J. Cell Sri. i i , 295-303 (1972) 295Printed in Great Britain

HELICAL FILAMENTS IN THE GLIAL CELLS

OF THE LOCUST (SCHISTOCERCA GREG ARIA)

M. P. OSBORNE

Department of Zoology and Comparative Physiology, University of Birmingham,Birmingham 15, England

SUMMARY

Organelles consisting of a spirally wound filament are present in the cytoplasm of glial cellsin Schistocerca gregaria. The diameter of the helix is 32-5 ran with an average pitch length of23 o ran. The filament is comprised of only a single strand about 6-0 nm in diameter whichappears to be made up of a chain of globular subunits. In order to distinguish between thesehelical structures and organelles made up of more than one strand, i.e. microtubules andmicrofilaments, it is suggested that they be termed 'microhelices'.

The structural features of microtubules, microfilaments and microhelices are compared, andthe possible functions of microhelices are discussed in the light of what is known about thephysiological properties of microtubules.

It is pointed out that the microhelix is structurally very similar to the internal ribonucleo-protein component of the influenza virus, and that the possibility cannot be discounted that themicrohelix might be a viral particle.

INTRODUCTION

Schmitt (1968) has recently reviewed the structure and function of neuronalfibrous proteins. Two structural configurations of fibrous protein are discernible, themicrofilament (neurofilament) and microtubule (neurotubule). Microtubules have adiameter of about 24-0 nm, a wall thickness of 5-0 nm and a central bore of 14-0 nmdiameter. Microfilaments are about 8-10 nm in diameter and may have a central coreabout 3-0 nm across. Both microfilaments and microtubules are made up of strandsformed from polymerized globular protein subunits. There are estimated to be 5 or 6subunit strands in the microfilament and up to 13 subunit strands in the wall of themicrotubule (Porter, 1968). The purpose of this paper is to describe helical filamentsin the cytoplasm of glial cells in the locust which, unlike the microfilament or micro-tubule, appear to be made up of only a single subunit strand.

MATERIAL AND METHODS

Adults of the locust Schistocerca gregaria were used for this study. They were taken from aculture that has been maintained in this Department for several years and came originally fromthe Anti-Locust Research Centre, London (see Harry, 1970). The locusts are kept in cages attemperatures ranging from 25 to 30 °C with a relative humidity of 70%, and are fed daily onfresh grass collected from a site on the University known to be free of insecticides and artificialfertilizers.

Peripheral nerves from this insect were fixed for 1 h in 25 % glutaraldehyde at room tem-perature and posrfixed at the same temperature for 0-5 h in 2 % osmium tetroxide. Bothfixatives were phosphate-buffered at pH 7 4 and made up to an osmotic concentration of 045 M

296 M. P. Osborne

by the addition of sucrose. Dehydration was carried out in ethanol followed by embedding inEpon (Manton, 1964). Sections were double-stained in uranyl acetate and lead citrate. Electronmicrographs used for estimating the dimensions of the helices were taken at an electron opticalmagnification of 60000.

RESULTS

Low-power electron micrographs (Fig. 1) of locust peripheral nerves show that thecytoplasm of the glial cells contains amorphous material. Interspersed amongst theamorphous material in addition to microtubules are filaments organized into a helicalconfiguration. Unlike the microtubules, which are oriented mainly in a directionparallel to the axons, the helices have no preferred direction of orientation but runboth transversely and longitudinally within the plane of a single section (Fig. 1,inset).

Higher magnification electron micrographs clearly demonstrate the helical arrange-ment of the filaments (Fig. 2). The helices do not run in a straight line but pursue awandering course, often curving through angles of at least 900 (Fig. 2). Consequentlyit is difficult to estimate their maximum length, but they are certainly at least 1 /tmlong. At these higher magnifications the amorphous cytoplasmic material bears someresemblance to that of the helical filaments. Thus it is conceivable that the helicesrepresent a more orderly condensation of the amorphous material. In some regionsthe helices or parts of helices (Figs. 1 (inset), 2) appear to be associated with theplasma membrane of the glial cell.

The diameter of the helix lies between 30 and 35 nm with a pitch of about 23-0 nmand an average pitch angle of about 23°, but as can be readily seen (Fig. 2), the pitchangle is very variable, ranging from about io° to 450. In high-resolution pictures(Fig. 2), more clearly definable regions of the helical strand show that it may beformed from globular subunits about 5-0-7-0 nm in diameter, with an electron-lucent core about 3-5 nm across.

DISCUSSION

The helical filaments described in this paper have been reported previously in thecytoplasm of sensory neurons of cephalic airflow receptors (Guthrie, 1966) andaccessory male reproductive glands (Odhiambo, 1969) of Schistocerca gregaria.Thus, as far as I am aware, these helices have been described only in this singlespecies of insect. Similar helical filaments but with smaller dimensions, i.e. a diameterof 14-0 nm and a pitch of 12-0 nm, have been reported in rat astrocytes (Mugnaini,1964), but in the rat they are situated within mitochondria and not in the cytoplasm.Odhiambo (1969) termed these helical filaments 'microtubules'. This term should berestricted to the types of tubule with ' solid' walls made up of tightly packed subunitstrands, although even now at least four types of microtubule have been documented(Behnke & Forer, 1967). The helical filaments described here and by Odhiambo(1969) are clearly open spirals and only by the very loosest use of terms could they becalled tubules. In order to avoid confusion between microtubules with solid wallsand these open helical structures it is proposed to call the latter 'microhelices'.

Helical filaments in glial cells 297

Odhiambo (1969) also implied that the microhelix is similar in structure to micro-tubules that exhibit cross-bandings or striations in longitudinal section (see, forexample, Grimstone & Cleveland, 1965; Satir & Stuart, 1965). It is clear, however,that the microhelix is quite distinct from the 'striated' microtubules, which presum-ably are formed from some molecular modification or addition to the basic micro-tubular wall structure.

Guthrie (1966), in his description of locust cephalic airflow receptor neurons,mentions that in transversely sectioned dendrites the microtubules vary markedly inelectron density. A glance at Fig. 1 (inset) shows that the microtubules have a muchgreater electron density than the microhelices. This is to be expected, since the micro-tubule is formed from about 13 times as many subunit strands as the microhelix andconsequently in transverse section will be much more opaque to electrons. Thus thedifference in microtubule density could be attributed to the presence of both micro-tubules and microhelices within the dendrite, especially since Guthrie mentions thatmicrohelices are present in the cell body.

Tilney & Porter (1967) have described an unusual microtubule in Heliozoa with adiameter similar to that of the microhelix at 34-0 nm. The 34-o-nm microtubuleappears in the cytoplasm only at temperatures of about 4 °C. At higher temperaturesonly microtubules of the more usual form with a diameter of 22-0 nm were present.Tilney & Porter (1967) suggested that the 22-o-nm tubule is transformed into the34-o-nm tubule at low temperatures and vice versa. Because there were fewer 34-0-than 22-o-nm tubules it was thought that some of the 22-o-nm tubules becamedepolymerized, which would account for the amorphous cytoplasmic material thatalso appeared along with the 34-o-nm tubules at low temperatures.

It was postulated that both types of microtubule have 13 subunit strands and thatit is only necessary for the subunit strands to slide 5 nm relative to one another totransform the smaller 22-o-nm tubule into the larger 34-o-nm tubule. The pitch ofthe helix of the 34-o-nm tubule is calculated to be 45°. Thus the spatial organizationof a single subunit strand of this 34-o-nm tubule closely conforms to the arrangementof the single subunit strand of the microhelix. However, the mocrohelix subunitstrand has a diameter of 5-0—7-0 nm, which is slightly larger than the 5-o-nm wallthickness of the microtubule, and possibly then is structurally different. The subunitstrand it most closely resembles in terms of diameter is that of the 6-o-nm strandproposed by Davison (1968) for the squid neurofilament. Clearly the major differencebetween the microhelix and microtubule and microfilament is that the microhelixconsists of only a single subunit strand, and this seems reason enough to suggestthat it could be a third type of configuration of cytoplasmic fibrous protein.

Functions of microtubules have been surveyed recently by Odhiambo (1969) andTilney (1971). The outcome of this is that there are 3 major functions assigned tomicrotubules. The first is that they are simply cytoskeletal elements, since micro-tubules are considered to have high rigidity (Behnke & Zelander, 1967), the second isthat they are involved in intracellar transport, and the third is that they are implicatedin cellular motility.

The microhelix will presumably have little or no mechanical rigidity in view of its

298 M. P. Osborne

meandering course through the cytoplasm and because of the considerable variationin pitch of the helix. Microhelices may, however, confer some elastic restoring forcewhich would return any mechanically deformed cell to its original shape. If themicrohelices are involved in intracellular transport it is difficult to reconcile theirstructure with the saltatory transport model proposed by Schmitt (1968), for micro-tubules, since the very acute pitch of the helix would necessitate a very lengthy trans-port pathway, and in any case the mocrohelices appear to be randomly oriented in thecytoplasm. This latter observation, coupled with that fact that there are no orderlycross-bridges between the microhelices, would also appear to exclude them frombeing involved in cell motility, since all microtubular systems that apparently playsuch a role are interconnected by systems of cross-bridges (Tilney, 1971).

There remains the possibility that the microhelix is not related to microtubules ormicrofilaments. Indeed it may not be a fibrous protein at all. There is a remarkablesimilarity between the microhelix and the internal ribonucleoprotein (RNP) coils ofthe influenza virus (Almeida & Waterson, 1970). These coils are open helical struc-tures, 37-o—67-0 nm in diameter, composed of a single strand 6-0 nm in diameter.The helices have a variable pitch and are apparently flexible, since they are oftenbent on emergence from the viral capsule (Almeida & Waterson, 1970). Thus themicrohelix and the viral RNP coil seem morphologically identical in every wayapart from the overall diameter of the helix, 37-0-67-0 n m f°r t n e RNP coil and30-0-35-0 nm for the microhelix, although the difference in diameter could be due tothe different methods of specimen preparation. Consequently the microhelices in thelocust might be comprised of RNP and, if this is the case, could be viral particles. Inthis context Hawkes (1968) has recently described tightly coiled spiral structuresabout 26 nm in diameter with a pitch of 18-0 nm in the spermatocytes of Schistocercagregaria, and concluded that they might be viral in nature. Hawkes suggested that the'virus' was contracted from the bran or lettuce upon which the locusts were fed. Shealso reported that the locusts were apparently in 'bad health'.

The microhelices described in this paper and by Guthrie (1966) and Odhiambo(1969) are very similar if not identical to the 'viruses' reported by Hawkes. None ofthese reports is in agreement over the dimensions of the helix, but this need notnecessarily preclude them from being identical structures, since there is considerablevariation in the diameter and pitch of RNP coils in the influenza virus (Almeida &Waterson, 1970). If all of these helical structures are viruses, they are not restrictedto the reproductive system, as was thought by Hawkes (1968), because they are alsofound in the nervous system. Hawkes's suggestion that the 'virus' was contractedfrom bran or lettuce and was responsible for the unhealthy state of his locusts is notentirely tenable, since in this study the locusts appeared to be in good health and werefed on grass.

I am most grateful to Professor L. H. Finlayson of the Department of Zoology and Compara-tive Physiology and to Dr N. Crawford of the Biochemistry Department for their criticisms ofthe manuscript. I am also indebted to Professor P. Wildy of the Department of Virology and toDr T. H. Flewett of the East Birmingham hospital for helpful discussions. I wish to thankMrs J. Birch and Mrs L. Tomkins for their skilled technical assistance.

Helical filaments in glial cells 299

REFERENCES

ALMEIDA, J. D. & WATERSON, A. P. (1970). Two morphological aspects of influenza virus. InThe Biology of Large R.N.A. Viruses (ed. R. D. Barry & B. W. J. Mahy), pp. 27-52. Londonand New York: Academic Press.

BEHNKE, O. & FORER, A. (1967). Evidence for four classes of microtubules in individual cells.J. Cell Sd. 2, 169-192.

BEHNKE, O. & ZELANDER, T. (1967). Filamentous substructure of microtubules of the marginalbundle of mammalian blood platelets. J. Ultrastruct. Res. 19, 147-165.

DAVISON, P. F. (1968). Physicochemical properties of neurofilaments. Neurosciences Res. Prog.Bull. 6, 176-179.

GRIMSTONE, A. V. & CLEVELAND, L. R. (1965). The fine structure and function of the con-tractile axostyles of certain flagellates. J. Cell Biol. 24, 387-400.

GUTHRIE, D. M. (1966). The function and fine structure of the cephalic airflow receptor inScltistocerca gregaria. J. Cell Sci. 1, 463-470.

HARRY, O. G. (1970). Gregarines: their effect on the growth of the desert locust (Sckistocercagregaria). Nature, Lond. 225, 964—966.

HAWKES, F. (1968). A virus-like structure in the desert locust. Naturwissenschaften 55, 547.MANTON, I. (1964). The possible significance of some details of flagellar bases in plants. Jl R.

microsc. Soc. 82, 279-285.MUGNAINI, F. (1964). Helical filaments in astrocytic mitochondria of the corpus striatum in the

rat. J. Cell Biol. 23, 173-182.ODHIAMBO, T. R. (1969). The architecture of the accessory reproductive glands of the male

desert locust. Tissue £f Cell 1, 325-340.PORTER, K. R. (1968). General morphology of microtubules. Neurosciences Res. Prog. Bull. 6,

145-148.SATIR, P. & STUART, A. M. (1965). A new apical microtubule-associated organelle in the sternal

gland of Zootermopsis neradensis (Hagen), Isoptera. J. Cell Biol. 24, 277-283.SCHMITT, F. O. (1968). The molecular biology of neuronal fibrous proteins. Neurosciences Res.

Prog. Bull. 6, 119-144.TILNEY, L. G. (1971). Origin and continuity of microtubules. In Origin and Continuity of Cell

Organelles (ed. J. Reinert & H. Urspring), pp. 222-260. Berlin and New York: Springer-Verlag.

TILNEY, L. G. & PORTER, K. R. (1967). Studies on the microtubules in Heliozoa. II . The effectof low temperature on these structures in the formation and maintenance of the axopodia. J.Cell Biol. 34, 327-343.

{Received 19 November 1971)

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Fig. i. Transverse section of a peripheral motor nerve of Sclustocerca gregaria. Theaxons (a) are enveloped by the Schwann cell (sc). Helices (short black arrows) are scat-tered throughout the cytoplasm of the Schwann cell. In some places (long black arrow)ring profiles are present which are thought to be helices cut transversely. These ringprofiles are less electron-dense than microtubules seen in the Schwann cell cytoplasm(clear arrow) and axons (m). The inset shows helices cut in various planes, the trans-verse profiles (short dark arrows) being less electron-dense than microtubules (cleararrow). The long black arrow shows an area where amorphous material comes intoclose association with the plasma membrane of the Schwann cell.

Helical filaments in glial cells

302 M. P. Osborne

Fig. 2. Helices cut in longitudinal section. Note the variation in pitch of the spiral andthe meandering of the helices through the cytoplasm. Amorphous material (am) ispresent throughout the glial cell and it is possible that the helices represent a moreorderly arrangement of this material. In some places fragments of helices appear toadhere to the glial cell plasma membrane (arrows). The inset shows a part of a micro-helix at higher magnification. The helical filament appears to be made of globularsubunits (arrow).

Helical filaments in glial cells

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