Serial reconstruction of microtubular arrays within dendrites of the cat retinal ganglion cell: The cytoskeleton of a vertebrate dendrite

Brain Research, 259 (1983) 193-206

193

Elsevier Biomedical Press

Serial Reconstruction of Microtubular Arrays within Dendrites of the Cat Retinal Ganglion Cell" The Cytoskeleton of a Vertebrate Dendrite SHARON SASAKI, JOHN K. STEVENS and NEIL BODICK Playfair Neuroscience Unit, University of Toronto, Physiology Dept. and Toronto Western Hospital, 399 Bathurst Street, Toronto, Ont. MST 2S8 (Canada) and ( N.B.) Rose F. Kennedy Center For Research in Mental Retardation, Albert Einstein College of Medicine, Bronx, N Y 10461 (U.S.A.)

(Accepted April 1st, 1982) Key words: dendrite - - microtubules - - microtubule-associated proteins - - cytoskeleton

Serial reconstruction at the EM level of cat retinal ganglion cell dendrites reveals that: (1) the microtubular array is discontinuous, (2) microtubular endings are associated with smooth endoplasmic reticulum (SER), mitochondria, and plasma membrane, (3) individual microtubules always maintain a minimum distance from other microtubules (87 nm), SER (43 nm) and plasma membrane (69 nm), and (5) individual microtubules can 'wander' independent of adjacent microtubules throughout the dendritic volume. These observations, taken with some recent biochemical and immunohistochemical data by other workers, suggest that the microtubules are surrounded by a coat of high molecular weight, microtubular-associated proteins (HMW MAPs), which effectively creates a 90 nm tube around a central microtubular core. Our results suggest that bundles of these 'MAP-tubes' may serve as a major component of the dendritic cytoskeleton in the cat ganglion cells. INTRODUCTION It is well documented that dendritic shape plays a direct role in how information is processed within a given neuron7,9, 26. It is also well known that dendritic geometry plays a major developmental role in determining local microcircuitry within the central nervous system of both vertebrates and invertebratesg, 21,24,25, and it may actually represent an important mechanism for the direct genetic control of these neural circuits25, 2~. Central to any characterization of the underlying cellular mechanism responsible for controlling and generating dendritic shape will be a comprehensive account of the dendrites' 'cytoskeleton'. Much experimental and anatomical work has recently appeared on the cytoskeleton of non-neuronal cells (see refs. 18, 19, 23 and 35), and a number of studies have appeared describing the axonal cytoskeleton1,10,1~,16,20,~2,27,~s, a4,s6,a7 and the cytoskeleton of invertebrate neurites 3. However, to date, very little information pertaining to the cytoskeleton of the vertebrate dendrite, even at the most basic descriptive level, is available 0006-8993/83/0000-0000/$03.00 © 1983 Elsevier Biomedical Press

(see refs. 9 and 33). Our purpose here is to describe what might be called the 'three-dimensional microanatomy' of a dendrite from a well-studied vertebrate cell, the cat retinal ganglion cell. Using recently developed computer methods a0,a2 we have reconstructed, from serial electron micrographs, portions of 5 dendrites including the microtubules, smooth endoplasmic reticulum (SER), and mitochondria. These data have given us a new and novel view of how these various intracellular components interact to form a dendritic cytoskeleton and thus contribute to the final dendritic shape. METHODS Cat retinas used in this study were all taken f r o m adult animals, perfused through the heart with a mixture of 1% paraformaldehyde and 1.25 ~o glutaraldehyde (200 ml), followed by 4~o paraformaldehyde and 5% glutaraldehyde (1000 ml). Both solutions were buffered with a 0.1 M sodium cacodylate buffer stabilized to a p H of 7.4. The entire

194 head was refrigerated overnight, and the following day the retina was carefully removed, placed in a 2 % buffered solution of OsO4 for 2 h, dehydrated with methanol and embedded in Epon. Additional details may be found in previous publications3L 32. A 400 ~< 200/zm mesa was cut on the face of the Epon block and serial sections were cut using the methods described by Stevens and co-workersa0, ~2. Since the section ribbons had a pale gold interference color, we have assumed a section thickness of 0. l # m in our calculations of microtubule length and in the computer displays of reconstructed cells. The material was stained with uranyl acetate and lead citrate, and photographed in a JEOL 100B or 120 CX electron microscop~ on 3.25 × 4.0 in. film at a final magnification of 8000 times. Identification of the ganglion cell dendrites within these series was based upon morphological criteria developed from our experience using more complete reconstructions at lower magnifications 31. The negatives were copied onto 35 mm film and analyzed on the computer reconstruction system described by Stevens and co-workersa0, 32. In the early stages of this work, we attempted to use the alignment of the dendritic membrane contour itself as a reference for the microtubular array. However, we found that the distortion due to cutting, photography, etc., was so great, that even within the 1 ~um diameter of the dendrite, the individual microtubules could not be simply connected from one section to the next. Thus, to establish continuity from section to section, we had to 'microalign' (see refs. 30 and 32) each microtubule through the entire series. The quantitative measures of distances between microtubules and other organelles described below were carried out on three separate series, all from different cats. The two major reconstructions illustrated in this paper came from a single series, 58 sections in length. The results of these reconstructions have been confirmed in 3 other ganglion cell dendrite reconstructions, two of which came from an additional series of 64 sections. Finally, the major results from these reconstructions have also been confirmed in other reconstructions of three AII amacrine dendrites, as well as in dendrites of mouse cortical cells (N. Bodick et al., in press, Brain Research).

RESULTS The anatomy, physiology, and microcircuitry of the cat retinal ganglion cell has been studied in some detail (see ref. 31) and it is particularly well suited as a model of the vertebrate neuron. The axons project centrally and are known to produce action potentials and the dendrites are thought to be passive and totally post-synaptic to bipolar and amacrine synaptic inputs. Moreover, the cat ganglion cell has an archetype neuronal dendrite in that it is a non-varicose, tapered structure containing SER, mitochondria and a tightly packed array of microtubules, while neurofilaments and synaptic vesicles are totally absent al. We have reconstructed a total of 238 individual microtubules, 46 SER, and 12 mitochondria within 5 retinal dendritic segments. Two examples of these reconstructions are illustrated in Figs. 1, 2, 4, 5 and 6. Figs. 1, 2 and 6 illustrate a 12.0 by 1.0 #m, longitudinally-sectioned dendritic segment, while Figs. 4 and 5 represent a 2.3 x 1.5 #m, transversely-cut dendritic segment. The other 3 reconstructions not shown here were all transversely-cut dendrites. These 5 reconstructions have revealed a number of interesting relationships between the microtubules themselves, and between the microtubules, plasma membrane, SER and mitochondria. Details of our findings are presented below. The dendritic microtubular array is not continuous

In all of our reconstructed material, microtubules start and stop within tile length of the series. We must emphasize that we were unable to establish with any confidence the location of the soma for any of our 5 reconstructed dendritic segments and thus the words 'start' and 'stop' are used only to describe the endings of microtubules with respect to the beginning or end of the series. Table I summarizes these stops and starts within the reconstruction illustrated in Figs. 1, 2 and 6. Out of 125 microtubules reconstructed there were only 2 microtubtdes that started on the first section and stopped on the last section. The remaining 123 microtubules were all discontinuous. In Table I, the numbers listed in brackets show the ranges of microtubule lengths (in btm) for the dendrite. One could argue that these endings represent an

195 TABLE I

Classification of microtubule ( M T ) endings in a reconstructed ganglion dendrite Total number of microtubules reconstructed = 125. Numbers outside brackets refer to numbers of microtubules. Numbers in brackets give the range of lengths of the microtubules in each category in microns.

M T starts (towards

M T stops (towards end of the series)

start of series)

Plasma membrane

SER

Mitochondria

Art. stops*

Cytoplasm

Last section

First Section Plasma membrane SER

39 (0.14-11.10) 4 ( 1.30-7.88) 14 (0.61-4.88)

24 (0.47-9.99) 0

10 (0.65-8.41) 1 (3.81 ) 0

4 (2.18-2.76) 1 (2.72) 0

0

2 (7.13-11.35) 0

0

0

0

0

1

0

0

0

0

(1.78) 1 (3.80)

0

0

0

0

Mitochondria Art. starts* Cytoplasm

5 (2.81-5.63)

1

1

(3.85) 0

(10.76)

0

1 (4.19) 3 (4.19-5.05)

13 (1.(~)-10.92)

* Art. = Artifactual. Some microtubules could not be followed due to problems with the material (i.e. details were obscured by folds or dirt) making reconstruction impossible.

l

Fig. I. External surface view of a reconstructed ganglion cell dendrite. Microtubule starts (bottom, triangles) and stops (top, stars) are shown to demonstrate that little, if any, correlation exists between the two. Note: that starts and stops are with respect to the beginning and end of the series. Bar = 1/~rn.

196 artifactual break, inflicted upon the cell either by our buffers or fixatives3,S,~.~, 35. However, one would then expect to see some correspondence between each start and stop. That is, if a continuous microtubule array were simply broken or depolymerized at points along its length by either the buffer or the fixative, one might expect to see starts o f new microtubules close to the stops o f preceding, broken microtubules. We therefore have drawn in Fig. 1 all o f the starts and stops, excluding artifactual endings, and endings found on the first and last sections (see Table I), to see if any correlation might exist. Starts are represented by triangles on the lower display, and stops are symbolized by stars in the upper dis-

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play. There appears to be little correspondence between the stars and triangles o f Fig. I, suggesting that our discontinuous microtubular array cannot be totally discounted as a fixation artifact. Additional observations supporting this conclusion will be presented in the Discussion.

Microtubule endings are associated with ,specific intracellular organelles We consistently f o u n d microtubule endings to be associated with SER, mitochondria, and the plasma membrane. For example, there are a total of 125 reconstructed microtubules shown in Figs. 2 and 6. O f these, 58 had endings near plasma membrane, 32

-

Fig. 2. Same dendrite as seen in Fig. I. Bottom shows microtubule endings on plasma membrane (black stars), middle is a magnified view of the same dendrite tilted 90° (edge view) with microtubules superimposed as black lines. The top photos are the actual micrographs corresponding to the sections immediately below arrows (sections 7 and 8 from bottom). This figure illustrates microtubules ending on a piece of 'terminating' membrane, corresponding to a large change in dendritic volume. Bar : 1 ,m.

197

(Colour Figures 4-6 overleaf)

198

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199

Fig. 3. Three serial micrographs of the reconstructed dendrite shown in Figs. 4 and 5. The small black arrowheads point to a microtubule on the left hand micrograph which ends on the smooth endoplasmic reticulum seen in middle and far right micrographs. The left micrograph also demonstrates that ending microtubules appear to have a dense core as described by Chalfie and Thomson3 in nematode. Note this microtubule was followed for the next 9 sections without major changes in diameter. Bar = 1/~m.

ended near small SER vesicles within the cytoplasm, 11 ended near mitochondria, and only 4 ended free within the cytoplasm (Table I). Thus, microtubule endings seem to have specific associations within the cell. It should be emphasized that these endings are in very close proximity to these intracellular components, but never actually come in direct contact with either organelles or other microtubules. In m a n y cases the microtubule endings near plasma membrane were clearly associated with dramatic changes in the overall geometry of the cell. This is illustrated in Fig. 2 where we show the same dendritic reconstruction (rotated 180 °) as seen in Figs. 1 and 6. The lower plot shows the microtubule endings associated with the plasma membrane, as black stars. The same dendrite is illustrated above in

the middle plot as a magnified and rotated (90 °) side view with the microtubular array superimposed as black lines. The lowest horizontal line of the middle figure represents an edge view of the first section of the series. Eight microtubules stop at the eighth section and correspond to the tight cluster of stars in the lower plot. The electron micrograph of the eighth section (right arrow) is shown in the upper right corner of Fig. 2; the left micrograph represents the preceding or seventh section (left arrow). It is clear that the eight microtubules seen in section 7 must end on section 8 simply because the plasma membrane has been sheared away. With the loss of these microtubules, there is a dramatic decrease in the dendrite's overall volume and a marked change in shape.

Fig. 4. (Top) Stereo pair of transversely-cut dendrite. Blue contours represent external plasma membrane; red contours represent internal plasma membrane; green contours represent smooth endoplasmic reticulum. Black lines are microtubules. Bar = 1/~m, for Figs. 4-6. Fig. 5. (Middle) Same reconstruction illustrated in Fig. 4 but plasma membrane has been removed. Fig. 6. (Bottom) Longitudinal cut series of reconstructed ganglion cell dendrite. Top plot is outer limiting membrane of the dendrite (red) with microtubular array superimposed (black). Middle plot is same view of outer limiting membrane with mitochondria (blue) and SER (green) inside. Bottom plot shows all the microtubules that have one node associated with SER (green triangles) and the other node associated with plasma membrane (black stars). Note the marked polarity of these microtubules.

200 In other cases the endings were associated with the appearance of either a mitochondrion or SER. An example of this is illustrated in the series of micrographs shown in Fig. 3. These micrographs are taken from the series used to reconstruct the dendrite in Figs. 4 and 5. The small arrow in the left micrograph points to a microtubule; the arrows in the middle and right micrographs point to the same location where a SER membrane now appears. In contrast to this example, most microtubules seem to simply pass around intracellular organelles rather than end near them. Again this is illustrated in Fig. 3, where the 6 microtubules surrounding the ending microtubute are 'deflected' around the SER. This is also illustrated in the reconstructions shown in Figs. 5 and 6, and will be discussed in more detail below. Certainrelationships seem to occur consistently between these organelles and microtubular endings while others seem to be forbidden. For example, all of the microtubules that have one ending associated with SER and the other ending associated with the plasma membrane (total count of 14, see Table 1), are oriented in the same direction. We illustrate this polarization in the lowest plot of Fig. 6. The green triangles represent microtubule endings near SER; the blue stars represent endings near plasma membrane. Again, it is clear that all these microtubules 'point' in the same direction. It is also clear from Table I, however, that many microtubules which start on SER also end on SER, and quite a few microtubules (count of 24) with unknown origins also seem to end on SER. Few microtubules (2) start on mitochondria while only 4 start on plasma membrane. Thus, SER appears to be the only site where starts and stops are equally likely, while mitochondria and plasma membrane seem to be locations largely reserved for stops. This conclusion was also corroborated in the other reconstructions. Although we cannot state with certainty which side of the dendrite is proximal to the soma, the dendritic taper suggests that the SER starts at the bottom of Fig. 6 are proximal to the soma and plasma membrane stops are distal to the soma. Microtubules maintain a minimum distance between each other and organelles Microtubules always maintain a minimum dis-

tance from plasma membrane, intracellular organelles, and each other. In all of the retinal material examined to date, we have yet to find a single example of a microtubule coming into direct contact with another microtubule, plasma membrane, or any intracellular organelle in normal adult material. The microtubules always appear to maintain a minimum separation. We have quantified this minimum separation in a histogram of the measured distance from the center of a given microtttbule to the center of the nearest adjacent microtubule or to the membrane of other intracellular organelles. These histograms are similar to methods developed by a number of other workers12,19,28,a7. The minimum distance between the centers of adjacent microtubules was determined from transversely-cut cross-sections apd plotted as a first-order tube-tube histogram. We illustrate the results for the series seen in Figs. 4 and 5 in Fig. 8. The tube-tube histogram as shown at the top of Fig. 8 has a narrow

Section ÷ 2 3

Section ÷1

Fig. 7. Outlines of first and twenty-third sections from reconstruction shown in Figs. 4 and 5. The stars represent selected microtubules followed from section 1 to section 23. The cross-hatched areas are SER. This figure illustrates the independent nature of individual microtubules. Bar -: I ,urn.

201 Tube - Tube

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Fig. 8. First-order h i s t o g r a m s o f center to center distances between microtubules (top graph, m e a n 0.094 ~um, m o d e 0.087 /~m, a n d S.D. 0.022 # m , n = 961); m i c r o t u b u l e to p l a s m a m e m b r a n e (middle graph, m e a n 0.083 # m , m o d e 0.069/~m, a n d S.D. 0.032/~m, n = 374), a n d m i c r o t u b u l e to S E R ( B o t t o m graph, m e a n 0.067 # m , m o d e 0.043 p m , a n d S.D. 0.028 /~m, n = 502). T h e ordinate is total c o u n t a n d abscissa is the distance in microns.

peak with a mean distance between microtubules of 93 nm and a mode of 87 nm. The middle histogram of Fig. 8 represents the minimum distance from plasma membrane to the center of adjacent microtubules (tube-membrane distance). The mean was 83 nm while the mode was 69 nm. Finally, the lower histogram is a plot of the distance from microtubule centers to SER membrane (tube-SER distance). The mean was 67 nm, and the mode was 43 nm, about 49 ~ of the tubetube distance. We also found the same range between our mitochondria and microtubules. Thus, the tube-membrane distance is about 7 9 ~ that of the first-order tube-tube distance and the tube-SER distance is about half that of the first-order tube-tube distance. A similar relationship was found between tubetube, tube-membrane, and tube-SER distances in AII amacrine dendrites and the other ganglion cells; however, the absolute distances did differ from those presented above• In some cases these differences represent errors in calibration of the magnification, but in other cases we believe they may represent legitimate differences. A detailed presentation of comparisons is now being prepared. We stated above that microtubules often end at mitochondria or SER; however, the vast majority actually go around the intracellular organelles while maintaining this minimum separation distance. The net effect is that the volume of the dendrite actually increases due to the addition of such organelles. This effect can be seen in Figs. 4, 5 and 6. Mierotubules can wander independently within the dendrite In single cross-sections, the microtubules appear to be arranged in a rigid parallel array. However, when reconstructed, it becomes clear that the array is not parallel and individual microtubules appear to wander within the volume of the dendrite. This wandering creates a very characteristic weaving pattern apparent in all of our reconstructions (Figs. 2, 4-6). Fig. 7 is a more direct illustration of this effect. In the lower portion of this figure, we have drawn the dendritic outline of section one from the reconstruction shown in Figs. 4 and 5. We selected a subgroup of 9 central microtubttles and 6 peripheral microtubules from the 112 microtubules

202 contained oll section one, and labeled them with stars. Immediately above we show the same 15 microtubules as seen on section 23. The 3 outer microtubular pairs are widely dispersed and the inner 9 microtubules are also beginning to separate. Thus, nearest neighbor relationships between individual microtubules are not maintained and individual microtubules appear to be able to move independently through the cytoplasm, yet as described above the minimum separation distance between microtubule pairs is always maintained. This observation has been confirmed in the other ganglion cell dendritic reconstructions, as well as in reconstructions of AII amacrine dendrites and mouse cortical dendrites (N. Bodick et al., in press, Brain Research). DISCUSSION At the outset we must emphasize that our observations on vertebrate dendrites cannot be directly extrapolated to other microtubttlar systems found in fibroblasts, mitotically active cells, neuroblastoma neurites or even to neuronal axons. As we outlined in the introduction very little is known about the basic descriptive anatomy of the dendritic cytoskeleton, and even less has been reported on the dendrite's cellular physiology. Such basic questions as: 'Are mitochondria and SER transported along the length of a mature dendrite?', or 'Is there a dendritic equivalent to axonal slow and fast transport in mature neurons?', have yet to be clearly answered 33. Recent work by Matus et al. 17 further underlines the need for caution. These workers have demonstrated via immunohistochemistry that certain high. molecular weight microtubular-associated proteins, or H M W MAPs, are found in very specific groups of neuronal dendrites to the exclusion of glia, neuronal axons, and processes of other cells. Others4,11 have also demonstrated that neuronal H M W MAPs do not appear to be present in nonneuronal systems. Thus, although we do make comparisons to other well studied microtubular systems, the discussion that follows should be taken quite narrowly as it applies to the cytoskeleton of a passive, vertebrate, neuronal dendrite.

Other microtubular systems Chalfie and Thomson have shown that micro-

tubules are discontinuous in invertebrate neuritesa; Bray and Bunge ~, Nadelhaft 22 and Zenker and Hohberg 37 have demonstrated that the microtubules within axons are discontinuous, and many reports have appeared suggesting that microtubules are discontinuous in non-neuronal cellslS, 2a. Although, Weiss and Mayer suggest in an early study 34 that axonal microtubules were continuous, they based this conclusion on total counts before and after branch points. Our own work and that of others demonstrates that constancy of microtubule counts does not guarantee an unbroken array z. We also find that the microtubules within the cat ganglion cell dendrite are discontinuous. We do not think these discontinuities can easily be attributed to artifacts associated either with fixation or the buffers for a number of reasons. First, although other investigators have provided us with evidence that certain buffers may break microtubules (see refs. 3, 8, 15 and 35) it seems unlikely th.at this artifact would create a microtubular array with totally unrelated starts and stops, similar to those seen in Fig. 1. Second, we would not expect to see such a large number of microtubule endings associated with organelles and plasma membrane (Table 1) as compared to those ending free in the cytoplasm. Finally, it would be unlikely that a fixation artifact could produce the polarity seen between SER starts and membrane stops as illustrated at the bottom of Fig. 6. In general, our reconstructions and analysis reveal an ordered, repeatable organization that is not consistent with changes expected from either fixation or buffers, but quite consistent with observed changes in the internal and external geometry and volume of the dendrite as well as with what might be expected from work on other systems. Thus, as has been reported in these other systems, the vertebrate dendrite also appears to have a discontinuous microtubule array. Chalfie and Thomson 3 also report that the microtubules in their nematode neurites have endings which appear to be polarized. They find that microtubules ending in association with plasma membrane are distal to the soma and are associated with a diffuse patch of stained material while proximal endings have darkly stained cores. Others (see ref. 3) have reported similar polarized endings in non-neuronal cells. We suspect that these same diffuse and

203 dense endings exist in our own material and they can be seen in Fig. 2 (diffuse on membrane) and Fig. 3 (dense core on SER); however, additional data at a higher magnification must be analyzed to be certain whether these relationships are entirely consistent. Bray and Bunge 1 report similar endings in their axons, but state that what appear to be microtubules with dense core terminations may actually be continuous SER. We do not find continuous SER in our dendritic material but we find that, on occasion, in transversely-cut dendritic sections, the SER does end in a densely stained spot resembling a microtubule. It might, therefore, be possible to make such errors in single cross-sections. Although recent work demonstrates that by using specialized chemical conditions it is possible to form long, 1/~m diameter tubes from phospholipids 14, it is unlikely that 24 nm phospholipid tubes similar to microtubules could be formed using in vivo conditions (H. M. McConnel, personal communication). Moreover, all of the starts and stops listed in Table I came from the longitudinally cut series where continuity between presumed microtubules and SER would be obvious. We find an additional suggestion of polarization in that all of the complete SER to membrane microtubules in the series shown in Fig. 6, point in the same direction. We also find that microtubules preferentially 'stop' on plasma membrane or mitochondria, but may 'start' or 'stop' on SER. Unfortunately we do not know the location of the soma with respect to these starts and stops. Without additional reconstructions as well as experimental studies specifically aimed at the mature, vertebrate dendrite, any comments pertinent to these observations would be highly speculative. ' H M W MAPs and microtubules as the dendritic cytoskeleton' The two observations that were most intr;gulng to us also seemed a bit paradoxical. We found that dendritic microtubules always maintain a minimum distance between each other and intracellular organelles, consistent with a lattice or cross-bridge cytoskeleton similar to that suggested by Wolosewick and Porter 2a,as for the fibroblast system. Yet, in serial sections, the microtubules seem to move within the dendrite as if they were independent, long, flexible tubes. Although one can contrive a number

of complex schemes to explain such behavior, these data, taken together with some recent biochemical and immunohistochemical findings, suggest a simple model for the ganglion cell dendrite that is surprisingly consistent with both a 'lattice' and a 'tube' cytoskeleton. It is well documented that a number of different high molecular weight proteins ( > 200,000 daltons) are associated with microtubules in general 2,4,5,6,11, 12,1~,~7, and a number of studies indicate tb_at a specific subgroup of these proteins may be uniquely associated with neurons 2,5,11,17. One recent study of Matus et al. 17 has demonstrated that some brain high molecular weight microtubular-associated proteins (HMW MAPs) may be specifically associated only with microtubules in dendrites of certain cells. These H M W MAPs are known to promote microtubule initiation and elongation in vitro, however, their function in vivo is really not knownZ,lL Kim et al. 12 have demonstrated that one of these H M W MAPs in brain, MAP2s, are attached directly to the surface of the microtubules with a periodicity of about 32 nm and extend laterally about 33 nm beyond the surface. The total distance from a center of a microtubule (24 nm in diameter) to the end of its MAPs would, therefore, be about 45 nm. They also demonstrate that when MAPs are removed the microtubules no longer maintain a minimum distance between each other, and can actually come into direct contact. Moreover, immunolabeling by Connolly and Kalnins 4,5 and Izant and Mclntosh 11 have demonstrated that H M W MAPs in neurons form a protein coat around individual microtubules and follow the detailed spatial course of the microtubule array. Thus, all of these data, largely on nonaldehyde-fixed material, suggest that in effect the H M W MAPs form a protein coat or 'cylinder' around the microtubule with a radius of about 45 rim.

H M W MAPs are not easily detected at the EM level in conventional, aldehyde-fixed, Eponembedded materialS,12,a5. If H M W MAPs do surround each microtubule as an electron transparent coat, we nevertheless would expect to see an average distance between nearest neighbor microtubule centers equal to twice the 45 nm radius reported by Kim et al. 1~, or about 90 rim. Furthermore, we would expect a distance approximately equal to half this

204

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Fig. 9. Artist's drawing of the MAP-microtubule system. Each dark center represents a microtubule as seen in an electron micrograph. The cylinders surrounding the microtubules represent the effective radius of a protein coat which is not seen in conventional aldehyde-fixed material. It is thought that this protein coat consists of HMW MAPs with a periodicity of about 30 nm that extends 30-35 nm beyond the microtubule surface 1~, as illustrated in the top inset. We propose that the MAP to microtubule bond is very strong and the MAP-MAP bond between adjacent microtubules is weak. Thus, this 'MAP-microtubule unit' behaves as if it were a large cylinder or tube as illustrated in the figure. These weak lateral bonds can create at certain points, a lattice-like structure which is capable of opposing the osmotic and surface tension forces of the outer limiting membrane. Organelles may also be embedded between these MAP-microtubules to create additional volume. The black inner coat on the plasma membrane represents an additional protein coat. value for m i c r o t u b u l e s adjacent to n o n - p r o t e i n -

predictions as illustrated in Fig. 8. The first-order

c o a t e d intracellular organeUes since only one cylinder radius w o u l d be involved. Finally, we w o u l d

distance (mode) between m i c r o t u b u l e centers was 87

expect to find an i n t e r m e d i a t e distance for microtubules adjacent to other p r o t e i n - c o a t e d c o m p o -

nm, the m o d e between m i c r o t u b u l e centers and S E R was 43 nm, and the m o d e between m i c r o t u b u l e

nents (e.g. p l a s m a m e m b r a n e ) . W i t h i n experimental

centers a n d p l a s m a m e m b r a n e was 69 nm. M o r e over, if the m i c r o t u b u l a r array were viewed as a

error, our own data are totally consistent with tb.ese

bundle o f 90 n m d i a m e t e r cylinders, each with a

205 24 nm central core (the visible microtubule), we can then easily explain both the observed constant distance between microtubules and the wandering behavior that so characterized all of our reconstructed dendrites (Figs. 6 and 7). We have illustrated this scheme in Fig. 9. At the heart of this theory is the assumption that the MAPs are attracted to the microtubule with very strong forces, while the distal ends of the MAPs are attracted to each other by relatively weak forces. This weak attraction between distal ends would prevent the MAPs on one microtubule from falling between MAPs on an adjacent microtubule, yet would allow adjacent microtubules the freedom to wander within the cytoplasm and allow SER, mitochondria, etc., to push their way through the cytoplasm between the microtubular array. At a given moment (or at a point in space), adjacent microtubules might appear to be interconnected by a cross-linked lattice, while at other locations the MAPs might be totally unattached. This connection between M A P distal ends also explains why crossbridges can occasionally be seen between microtubules in aldehyde-fixed materiaP2,zT,2s suggesting a lattice cytoskeleton but, at the same time, M A P immtmofluorescent studies reveal2,4,5,11 a tight coat

of protein surrounding each microtubule. Thus, these data suggest that the microtubules in our dendrites may not be suspended within a lattice substructure, but rather each microtubule might act as a support surface for H M W MAPs which in turn produce what is effectively a much larger cylinder or tube surrounding a microtubule core. These M A P microtubule tubes when packed closely together would be self-supporting and could actually create or define the internal volume of the dendrite. Other organelles could, of course, also contribute to the final shape of the dendrite by simply pushing these MAP-microtubules apart as we have seen in our reconstructions. Furthermore, if one takes into account that H M W MAPs appear to come in a wide variety of sizes and configurations~, 17,19, it becomes easy to imagine how a cell could control final dendritic cross-sectional geometry simply by producing the appropriate MAP.

REFERENCES

tion revealed in platinum replicas of freeze-dried cytoskeletons, J. Cell Biol., 86 (1980) 212-234. 9 Hillman, D. E., Neuronal shape parameters and substructures as a basis of neuronal form. In F. O. Schmitt and F. G. Worden (Eds.), The Neurosciences: Fourth Study Program, The MIT Press, Cambridge, MA, 1979, pp. 477--498. 10 Hodge, A. J. and Adelman, Jr., W. J., The neuroplasmic network in Loligo and Hermissenda neurons, J. Ultrastruct. Res., 70 (1980) 220-241. 11 Izant, J. G. and McIntosh, J. R., Microtubule-associated proteins, A monoclonal antibody to MAP2 binds to differentiated neurons, Proc. nat. Acad. Sci. U.S.A., 77 (1980) 4741-4745. 12 Klm, " H. L., Binder, ' I. and Rosenbaum, J. L., The periodic association of MAP2 with brain microtubules in vitro, J. Cell Biol., 80 (1979) 266-276. 13 Lasek, R. J., The dynamic ordering of neuronal cytoskeletons, Neurosci. Res. Prog. Bull., 19 (1981) 7-31. 14 Lin, K. E., Weis, R. E, and McConnell, H. M., Induction of helical liposomes by Ca2+-mediated intermembrane binding, Nature (Lond.), 296 (1982) 164-165. 15 Luftig, R. B., McMillan, P. N., Weatherbee, J. A. and Weibing, R. R., Increased visualization of microtubules by an improved fixation procedure, J. Histochem. Cytochem., 25 (1977) 175-187. 16 Matsumoto, G. and Sakai, H., Microtubules inside the

1 Bray, D. and Bunge, M. B., Serial analysis of microtubules in cultured rat sensory axons, J. Neurocytol., 10 (1981) 589-605. 2 Brinkley, B. R., Cox, S. and Fistel, S., Organizing centers of cell processes Neurosci. Res. Prog. Bull., 19 (1981) 108-124. 3 Chalfie, M. and Thomson, J., Organization of neuronal microtubules in the nematode Caenorhabditis elegans, J. Cell Biol., 82 (1979) 278-289. 4 Connolly, J. A. and Kalnins, V. I., The distribution of tau and HMW microtubule-associated proteins in different cell types, Exp. Cell Res., 27 (1980) 341-350. 5 Connolly, J. A. and Kalnins, V. I., Tau and HME microtubule-associated proteins have different microtubule binding sites in vivo, Europ. J. Cell Res., 21 (1980) 296300. 6 Dentler, W. L., Granett, S. and Rosenbaum, J. L., Ultrastructural localization of the high molecular weight proteins associated with in vitro assembled brain microtubules, J. Cell Biol., 65 (1975) 237-241. 7 Ellias, S. and Stevens, J. K., The dendritic varicosity: a mechanism for electrically isolating the dendrites of cat retinal amacrine cells? Brain Research, 196 (1980) 365-372. 8 Heuser, J. E. and Kirsclmer, M. W., Filament organiza-

ACKNOWLEDGEMENTS We thank C. Yeung, J. Connolly, R. Lasek, R. Jacobs and J. Trogadis for help and advice. This work was supported by Medical Research Council Grant MA-7345 to J. Stevens.

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18

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22

23

24

25

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plasma membrane of squid giant axons and their possible physiological function, J. Membrane Biol., 50 (1979) 1-14. Matus, A., Bernhardt, R. and Hugh-Jones, T., High molecular weight microtubule-associated proteins are preferentially associated with dendritic microtubules in brain, Proc. nat. Acad. Sci. U.S.A., 78 (1981) 3010-3014. Mclntosh, J. R., McDonald, K. L., Edwards, M. K. and Ross, B. M., Three-dimensional structure of the central mitotic spindle of Diatoma vulgare, J. Cell BioL, 83 (1979) 428442. Mclntosh, J. R., Bridges between microtubules, J. Cell Biol., 61 (1974) 166-187. Metuzals, J. and Tasaki, I., Subaxolemmal filamentous network in the giant nerve fiber of the squid (Loligo pealei L.) and its possible role in excitability, J. Cell Biol., 78 (1978) 597-621. Muller, K. J. and McMahan, U. J., The shapes of sensory neuron and motor neurons and the distribution of their synapses in the ganglia of the leech: a study using intracellular injection of horseradish peroxidase, Proc. roy. Soc. B, 194 (1976) 481~499. Nadelhaft, I., Microtubule densities and total numbers in selected axons of the crayfish abdominal nerve cord, J. NeurocytoL, 3 (1974) 73-86. Porter, K. R., Byers, H. R. and Ellisman, M. H., The cytoskeleton. In F. O. Schmitt and F. G. Worden (Eds.), The Neurosciences: Fourth Study Program, The MIT Press, Cambridge, MA, 1979, pp. 703-722. Purves, D. and Hume, R. L., The relation of postsynaptic geometry to the number of presynaptic axons that innervate autonomic ganglion cells, J. Neurosci., 1 (1981) 441-452. Rakic, P., Genetic and epigenetic determinants of local neuronal circuits in the mammalian central nervous system. In F. O. Schmitt and F. G. Worden (Eds.), The Neurosciences: Fourth Study Program, The MIT Press, Cambridge, MA, 1979, pp. 109-128. Rail, W., Branching dendritic trees and motoneuron mem-

brane resistivity, Exp. Neurol., 1 (1959) 491-527. 27 Rice, R. V., Roslansky, P. F., Pascoe, N. and Houghton, S. M., Bridges between microtubules and neurofilaments visualized by stereoelectron microscopy, J. Ultrastruct. Res., 71 (1980) 303-310. 28 Smith, D. S., On the significance of cross-bridges between microtubules and synaptic vesicles, PhiL Trans. B, 261 (1971) 395-405. 29 Solomon, F. and Zurn, A., The cytoskeleton and specification of neuronal morphology, Neurosci. Res. Prog. Bull., 19 (1981) 100-107. 30 Stevens, J. K., Reconstructing neuronal microcircuitry: using computers to assemble three dimensional structures from montages of serial electron micrographs, Bull. Microscop. Soc. Canada, 8 (1980) 4-12. 31 Stevens, J. K., McGuire, B. A. and Sterling, P., Toward a functional architecture of the retina: serial reconstruction of adjacent ganglion cells, Science, 207 (1980) 317-319. 32 Stevens, J. K., Davis, T. L., Friedman, N. and Sterling, P., A systematic approach to reconstructing microcircuitry by electron microscopy of serial sections, Brain Res. Rev., 2 (1980) 265-293. 33 Thoenen, H. and Kreutzberg, G. W., The role of fast transport in the nervous system, Neurosci. Res. Prog. Bull., 20 (1981) 45-55. 34 Weiss, P. A. and Mayr, R., Neuronal organelles in neuroplasmic (axonal) flow II Neurotubules, Acta neuropath., 5, Suppl. (1971) 198-206. 35 Wolosewick, J. J. and Porter, K. R., Microtrabecular lattice of the cytoplasmic ground substance. Artifact or reality? J. CellBioL, 82 (1979) 114-139. 36 Yamada, K., Spooner, B. and Wessells, N., Ultrastructure and function of growth cones and axons of cultured nerve cells, J. Cell Biol., 49 (1971) 614-635. 37 Zenker, W. and Hohberg, E., A alpha-nerve fiber number of neurotubules in the stem fiber and in the terminal branches, J. Neurocytol., 2 (1973) 143-148.