J. Cell Sci. 4, 729-737 (1969) 729 Printed in Great Britain MITOCHONDRIAL EXTENSIONS ASSOCIATED WITH MICROTUBULES IN OUTGROWING PROCESSES FROM CHICK SPINAL CORD IN VITRO FELICITY GRAINGER AND D.W.JAMES Department of Anatomy, University College London, London, W.C. 1, England SUMMARY Electron-microscopic examination of processes growing out from chick spinal cord cultivated in vitro raises the possibility that mitochondria may be continuous with a system of membrane- bound profiles ramifying within the cytoplasm. These profiles are distinctive in appearance, and appear to establish a particular spatial relationship with microtubules. In this, dense material extends from the profiles to constitute a meshwork within whose interstices tubules lie. The suggestion is made that these appearances may reflect the utilization of the products of mitochondrial activity for transport by microtubules. INTRODUCTION When chick embryo spinal cord fragments are explanted in vitro upon a plastic substrate, the cell processes that grow outwards from them are commonly grouped together in bundles (Grainger, James & Tresman, 1968). Such bundles often pursue a nearly straight course for appreciable distances, and are thus well suited to serial ultrathin sectioning in the transverse plane. Since their constituent organelles are largely longitudinally oriented, they often remain well defined in adjacent sections. The purpose of this paper is to suggest the possibility that there may exist within such processes a system of membrane-bound channels originating from mitochondria and achieving a distinctive spatial relationship with microtubules. MATERIALS AND METHODS The techniques of culture and preparation for electron microscopy employed have been fully described elsewhere (Grainger et al. 1968). In brief, cultures of 6 to 8-day chick embryo spinal cord were grown upon a plastic ('Melinex', Imperial Chemical Industries) substrate in Maximov flying coverslip assemblies in hollow- ground slides. They were incubated at 37 °C for periods of up to 7 days, fixed in glutaraldehyde, stained with osmium tetroxide and embedded in Araldite in such a manner as to enable appropriate regions of the outgrowth to be selected for sectioning and electron-microscopic examination. Sections were cut with an LKB ultramicrotome, using a Du Pont diamond 46 Cell Sci. 4
Transcript
J. Cell Sci. 4, 729-737 (1969) 729Printed in Great Britain
MITOCHONDRIAL EXTENSIONS ASSOCIATED
WITH MICROTUBULES IN OUTGROWING
PROCESSES FROM CHICK SPINAL CORD
IN VITRO
FELICITY GRAINGER AND D.W.JAMES
Department of Anatomy, University College London,London, W.C. 1, England
SUMMARY
Electron-microscopic examination of processes growing out from chick spinal cord cultivatedin vitro raises the possibility that mitochondria may be continuous with a system of membrane-bound profiles ramifying within the cytoplasm. These profiles are distinctive in appearance,and appear to establish a particular spatial relationship with microtubules. In this, densematerial extends from the profiles to constitute a meshwork within whose interstices tubuleslie. The suggestion is made that these appearances may reflect the utilization of the productsof mitochondrial activity for transport by microtubules.
INTRODUCTION
When chick embryo spinal cord fragments are explanted in vitro upon a plasticsubstrate, the cell processes that grow outwards from them are commonly groupedtogether in bundles (Grainger, James & Tresman, 1968). Such bundles often pursuea nearly straight course for appreciable distances, and are thus well suited to serialultrathin sectioning in the transverse plane. Since their constituent organelles arelargely longitudinally oriented, they often remain well defined in adjacent sections.The purpose of this paper is to suggest the possibility that there may exist withinsuch processes a system of membrane-bound channels originating from mitochondriaand achieving a distinctive spatial relationship with microtubules.
MATERIALS AND METHODS
The techniques of culture and preparation for electron microscopy employedhave been fully described elsewhere (Grainger et al. 1968). In brief, cultures of6 to 8-day chick embryo spinal cord were grown upon a plastic ('Melinex', ImperialChemical Industries) substrate in Maximov flying coverslip assemblies in hollow-ground slides. They were incubated at 37 °C for periods of up to 7 days, fixed inglutaraldehyde, stained with osmium tetroxide and embedded in Araldite in sucha manner as to enable appropriate regions of the outgrowth to be selected forsectioning and electron-microscopic examination.
Sections were cut with an LKB ultramicrotome, using a Du Pont diamond46 Cell Sci. 4
730 F. Grainger and D. W. James
knife. They were stained on the grid with uranyl acetate and lead citrate (Reynolds,1963), and examined in a Siemens Elmiskop Mark II electron microscope. Bundleswere sectioned in both the transverse and horizontal planes. In the former case, serialor near-serial sections were obtained. Qualification is necessary, since sections weresometimes lost in the knife trough, while others were sometimes imperfectly positionedupon the grids.
RESULTS
Figures 1 and 2 typify the appearances of outgrowing bundles as seen in cross-section. In addition to mitochondria, microtubules, filaments and vesicles, a numberof other types of profile are present whose identification is less certain.
Among the latter is a group whose bounding membranes appear markedly dense,and whose shape and size in transverse section may undergo rapid changes withinshort distances. Thus the small dense profiles arrowed in Figs. 3 and 5 are dilated inFigs. 4 and 6 respectively. Comparable changes are shown in Figs. 7-10, where insuccessive sections a triangular profile diminishes first to an apparently solid triangular(Fig. 9) and then to a flattened form (Fig. 10). Both the dilated and constrictedportions of these profiles often approach a straight-sided configuration, the formereither triangular or rectangular (e.g. Figs. 6 and 8), the latter more commonlytriangular (e.g. Fig. 3). Appearances that correspond to this constriction/dilationpattern are identifiable not only in transverse but also in longitudinal sections (Figs.11 and 12) and appear to constitute a system distinct from the microtubular components.
Profiles of the kind described appear in places to be continuous with mitochondria.Thus in successive sections (Figs. 13-15) a mitochondrial extension gives rise toa small triangular profile some 30 nm in its maximum dimension. Further extensionsfrom mitochondria in cultured material are shown in Figs. 16-18. That they are notunique to chick embryo material in vitro is indicated in Fig. 19, where extensions ofa comparable kind are shown in intact goldfish spinal cord.
The profiles we describe appear in places to be related to a pattern of dense materialsurrounding microtubules (Figs. 20-25). In this, tubules, usually single, are sur-rounded by a relatively translucent zone within compartments whose boundariesare formed by dense material extending outwards from the profiles themselves.Whether this material is continuous with the lumen of the profiles is uncertain. Allthat can be said is that this occasionally appears to be the case (e.g. Fig. 21).
In no instance have we so far found evidence to suggest that profiles of the kindwe describe are continuous with the plasma membrane, although profiles of otherkinds possess such continuity. Thus, Figs. 26 and 27 demonstrate typical pinocytoticappearances, with a markedly radiate arrangement of electron-dense material onthe cytoplasmic aspects of the invaginations. Other invaginations exist (Figs. 28 and29) that appear to give rise to a tubular system whose membranes are not associatedwith material of this kind. In neither case, however, are profiles of the dense membranetype produced.
Relation of mitochondria to microtubules 731
DISCUSSION
The appearances we describe raise the possibility that there may exist within ourmaterial a system of profiles, clearly distinct from microtubules themselves, thatundergo marked changes in shape along their course. In places they appear as solidstructures as little as 15 run across. In other regions they dilate to form cisternae,frequently characterized by relative straight-side outlines, so that they appear eithertriangular or rectangular in transverse section.
Profiles of this kind are not continuous with the plasma membrane—or at anyrate no continuity has so far been discovered—and are different in appearance frommembrane-bounded systems for which continuity has been demonstrated (Figs.26-29). I* appears, however, that they may be continuous withmitochondria(Figs. 13-19). While in some places evaginations of the latter appear to consist of both innerand outer membranes (e.g. Fig. 18) this is not invariably the case (e.g. Fig. 16), andin our view it seems likely that the profiles we describe are derived from the outermitochondria] membrane. If this were not the case, and they were derived from theinner membrane, then outer and inner membranes must at some point fuse, and forthis we have found no evidence.
That a system of channels exists within the cytoplasm continuous with mitochondriais in no way a novel conception (see Robertson, i960). Interest attaches, therefore,not so much to the origin of the profiles we describe as to the relationship they appearto establish with microtubules. In this, dense material is seen to extend from theprofiles to surround translucent zones within which microtubules lie. It is notclear whether this dense material is continuous with the interior of the profilesthemselves, although this may be the case (cf. Fig. 21).
Since our present evidence is of a purely morphological nature, interpretationof the appearances we have described must remain speculative. However, it is amatter of some interest to consider what functions are subserved by mitochondriawithin outgrowing processes in vitro, or for that matter in axons in vivo. No ribosomesare present in these axons, so that the ATP produced by mitochondria is unlikely tobe utilized, as it is in the cell body, for major local protein synthesis. While admittedlythe functions that microtubules subserve are still a matter of debate, it has beenproposed that they may constitute a rapid intracellular transport system (see Weiss,1967). If this is the case, the system of channels we describe may play a part indirecting ATP towards tubules, where it may be utilized to provide the necessaryenergy for transport. In this respect it is of interest that while the electron-densematerial surrounds tubules, it does not come into direct contact with them. Con-ceivably, transport may occur on the perimeter of microtubules as well as withinthem.
We are particularly grateful to our colleague Professor E. G. Gray not only for his permissionto publish Fig. 19 from his own material, but for valuable discussion. We are grateful also toMiss E. Franke and Miss Z. Bienkowska for technical assistance, and to Mr A. Aldrich andMr D. Gunn for photography.
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REFERENCES
GRAINGER, F., JAMES, D. W. & TRESMAN, R. L. (1968). An electron-microscopic study of theearly outgrowth from chick spinal cord in vitro. Z. Zellforsch. viikrosk. Anat. 90, 1-18.
REYNOLDS, E. S. (1963). The use of lead citrate at high pH as an electron-opaque stain inelectron microscopy. J. Cell Biol. 17, 208-212.
ROBERTSON, J. D. (i960). Cell membranes and the origin of mitochondria. In Regional Neuro-chemistry: Regional Chemistry, Physiology and Pliarmacology of tlie Nervous System (ed.S. S. Kety and J. Elkes), pp. 497-530. New York and London: Pergamon.
Figs, i, 2. Transverse sections of a typical bundle growing out from cultured chickspinal cord. Within its constituent processes mitochondria, one of which (m) showsbranching, microtubules (mt), dense-cored vesicles (dv) and clear vesicles (en) canbe identified.Figs. 3-10. Marked changes in shape of profiles (arrowed) distinguished by theelectron-density of their bounding membranes are shown. The small profile arrowedin Fig. 3 is considerably enlarged in the adjacent section shown in Fig. 4. The ovalprofile arrowed in Fig. 5 achieves a dilated quadrilateral form in the adjacent sectionshown in Fig. 6. In Figs. 7-10 a further profile, readily identified by its relationshipwith two mitochondria, is seen to change from a triangular to a linear form.Figs. 11, 12. Longitudinal sections of outgrowing processes. In addition to micro-tubules (mt), additional profiles are arrowed. Their configuration is such as to suggestthat in transverse section appearances of the kind arrowed in Figs. 3-10 would beproduced.
Relation of mitochondria to microtubules 1»
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Figs. 13-15. An extension from a mitochondrion (arrowed) becomes continuous(Fig. 15) with an electron-dense profile of near-triangular form.Figs. 16-18. Figs. 16 and 17 show a mitochondrial extension (arrowed) in adjacentsections. A further example is shown in Fig. 18.Fig. 19. Extension from a mitochondrion (m) in goldfish spinal cord, clearly distin-guishable from microtubular and microfilamentous components. (This electronmicrograph was taken by Professor E. G. Gray from his own material.)
Relation of mitochondria to microtubules
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736 F. Grainger and D. W. James
Figs. 20—25. Profiles, bounded by dense membranes (dmp), are seen to be relatedto electron-dense material extending to enclose microtubules (mt) (Figs. 20-23).At higher magnification (Figs. 24, 25) this material appears in places (arrowed) topossess a bounding membrane. The enclosed tubules have a relatively electron-translucent zone around them.Figs. 26, 27. Typical pinocytotic appearances are arrowed, with radiating strandsof electron-dense material extending from the cytoplasmic aspects of the invaginatedmembrane.Figs. 28, 29. A membrane invagination gives rise to two membrane-bounded profilesin an adjacent section.
