Acta neuropath. (Berl.) 31, 13--19 (1975) 9 by Springer-Verlag 1975

Fenestrae in Golgi and Endoplasmic Reticulum Cisternae of Human Brain Tumours* Eiichi Tani, Toshio Ametani, K e n Nakano, Mi*sugu Nishiura, and N o b o r u Higashi Department of Neurosurgery, Ityogo College of Medicine, Nishinomiya, Hyogo, and Leprosy Research Laboratory and Institute for Virus Research, Kyoto University, Shogoin, Kyoto P~eceived July 22, 1974; Accepted October 10, 11974

Summary. Fenestrae were found in freeze-fractured cisternae of the Golgi apparatus and endoplasmie reticulum of glioblastoma, oligodendroglioma, ependymoma, medulloblastoma, medulloepithelioma, meningioma, cerebellar sarcoma, hemangioblastoma, and chromophobe adenoma. They were about 200--400 _~ in diameter and often diffusely distributed or concentrated in groups in Golgi cisternae, while they were around 300--600 _~in size and scattered in distribution in cisternae of endoplasmic reticulum. They appeared as conical protrusions or circular broken-off necks of face A and as circular holes on face B in tangential fractures, and as several constrictions of cisternae in cross fractures. Key words: Freeze-Fracture -- Brain Tumours -- Fenestrae -- Golgi Apparatus -Endoplasmic Reticulum. Golgi apparatus and endoplasmic reticulum (ER) have been well characterized by sectioned material for electron microscopy. The presence of fenestrae in Golgi and E R cisternae was already reported in the early development of electron microscopy (Palay and Palade, 1955; Palade, 1956). Thereafter, fenestrated Golgi cisternae were found in plant (Manton, 1960; Morr6 et al., 1965; Cunningham et al., 1966; Mollenhauer and Morr6, 1966a and b) and animal cells (Mollenhauer and Zebrun, 1960; Essner and Novikoff, 1962; Novikoff, 1964; Kessel and Beams, 1965; Lane, 1966; Flickinger, 1969). However, since sectioned materials give only a limited sampling of an entire Golgi and EI~ sheets, the n u m b e r and the distribution of the fenestrae could not be accurately assessed. The three-dimensional feature of fenes~rated E g cisternae was beautifully revealed in freezefractured liver (Orci et al., 1971) and pancreatic (Orci et al., 1972) cells. This communication presents fenestrae in Golgi and E R cisternae in h u m a n brain turnouts. Materials and Methods The materials in the present study were 6 glioblastomas, 3 oligodendrogliomas, 2 ependymomas, 3 medulloblastomas, 1 medulloepithelioma, 5 meningiomas, 1 cerebellar sarcoma, 3 hemangioblastomas, and 3 chromophobe adenomas. All specimens were taken at surgery before their blood supply was interrupted. They were diced immediately into small pieces and fixed for 2 hrs at 4 ~ C in 2.5~ glutaraldehyde solution buffered with 0.1 M phosphate (pH 7.4), * This study was partly supported by a grant-in-aid for scientific research from the Ministry of Education, Japan.

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and t h e n immersed at 4 ~ C overnight in 30~ glycerol solution containing 0.9% sodium chloride after a brief washing in the phosphate buffer. The HFZ-1 a n d I-IUS-4 freeze-etching apparatus were used. The specimen was mounted on a copper specimen holder, frozen rapidly in liquid Freon 12, t h e n transferred into liquid nitrogen, and fractured in the freeze-etching device in vaeuo (10-5 Torr). The fracture surface of the specimen was replicated b y platinum and carbon. The replica, after digesting the tissue in a commercial bleaching solution, was washed in distilled water, picked up on grid, a n d examined with a n ttU-11 A electron microscope.

Fig. 1. A Golgi apparatus in an ependymoma cell. Many conical protrusions (arrows I and 2) are visible on face A of Golgi cisternae, which directs towards the lumina and possesses m a n y m e m b r a n e particles. Large conical protrusions (arrow 1) are about 400 • in diameter and demonstrate a central crater-like depression, whereas small protrusions (arrow 2) are around 200 ~_ in size and ~conical in shape. Circular holes (arrow 3) are evident on face B of Golgi cisternae, which directs towards the cytoplasmic matrix and shows fewer particles. The periphery of holes reveals a sharply furrowed rim. Arrows A and B indicate faces A and B of the endoplasmic reticulum, respectively, and exhibit similar characteristics in n u m b e r of membrane particles to the fracture face of Golgi cisternae. • 51000

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Fig.2. A Golgi apparatus in a glioblastoma. Its fracture face A demonstrates three conical protrusions (arrow). • 56000 Fig. 3. A Golgi complex in a meningioma. Three conical protrusions, two of them showing a flat top (arrow 1), are found on face A of Golgi cisterna. Large (arrow 2) and small (arrow 3) circular holes are present on face B of Golgi cisterna, and reveal no sharply limited rim. Where the fracture plane jumps from face A to face B of Golgi cisternae a fenestra (arrow 4) involves both faces A and B at the fracture edge. Small area free of membrane particles is observed on face A. Arrow 5 is a nulcear pore on face B of the outer nuclear membrane. • 70000

Results The Golgi a p p a r a t u s in replicas was easily identified b y its c h a r a c t e r i s t i c l o c a t i o n n e a r nuclei a n d closely a p p l i e d s w a r m s of cisternae, vesicles a n d vacuoles (Figs. l - - 5 ) . E n d o p l a s m i e r e t i e u l u m , a l t h o u g h its a t t a c h e d ribosomes were u s u a l l y n o t visible in replicas, was c h a r a c t e r i z e d b y elongated, i r r e g u l a r l y - s h a p e d , bent, or b r a n c h e d profile (Figs. 1 a n d 6), which was n o t u s u a l l y so closely app r o x i m a t e d as t h e Golgi a p p a r a t u s . The a p p e a r a n c e of Golgi a n d E R c o m p o n e n t s d e p e n d e d u p o n w h e t h e r t h e f r a c t u r e p l a n e split t h e m e m b r a n e s l o n g i t u d i n a l l y or broke t h e m t r a n s v e r s e l y . The eisternae f r a c t u r e d t r a n s v e r s e l y h a d t h e conv e n t i o n a l a p p e a r a n c e s of Golgi a n d E R profiles seen in sectioned m a t e r i a l . W i t h this o r i e n t a t i o n of t h e f r a c t u r e plane, t h e t h r e e - d i m e n s i o n a l e x t e n s i o n of Golgi a n d E R could n o t be e s t i m a t e d w i t h m o r e precision t h a n with sectioned material. W h e n t h e f r a c t u r e p l a n e p a s s e d t a n g e n t i a l l y w i t h i n Golgi a n d E R m e m b r a n e s , it e x p o s e d wide Golgi a n d E R m e m b r a n e sheets. Golgi a n d E R m e m b r a n e sheets d e m o n s t r a t e d two differing features d e p e n d i n g u p o n t h e n u m b e r of m e m b r a n e particles (Figs. 1 - - 6 ) . F r a c t u r e face A was chara c t e r i z e d b y t h e presence of m a n y m e m b r a n e particles a n d d i r e c t e d t o w a r d s t h e l u m i n a of Golgi or E R cisternae, whereas face B possessed fewer particles a n d

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Fig.4. A large Golgi apparatus in a meningioma. Conical protrusions (arrow 1) and hole (arrow 2) are evident on faces A and B of Golgi eisternae, respectively. When the Golgi complex is fractured tangentially, a fenestra exhibits a constriction of a Golgi cisterna (arrow 3). Associated vesicles are fractured within ~heir limiting membranes, revealing face A (arrow 4) or face B (arrow 5). • 62000 directed to the cytoplasmic matrix. The membrane particles were about 70 to 110 _& in diameter and intercalated in Golgi or EI~ membrane sheet. They were usually distributed rather at random and, in addition, speckled areas free of the membrane particles were sometimes evident on face A of Golgi (Figs. 3--5) and E R membranes (Fig. 6). Fenestrae appeared as conical protrusions or circular broken-off necks on face A and as circular holes on face B of Golgi and E R membranes (Figs. 1--6). They were about 200--400~_ in diameter and often diffusely distributed or concentrated in groups in Golgi eisternae (Figs. 1--5), whereas they were around 300--600/~ in size and rather scattered in distribution in E R membrane (Fig. 6). The membrane particles were occasionally concentrated around the fenestrae. Particles were occasionally studded on the top of broken-off necks and in the bottom of holes. The periphery of some fenestrae was not so sharply ridged on face A (Figs. 1 and 5) or furrowed on face B (Fig.3), particularly in smaller fenestrae, as were the fenestrae of capillary endothelium (Tani et al., 1974). Consequently, the top of conical protrusions in such fenestrae was not usually depressed like a crater but purely conical in form. Most of the larger fenestrae revealed crater-like protrusions on face A and corresponding circular holes on face B, the rim of which was often sharply furrowed. Smaller fenestrae, therefore, appeared to be somewhat different in freeze-fracture morphology from larger fenestrae. The larger fenestrae usually demonstrated freeze-fracture characteristics of fenestrae (Tani et al., 1974). However, when the fracture plane passed in a

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Fig.5. A Golgi complex in a cerebellar sarcoma. Large (arrow 1) and small (arrow 2) conical protrusions arc found on face A of Golgi cisterna. When the fracture plane passes in a stepwise fashion from face A to face B of Golgi cistcrnae, both of large (arrow 3) and scmall (arrow d) fenestrae involve both faces A and B at the fracture edges. A and B indicate face A of ~he outer nuclear membrane and face B of the inner nuclear membrane, respectively. • 61000 stepwise fashion from face A into face B or vice versa, t h e fenestrae, larger or smaller in size, c h a r a c t e r i s t i c a l l y i n v o l v e d b o t h faces A a n d B a t t h e f r a c t u r e edges (Figs. 3 a n d 5). I n a d d i t i o n , when t h e Golgi a p p a r a t u s a n d E R were f r a c t u r e d t r a n s v e r s e l y , t h e fenestrae showed several constriction of t h e Golgi (Fig.4) a n d ER membranes. Ellipsoidal protrusions, a b o u t 150 • 100 _~ in size, were s o m e t i m e s visible on face B of El% cisternae (Fig. 6), a n d quite different in size a n d shape from t h e m e m b r a n e particles a n d t h e fenestrae.

Discussion Since t h e f r a c t u r e p l a n e passes b e t w e e n t h e b i m o l e c u l a r p h o s p h o l i p i d leaflets of t h e p l a s m a m e m b r a n e , face A is left frozen to t h e c y t o p l a s m a n d possesses m a n y 2 Acta neuropath. (Berl.) Bd. 31

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Fig. 6. Endoplasmie reticulum in a meningioma is irregularly-shaped and branched in form. A conical protrusion (arrow 1) and circular holes (arrow 2) are visible on faces A and B of endoplasmic reticulum. The membrane particles are distributed more densely on face A than on face B. In addition, ellipsoid protrusions (arrow 3) are found on face B. • 98000

membrane particles, whereas face B is adjacent to the extracellular space and showed fewer membrane particles (Branton, 1966). The fracture in G01gi and E R membranes proceeds in a similar plane to the plasma membrane, and, as a result face A was adjacent to the cytoplasm and direct to the cisternal lumina, and face B was adjacent to the lumina and directed towards the cytoplasm. The fenestrae in the Golgi apparatus and E R cisternae, although generally smaller in size than the fenestrae in capillary endothelium (Tani et al., 1974), possessed all of freeze-fracture characteristics necessary for definition of fenestrae. The functional significance of fenestrae in Golgi apparatus and E R is unknown, but the fenestrae would provide a wider range of functional capabilities than the classical sac-like eisternae. I t has been noted t h a t E R is closely related, topographically and in enzyme content, to the eisternae at one pole of the Golgi apparatus, and t h a t it might prove to be in continuity with the Golgi eisternae. Orci et al. (1972) proposed two hypotheses: a) communication channels from one intereisternal space to the other and b) compartmentalization of the intraeisternal space which could play a role in directing the flow of intracisternal material. I n addition, spherical or etlipsoidal structures localized to the cisternal pores in the Golgi apparatus were suggested to be involved in membrane differentiation (Sturgess et al., 1973). Further investigations of the correlation between structure and function m a y provide clues about their functional significance. The physiological significance of ellipsoidal protrusions on face B of E R eisternae is unknown at present.

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References Branton, D.: Fracture faces of frozen membranes. Proc. nat. Acad. Sei. (Wash.) 55, 1048--1056 (1966) Cunningham, W. P., MorrO, D.J., Mollenhauer, H. It. : Structure of isolated plant Golgi apparatus revealed by negative staining. J. Cell Biol. 28, 169--179 (1966) Essner, E., Novikoff, A. B. : Cytological studies on two functional hepatomas. Interrelations of endoplasmic reticulum, Golgi apparatus, and lysosomes. J. Cell Biol. 15, 289--312 (1962) Flickinger, C. J. : Fenestrated eisternae in the Golgi apparatus of the epididymis. Anat. l~ec. 163, 39--54 (1969) Kessel, P~. G., Beams, H. W.: An unusual configuration of the Golgi complex in pigmentproducing "test" cells of the ovary of the tunicate Styela. J. Cell Biol. 25, 55--67 (1965) Lane, N. J. : The fine-structural localization of phosphatases in neurosecretory cells within the ganglia of certain gastropod snails. Amer. Zool. 6, 139--157 (1966) 5~anton, I. : On a reticular derivative from Golgi bodies in the meristem of Anthoceros. J. biophys, biochem. Cytol. 8, 221--231 (1960) Mollenhauer, H. H., MorrO, D. J. : Tubular connections between dicytosomes and forming secretory vesicles in plant Golgi apparatus. J. Cell Biol. 29, 373--376 (1966a) Mollenhauer, H. H., MorrS, D. J.: Golgi apparatus and plant secretion. Ann. Rev. Plant Physiol. 17, 27--46 (1966b) Mollenhauer, H. H., Zebrun, W. : Permanganate fixation of the Golgi complex and other cytoplasmic structures of mammalian testes. J. biophys, biochem. Cytol. 8, 761--775 (1960) Morr6, D. J., Mollenhauer, tt. H., Chambers, J. E. : Glutaraldehyde stabilization as an aid to Golgi apparatus isolation. Exp. Cell Res. 38, 672--675 (1965) Novikoff, A. B. : GERL, its form and function in neurons of rat spinal ganglia. Biol. Bull. 127, 358 (1964) Orci, L., Matter, A., tLouiller, Ch.: A comparative study of freeze-etch replicas and thin sections of rat liver. J. Ultrastrnet. Res. 35, 1 - 1 9 (1971) 0rci, L., Perrelet, A., Like, A. A.: Fenestrae in the rough endoplasmic reticulum of the exocrine pancreatic cells. J. Cell Biol. 55, 245--249 (1972) Palade, G. E.: The endoplasmic reticulum. J. biophys, biochem. Cytol. 2 (Suppl.) 85--98 (t956) Palay, S. L., Palade, G. E.: The fine structure of neurons. J. biophys, biochem. Cytol. 1, 69--88 (1955) Sturgess, J. M., Moscarello, M. A., Vail, W. J. : Freeze fracture of the Golgi complex. J. Cell Biol. 59, 340a (1973) Tani, E., Ikeda, K., Kudo, S., Yamagata, S., Higashi, N., Fujihara, E. : Fenestrated vessels in human hemangioblastoma. J. Neurosurg. 40, 696--705 (1974) Dr. E. Tani Dept. of Neurosurgery ttyogo College of 5Iedicine Nishinomiya Hyogo Japan

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Fenestrae in golgi and endoplasmic reticulum cisternae of human brain tumours.

Acta neuropath. (Berl.) 31, 13--19 (1975) 9 by Springer-Verlag 1975 Fenestrae in Golgi and Endoplasmic Reticulum Cisternae of Human Brain Tumours* Ei...
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