J. Cell Sci. 85, 113-124 (1986)
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SPLITTING AND INTERNALIZATION OF THE
DESMOSOMES OF CULTURED KIDNEY EPITHELIAL
CELLS BY REDUCTION IN CALCIUM
CONCENTRATION
D. L. MATTEY AND D. R. GARROD
Cancer Research Campaign Medical Oncology Unit, Centre Block, CF99,
Southampton General Hospital, Southampton SO9 4XY, England
SUMMARY
Desmosome assembly may be induced in simple epithelial (MDBK and MDCK) cells maintained in low calcium medium (LCM: [Ca 2+ ] <0-05mM) by raising [Ca 2+ ] to that of standard
culture medium (SM: [Ca 2+ ] = l-8mM). Here it is shown that if cells in SM are simply returned
to LCM, their desmosomes split in the intercellular region within 15min and the desmosomal
halves are internalized within 30min. This is the first time that desmosome splitting has been
shown to occur in response to a reduction in [Ca 2+ ] rather than Ca 2+ chelation. Fluorescent
antibody staining shows that the desmosomal glycoproteins as well as the plaque constituents are
internalized, although a pool of the glycoproteins known as desmocollins remains at the cell
surface, apparently unassociated with other desmosomal components. Desmosomal halves that
have been recently internalized in response to LCM treatment do not return to the cell surface to
participate in new desmosome formation. MDCK cells are able to form new desmosomes rapidly
(15—30min) while old desmosomes continue to be internalized.
The desmosomes of MDBK cells remain sensitive to splitting and internalization in response to
reduction in [Ca 2+ ] for up to 14 days of culture in SM. In contrast, the desmosomes of MDCK
cells become resistant to reduction in [Ca 2 + ], as well as Ca 2+ chelation by EGTA, after 4-5 days
in SM. When treated with LCM or EGTA, MDCK cells with 'stabilized' desmosomes partially
separate but remain attached to each other at some points. Regions of attachment stain brightly
with anti-demosomal antibodies and are characterized by 'giant' desmosomes, up to 4;um long,
roughly 20 times larger than those formed in cells in SM. These giant desmosomes may form by
lateral fusion of small desmosomes.
INTRODUCTION
The purpose of this paper is to present some new observations on the breakdown
of desmosomes in response to reduction in [Ca 2+ ]. Previous work has shown that
the chelating agent EDTA causes the desmosomes of some intact tissues to split
in the intercellular region. This is the case with desmosomes in simple epithelia
(Borysenko & Revel, 1973). Desmosomes in stratified epithelia, on the other hand,
require trypsin digestion in order to split them (Borysenko & Revel, 1973; Overton,
1962). Many cultured cell types are routinely dissociated by treatment with a mixture
of trypsin and EDTA, although the desmosomes (and zonulae adhaerentes) of
MDBK cells may be split by treatment with the calcium-specific chelating agent
EGTA (Kartenbeck et al. 1982; Cowinef al. 1984). However, simple epithelial cells
Key words: desmosome, calcium, kidney cells, epithelial cells, cell adhesion.
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in culture may be induced to form desmosomes by raising the [Ca2+] from 0-05 mid
to 1 -8 mM. It is of interest, therefore, to determine whether desmosome splitting may
be induced simply by placing the cells at a [Ca2+] of 0-05 mM, or whether actual
calcium chelation is required, as implied by previous studies. Calcium-induced
desmosomes of human keratinocytes have been shown to remain susceptible to
splitting by EDTA treatment for 2h after their formation, but thereafter to become
resistant to splitting by this reagent (Watt et al. 1984).
A consequence of desmosomal splitting is that the cells internalize the halfdesmosomes, which are no longer linked by intercellular bonding (Overton, 1968,
1973; Overton & Culver, 1973; Fukuyamae* al. 1974; Shimono & Clementi, 1977;
Kartenbeck et al. 1982). Internalized desmosomes of MDBK cells have been shown
to react with antibodies to desmoplakins, the high molecular weight desmosomal
proteins (Kartenbeck et al. 1982). However, Cowin et al. (1984) showed that the
desmocollins, which are desmosomal glycoproteins, remain on the surface of MDBK
cells after EGTA treatment. Clearly, a fuller study of the behaviour of desmosomal
components during internalization is required, especially since the fate of the other
major desmosomal components is unknown.
In this paper we show that the desmosomes of MDBK and MDCK cells may be
split simply by reducing [Caz+] from l-8mM to 0-05 mM. This remains true for
MDBK cells for at least 14 days after desmosome formation. However, the desmosomes of MDCK cells become resistant to reduction in [Ca2+] and even to
divalent cation chelation, after approximately 4 days in culture. Fluorescent antibody
staining is used to study the behaviour of various desmosomal components during
internalization of half-desmosomes. We also show that cells can internalize 'old'
desmosomes and form 'new' ones simultaneously.
MATERIALS AND METHODS
The cells, antibodies and techniques used in the present paper are identical to those used in the
accompanying paper (Mattey & Garrod, 1986).
RESULTS
Internalization of desmosomes
Electron microscopy. MDBK and MDCK cells were placed in standard medium
(SM: [Ca2+] = 1-8mM) for 24-48h, ample time for desmosome formation (Mattey
& Garrod, 1986). They were then transferred into low calcium medium (LCM:
[Ca + ] = 0-05 mM) and fixed for electron microscopy at various times. In both cell
types desmosome splitting occurred within 15min (Fig. 1A). After 30min many of
Fig. 1. Electron micrographs showing splitting and internalization of desmosomes of
MDCK cells transferred from SM to LCM for 15min (A), 30min (B) and 3h (C,D).
A. Note fibrillar intercellular material at splitting desmosomes. The desmosomal intercellular space is much wider than'that of mature desmosomes of cells in SM (Mattey &
Garrod, 1986, fig. 6C). B. Note close association of internalized desmosomes (arrows)
with intermediate filaments. C,D. Higher-power photographs showing typical examples
of internalized desmosomes. Bars: A, 0-3 ^m; B, 0-4jUm; C, 0-2 fim; D,
Desmosome intemalization in kidney cells
III
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D.L. Mattey and D. R. Garrod
these half-desmosomes had become internalized in vesicular structures (Fig. IB).
Plaque material and tonofilaments remained attached to the cytoplasmic side of the
internalized membranes. With further time in LCM these internalized structures
became detectable deep within the cytoplasm of the cells in close association with
bundles of intermediate filaments.
Internalized desmosomes showed a variety of forms. These included circular and
crescent-shaped segments of membrane associated with tonofilaments and aggregates
of electron-dense material (Fig. 1C,D) or, more rarely, paired plaque structures (see
Mattey & Garrod, 1986, fig. 4).
Immunofluorescence microscopy. The staining patterns produced by the various
anti-desmosomal antibodies on MDBK and MDCK cells maintained in SM are
described in the accompanying paper (Mattey & Garrod, 1986). In both cell types
the boundaries between cells in contact are stained by the antibodies. This staining
may appear punctate with anti-desmoplakin (figs 1C, 6C, Mattey & Garrod, 1986)
and anti-175-164K, or linear with anti-desmocollin (fig. 6L, Mattey & Garrod,
1986) and anti-83/75 K (K represents 103Mr).
The sequence of changes that occurred when MDCK cells that had been maintained in SM for less than 3 days were transferred into LCM, as revealed by antidesmoplakin staining, is shown in Fig. 2. At 5 min some gaps appeared between
the cells and the separated surfaces stained brightly, the staining presumably
representing separated desmosomal halves (Fig. 2A). By 15 min the bright ring
of desmoplakin staining in each cell was separated from that of its neighbours
(Fig. 2B). One or two regions of contact persisted. By 30 min each fluorescent ring
became much smaller in diameter, in some cases apparently surrounding the cell
nucleus (Fig. 2C). The rings also began to fragment into dots of staining. Electron
microscopy showed that internalization of desmosomal remnants had occurred by
this time. By 4 h staining was rarely present in the form of a ring. Instead there were
many small dots together with one or two larger bright spots adjacent to the nucleus
(Fig. 2D). The cells at this stage resembled cells that had been plated into LCM
directly (Mattey & Garrod, 1986). Similar patterns of invagination were seen with
other desmosomal components (Fig. 2E,F,G). However, as well as an internalized
ring, anti-desmocollin staining of living or formaldehyde-fixed cells revealed that
some of these antigens persisted on the surface (Fig. 2H). The internalized ring
of desmoplakin staining was co-localized with a ring of anti-cytokeratin staining
(Fig. 3A,B).
In MDBK cells a similar sequence of events occurred, but was rather less striking
because there were fewer desmosomes. Peripheral rows of anti-desmoplakin or anti175-164 staining dots were present on separated cells after 10-15 min, presumably
representing half-desmosomes (Fig. 4A). Such dots were found within the cytoplasm of cells that had been treated with LCM for longer periods (Fig. 4B). However, they did not accumulate in a juxtanuclear spot. Internalization of anti-83/75
and anti-desmocollin staining occurred initially as a continuous ring (Fig. 4C,D).
Some punctate cytoplasmic staining was also seen with anti-83/75 K (Fig. 4D)
Desmosome internalization in kidney cells
117
Fig. 2. Fluorescence photomicrographs showing changes in patterns of staining with
anti-desmosomal antibodies in MDCK cells transferred from SM to LCM for: A, 5 min;
B, 15 min; C,E,F,G,H, 30 min; and D, 4h. A-G. Methanol fixation showing cytoplasmic staining; H, formaldehyde fixation showing surface staining. Antibodies used
were: A-D, anti-desmoplakin; E, anti-17S-164K; F, anti-83/75 K; G,H, anti-desmocollin. Bar, 20|Um.
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D. L. Mattey and D. R. Garrod
but not with anti-desmocollin. Cytoplasmic staining for desmocollins became undetectable after 4-6 h. However, desmocollin staining persisted on the cell surface in
a diffuse distribution, similar to that shown by MDBK cells that have been cultured
in LCM (fig. 7A, Mattey & Garrod, 1986).
Re-formation of desmosomes
MDBK and MDCK cells cultured for 48 h in SM were permitted to internalize
their desmosomes by switching the cells to LCM for 45 min. Cells were then
switched back to SM and fixed in methanol at various times before staining with antidesmoplakin antibody.
In MDCK cells desmoplakins became detectable at cell boundaries within
20-30 min of switching back into SM. The previously internalized desmoplakins
remained as a perinuclear ring in the cytoplasm. The desmoplakins thus appeared as
a double ring with staining at the cell boundaries due to newly formed desmosomes
and intra-cytoplasmic staining due to internalized desmosomes (Fig. 5A). The
internalized components accumulated in a juxtanuclear region (Fig. 5B) and eventually disappeared after 4 or 5h (Fig. 5C). In contrast, desmoplakin staining in
MDBK cells was first seen at cell boundaries only after 3-4 h of re-switching into
SM. By 6h the cell-boundary staining was more pronounced although dotted
staining of internalized desmosomal components was still present in the cytoplasm
well away from the cell periphery (Fig. 5D).
Stabilization of desmosomes
MDBK cells cultured for up to 14 days in SM always showed internalization of
desmoplakins within 2h of switching from SM to LCM. However, the same was
Fig. 3. Fluorescence micrographs showing co-localization of internalized desmoplakin
staining with a ring of cytokeratin staining in an MDCK cell transferred from SM to
LCM for 30 min. A. Stained with monoclonal antibody to desmoplakin I; B, stained with
guinea-pig anti-cytokeratin. Methanol fixation. Bar, 20 jum.
Desmosome internalization in kidney cells
119
Fig. 4. Fluorescence micrographs showing changes in patterns of staining with antidesmosomal antibodies in MDBK cells transferred from SM to LCM for: A, 15min;
B, 24h; C,D, 45 min. Antibodies used are: A,B, anti-desmoplakin; C, anti-desmocollin;
D, anti-83/75 K. Methanol fixation. Bar, 20 Jim.
true for MDCK cells for only 3 days after plating. After 4—5 days of culture cells
switched into LCM pulled apart from each other to some extent, but remained
attached at regions around the periphery. There was some internalization of desmosomal material as demonstrated by desmoplakin staining, but desmoplakin
staining persisted at the regions of cell contact, even after 24 h in LCM (Fig. 6A).
The cytokeratin filaments became organized into large bundles at those regions of
contact (Fig. 6C,D). After 7 days in culture there was little or no internalization of
desmoplakin-staining material, but instead very brightly staining concentrations
of desmosomal material were found at persistent regions of cell contact (Fig. 6B).
Examination of the regions of contact between these cells by electron microscopy
revealed that they consist of 'giant' desmosomes (Fig. 6E) up to 4^m long, and
therefore about 20 times larger than those found in SM.
The time of onset of desmosomal stability was dependent upon cell seeding
density. The above remarks apply to cells seeded at a density of 104cm~2. Cells
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D. L. Mattey and D. R. Garrod
Fig. 5. Fluorescence micrographs showing simultaneous formation of new desmosomes
and internalization of old desmosomes in MDCK cells (A,B,C) and MDBK cells (D)
transferred from SM to LCM for 45min and then returned to SM for: A, 1 h; B, 3h;
C, 5h; D, 6h. Staining of methanol-fixed cells with guinea-pig anti-desmoplakin.
Internalized material marked with arrowheads and staining at cell periphery with arrows.
The punctate staining areas in C (arrowheads) are regions of intercellular contact viewed
obliquely, not internalized material. Bar, 20 /zm.
Desmosome internalization in kidney cells
f*s
Fig. 6. A - D . Fluorescence micrographs showing desmosomal material in MDCK cells
maintained in SM for 4 days (A,C,D) or 7 days (B) and transferred into LCM for 24 h.
Methanol fixation staining with guinea-pig anti-desmoplakin (A,B), monoclonal antidesmoplakin I (C) and guinea-pig anti-cytokeratin (D). C,D. Showing double staining of
the same cells. E. Electron micrograph showing 'giant' desmosome in region of contact
between MDCK cells maintained in SM for 4 days and transferred to LCM for 24 h.
Bars: A - D , 20jUm; E, 0-3 fim.
121
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D. L. Mattey and D. R. Garrod
seeded at lower density took about 24 h longer to acquire desmosomal stability.
However, cells seeded at confluent density initially still took about 4-5 days to
acquire stability.
The stabilized desmosomes were not only resistant to reduction in [Ca z + ], but
also not broken down by treatment with 1 mM-EGTA for 1 h. EGTA treatment
eventually caused the cells to detach from the substratum as a sheet.
DISCUSSION
Desmosome splitting and desmosome stability
Our results show that desmosomes formed by MDBK and MDCK cells in the
presence of 1-8 mM-Caz+ may be split and internalized simply as a result of reduction
in [Ca 2+ ] to 0-05 mM. Splitting does not require treatment with chelating agents
such as EDTA or EGTA. The desmosomes of MDBK cells remain susceptible to
splitting by reduced [Ca 2+ ] for at least 14 days in culture. However, the desmosomes
of MDCK cells acquire resistance to splitting, even by treatment with EGTA, from
about 4 days of culture in SM. The desmosomes are then susceptible to splitting by
trypsin plus EDTA as used in routine passaging.
We have previously shown that keratinocytes acquire resistance to desmosomal
splitting by EDTA within 3 h of transfer into high [Ca 2+ ], and consequently we have
suggested that keratinocyte desmosomes undergo stabilization (Watt et al. 1984).
MDCK desmosomes may also undergo stabilization, but initiated later and taking a
considerably longer time to occur. We have no indication of the biochemical nature of
the stabilizing event.
The effect of reduced [Ca 2+ ] on MDBK cells and on MDCK cells cultured for less
than 4 days appears to be twofold. First the desmosomes split, and second the cells
round up. The observation that desmosomes are split at [Ca 2+ ] of 0-05 mM may
indicate that the adhesive bond in the desmosomes is extremely sensitive to [Ca 2 + ].
Whatever the nature of the stabilization that occurs in MDCK cells, it appears to
overcome this sensitivity to [Ca ] : in cells with stable desmosomes, transfer to
LCM still appears to have the same general effect on cell rounding, but the
desmosomes do not split. The alteration in cell shape probably involves both the
cytoskeleton and the splitting of intercellular junctions other than desmosomes.
However, we have not studied these in detail.
The most striking feature of MDCK cells with stable desmosomes in LCM is that
as the cells round up desmosomal staining accumulates in remaining regions of the
intercellular contact. These regions are found to consist of massive desmosomes on
electron-microscopical examination. We suggest that as the cells round up and pull
apart from each other, the desmosomes, which must be mobile within the cell
membrane, are drawn together and fuse laterally. The cytokeratin filament bundles
that are attached to the desmosomal plaques are thereby also drawn together
into larger bundles. Mobility of desmosomes within the cell membrane has been
suggested previously on the basis of indirect evidence by Klymkowsky et al. (1983)
who showed that if the cytokeratin system of one of an attached pair of cells was
Desmosome internalization in kidney cells
123
disrupted by injection of anti-cytokeratin antibody, the cytokeratin filaments in the
other cell became bundled together at a point in the region of contact between the
two. They inferred that the desmosomes had accumulated at this point. Our present
results suggest that the distribution of desmosomes is determined by some aspect of
overall cellular shape organization, possibly involving the cytoskeleton. Furthermore, the size of desmosomes appears to be determined in relation to their distribution on the cell surface rather than by some size-determining mechanism intrinsic
to them. Thus, when cellular shape organization is disrupted, desmosomes are able
to accumulate and to fuse, generating giant desmosomes. It may be significant that
the desmosomes of carcinoma cells are sometimes found to be enlarged compared
with those of their normal counterparts (Pauli et al. 1978; Hand, Garrod & Parry,
unpublished observations). This may reflect lateral desmosomal fusion as a result of
cell shape disorganization.
Desmosome internalization and re-formation
Internalization of desmosomes in MDBK cells has been shown previously to
involve internalization of desmoplakins (Kartenbeck et al. 1982), while some desmocollin staining remains at the cell surface (Cowin et al. 1984). Here we confirm
that desmocollin staining persists at the cell surface, but that some internalization
of desmocollin staining also occurs, together with staining for all other desmosomal
antigens. This is the case for both MDBK and MDCK cells.
Apart from the greater number of desmosomes and therefore greater quantity of
staining in MDCK cells, there are two principal differences between the two cell
types. First, the tendency was for internalized components to collect in one or more
juxtanuclear spots in MDCK cells, whereas in MDBK cells they were dispersed as
discrete dots within the cytoplasm. Second, the internalized components persisted
in the cytoplasm of MDCK cells even when they were cultured for long periods in
LCM, whereas with MDBK cells, staining for internalized components disappeared
after about 4 days in LCM.
In the accompanying paper (Mattey & Garrod, 1986) we suggested that calciuminduced desmosome formation in MDCK cells did not seem to involve rapid
movement of cytoplasmic desmosomal particles from the nuclear region to the
cell periphery as has been suggested by Jones & Goldman (1985) for mouse
keratinocytes. This view is reinforced by the observation that 'old' desmosomes
continue to be internalized while 'new' desmosomes form. This raises the question of
the origin of the components that form the new desmosomes. Presumably, pools of
unassembled desmosomal proteins are available within these cells. However, they are
apparently not detectable by fluorescent antibody staining until they reach the cell
periphery to participate in desmosome formation.
Lastly, the appearance of MDBK cells shortly after being transferred from SM to
LCM is different from that of cells cultured for longer periods in LCM (Mattey &
Garrod, 1986). The latter have peripheral rings of anti-desmocollin and anti-83/75
staining where cells are in contact. We assume that the appearance of these antigens
at the cell periphery involves a 'recovery' phase, a type of adaptation to culture in
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D. L. Mattey and D. R. Garrod
LCM in which cell contact is re-established. Zonulae adhaerentes junctions, but not
desmosomes, are able to form under these conditions (Mattey & Garrod, 1986). As
discussed in the accompanying paper, further investigations are needed into the
possibility of an association between the desmocollins, the 83/75 K protein and the
zonulae adhaerentes in these cells.
We thank Dr Terry Kenny for suggesting improvements to the manuscript. This work was
supported by the Cancer Research Campaign.
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(Received 12 February 1986 -Accepted 23 May 1986)