Intercisternal parallel filaments in the endoplasmic reticulum in cells of the rat subcommissural organ

© 1971 by Academic Press, Inc.

j. ULTRASTRUCTURE RESEARCH 37, 401-410 (1971)

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Intercisternal Parallel Filaments in the Endoplasmic Reticulum in Cells of the Rat Subcommissural Organ HuM-SAN giN and I-LI CHEN

Department of Anatomy, College of Medicine, National Taiwan University, Taipei, Taiwan, Republic of China Received April 19, 1971 A unique type of filaments about 12 nm thick has been found intervening in orderly arrangement between rather closely apposed cisternae of the granular endoplasmic reticulum in ependymal cells of the rat subcommissural organ. The filaments are aligned parallel to one another and spaced at uniform intervals, their course being either straight or slightly curved. Their assemblage may display a hexagonal array in end-on views. Their occurrence is confined to intercisternal regions of the cytoplasmic matrix, other cytoplasmic structures being excluded from such regions. In the present study the parallel filaments have been observed only in rats over 5 months of age, but not in the younger animals. The observations suggest that they may represent a special substance of the cytoplasm produced in association with aging of the cells. Ependymal cells of the subcommissural organ are characterized by a profusion of the endoplasmic reticulum, the cisternae of which are pleomorphic and usually distended with a flocculent material considered to be a secretory substance (8, 16). The physiological significance of this circumventricular organ is still enigmatic. In the course of studying the rat subcommissural organ, a crystalloid structure composed of rather thick filaments has sometimes been observed intervening between apposed profiles of the endoplasmic reticulum in the ependymal cells of the organ. This paper reports an electron microscopical study of this unique type of filament in subcommissural cells based on a survey of a number of materials taken from rats at different ages. MATERIALS AND METHODS Long-Evans strain rats of both sexes at ages ranging from newborn to over 2 years were used. Specimens were prepared for electron microscopy by the procedures described in the previous report (9). In brief, tissue blocks including the subcommissural organ were obtained from the brains fixed, mostly by vascular perfusion, with buffered glutaraldehyde solution, and then were postosmicated. They were embedded in Epon after dehydration. The sections were stained first in uranyl acetate and then in lead citrate solution. They were examined with a Hitachi HU 11 electron microscope.

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FIG. 1. Several distended cisternae of the endoplasmic reticulum containing a flocculent material in a subcommissural cell are seen closely apposed. A single row of particles uniformly spaced (A) and a slightly curved filament (B) are interposed between the cisternae. The particles (A) are interpreted as being cross sections of such filamentous structures as that labeled B. Another narrow intercisternal cleft containing parallel filaments in this figure is seen in subtangential section (C). Notice the lower density (at B) and smaller size (at A) of the filaments in comparison with the cisternal limiting membranes and ribosomes (R), respectively. 17-Month-old rat. x 50 000.

OBSERVATIONS E p e n d y m a l cells of the a d u l t s u b c o m m i s s u r a l organ are extremely h i g h c o l u m n a r in shape, their nuclei being situated near the base. N u m e r o u s cisternae of the e n d o plasmic reticulum are d i s t r i b u t e d t h r o u g h o u t the c y t o p l a s m (8, 16). T h e y usually contain a flocculent m a t e r i a l of m o d e r a t e electron density. The cisternae, distended to varying degrees, r a n d o m l y c o m m u n i c a t e with one another, thus affording p l e o m o r phic configurations to the reticulum. A s the e n d o p l a s m i c reticulum a p p r o a c h e s the apical p o r t i o n of cytoplasm, m e m b r a n e - a s s o c i a t e d r i b o s o m e s b e c o m e sparser t h a n in the deeper portion. R i b o s o m e s are lacking f r o m the surface of cisternal m e m b r a n e s which are m o r e o r less in close j u x t a p o s i t i o n . I n such regions where cisternae are

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Fio. 2. A low power electron micrograph of portions of subcommissural cells showing two enormously expanded cisternae of the endoptasmic reticulum filled with a flocculent precipitate in different degrees of concentration. The striped band intervening between the two may be seen to better advantage at higher magnification in Fig. 3. N, nucleus. 7-Month-old rat. × 26 000. FIo. 3. A higher magnification of part of Fig. 2. The striped band at the center is interpreted as a parallel array of filaments intervening between closely apposed membranes of the endoplasmic reticulum, along which the plane of section has grazed. 7-Month-old rat. x 56 000.

closely a p p o s e d to each other, well-ordered arrays of stripes or dots are sometimes observed in the c y t o p l a s m i c matrix, intervening between the m e m b r a n e s (Figs. 1-5). The width of the stripes a n d the d i a m e t e r of the dots are of the same magnitude, a b o u t 12 nm, a n d b o t h structures show similar m o d e r a t e electron density. The stripes are i n t e r p r e t e d as representing l o n g i t u d i n a l l y oriented profiles of filamentous structures, whereas the dots represent the cut ends of the same structures. The filaments are always aligned in p a r a l l e l a r r a y with a center-to-center spacing of a b o u t 24 nm. The course of the parallel filaments is either r a t h e r straight o r slightly curved (Figs. 1, 11-13). I n the present study straight p a t h s of the filaments c o u l d be t r a c e d as far as 6.3 # m in thin sections. A linear a r r a y of p u n c t a t e profiles of the filaments in cross section can be distinguished f r o m m e m b r a n e - a s s o c i a t e d r i b o s o m e s b y the fact 26 - 711825 J . Ult~'astruc~u~'e Rese~'e~

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FIGS. 4 and 5. Intercisternal filaments in parallel array are seen to be confined to the interfacial regions of cisternae of endoplasmic reticulum. Fig. 4, 9-Month-old rat, × 50 000; Fig. 5, 7-monthold rat. x 92 000. t h a t the latter are larger in size, higher in electron opacity, a n d usually a p p e a r to be interspersed on the m e m b r a n e s in d i s o r d e r (Figs. 1 a n d 12). A r r a y s of the parallel filaments intervening between cisternal m e m b r a n e s m a y be single, double, o r multiple (Figs. 1, 6-12). The transverse section of an assemblage of parallel filaments, in which neither b r a n c h i n g n o r crossing of filaments has been found, displays a well o r d e r e d h e x a g o n a l a r r a n g e m e n t (Figs. 6-9). The filaments are never closely p a c k e d in an assemblage, b u t are e m b e d d e d in, a n d equidistantly sepa r a t e d f r o m one a n o t h e r by, an a m o r p h o u s m a t e r i a l of low electron density n o t dissimilar to the general g r o u n d substance of the cytoplasm. F i l a m e n t s between greatly e x p a n d e d cisternae tend to be aligned in a single r o w (Fig. 1), whereas those l o c a t e d between flattened or collapsed cisternae are often seen a r r a n g e d in d o u b l e FIGS. 6-9. Assemblages of the orderly aligned filaments in transverse section are found to occupy interstices of pleomorphic cisternae of the endoplasmic reticulum. It is obvious that the filaments are hexagonally arranged and maintain a uniform distance from one another. Apparently they are not attached to the membranes. Fig. 6, 7-month-old rat; Figs. 7 and 9, 26-month-old rat; Fig. 8, 16-month-old rat. × 91 000.

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(Figs. 11 and 12) or multiple rows (Figs. 6-9). The occurrence of the parallel filaments is confined to the cytoplasmic matrix interposed between, and usually coextensive with, apposed cisternal membranes. They have never been observed elsewhere away from the endoplasmic reticulum. Other cytoplasmic structures, including ribosomes, are excluded from the filament-containing regions. Most frequently, the filaments appear in the vicinity of the nucleus. In some instances they are lodged in a narrow cleft between the outer leaf of the nuclear envelope and the membrane of endoplasmic reticulum (Fig. 10). They have never been encountered in the apical cytoplasm. The parallel filaments have been observed in rats of both sexes over 5 months old, but not in younger ones. The filaments appear to increase in incidence and in dimensions of their assemblage with advancing age, although the degree of their development seems to vary considerably from rat to rat. Conventional cytoplasmic filaments of about 5 nm thickness and microtubules are much more common structures found in the cytoplasm of rat subcommissural cells. Both these structures do not display such positioning and alignment as are shown by the parallel filaments described here. Distinct structural differences between the orderly arranged filaments and microtubules as well as general cytoplasmic filaments are obvious, as illustrated in the accompanying figures (Figs. 11 and 12). DISCUSSION Two classes of fibrous structures, general cytoplasmic filaments and microtubules, are known to be present in subcommissural cells. The present paper reports a third type comprising the medium diameter complement of the fibrous population in the cells. Since the parallel thick filaments described here have not been found in rats under 5 months of age, it is likely that they might be formed in association with aging of subcommissural cells, which are known not to renew themselves (9, 14). As the parallel filaments occur only in localized regions intervening between cisternae of the endoplasmic reticulum, it is conceivable that the regions may have changed in physical state different from the general cytoplasmic matrix. Multifarious profiles of the endoplasmic reticulum in subcommissural cells observed among different cells as well as in different rats appear to reflect the occurrence of dynamic or repeating processes of waxing and waning in cisternal contents. It is presumable that mechanical processes of the continuous cisternal fluctuation may be exerted upon the intercisterhal substance to deposit filaments through induction of a molecular rearrangement in the matrix of such a particular region. Straight and curved courses of the parallel filaments suggest a tensile strength. Thus, the filaments may confer mechanical strength to a certain degree on the everchanging endoplasmic reticulum, whether this is favorable or not.

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F i t . 10. An expanded cisterna is found in close juxtaposition with the nuclear envelope. In the narrow interfacial space between both structures lies an array of particles spaced at rather uniform intervals, which are identified as cross sections of parallel filaments. The filaments appear in contact with neither the limiting membrane of the cisterna nor the outer leaf of the nuclear envelope. R, ribosomes. 6-Month-old rat. x 92 000. F~6. 1l. Along the nuclear envelope is a couple of confronting flattened cisternae (arrows). Coursing in the narrow intercisternal cleft are double parallel filaments that display a lower electron density than the membranes of the cisternae, and extend as far as both ends of the cisternae. Ribosomes are found in limited numbers on the outer aspect of the cisternae but never on the membrane facing the filaments. Mt, microtubules in an adjacent cell; X, apposing plasma membranes. 7-Month-old rat. x 46 000.

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The propensity that single rows of the filaments are associated with expanded cisternae and hexagonal arrays of the filaments with less distended cisternae may lead one to assume that the filaments are embedded in a rather plastic material, instead of being held together as a fixed assemblage, so that their arrangement is changeable from the state of single rows to that of multiple arrays, or vice versa, depending upon the degree of distension of cisternae. Alternatively, it may be argued that the hexagonal arrangement of filaments represents an accumulation of single arrays of filaments, since the former type of assembly of filaments appears to be more frequent in the cells of older rats than in those of younger ones. Closely apposed cisternae of the endoplasmic reticulum are usually not provided with ribosomes on their confronting surfaces (1, 3-7, i2, 13, 17). In such situations the narrow intercisternal space often contains a moderately dense material which, nevertheless, does not yield such well-ordered arrays of filaments as described here in subcommissural cells. Repeating septate subunits between plasma membranes or between mitochondria have been reported in various cell types of a variety of animals (2, 10, 11, 15, 18, 19). They are of about 15 nm periodicity and similar in appearance. It has been assumed that they may result from an interaction between adjacent membranes and probably represent a complete reorganization or recrystallization of membrane components (2, 11). Although those examples of repeating septate subunits between cellular membranes are somewhat comparable to the filaments aligned in a single row described here, the subunits in the former structures are smaller in width than the filaments, being intimately adjoined to the membranes. Such septate subunits have never been reported to become assembled to show such a hexagonal arrangement as that displayed by the end-on views of the parallel filaments. Nevertheless, it is premature to exclude the possibility that the membrane-associated repeating subunits may represent an early phase of patterns which will later develop into the arrayed filaments described in this paper, because profiles comparable with such repeating subunits have been encountered between very closely apposed membranes of cisternae in subcommissural cells (unpublished observations). Further studies are needed to clarify the relationship between both structures. FIG. 12. A stack of three lamellar cisternae (arrows) uniformly spaced embraces a pair of filaments in each of the intercisternal clefts. The filaments course parallel to each other and to the surface of the lamellar cisternae. Again, it is apparent that the filaments appear less dense than the membranes, not being in contact with the latter, and ribosomes are present in limited numbers on the outer surface of the lamellar stack but not on the confronting aspect of the cisternae. The inset shows a bundle of conventional cytoplasmic filaments (F) and a few microtubules (Mr) from another portion of the same cell at the same magnification. 7-Month-old rat. x 65 000. FIG. 13. A low power electron micrograph of a juxtanuclear portion of a subcommissuralcell showing an extensive profile of parallel filaments closely associated with the cisternae of endoplasmic reticulum (*). The surface of the cistemae in many places is oblique or parallel to the plane of section so that the limiting membranes are hardly visible. 15-Month-old rat. x 35 000.

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Another example of patterns comparable to the intercisternal single array of illaments described here may be "narrow pillars" of 8-10 nm thickness lying between apposed cisternal membranes of the granular reticulum reported in hamster oocytes (17). However, it is disputable that the "pillars" are homologous with the parallel filaments, because the former show a far less orderly arrangement than the latter and there is an additional structure of one or two central lines between the cisternae, where "pillars" occur. It is likely that the parallel filaments intimately associated with the endoplasmic reticulum may represent a unique type of intercisternal substance not reported hitherto. This study was supported by grants from the National Science Council, the Republic of China, and the China Medical Board of New York, Inc. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.

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