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Cytoplasmic microtubules in mammalian cells
J. ULTRASTRUCTURERESEARCH 11, 123-138 (1964)
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Cytoplasmic Microtubules in Mammalian Cells E. SANDBORN,1 PATRICK F. KOEN, J. D. MCNABB, a n d G. MOORE
Faculty of Medicine, McGill University, Montreal, Canada Received February 13, 1964 Cytoplasmic microtubules can be shown to be a constant finding in tissues of the rat which have been fixed in aldehydes followed by osmium tetroxide. The aldehydes employed were acrolein and glutaraldehyde, which were combined to give both rapid penetration and stabilization of the ultrastructural constituents of the cell. By this method microtubules are seen to be common to all areas of the cytoplasm. Connections to other membranes are illustrated. Possible functional roles for the cytoplasmic microtubules are discussed.
C y t o p l a s m i c microtubules, which have been r e p o r t e d occasionally in p l a n t a n d a n i m a l cells, have n o t been shown to be a c o n s t a n t finding in m a m m a l i a n cells. By the use of a l d e h y d e s (32, 33) followed b y o s m i u m tetroxide (19) fixation of tissues, t u b u l a r structures of v a r y i n g d i m e n s i o n were f o u n d in all types of cells examined. F u r t h e r m o r e , evidence of continuity between c y t o p l a s m i c organelles was seen repeatedly. The p u r p o s e of this r e p o r t is to describe the m i c r o t u b u l e a n d to illustrate t h a t it is a frequent finding in the cells of the rat.
MATERIALS AND METHODS By a modification of Palay's technique (21) young adult rats were perfused with balanced salt to remove the circulating blood followed by a fixative solution containing 2% acrolein and 6.5% glutaraldehyde in a phosphate buffer (32, 33). The perfusion of fixative was continued for 20 minutes; the tissues were then removed from several organs, diced, and fixed for an additional hour in the same fixative. After fixation in the aldehyde solution, the blocks were washed in Veronal acetate buffer (10) for 15 minutes and then postfixed in 2% Veronal acetate-buffered osmium (•9) for 2 hours. The tissues were dehydrated in graded ethanol, then transferred to propylene oxide and embedded in Epon as described by Luft (16). The blocks were sectioned on a Porter-Blum microtome. Sections 0.5-1.0 # in thickness were stained with 1% aqueous toluidine blue at p H 8.5. Thin sections were stained with uranyl acetate (44), lead hydroxide (14), or a combined stain of lead hydroxide followed by 1% uranyl acetate (14, 44). The sections were examined in a Siemens Elmiskop I electron microscope. 1 Present address: D6partement d'Anatomie, Universit6 de Montr6al, Montr6al, Canada.
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E. SANDBORN, P. F. KOEN, J. D. MCNABB AND G. MOORE OBSERVATIONS
I n light microscopy all cell features ordinarily seen were demonstrated. Some features were r e m a r k a b l y accentuated, a n d others that ordinarily can be d e m o n strated only by special techniques were seen quite clearly. By light microscopy, m i t o c h o n d r i a were more distinct t h a n with o r d i n a r y techniques a n d the cell web (25) was clearly seen in intestine a n d kidney. Densely.stained granules, in larger n u m b e r s t h a n observed by other techniques, were seen in cells of the thyroid follicles, the nervous system, a n d kidney tubules. The elastic l a m i n a of arterioles stained vividly with toluidine blue. I n the electron microscope, the most striking new finding was a complex n e t w o r k of tubules within all cells examined. The tubules were seen in n e u r o n s a n d neuroglia of the cerebellum, cerebral m o t o r cortex, a n d semilunar ganglion, the cells of p a n creatic acinus, thyroid follicle, capillary endothelium, intestinal epithelium, p r o x i m a l a n d distal convoluted tubules of the kidney, the a d r e n a l medulla, a n d those of b o t h the alveolus a n d duct of the m a m m a r y gland. The tubules are of varying dimensions; the most c o m m o n of these is a long, relatively straight tubule approximately 220 A in diameter (Figs. 1 a n d 2). This type of tubule is seen in all the cells examined a n d often traverses the whole of the field seen in the microscope. The dimensions of this tubule are approximately the same in the n e u r o n (Figs. 1-4), secretory (Figs. 8, 10 a n d 11), or absorptive (Figs. 5 a n d 6) epithelial a n d endothelial (Fig. 13) cells. A similar structure in h y d r a was referred to b y Slautterback (42) as a cytoplasmic microtubule. The microtubules seen i n the axons of peripheral nerves of the rat have walls 60 A in thickness s u r r o u n d i n g a l u m e n approximately 100 A in diameter. The wall is seen to be a t r i l a m i n a r m e m b r a n e (28, 36, 37, 38-40, 41). I n addition, dark b a n d s bridge the space between the i n n e r a n d outer l a m i n a e of the m e m b r a n e at a periodiFIG. 1. A longitudinally sectioned axon from the semilunar ganglion of the rat. The microtubules range. The tubules are seen to branch (arrow) and continuity of filaments with tubules is suggested (arrow). The filaments are about 60 /~ in diameter. At the upper center is a mitochondrion with tubular appearing cristae as well as the flattened type. At the upper left a large tubule of the agranular reticulum (ar) with a diameter of 600 ~ + is illustrated. Its membranes are continuous with the outer mitochondrial membrane, x 66,000. FIG. 2. A cross section of a small myelinated axon in the semilunar ganglion of the rat. The layers of myelin have a spacing of 120 A. The separation of the interperiod line into two layers is visible for considerable distances. The plasma membrane of the axon is trilaminar and is 60/~ in thickness. Interconnecting bands between the laminae of the unit membrane are apparent at a periodicity of 50-70/~. Cytoplasmic microtubules (neurotubules) (cm) are seen to be surrounded by a wall made up of a trilaminar membrane similar to that of the plasma membrane. Bands occur between the outer and inner laminae imparting to the membrane the appearance of a series of globules. The wall of the neurotubules is 60/~ in thickness and there is a 100 ~ lumen. The neurofilaments appear o be made up of globular subunits, x 207,000.
(cm) and filaments (f) are seen in abundance. The diameter of the microtubules is in the 220 ~
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E. SANDBORN~ P. F. KOEN, J. D. MCNABB AND G. MOORE
city of a b o u t 60-70 A (Fig. 2), as recently described b y Sj/Sstrand (39). This is also seen in the p l a s m a m e m b r a n e of the a x o n (Fig. 2). V a r i a b l e a m o u n t s of d a r k l y stained content are seen in the l u m e n of the tubule. L a r g e r tubules are also se'en in the axon. T h e y r e a c h a d i a m e t e r of 700 A (Figs. 1 a n d 3), are slightly t o r t u o u s a n d c o n t a i n irregular, d a r k l y stained material, These structures are considered to be the c o n v e n t i o n a l a g r a n u l a r reticulum b u t are t u b u l a r r a t h e r t h a n vesicular in nature. These will be referred to as the a g r a n u l a r o r t u b u l a r a g r a n u l a r reticulum. The c o n t e n t of these larger tubules is generally m o r e densely stained t h a n in the microtubule. C o n t i n u i t y of the m e m b r a n e of the a g r a n u l a r retic u l u m with the outer m i t o c h o n d r i a l m e m b r a n e is seen (Fig. 1). F i l a m e n t s are f o u n d in a b u n d a n c e in the axon. The filaments have a d i a m e t e r of a p p r o x i m a t e l y 60 A. T h e y a p p e a r to be m a d e up of subunits as described b y Schmitt a n d G e r e n (34). The presence of a l u m e n is p r o b l e m a t i c a l ; in some there is a suggestion of a l u m e n while in m o s t this is n o t visible (Fig. 2). The l a m e l l a t e d n a t u r e of the nerve myelin sheath (35) of axons is illustrated well b y this fixative. The myelin has a p e r i o d i c i t y of 120 A. The two layers of the interp e r i o d line (26) are seen over considerable distances (Fig. 2). The c o n t a c t between the two m e m b r a n e s in the dense line is also discernible. The m i t o c h o n d r i a in the a x o n are seen to c o n t a i n d a r k l y stained m a t e r i a l between the inner a n d o u t e r m e m b r a n e s a n d between the two layers of the cristae. I n the p e r i k a r y o n of ganglionic cells of the peripheral nervous system (Fig. 3) a n d in neurons of the cerebral cortex a n d cerebellum, m a n y filaments, microtubules, a n d larger tubules of the a g r a n u l a r r e t i c u l u m are found. These are of the same dimensions as seen in the axon. The t u b u l a r a n d filamentous structures invade all areas of the cytoplasm. As in the axon, there is a variable a m o u n t of d a r k l y stained content within the l u m e n of the microtubules, the a g r a n u l a r reticulum, a n d the space between the m i t o c h o n d r i a l m e m b r a n e s . I n the a x o n of m y e l i n a t e d s y m p a t h e t i c nerve fibers in the a d r e n a l m e d u l l a (Fig. 4), m i c r o t u b u l e s are seen to be as n u m e r o u s as in the axons of peripheral nerves (Figs. 1 a n d 2). The Schwann cells (Fig. 4) are seen to c o n t a i n m i c r o t u b u l e s as in the neuron. Branching of the m i c r o t u b u l e s is evident.
FIG. 3. Electron micrograph of the cytoplasm of the large, pale neuron of the semilunar ganglion of the rat. Mitochondria with darkly stained material between the membranes of the cristae are present in abundance. Microtubules (cm), 220 A in diameter, are seen to cross in straight lines while larger tubules of the agranular reticulum (ar) with more irregular outline are also seen. These larger tubules contain darkly stained material similar to that seen in the mitochondria. The tubular network within the neuron is very complex. Continuity of membrane structure between the microtubules, the reticulum, and mitochondria is suggested, x 47,500. Fro. 4. A myelinated axon of a sympathetic autonomic neuron in the adrenal medulla with a Schwann cell. The outer and inner connections of the myelin with the plasma membrane can be seen. Within the axon mitochondria and microtubules can be seen. In the Schwann cell cytoplasm microtubules (cm) are present and branching of the tubules (arrow) is demonstrated. × 37,500.
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I n the absorptive cells of the intestinal epithelium, n u m e r o u s microtubules a n d filaments are found. The microtubules are seen to be vertically (Fig. 5) as well as h o r i z o n t a l l y (Fig. 6) oriented. Large n u m b e r s are seen horizontally disposed in the cell web. I n t e r m i n g l e d with the microtubules are m a n y filaments extending into the microvilli. Both microtubules a n d filaments reach the apical a n d the lateral cell m e m b r a n e s . These areas of contact are particularly c o m m o n at the desmosomes. Microtubules pass for considerable distances in the cytoplasm. The dimensions of the microtubules a n d filaments are similar to those in the n e u r o n a n d its processes. I n the p r o x i m a l convoluted tubule of the kidney (Fig. 7), microtubules similar to those in the other cell types described are seen in the cytoplasm. M i c r o t u b u l e s are closely associated with m i t o c h o n d r i a a n d m e m b r a n e - b o u n d secretory granules. I n the follicular cells of the thyroid gland (Figs. 8 a n d 9), there is a n e t w o r k of microtubules a n d filaments t h r o u g h o u t the cytoplasm. The greatest c o n c e n t r a t i o n of t u b u l a r elements is i n the cell web area of the apex a n d along the lateral borders of the cell. M i c r o t u b u l e s are particularly concentrated at the desmosomes. M e m b r a n e b o u n d secretory granules are seen from the base to the apex of these cells in sections prepared for b o t h light a n d electron microscopy. Microtubules are seen in close c o n t a c t with these granules. I n the light cells of the thyroid gland (Fig. 9), microtubules, even more n u m e r o u s t h a n in the follicular cells, are found. M a n y of these tubules are seen to be arranged parallel to one another, to t u b u l a r a g r a n u l a r reticulum, a n d to the m a j o r i t y of the m i t o c h o n d r i a . The microtubules often are in close contact with these other structures. I n the m i t o c h o n d r i a of the light cells (Fig. 9), granules can be distinguished along the i n n e r m i t o c h o n d r i a l m e m b r a n e (22, 23, 43). I n the cells of pancreatic acini (Fig. 10), microtubules are f o u n d arranged as in the intestine a n d thyroid. There is a c o n c e n t r a t i o n of m i c r o t u b u l e s in the apical cell web area a n d a l o n g the lateral b o r d e r of the cell. The tubules are intermingled with filaments in the apex of the cell. Both microtubules a n d filaments make contact with
Fra. 5. A segment of the cytoplasm of a duodenal crypt absorptive cell of the rat. Microtubules (cm) and filaments (f)in large numbers are found in a vertical direction approaching the microvillus border. Microtubules and filaments are similar in dimension to those in the neuron and its processes. Dark granules, probably glycogen, are seen in the cytoplasm. × 47,500. FIo. 6. A section of duodenal crypt epithelium showing the large number of microtubules arranged in a horizontal direction in the apical web of the cell. The microtubules, cut transversely or obliquely, are similar in size to those found in the neuron, x 47,500. FIG. 7. An electron micrograph of the proximal convoluted tubule of the kidney showing mitochondria, plasma membranes, microtubules (cm), and a secretory granule. The mitochondria contain the dense granules, well known in some cells, in addition to finer granules (arrow) attached to the cristae. Tubular cristae are seen frequently. In the cytoplasm microtubules (cm) of a dimension similar to those seen in other cells are present. Microtubular stems on the secretory granules are seen. Dark glycogen granules are present in the cytoplasm, x 50,000.
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apical and lateral cell plasma membranes. The greatest concentration of tubules appears at the desmosome in association with the typical darkly stained material in this area. There is suggestive evidence that some of the microtubules m a y bridge the intercellular space. Some microtubules make contact with the z y m o g e n granules. In the alveolus of the lactating mammary gland (Fig. 11), microtubules are seen in vertical and horizontal directions t h r o u g h o u t the cell, but again the apex and the borders of the cell have a greater concentration of these'structures. Microtubules a p p r o a c h the apical m e m b r a n e and can be seen to f o r m stems on apical vesicles. These stems make contact with the apical plasma membrane. In transverse sections of the microvilli, circular filamentous structures cut in cross section can be seen. The protein granules (pr) in the alveolar lumen exhibit a subunit structure (Fig. 11). In the columnar epithelial cells of the intralobular duct of the mammary gland (Fig. 12), microtubules can be seen f r o m the apex deep into the cytoplasm. At the terminal bar (Fig. 12), the m e m b r a n o u s wall of the microtubules is seen to be continuous with the plasma membrane. M a n y microtubules are found in this area. In the capillary endothelium (Fig. 13), cytoplasmic microtubules, m e m b r a n o u s circular profiles, "pinocytotic vesicles," and mitochondria are numerous. The microtubules pass in an oblique direction f r o m the luminal surface toward the base of the cell. Near the base of the cell, contact is seen between the microtubules and the larger circular profiles in several areas. Tubular connections between these profiles are seen. This thicker section shows superimposition of microtubules. Microtubules are in close contact with the mitochondrion.
DISCUSSION T1/e observer cannot avoid being impressed by the lack of any organized structure in some areas of cells prepared by conventional means For electron microscopy. This applies in particular to cells of the nervous system where R a m d n y Cajal by the use of silver staining technique (26) demonstrated in light microscopy a dense network of filaments that have not been reproduced by electron m i c r o s c o p y .
FI6. 8. Apex of a thyroid follicular cell showing the microvilli (my) containing filaments. In the area of the apical cell web we see "apical vesicles" and microtubules (cm). The vesicular content varies from dark secretory granule to one with a moderate density. Microtubules (era)are interlaced among the apical vesicles and appear to form stems (arrow) On some of the vesicles, x 82,000. F~6. 9. Differing types of thyroid follicular cell cytoplasm; below is that of a "light-type" cell and above two ordinary follicular cells. Many microtubules (cm) are seen in the cytoplasm of the light cell. In the ordinary follicular cell these microtubules are not as numerous. Larger irregular tubules of the agranular reticulum (ar) are seen to be continuous with microtubules and mitochondrial membranes (arrows). x 64,000.
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E. SANDBORN, P. F. KOEN, J. D. MCNABB AND G. MOORE
Another instance is the "cell web" structure of Puchtler and Leblond (25) which has recently been shown by l~arquhar and Palade (10) and SjSstrand (38) in electron microscopy. However, their illustrations do not demonstrate a structure of the extent indicated by light microscopy. In the electron micrographs of cells fixed by aldehyde perfusion, an increase in filamentous and tubular structures was seen. Considerable modifications in techniques were necessary to demonstrate them to best advantage. Tubules and filaments in the cytoplasm are not a new finding. In simple organisms tubules have been demonstrated on occasion (42). In plants, Buvat (7) and Ledbetter and Porter (15) have shown the existence of tubules. In mammals, Fawcett and his co-workers (5, 6, 8, 9) have demonstrated large numbers of tubules in studies of testis. Gray (4, 11, 12), Robertson (27, 29, 30), Palay (20), Rosenbluth (31), and others have demonstrated tubules in dendrites, axons, and even in the neuron. Sarcotubules have been demonstrated repeatedly (1, 2, 18, 24), and Anderson-Cedergren (2) has analyzed their content. Robertson (29) described larger tubules and showed continuity with mitochondria. From this he proposed a theory for the origin of mitochondria. Slautterback (42) termed a longer straight tubule (180 A) in Hydra a "cytoplasmic microtubule." From his review of the literature (42) one factor seems to have some possible significance; the tissues where tubules have been demonstrated have ordinarily been readily accessible to the fixative. In simple organisms such as Hydra, access to the osmium fixative is unobstructed. In the testis, with very little interstitial tissue, the same holds true. In muscle, direct fixation of the exposed living fibers is routinely done; in nervous tissue, Palay (21) overcame its inaccessibility by perfusing the animal with fixative. By negative staining procedures, tubular structures in cytoplasm have alsobeen demonstrated (22, 23, 43). In perfusion experiments with acrolein in a phosphate buffer followed by osmic acid fixation, a considerable increase in the number of filaments and tubules was seen in neurons of the semilunar ganglion and the cerebellum. In replacing the acrolein by glutaraldehyde fixation (13, 15, 32, 33), an increase in both tubular structures and zymogen-like granules appeared. By combining the acrolein and glutaraldehyde, tubular and filamentous structures were accentuated and secretory granules were retained in increased quantity over any method previously employed. In this publication, the definition "cytoplasmic microtubule" or microtubule is
FIG. 10. The apex of a pancreatic acinar cell showing a microvillus border and zymogen granules (z) within a limiting membrane. The microvilli (my), terminal bar (tb), desmosomes (de), and the three layers of the unit membrane can be seen distinctly. Cytoplasmic microtubules (cm) are seen throughout the cytoplasm with a greater number in the area of the apical "cell web" and the lateral borders. The tubules are seen to make contact with the plasma membranes at the apex of the cell, at the terminal bar and at the desmosomes (arrows). Stems are seen on zymogen granules. × 65,000.
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limited to the long t u b u l e 2 2 0 / ~ in d i a m e t e r described b y S l a u t t e r b a c k (42) as distinct f r o m the larger tubules which are c o n s i d e r e d to be the a g r a n u l a r reticulum. The s a r c o t u b u l a r system previously described a n d o t h e r irregular t u b u l o m e m b r a n o u s structures (3) p r o b a b l y fall into this l a t t e r g r o u p of larger tubules. The larger t u b u l a r extensions to m i t o c h o n d r i a l m e m b r a n e s 500 A in d i a m e t e r shown b y Parsons (22, 23) e m p l o y i n g negative staining techniques p r o b a b l y are the larger a g r a n u l a r t y p e tubule. The d i a m e t e r of the tubules illustrated in his p u b l i c a t i o n s considerably. exceed t h a t of the microtubule. I t w o u l d a p p e a r t h a t the negative staining m e t h o d s have n o t preserved this smaller class of tubules. The granules on inner m i t o c h o n d r i a l m e m b r a n e s described by Parsons (22, 23) a n d Stockenius (43) are sometimes visible in this study. There has been no previous suggestion t h a t m i c r o t u b u l e s are a frequent a n d c o n s t a n t inclusion in m a m m a l i a n cytoplasm. The n u m b e r s of m i e r o t u b u l e s within the c y t o p l a s m of these cells are m u c h higher t h a n previously suspected. T h e y are a c o m m o n finding in all the cells t h a t we have examined; this leads us to suggest t h a t t h e y are p r o b a b l y present in considerable n u m b e r s in all m a m m a l i a n cells a n d p o s s i b l y in all cells. The m i c r o t u b u l e s 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 of all of the cells examined; there are localized ' c o n c e n t r a t i o n s in specific areas such as the cell web a n d n e a r the d e s m o s o m e s . The m i c r o t u b u l e s are n o t a r r a n g e d in a n y one direction within a cell except in specific instances such as in the a x o n a n d dendrite, where m o s t are a r r a n g e d in the l o n g i t u d i n a l axis of the process. The presence of the m i c r o t u b u l e s in such areas as the cell web is n o t evident in recent p u b l i c a t i o n s (10, 38). I n m a t e r i a l p r e p a r e d in the m a n n e r described here, there are large n u m b e r s of m i c r o t u b u l e s in the cell web zone. M o s t of these are horizontally arranged, b u t m a n y vertical a n d oblique m i c r o t u b u l e s r e a c h the apical cell
FIG. 11. An electron micrograph of the apices of alveolar cells of the mammary gland of the rat on the 10th day of lactation. The cells show a microvillus border with protein granules (pr) in the lumen. The protein granules show a subunit structure. In longitudinal and transverse sections of the microvilli (my) filaments are seen. Within the apex of the cell we see numerous microtubules (cm) in longitudinal and cross sections. Connections between "apical vesicles" and the plasma membrane by means of microtubules are seen (arrow). x 61,500. FIG. 12. A columnar type of cell of an intralobular duct of the lactating mammary gland illustrating cytoplasmic microtubules (cm) passing for long distances toward the apex of the cell. Others are seen parallel to the apical surface in the cell web area. At the terminal bar (tb) microtubules are seen to be continuous with the plasma membrane (arrow). Microtubular stems are seen on mitochondria and the agranular reticulum (arrows). × 50,000. FIG. 13. A capillary endothelial cell in the thyroid gland of the rat with numerous "pinocytotic vesicles" (v) and microtubules (cm). Although in this photograph clear-cut continuity between the "pinocytotic vesicle" and the microtubules is not seen, the contact between these structures suggests likely continuity. The oblique direction of the microtubule from one surface of the capillary endothelial cell to the other is evident. × 95,000.
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membrane either between or within microvilli. The horizontal microtubules make frequent contact with cell membranes at desmosomes and the apical terminal bar (Figs. 10-12). The results of this investigation indicate that cytoplasmic microtubules (42) are a constant finding in the cells of the rat. The large numbers of these microtubules and their contacts with other structures suggest several possible functions. These may be: (a) supportive; (b) contractile; and (c) serve to transport fluids and suspended solids. It is highly unlikely that structures so frequently seen are of no functional importance. The inability of investigators to preserve these structures previously has created a situation wherein the various cytoplasmic inclusions had to be considered as separate functional units. It is suggested that tubular structures m a y have been broken down by enzymatic digestion during the slow penetration by the osmium fixative. This theory gains some support from the findings of Marchese and Barrnett (17) that enzymatic activity occurs in pinocytotic vesicles. Our findings suggest that the vesicles m a y only be dilatations in the microtubular system (Figs. 11 and 13). The nature of the microtubular wall has not been reported. SjSstrand (39) recently illustrated some membranes with a globular appearance due to bands connecting the outer and inner laminae at regular intervals. In our findings, the microtubule has this globular appearance with dark bands between the laminae at regular intervals. Furthermore, the plasma membrane of the axon has this type of structure. The staining characteristics of the content of the lumen of the microtubule are similar to those of the lumen of the tubular agranular reticulum. It is also similar to the content of the interspace between the inner and outer membranes of the mitochondria and between the membranes of the mitochondrial cristae. The recorded intensity of stain of the luminal content in the microtubule is limited in longitudinal view by the thickness available within any one section. This limit is the 100 A diameter of its lumen while larger structures are recorded through the full thickness of the section. The connection of these tubular structures with other membranes warrants further study. It is suggested that the microtubules may serve as connecting channels between previously described organelles and the plasma membrane, creating a continuous membrane system. If so, the content of this microcirculatory system would in effect be extracellular since the microtubular membranes are continuous with the plasma membrane. The authors wish to thank Dr. S. A. Bencosme for valuable instruction and encouragement during the early stages of this work, Dr. C. P. Leblond for his constant support, and Dr. H. Sheldon for valuable advice in the preparation of the manuscript. This work was done with the support of grants from the Medical Research Council of Canada.
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REFERENCES 1. ANDERSSON,E., Electron Microscopy Proc. Stockholm Conf. 1956, p. 208. Almqvist and Wiksells, Stockholm, and Academic Press, New York (1957). 2. ANDERSSON-CEDERGREN,E. and MUSCATELLO, U., J. Ultrastruct. Res. 8, 391-401
(1963). 3. BATTIG, C. G. and CLEVINGER,M. R., Anat. Record 193, 328 (1961). 4. BOYCOTT,B. B., GRAY, E. G. and GUILLERY,R. W., Proc. Roy. Soc. B154, 151 (1961). 5. BURGOS,N. H. and FAWCETT, D. W., Y. Biophys. Bioehem. Cytol. 1, 287 (1955). 6. - ibid. 2, 223 (1956). 7. BUVAT, R., Ann. Sci. Nat. Botan. Biol. Vegemle [11] 19, 9, 123 (1958). 8. CHRISTENSEN,A. K., Biol. Bull. 121, 416 (1961). 9. CHRISTENSEN,A. K. and FAWCETT, D. W., Y. Biophys. Biochem. Cytol. 9, 653 (1961). 10. FARQUHAR,M. G., and PALADE, G. F., J. Cell Biol. 17, 375 (1963). 11. GRAY, E. G., J. Anat. 93, 420 (1959). 12. - J. Physiol. (London) 145, 25 (1959). 13. HORRIGAN,T. R. and GOOD, R. A., Proe. Am. Assoc. Anat. (1963). 14. KARNOVSKY,M. J., Y. Biophys. Bioehem. Cytol. 11, 729 (1961). 15. LEDBETTER,M. C. and PORTER, K. R., J, Cell Biol. 19, 239 (1963). t6. LUET, J. H., J. Biophys. Biochem. Cytol. 9, 409 (1961). 17. MARCHESI,V. T. and BARRNETT,R. J., J. Cell Biol. 17, 547 (1963). 18. NORTH, R. J., J. Ultrastruct. Res. 8, 206 (1963). 19. PALADE,G. F., Y. Exptl. Med. 95, 285 (1952). 20. PALAY, S. L., Anat. Record 138, 417 (1960). 21. PALAY,S. L., MCGEE-RUSSELL,S. M., GORDON, S. and GRILLO, M. A., J. Cell Biol. 12, 385 (1962). 22. PARSONS,D. F., Science 140, 985 (1963). 23. - J. Cell Biol. 16, 620 (1963). 24. PUCCHt, I. and AEZELIUS,B. A., J. Ultrastruct. Res. 7, 210-224 (1962). 25. PUCHTLER,H. and LEBLOND,C. P., Am. J. Anat. 102, 1 (1958). 26. RAMON Y CAJAL, Histologie du syst6me nerveux de l'homme et des vertGbrGs. A. Maloine, Paris, 1909. 27. ROBERTSON,J. D., J. Biophys. Biochem. Cytol. 2, 381-394 (1956). 28. - Intern. Kongr. Elektronenmikroskopie 4, Berlin 1958, Verhandl. Vol. 2, p. 139 (1960). Springer, Berlin. 29. - J. Physiol. (London) 153, 58 (1960). 30. ROBERTSON,J. D., BODENHEIMER,T. S. and STAGE, D. E., J. Cell Biol. 19, 159 (1963). 31. ROSENBLUTH,J., J. Cell Biol. 12, 329 (1962). 32. SABATINI,D. D., BENSCH, K. G. and BARRNETT, R. J., J. Histochem. Cytochem. 10, 652 (1962). 33. - J. Cell Biol. 17, 19 (1963). 34. SCHMITT,F. O. and GEREN, B. B., J. Exptl. Med. 91, 499 (1950). 35. SJOSTRAND.F. S., Experientia 9, 68 (1953). 36. - Nature 171, 30 (1953). 37. - ibid. 171, 31 (1953).
138 38. 39. 40. 41. 42. 43. 44.
E. SANDBORN, P. F. KOEN, J. D. MCNABB AND G. MOORE J. Ultrastruct. Res. 8, 517 (1963). ibid. 9, 340 (1963). SJ6STRANO, F. S. and ELFVlN, L.-G., Y. Ultrastruct. Res. 7, 504 (1962). SJ6STRANI~,F. S. and HERZOG, V., Exptl. Cell Res. 7, 393 (1954). SLAUTTERBACK,D. B., J. Cell Biol. 18, 367 (1963). STOCKENIUS,W . , J. Cell Biol. 17, 443 (1963). WATSON, M. L., d. Biophys. Biochem. Cytol. 4, 475 (1958).
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123
Cytoplasmic Microtubules in Mammalian Cells E. SANDBORN,1 PATRICK F. KOEN, J. D. MCNABB, a n d G. MOORE
Faculty of Medicine, McGill University, Montreal, Canada Received February 13, 1964 Cytoplasmic microtubules can be shown to be a constant finding in tissues of the rat which have been fixed in aldehydes followed by osmium tetroxide. The aldehydes employed were acrolein and glutaraldehyde, which were combined to give both rapid penetration and stabilization of the ultrastructural constituents of the cell. By this method microtubules are seen to be common to all areas of the cytoplasm. Connections to other membranes are illustrated. Possible functional roles for the cytoplasmic microtubules are discussed.
C y t o p l a s m i c microtubules, which have been r e p o r t e d occasionally in p l a n t a n d a n i m a l cells, have n o t been shown to be a c o n s t a n t finding in m a m m a l i a n cells. By the use of a l d e h y d e s (32, 33) followed b y o s m i u m tetroxide (19) fixation of tissues, t u b u l a r structures of v a r y i n g d i m e n s i o n were f o u n d in all types of cells examined. F u r t h e r m o r e , evidence of continuity between c y t o p l a s m i c organelles was seen repeatedly. The p u r p o s e of this r e p o r t is to describe the m i c r o t u b u l e a n d to illustrate t h a t it is a frequent finding in the cells of the rat.
MATERIALS AND METHODS By a modification of Palay's technique (21) young adult rats were perfused with balanced salt to remove the circulating blood followed by a fixative solution containing 2% acrolein and 6.5% glutaraldehyde in a phosphate buffer (32, 33). The perfusion of fixative was continued for 20 minutes; the tissues were then removed from several organs, diced, and fixed for an additional hour in the same fixative. After fixation in the aldehyde solution, the blocks were washed in Veronal acetate buffer (10) for 15 minutes and then postfixed in 2% Veronal acetate-buffered osmium (•9) for 2 hours. The tissues were dehydrated in graded ethanol, then transferred to propylene oxide and embedded in Epon as described by Luft (16). The blocks were sectioned on a Porter-Blum microtome. Sections 0.5-1.0 # in thickness were stained with 1% aqueous toluidine blue at p H 8.5. Thin sections were stained with uranyl acetate (44), lead hydroxide (14), or a combined stain of lead hydroxide followed by 1% uranyl acetate (14, 44). The sections were examined in a Siemens Elmiskop I electron microscope. 1 Present address: D6partement d'Anatomie, Universit6 de Montr6al, Montr6al, Canada.
124
E. SANDBORN, P. F. KOEN, J. D. MCNABB AND G. MOORE OBSERVATIONS
I n light microscopy all cell features ordinarily seen were demonstrated. Some features were r e m a r k a b l y accentuated, a n d others that ordinarily can be d e m o n strated only by special techniques were seen quite clearly. By light microscopy, m i t o c h o n d r i a were more distinct t h a n with o r d i n a r y techniques a n d the cell web (25) was clearly seen in intestine a n d kidney. Densely.stained granules, in larger n u m b e r s t h a n observed by other techniques, were seen in cells of the thyroid follicles, the nervous system, a n d kidney tubules. The elastic l a m i n a of arterioles stained vividly with toluidine blue. I n the electron microscope, the most striking new finding was a complex n e t w o r k of tubules within all cells examined. The tubules were seen in n e u r o n s a n d neuroglia of the cerebellum, cerebral m o t o r cortex, a n d semilunar ganglion, the cells of p a n creatic acinus, thyroid follicle, capillary endothelium, intestinal epithelium, p r o x i m a l a n d distal convoluted tubules of the kidney, the a d r e n a l medulla, a n d those of b o t h the alveolus a n d duct of the m a m m a r y gland. The tubules are of varying dimensions; the most c o m m o n of these is a long, relatively straight tubule approximately 220 A in diameter (Figs. 1 a n d 2). This type of tubule is seen in all the cells examined a n d often traverses the whole of the field seen in the microscope. The dimensions of this tubule are approximately the same in the n e u r o n (Figs. 1-4), secretory (Figs. 8, 10 a n d 11), or absorptive (Figs. 5 a n d 6) epithelial a n d endothelial (Fig. 13) cells. A similar structure in h y d r a was referred to b y Slautterback (42) as a cytoplasmic microtubule. The microtubules seen i n the axons of peripheral nerves of the rat have walls 60 A in thickness s u r r o u n d i n g a l u m e n approximately 100 A in diameter. The wall is seen to be a t r i l a m i n a r m e m b r a n e (28, 36, 37, 38-40, 41). I n addition, dark b a n d s bridge the space between the i n n e r a n d outer l a m i n a e of the m e m b r a n e at a periodiFIG. 1. A longitudinally sectioned axon from the semilunar ganglion of the rat. The microtubules range. The tubules are seen to branch (arrow) and continuity of filaments with tubules is suggested (arrow). The filaments are about 60 /~ in diameter. At the upper center is a mitochondrion with tubular appearing cristae as well as the flattened type. At the upper left a large tubule of the agranular reticulum (ar) with a diameter of 600 ~ + is illustrated. Its membranes are continuous with the outer mitochondrial membrane, x 66,000. FIG. 2. A cross section of a small myelinated axon in the semilunar ganglion of the rat. The layers of myelin have a spacing of 120 A. The separation of the interperiod line into two layers is visible for considerable distances. The plasma membrane of the axon is trilaminar and is 60/~ in thickness. Interconnecting bands between the laminae of the unit membrane are apparent at a periodicity of 50-70/~. Cytoplasmic microtubules (neurotubules) (cm) are seen to be surrounded by a wall made up of a trilaminar membrane similar to that of the plasma membrane. Bands occur between the outer and inner laminae imparting to the membrane the appearance of a series of globules. The wall of the neurotubules is 60/~ in thickness and there is a 100 ~ lumen. The neurofilaments appear o be made up of globular subunits, x 207,000.
(cm) and filaments (f) are seen in abundance. The diameter of the microtubules is in the 220 ~
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126
E. SANDBORN~ P. F. KOEN, J. D. MCNABB AND G. MOORE
city of a b o u t 60-70 A (Fig. 2), as recently described b y Sj/Sstrand (39). This is also seen in the p l a s m a m e m b r a n e of the a x o n (Fig. 2). V a r i a b l e a m o u n t s of d a r k l y stained content are seen in the l u m e n of the tubule. L a r g e r tubules are also se'en in the axon. T h e y r e a c h a d i a m e t e r of 700 A (Figs. 1 a n d 3), are slightly t o r t u o u s a n d c o n t a i n irregular, d a r k l y stained material, These structures are considered to be the c o n v e n t i o n a l a g r a n u l a r reticulum b u t are t u b u l a r r a t h e r t h a n vesicular in nature. These will be referred to as the a g r a n u l a r o r t u b u l a r a g r a n u l a r reticulum. The c o n t e n t of these larger tubules is generally m o r e densely stained t h a n in the microtubule. C o n t i n u i t y of the m e m b r a n e of the a g r a n u l a r retic u l u m with the outer m i t o c h o n d r i a l m e m b r a n e is seen (Fig. 1). F i l a m e n t s are f o u n d in a b u n d a n c e in the axon. The filaments have a d i a m e t e r of a p p r o x i m a t e l y 60 A. T h e y a p p e a r to be m a d e up of subunits as described b y Schmitt a n d G e r e n (34). The presence of a l u m e n is p r o b l e m a t i c a l ; in some there is a suggestion of a l u m e n while in m o s t this is n o t visible (Fig. 2). The l a m e l l a t e d n a t u r e of the nerve myelin sheath (35) of axons is illustrated well b y this fixative. The myelin has a p e r i o d i c i t y of 120 A. The two layers of the interp e r i o d line (26) are seen over considerable distances (Fig. 2). The c o n t a c t between the two m e m b r a n e s in the dense line is also discernible. The m i t o c h o n d r i a in the a x o n are seen to c o n t a i n d a r k l y stained m a t e r i a l between the inner a n d o u t e r m e m b r a n e s a n d between the two layers of the cristae. I n the p e r i k a r y o n of ganglionic cells of the peripheral nervous system (Fig. 3) a n d in neurons of the cerebral cortex a n d cerebellum, m a n y filaments, microtubules, a n d larger tubules of the a g r a n u l a r r e t i c u l u m are found. These are of the same dimensions as seen in the axon. The t u b u l a r a n d filamentous structures invade all areas of the cytoplasm. As in the axon, there is a variable a m o u n t of d a r k l y stained content within the l u m e n of the microtubules, the a g r a n u l a r reticulum, a n d the space between the m i t o c h o n d r i a l m e m b r a n e s . I n the a x o n of m y e l i n a t e d s y m p a t h e t i c nerve fibers in the a d r e n a l m e d u l l a (Fig. 4), m i c r o t u b u l e s are seen to be as n u m e r o u s as in the axons of peripheral nerves (Figs. 1 a n d 2). The Schwann cells (Fig. 4) are seen to c o n t a i n m i c r o t u b u l e s as in the neuron. Branching of the m i c r o t u b u l e s is evident.
FIG. 3. Electron micrograph of the cytoplasm of the large, pale neuron of the semilunar ganglion of the rat. Mitochondria with darkly stained material between the membranes of the cristae are present in abundance. Microtubules (cm), 220 A in diameter, are seen to cross in straight lines while larger tubules of the agranular reticulum (ar) with more irregular outline are also seen. These larger tubules contain darkly stained material similar to that seen in the mitochondria. The tubular network within the neuron is very complex. Continuity of membrane structure between the microtubules, the reticulum, and mitochondria is suggested, x 47,500. Fro. 4. A myelinated axon of a sympathetic autonomic neuron in the adrenal medulla with a Schwann cell. The outer and inner connections of the myelin with the plasma membrane can be seen. Within the axon mitochondria and microtubules can be seen. In the Schwann cell cytoplasm microtubules (cm) are present and branching of the tubules (arrow) is demonstrated. × 37,500.
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E. SANDBORN, P. F. KOEN~ J. D. MCNABB AND G. MOORE
I n the absorptive cells of the intestinal epithelium, n u m e r o u s microtubules a n d filaments are found. The microtubules are seen to be vertically (Fig. 5) as well as h o r i z o n t a l l y (Fig. 6) oriented. Large n u m b e r s are seen horizontally disposed in the cell web. I n t e r m i n g l e d with the microtubules are m a n y filaments extending into the microvilli. Both microtubules a n d filaments reach the apical a n d the lateral cell m e m b r a n e s . These areas of contact are particularly c o m m o n at the desmosomes. Microtubules pass for considerable distances in the cytoplasm. The dimensions of the microtubules a n d filaments are similar to those in the n e u r o n a n d its processes. I n the p r o x i m a l convoluted tubule of the kidney (Fig. 7), microtubules similar to those in the other cell types described are seen in the cytoplasm. M i c r o t u b u l e s are closely associated with m i t o c h o n d r i a a n d m e m b r a n e - b o u n d secretory granules. I n the follicular cells of the thyroid gland (Figs. 8 a n d 9), there is a n e t w o r k of microtubules a n d filaments t h r o u g h o u t the cytoplasm. The greatest c o n c e n t r a t i o n of t u b u l a r elements is i n the cell web area of the apex a n d along the lateral borders of the cell. M i c r o t u b u l e s are particularly concentrated at the desmosomes. M e m b r a n e b o u n d secretory granules are seen from the base to the apex of these cells in sections prepared for b o t h light a n d electron microscopy. Microtubules are seen in close c o n t a c t with these granules. I n the light cells of the thyroid gland (Fig. 9), microtubules, even more n u m e r o u s t h a n in the follicular cells, are found. M a n y of these tubules are seen to be arranged parallel to one another, to t u b u l a r a g r a n u l a r reticulum, a n d to the m a j o r i t y of the m i t o c h o n d r i a . The microtubules often are in close contact with these other structures. I n the m i t o c h o n d r i a of the light cells (Fig. 9), granules can be distinguished along the i n n e r m i t o c h o n d r i a l m e m b r a n e (22, 23, 43). I n the cells of pancreatic acini (Fig. 10), microtubules are f o u n d arranged as in the intestine a n d thyroid. There is a c o n c e n t r a t i o n of m i c r o t u b u l e s in the apical cell web area a n d a l o n g the lateral b o r d e r of the cell. The tubules are intermingled with filaments in the apex of the cell. Both microtubules a n d filaments make contact with
Fra. 5. A segment of the cytoplasm of a duodenal crypt absorptive cell of the rat. Microtubules (cm) and filaments (f)in large numbers are found in a vertical direction approaching the microvillus border. Microtubules and filaments are similar in dimension to those in the neuron and its processes. Dark granules, probably glycogen, are seen in the cytoplasm. × 47,500. FIo. 6. A section of duodenal crypt epithelium showing the large number of microtubules arranged in a horizontal direction in the apical web of the cell. The microtubules, cut transversely or obliquely, are similar in size to those found in the neuron, x 47,500. FIG. 7. An electron micrograph of the proximal convoluted tubule of the kidney showing mitochondria, plasma membranes, microtubules (cm), and a secretory granule. The mitochondria contain the dense granules, well known in some cells, in addition to finer granules (arrow) attached to the cristae. Tubular cristae are seen frequently. In the cytoplasm microtubules (cm) of a dimension similar to those seen in other cells are present. Microtubular stems on the secretory granules are seen. Dark glycogen granules are present in the cytoplasm, x 50,000.
I
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E. SANDBORN, P. F. KOEN~ J. D. MCNABB AND G. MOORE
apical and lateral cell plasma membranes. The greatest concentration of tubules appears at the desmosome in association with the typical darkly stained material in this area. There is suggestive evidence that some of the microtubules m a y bridge the intercellular space. Some microtubules make contact with the z y m o g e n granules. In the alveolus of the lactating mammary gland (Fig. 11), microtubules are seen in vertical and horizontal directions t h r o u g h o u t the cell, but again the apex and the borders of the cell have a greater concentration of these'structures. Microtubules a p p r o a c h the apical m e m b r a n e and can be seen to f o r m stems on apical vesicles. These stems make contact with the apical plasma membrane. In transverse sections of the microvilli, circular filamentous structures cut in cross section can be seen. The protein granules (pr) in the alveolar lumen exhibit a subunit structure (Fig. 11). In the columnar epithelial cells of the intralobular duct of the mammary gland (Fig. 12), microtubules can be seen f r o m the apex deep into the cytoplasm. At the terminal bar (Fig. 12), the m e m b r a n o u s wall of the microtubules is seen to be continuous with the plasma membrane. M a n y microtubules are found in this area. In the capillary endothelium (Fig. 13), cytoplasmic microtubules, m e m b r a n o u s circular profiles, "pinocytotic vesicles," and mitochondria are numerous. The microtubules pass in an oblique direction f r o m the luminal surface toward the base of the cell. Near the base of the cell, contact is seen between the microtubules and the larger circular profiles in several areas. Tubular connections between these profiles are seen. This thicker section shows superimposition of microtubules. Microtubules are in close contact with the mitochondrion.
DISCUSSION T1/e observer cannot avoid being impressed by the lack of any organized structure in some areas of cells prepared by conventional means For electron microscopy. This applies in particular to cells of the nervous system where R a m d n y Cajal by the use of silver staining technique (26) demonstrated in light microscopy a dense network of filaments that have not been reproduced by electron m i c r o s c o p y .
FI6. 8. Apex of a thyroid follicular cell showing the microvilli (my) containing filaments. In the area of the apical cell web we see "apical vesicles" and microtubules (cm). The vesicular content varies from dark secretory granule to one with a moderate density. Microtubules (era)are interlaced among the apical vesicles and appear to form stems (arrow) On some of the vesicles, x 82,000. F~6. 9. Differing types of thyroid follicular cell cytoplasm; below is that of a "light-type" cell and above two ordinary follicular cells. Many microtubules (cm) are seen in the cytoplasm of the light cell. In the ordinary follicular cell these microtubules are not as numerous. Larger irregular tubules of the agranular reticulum (ar) are seen to be continuous with microtubules and mitochondrial membranes (arrows). x 64,000.
132
E. SANDBORN, P. F. KOEN, J. D. MCNABB AND G. MOORE
Another instance is the "cell web" structure of Puchtler and Leblond (25) which has recently been shown by l~arquhar and Palade (10) and SjSstrand (38) in electron microscopy. However, their illustrations do not demonstrate a structure of the extent indicated by light microscopy. In the electron micrographs of cells fixed by aldehyde perfusion, an increase in filamentous and tubular structures was seen. Considerable modifications in techniques were necessary to demonstrate them to best advantage. Tubules and filaments in the cytoplasm are not a new finding. In simple organisms tubules have been demonstrated on occasion (42). In plants, Buvat (7) and Ledbetter and Porter (15) have shown the existence of tubules. In mammals, Fawcett and his co-workers (5, 6, 8, 9) have demonstrated large numbers of tubules in studies of testis. Gray (4, 11, 12), Robertson (27, 29, 30), Palay (20), Rosenbluth (31), and others have demonstrated tubules in dendrites, axons, and even in the neuron. Sarcotubules have been demonstrated repeatedly (1, 2, 18, 24), and Anderson-Cedergren (2) has analyzed their content. Robertson (29) described larger tubules and showed continuity with mitochondria. From this he proposed a theory for the origin of mitochondria. Slautterback (42) termed a longer straight tubule (180 A) in Hydra a "cytoplasmic microtubule." From his review of the literature (42) one factor seems to have some possible significance; the tissues where tubules have been demonstrated have ordinarily been readily accessible to the fixative. In simple organisms such as Hydra, access to the osmium fixative is unobstructed. In the testis, with very little interstitial tissue, the same holds true. In muscle, direct fixation of the exposed living fibers is routinely done; in nervous tissue, Palay (21) overcame its inaccessibility by perfusing the animal with fixative. By negative staining procedures, tubular structures in cytoplasm have alsobeen demonstrated (22, 23, 43). In perfusion experiments with acrolein in a phosphate buffer followed by osmic acid fixation, a considerable increase in the number of filaments and tubules was seen in neurons of the semilunar ganglion and the cerebellum. In replacing the acrolein by glutaraldehyde fixation (13, 15, 32, 33), an increase in both tubular structures and zymogen-like granules appeared. By combining the acrolein and glutaraldehyde, tubular and filamentous structures were accentuated and secretory granules were retained in increased quantity over any method previously employed. In this publication, the definition "cytoplasmic microtubule" or microtubule is
FIG. 10. The apex of a pancreatic acinar cell showing a microvillus border and zymogen granules (z) within a limiting membrane. The microvilli (my), terminal bar (tb), desmosomes (de), and the three layers of the unit membrane can be seen distinctly. Cytoplasmic microtubules (cm) are seen throughout the cytoplasm with a greater number in the area of the apical "cell web" and the lateral borders. The tubules are seen to make contact with the plasma membranes at the apex of the cell, at the terminal bar and at the desmosomes (arrows). Stems are seen on zymogen granules. × 65,000.
CYTOPLASMIC MICROTUBULES IN MAMMALIAN CELLS
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E. SANDBORN~ P. F. KOEN, J. D. MCNABB AND G. MOORE
limited to the long t u b u l e 2 2 0 / ~ in d i a m e t e r described b y S l a u t t e r b a c k (42) as distinct f r o m the larger tubules which are c o n s i d e r e d to be the a g r a n u l a r reticulum. The s a r c o t u b u l a r system previously described a n d o t h e r irregular t u b u l o m e m b r a n o u s structures (3) p r o b a b l y fall into this l a t t e r g r o u p of larger tubules. The larger t u b u l a r extensions to m i t o c h o n d r i a l m e m b r a n e s 500 A in d i a m e t e r shown b y Parsons (22, 23) e m p l o y i n g negative staining techniques p r o b a b l y are the larger a g r a n u l a r t y p e tubule. The d i a m e t e r of the tubules illustrated in his p u b l i c a t i o n s considerably. exceed t h a t of the microtubule. I t w o u l d a p p e a r t h a t the negative staining m e t h o d s have n o t preserved this smaller class of tubules. The granules on inner m i t o c h o n d r i a l m e m b r a n e s described by Parsons (22, 23) a n d Stockenius (43) are sometimes visible in this study. There has been no previous suggestion t h a t m i c r o t u b u l e s are a frequent a n d c o n s t a n t inclusion in m a m m a l i a n cytoplasm. The n u m b e r s of m i e r o t u b u l e s within the c y t o p l a s m of these cells are m u c h higher t h a n previously suspected. T h e y are a c o m m o n finding in all the cells t h a t we have examined; this leads us to suggest t h a t t h e y are p r o b a b l y present in considerable n u m b e r s in all m a m m a l i a n cells a n d p o s s i b l y in all cells. The m i c r o t u b u l e s 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 of all of the cells examined; there are localized ' c o n c e n t r a t i o n s in specific areas such as the cell web a n d n e a r the d e s m o s o m e s . The m i c r o t u b u l e s are n o t a r r a n g e d in a n y one direction within a cell except in specific instances such as in the a x o n a n d dendrite, where m o s t are a r r a n g e d in the l o n g i t u d i n a l axis of the process. The presence of the m i c r o t u b u l e s in such areas as the cell web is n o t evident in recent p u b l i c a t i o n s (10, 38). I n m a t e r i a l p r e p a r e d in the m a n n e r described here, there are large n u m b e r s of m i c r o t u b u l e s in the cell web zone. M o s t of these are horizontally arranged, b u t m a n y vertical a n d oblique m i c r o t u b u l e s r e a c h the apical cell
FIG. 11. An electron micrograph of the apices of alveolar cells of the mammary gland of the rat on the 10th day of lactation. The cells show a microvillus border with protein granules (pr) in the lumen. The protein granules show a subunit structure. In longitudinal and transverse sections of the microvilli (my) filaments are seen. Within the apex of the cell we see numerous microtubules (cm) in longitudinal and cross sections. Connections between "apical vesicles" and the plasma membrane by means of microtubules are seen (arrow). x 61,500. FIG. 12. A columnar type of cell of an intralobular duct of the lactating mammary gland illustrating cytoplasmic microtubules (cm) passing for long distances toward the apex of the cell. Others are seen parallel to the apical surface in the cell web area. At the terminal bar (tb) microtubules are seen to be continuous with the plasma membrane (arrow). Microtubular stems are seen on mitochondria and the agranular reticulum (arrows). × 50,000. FIG. 13. A capillary endothelial cell in the thyroid gland of the rat with numerous "pinocytotic vesicles" (v) and microtubules (cm). Although in this photograph clear-cut continuity between the "pinocytotic vesicle" and the microtubules is not seen, the contact between these structures suggests likely continuity. The oblique direction of the microtubule from one surface of the capillary endothelial cell to the other is evident. × 95,000.
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E. SANDBORN, P. F. KOEN, J. D. MCNABB AND G. MOORE
membrane either between or within microvilli. The horizontal microtubules make frequent contact with cell membranes at desmosomes and the apical terminal bar (Figs. 10-12). The results of this investigation indicate that cytoplasmic microtubules (42) are a constant finding in the cells of the rat. The large numbers of these microtubules and their contacts with other structures suggest several possible functions. These may be: (a) supportive; (b) contractile; and (c) serve to transport fluids and suspended solids. It is highly unlikely that structures so frequently seen are of no functional importance. The inability of investigators to preserve these structures previously has created a situation wherein the various cytoplasmic inclusions had to be considered as separate functional units. It is suggested that tubular structures m a y have been broken down by enzymatic digestion during the slow penetration by the osmium fixative. This theory gains some support from the findings of Marchese and Barrnett (17) that enzymatic activity occurs in pinocytotic vesicles. Our findings suggest that the vesicles m a y only be dilatations in the microtubular system (Figs. 11 and 13). The nature of the microtubular wall has not been reported. SjSstrand (39) recently illustrated some membranes with a globular appearance due to bands connecting the outer and inner laminae at regular intervals. In our findings, the microtubule has this globular appearance with dark bands between the laminae at regular intervals. Furthermore, the plasma membrane of the axon has this type of structure. The staining characteristics of the content of the lumen of the microtubule are similar to those of the lumen of the tubular agranular reticulum. It is also similar to the content of the interspace between the inner and outer membranes of the mitochondria and between the membranes of the mitochondrial cristae. The recorded intensity of stain of the luminal content in the microtubule is limited in longitudinal view by the thickness available within any one section. This limit is the 100 A diameter of its lumen while larger structures are recorded through the full thickness of the section. The connection of these tubular structures with other membranes warrants further study. It is suggested that the microtubules may serve as connecting channels between previously described organelles and the plasma membrane, creating a continuous membrane system. If so, the content of this microcirculatory system would in effect be extracellular since the microtubular membranes are continuous with the plasma membrane. The authors wish to thank Dr. S. A. Bencosme for valuable instruction and encouragement during the early stages of this work, Dr. C. P. Leblond for his constant support, and Dr. H. Sheldon for valuable advice in the preparation of the manuscript. This work was done with the support of grants from the Medical Research Council of Canada.
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