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Filamentous substructure of microtubules of the marginal bundle of mammalian blood platelets
1967 by Academic Press Inc.
s. ULTRASTRUCTURERES~ARCrI19, 147-165 (1967)
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Filamentous Substructure of Microtubules of the Marginal Bundle of Mammalian Blood Platelets O. BEHNKE AND T. ZELANDER
Department of Anatomy, The Royal Dental College, Copenhagen, Denmark Received November 29, 1966 Cytoplasmic microtubules from the marginal bundle in human and rat blood platelets were prepared by negative staining and shadow-casting, and studied in the electron microscope. The microtubules from both species gave the same results: their walls appear to be constructed of 35 A-thick filaments evenly spaced in the wall and with a center-to-center distance of 60 &. The total number of filaments in each microtubule is probably 12. Microtubules which disintegrate during the preparative procedures break into short pieces of filaments, or globules, the latter being 35 A in diameter. While some experimental conditions gave preparations which revealed a filamentous substructure of the microtubule wall, others showed staining of the microtubule "core." The experiments substantiate the hypothesis that microtubules are rigid structures. Some of the information obtained strongly suggests that the marginal bundle consists of but one long, rolled-up microtubule.
The term microtubule, which was introduced by Slautterback (34), is currently used as a designation for a class of cellular structures which, in sectioned material, appear as "hollow" tubules with electron dense walls and a less dense interior. They range in outer diameter from about 180 • to 300 tk. Microtubules have, since the introduction of glutaraldehyde as a fixative (30) been recognized as a cytoplasmic component of a wide variety of cells of plant and animal origin, and it seems at present reasonable to assume that the microtubule is a universal cellular organelle. Conventional osmium tetroxide fixation preserves some microtubules, for example the axial filament complex of cilia and flagella [see review by Fawcett (10)], neurotubules (27), and the marginal bundle of nucleated red cells (11, 22). Other microtubules are more labile and require fixation in glutaraldehyde to be properly preserved (3, 9, 19, 29, 31). The morphological similarity in sectioned material between "osmium-preservable" and "glutaraldehyde-preservable" tubules is indisputable. We think, however, that
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it is justified, until better criteria are at hand, to discriminate between two classes of microtubules: those which are preserved by conventional osmium tetroxide (referred to as O-microtubules in the following), and the labile microtubules which require fixation in glutaraldehyde (referred to as G-microtubules). Filamentous substructure in O-microtubules has been reported. Pease (28) and Andr6 and Thi6ry (1), investigating the microtubules of the axial filament complex of rat and h u m a n sperm tails with the negative-stain technique, reported the presence of 10 or 11 filaments in the walls of these tubules. The filaments were named protofibrils by Andr6 and Thi6ry. Nielsen (26) found a similar substructure in the O-microtubules of flagella and axostyles in Trichornonas vaginalis and recently Grimstone and Klug (16) have made similar observations on cilia f r o m Trichonympha. Gall (12), studying the O-microtubules of the marginal band of nucleated red cells, estimated the presence of 12-14 filaments in the walls of these tubules. The presence of a substructure in G-microtubules was suggested by Ledbetter and Porter in their first report on microtubules in plants (19), and, based on M a r k h a m rotation tests performed on fixed and sectioned material, they concluded that the wall of G-microtubules probably has 13 substructures spaced evenly around the circumference (20). The substructures were described as being circular in outline and having a center-to-center distance of 45/~. Most of the information on G-microtubules so far obtained has come from the study of thin sections. Two recent reports (2, 18) have described the presence of a filamentous substructure in microtubules of the mitotic spindle. To our knowledge no detailed report has been published concerning the substructure of cytoplasmic G-microtubules of interphase mammalian cells. It is the purpose of this paper to relate some observations on the substructure of mammalian G-microtubules as obtained by the negative staining technique. Since blood platelets of mammals contain a conspicuous marginal bundle of Gmicrotubules (4, 17, 32, 33) encircling the circumference of the platelets just beneath the plasma membrane, and since they have no O-microtubules, they are well suited for such a study.
MATERIAL AND METHODS Blood was obtained from adult humans and albino rats by venipuncture, using either 5 % EDTA in 0.9 % saline or a 2 % sodium citrate solution as anticoagulants. The anticoagulants were used at room temperature and were adjusted to pH 6. Three milliliters of anticoagulant were used for 7 ml of blood. Red cells and leukocytes were sedimented by centrifugation at 300 g at room temperature for 15 minutes. The supernatant platelet rich plasma was then centrifuged at 1500 g for 15-20 minutes. This procedure yields pellets consisting almost entirely of platelets.
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A small amount of platelets were transferred with the tip of a small glass rod to the surface of a drop of distilled water. After 30-60 seconds the spread and ruptured platelets were picked up by touching the water surface with carbon-coated specimen grids. Some grids were subsequently floated briefly on a drop of 2 % glutaraldehyde to fix the platelet debris before further processing; other specimens were processed unfixed. Shadow casting. Fixed and unfixed specimens were shadowed with platinum or platinum and carbon at various angles at a filament-specimen distance of 11-15 cm. Negative staining procedure. Fixed and unfixed specimens were negatively stained (8) with 2 % potassium phosphotungstate at pH 4, 5, 6, and 7, or with 2 % uranyl acetate at pH 4.1 (unadjusted) or adjusted to pH 5 with potassium hydroxide. The specimens were examined in a Siemens Elmiskop I, operated at 60 kV with double condensor and 50-# objective aperture. The direct magnifications ranged from 15,000 to 60,000. The microscope was calibrated by using a grating replica. OBSERVATIONS
General remarks The preparations of microtubules from rat and human blood platelets showed no morphological differences, and the observations described relate to both. Negative-staining images of the microtubules of the marginal bundle were regularly obtained with the stated procedure. For some unknown reason, however, the yield was higher in rat platelets than in platelets of human origin. Microtubules were never found if the isolation of platelets had been carried out in the cold (0-4°C). This observation, which probably indicates that the microtubules of platelets are not stable in the cold, is in agreement with observations by Tilney (35) on G-microtubules in Actinosphaeriurn and by Behnke and Forer (6) on cytoplasmic microtubules in crane fly, Nephrotoma suturalis (Loew), sperm and spermatids. The platelet membranes generally disappeared during the preparatory procedure. In some specimens, however, unruptured platelets which had adhered to and flattened on the grid were observed. The intact bundle of microtubules could sometimes be identified in these platelets (Fig. 11), leaving no doubt as to the identification of microtubules lying free on the supporting film. Not infrequently the microtubules of the marginal bundle had retained their original spatial relationship and the bundle could be observed in toto as a ring-like structure (Fig. 1). Thus, depriving the blood platelet of its plasma membrane does not necessarily cause the marginal bundle to break up. In those cases where the bundle as such could be observed, the microtubules showed the curved shape they have in intact platelets. In most cases, however, the bundle had been disrupted during the preparative procedure, and pieces of microtubules become freely exposed on the grid (Fig. 6). "Free" microtubules were straight or only gently curved. Many of them showed fractures (Fig. 7), supporting the hypothesis that microtubules are relatively rigid structures.
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C o m m o n l y , " f r e e " m i c r o t u b u l e s were seen u n c o n n e c t e d to m e m b r a n e fragments. It is n o t k n o w n w h e t h e r the m a r g i n a l b u n d l e in platelets is c o m p o s e d of i n d i v i d u a l m i c r o t u b u l e s f o r m i n g c o m p l e t e l o o p s a r o u n d the entire circumference, or w h e t h e r the m a r g i n a l b u n d l e consists of b u t one coiled-up microtubule. O u r o b s e r v a t i o n s o n s h a d o w e d a n d negatively stained p r e p a r a t i o n s of intact m a r g i n a l bundles have n o t solved this p r o b l e m , because i n d i v i d u a l m i c r o t u b u l e s could n o t be followed w i t h o u t a m b i g u i t y for m o r e t h a n a fraction of the circumference due to o v e r l a p p i n g of structures. I n several p r e p a r a t i o n s it was observed, however, t h a t two "free e n d s " of m i c r o t u b u l e s p r o j e c t e d f r o m an otherwise intact bundle, suggesting t h a t the b u n d l e was constructed of one coiled-up m i c r o t u b u l e with two free ends (Fig. 1). U n d e r some e x p e r i m e n t a l c o n d i t i o n s m i c r o t u b u l e s were unstable. It has a l r e a d y been n o t e d that the m i c r o t u b u l e s d i s a p p e a r e d if the platelets were subjected to cold. A p p l i c a t i o n of the negative stain to unfixed m i c r o t u b u l e s revealed t h a t the preserv a t i o n of their integrity was also d e p e n d a n t of the p H of the negative stain. M i c r o tubules were never o b s e r v e d if the stain was a p p l i e d to unfixed m i c r o t u b u l e s at p H 6 a n d 7. The effects of the different negative stains, of their p H , a n d of fixation o n the a p p e a r a n c e of substructure in the m i c r o t u b u l e s are s u m m a r i z e d in T a b l e I. A s indicated, some e x p e r i m e n t a l c o n d i t i o n s p r o d u c e d a filamentous substructure of m i c r o tubules, whereas others revealed a central " c o r e . " Filamentous substructure
U n d e r suitable c o n d i t i o n s of staining (see T a b l e I) the m i c r o t u b u l e s p r e s e n t e d a l o n g i t u d i n a l striation due to the presence of fine filaments in the m i c r o t u b u l e wall FIG. 1. Electron micrograph of a shadowed preparation of a complete marginal bundle of microtubules (mb) from a ruptured rat blood platelet. The bundle was not fixed before shadowing with platinum. Because of overlapping it is not possible to follow the course of individual microtubules for long distances. Two microtubules have swung out from the bundle and have assumed a straight course (arrows). Membrane fragments are seen at m. g, platelet dense granules. Reversed print. x 38,000. FIG. 2. Part of a microtubule (mt) from a rat blood platelet. The preparation was fixed with glutaraldehyde before shadowing with platinum. The irregular background is due to shadowed cytoplasmic material from the platelet, x 109,000. FIG. 3. Glutaraldehyde-fixed microtubules stained with potassium phosphotungstate at pH 4. The outline of the microtubules is somewhat irregular. The core shows up as if positively stained (arrow). There is no indication of substructure, x 200,000. FIG. 4. Glutaraldehyde-fixed microtubules stained with uranyl acetate at pH 4.1. The core is positively stained. The somewhat irregular light lines (arrow) are interpreted as the unstained wall. x 165,000. F~G. 5. Unfixed microtubule stained with potassium phosphotungstate at pH 4. The core appears positively stained, but at some places there is a slight indication of filaments. × 198,000. Fro. 6. Unfixed microtubules negatively stained with potassium phosphotungstate at pH 5. The individual microtubules show 6 filaments which at some places show an indication of beading. One microtubule has undergone depolymerization at one end before (or during) application of the negative stain (d). In another microtubule two of the filaments are absent in a part of the microtubule (arrow). × 240,000.
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Z
N t~
Z
.o
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O. BEHNKE AND T. ZELANDER TABLE I EFFECTS OF FIXATION AND OF P H OF NEGATIVE STAIN Uranyl Acetate
Potassium Phosphotungstate Fixed Microtubules pH of r ^ Negative Core Stain Staining Filaments 4
5
+
+-
UnfixedMicrotubules Core Staining
A few s h o w trace of filaments +-
Filaments
+
A few s h o w trace of filaments
-
+
Fixed Microtubules Core Staining
Filaments
+ _ a
+ _
+-
+-
6
-
+
N o microtubules observed
7
-
+
N o microtubules observed
UnfixedMicrotubules Core Staining
Filaments
_
+-
a + _ indicates that some microtubules showed filaments and others core staining in the preparation.
+
+-
same
(Figs. 6, 7, 9, 1 I, and 12). The filaments measured about 35 A in width and were spaced with a center-to-center distance of about 60 A. In some filaments a beading could be distinguished (Figs. 6, 7, and 8), but this was not a constant finding. Beading was observed both in fixed and unfixed microtubules. In the majority of microtubules the number of filaments that could be counted was 6 (Fig. 6), but occasionally 7 were observed, and one and the same microtubule sometimes showed different numbers at different places (Figs. 6 and 12). Sometimes a microtubule was observed which had apparently split and been unfolded, like a ribbon, on the supporting film (Fig. 12) during the preparative procedure. In these cases the number of filaments was 10-12, although the exact number could often not be counted without ambiguity. Fractured microtubules and short fragments of microtubules showed the same filamentous substructure as unbroken ones. Microtubules in various stages of disintegration were frequently seen, both in preparations fixed before application of the negative stain (Fig. 8) and in unfixed preparations (Fig. 6). Small pieces of filaments and small granules, approximately 35 A in diameter, were seen at the sites of disintegration, which may be located either
FIG. 7. Unfixed microtubules stained with potassium p h o s p h o t u n g s t a t e at p H 5. At some places the filamentous substructures have a beaded appearance (b). One microtubule shows two fracture sites (arrows). x 139,000. Fro. 8. Microtubules fixed in glutaraldehyde before being negatively stained with potassium phosphotungstate at p H 7. The microtubules were apparently in a process of depolymerization before they were fixed. The filaments are broken into small pieces and granules (arrows). Accumulations of depolymerized filaments, mainly consisting of a fine granular material, are seen in the lower part of the figure (asterisks). x 149,000.
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at the "ends" of microtubules (Figs. 6 and 8) or at any place along their course on the grid (Fig. 8). We interpret this disintegration as probably being due to depolymerization. Sometimes gaps were observed in the continuity of filaments in otherwise intact microtubules. These gaps were often seen in the "lateral" (or "outer") filaments (Fig. 6). In only a few instances were longer fragments of filaments observed, and it was apparent that disintegrating microtubules did not split into filaments, but that the filaments themselves broke into small pieces and granules (Fig. 8). N o t even in cases where a microtubule had been unfolded like a ribbon, did the filaments separate. This observation strongly suggests that cross links between the individual filaments do exist, although oblique or transverse striations connecting the filaments were not revealed, either in fixed or in unfixed preparations. If disintegration of microtubules occurs in a similar way in intact cells, identification of depolymerized pieces of microtubule filaments in sectioned material would probably be extremely difficult if not impossible.
The "core" of microtubules In sectioned material stained with uranyl acetate and lead citrate microtubules appear "hollow," having unstained cores. In negatively stained preparations, in which the filamentous substructure of microtubules was displayed, the appearance of the microtubules did not seem to be consistent with the conception that they are hollow structures (Figs. 6-9 and 12). Under some experimental conditions, however, filaments were less evident or not visible at all (see Table I), and in these cases the "core" was stained. This phenomenon was seen both in fixed (Figs. 3 and 4) and in unfixed (Fig. 5) microtubules. The stained cores were often rather sharply outlined and were delineated by light zones (Figs. 3 and 4) which probably represented the walls of the microtubules.
Other filaments In some preparations long filaments, approximately 5 0 / ~ in diameter, were observed. These filaments were most often encountered in fixed preparations, especially where intact marginal bundles and much platelet debris were present (Fig. 9). These filaments had a more uneven course and did not seem to be of microtubular origin.
FIG. 9. Four microtubules from a complete marginal bundle. The microtubules have retained the curved shape they have in intact platelets. Six or seven filaments are seen in each microtubule. There is no indication of beading of the filaments. Other somewhat coarser filaments (arrows) are also seen (see text). Preparation fixed with glutaraldehyde and negativelystained with potassium phosphotungstate at pH 7. x 160,000. FIG. 10. Unfixed microtubule stained with uranyl acetate adjusted to pH 5 with potassium hydroxide. At some places filamentous substructures are seen (arrows). x 230,000.
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o. BEHNKE AND T. ZELANDER
Shadow-casting experiments In sectioned material the microtubules of the marginal bundle appear as "hollow" structures with an outer diameter of approximately 250 A. There is a difference in size between sectioned and negatively stained microtubules, the width of microtubules in the negative stain image measuring about 400 A (370-430 A). This difference may be due to some shrinkage of microtubules during fixation and embedding procedures. It seems likely, however, that at least some of the augmentation in width of microtubules in the negatively stained preparations can be accounted for by flattening of microtubules on the supporting film before the fixative or the negative stain is applied. In order to obtain some information on the degree of collapse taking place when microtubules adhere to the supporting film, preparations were shadowed with platinum or platinum-carbon (Figs. 1 and 2). The average height of both fixed and unfixed microtubules was calculated to be about 90 A, and the mean width of shadowed microtubules measured about 370 A. If we assume that the walls of the microtubules were collapsed, but otherwise intact, and compare this figure with the diameter of 250 A in sectioned platelets, it follows that the microtubules had collapsed 60-70 % when adhering to the grid and that the interior or "lumen" of the microtubules was nearly obliterated.
DISCUSSION
General remarks A brief survey of some of the observations hitherto reported on negatively stained preparations of microtubules from various sources is given in Table II. It is seen that the majority of the data concern observations on microtubules that are preservable in osmium tetroxide. Comparison of the various data should, of course, be made with caution because of the different techniques used by the various authors and the inherent limitations of the negative staining technique. It is obvious from Table II, however, that the substructure of glutaraldehyde preservable microtubules is essentially comparable to that of the osmium preservable ones. Both exhibit a wall constructed of fine filaments which regularly (16, 18) or sometimes (1, 28, present work) have a beaded appearance. Based on studies with the freeze-etching technique on yeast cells, Moor (23, 24) has proposed a microtubule model with the wall consisting of 40 A globular subunits arranged in a double helix. The globular subunits are also arranged in rows parallel to the long axis of the microtubule (see Fig. 8 in reference 23) and would correspond to what in the negative stain image is seen as beaded filaments. The evident morphological similarities in substructure between G- and O-micro-
Axial filament complex, rat sperm tails
Trichomonas vaginalis
Pease (28)
Nielsen, M. (26)
Spindle microtubules
Marginal bundle, blood platelets
Barnicot (2)
Present work
Trichonympha
35 A (beads)
Glutaraldehyde 35 N (beads)
Glutaraldehyde 35/~ (beads)
Osminm
Flagella from
Grinastone and Klug (16)
Not given
Osmium
Marginal band, nucleated red cells
Gall (12)
24 A 24 A
35-40 A (beads)
35 A (beads)
Glutaraldehyde 33 A (beads)
Osmium
Osmium
Osmium
Whether Diameter of Glutaraldehyde or Filaments Osmium preservable (beads)
Kiefer et al. (18) Spindle microtubules, sea urchin eggs
Flagellum Axostyle
Axial filament complex, rat and human sperm tails
Source of Microtubules
Andr6 and Thi6ry (1)
Author
T A B L E II
60 A
50 ~
40-50 ~
50/~
45 A
41 A 41 ~
55-60 ,~
60-80 A
Lateral Spacing of Filaments
Noted in some preparations
Noted
Core Staining
Noted
Noted
Uranyl acetate, Noted 4.8; armnonium molybdate, 6.5 and 5.2; PTA, 6.5 PTA, 4,5,6,7; In some preuranyl acetate, parations 4 and 5
Uranyl acetate and PTA; pH not given PTA 7
Uranyl acetate, 4 Noted
Ammonium molybdate
PTA 6-7.4
PTA "neutralis6"
Stair~ Used and pH at Which Used
SURVEY OF REPORTED OBSERVATIONS ON NEGATIVELY STAINED MICROTUBULES
12
Uncertain, range 9-12
Not less than 12
12-14
12-13
10-12
10
10-11 in central tubules
Total Number of Filaments
M~
O
o
~
160
o . B E H N K E A N D T. Z E L A N D E R
tubules should not be overemphasized to the extent of disregarding the obvious dissimilarities. Thus, as mentioned previously, the axial complex of cilia and flagella is readily preserved by osmium tetroxide fixation under neutral or slightly alkaline conditions, whereas the G-microtubules of mammalian cells generally require fixation in glutaraldehyde to be preserved. Likewise, G-microtubules disintegrate rapidly if attempts are made to produce negatively stained preparations at neutral pH, whereas Table II shows that a neutral or slightly alkaline negative stain was used in most studies of axial complex microtubules. In two studies on the substructure of spindle (G) microtubules (2, 18) the information was obtained by using a negative stain at low pH. It is particularly interesting in this connection to note that in the recent report by Barnicot (2), the author stated that when ammonium molybdate was used at p H 6.5 "very few spindle fibres were found and these were so much damaged that they were hardly recognizable as such." At p H 5.2, however, the author found "fairly well preserved fibres." Nor did the author detect any spindle fibers with PTA employed at p H 6.5. These observations apparently provide further evidence that G-microtubules are sensitive to p H changes. Evidence has been presented suggesting that differences in stability exist between the peripheral doublets and the central pair of microtubules even among the O-tubules of the axial filament complex of cilia and flagella (6, 13, 14, 16, 25). It is likely that the dissimilarities in behavior toward chemicals, temperature, and p H changes reflect physicochemical differences in the various microtubules despite similarities in morphology. If so, this in all probability indicates differences also in their function. The interpretation of the function of marginal band microtubules is still hindered by the fact that very little is known about their chemistry. Cytoplasmic microtubules in most cells are straight or only slightly curved and thus possibly possess an inherent ridigity. It has been speculated that the curved microtubules in the marginal bundle in platelets are under tension (4) and may be responsible for the maintenance of the discoid shape of platelets (4, 11) and nucleated red cells (11, 22). Evidence obtained in the present work corroborates this idea. Fragments of microtubules from ruptured bundles were nearly always completely straight (Figs. 5, 6, 7, and 10), but if the integrity of the bundle had been preserved the microtubules retained the curved shape they have in intact platelets (Figs. 1 and 9). FIa. 11. Part of an unbroken platelet which had adhered to the grid. A rim of the platelet membrane is seen atp. The marginal bundle of microtubules (rob) was intact, and, even at this low magnification, the filamentous substructure of the individual microtubules can be discerned. Specimen fixed in glutaraldehyde before being negatively stained with potassium phosphotungstate at pH 7. × 63,000. Fro. 12. Microtubule showing split and spread (s) and unsplit (u) portions. In the spread portions 10 or 11 filaments are seen; in the unsplit portions only 6 can be counted. Fixed in glutaraldehyde before being negatively stained with potassium phosphotungstate at pH 7. x 195,000.
SUBSTRUCTURE
] I - 6 7 1 8 2 5 J . Ultrastructure Research
OF MICROTUBULES
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It is known that platelets attain a spherical shape in the cold (37). This change in shape might be explained, at least in part, by the sensitivity of G-microtubules to cold (6, 35) and is probably related to the fact that negatively stained preparations of platelets isolated in the cold revealed no microtubules. The special arrangement of microtubules in platelets makes it unlikely that they function in intracellular transport mechanisms. The hypothesis that they are cytoskeletal structures seems more appropriate.
Number of filaments in the microtubule wall The majority of microtubules showed 6 filaments but occasionally 7 could be counted. In a few instances short segments of microtubules were observed which had apparently split and unfolded on the supporting film. These microtubules showed 10 to 12 filaments. A likely interpretation of these images is that the microtubules showing 6 or 7 filaments are intact but flattened. The flattening of microtubules when they adhere to and dry down on the supporting film was estimated from the shadow cast specimens to be about 60-70 %. Since the width of the microtubules was about the same whether they were negatively stained or shadowed (400 A as against 370 A), it seems probable that the negatively stained microtubules were flattened to about the same degree. The negative contrast image of filaments in the wall of microtubules can best be explained by assuming that the stain had penetrated into the space between the individual filaments. Whether this space contains a positively stainable interfilament substance or is passively occupied by the stain, cannot be decided on the available evidence. However, the fact that filaments were observed in microtubules in flattened but unruptured platelets speaks in favor of a positively stainable interfllament substance. If it is assumed that the stain penetrates both the "free" (freely exposed) wall of the flattened tubule and the wall which is adherent to the grid, then the appearance of the substructure must be dependent on the superimposition of the one wall upon the other. When filaments of the "free" wall are directly superimposed on the filaments of the "deep" wall the filamentous substructure would be enhanced. Alternatively, when the filaments of the free wall are staggered to correspond to the spaces between the filaments of the deep wall, a blurred image of the substructure would be encountered. Since areas of both enhanced and of blurred substructure were evident in some preparations this also seems to indicate that both the free wall and the deep wall are penetrated by stain. If, then, the filaments of the free and the deep wall are in register, the observation of 6 filaments in the majority of the tubules seen suggests that the total number of filaments can hardly be less than 12. The occasional observation of 7 filaments in
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some tubules may also be explained from a model with a total number of 12. A model with a total number of 11 filaments would allow the observation of 6 filaments (one filament at either edge being "single") but would hardly explain the observation of 7 filaments if the tubule is intact. With a center to center distance of 60 A a total number of 12 filaments would correspond to a diameter of about 240 A in an unflattened tubule. This figure corresponds well with the diameter measured in fixed and embedded specimens. Irrespective of the above discussion, it should be borne in mind that it is not inconceivable that the number of filaments in individual microtubules may vary. Since the diameter of microtubules as reported in the literature varies from 180 to 300/~ this might well be the case (see also 23, 24).
"Core" staining It might be argued that staining of the core was due to "central filling" of a hollow microtubule, and this has been the explanation of the phenomenon given by some authors (see Table II). Glauert et al. (15) in studying negatively stained bacterial flagella observed "central filling" only in flagella disrupted by ultrasonication and argued that the interior of the flagella were filled with stain entering at the site of disruption. Lowy and Hanson (21), however, in their elegant study on flagella from various bacteria, did not observe core staining or central filling. In the present material staining of the core did not seem to be dependent on mechanical disturbance of the microtubules. The phenomenon was seen in short pieces of microtubules, in intact marginal bundles, and in microtubules which were continuous for long distances. Furthermore, the observation that staining of the core in marginal band microtubules seemed to be dependent upon the state of fixation, the negative stain and the p H at which it was used, makes it unlikely that it was due to a mere mechanical filling of a hollow tube. Our results suggest that the wall of the tubule is permeable to the stain under special conditions. Whether a hypothethical core material is positively stained, or the space of the core is passively filled with the stain, cannot be decided from our observations. However, the variation of the staining of the core seems to be dependent on chemical differences of application, and whatever the explanation may turn out to be, the possibility of the presence of a core material seems to be of relevance. It may thus be suggested that the microtubules are two-component structures.
Other filaments observed The filaments of approximately 50 • width which were observed in some preparations probably correspond to the filaments which appear in platelets when they stick
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O. BEHNKE AND T. ZELANDER
to a glass surface (7) or aggregate in thrombi (5). Platelets fixed intravascularly, or instantaneously upon withdrawal from a vessel, have a finely granular cytoplasm and filaments are usually not discernible. Their presence in some specimens probably means that unruptured platelets occasionally stick to the grid and subsequently burst. The filaments are not considered to be of microtubular origin because their diameter is bigger than that of the filamentous substructures of microtubules. In addition, platelets which have stuck to a surface or aggregated in thrombi possess both filaments of this type, and microtubules. Finally, evidence given in this paper indicates that when G-microtubules depolymerize they do not split into filaments, but the filaments themselves break into short pieces and granules (Fig. 8). This work was supported by grants from the Danish State Research Foundation and the Carlsberg Foundation.
REFERENCES 1. AN1)R~, J. and Trtr~RY, J.-P., J. Microscopie 2, 71 (1963). 2. BARNICOT,N. A., J. Cell Sci. 1, 217 (1966). 3. BErtNKE, 0., J. Ultrastruct. Res. 11, 139 (1964). 4. - ibid. 13, 469 (1965). 5. - Scand. J. HaematoL 3, 136 (1966). 6. BEHNKE,O. and FOILER,A. F., J. Cell Sci., in press. 7. BESSIS,M. and BRETON-GORrUS,J., Nouvelle Rev. Franc. Hdmatol. 5, 657 (1965). 8. BRENNER,S. and HORNE, R. W., Biochim. Biophys. Acta 34, 103 (1959). 9. DE-THe, G., J. Cell BioL 23, 265 (1964). 10. FAWCETT, D. W., in BRACI-mT, J. and MmSKY, A. E. (Eds.), The Cell, Vol. II, 217. Academic Press, New York, 1961. 11. FAWCETT,D. W. and WITEBSKY,F., Z. Zellforsch. 62, 785 (1964). 12. GALL, J. G., J. CellBiol. 27, 32 A (1965). 13. GIBBONS,I. R., Proc. Natl. Acad. Sci. U.S. 50, 1002 (1963). 14. - Arch. Biol. (Liege) 76, 317 (1965). 15. GLAU~RT,A. M., KERRIDGE,D. and HORNE, R. W., or. Cell Biol. 18, 327 (1963). 16. GRIMSTONE,A. V. and KLUG, A., J. Cell Sci. 1, 351 (1966). 17. HAYDON,G. B. and TAYLOR,D. A., or. CellBiol. 26, 673 (1965). 18. KtEFER, B., SAKAr, H., SOLARr, A. J. and MAZlA, D., J. MoL Biol. 20, 75 (1966). 19. LEDBETTER,M. C. and PORTER, K. R., J. Cell BioL 19, 329 (1963). 20. - Science 144, 872 (1964). 21. LowY, J. and HANSON, J., or. Mol. BioL 11, 293 (1965). 22. MASER, M. D. and PrIILPOTT,C. W., Anat. Recordl50, 365 (1964). 23. MOOR, H., Balzers High Vacuum Rep. 9, 1 (1966). 24. - Protoplasma in press. 25. NAGANO,T., J. Cell BioL 18, 337 (1963). 26. N~ELSEN,M., personal communication. 27. PALAY, S. L., J. Biophys. Biochem. CytoL 2, Suppl., 193 (1956).
SUBSTRUCTURE OF MICROTUBULES 28. 29. 30. 31. 32. 33. 34. 35. 36. 37.
165
PEASE, D. C., or. Cell Biol. 18, 313 (1963). ROBBINS,E. and GONATAS,N. K., J. Cell Biol. 21, 429 (1964). SABATINI,D. D., BENSCrt, K. G. and BARRNETT,R. J., J. Cell Biol. 17, 19 (1963). SANDBORN,E., KOEN, P. F., MCNABB, J. D. and MOORE, G., J. Ultrastruct. Res. ll, 123 (1964). SANDBORN,E. B., LEBtns, J.-J. and Bois, P., Blood 27, 247 (1966). SILVER,M. D., Nature 209, 1048 (1965). SLAUTTERBACK,D., Or. Cell Biol. 18, 367 (1963). TILNEY, L. G., Anat. Record 151, 426 (1965). TILNEY, L. G. and PORTER, K. R., Protoplasma 60, 317 (1965). ZUCKER, M. B. and BORELLI,J., Blood 9, 602 (1954).
s. ULTRASTRUCTURERES~ARCrI19, 147-165 (1967)
147
Filamentous Substructure of Microtubules of the Marginal Bundle of Mammalian Blood Platelets O. BEHNKE AND T. ZELANDER
Department of Anatomy, The Royal Dental College, Copenhagen, Denmark Received November 29, 1966 Cytoplasmic microtubules from the marginal bundle in human and rat blood platelets were prepared by negative staining and shadow-casting, and studied in the electron microscope. The microtubules from both species gave the same results: their walls appear to be constructed of 35 A-thick filaments evenly spaced in the wall and with a center-to-center distance of 60 &. The total number of filaments in each microtubule is probably 12. Microtubules which disintegrate during the preparative procedures break into short pieces of filaments, or globules, the latter being 35 A in diameter. While some experimental conditions gave preparations which revealed a filamentous substructure of the microtubule wall, others showed staining of the microtubule "core." The experiments substantiate the hypothesis that microtubules are rigid structures. Some of the information obtained strongly suggests that the marginal bundle consists of but one long, rolled-up microtubule.
The term microtubule, which was introduced by Slautterback (34), is currently used as a designation for a class of cellular structures which, in sectioned material, appear as "hollow" tubules with electron dense walls and a less dense interior. They range in outer diameter from about 180 • to 300 tk. Microtubules have, since the introduction of glutaraldehyde as a fixative (30) been recognized as a cytoplasmic component of a wide variety of cells of plant and animal origin, and it seems at present reasonable to assume that the microtubule is a universal cellular organelle. Conventional osmium tetroxide fixation preserves some microtubules, for example the axial filament complex of cilia and flagella [see review by Fawcett (10)], neurotubules (27), and the marginal bundle of nucleated red cells (11, 22). Other microtubules are more labile and require fixation in glutaraldehyde to be properly preserved (3, 9, 19, 29, 31). The morphological similarity in sectioned material between "osmium-preservable" and "glutaraldehyde-preservable" tubules is indisputable. We think, however, that
148
O. B E H N K E A N D T. Z E L A N D E R
it is justified, until better criteria are at hand, to discriminate between two classes of microtubules: those which are preserved by conventional osmium tetroxide (referred to as O-microtubules in the following), and the labile microtubules which require fixation in glutaraldehyde (referred to as G-microtubules). Filamentous substructure in O-microtubules has been reported. Pease (28) and Andr6 and Thi6ry (1), investigating the microtubules of the axial filament complex of rat and h u m a n sperm tails with the negative-stain technique, reported the presence of 10 or 11 filaments in the walls of these tubules. The filaments were named protofibrils by Andr6 and Thi6ry. Nielsen (26) found a similar substructure in the O-microtubules of flagella and axostyles in Trichornonas vaginalis and recently Grimstone and Klug (16) have made similar observations on cilia f r o m Trichonympha. Gall (12), studying the O-microtubules of the marginal band of nucleated red cells, estimated the presence of 12-14 filaments in the walls of these tubules. The presence of a substructure in G-microtubules was suggested by Ledbetter and Porter in their first report on microtubules in plants (19), and, based on M a r k h a m rotation tests performed on fixed and sectioned material, they concluded that the wall of G-microtubules probably has 13 substructures spaced evenly around the circumference (20). The substructures were described as being circular in outline and having a center-to-center distance of 45/~. Most of the information on G-microtubules so far obtained has come from the study of thin sections. Two recent reports (2, 18) have described the presence of a filamentous substructure in microtubules of the mitotic spindle. To our knowledge no detailed report has been published concerning the substructure of cytoplasmic G-microtubules of interphase mammalian cells. It is the purpose of this paper to relate some observations on the substructure of mammalian G-microtubules as obtained by the negative staining technique. Since blood platelets of mammals contain a conspicuous marginal bundle of Gmicrotubules (4, 17, 32, 33) encircling the circumference of the platelets just beneath the plasma membrane, and since they have no O-microtubules, they are well suited for such a study.
MATERIAL AND METHODS Blood was obtained from adult humans and albino rats by venipuncture, using either 5 % EDTA in 0.9 % saline or a 2 % sodium citrate solution as anticoagulants. The anticoagulants were used at room temperature and were adjusted to pH 6. Three milliliters of anticoagulant were used for 7 ml of blood. Red cells and leukocytes were sedimented by centrifugation at 300 g at room temperature for 15 minutes. The supernatant platelet rich plasma was then centrifuged at 1500 g for 15-20 minutes. This procedure yields pellets consisting almost entirely of platelets.
S U B S T R U C T U R E OF M I C R O T U B U L E S
149
A small amount of platelets were transferred with the tip of a small glass rod to the surface of a drop of distilled water. After 30-60 seconds the spread and ruptured platelets were picked up by touching the water surface with carbon-coated specimen grids. Some grids were subsequently floated briefly on a drop of 2 % glutaraldehyde to fix the platelet debris before further processing; other specimens were processed unfixed. Shadow casting. Fixed and unfixed specimens were shadowed with platinum or platinum and carbon at various angles at a filament-specimen distance of 11-15 cm. Negative staining procedure. Fixed and unfixed specimens were negatively stained (8) with 2 % potassium phosphotungstate at pH 4, 5, 6, and 7, or with 2 % uranyl acetate at pH 4.1 (unadjusted) or adjusted to pH 5 with potassium hydroxide. The specimens were examined in a Siemens Elmiskop I, operated at 60 kV with double condensor and 50-# objective aperture. The direct magnifications ranged from 15,000 to 60,000. The microscope was calibrated by using a grating replica. OBSERVATIONS
General remarks The preparations of microtubules from rat and human blood platelets showed no morphological differences, and the observations described relate to both. Negative-staining images of the microtubules of the marginal bundle were regularly obtained with the stated procedure. For some unknown reason, however, the yield was higher in rat platelets than in platelets of human origin. Microtubules were never found if the isolation of platelets had been carried out in the cold (0-4°C). This observation, which probably indicates that the microtubules of platelets are not stable in the cold, is in agreement with observations by Tilney (35) on G-microtubules in Actinosphaeriurn and by Behnke and Forer (6) on cytoplasmic microtubules in crane fly, Nephrotoma suturalis (Loew), sperm and spermatids. The platelet membranes generally disappeared during the preparatory procedure. In some specimens, however, unruptured platelets which had adhered to and flattened on the grid were observed. The intact bundle of microtubules could sometimes be identified in these platelets (Fig. 11), leaving no doubt as to the identification of microtubules lying free on the supporting film. Not infrequently the microtubules of the marginal bundle had retained their original spatial relationship and the bundle could be observed in toto as a ring-like structure (Fig. 1). Thus, depriving the blood platelet of its plasma membrane does not necessarily cause the marginal bundle to break up. In those cases where the bundle as such could be observed, the microtubules showed the curved shape they have in intact platelets. In most cases, however, the bundle had been disrupted during the preparative procedure, and pieces of microtubules become freely exposed on the grid (Fig. 6). "Free" microtubules were straight or only gently curved. Many of them showed fractures (Fig. 7), supporting the hypothesis that microtubules are relatively rigid structures.
150
O. BEHNKE AND T. ZELANDER
C o m m o n l y , " f r e e " m i c r o t u b u l e s were seen u n c o n n e c t e d to m e m b r a n e fragments. It is n o t k n o w n w h e t h e r the m a r g i n a l b u n d l e in platelets is c o m p o s e d of i n d i v i d u a l m i c r o t u b u l e s f o r m i n g c o m p l e t e l o o p s a r o u n d the entire circumference, or w h e t h e r the m a r g i n a l b u n d l e consists of b u t one coiled-up microtubule. O u r o b s e r v a t i o n s o n s h a d o w e d a n d negatively stained p r e p a r a t i o n s of intact m a r g i n a l bundles have n o t solved this p r o b l e m , because i n d i v i d u a l m i c r o t u b u l e s could n o t be followed w i t h o u t a m b i g u i t y for m o r e t h a n a fraction of the circumference due to o v e r l a p p i n g of structures. I n several p r e p a r a t i o n s it was observed, however, t h a t two "free e n d s " of m i c r o t u b u l e s p r o j e c t e d f r o m an otherwise intact bundle, suggesting t h a t the b u n d l e was constructed of one coiled-up m i c r o t u b u l e with two free ends (Fig. 1). U n d e r some e x p e r i m e n t a l c o n d i t i o n s m i c r o t u b u l e s were unstable. It has a l r e a d y been n o t e d that the m i c r o t u b u l e s d i s a p p e a r e d if the platelets were subjected to cold. A p p l i c a t i o n of the negative stain to unfixed m i c r o t u b u l e s revealed t h a t the preserv a t i o n of their integrity was also d e p e n d a n t of the p H of the negative stain. M i c r o tubules were never o b s e r v e d if the stain was a p p l i e d to unfixed m i c r o t u b u l e s at p H 6 a n d 7. The effects of the different negative stains, of their p H , a n d of fixation o n the a p p e a r a n c e of substructure in the m i c r o t u b u l e s are s u m m a r i z e d in T a b l e I. A s indicated, some e x p e r i m e n t a l c o n d i t i o n s p r o d u c e d a filamentous substructure of m i c r o tubules, whereas others revealed a central " c o r e . " Filamentous substructure
U n d e r suitable c o n d i t i o n s of staining (see T a b l e I) the m i c r o t u b u l e s p r e s e n t e d a l o n g i t u d i n a l striation due to the presence of fine filaments in the m i c r o t u b u l e wall FIG. 1. Electron micrograph of a shadowed preparation of a complete marginal bundle of microtubules (mb) from a ruptured rat blood platelet. The bundle was not fixed before shadowing with platinum. Because of overlapping it is not possible to follow the course of individual microtubules for long distances. Two microtubules have swung out from the bundle and have assumed a straight course (arrows). Membrane fragments are seen at m. g, platelet dense granules. Reversed print. x 38,000. FIG. 2. Part of a microtubule (mt) from a rat blood platelet. The preparation was fixed with glutaraldehyde before shadowing with platinum. The irregular background is due to shadowed cytoplasmic material from the platelet, x 109,000. FIG. 3. Glutaraldehyde-fixed microtubules stained with potassium phosphotungstate at pH 4. The outline of the microtubules is somewhat irregular. The core shows up as if positively stained (arrow). There is no indication of substructure, x 200,000. FIG. 4. Glutaraldehyde-fixed microtubules stained with uranyl acetate at pH 4.1. The core is positively stained. The somewhat irregular light lines (arrow) are interpreted as the unstained wall. x 165,000. F~G. 5. Unfixed microtubule stained with potassium phosphotungstate at pH 4. The core appears positively stained, but at some places there is a slight indication of filaments. × 198,000. Fro. 6. Unfixed microtubules negatively stained with potassium phosphotungstate at pH 5. The individual microtubules show 6 filaments which at some places show an indication of beading. One microtubule has undergone depolymerization at one end before (or during) application of the negative stain (d). In another microtubule two of the filaments are absent in a part of the microtubule (arrow). × 240,000.
SUBSTRUCTURE OF MICROTUBULES
151
Z
N t~
Z
.o
SUBSTRUCTURE OF MICROTUBULES
153
154
O. BEHNKE AND T. ZELANDER TABLE I EFFECTS OF FIXATION AND OF P H OF NEGATIVE STAIN Uranyl Acetate
Potassium Phosphotungstate Fixed Microtubules pH of r ^ Negative Core Stain Staining Filaments 4
5
+
+-
UnfixedMicrotubules Core Staining
A few s h o w trace of filaments +-
Filaments
+
A few s h o w trace of filaments
-
+
Fixed Microtubules Core Staining
Filaments
+ _ a
+ _
+-
+-
6
-
+
N o microtubules observed
7
-
+
N o microtubules observed
UnfixedMicrotubules Core Staining
Filaments
_
+-
a + _ indicates that some microtubules showed filaments and others core staining in the preparation.
+
+-
same
(Figs. 6, 7, 9, 1 I, and 12). The filaments measured about 35 A in width and were spaced with a center-to-center distance of about 60 A. In some filaments a beading could be distinguished (Figs. 6, 7, and 8), but this was not a constant finding. Beading was observed both in fixed and unfixed microtubules. In the majority of microtubules the number of filaments that could be counted was 6 (Fig. 6), but occasionally 7 were observed, and one and the same microtubule sometimes showed different numbers at different places (Figs. 6 and 12). Sometimes a microtubule was observed which had apparently split and been unfolded, like a ribbon, on the supporting film (Fig. 12) during the preparative procedure. In these cases the number of filaments was 10-12, although the exact number could often not be counted without ambiguity. Fractured microtubules and short fragments of microtubules showed the same filamentous substructure as unbroken ones. Microtubules in various stages of disintegration were frequently seen, both in preparations fixed before application of the negative stain (Fig. 8) and in unfixed preparations (Fig. 6). Small pieces of filaments and small granules, approximately 35 A in diameter, were seen at the sites of disintegration, which may be located either
FIG. 7. Unfixed microtubules stained with potassium p h o s p h o t u n g s t a t e at p H 5. At some places the filamentous substructures have a beaded appearance (b). One microtubule shows two fracture sites (arrows). x 139,000. Fro. 8. Microtubules fixed in glutaraldehyde before being negatively stained with potassium phosphotungstate at p H 7. The microtubules were apparently in a process of depolymerization before they were fixed. The filaments are broken into small pieces and granules (arrows). Accumulations of depolymerized filaments, mainly consisting of a fine granular material, are seen in the lower part of the figure (asterisks). x 149,000.
SUBSTRUCTURE OF MICROTUBULES
155
156
o . B E H N K E A N D T. Z E L A N D E R
at the "ends" of microtubules (Figs. 6 and 8) or at any place along their course on the grid (Fig. 8). We interpret this disintegration as probably being due to depolymerization. Sometimes gaps were observed in the continuity of filaments in otherwise intact microtubules. These gaps were often seen in the "lateral" (or "outer") filaments (Fig. 6). In only a few instances were longer fragments of filaments observed, and it was apparent that disintegrating microtubules did not split into filaments, but that the filaments themselves broke into small pieces and granules (Fig. 8). N o t even in cases where a microtubule had been unfolded like a ribbon, did the filaments separate. This observation strongly suggests that cross links between the individual filaments do exist, although oblique or transverse striations connecting the filaments were not revealed, either in fixed or in unfixed preparations. If disintegration of microtubules occurs in a similar way in intact cells, identification of depolymerized pieces of microtubule filaments in sectioned material would probably be extremely difficult if not impossible.
The "core" of microtubules In sectioned material stained with uranyl acetate and lead citrate microtubules appear "hollow," having unstained cores. In negatively stained preparations, in which the filamentous substructure of microtubules was displayed, the appearance of the microtubules did not seem to be consistent with the conception that they are hollow structures (Figs. 6-9 and 12). Under some experimental conditions, however, filaments were less evident or not visible at all (see Table I), and in these cases the "core" was stained. This phenomenon was seen both in fixed (Figs. 3 and 4) and in unfixed (Fig. 5) microtubules. The stained cores were often rather sharply outlined and were delineated by light zones (Figs. 3 and 4) which probably represented the walls of the microtubules.
Other filaments In some preparations long filaments, approximately 5 0 / ~ in diameter, were observed. These filaments were most often encountered in fixed preparations, especially where intact marginal bundles and much platelet debris were present (Fig. 9). These filaments had a more uneven course and did not seem to be of microtubular origin.
FIG. 9. Four microtubules from a complete marginal bundle. The microtubules have retained the curved shape they have in intact platelets. Six or seven filaments are seen in each microtubule. There is no indication of beading of the filaments. Other somewhat coarser filaments (arrows) are also seen (see text). Preparation fixed with glutaraldehyde and negativelystained with potassium phosphotungstate at pH 7. x 160,000. FIG. 10. Unfixed microtubule stained with uranyl acetate adjusted to pH 5 with potassium hydroxide. At some places filamentous substructures are seen (arrows). x 230,000.
o
©
158
o. BEHNKE AND T. ZELANDER
Shadow-casting experiments In sectioned material the microtubules of the marginal bundle appear as "hollow" structures with an outer diameter of approximately 250 A. There is a difference in size between sectioned and negatively stained microtubules, the width of microtubules in the negative stain image measuring about 400 A (370-430 A). This difference may be due to some shrinkage of microtubules during fixation and embedding procedures. It seems likely, however, that at least some of the augmentation in width of microtubules in the negatively stained preparations can be accounted for by flattening of microtubules on the supporting film before the fixative or the negative stain is applied. In order to obtain some information on the degree of collapse taking place when microtubules adhere to the supporting film, preparations were shadowed with platinum or platinum-carbon (Figs. 1 and 2). The average height of both fixed and unfixed microtubules was calculated to be about 90 A, and the mean width of shadowed microtubules measured about 370 A. If we assume that the walls of the microtubules were collapsed, but otherwise intact, and compare this figure with the diameter of 250 A in sectioned platelets, it follows that the microtubules had collapsed 60-70 % when adhering to the grid and that the interior or "lumen" of the microtubules was nearly obliterated.
DISCUSSION
General remarks A brief survey of some of the observations hitherto reported on negatively stained preparations of microtubules from various sources is given in Table II. It is seen that the majority of the data concern observations on microtubules that are preservable in osmium tetroxide. Comparison of the various data should, of course, be made with caution because of the different techniques used by the various authors and the inherent limitations of the negative staining technique. It is obvious from Table II, however, that the substructure of glutaraldehyde preservable microtubules is essentially comparable to that of the osmium preservable ones. Both exhibit a wall constructed of fine filaments which regularly (16, 18) or sometimes (1, 28, present work) have a beaded appearance. Based on studies with the freeze-etching technique on yeast cells, Moor (23, 24) has proposed a microtubule model with the wall consisting of 40 A globular subunits arranged in a double helix. The globular subunits are also arranged in rows parallel to the long axis of the microtubule (see Fig. 8 in reference 23) and would correspond to what in the negative stain image is seen as beaded filaments. The evident morphological similarities in substructure between G- and O-micro-
Axial filament complex, rat sperm tails
Trichomonas vaginalis
Pease (28)
Nielsen, M. (26)
Spindle microtubules
Marginal bundle, blood platelets
Barnicot (2)
Present work
Trichonympha
35 A (beads)
Glutaraldehyde 35 N (beads)
Glutaraldehyde 35/~ (beads)
Osminm
Flagella from
Grinastone and Klug (16)
Not given
Osmium
Marginal band, nucleated red cells
Gall (12)
24 A 24 A
35-40 A (beads)
35 A (beads)
Glutaraldehyde 33 A (beads)
Osmium
Osmium
Osmium
Whether Diameter of Glutaraldehyde or Filaments Osmium preservable (beads)
Kiefer et al. (18) Spindle microtubules, sea urchin eggs
Flagellum Axostyle
Axial filament complex, rat and human sperm tails
Source of Microtubules
Andr6 and Thi6ry (1)
Author
T A B L E II
60 A
50 ~
40-50 ~
50/~
45 A
41 A 41 ~
55-60 ,~
60-80 A
Lateral Spacing of Filaments
Noted in some preparations
Noted
Core Staining
Noted
Noted
Uranyl acetate, Noted 4.8; armnonium molybdate, 6.5 and 5.2; PTA, 6.5 PTA, 4,5,6,7; In some preuranyl acetate, parations 4 and 5
Uranyl acetate and PTA; pH not given PTA 7
Uranyl acetate, 4 Noted
Ammonium molybdate
PTA 6-7.4
PTA "neutralis6"
Stair~ Used and pH at Which Used
SURVEY OF REPORTED OBSERVATIONS ON NEGATIVELY STAINED MICROTUBULES
12
Uncertain, range 9-12
Not less than 12
12-14
12-13
10-12
10
10-11 in central tubules
Total Number of Filaments
M~
O
o
~
160
o . B E H N K E A N D T. Z E L A N D E R
tubules should not be overemphasized to the extent of disregarding the obvious dissimilarities. Thus, as mentioned previously, the axial complex of cilia and flagella is readily preserved by osmium tetroxide fixation under neutral or slightly alkaline conditions, whereas the G-microtubules of mammalian cells generally require fixation in glutaraldehyde to be preserved. Likewise, G-microtubules disintegrate rapidly if attempts are made to produce negatively stained preparations at neutral pH, whereas Table II shows that a neutral or slightly alkaline negative stain was used in most studies of axial complex microtubules. In two studies on the substructure of spindle (G) microtubules (2, 18) the information was obtained by using a negative stain at low pH. It is particularly interesting in this connection to note that in the recent report by Barnicot (2), the author stated that when ammonium molybdate was used at p H 6.5 "very few spindle fibres were found and these were so much damaged that they were hardly recognizable as such." At p H 5.2, however, the author found "fairly well preserved fibres." Nor did the author detect any spindle fibers with PTA employed at p H 6.5. These observations apparently provide further evidence that G-microtubules are sensitive to p H changes. Evidence has been presented suggesting that differences in stability exist between the peripheral doublets and the central pair of microtubules even among the O-tubules of the axial filament complex of cilia and flagella (6, 13, 14, 16, 25). It is likely that the dissimilarities in behavior toward chemicals, temperature, and p H changes reflect physicochemical differences in the various microtubules despite similarities in morphology. If so, this in all probability indicates differences also in their function. The interpretation of the function of marginal band microtubules is still hindered by the fact that very little is known about their chemistry. Cytoplasmic microtubules in most cells are straight or only slightly curved and thus possibly possess an inherent ridigity. It has been speculated that the curved microtubules in the marginal bundle in platelets are under tension (4) and may be responsible for the maintenance of the discoid shape of platelets (4, 11) and nucleated red cells (11, 22). Evidence obtained in the present work corroborates this idea. Fragments of microtubules from ruptured bundles were nearly always completely straight (Figs. 5, 6, 7, and 10), but if the integrity of the bundle had been preserved the microtubules retained the curved shape they have in intact platelets (Figs. 1 and 9). FIa. 11. Part of an unbroken platelet which had adhered to the grid. A rim of the platelet membrane is seen atp. The marginal bundle of microtubules (rob) was intact, and, even at this low magnification, the filamentous substructure of the individual microtubules can be discerned. Specimen fixed in glutaraldehyde before being negatively stained with potassium phosphotungstate at pH 7. × 63,000. Fro. 12. Microtubule showing split and spread (s) and unsplit (u) portions. In the spread portions 10 or 11 filaments are seen; in the unsplit portions only 6 can be counted. Fixed in glutaraldehyde before being negatively stained with potassium phosphotungstate at pH 7. x 195,000.
SUBSTRUCTURE
] I - 6 7 1 8 2 5 J . Ultrastructure Research
OF MICROTUBULES
161
162
O. B E H N K E A N D T. Z E L A N D E R
It is known that platelets attain a spherical shape in the cold (37). This change in shape might be explained, at least in part, by the sensitivity of G-microtubules to cold (6, 35) and is probably related to the fact that negatively stained preparations of platelets isolated in the cold revealed no microtubules. The special arrangement of microtubules in platelets makes it unlikely that they function in intracellular transport mechanisms. The hypothesis that they are cytoskeletal structures seems more appropriate.
Number of filaments in the microtubule wall The majority of microtubules showed 6 filaments but occasionally 7 could be counted. In a few instances short segments of microtubules were observed which had apparently split and unfolded on the supporting film. These microtubules showed 10 to 12 filaments. A likely interpretation of these images is that the microtubules showing 6 or 7 filaments are intact but flattened. The flattening of microtubules when they adhere to and dry down on the supporting film was estimated from the shadow cast specimens to be about 60-70 %. Since the width of the microtubules was about the same whether they were negatively stained or shadowed (400 A as against 370 A), it seems probable that the negatively stained microtubules were flattened to about the same degree. The negative contrast image of filaments in the wall of microtubules can best be explained by assuming that the stain had penetrated into the space between the individual filaments. Whether this space contains a positively stainable interfilament substance or is passively occupied by the stain, cannot be decided on the available evidence. However, the fact that filaments were observed in microtubules in flattened but unruptured platelets speaks in favor of a positively stainable interfllament substance. If it is assumed that the stain penetrates both the "free" (freely exposed) wall of the flattened tubule and the wall which is adherent to the grid, then the appearance of the substructure must be dependent on the superimposition of the one wall upon the other. When filaments of the "free" wall are directly superimposed on the filaments of the "deep" wall the filamentous substructure would be enhanced. Alternatively, when the filaments of the free wall are staggered to correspond to the spaces between the filaments of the deep wall, a blurred image of the substructure would be encountered. Since areas of both enhanced and of blurred substructure were evident in some preparations this also seems to indicate that both the free wall and the deep wall are penetrated by stain. If, then, the filaments of the free and the deep wall are in register, the observation of 6 filaments in the majority of the tubules seen suggests that the total number of filaments can hardly be less than 12. The occasional observation of 7 filaments in
SUBSTRUCTURE OF MICROTUBULES
163
some tubules may also be explained from a model with a total number of 12. A model with a total number of 11 filaments would allow the observation of 6 filaments (one filament at either edge being "single") but would hardly explain the observation of 7 filaments if the tubule is intact. With a center to center distance of 60 A a total number of 12 filaments would correspond to a diameter of about 240 A in an unflattened tubule. This figure corresponds well with the diameter measured in fixed and embedded specimens. Irrespective of the above discussion, it should be borne in mind that it is not inconceivable that the number of filaments in individual microtubules may vary. Since the diameter of microtubules as reported in the literature varies from 180 to 300/~ this might well be the case (see also 23, 24).
"Core" staining It might be argued that staining of the core was due to "central filling" of a hollow microtubule, and this has been the explanation of the phenomenon given by some authors (see Table II). Glauert et al. (15) in studying negatively stained bacterial flagella observed "central filling" only in flagella disrupted by ultrasonication and argued that the interior of the flagella were filled with stain entering at the site of disruption. Lowy and Hanson (21), however, in their elegant study on flagella from various bacteria, did not observe core staining or central filling. In the present material staining of the core did not seem to be dependent on mechanical disturbance of the microtubules. The phenomenon was seen in short pieces of microtubules, in intact marginal bundles, and in microtubules which were continuous for long distances. Furthermore, the observation that staining of the core in marginal band microtubules seemed to be dependent upon the state of fixation, the negative stain and the p H at which it was used, makes it unlikely that it was due to a mere mechanical filling of a hollow tube. Our results suggest that the wall of the tubule is permeable to the stain under special conditions. Whether a hypothethical core material is positively stained, or the space of the core is passively filled with the stain, cannot be decided from our observations. However, the variation of the staining of the core seems to be dependent on chemical differences of application, and whatever the explanation may turn out to be, the possibility of the presence of a core material seems to be of relevance. It may thus be suggested that the microtubules are two-component structures.
Other filaments observed The filaments of approximately 50 • width which were observed in some preparations probably correspond to the filaments which appear in platelets when they stick
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to a glass surface (7) or aggregate in thrombi (5). Platelets fixed intravascularly, or instantaneously upon withdrawal from a vessel, have a finely granular cytoplasm and filaments are usually not discernible. Their presence in some specimens probably means that unruptured platelets occasionally stick to the grid and subsequently burst. The filaments are not considered to be of microtubular origin because their diameter is bigger than that of the filamentous substructures of microtubules. In addition, platelets which have stuck to a surface or aggregated in thrombi possess both filaments of this type, and microtubules. Finally, evidence given in this paper indicates that when G-microtubules depolymerize they do not split into filaments, but the filaments themselves break into short pieces and granules (Fig. 8). This work was supported by grants from the Danish State Research Foundation and the Carlsberg Foundation.
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