, irregularly shaped, 10- 15nm particles, many of which
are attached or related to enlongate ridge-like structuresof approximately the same or smaller caliber (arrows).In the upper
replica, the desmosomal E-face (Elshows a slight indication of a finer particulate “pebbling.” (IS), intercellular space.
x 117,000
Fig. 4. Stereo-pair showing the E-face aspect of a desmosomal territory (E).A distinct pebbling is evident, the individual
particles of which are smaller in diameter and ofmore irregular shape than either the general or desmosomal intramembranous particles found adherent to the P-face (P) of the split membrane of the adjacent cell. The intercellular space of the
desmosome displays linear profiles (arrows), some of which seem to enter the two desmosomal membranes. x 72,700
Fig. 5. Stereo-imagesof a replica showing the apical junctional region between two surface epidermal cells. The apical
epithelial surface is at the bottom of the picture, and next to it a tight junction (TJ)is visible in the replica. Two desmosomal
territories (outlined) are identifable by the typical particulate aggregates along the P-face (P) of the cell on the right.
to the line where fractured cytoplasm intersects the
Traversing filaments (TR)are visible coursing from tonofilaments (TO)
cytoplasmic leaflet of the desmosomal membrane. They appear to penetrate that leaflet and relate to the P-face desmosomal
ridges or particles. Linear profiles in the intercellular space (arrows) may also relate to the particles after penetrating the
external desmosomal membrane leaflet. (E), E-face. x 72,700
DESMOSOMAL TRAVERSING FILAMENTS
7
8
DOUGLAS E. KELLY AND AILEEN M. KUDA
E-face desmosomal territories in which a distinct finer “pebbling” is apparent (compare
Figs. 2, 3, 4 and 6). The size of these E-face
particles is quite variable, but nearly always
smaller than 8 nm. The typical P-face particulation is regularly seen in all desmosomal territories whether or not exposed E-faces of the
same desmosome appear smooth or pebbled.
Therefore, E-face pebbling does not seem to be
the result of transfer of entire particles from the
P-face. The reason for appearance or nonappearance of E-face pebbling is unclear; it is
possible that the structure underlying this particulation is often obscured by the thickness of
the replica or by the angle of metal deposition.
Thus far no close correlation has been possible
between P-face particles and the E-face pebbling. It is difficult to determine if E-face pebbling is due only to elevated particles or is in
part representative of minute indentations.
Cytoplasmic filaments are replicated distinctly and with less distortion in fixed preparations (see also McNutt, ’76). This is true of
both intermediate filaments of 10-12 nm diameter and finer microfilaments (less than 8 nm),
which occupy desmosomal as well as other
peripheral regions of the epithelial cells studied (Figs. 1, 5 and 6). Interestingly, both
categories of filaments appear as elevations in
the replicas and never as pits, a behavior in the
freeze-fracture process that is not unlike that
shown by myosin filaments when skeletal muscle is fractured (Bullivant et al., ’72). Apparently, during fracturing such filaments are
plucked slightly out of the frozen cytoplasm on
both faces of the fracture. Moreover, the resulting images do not show precise alignment of
filaments on the opposite fracture faces, indicating that distortion produced by fracturing is
random. These are particularly evident when
complementary replicas are studied (Fig. 2).
Groups of intermediate filaments (commonly
identified as tonofilaments in epidermis) make
looping excursions past the cytoplasmic
plaques of desmosomes or hemidesmosomes. It
does not appear that these tonofilaments terminate a t the membranes or plaques of desmosomes or hemidesmosomes. Rather, a second
population of filament appears to intervene between tonofilament loops and desmosomal or
hemidesmosomal membranes. These “traversing” filaments are clearly visible in the present
replicas of fixed desmosomes as narrow aggregated elevations, each somewhat less than 8
nm in diameter (Figs. 2, 5 and 6). Like tonofilaments, each traversing filament appears as
an elevation, even in complementary replicas,
and corresponding depressions are not observed. Profiles of traversing filaments are distinct and regular in these preparations.
Though elevated from the fracture surface,
they do not display the excessive plastic deformity that has been encountered in the same
areas of unfixed material (Kelly and Shienvold,
’76). As in those previous studies, traversing
filaments are occasionally seen to extend from
the cytoplasm in the desmosomal plaque region
onto the E-face of a split membrane (see Figs. 1
and 2). Such images may represent exposed,
but not displaced, insertions of the traversing
filaments into the membrane interior. Alternately, they may be severed and extruded filament tips that fall back on the replica surface
after the fracture process. More important, the
present studies disclose images in which a
given traversing filament profile can be seen to
extend through the cross-fractured cytoplasmic
leaflet to end in relationship to a P-face ridge or
particle (Figs. 5, 6 and 7). Images observed to
date do not disclose with certainty whether
traversing filaments extend beyond the particles to penetrate completely the desmosomal
membrane. Nor is their nature within the
plaque region, or the architecture of their attachment to tonofilament loops, yet discernible.
Fig. 6. Higher magnification stereo-pair depicting freeze-fractured desmosomal membrane faces (P and E). The P-face
particulation is typical, and a faint indication of E-face pebbling is apparent in this replica. Traversingfilaments (TR)are
visible near and within the cross-fractured desmosomal cytoplasmic membrane leaflet. Stereo-imaging reveals the close
relationship or continuity that exists between the P-face desmosomal ridges and particles and the traversing filaments.
Linear images within the intercellular space appear to reach the P-face (arrows).Such images are suggestive of filamentous
components extending into or across the intercellularspace, but in our studies these have been seen with less frequency than
images of traversing filaments penetrating from the cytoplasm to join the P-face particles. X 72,700
Fig. 7. High magnification stereo-pair showing a free%-fractured desmosome in which both cytoplasmic and intramembranous components are visible in each of the participant cells. Typical desmosomal P-face particulation is apparent
within the membrane of the cell on the left. Traversing filaments (TR)extend from the region of tonofilaments(TO) and in
some instances (arrows) can be traced across the fractured cytoplasmic leaflet where they display continuity with P-face
P-face; (E), E-face. x 122,000
particles. (P),
DESMOSOMAL TRAVERSING FILAMENTS
9
10
DOUGLAS E. KELLY AND AILEEN M. KUDA
Some of the replicas provide images suggesting a filamentous structure within the intercellular desmosomal gap (see Figs. 4,5 and 6).
Occasionally these extracellular linear profiles
appear to penetrate the extracellular leaflet
and end on the P-face. The images examined to
date, however, do not reveal a special relationship of these filaments with the P-face particles.
The basalmost cells of larval newt epidermis
display prominent hemidesmosomes. These
can often be studied in the same replicas in
which desmosomes appear. As in the case of
desmosomes, the clarity of hemidesmosomal
components has been improved with the newer
techniques. In our prior studies (Shienvold and
Kelly, '76) some larval newt epidermal hemidesmosomal intramembranous particles were
noted to be much larger than the population in
desmosomes. These 20-30 nm particles were
observed to cling with equal frequency to P- and
E-facesin fixed preparations, while adhering to
the P-face almost exclusively in unfixed material (a pattern unlike that of desmosomes).The
present observations of fixed preparations confirm the size difference for most hemidesmosoma1 particles, but comparisons of complementary replicas (Fig. 8) suggest a slightly
different interpretation regarding distribution.
The largest (20-30 nm) hemidesmosomal particles are quite irregular in shape, are somewhat widely separated and tend to cling almost
exclusively to the P-face in these fixed preparations. The E-face hemidesmosomal territory
does disclose particles, but for the most part
these are smaller, even more widely separated,
and quite similar to the 10-15nm desmosomal
particles. Moreover, close inspection reveals
that the smaller particles are also found on the
P-face territory scattered among the large particles. In addition, the hemidesmosomal P-face
displays numerous longer, 10 nm caliber, ridgeor thread-like profiles that also resemble those
of the desmosomal P-face. These are rare on the
E-face. The hemidesmosomal E-face also displays a somewhat roughened or pitted background, the depressions of which do not precisely correlate withparticles, large or small, of
the complementary P-face.
Like the situation in desmosomes, traversing
filaments are a n integral component of hemidesmosomes, spanning the region between
tonofilament bundles and the hemidesmosomal
membrane. However, a relationship of traversing filaments to the special large particles of
the hemidesmosomes is not indicated clearly in
the images obtained thus far. Nor have we yet
been able to demonstrate a connectionbetween
traversing filaments and the smaller particles
seen widely scattered on both P- and E-face
hemidesmosomal territories.
DISCUSSION
Desmosomes and hemidesmosomes a r e
highly complex structures in their intramembranous and cytoplasmic organization. While
much remains to be worked out concerning the
details of this complexity, some new insight is
provided by the techniques and images described here.
First of all, the intramembranous organization within desmosomal territories is structurally more complicated than generally perceived. Our findings confirm previous observations concerning the characteristic 10-15 nm
Fig. 8. Complementaryreplicas (top and bottom) shown
in stereo-pairs and depicting hemidesmosomal territories
along the basalmost cell membrane of larval newt epidermis.
Abundant tonofilaments (TO)are seen in the cleaved cytoplasm to the right. Characteristic large (20-30 nm) irregularly shaped particles are loosely clustered in each of many
hemidesmosomal domains distributed over the P-face (P).
Smaller (10-15 nm) particles are seen amongthe larger ones
and also on the complementary E-face hemidesmosomalterritories. Elongate linear profiles are also found on the P-face
(arrows), in some instances in close proximity to the large
P-face particles. The hemidesmosomal domains of the complementary E-face (E) display a dispersed, finer particulation as well as an array of pits in these fixed preparations. It
has not been possible to relate the pits precisely to any
particulate components of the complementary P-face.
Traversing filaments (TR)are bunched in the cytoplasm,
where they approach the hemidesmosomal membrane. The
caliber of traversing filaments is characteristically about
one-half that of tonofilaments. x 70,000. Hemidesmosomal
components are illustrated in the above micrograph of
sectioned basal epithelial cell surface and underlying connective tissue. The approximate fracture route observed in
the replicas is indicated by the dotted line.
DESMOSOMAL TRAVERSING FILAMENTS
11
12
DOUGLAS E. KELLY AND AILEEN M. KUDA
irregular desmosomal intramembranous particles. They are closely packed and, in larval
newt epidermis, cling almost exclusively to the
P-face in fixed material and remain with the
E-face when fixation is omitted (Kelly and
Shienvold, '76). In addition, however, a bed of
finer particulation can be found on the E-face of
fixed desmosomes that should not be confused
with cases of E-face particulation previously
reported by other authors. Breathnach, et al.
('761, for example, noted that the typical 10-15
nm desmosomal particles are found scattered
over both fracture faces in replicas of unfixed
rat buccal epithelium, but their published images do not reveal the finer E-face pebbling
noted here. Leloup et al. ('79), studying fixed
calf muzzle stratum spinosum, described typical particles on desmosomal E-faces that were
similar to, though less prominent than, those
seen on the complementary P-faces (see also
Ishimura et a]., '79; Pauli et al., '79). Thus far,
the fine E-face particles seen in our study do not
seem to display any complementary relationship to the larger desmosomal particles of the
opposite face. They are seen to best advantage
in thin replicas where platinum deposition is
minimal and its angle is shallow, a relatively
infrequent combination of circumstances.
Thicker replication probably obscures the finer
details so that the desmosomal E-face is similar
to the surrounding territory. Caputo and Peluchetti ('77) reported a n elevated "plaque" over
E-faces of fixed desmosomes of normal human
epidermis, a feature limited to the stratum corneum. This plaque bears only superficial resemblance to the non-elevated E-face particulation reported here. Moreover, the replicas in
the Caputo and Peluchetti study appear rather
thick and hence are not apt to reveal the E-face
particulation, if present, in their system. No
functional significance for the finer E-face particulation can be adduced from our present evidence. However, one possibility is that they
reflect sites of filament penetration to the extracellular space, a concept to be discussed further below.
The present observations, combined with
those ofMcNutt and Weinstein ('73) and Leloup
et al. ('791, lead to a greater confidence in the
reality of traversing filaments as an essential
structural component of both desmosomes and
hemidesmosomes. Traversing filament images
were not obvious in our prior studies of fixed
larval newt epidermis (Kelly and Shienvold,
'76). With the fixation techniques now employed, the filaments are crisp and regular.
Moreover, they are now revealed with greater
precision than in our prior studies utilizing unfixed material, wherein small-caliber filament
images were apparent, but equivocal, due to
plastic distortion, which exaggerated their dimensions and obscured their directions.
The traversing filaments regularly display a
smaller caliber (5-8 nm) compared to nearby
intermediate filaments (10-12 nm), and they
occupy the region of the desmosomal plaque,
although the plaque itself is not distinct in
freeze-fracture preparations (see also Caputo
and Peluchetti, '77). The traversing filaments
appear to pierce the cytoplasmic leaflet of the
cell membrane within the junctional territory;
this is especially clear in desmosomal replicas
where the cytoplasmic leaflet has been crossfractured. (Figs. 5, 6 and 7). More important,
after penetration, the filaments appear to fuse
with desmosomal particles (Fig. 7). It now
seems likely that the irregular shapes of the
desmosomal intramembranous particles are a
reflection of their contiguity with inserted
traversing filaments. Significantly, the smallest diameter of the particles (10 nm) is only
slightly larger than the diameter of traversing
filaments. Thus, the available evidence appears to favor the earlier suggestion regarding
an essential role for the desmosomal particles
in the anchorage of the traversing filaments
into the desmosomal cell membrane (McNutt
and Weinstein, '73; Skerrow and Matoltsy, '74;
and Staehelin, '74).
Alternatively, Leloup et al. ('79) have suggested that traversing filaments penetrate
the cell membrane, but en route they attach to
the cytoplasm-facingsurface of the cytoplasmic
leaflet, forming a small bulge on the P-face, the
desmosomal particle. This interpretation
seems to point up the difficulty in discerning
accurately whether it is the intramembranous
particle or the traversing filament that pierces
the cytoplasmic leaflet, or if the two components are in reality parts of the same entity. In
our images, we interpret the continuity of the
traversing filaments through the cytoplasmmembrane interface plus the elongate nature of
many of the desmosomal particles as favoring
the incorporation of the filament tips into the
membrane and a n intramembranous association with the particles. The degree to which
desmosomal particles may reach the cytoplasmic or extracellular surfaces of the membrane
is unclear (see Lempert and Elias, '79).
The cytoplasmic extent reached by a given
traversing filament also remains problematical. Staehelin and Hull ('78) have generalized that the intermediate filaments of
DESMOSOMAL TRAVERSING FILAMENTS
desmosomes loop through the cytoplasmic
desmosomal plaque where they are in some
manner associated with traversing filaments
(which they term “linkers”).In newt epidermal
desmosomes, tonofilament loops occur for the
most part 20-40 nm away from the desmosomal
plaque, a structure seen best in sectioned material (see micrograph accompanying Fig. 1;
Kelly, ’66).In our freeze-fracture replicas, the
margin of the zone rich in traversing filament
profiles is found in association with the intermediate filament loops. Therefore, the traversing filaments must extend beyond the desmosomal plaque to reach the level of intermediate
filaments; the substance of the plaque must be
in part traversing filaments and in part another material that is packed around the filaments and not revealed in these preparations.
The mode of attachment between intermediate filaments and traversing filaments is not
clarified by the present evidence. In their study
of calf muzzle epidermis, Leloup ekal. (‘79)have
described profiles, roughly equivalent to our
traversing filaments, from freeze-fracture images in which considerable etching is apparent.
They related these images to prior biochemical
studies of isolated desmosomal complexes
(Drochmans et al., ’78), suggesting that intermediate filaments are composed of subfilaments surrounding a hollow central core. The
tips of broken intermediate filaments in such
preparations appear unraveled. Leloup et al.
(‘79) have therefore interpreted the traversing
filaments to be unraveled protofilaments from
terminating intermediate filaments. This
model is difficult to reconcile with the distinct
non-terminating, non-raveling looping profiles
of intermediate filaments that have been seen
clearly in stereo-electron microscopic studies of
sectioned newt epidermal desmosomes (Kelly,
’66).Moreover, definitive biochemical evidence
as to the molecular structure of any of the several known types of intermediate filaments is
yet lacking. In any case, it remains unclear how
tensile forces might be transmitted from the
cytoskeletal intermediate filaments to traversing filaments a t desmosomes.
Similarly, the question of the mechanism by
which adjacent membranes of the desmosomal
system are interconnected across the intercellular space is unresolved. Staehelin and
Hull (’78)envisioned individual traversing filaments (“linkers”) as extending from the cytoplasmic desmosomalplaque of one cell to that of
the next, penetrating both leaflets of both cell
membranes and incorporating a lattice-like
array along the intercellular midline. In their
13
diagram a shorter form of traversing filament
appears to attach intermediate filaments to the
plaques. Leloup et al. (‘791, by contrast, diagrammed a hypothetical midline population
of extracellular particles from which a second,
extracellular type of thin filament radiates and
extends from the extracellular space through
the membrane leaflets, connecting en route
with the desmosomal particles. Shimono and
Clementi (‘77) have postulated that desmosomal external material is in the form of “side
arms” that radiate from E-face particles within
the membrane. Since this moiety is trypsin
sensitive, unlike the mechanism that attaches
tonofilaments to the membrane (see also
Borysenko and Revel, ’73),the evidence favors
the concept of different proteins/filaments on
the two sides of each desmosomal membrane.
Our images do not permit development of a
more definitive model on this question, but
they do provide evidence that cytoplasmic filaments penetrate the cytoplasmic leaflet and
relate within the membrane to the desmosomal
intramembranous particles. Some of the images of cross-fractured desmosomal external
leaflets suggest also the possibility of filament
penetration of that leaflet and a filamentous
character for extracellular bridging material.
These images are infrequent and less distinct
compared to images of traversing filaments
penetrating the cytoplasmic leaflet. In support
of the idea of insertion of extracellular filaments, the fine “pebbling” noted on the desmosomal E-face and described above might be
interpreted as a consequence of the external
leaflet being penetrated.
There is scant firm evidence published thus
far to indicate that a single filament courses
through both leaflets, much less that the same
continuous filament is shared across the membranes of both adherent cells. Although this
remains a possibility, we deem it at least
equally feasible that different filamentous proteins are involved with intercellular linkage on
the one hand and as an intermediate filamentto-cell membrane linkage on the other.
This concept is supported by the observation
that hemidesmosomes of the same epithelium
display markedly different, larger intramembranous particles in addition to the 10-15 nm
ones. If one assumes that the attachment of
intermediate filaments to the cell membrane
requires a mechanism in hemidesmosomesthat
is similar to that in desmosomes, it follows that
the larger hemidesmosomal intramembranous
components might be more related to the function of adhesion between cell membrane and
14
DOUGLAS E. KELLY AND AILEEN M. KUDA
extracellular connective tissue components
(basal lamina, collagen, etc.). Then it is plausible to relate the 10-15 nm particles of both
desmosomes and hemidesmosomesto the insertion of cytoplasmic traversing filaments, and
the 20-30 nm hemidesmosomal particles to insertion of extracellular attachment proteins
peculiar to the hemidesmosomes. Two hemidesmosomal filament protein species should
logically be involved; one (the traversing filaments) mediating attachment of intermediate
filaments to the cell membrane; and the other
(an extracellular vehicle) serving membraneto-connective tissue adhesion. Desmosomes,
perhaps, incorporate a different extracellular
vehicle that mediates membrane-to-membrane
adhesion and does not involve a n intramembranous particle distinguishable from those
connected to traversing filaments. This line of
reasoning would be strengthened if a connection could be demonstrated between traversing
filaments and small particles or ridges in
hemidesmosomal membranes, a n aspect not
accomplished in the current study or in prior
work with unfixed hemidesmosomes (Shienvold and Kelly, '76).
The above speculations and questions stress
the desirability of efforts to define in biochemical terms the differences in the various filamentous and particulate components of desmosomes and hemidesmosomes now that some
of their anatomic relationships are becoming
more clearly understood.
ACKNOWLEDGMENTS
The authors are grateful to Dr. Richard L.
Wood for critical review of the manuscript,
and to Mrs. Delcina McMillan for expert secretarial assistance. This research was supported
by a research grant (PCM 76-18755) from the
National Science Foundation.
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