JOURNAL OF ULTRASTRUCTURERESEARCH50, 253-263 (1975)
Ultrastructure of Gliosomes in Ependymal Cells of the Lizard G.
DONELLI, 1 V.
D'UvA,
2 AND
L.
PAOLETTI I
Physics Laboratory, lstituto Superiore di Sanith, Rome 1, and Institute of Histology and Embryology, Faculty
of Science, Naples 2, Italy Received July 2, 1973, and in revised form February 21, 1974 Gliosomes similar to those already described by other authors in amphibia and mammalia w e r e found in the hypothalamic region ofLacerta s. sicula Raf. The internal matrix of these particular organelles, which closely resemble transformed mitochondria, is in a more or less advanced state of crystallization. The dimensions of the unit cell, the symmetry and the space group of these mitochondrial crystals were determined by analyzing electron micrographs of ultrathin sections of gliosomes by means of the optical transform technique. An evaluation of the molecular weight of the single asymmetrical subunit of the crystal lattice was obtained. INTRODUCTION Crystalline and paracrystalline inclus i o n s h a v e b e e n d e s c r i b e d in m i t o c h o n d r i a of v a r i o u s t y p e s of cells in b o t h n o r m a l a n d p a t h o l o g i c a l t i s s u e s (20). I n c l u s i o n s of a p a r t i c u l a r t y p e are p r e s e n t in o r g a n e l l e s k n o w n as g l i o s o m e s considered to be modified mitochondria. These gliosomes, described by several aut h o r s (6, 7, 14, 18) in g l i a l f i b e r s of s o m e a n i m a l s p e c i e s , were o b s e r v e d a n d s t u d i e d b y one of us in e p e n d y m a l cells of L a c e r t a s. sicula R a f . (3, 4). T o d a t e l i t t l e is k n o w n of t h e f u n c t i o n a l a n d p h y s i o l o g i c a l s i g n i f i c a n c e of t h e s e s t r u c t u r e s . B y m e a n s of a n a n a l y s i s of electron micrographs with the optical Four i e r t r a n s f o r m t e c h n i q u e (2, 9, 11), m o r e s p e c i f i c a l l y w i t h t h e m e t h o d of r e c o n s t r u c t i o n of t h e r e c i p r o c a l l a t t i c e of t h e c r y s t a l p r o p o s e d b y B e r g e r (1, 19), i t is p o s s i b l e to o b t a i n a m u c h m o r e c o m p l e t e s e t of s t r u c t u r a l d a t a t h a n c a n g e n e r a l l y b e f o u n d in t h e l i t e r a t u r e ; t h e r e f o r e we h a v e c o n s i d e r e d it i n t e r e s t i n g , in t h i s work, to a p p l y t h e s e m e t h o d s in t h e s t u d y of g l i o s o m e s . The morphological and ultrastructural d a t a o b t a i n e d d i d n o t p e r m i t us to c o m p l e t e l y d e f i n e t h e f u n c t i o n a l s i g n i f i c a n c e of these formations. However, reconstruction
of t h e c r y s t a l l a t t i c e a n d d e t e r m i n a t i o n of t h e p a r a m e t e r s of t h e u n i t cell w i t h t h e a b o v e t e c h n i q u e s m a d e it p o s s i b l e to p u t f o r t h a n u m b e r of h y p o t h e s e s c o n c e r n i n g t h e n a t u r e of t h e o b s e r v e d c r y s t a l l i n e inclusions. MATERIAL AND' METHODS Electron micrographs of the hypothalmic region of
Lacerta s. sicula brain were studied by optical Fourier transforms. The hypothalmic region was removed from the brain with the aid of a dissection microscope, reduced to small pieces with a diameter of 2-3 mm, and fixed, part in 2.5% glutaraldehyde in phosphate buffer pH 7.4 and part in formaldehyde-glutaraldehyde in phosphate buffer pH 7.4. The sections were then postfixed in 2% osmium tetroxide in phosphate buffer, dehydrated with increasing concentrations of alcohol and embedded in Epon-Araldite (15). Thin sections were stained with toluidine blue and observed with the optical microscope. Ultrathin sections obtained with an LKB microtome (Ultratome III) were stained with uranyl acetate and lead citrate (8) and observed with a Siemens IA electron microscope. Apertures of 50 #m for the objective lens and 400 #m for the condenser lenses were used. The accelerating voltage was 80 KV and the micrographs were taken at a magnification between 10,000 and 40,000, the second pole piece being used for the second projector lens. The diffraction patterns were obtained with a Polaron optical diffractometer equipped with a Spectra Physics 2.0 mW helium-neon laser. 253
Copyright © 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
254
DONELLI, D'UVA AND PAOLETTI
EXPERIMENTAL RESULTS In the basal zone of the ependymal cells, however, mainly LMG are present. (1) Morphological Observations The ultrastructural aspects of gliosomes (2) Optical Transforms of Electron Microof the species studied are similar to those graphs described by Srebro in Amphibia (18). Electron micrographs of gliosomes of Gliosomes are, as a rule, elongated structures, ranging in length from 1 to 10 ttm both the first and the second type were analyzed. While it was possible to deterand in thickness from 0.4 to 0.6 ttm. The external wall is somewhat similar to mine the structure of the inclusions in the the mitochondrial wall; it is constituted by GMG, it was not possible to detect any two cytomembranes separated by a small "crystalline" structure of the interlamellar electron transparent space; the inner cyto- matrix in the LMG. For the GMG, the analysis was carried membrane is introflected, and often forms out on about a hundred electron microcanaliculi of cristae 180/~ in width. In cross graphs corresponding to sections whose section the latter appear as small circular, thickness could be estimated equal to or or in some cases triangular, profiles, which less than 1000 fl,. are, as a rule, located in the peripheral zone With preliminary measurements it was of the matrix of the gliosome, but which can also be found further towards the in- possible to estimate the lattice spacings of the crystal as about 100 A: on the basis of side. On the basis of the ultrastructural orga- this evaluation the number of lattice nization of the matrix, two types of gli- planes contained in the thickness of the osomes can be recognized: (a) gliosomes sections can be calculated as lying between with lamellar matrix (LMG) and (b) gli- 5 and 10. The electron micrographs can osomes with granular matrix (GMG) (Fig. therefore be considered, with close approximation, projections of the crystal onto the 1a-b). plane of the photographic plate and their In gliosomes with a granular matrix optical transforms sections of the recipro(GMG), at high magnification (× 60 000), cal lattice parallelly oriented to the projecthe matrix is resolved in a crystal lattice of granules 80-90 A in width, located at tion plane. For the effective construction of the intervals of 100-110 A. Very electron-dense granules are also crystal and the singling out of the elements present in the matrix, these having an of symmetry, the "bidimensional" diffracaverage diameter of 450 A and resembling tion patterns were used, that is, those which correspond to sections of the reciprothose found in the mitochondria (5). In gliosomes with a lamellar matrix cal lattice passing through the lattice (LMG) the lamellae are arranged longitu- planes (1). In quantitative terms these patterr~s dinally to the gliosomes with an apparent periodical array. Fusion between two or represent about 40% of all observed patmore gliosomes of the same type is often terns. For a quantitative analysis of the diffracobserved (Fig. la). tion patterns the various sources of error The glial fibres contain what are probawere isolated and their relative sizes were bly glycogen granules and sheafs of filaestimated. The results can be summarized ments; the gliosomes of the first and secas follows: ond type are present in equal proportions; (1) Compression and distortion of the the filaments themselves are particularly numerous where gliosomes are to be found structure of the crystal during cutting o[ the section. The incidence of this error was (Fig. 2c).
ULTRASTRUCTURE OF GLIOSOMES
255
Fio. 1. (a) Gliosome with lameIlar matrix. Note the fusion or splitting between gliosomes (arrow) x 15 000 (b) Gliosome with granular matrix. × 16 000. m i n i m i z e d b y c h o o s i n g for t h e a n a l y s i s of the data those sections whose structure was a p p a r e n t l y n o t d a m a g e d . T h e close a g r e e m e n t of t h e d a t a , w i t h i n t h e final s p r e a d i n g c a l c u l a t e d on t h e b a s i s of t h e
v a r i o u s s o u r c e s of error, s u p p l i e d t h e conf i r m a t i o n a p o s t e r i o r i t h a t t h e c r i t e r i a of s e l e c t i o n a d o p t e d were s u f f i c i e n t to m a k e this error negligible. (2) Magnification of electron micro-
FIG. 2. (a) A gliosome of first type (LMG) in an e p e n d y m a l cell x 14 000 (b) A gliosome of t h e s a m e type at higher m a g n i f i c a t i o n × 41 000 (c) Gliosome with g r a n u l a r m a t r i x in a glial fiber. T h e sheafs of f i l a m e n t which are particularly n u m e r o u s near gliosomes are evident. × 24 000. 256
ULTRASTRUCTURE OF GLIOSOMES
graphs. In our working conditions, the relative error of the calibration with standard samples, was estimated to be not greater than 10%. (3) Diffraction constant. Calibration of the optical bench, in working conditions, with standard lattices, made it possible to estimate the relative error as less than 1%. (4) Tilting of the crystal with respect to
the plane of the section and tilting of the section with respect to the plane normal to the projection line (electron beam). This causes a spreading of the measurements on the diffraction patterns. In fact to every different degree of tilting of the crystal with respect to the projection line corresponds a different tilting of the sections of the reciprocal lattice. For the finite dimensions of the crystal, it is not possible to interpret Fourier transforms of the micrographs in terms of sections of a reciprocal lattice of points without dimensions, but rather of a lattice whose points are considered "expanded" in the reciprocal space. For this reason we expect a broad range of sections which intersect, with different orientations, "expanded points" of the same plane of the reciprocal lattice. The measurements of the spacings and angles relative to this range of sections correspond, therefore, to a spreading of values whose amplitude can be evaluated with the equation: e/p = 2 P/D (1), where e and p are the "expansion" of the points of the reciprocal lattice in a given direction and their spacing in the considered direction, respectively; P and D, the corresponding spacing and the size of the diffraction latrice in the same direction, respectively. The spreading of the spacing and angle measurements was in this way evaluated to be of the order of 5-10%. The above discussion of the possible errors implies that a total spreading of about 15-20% in the spacing and angle measurements on the diffraction patterns must be expected. On the basis of the elements of symme-
257
try and the spacing between the spots present, the bidimensional diffraction patterns obtained were classified into three main types. Type A: (Fig. 3a-b), is characterized by an arrangement of the spots on a rectangular lattice. The corresponding section of the reciprocal lattice and the main spacings Pl and P2 are given in Fig. 4a. Type B: (Fig. 3c-d), is characterized by an arrangement of the spots on a pseudosquare lattice. The corresponding section of the reciprocal lattice and the main spacingspl and Pa are shown in Fig. 4b. Type C: (Fig. 3e-f), is characterized by an arrangement of the spots on a pseudohexagonal lattice. The corresponding section of the reciprocal lattice and the spacings p2 and ps are given in Fig. 4c. Apart from the three types mentioned above, there is a limited number of the patterns, which can be in turn classified into two types, on which systematic extinctions are evident in some classes of diffraction spots. Type A 1: (Fig. 5a-b). Along the meridian systematic extinctions of a number of spots are evident and the condition for the nonextinction of the Kth is K=2n, with n a whole number. This implies the existence of a crystallographic axis with 2-fold screw symmetry. In that the spacings are pJ2 and P2, respectively, while the spacing of the spots along the meridian is p~ (Fig. 6a), it is likely that type A1 patterns coincide with type A patterns, but correspond to micrographs on which the resolution has made it possible to show up the axis of screw symmetry. That is, it is possible to distinguish the asymmetric subunits of the crystal rotated by ~.
258
DONELLI, D'UVA AND PAOLETTI
m FIG. 3. (a) Section of GMG corresponding to type A bidimensional pattern, x 72 000. (b) Type A diffraction pattern. (c) Section of GMG corresponding to type B bidimensional pattern. × 72 000. (d) Type B diffraction pattern. (e) Section of GMG corresponding to type C bidimensional pattern. × 72 000. (f) Type C diffraction pattern. The diffraction patterns are presented at the same magnification and are aligned with respect to the crystal images.
ULTRASTRUCTURE OF GLIOSOMES
a)
•
•
Q
P3
bl
c)
Fxa. 4. (a) Section of the reciprocal lattice corresponding to type A diffraction pattern. (b) Section of the reciprocal lattice corresponding to type B diffraction pattern. (c) Section of the reciprocal lattice corresponding to type C diffraction pattern.
Type BI: (Fig. 5c-d). S y s t e m a t i c extinctions of spots are evident along the meridian. T h e condition for nonextinction of the K t h is still K = 2n, with n a whole number, confirming the presence of the axis of 2-fold screw s y m m e t r y . In t h a t the spacings are p J 2 and p~, respectively, while the spacing along the meridian is p~ (Fig. 6b), it is likely t h a t type B1 coincides with type B, corresponding however, to micrographs on which the resolving power is such as to show up the axis of screw symmetry. T h e types of bidimensional patterns singled out together with the spacings measured on t h e m are s u m m a r i z e d in Table I. (3) Reconstruction of Crystal Lattice In order to determine the most likely value of the spacings of T a b l e I, t h a t is, to
259
d e t e r m i n e the dimensions of the unit cell of the reciprocal lattice, a statistical analysis of the m e a s u r e m e n t s was made. T h e results of this analysis are s u m m a r i z e d in the histogrammes of Fig. 7, constructed on the basis of tl~e frequency with which the values of the spacings between the spots in the m e a s u r e m e n t s taken on 41 bidimensional patterns were obtained. On every pattern the two or three main spacings were measured. These are shown in the histogrammes of Fig. 7 and in Table I. The sources of the measured spacings are m a r k e d with capital letters. T h e distributions obtained show halfspreadings which represent 12-13% of the corresponding m e a n value, in good agreem e n t with the spreading of the measurements previously evaluated. T h e relative a b u n d a n c e of the different types of patterns resulted as follows: T y p e A = 23, type B = 11, type C - 7. For the spacings of T a b l e I, the mean values of the corresponding peaks, with an error equal to the half-width of the peaks themselves were taken as the most probable values (Table II). T h e unit cell of the reciprocal lattice was reconstructed by combining the bidimensional patterns along the lines on which the same spacing of the spots was present. In this way a monoclinic unit cell was obtained, the dimensions of which were taken as follows: a* = P2, b* = pl/2c* - p3 with the ~* angle (between the a* and the c* axes) equal to 63 ° ± 12% (Fig. 8). The Miller indices of the lattice planes corresponding to the various types of patterns are listed in Table I. It is i m p o r t a n t to note t h a t the separation between the mean values of the spacings p2 and ps is lower t h a n the error ascribed to the values themselves. Within the precision of m e a s u r e m e n t it is not possible to exclude t h a t p 2 and p a may have the same value. In this case the reciprocal lattice and the crystal itself would be
260
DONELLI, D'UVA AND PAOLETTI
FIG. 5. (a) Section of GMG corresponding to type A 1bidimensional pattern. × 72 000. (b) Type A 1diffraction pattern. (c) Section of GMG corresponding to type B1 bidimensional pattern. × 72 000. (d) Type B1 diffraction pattern. The diffraction patterns are presented at the same magnification and are aligned with respect to the crystal images. hexagonal. It m u s t be considered, however, t h a t the separation between P2 and P3 is slightly smaller t h a n the half-spreading of the measurements, and that, moreover, if p~ and P3 should have the same spacing, the c o r r e s p o n d i n g m e a s u r e m e n t w o u l d have an error of a b o u t 20%, which is s o m e w h a t higher t h a n the error of the m e a s u r e m e n t of pl, 12%. It would be difficult to explain this difference in the precision of two m e a s u r e m e n t s obtained with the same procedure and from the same set of patterns. For these reasons it could be more correct to interpret the data according to the hypothesis of two different spac-
ings P2 and Ps of a pseudohexagonal monoclinic lattice. The unit cell of the crystal, on the basis of the data relative to the reciprocal lattice, is monoclinic with dimensions o f a = 110 423A, b = 2 4 0 ± 3 1 A , c - 120± 16/~with the ~ angle (between the a and the c axes) equal to 117 ° ± 14 ° and with a vol of V = 2.8-10 6 ± 1.5-10 6/~3. The systematic extinctions of the diffraction spots, observed on the p a t t e r n s (1, 0, 0) and (0, 0, 1), corresponding to the class of reflexions 0, K, 0 for the indices K = 2n, with n a whole number, imply the presence of a screw axis perpendicular to
261
ULTRASTRUCTURE OF GLIOSOMES
the lattice planes (0, 1, 0) with component b/2. These results make it possible to single out the space group of the crystal as the group P 21, with which two equivalent asymmetrical subunits per unit cell (Fig. 9) are compatible. DISCUSSION
The problem of the meaning of the two types of gliosomes, i.e., whether they are the result of different processes of transformation of the mitochondria or of different stages of the same process, is still completely unsolved. In any case, special stress must be laid on the absence of "hybrid" arrays which can be interpreted in terms of transformation from one type of gliosome to the other. Crystallographic data prove that GMG have a crystalline structure, very different from the one displayed by the matrix of the LMG, thus showing a new differential character, which strengthens the hypothesis of the existence of two types of gliosomes. In GMG the crystalline arrangement of the matrix is generally present with a definite orientation with respect to the gliosomes. In particular cross sections of the gliosomes have always given patterns of type C (Fig. 3e), that is, the longitudinal axis of the gliosome and the lattice axis [0, 1, 0] are, with close approximation, parallel. This supports the hypothesis of some interaction between the structures of the gliosome (cytomembranes, cristae, etc.) and the crystalline matrix. The triangular profiles of crystae sections in gliosomes and the existence of prismatic cristae in mitochondria with crystalline inclusions (13) have been interpreted, also by other authors, as effects of crystallization of the matrix in these structures (10). In the hypothesis that, as found by Srebro in amphibia (17), the crystalline inclusions studied are protein crystals, it is possible to estimate the molecular weight
of each of the two equivalent asymmetrical subunits "per" unit cell of the crystal, using the volume V of the unit cell itself. In most protein crystals (I2) the volume of the unit cell per unit of molecular weight (Vm) lies in the interval between V m = 1.5 AVdalton and V m = 3.5 AVdalton. Therefore for the crystal studied, whose unit cell is monoclinic with a vol V = 2.8-10 ~ ha, the molecular weight M W = V/2Vm of the single asymmetrical subunit would lie between M W = 4.10 5 and M W = 9. l0 B. The protein material included and crystallized in GMG can:
a)
T,pl/2 I pl
• -- t-
o
Q
--
O
b) FIG. 6. (a) Spacings present on type A~ diffraction pattern. (b) Spacing present on type B1 diffraction pattern. TABLEI Patterns
A B C A~ B1
Spacings
Pl Pl P2 pj2 pJ2
; ; ; ; ;
P2 P~ P3 P2 p~
Spacings corresponding of systematic extinctions
Pl
Pl
Miller indices of corresponding planes
(o, o, 1) (1, o,o) (o, 1, o) (o, o, 1) (1,o,o)
262
DONELLI, D'UVA AND PAOLETTI
,0
°'
LI
A _
A
i._ I.L
BA__
i_
_
~
S_
5~-
ICAC
BAA
A AA
BAAAA
I
03
CC
BA
1/1/,0
'i
p2
B
C
11120
1/100
[BB
II
AAA
I
11110
1/97
1/80
1/125
11106
Spot
1/90
spacing (/~-~)
Fro. 7. Experimental distributions of the spacing between the spots in measurements on bidimensional diffraction patterns. Each histogramme shows the types of diffraction patterns from which the measurements come. /~J
/
S
~ ' - - - -
.j~ --
I I
J J
/
D
/7q-
I I
JJ
, [
/
~ / j J
I
J
I I
/
.J /
C~
l/
Fro. 8. The reciprocal lattice and the monoclinic unit cell. TABLE II Reciprocal lattice spacings
A-
Pl P2 p~
1/120 ± 13% 1/97 - 14% 1/106 -~ 13%
(a) be of external origin, (b) derive from the different organization of protein components present in mitochondria in the course of their transformation into gliosomes. In the latter hypothesis it needs to be taken into account that most protein components present also in normal mitochondria have a molecular weight (16, 19) which is from two to ten times less than the minimum molecular weight calculated by us for the asymmetrical subunit. If it is considered that in the majority of protein crystals the number of equal molecules per asymmetrical subunit is n = 1
Fla. 9. The unit cell of the Cristal. The screw axis is shown. In the figure, the directions along which the projections of the lattice were obtained, which gave rise to the patterns in Table 1, can be easily singled out; in fact these projections have indices which are equal to the indices, within the reciprocal space, of the corresponding types of patterns listed in Table I.
(12, 21) it seems more probable that each asymmetrical subunit is made up of a multimolecular complex. It could be imagined that the asymmetrical subunit is one of the multienzymatic complexes which make up the structural elements of the model of the mitochondrial membranes proposed by SjSstrand and Barajas (16). The observations made by one of the authors (3, 4) on the presence of gliosomes at different stages of change of cristae and of organization of the crystalline matrix support the hypothesis that these structures derive from mitochondria. As far as the functional significance is
ULTRASTRUCTURE OF GLIOSOMES
263
Sanit& 8, 197, (1972). concerned, Hashimoto claims (7) that in 3. D'UvA, V., Boll. Zool. 36, (1969). newborn cats gliosomes play the role of 4. D'UvA, V., Septi~me Congr~s International de protecting the glial fibres from the comMicroscopie Electronique, Grenoble, 131, pression of surrounding tissues, while the (1970). gliofilaments, always numerous and often 4. GREENAWALT,J. W., RossI, C. S., ANDLEHMNGER, A. L. J. Cell Biol. 23, 21, (1964). in direct contact with them, strengthen the 6. HASHIMOTO, P. H., VI Int. Congr. Electron Mistructures. cros, Kyoto, 467, (1966). Our data on adult animals do not give 7. HASHIMOTO, P. H., J. Comp. Neurol. 137, 251, support to this hypothesis. (1969). 8. KARNOVSKY, M. J., J. Cell Biol. 27, Abstracts The fusion and the fragmentation Fifth Annual Meeting American Society for pointed out by us indicate a remarkable Ceil Biology, 137 A, (1965). structural mobility in these organelles, and 9. KLUG, A., ANDBERGER,J. E., J. Mol. Biol. 10, 565, the same consideration previously made on (1964). the crystalline lattice of the matrix leads us 10. KORMAN, E. F., HASSIS, R. A., WILLIAMS, C. H., WAKAPAYASHI,T., GREEN, D. E., AND VALDIVIA, to think that it may be an array of part of E., Bioenergetics l, 387, (1970). the mitochondrial population due to a 11. LIPSON, H. S., (Ed.), Optical Transforms. Acaparticular functional stage of cells and glial demic Press, London and New York, 1972. fibres. 12. MATTHEWS,B. W., J. Mol. Biol. 33, 491, (1968). The electron microscope observations 13. MORALES, R., AND DUNCAN, D., Anat. Rec. 171, 545, (1971). were carried out in Naples at the Electron Microscopy Center of the Faculty of 14. PmA, R. L., NISHIOKA, R. S., AND BERN, U., J. Ultrastruct. Res. 6, 164, (1962). Science. 15. REYNOLDS, E. S., J. Cell Biol. 17, 208, (1963).
The authors are indebted to Prof. D. Steve Bocciarelli and Prof. G. Ghiara for critical reading of the manuscript. The authors thank Mr. G. Monteleone, Mr. G. Falcone, and Mr. B. Scorza tbr their valuable photographic assistance; Mr. L. Pierangeli for the careful execution of drawings, Mr. G. Cafiero, and Mr. G. Orsello for their very capable technical assistance. REFERENCES 1. BERGER,J. E., J. Cell Biol. 43, 442, (1969). 2. DONELLI, G., AND PAOLETTI, L., Ann. Ist. Super.
16. SJOSTRAND, F. S., AND BARAJAS,L., J. Ultrastruct. Res. 32, 293, (1970). 17. SREBRO,Z., AND BORZEDOWSKA,E., Folia Biol. 12, 313, (1964). 18. SREBRO, Z., J. Cell Biol. 26, 313, (1965). 19. STERNLIEB, I., ANDBERGER, J. E., J. Cell Biol. 43, 448, (1969). 20. SUZUKI,T., AND MUSTOKI, F., J. Cell Biol. 33, 605, (1967). 21. VAINSHTEIN, B. K., in Straub, F. B., and Friedrich, P., (Eds.), Symposium on Modern Methods in the Investigation of Protein Structure, p. 29. Akad~miai Kiad6, Budapest, 1967.
Ultrastructure of Gliosomes in Ependymal Cells of the Lizard G.
DONELLI, 1 V.
D'UvA,
2 AND
L.
PAOLETTI I
Physics Laboratory, lstituto Superiore di Sanith, Rome 1, and Institute of Histology and Embryology, Faculty
of Science, Naples 2, Italy Received July 2, 1973, and in revised form February 21, 1974 Gliosomes similar to those already described by other authors in amphibia and mammalia w e r e found in the hypothalamic region ofLacerta s. sicula Raf. The internal matrix of these particular organelles, which closely resemble transformed mitochondria, is in a more or less advanced state of crystallization. The dimensions of the unit cell, the symmetry and the space group of these mitochondrial crystals were determined by analyzing electron micrographs of ultrathin sections of gliosomes by means of the optical transform technique. An evaluation of the molecular weight of the single asymmetrical subunit of the crystal lattice was obtained. INTRODUCTION Crystalline and paracrystalline inclus i o n s h a v e b e e n d e s c r i b e d in m i t o c h o n d r i a of v a r i o u s t y p e s of cells in b o t h n o r m a l a n d p a t h o l o g i c a l t i s s u e s (20). I n c l u s i o n s of a p a r t i c u l a r t y p e are p r e s e n t in o r g a n e l l e s k n o w n as g l i o s o m e s considered to be modified mitochondria. These gliosomes, described by several aut h o r s (6, 7, 14, 18) in g l i a l f i b e r s of s o m e a n i m a l s p e c i e s , were o b s e r v e d a n d s t u d i e d b y one of us in e p e n d y m a l cells of L a c e r t a s. sicula R a f . (3, 4). T o d a t e l i t t l e is k n o w n of t h e f u n c t i o n a l a n d p h y s i o l o g i c a l s i g n i f i c a n c e of t h e s e s t r u c t u r e s . B y m e a n s of a n a n a l y s i s of electron micrographs with the optical Four i e r t r a n s f o r m t e c h n i q u e (2, 9, 11), m o r e s p e c i f i c a l l y w i t h t h e m e t h o d of r e c o n s t r u c t i o n of t h e r e c i p r o c a l l a t t i c e of t h e c r y s t a l p r o p o s e d b y B e r g e r (1, 19), i t is p o s s i b l e to o b t a i n a m u c h m o r e c o m p l e t e s e t of s t r u c t u r a l d a t a t h a n c a n g e n e r a l l y b e f o u n d in t h e l i t e r a t u r e ; t h e r e f o r e we h a v e c o n s i d e r e d it i n t e r e s t i n g , in t h i s work, to a p p l y t h e s e m e t h o d s in t h e s t u d y of g l i o s o m e s . The morphological and ultrastructural d a t a o b t a i n e d d i d n o t p e r m i t us to c o m p l e t e l y d e f i n e t h e f u n c t i o n a l s i g n i f i c a n c e of these formations. However, reconstruction
of t h e c r y s t a l l a t t i c e a n d d e t e r m i n a t i o n of t h e p a r a m e t e r s of t h e u n i t cell w i t h t h e a b o v e t e c h n i q u e s m a d e it p o s s i b l e to p u t f o r t h a n u m b e r of h y p o t h e s e s c o n c e r n i n g t h e n a t u r e of t h e o b s e r v e d c r y s t a l l i n e inclusions. MATERIAL AND' METHODS Electron micrographs of the hypothalmic region of
Lacerta s. sicula brain were studied by optical Fourier transforms. The hypothalmic region was removed from the brain with the aid of a dissection microscope, reduced to small pieces with a diameter of 2-3 mm, and fixed, part in 2.5% glutaraldehyde in phosphate buffer pH 7.4 and part in formaldehyde-glutaraldehyde in phosphate buffer pH 7.4. The sections were then postfixed in 2% osmium tetroxide in phosphate buffer, dehydrated with increasing concentrations of alcohol and embedded in Epon-Araldite (15). Thin sections were stained with toluidine blue and observed with the optical microscope. Ultrathin sections obtained with an LKB microtome (Ultratome III) were stained with uranyl acetate and lead citrate (8) and observed with a Siemens IA electron microscope. Apertures of 50 #m for the objective lens and 400 #m for the condenser lenses were used. The accelerating voltage was 80 KV and the micrographs were taken at a magnification between 10,000 and 40,000, the second pole piece being used for the second projector lens. The diffraction patterns were obtained with a Polaron optical diffractometer equipped with a Spectra Physics 2.0 mW helium-neon laser. 253
Copyright © 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
254
DONELLI, D'UVA AND PAOLETTI
EXPERIMENTAL RESULTS In the basal zone of the ependymal cells, however, mainly LMG are present. (1) Morphological Observations The ultrastructural aspects of gliosomes (2) Optical Transforms of Electron Microof the species studied are similar to those graphs described by Srebro in Amphibia (18). Electron micrographs of gliosomes of Gliosomes are, as a rule, elongated structures, ranging in length from 1 to 10 ttm both the first and the second type were analyzed. While it was possible to deterand in thickness from 0.4 to 0.6 ttm. The external wall is somewhat similar to mine the structure of the inclusions in the the mitochondrial wall; it is constituted by GMG, it was not possible to detect any two cytomembranes separated by a small "crystalline" structure of the interlamellar electron transparent space; the inner cyto- matrix in the LMG. For the GMG, the analysis was carried membrane is introflected, and often forms out on about a hundred electron microcanaliculi of cristae 180/~ in width. In cross graphs corresponding to sections whose section the latter appear as small circular, thickness could be estimated equal to or or in some cases triangular, profiles, which less than 1000 fl,. are, as a rule, located in the peripheral zone With preliminary measurements it was of the matrix of the gliosome, but which can also be found further towards the in- possible to estimate the lattice spacings of the crystal as about 100 A: on the basis of side. On the basis of the ultrastructural orga- this evaluation the number of lattice nization of the matrix, two types of gli- planes contained in the thickness of the osomes can be recognized: (a) gliosomes sections can be calculated as lying between with lamellar matrix (LMG) and (b) gli- 5 and 10. The electron micrographs can osomes with granular matrix (GMG) (Fig. therefore be considered, with close approximation, projections of the crystal onto the 1a-b). plane of the photographic plate and their In gliosomes with a granular matrix optical transforms sections of the recipro(GMG), at high magnification (× 60 000), cal lattice parallelly oriented to the projecthe matrix is resolved in a crystal lattice of granules 80-90 A in width, located at tion plane. For the effective construction of the intervals of 100-110 A. Very electron-dense granules are also crystal and the singling out of the elements present in the matrix, these having an of symmetry, the "bidimensional" diffracaverage diameter of 450 A and resembling tion patterns were used, that is, those which correspond to sections of the reciprothose found in the mitochondria (5). In gliosomes with a lamellar matrix cal lattice passing through the lattice (LMG) the lamellae are arranged longitu- planes (1). In quantitative terms these patterr~s dinally to the gliosomes with an apparent periodical array. Fusion between two or represent about 40% of all observed patmore gliosomes of the same type is often terns. For a quantitative analysis of the diffracobserved (Fig. la). tion patterns the various sources of error The glial fibres contain what are probawere isolated and their relative sizes were bly glycogen granules and sheafs of filaestimated. The results can be summarized ments; the gliosomes of the first and secas follows: ond type are present in equal proportions; (1) Compression and distortion of the the filaments themselves are particularly numerous where gliosomes are to be found structure of the crystal during cutting o[ the section. The incidence of this error was (Fig. 2c).
ULTRASTRUCTURE OF GLIOSOMES
255
Fio. 1. (a) Gliosome with lameIlar matrix. Note the fusion or splitting between gliosomes (arrow) x 15 000 (b) Gliosome with granular matrix. × 16 000. m i n i m i z e d b y c h o o s i n g for t h e a n a l y s i s of the data those sections whose structure was a p p a r e n t l y n o t d a m a g e d . T h e close a g r e e m e n t of t h e d a t a , w i t h i n t h e final s p r e a d i n g c a l c u l a t e d on t h e b a s i s of t h e
v a r i o u s s o u r c e s of error, s u p p l i e d t h e conf i r m a t i o n a p o s t e r i o r i t h a t t h e c r i t e r i a of s e l e c t i o n a d o p t e d were s u f f i c i e n t to m a k e this error negligible. (2) Magnification of electron micro-
FIG. 2. (a) A gliosome of first type (LMG) in an e p e n d y m a l cell x 14 000 (b) A gliosome of t h e s a m e type at higher m a g n i f i c a t i o n × 41 000 (c) Gliosome with g r a n u l a r m a t r i x in a glial fiber. T h e sheafs of f i l a m e n t which are particularly n u m e r o u s near gliosomes are evident. × 24 000. 256
ULTRASTRUCTURE OF GLIOSOMES
graphs. In our working conditions, the relative error of the calibration with standard samples, was estimated to be not greater than 10%. (3) Diffraction constant. Calibration of the optical bench, in working conditions, with standard lattices, made it possible to estimate the relative error as less than 1%. (4) Tilting of the crystal with respect to
the plane of the section and tilting of the section with respect to the plane normal to the projection line (electron beam). This causes a spreading of the measurements on the diffraction patterns. In fact to every different degree of tilting of the crystal with respect to the projection line corresponds a different tilting of the sections of the reciprocal lattice. For the finite dimensions of the crystal, it is not possible to interpret Fourier transforms of the micrographs in terms of sections of a reciprocal lattice of points without dimensions, but rather of a lattice whose points are considered "expanded" in the reciprocal space. For this reason we expect a broad range of sections which intersect, with different orientations, "expanded points" of the same plane of the reciprocal lattice. The measurements of the spacings and angles relative to this range of sections correspond, therefore, to a spreading of values whose amplitude can be evaluated with the equation: e/p = 2 P/D (1), where e and p are the "expansion" of the points of the reciprocal lattice in a given direction and their spacing in the considered direction, respectively; P and D, the corresponding spacing and the size of the diffraction latrice in the same direction, respectively. The spreading of the spacing and angle measurements was in this way evaluated to be of the order of 5-10%. The above discussion of the possible errors implies that a total spreading of about 15-20% in the spacing and angle measurements on the diffraction patterns must be expected. On the basis of the elements of symme-
257
try and the spacing between the spots present, the bidimensional diffraction patterns obtained were classified into three main types. Type A: (Fig. 3a-b), is characterized by an arrangement of the spots on a rectangular lattice. The corresponding section of the reciprocal lattice and the main spacings Pl and P2 are given in Fig. 4a. Type B: (Fig. 3c-d), is characterized by an arrangement of the spots on a pseudosquare lattice. The corresponding section of the reciprocal lattice and the main spacingspl and Pa are shown in Fig. 4b. Type C: (Fig. 3e-f), is characterized by an arrangement of the spots on a pseudohexagonal lattice. The corresponding section of the reciprocal lattice and the spacings p2 and ps are given in Fig. 4c. Apart from the three types mentioned above, there is a limited number of the patterns, which can be in turn classified into two types, on which systematic extinctions are evident in some classes of diffraction spots. Type A 1: (Fig. 5a-b). Along the meridian systematic extinctions of a number of spots are evident and the condition for the nonextinction of the Kth is K=2n, with n a whole number. This implies the existence of a crystallographic axis with 2-fold screw symmetry. In that the spacings are pJ2 and P2, respectively, while the spacing of the spots along the meridian is p~ (Fig. 6a), it is likely that type A1 patterns coincide with type A patterns, but correspond to micrographs on which the resolution has made it possible to show up the axis of screw symmetry. That is, it is possible to distinguish the asymmetric subunits of the crystal rotated by ~.
258
DONELLI, D'UVA AND PAOLETTI
m FIG. 3. (a) Section of GMG corresponding to type A bidimensional pattern, x 72 000. (b) Type A diffraction pattern. (c) Section of GMG corresponding to type B bidimensional pattern. × 72 000. (d) Type B diffraction pattern. (e) Section of GMG corresponding to type C bidimensional pattern. × 72 000. (f) Type C diffraction pattern. The diffraction patterns are presented at the same magnification and are aligned with respect to the crystal images.
ULTRASTRUCTURE OF GLIOSOMES
a)
•
•
Q
P3
bl
c)
Fxa. 4. (a) Section of the reciprocal lattice corresponding to type A diffraction pattern. (b) Section of the reciprocal lattice corresponding to type B diffraction pattern. (c) Section of the reciprocal lattice corresponding to type C diffraction pattern.
Type BI: (Fig. 5c-d). S y s t e m a t i c extinctions of spots are evident along the meridian. T h e condition for nonextinction of the K t h is still K = 2n, with n a whole number, confirming the presence of the axis of 2-fold screw s y m m e t r y . In t h a t the spacings are p J 2 and p~, respectively, while the spacing along the meridian is p~ (Fig. 6b), it is likely t h a t type B1 coincides with type B, corresponding however, to micrographs on which the resolving power is such as to show up the axis of screw symmetry. T h e types of bidimensional patterns singled out together with the spacings measured on t h e m are s u m m a r i z e d in Table I. (3) Reconstruction of Crystal Lattice In order to determine the most likely value of the spacings of T a b l e I, t h a t is, to
259
d e t e r m i n e the dimensions of the unit cell of the reciprocal lattice, a statistical analysis of the m e a s u r e m e n t s was made. T h e results of this analysis are s u m m a r i z e d in the histogrammes of Fig. 7, constructed on the basis of tl~e frequency with which the values of the spacings between the spots in the m e a s u r e m e n t s taken on 41 bidimensional patterns were obtained. On every pattern the two or three main spacings were measured. These are shown in the histogrammes of Fig. 7 and in Table I. The sources of the measured spacings are m a r k e d with capital letters. T h e distributions obtained show halfspreadings which represent 12-13% of the corresponding m e a n value, in good agreem e n t with the spreading of the measurements previously evaluated. T h e relative a b u n d a n c e of the different types of patterns resulted as follows: T y p e A = 23, type B = 11, type C - 7. For the spacings of T a b l e I, the mean values of the corresponding peaks, with an error equal to the half-width of the peaks themselves were taken as the most probable values (Table II). T h e unit cell of the reciprocal lattice was reconstructed by combining the bidimensional patterns along the lines on which the same spacing of the spots was present. In this way a monoclinic unit cell was obtained, the dimensions of which were taken as follows: a* = P2, b* = pl/2c* - p3 with the ~* angle (between the a* and the c* axes) equal to 63 ° ± 12% (Fig. 8). The Miller indices of the lattice planes corresponding to the various types of patterns are listed in Table I. It is i m p o r t a n t to note t h a t the separation between the mean values of the spacings p2 and ps is lower t h a n the error ascribed to the values themselves. Within the precision of m e a s u r e m e n t it is not possible to exclude t h a t p 2 and p a may have the same value. In this case the reciprocal lattice and the crystal itself would be
260
DONELLI, D'UVA AND PAOLETTI
FIG. 5. (a) Section of GMG corresponding to type A 1bidimensional pattern. × 72 000. (b) Type A 1diffraction pattern. (c) Section of GMG corresponding to type B1 bidimensional pattern. × 72 000. (d) Type B1 diffraction pattern. The diffraction patterns are presented at the same magnification and are aligned with respect to the crystal images. hexagonal. It m u s t be considered, however, t h a t the separation between P2 and P3 is slightly smaller t h a n the half-spreading of the measurements, and that, moreover, if p~ and P3 should have the same spacing, the c o r r e s p o n d i n g m e a s u r e m e n t w o u l d have an error of a b o u t 20%, which is s o m e w h a t higher t h a n the error of the m e a s u r e m e n t of pl, 12%. It would be difficult to explain this difference in the precision of two m e a s u r e m e n t s obtained with the same procedure and from the same set of patterns. For these reasons it could be more correct to interpret the data according to the hypothesis of two different spac-
ings P2 and Ps of a pseudohexagonal monoclinic lattice. The unit cell of the crystal, on the basis of the data relative to the reciprocal lattice, is monoclinic with dimensions o f a = 110 423A, b = 2 4 0 ± 3 1 A , c - 120± 16/~with the ~ angle (between the a and the c axes) equal to 117 ° ± 14 ° and with a vol of V = 2.8-10 6 ± 1.5-10 6/~3. The systematic extinctions of the diffraction spots, observed on the p a t t e r n s (1, 0, 0) and (0, 0, 1), corresponding to the class of reflexions 0, K, 0 for the indices K = 2n, with n a whole number, imply the presence of a screw axis perpendicular to
261
ULTRASTRUCTURE OF GLIOSOMES
the lattice planes (0, 1, 0) with component b/2. These results make it possible to single out the space group of the crystal as the group P 21, with which two equivalent asymmetrical subunits per unit cell (Fig. 9) are compatible. DISCUSSION
The problem of the meaning of the two types of gliosomes, i.e., whether they are the result of different processes of transformation of the mitochondria or of different stages of the same process, is still completely unsolved. In any case, special stress must be laid on the absence of "hybrid" arrays which can be interpreted in terms of transformation from one type of gliosome to the other. Crystallographic data prove that GMG have a crystalline structure, very different from the one displayed by the matrix of the LMG, thus showing a new differential character, which strengthens the hypothesis of the existence of two types of gliosomes. In GMG the crystalline arrangement of the matrix is generally present with a definite orientation with respect to the gliosomes. In particular cross sections of the gliosomes have always given patterns of type C (Fig. 3e), that is, the longitudinal axis of the gliosome and the lattice axis [0, 1, 0] are, with close approximation, parallel. This supports the hypothesis of some interaction between the structures of the gliosome (cytomembranes, cristae, etc.) and the crystalline matrix. The triangular profiles of crystae sections in gliosomes and the existence of prismatic cristae in mitochondria with crystalline inclusions (13) have been interpreted, also by other authors, as effects of crystallization of the matrix in these structures (10). In the hypothesis that, as found by Srebro in amphibia (17), the crystalline inclusions studied are protein crystals, it is possible to estimate the molecular weight
of each of the two equivalent asymmetrical subunits "per" unit cell of the crystal, using the volume V of the unit cell itself. In most protein crystals (I2) the volume of the unit cell per unit of molecular weight (Vm) lies in the interval between V m = 1.5 AVdalton and V m = 3.5 AVdalton. Therefore for the crystal studied, whose unit cell is monoclinic with a vol V = 2.8-10 ~ ha, the molecular weight M W = V/2Vm of the single asymmetrical subunit would lie between M W = 4.10 5 and M W = 9. l0 B. The protein material included and crystallized in GMG can:
a)
T,pl/2 I pl
• -- t-
o
Q
--
O
b) FIG. 6. (a) Spacings present on type A~ diffraction pattern. (b) Spacing present on type B1 diffraction pattern. TABLEI Patterns
A B C A~ B1
Spacings
Pl Pl P2 pj2 pJ2
; ; ; ; ;
P2 P~ P3 P2 p~
Spacings corresponding of systematic extinctions
Pl
Pl
Miller indices of corresponding planes
(o, o, 1) (1, o,o) (o, 1, o) (o, o, 1) (1,o,o)
262
DONELLI, D'UVA AND PAOLETTI
,0
°'
LI
A _
A
i._ I.L
BA__
i_
_
~
S_
5~-
ICAC
BAA
A AA
BAAAA
I
03
CC
BA
1/1/,0
'i
p2
B
C
11120
1/100
[BB
II
AAA
I
11110
1/97
1/80
1/125
11106
Spot
1/90
spacing (/~-~)
Fro. 7. Experimental distributions of the spacing between the spots in measurements on bidimensional diffraction patterns. Each histogramme shows the types of diffraction patterns from which the measurements come. /~J
/
S
~ ' - - - -
.j~ --
I I
J J
/
D
/7q-
I I
JJ
, [
/
~ / j J
I
J
I I
/
.J /
C~
l/
Fro. 8. The reciprocal lattice and the monoclinic unit cell. TABLE II Reciprocal lattice spacings
A-
Pl P2 p~
1/120 ± 13% 1/97 - 14% 1/106 -~ 13%
(a) be of external origin, (b) derive from the different organization of protein components present in mitochondria in the course of their transformation into gliosomes. In the latter hypothesis it needs to be taken into account that most protein components present also in normal mitochondria have a molecular weight (16, 19) which is from two to ten times less than the minimum molecular weight calculated by us for the asymmetrical subunit. If it is considered that in the majority of protein crystals the number of equal molecules per asymmetrical subunit is n = 1
Fla. 9. The unit cell of the Cristal. The screw axis is shown. In the figure, the directions along which the projections of the lattice were obtained, which gave rise to the patterns in Table 1, can be easily singled out; in fact these projections have indices which are equal to the indices, within the reciprocal space, of the corresponding types of patterns listed in Table I.
(12, 21) it seems more probable that each asymmetrical subunit is made up of a multimolecular complex. It could be imagined that the asymmetrical subunit is one of the multienzymatic complexes which make up the structural elements of the model of the mitochondrial membranes proposed by SjSstrand and Barajas (16). The observations made by one of the authors (3, 4) on the presence of gliosomes at different stages of change of cristae and of organization of the crystalline matrix support the hypothesis that these structures derive from mitochondria. As far as the functional significance is
ULTRASTRUCTURE OF GLIOSOMES
263
Sanit& 8, 197, (1972). concerned, Hashimoto claims (7) that in 3. D'UvA, V., Boll. Zool. 36, (1969). newborn cats gliosomes play the role of 4. D'UvA, V., Septi~me Congr~s International de protecting the glial fibres from the comMicroscopie Electronique, Grenoble, 131, pression of surrounding tissues, while the (1970). gliofilaments, always numerous and often 4. GREENAWALT,J. W., RossI, C. S., ANDLEHMNGER, A. L. J. Cell Biol. 23, 21, (1964). in direct contact with them, strengthen the 6. HASHIMOTO, P. H., VI Int. Congr. Electron Mistructures. cros, Kyoto, 467, (1966). Our data on adult animals do not give 7. HASHIMOTO, P. H., J. Comp. Neurol. 137, 251, support to this hypothesis. (1969). 8. KARNOVSKY, M. J., J. Cell Biol. 27, Abstracts The fusion and the fragmentation Fifth Annual Meeting American Society for pointed out by us indicate a remarkable Ceil Biology, 137 A, (1965). structural mobility in these organelles, and 9. KLUG, A., ANDBERGER,J. E., J. Mol. Biol. 10, 565, the same consideration previously made on (1964). the crystalline lattice of the matrix leads us 10. KORMAN, E. F., HASSIS, R. A., WILLIAMS, C. H., WAKAPAYASHI,T., GREEN, D. E., AND VALDIVIA, to think that it may be an array of part of E., Bioenergetics l, 387, (1970). the mitochondrial population due to a 11. LIPSON, H. S., (Ed.), Optical Transforms. Acaparticular functional stage of cells and glial demic Press, London and New York, 1972. fibres. 12. MATTHEWS,B. W., J. Mol. Biol. 33, 491, (1968). The electron microscope observations 13. MORALES, R., AND DUNCAN, D., Anat. Rec. 171, 545, (1971). were carried out in Naples at the Electron Microscopy Center of the Faculty of 14. PmA, R. L., NISHIOKA, R. S., AND BERN, U., J. Ultrastruct. Res. 6, 164, (1962). Science. 15. REYNOLDS, E. S., J. Cell Biol. 17, 208, (1963).
The authors are indebted to Prof. D. Steve Bocciarelli and Prof. G. Ghiara for critical reading of the manuscript. The authors thank Mr. G. Monteleone, Mr. G. Falcone, and Mr. B. Scorza tbr their valuable photographic assistance; Mr. L. Pierangeli for the careful execution of drawings, Mr. G. Cafiero, and Mr. G. Orsello for their very capable technical assistance. REFERENCES 1. BERGER,J. E., J. Cell Biol. 43, 442, (1969). 2. DONELLI, G., AND PAOLETTI, L., Ann. Ist. Super.
16. SJOSTRAND, F. S., AND BARAJAS,L., J. Ultrastruct. Res. 32, 293, (1970). 17. SREBRO,Z., AND BORZEDOWSKA,E., Folia Biol. 12, 313, (1964). 18. SREBRO, Z., J. Cell Biol. 26, 313, (1965). 19. STERNLIEB, I., ANDBERGER, J. E., J. Cell Biol. 43, 448, (1969). 20. SUZUKI,T., AND MUSTOKI, F., J. Cell Biol. 33, 605, (1967). 21. VAINSHTEIN, B. K., in Straub, F. B., and Friedrich, P., (Eds.), Symposium on Modern Methods in the Investigation of Protein Structure, p. 29. Akad~miai Kiad6, Budapest, 1967.
