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Changes in the distribution of filament-containing septate junctions as coelenterate myoepithelial cells change shape
TISSUE & CELL 1985 17 (1) 1-11 fQ 1985 Longman Group Ltd
M. C. HOLLEY
CHANGES IN THE DISTRIBUTION OF FILAMENTCONTAINING SEPTATE JUNCTIONS AS COELENTERATE MYOEPITHELIAL CELLS CHANGE SHAPE Key words: Coelenterate,
septate
junction,
cell shape, microvilli,
myoepithelial
cell
ABSTRACT. This paper describes the redistribution of septate junctions during an increase in diameter of myoepithelial cells from mesenteries of the sea anemone Merridium senile (L). Each septum was composed of a filament core, 9.5-10.2 nm in diameter, which had a double row of lateral projections from each side to the adjacent cell membrane. Septa were arranged in patches in which neighbouring septa lay parallel, 28-33 nm apart. When anaesthetized mesenteries were stretched, myoepithelial cell layers decreased from a mean of 32 to 8 pm thick; each cell shortened and its apical diameter increased. The integrity of the septate junctions was, however, maintained. The mean perimeter of septate junctions, corresponding to that of the cells, increased from 20 to 31 pm; mean depth decreased from 3.7 lo 2.1 pm. There was no significant change in spacing between septa. Patches of septa, free to move in a fluid matrix of junction cell membranes, may form mobile attachment sites betw-een cells, thus allowing those cells to change shape. Number and distribution density of microvilli decreased when cell diameter increased. This implies that the microvilli contribute membrane to the cell surface as its surface area increases. Gastrodermal cells are compared with epidermal cells that do not undergo dramatic changes in diameter.
cent to the mesogloea. On one surface of a mesentery the muscle processes are arranged longitudinally with respect to the animal, and on the other they are arranged radially. Septate junctions form belts around the cell apices (Grimstone et al., 1958; Green and Flower, 1980). The response of an extended mesentery to contraction of its muscle fibres can be divided into three stages. In the first stage the epithelial surfaces of the mesentery remain flat, but the two epithelial layers and the mesogloea thicken. This stage involves a decrease in surface area of the epithelia and is accompanied by cell elongation and a decrease in cell diameter. The cells, originally resembling a pavement epithelium, then resemble a columnar epithelium (Robson, 1957). Continued contraction leads to buckling of the two epithelia either side of the mesogloea. The enithelial surfaces then appear-to be corrugated although the mesentery itself is still flat. In the last stage the entire mesentery buckles (Batham and Pantin, 1951).
Introduction
This paper reports an experiment designed to examine the distribution of septate junctions during shape changes of myoepithelial cells from mesenteries of the sea anemone Metridium senile (L.). Sea anemone mesenteries are highly contractile. Their structure and function have already been considered in some detail (Hertwig and Hertwig, 1879; Batham and Pantin, 1951; Robson, 1957; Grimstone et al., 1958; 1960). A mesentery is composed of a sheet of mesogloea which has a single layer of myoepithelial cells upon each surface. Each myoepithelial cell has an epitheIial portion containing single large nucleus, and muscle processes projecting laterally from the base; the two are joined by thin cytoplasmic strands. The muscle processes are integrated to form a muscle layer adjaDepartment of
Zoology,
South
Parks
Road,
Oxford
OX13PS. Received 1
1 October
1984. 1
2
HOLLEY
The sentate iunctions must be able to accommodate changes in myoepithelial cell diameter. Septate junctions have been studied extensively from Hydra (Wood, 1959, 1977) and occur in many other invertebrates (Staehelin, 1974; Lane and Skaer, 1980; Green and Bergquist, 1982). One of the main proposed functions of the septate junctions in Hydra is to maintain the topographical relationship between the two epithelial sheets as the individual cells change shape during concentration and relaxation of the animal (Hand and Gobel, 1972). The function of the septate junctions in sea anemone gastrodermal cells may be similar, depending upon the nature of cell shape changes, but the structure of the septa is different (Green and Flower, 1980). The experiment presented in this paper was designed to test the idea that the area of cell occupied by the septate junction remains unchanged during relaxation of the mesentery; that the junction is simply redistributed around the cell. It is predicted, therefore, that as each cell increases its perimeter and decreases in depth, so too does the septate junction.
Materials and Methods Live hf. senile were obtained from the Marine Biology Laboratory, Millport, and maintained in a closed circulation sea water aquarium at 12°C. Four animals were placed in a solution of isosmotic magnesium chloride and sea water (1000 ml 7.5% MgC12.6H20 and 140 ml sea water) for 5 hr. Anaesthetic solution was pipetted into the coelenteron hourly to ensure relaxation of the mesenteries. Six directive mesenteries were then dissected from the four animals. Each mesentery was pinned flat on a wax dissecting dish with the longitudinal muscle surface uppermost. Each was then cut in half transversely; one half was stretched, over a period of about 1 min, to two to four times its original surface area and then pinned again to the wax. Mesentery extension occurs in a similar fashion under normal conditions. M. senile expands by relaxing its musculature when maintaining the hydrostatic pressure within the coelenteron (Batham and Pantin. 1950). During this process the mesenteries
are stretched and may become they are transparent.
so thin that
TEM Mesenteries, pinned to the wax sheets, were fixed for 3 hr at 4°C in glutaraldehyde (2.5%) with sucrose (16.6%) and buffered with sodium cacodylate (0.05 M, pH 7.2). They were then washed with buffer overnight, treated for 1 hr with 0~0~ (1%). dehydrated in ethanol, detached from the wax and embedded in Araldite. Since whole mesenteries were embedded, it was possible to cut blocks for sectioning from selected areas of individual mesenteries. Areas from two halves of a given mesentery were selected so that the two subsequent samples were from approximately the same area of the intact mesentery. Thin sections were prepared on a Reichert OMU4 ultramicrotome, stained with alcoholic uranyl acetate (30 min) and lead citrate (10 min), and examined with a Philips EM 400T electron microscope at 60 kV. Samples were taken only from the longitudinal muscle surface of directive mesenteries (see Batham and Pantin, 1951). Each mesentery. and the component epithelial cells. were initially sectioned longitudinally. The cut blocks were then turned so that remaining epithelial cells could be sectioned transversely. There were six pairs of results. Each pair corresponded to a piece of relaxed and a piece of stretched epithelium from the same mesentery; in both cases the epithelial cells had been sectioned longitudinally and transversely. The value ‘T’ for the Wilcoxon matched-pairs signed-ranks test (Siegal, 1956) was calculated to test the results. Measurement of septatr junctions Micrographs were taken at the same magnification and printed to a total magnification of x6000. To avoid analysis of different sections of the same cells, only one section in each plane was photographed from each block. The distribution of a septate junction can be represented as a cylinder (Fig. 1). Depth (d) was measured from longitudinal sections of epithelial cells; perimeter (p) and crosssectional area (a) from transverse sections.
SEI’TATE JUNCTIONS
AND CELL SHAPE CHANGES
-
d
sa
-
Fig 1. A septate junction represented as a cylinder. a, .. .
Cell
CrOSS-SeCtIOnal area;
d,
depth;
p,
pcrlmeter;
sa,
surfacearea of cell occupied by the septate junction.
Surface area (sa) was calculated by the product of perimeter and depth. Measurements were made using an IBAS interactive image analysis system with a digitizing pad. Cell cross-sectional areas were automatically calculated from perimeter measurements. Measurements of spacing between septa were made from micrographs printed to a magnification of x40,000. Microvilli
Transverse sections of microvilli were counted in a measured surface area from sections that grazed the surfaces of relaxed and stretched epithelia. The number of microvilli per cell was calculated from density estimates and measurements of cell cross-sectional area. Results Structure of the septate junction
The septate junction was easily recognized in all planes of section because it was densely stained and associated with amorphous electron-dense material on the cytoplasmic surfaces of the adjacent cell membranes. Cross-sectioned septa were frequently observed in cells sectioned longitudinally
3
(Figs. 2-4), but rarely observed in cells sectioned transversely (Fig. 5). this suggested that septa were organized predominantly in a horizontal plane around the circumference of each cell. Each cross-sectioned septum was composed of a filament core, 9.5-10.2 nm in diameter, which was apparently linked to the adjacent membranes by a pair of lateral projections from each side (inset Fig. 3). Similar lateral projections, 6-8 nm apart, were observed in transverse sections of cells (inset Fig. 5) and in tangential sections of the junctions (inset Fig. 6). The cell membranes on either side of a cross-sectioned septum appeared to be concave. In three dimensions neighbouring cell membranes would, therefore, resemble corrugated sheets, opposed to form series of semi-tubular canals each containing a single septum. On the cytoplasmic surfaces of the cell membranes amorphous patches of electron-dense material, 10-20 nm in diameter, were clearly associated with many of the septa (Fig. 2). Similar patches were identified in cross-sectioned cells (Fig. 5), which suggested that they were spherical and arranged in lines corresponding to the position of each septum. Their spacing of 40-70 nm meant that, in thin sections, at least one would nearly always be visible on each side of a cross-sectioned septum. Neighbouring septa were generally spaced 28-33 nm apart. Frequently, however, one or two septa of a series were apparently missing, and much larger spaces were occasionally observed up to a maximum of about 380 nm (Fig. 3). There appeared, therefore, to be numerous series of regularly spaced septa; in one case there were 14 septa in a single series (Fig. 2). Cross-sectioned septa were rarely observed down the full depth of any single septate junction. They could, however, be observed in any region of the junction but not between neighbouring cell membranes above or below it. Two major patterns were observed in obliquely sectioned membranes (Fig. 4), namely that of septa, which appeared as densely stained bars, and that of corrugated cell membranes. Tangential sections of the intercellular space revealed a complex array of septa (Figs. 68). Each septum was 9-10 nm
4
HOLLEY
across and apparently composed of two separate electron-dense lines 6-7 nm apart. Septa were straight or curved, and the maximum length recorded was about 830 nm. In patches, they were organized in regularly spaced series that corresponded directly to the patterns of cross-sectioned septa described above. In such cases they were separated by 28-33 nm spaces. Neighbouring patches of septa abutted one another at angles of up to 90” but there was little evidence that septa from different patches were continuous at such junctions (see Figs. 7, 8). These measurements, coupled with observations from different planes of section through the same region of the junction (Figs. 2, 6), strongly imply that the longitudinally and cross-sectioned septa were the same structures. Relaxed and stretched epithelia Table 1 summarizes the paired results of measurements from six mesenteries taken from four animals. Figs. 9-12 illustrate differences between relaxed and stretched epithelia from mesentery 4B.
The thickness of the longitudinal myoepithelial cell layer was significantly reduced after the mesenteries had been stretched (T=O, P <0.025). As predicted, the mean depth of the septate junctions decreased, and the perimeter increased, after the mesenteries had been stretched (T=O, RO.025). The surface area calculation, however, showed no consistent change although there was considerable variation in the rest&s. There was no systematic change in spacing between septa. The mean values of between 32 and 41 nm were greater than the 28-33 nm estimate from regularly spaced parallel septa; this was because all spaces of up to 100 nm were included in the sampling. Microvilli (Table 2) The distribution density of microvilli was decreased dramatically when the epithelia had been stretched (Figs. 9-12). Furthermore, the estimated number of microvilli per cell decreased significantly (T=O. RO.05).
Fig. 2. Transverse sections of septa between two cells aectmned longitudinally The epithelial surface is at the top of the micrograph. A continuous series of 14 cross-secttoned x x4,ootl. septa is bracketed. Arrows indicate spherical densities adjacent to each septum. Fig. 3. As Fig. 2. Regular sertes of cross-sectioned aepta were often interrupted by space\ which were variable in size (arrows and brackets). The insert (x230.000) shows that each septum was composed of a filament core with pairs of lateral projections from each srde (arrows). ~84,000 Fig. 4. As Ftg. 2. Oblique sections through septate juncttons gave rtse to two patterns; one relating to intracellular filaments (lines), the other to membrane corrugatton (arrows). x84,OtXI. Fig. 5. Septate junction regton of a cell sectioned transversely. Arrows indrcatc spherical cytoplasmic densities equivalent to those illustrated m Fig. 2. Cross-secttoned septa were rarely observed in this plane. The inset (X 160,000) shows a longitudmally sectroned septum with lateral projections 6-8 nm apart (arrows). x94.000. Fig. 6. Tangential section of a septate junctton. The epithelial surface is at the top of the micrograph. Arrows indicate lateral processes from a longitudinally sectioned septum (see inset, ~165,000). This micrograph corresponds to the upper part of Fig. 2 x94.000. Fig. 7. As Fig. 6. Septa had a tramline appearance when sectioned longitudinally. Patches x94.000 of parallel septa were often arranged at angles of up to 90” to each other (arrows). Fig. 8. As Fig. 7.
~94,000.
i
SEPTATE
JUNCTIONS
7
AND CELL SHAPE CHANGES
Table 2. Number and distribution density of microvilli Number of microvilli per cell
Microvilh per j.4mZ Mesentry
R
s
R
S
1A 1B 2 3 4A 4B
14 (1145) 31 (2574) 18 (1435) 27 (2184) 45 (3229) 26 (959)
5 (561) 10 (576) 4 (148) 3 (160) 7 (489) 9 (449)
307 396 299 385 633 271
173 230 248 137 331 258
Figures in brackets indicate the number of microvilli counted. Number of microvilli per cell was calculated from density and cell crosssectional area (Table 1).
Discussion Structure of the septate junction
The septate junction from mesenteric myoepithelial cells of M. senile is almost certainly the same as the double-septate junction described from endodermal (gastrodermal) cells of other sea anemones (Green and Flower, 1980). The double septum is interpreted here as a filament core, 9.5-10.2 nm in diameter, with a double row of lateral projections along each side which link the filament core to the adjacent cell membranes (Fig. 13). Evidence for a filament core comes mainly from longitudinal sections of cells in which the septa are cross-sectioned. Green and Flower (1980) did not illustrate such sections; they described the junctions on the basis of freeze-fracture replicas, and tangential sections following lanthanum impregnation. Their lanthanum results, which illustrate lateral projections particularly well, are very similar to those from the tangential sections illustrated here. The array of broad, shallow grooves on the E-face of their freeze-fracture replicas may correspond to membrane corrugations on either side of each septum. Adaptation of the septate junction shape changes
to cell
This paper shows that the septate junction complex is fluid and can readily be redistributed during changes in cell diameter.
SEPTATE
JUNCTIONS
AND CELL
SHAPE
CHANGES
9
The results support the prediction that the surface area of a cell occupied by a septate junction remains constant during an increase in cell diameter; an increase in perimeter is clearly accompanied by a decrease in depth. Accurate measurements of the surface areas of septate junctions are difficult because there is a wide range of cell size within a single mesentery and it is not possible to take all measurements from the same cells. Furthermore, the distribution of the junction around a cell is not perfectly cylindrical; buckling and unbuckling of the cell membrane could permit some change in cell depth and diameter without redistribution of the membrane. This may explain the result in mesentery 1A (Table l), where there is little change in perimeter but considerable increase in cell cross-sectional area. Constant spacing between neighbouring septa from relaxed and stretched mesenteries might be expected if the surface area of the septate junction remains constant, and there is no breakdown or synthesis of septa during stretching. But how can this observation be explained in terms of junction redistribution? One explanation is that septa are arranged in patches that form mobile attachment sites between cells. There may be irregular spaces between patches, but septa in each one are arranged in parallel, 28-33 nm apart. Patch length cannot easily be determined although some might be as long as the longest septum measured; that is about 830 nm. As cell diameter changes, patches move independently so that they
l-15-17-(
3
Fig. 13. Diagram of septa and adjacent cell membranes. Septa are composed of a filament core with a double row of lateral projections from each side. The cell membranes are corrugated. The black circles represent cross-sectioned filament cores and the hatched circles represent electron-dense spheres on the cytoplasmic surfaces of the membranes. All measurements are m nanometres.
Fig. 9. Relaxed mesentery 4B. Cells septate junction (arrows). X4400. Fig. 10. Relaxed junctions. X4400.
mesentery
4B.
sectioned
Cells
longitudmally
sectioned
transversely
Fig. 11. Stretched mesentery 4B; as Fig. 9. Note the decrease the cell surfaces. mu, muscle layer; me, mesogloea. x4400. Fig. 12. Stretched mesentery 4B; as Fig. 10. Note sectioned microvilli (top left hand corner of micrograph).
to show the depth
through
in number
the decrease x4400.
of the
septate
of microvilli
in number
on
of CTOSS-
10
IIOLLEI
are redistributed but remain close to one another. Spacing between septa would not change within patches although there may be some variation in spacing between patches; the latter was not detected in this study. The pattern of septa may change predictably as cell diameter increases. In relaxed mesenteries patches might be organized in a zigzag pattern which is straightened out during stretching (Fig. 14, also see Figs. 7. 8). This idea could be tested by freezefracture, or by counting the number of cross-sectioned septa in transversely sectioned cells from relaxed and stretched mesenteries; the number should be greater in cells from relaxed mesenteries. Tangential sections of junctions do not always indicate such a regular pattern, and the organization of septa may be more regular at the top of the junction, near to the cell surface, than it is lower down (Green and
Ii
P
Flower, 1980). Nevertheless. however the redistribution of septa occurs, they remain predominantly in a horizontal plane around the circumference of the cell. Patches of septa may be variable in size, coupling and uncoupling according to the state of the cell. During cell shape changes the patches may be uncoupled to increase the fluidity of the junction complex, but they may otherwise reform to increase epithelial stability (see below). Temporary links might explain the irregular phenomenon of anastomosis between filaments (Green and Flower, 1980). The time course of cell shape changes is unknown. Full contraction of a mesentery takes several seconds but the epithelial portions of cells are to some extent protected from the direct mechanical influence of their muscle processes by subepithelial fluid (Robson, 1957); cell shape changes may, therefore, be delayed. Epidermal
>I
and gastrodermal
cells
There are several interesting differences between gastrodermal and epidermal cells in sea anemones. In Calliactis parasitica, the apices of pharynx epidermal cells buckle reversibly when the animals contract (Holley, 1982); the cells do not have muscle d
IMnJu -b
(1
c
(:I -
d
ilj
H
Fig. 14. Idealized diagram to illustrate distribution of patches of septa in relaxed (a) and stretched (b) epithelia. Each block of parallel lines represents a patch of septa. When mesenteries are stretched the perimeter (p) of a septate junction increases and the depth (d) decreases. Patches can be redistributed without variation in spacing between septa.
-Ipm
Fig. 15. Comparison ot shape changes ot the dp~cal junction region from mesentenc myoepithehal cells of M. senrk (a-b). and pharynx epidermal cells of C. parasitica (c-d). ‘a’ and ‘d’ represent cells from contracted animals. and ‘b’ and ‘c’ from inflated animals. m. microvilli.
SEPTATE
JUNCTIONS
AND
CELL
SHAPE
11
CHANGES
nor do they undergo great changes in diameter (Fig. 15). Epidermal cells have a different kind of septate junction; their septa are long, wavy structures rather than the shorter, straight septa characteristic of gastrodermal cells (Green and Flower, 1980). Longer septa may increase the mechanical stability of cells, thus helping them to maintain their shape. The cytoskeleton is well developed in pharynx epidermal cells in C. parasitica (Holley, 1982). Intermediate filaments and microfilaments cross the cell apices in a pattern that would resist any increase in cell diameter. The cytoskeleton associated with the apices of myoepithelial cells from mesenteries of M. senile, however, is less extensive, with filaments apparently limited to the cell periphery. It obviously accommodates changes in cell diameter, and if it is contractile then it may even assist those changes. processes,
Microvilli
Microvilli may act as membrane stores that contribute to the extra cell surface membrane required as the cells increase in
diameter. Distribution density is expected to decrease as cell surface area increases, assuming that the number of microvilli per cell is constant. The results indicate, however, that the number of microvilli per cell decreases. Nevertheless, the measurements do not take into account microvillar length, nor do they distinguish between local and general changes of the microvillar field. Acknowledgements
I thank Dr C. R. Green for advice concerning the structure of the septate junctions and for reading the manuscript, and Dr E. A. Robson for very helpful discussion. I also thank Drs N. J. Lane, H. Saibil, G. M. Sainsbury, P. Willmer, and Professor D. S. Smith for their comments. Mr M. Lomas prepared the thin sections. Facilities for image analysis were generously provided by Dr S. Bradbury and Mrs A. Stanmore from the Department of Human Anatomy, Oxford. I am grateful to the fellows of the Queen’s College, Oxford, and to the Department of Zoology for their support.
References Batham, E. J. and Pantin, C. F. A. 1950. Muscular J. up. Biol., 21, 264289.
and hydrostatic
action in the sea anemone
Metridium senile (L).
Batham, E. J. and Pantm, C. F. A. 1951. The organisatmn of the muscular system of Metridium se&e. Q. II microsc. Sci., 92, 27-M. Batham, E. J. 1960. The fine structure of epithelium and mesogloea in a sea anemone. Q. II Microsc. Sci., 101, 481-486. Green, C. R. and Bergquist, P. R. 1982. Phylogenetic relationships within the invertehrata in relation to the structure of septate junctions and the development of ‘occluding’ junctional types. J. Cell Sci., 53, 279-305. Green, C. R. and Flower, N. E. 1980. Two new septate junctions in the pylum Coelenterata, J. Cell Sci., 42, 4%59. Grimstone, A. V., Home, R. W., Pantin, C. F. A. and Robson, E. A. 1958. The fine structure of the mesenteries of the sea anemone Metridium senile (L.). Q. II microsc. Sci., 99, 523-540. Hand, A. R. and Gohel, G. 1972. The structural organisation of the septate and gap junctions of Hydra. J. Cell Biol., 52, 397-408. Hertwig. 0. and Hertwig, R. 1879. Stud& zur Blattertheorie I. Die Actinien. Jena (Fischer). Holley, M. C. 1982. Control of anthozoan cilia by the basal apparatus. Tissue & Cell, 14, 607-620. Lane, N. J. and Skaer, H. Le B. (1980). Intercellular junctions in insect tissues. Adv. Insect Physiol. 15, 35-213. Robson, E. A. 1957. The structure and hydromechanics of the musculoepithelium in Metridium. Q. Jl microsc. Sci., 98, 265-278. Siegal, S. 1956. Non-parametric Sraristics. McGraw-Hill. Staehelin, L. A. 1974. Structure and function of intercellular junctions. ht. Rev. Cytok, 39, 191-283. Wood, R. L. 1959. Intercellular attachment in the epithelium of Hydra as revealed by electron microscopy. J. biophys. biochem. Cytol., 6, 34S352. Wood, R. L. 1977. The cell junctions of Hydra as viewed by freeze-fracture replication. J. Ultrastruct. Res., 58, 29’%315.
M. C. HOLLEY
CHANGES IN THE DISTRIBUTION OF FILAMENTCONTAINING SEPTATE JUNCTIONS AS COELENTERATE MYOEPITHELIAL CELLS CHANGE SHAPE Key words: Coelenterate,
septate
junction,
cell shape, microvilli,
myoepithelial
cell
ABSTRACT. This paper describes the redistribution of septate junctions during an increase in diameter of myoepithelial cells from mesenteries of the sea anemone Merridium senile (L). Each septum was composed of a filament core, 9.5-10.2 nm in diameter, which had a double row of lateral projections from each side to the adjacent cell membrane. Septa were arranged in patches in which neighbouring septa lay parallel, 28-33 nm apart. When anaesthetized mesenteries were stretched, myoepithelial cell layers decreased from a mean of 32 to 8 pm thick; each cell shortened and its apical diameter increased. The integrity of the septate junctions was, however, maintained. The mean perimeter of septate junctions, corresponding to that of the cells, increased from 20 to 31 pm; mean depth decreased from 3.7 lo 2.1 pm. There was no significant change in spacing between septa. Patches of septa, free to move in a fluid matrix of junction cell membranes, may form mobile attachment sites betw-een cells, thus allowing those cells to change shape. Number and distribution density of microvilli decreased when cell diameter increased. This implies that the microvilli contribute membrane to the cell surface as its surface area increases. Gastrodermal cells are compared with epidermal cells that do not undergo dramatic changes in diameter.
cent to the mesogloea. On one surface of a mesentery the muscle processes are arranged longitudinally with respect to the animal, and on the other they are arranged radially. Septate junctions form belts around the cell apices (Grimstone et al., 1958; Green and Flower, 1980). The response of an extended mesentery to contraction of its muscle fibres can be divided into three stages. In the first stage the epithelial surfaces of the mesentery remain flat, but the two epithelial layers and the mesogloea thicken. This stage involves a decrease in surface area of the epithelia and is accompanied by cell elongation and a decrease in cell diameter. The cells, originally resembling a pavement epithelium, then resemble a columnar epithelium (Robson, 1957). Continued contraction leads to buckling of the two epithelia either side of the mesogloea. The enithelial surfaces then appear-to be corrugated although the mesentery itself is still flat. In the last stage the entire mesentery buckles (Batham and Pantin, 1951).
Introduction
This paper reports an experiment designed to examine the distribution of septate junctions during shape changes of myoepithelial cells from mesenteries of the sea anemone Metridium senile (L.). Sea anemone mesenteries are highly contractile. Their structure and function have already been considered in some detail (Hertwig and Hertwig, 1879; Batham and Pantin, 1951; Robson, 1957; Grimstone et al., 1958; 1960). A mesentery is composed of a sheet of mesogloea which has a single layer of myoepithelial cells upon each surface. Each myoepithelial cell has an epitheIial portion containing single large nucleus, and muscle processes projecting laterally from the base; the two are joined by thin cytoplasmic strands. The muscle processes are integrated to form a muscle layer adjaDepartment of
Zoology,
South
Parks
Road,
Oxford
OX13PS. Received 1
1 October
1984. 1
2
HOLLEY
The sentate iunctions must be able to accommodate changes in myoepithelial cell diameter. Septate junctions have been studied extensively from Hydra (Wood, 1959, 1977) and occur in many other invertebrates (Staehelin, 1974; Lane and Skaer, 1980; Green and Bergquist, 1982). One of the main proposed functions of the septate junctions in Hydra is to maintain the topographical relationship between the two epithelial sheets as the individual cells change shape during concentration and relaxation of the animal (Hand and Gobel, 1972). The function of the septate junctions in sea anemone gastrodermal cells may be similar, depending upon the nature of cell shape changes, but the structure of the septa is different (Green and Flower, 1980). The experiment presented in this paper was designed to test the idea that the area of cell occupied by the septate junction remains unchanged during relaxation of the mesentery; that the junction is simply redistributed around the cell. It is predicted, therefore, that as each cell increases its perimeter and decreases in depth, so too does the septate junction.
Materials and Methods Live hf. senile were obtained from the Marine Biology Laboratory, Millport, and maintained in a closed circulation sea water aquarium at 12°C. Four animals were placed in a solution of isosmotic magnesium chloride and sea water (1000 ml 7.5% MgC12.6H20 and 140 ml sea water) for 5 hr. Anaesthetic solution was pipetted into the coelenteron hourly to ensure relaxation of the mesenteries. Six directive mesenteries were then dissected from the four animals. Each mesentery was pinned flat on a wax dissecting dish with the longitudinal muscle surface uppermost. Each was then cut in half transversely; one half was stretched, over a period of about 1 min, to two to four times its original surface area and then pinned again to the wax. Mesentery extension occurs in a similar fashion under normal conditions. M. senile expands by relaxing its musculature when maintaining the hydrostatic pressure within the coelenteron (Batham and Pantin. 1950). During this process the mesenteries
are stretched and may become they are transparent.
so thin that
TEM Mesenteries, pinned to the wax sheets, were fixed for 3 hr at 4°C in glutaraldehyde (2.5%) with sucrose (16.6%) and buffered with sodium cacodylate (0.05 M, pH 7.2). They were then washed with buffer overnight, treated for 1 hr with 0~0~ (1%). dehydrated in ethanol, detached from the wax and embedded in Araldite. Since whole mesenteries were embedded, it was possible to cut blocks for sectioning from selected areas of individual mesenteries. Areas from two halves of a given mesentery were selected so that the two subsequent samples were from approximately the same area of the intact mesentery. Thin sections were prepared on a Reichert OMU4 ultramicrotome, stained with alcoholic uranyl acetate (30 min) and lead citrate (10 min), and examined with a Philips EM 400T electron microscope at 60 kV. Samples were taken only from the longitudinal muscle surface of directive mesenteries (see Batham and Pantin, 1951). Each mesentery. and the component epithelial cells. were initially sectioned longitudinally. The cut blocks were then turned so that remaining epithelial cells could be sectioned transversely. There were six pairs of results. Each pair corresponded to a piece of relaxed and a piece of stretched epithelium from the same mesentery; in both cases the epithelial cells had been sectioned longitudinally and transversely. The value ‘T’ for the Wilcoxon matched-pairs signed-ranks test (Siegal, 1956) was calculated to test the results. Measurement of septatr junctions Micrographs were taken at the same magnification and printed to a total magnification of x6000. To avoid analysis of different sections of the same cells, only one section in each plane was photographed from each block. The distribution of a septate junction can be represented as a cylinder (Fig. 1). Depth (d) was measured from longitudinal sections of epithelial cells; perimeter (p) and crosssectional area (a) from transverse sections.
SEI’TATE JUNCTIONS
AND CELL SHAPE CHANGES
-
d
sa
-
Fig 1. A septate junction represented as a cylinder. a, .. .
Cell
CrOSS-SeCtIOnal area;
d,
depth;
p,
pcrlmeter;
sa,
surfacearea of cell occupied by the septate junction.
Surface area (sa) was calculated by the product of perimeter and depth. Measurements were made using an IBAS interactive image analysis system with a digitizing pad. Cell cross-sectional areas were automatically calculated from perimeter measurements. Measurements of spacing between septa were made from micrographs printed to a magnification of x40,000. Microvilli
Transverse sections of microvilli were counted in a measured surface area from sections that grazed the surfaces of relaxed and stretched epithelia. The number of microvilli per cell was calculated from density estimates and measurements of cell cross-sectional area. Results Structure of the septate junction
The septate junction was easily recognized in all planes of section because it was densely stained and associated with amorphous electron-dense material on the cytoplasmic surfaces of the adjacent cell membranes. Cross-sectioned septa were frequently observed in cells sectioned longitudinally
3
(Figs. 2-4), but rarely observed in cells sectioned transversely (Fig. 5). this suggested that septa were organized predominantly in a horizontal plane around the circumference of each cell. Each cross-sectioned septum was composed of a filament core, 9.5-10.2 nm in diameter, which was apparently linked to the adjacent membranes by a pair of lateral projections from each side (inset Fig. 3). Similar lateral projections, 6-8 nm apart, were observed in transverse sections of cells (inset Fig. 5) and in tangential sections of the junctions (inset Fig. 6). The cell membranes on either side of a cross-sectioned septum appeared to be concave. In three dimensions neighbouring cell membranes would, therefore, resemble corrugated sheets, opposed to form series of semi-tubular canals each containing a single septum. On the cytoplasmic surfaces of the cell membranes amorphous patches of electron-dense material, 10-20 nm in diameter, were clearly associated with many of the septa (Fig. 2). Similar patches were identified in cross-sectioned cells (Fig. 5), which suggested that they were spherical and arranged in lines corresponding to the position of each septum. Their spacing of 40-70 nm meant that, in thin sections, at least one would nearly always be visible on each side of a cross-sectioned septum. Neighbouring septa were generally spaced 28-33 nm apart. Frequently, however, one or two septa of a series were apparently missing, and much larger spaces were occasionally observed up to a maximum of about 380 nm (Fig. 3). There appeared, therefore, to be numerous series of regularly spaced septa; in one case there were 14 septa in a single series (Fig. 2). Cross-sectioned septa were rarely observed down the full depth of any single septate junction. They could, however, be observed in any region of the junction but not between neighbouring cell membranes above or below it. Two major patterns were observed in obliquely sectioned membranes (Fig. 4), namely that of septa, which appeared as densely stained bars, and that of corrugated cell membranes. Tangential sections of the intercellular space revealed a complex array of septa (Figs. 68). Each septum was 9-10 nm
4
HOLLEY
across and apparently composed of two separate electron-dense lines 6-7 nm apart. Septa were straight or curved, and the maximum length recorded was about 830 nm. In patches, they were organized in regularly spaced series that corresponded directly to the patterns of cross-sectioned septa described above. In such cases they were separated by 28-33 nm spaces. Neighbouring patches of septa abutted one another at angles of up to 90” but there was little evidence that septa from different patches were continuous at such junctions (see Figs. 7, 8). These measurements, coupled with observations from different planes of section through the same region of the junction (Figs. 2, 6), strongly imply that the longitudinally and cross-sectioned septa were the same structures. Relaxed and stretched epithelia Table 1 summarizes the paired results of measurements from six mesenteries taken from four animals. Figs. 9-12 illustrate differences between relaxed and stretched epithelia from mesentery 4B.
The thickness of the longitudinal myoepithelial cell layer was significantly reduced after the mesenteries had been stretched (T=O, P <0.025). As predicted, the mean depth of the septate junctions decreased, and the perimeter increased, after the mesenteries had been stretched (T=O, RO.025). The surface area calculation, however, showed no consistent change although there was considerable variation in the rest&s. There was no systematic change in spacing between septa. The mean values of between 32 and 41 nm were greater than the 28-33 nm estimate from regularly spaced parallel septa; this was because all spaces of up to 100 nm were included in the sampling. Microvilli (Table 2) The distribution density of microvilli was decreased dramatically when the epithelia had been stretched (Figs. 9-12). Furthermore, the estimated number of microvilli per cell decreased significantly (T=O. RO.05).
Fig. 2. Transverse sections of septa between two cells aectmned longitudinally The epithelial surface is at the top of the micrograph. A continuous series of 14 cross-secttoned x x4,ootl. septa is bracketed. Arrows indicate spherical densities adjacent to each septum. Fig. 3. As Fig. 2. Regular sertes of cross-sectioned aepta were often interrupted by space\ which were variable in size (arrows and brackets). The insert (x230.000) shows that each septum was composed of a filament core with pairs of lateral projections from each srde (arrows). ~84,000 Fig. 4. As Ftg. 2. Oblique sections through septate juncttons gave rtse to two patterns; one relating to intracellular filaments (lines), the other to membrane corrugatton (arrows). x84,OtXI. Fig. 5. Septate junction regton of a cell sectioned transversely. Arrows indrcatc spherical cytoplasmic densities equivalent to those illustrated m Fig. 2. Cross-secttoned septa were rarely observed in this plane. The inset (X 160,000) shows a longitudmally sectroned septum with lateral projections 6-8 nm apart (arrows). x94.000. Fig. 6. Tangential section of a septate junctton. The epithelial surface is at the top of the micrograph. Arrows indicate lateral processes from a longitudinally sectioned septum (see inset, ~165,000). This micrograph corresponds to the upper part of Fig. 2 x94.000. Fig. 7. As Fig. 6. Septa had a tramline appearance when sectioned longitudinally. Patches x94.000 of parallel septa were often arranged at angles of up to 90” to each other (arrows). Fig. 8. As Fig. 7.
~94,000.
i
SEPTATE
JUNCTIONS
7
AND CELL SHAPE CHANGES
Table 2. Number and distribution density of microvilli Number of microvilli per cell
Microvilh per j.4mZ Mesentry
R
s
R
S
1A 1B 2 3 4A 4B
14 (1145) 31 (2574) 18 (1435) 27 (2184) 45 (3229) 26 (959)
5 (561) 10 (576) 4 (148) 3 (160) 7 (489) 9 (449)
307 396 299 385 633 271
173 230 248 137 331 258
Figures in brackets indicate the number of microvilli counted. Number of microvilli per cell was calculated from density and cell crosssectional area (Table 1).
Discussion Structure of the septate junction
The septate junction from mesenteric myoepithelial cells of M. senile is almost certainly the same as the double-septate junction described from endodermal (gastrodermal) cells of other sea anemones (Green and Flower, 1980). The double septum is interpreted here as a filament core, 9.5-10.2 nm in diameter, with a double row of lateral projections along each side which link the filament core to the adjacent cell membranes (Fig. 13). Evidence for a filament core comes mainly from longitudinal sections of cells in which the septa are cross-sectioned. Green and Flower (1980) did not illustrate such sections; they described the junctions on the basis of freeze-fracture replicas, and tangential sections following lanthanum impregnation. Their lanthanum results, which illustrate lateral projections particularly well, are very similar to those from the tangential sections illustrated here. The array of broad, shallow grooves on the E-face of their freeze-fracture replicas may correspond to membrane corrugations on either side of each septum. Adaptation of the septate junction shape changes
to cell
This paper shows that the septate junction complex is fluid and can readily be redistributed during changes in cell diameter.
SEPTATE
JUNCTIONS
AND CELL
SHAPE
CHANGES
9
The results support the prediction that the surface area of a cell occupied by a septate junction remains constant during an increase in cell diameter; an increase in perimeter is clearly accompanied by a decrease in depth. Accurate measurements of the surface areas of septate junctions are difficult because there is a wide range of cell size within a single mesentery and it is not possible to take all measurements from the same cells. Furthermore, the distribution of the junction around a cell is not perfectly cylindrical; buckling and unbuckling of the cell membrane could permit some change in cell depth and diameter without redistribution of the membrane. This may explain the result in mesentery 1A (Table l), where there is little change in perimeter but considerable increase in cell cross-sectional area. Constant spacing between neighbouring septa from relaxed and stretched mesenteries might be expected if the surface area of the septate junction remains constant, and there is no breakdown or synthesis of septa during stretching. But how can this observation be explained in terms of junction redistribution? One explanation is that septa are arranged in patches that form mobile attachment sites between cells. There may be irregular spaces between patches, but septa in each one are arranged in parallel, 28-33 nm apart. Patch length cannot easily be determined although some might be as long as the longest septum measured; that is about 830 nm. As cell diameter changes, patches move independently so that they
l-15-17-(
3
Fig. 13. Diagram of septa and adjacent cell membranes. Septa are composed of a filament core with a double row of lateral projections from each side. The cell membranes are corrugated. The black circles represent cross-sectioned filament cores and the hatched circles represent electron-dense spheres on the cytoplasmic surfaces of the membranes. All measurements are m nanometres.
Fig. 9. Relaxed mesentery 4B. Cells septate junction (arrows). X4400. Fig. 10. Relaxed junctions. X4400.
mesentery
4B.
sectioned
Cells
longitudmally
sectioned
transversely
Fig. 11. Stretched mesentery 4B; as Fig. 9. Note the decrease the cell surfaces. mu, muscle layer; me, mesogloea. x4400. Fig. 12. Stretched mesentery 4B; as Fig. 10. Note sectioned microvilli (top left hand corner of micrograph).
to show the depth
through
in number
the decrease x4400.
of the
septate
of microvilli
in number
on
of CTOSS-
10
IIOLLEI
are redistributed but remain close to one another. Spacing between septa would not change within patches although there may be some variation in spacing between patches; the latter was not detected in this study. The pattern of septa may change predictably as cell diameter increases. In relaxed mesenteries patches might be organized in a zigzag pattern which is straightened out during stretching (Fig. 14, also see Figs. 7. 8). This idea could be tested by freezefracture, or by counting the number of cross-sectioned septa in transversely sectioned cells from relaxed and stretched mesenteries; the number should be greater in cells from relaxed mesenteries. Tangential sections of junctions do not always indicate such a regular pattern, and the organization of septa may be more regular at the top of the junction, near to the cell surface, than it is lower down (Green and
Ii
P
Flower, 1980). Nevertheless. however the redistribution of septa occurs, they remain predominantly in a horizontal plane around the circumference of the cell. Patches of septa may be variable in size, coupling and uncoupling according to the state of the cell. During cell shape changes the patches may be uncoupled to increase the fluidity of the junction complex, but they may otherwise reform to increase epithelial stability (see below). Temporary links might explain the irregular phenomenon of anastomosis between filaments (Green and Flower, 1980). The time course of cell shape changes is unknown. Full contraction of a mesentery takes several seconds but the epithelial portions of cells are to some extent protected from the direct mechanical influence of their muscle processes by subepithelial fluid (Robson, 1957); cell shape changes may, therefore, be delayed. Epidermal
>I
and gastrodermal
cells
There are several interesting differences between gastrodermal and epidermal cells in sea anemones. In Calliactis parasitica, the apices of pharynx epidermal cells buckle reversibly when the animals contract (Holley, 1982); the cells do not have muscle d
IMnJu -b
(1
c
(:I -
d
ilj
H
Fig. 14. Idealized diagram to illustrate distribution of patches of septa in relaxed (a) and stretched (b) epithelia. Each block of parallel lines represents a patch of septa. When mesenteries are stretched the perimeter (p) of a septate junction increases and the depth (d) decreases. Patches can be redistributed without variation in spacing between septa.
-Ipm
Fig. 15. Comparison ot shape changes ot the dp~cal junction region from mesentenc myoepithehal cells of M. senrk (a-b). and pharynx epidermal cells of C. parasitica (c-d). ‘a’ and ‘d’ represent cells from contracted animals. and ‘b’ and ‘c’ from inflated animals. m. microvilli.
SEPTATE
JUNCTIONS
AND
CELL
SHAPE
11
CHANGES
nor do they undergo great changes in diameter (Fig. 15). Epidermal cells have a different kind of septate junction; their septa are long, wavy structures rather than the shorter, straight septa characteristic of gastrodermal cells (Green and Flower, 1980). Longer septa may increase the mechanical stability of cells, thus helping them to maintain their shape. The cytoskeleton is well developed in pharynx epidermal cells in C. parasitica (Holley, 1982). Intermediate filaments and microfilaments cross the cell apices in a pattern that would resist any increase in cell diameter. The cytoskeleton associated with the apices of myoepithelial cells from mesenteries of M. senile, however, is less extensive, with filaments apparently limited to the cell periphery. It obviously accommodates changes in cell diameter, and if it is contractile then it may even assist those changes. processes,
Microvilli
Microvilli may act as membrane stores that contribute to the extra cell surface membrane required as the cells increase in
diameter. Distribution density is expected to decrease as cell surface area increases, assuming that the number of microvilli per cell is constant. The results indicate, however, that the number of microvilli per cell decreases. Nevertheless, the measurements do not take into account microvillar length, nor do they distinguish between local and general changes of the microvillar field. Acknowledgements
I thank Dr C. R. Green for advice concerning the structure of the septate junctions and for reading the manuscript, and Dr E. A. Robson for very helpful discussion. I also thank Drs N. J. Lane, H. Saibil, G. M. Sainsbury, P. Willmer, and Professor D. S. Smith for their comments. Mr M. Lomas prepared the thin sections. Facilities for image analysis were generously provided by Dr S. Bradbury and Mrs A. Stanmore from the Department of Human Anatomy, Oxford. I am grateful to the fellows of the Queen’s College, Oxford, and to the Department of Zoology for their support.
References Batham, E. J. and Pantin, C. F. A. 1950. Muscular J. up. Biol., 21, 264289.
and hydrostatic
action in the sea anemone
Metridium senile (L).
Batham, E. J. and Pantm, C. F. A. 1951. The organisatmn of the muscular system of Metridium se&e. Q. II microsc. Sci., 92, 27-M. Batham, E. J. 1960. The fine structure of epithelium and mesogloea in a sea anemone. Q. II Microsc. Sci., 101, 481-486. Green, C. R. and Bergquist, P. R. 1982. Phylogenetic relationships within the invertehrata in relation to the structure of septate junctions and the development of ‘occluding’ junctional types. J. Cell Sci., 53, 279-305. Green, C. R. and Flower, N. E. 1980. Two new septate junctions in the pylum Coelenterata, J. Cell Sci., 42, 4%59. Grimstone, A. V., Home, R. W., Pantin, C. F. A. and Robson, E. A. 1958. The fine structure of the mesenteries of the sea anemone Metridium senile (L.). Q. II microsc. Sci., 99, 523-540. Hand, A. R. and Gohel, G. 1972. The structural organisation of the septate and gap junctions of Hydra. J. Cell Biol., 52, 397-408. Hertwig. 0. and Hertwig, R. 1879. Stud& zur Blattertheorie I. Die Actinien. Jena (Fischer). Holley, M. C. 1982. Control of anthozoan cilia by the basal apparatus. Tissue & Cell, 14, 607-620. Lane, N. J. and Skaer, H. Le B. (1980). Intercellular junctions in insect tissues. Adv. Insect Physiol. 15, 35-213. Robson, E. A. 1957. The structure and hydromechanics of the musculoepithelium in Metridium. Q. Jl microsc. Sci., 98, 265-278. Siegal, S. 1956. Non-parametric Sraristics. McGraw-Hill. Staehelin, L. A. 1974. Structure and function of intercellular junctions. ht. Rev. Cytok, 39, 191-283. Wood, R. L. 1959. Intercellular attachment in the epithelium of Hydra as revealed by electron microscopy. J. biophys. biochem. Cytol., 6, 34S352. Wood, R. L. 1977. The cell junctions of Hydra as viewed by freeze-fracture replication. J. Ultrastruct. Res., 58, 29’%315.