0270.6474/0503-0705!EO2.00/0 The Journal of Neuroscience Copyright Q Society for Neuroscience Vol. 5. No. 3, pp. 705-714 Printed in U.S.A. March 1985 A Regional Analysis of wspectrin in the Isolated Mauthner Neuron and in Isolated Axons of the Goldfish and Rabbit’ EDWARD KOENIG* AND ELIZABETH REPASKY* State University of New York, Department of Physiology, Division of Neurobiology, Buffalo, New York 14214 and *Department of Molecular Immunology, Roswell Park Memorial Institute, Buffalo, New York 14263 Abstract In brain tissue, however, spectrin-like protein was described and isolated as a result of independent lines of investigation. In one case, Isolated dendrites, somata, and desheathed axons of the an analogous protein was first noted as a high molecular weight goldfish Mauthner neuron (M-cell), in addition to other iso- doublet undergoing axoplasmic transport (Lorenz and Willard, 1978) lated myelin sheath-free axons of the goldfish spinal cord and, when later isolated and characterized, it was called fodrin and of rabbit lumbar ventral roots, were shown by immuno- (Levine and Willard, 1981). In the second case, an investigation of chemical and immunofluorescence techniques to contain CX- calmodulin-binding activity in brain also led to the isolation of the spectrin (fodrin). a-Spectrin appeared to be organized as a homologous protein, named calspectin (Kakiuchi et al., 1982). Cur- randomly distributed reticular network, localized to the sur- rent evidence indicates that fodrin (calspectin) is related to erythro- face of isolated neuronal cellular structures. In addition, (Y- cyte spectrin in structure, in cellular localization and, in F-actin- and spectrin was also distributed nonrandomly at specialized calmodulin-binding properties, as well as in immunological cross- cellular sites. These sites included synaptic junctions and reactivity. This has been used as a compelling argument for the view morphologically differentiated nodes of Ranvier (i.e., rabbit that spectrin-like polypeptides constitute a family of proteins with axons, but not goldfish axons). At the latter sites, it is pos- similar functions, a circumstance analogous to that of myosin, and sible to demonstrate that a-spectrin is co-localized with F- that, therefore, spectrin should be retained as a generic name. actin, as indicated by a striking correspondence of fluores- Differences, nonetheless, between erythrocyte and brain spectrins cent images due to double labeling, using the indirect im- have been reported, most notable of which are differences in the munofluorescence technique with cY-spectrin antiserum, and subunit composition and degree of structural homology (Bennett et direct binding of F-actin by rhodamine-conjugated palloidin. al., 1982a, b; Burridge et al., 1982; Glenny et al., 1982a, b). However, the spectrin-actin network at synaptic junctions In this paper, we utilize the isolated Mauthner neuron of the appears to be distributed over the entire area of junctional goldfish and selected desheathed axons isolated from the goldfish contact and is not just restricted to postsynaptic densities. brainstem and spinal cord and from lumbar ventral roots of the The possibility of a duality of roles of spectrin in membrane- rabbit to examine the regional content and localization of a polypep- related motile and anchorage functions is discussed. tide that cross-reacts with chicken erythrocyte cu-spectrin antiserum. The Mauthner neuron, which is electrophysiologically and morpho- logically identifiable, is among the largest of vertebrate neurons, and Spectrin is a high molecular weight heterodimer originally identified its size lends itself to microanalytical and cytochemical studies of its as a membrane-associated protein in erythrocyte ghosts (Marchesi cellular subdivisions. We confirm that a cytoskeletal network con- and Steers, 1968; Clarke, 1971; Fairbanks et al., 1971). Later, it was taining cY-spectrin is distributed throughout the soma, dendrites, and shown that spectrin was an F-actin-binding protein and that short, axon of the Mauthner neuron. In general, the network is distributed oligomeric actin segments, cross-linked by spectrin, probably randomly and localized near the surface; however, the spectrin formed a two-dimensional cytoskeletal framework underlying the network is also distributed in a nonrandom manner at synapses and etythrocyte membrane (Hainfeld and Steck, 1977; Brenner and Korn, nodes of Ranvier. In addition, we demonstrate by indirect means 1979, 1980). Largely because there was an apparent lack of immu- that cY-spectrin is co-localized with F-actin at specialized cellular nological labeling of other cell types by antisera to mammalian sites. erythrocyte spectrin (Painter et al., 1975; Hiller and Weber, 1977), a decade passed after its initial discovery before it became evident Materials and Methods that spectrin-like proteins were, indeed, widely distributed among many nonerythroid cells, including cells of the nervous system &$ectrin antiserum. Details regarding the preparation and characteri- (Goodman et al., 1981; Bennett et al., 1982b; Burridge et al., 1982; zation of a-spectrin antiserum have been described previously (Repasky et Glenny et al., 1982a, b; Palfrey et al., 1982; Repasky et al., 1982). al., 1982). Briefly, a chicken erythrocyte cyioskeletal residue was solubilized after cell lysis and fractionated by two-dimentional polyacrylamide gel elec- trophoresis. The cu-spectrin was cut out of the gel, emulsified in Freund’s complete adjuvant, and used to immunize a rabbit. lmmunoradiography and Received May 25, 1984; Revised August 6, 1984; immunofluorescence indicated cross-reactivity of the antiserum with tissues Accepted August 10. 1984. of the CNS and PNS of the mouse and chicken, in addition to other nonerythroid tissues (Repasky et al., 1982). ’ We thank Ms. Terry Crumb for her technical assistance, Mrs. Jean Seiler lsolafion of microscopic cellular samples. Experiments reported here were for typing, and Mrs. Julie Lakatos for photography. In addition, we thank Drs. conducted exclusively on cellular material that was isolated by hand. The Donald Faber and Malcolm Wood for the benefit of their discussion. This two Mauthner neurons (M-cells), consisting of the soma and the large lateral research was supported by Grant BNS 81-17219 from the National Science and ventral dendrites, were dissected from a goldfish brainstem tissue slice. Foundation. The slice included the rostra1 half of the cerebellar crests; i.e., a segment ’ To whom correspondence should be addressed. extending from midway between the facial lobe and cerebellum to the caudal 705 706 Koenig and Repasky Vol. 5, No. 3, Mar. 1985 half of the cerebellar peduncles. The Mauthner cell axon (M-axon) was segment of the soma-dendrite transition zone with the solvating medium (see isolated by translating the axon out of Its myelin sheath from a sprnal cord- below), using the micropipette tip to form a cleavage channel through the brarnstem segment of vanable length (e.g., 1 to 3 cm) with a pair of no. 5 cellular process (Fig. 1). Dendrites or somata were then wetted sufficiently mrcrotweezers (Koenrg, 1965, 1978). The same technique was used to to dislodge them in order to transfer them to separate locations for solubili- Isolate axons, free of myelrn sheath, from goldfish brainstem-spinal cord zation. segments and from rabbrt lumbar ventral root segments (0.5 to 1 cm). Microscopic samples were solubilized with 1% SDS, 0.05 M Tris borate, 1 Cellular samples were Isolated rn one of the followrng zinc-containing solu- mM EDTA, and 0.02 M dithiothreitol. Brief heating (2 min) was used to facilitate trons: (a) 5 mM zinc acetate, 135 M NaCI, 5 mM KCI, 1.9 mM MgC12, 20 mM solubilization of axon clumps. Aliquot volumes were taken up by micropipette HEPES (pH 7.3 with Tns); or (b) 5 mM zinc acetate, 0.1 M guanrdrne and expelled into slits of a specialized gel microslab system (22 x 30 x 0.3 hydrochloride (Schwarz-Mann), and 0.1 M taunne (Sigma) (pH 6.5). As mm) for SDS microelectrophoresis (E. Koenig, unpublished procedure; details prevtously noted (Koenig, 1978) zinc inhibits endogenous calcium-activated will be described elsewhere). The microslab gels were composed of 0.25% protease activity and stabrlrzes cytoskeletal structure. Solution a was generally agarose and 7% polyacrylamide; they were equilibrated and stored in 0.04 used to Isolate samples for rmmunocytochemrcal experiments because the M Tris, 0.04 M glycine, 0.2% SDS, 30% urea, and 10% T-10 dextran cellular material IS more compliant and sticks more readily to a glass surface, (Pharmacia). In preparation for a run, a gel microslab was removed from the and solution b was generally used to isolate samples for microanalytical storage buffer, blotted on glassine powder paper (Lilly) until the surface purposes because it facilitates isolation, particularly of long segments of buffer was removed, laid onto a no. 2 coverslip (22 x 30 mm), and placed axons. over the well of an oil chamber. Slits were formed in the gel with a microneedle Preparative procedure for sodium dodecyl sulfate mrcroelectrophoresrs. to accommodate solubilized samples. The gel microslab was placed over Isolated mrcroscoprc samples used for sodium dodecyl sulfate (SDS) microe- an oil well of a specially designed chamber for microelectrophoresis. After lectrophoresis were deposited onto the surface of a split no. 2 coverslip (1 .l microelectrophoresis the gel was fixed by immersion in 30% ethanol. x 4 cm). M-cells were deposited with dendrites extended in a droplet of Immunoradiography. lmmunoradiography (Burridge, 1978) was performed solution b, and excess fluid was removed. Axons in suspension were plucked as described by Granger and Lazarides (1980). except that the gels were out into air by their ends and were collected as a clump on the tip of a 50- washed for 1 day after removal of antiserum or radioiodinated protein A. pm nrchrome wire. The axon clump was deposited onto a coverslip and After immunolabeling and reaction with ‘251-protein A (New England Nuclear), wetted briefly wtth drstrlled water so that it would stick, after which the fluid the gels were stained with silver according to the method of Switzer et al.