Subaxolemmal cytoskeleton in squid giant axon. I. Biochemical analysis of microtubules, microfilaments, and their associated high-molecular- weight proteins

Using the squid giant axon, we analyzed biochemically the molecular organization of the axonal cytoskeleton underlying the axolemma (subaxolemmal cytoskeleton). The preparation enriched in the subaxolemmal cytoskeleton was obtained by squeezing out the central part of the axoplasm using a roller. The electrophoretic banding pattern of the subaxolemmal cytoskeleton was characterized by large amounts of two high-molecular-weight (HMW) proteins (260 and 255 kD). The alpha, beta-tubulin, actin, and some other proteins were also its major constituents. The 260-kD protein is known to play an important role in maintaining the excitability of the axolemma (Matsumoto, G., M. Ichikawa, A. Tasaki, H. Murofushi, and H. Sakai, 1983, J. Membr. Biol., 77:77-91) and was recently designated "axolinin" (Sakai, H., G. Matsumoto, and H. Murofushi, 1985, Adv. Biophys., 19:43-89). We purified axolinin and the 255-kD protein in their native forms and further characterized their biochemical properties. The purified axolinin was soluble in 0.6 M NaCl solution but insoluble in 0.1 M NaCl solution. It co-sedimented with microtubules but not with actin filaments. In low-angle rotary-shadowing electron microscopy, the axolinin molecule in 0.6 M NaCl solution looked like a straight rod approximately 105 nm in length with a globular head at one end. On the other hand, the purified 255-kD protein was soluble in both 0.1 and 0.6 M NaCl solution and co-sedimented with actin filaments but not with microtubules. The 255-kD protein molecule appeared as a characteristic horseshoe-shaped structure approximately 35 nm in diameter. Furthermore, the 255-kD protein showed no cross-reactivity to the anti-axolinin antibody. Taken together, these characteristics lead us to conclude that the subaxolemmal cytoskeleton in the squid giant axon is highly specialized, and is mainly composed of microtubules and a microtubule-associated HMW protein (axolinin), and actin filaments and an actin filament-associated HMW protein (255-kD protein).     

Subaxolemmal cytoskeleton in squid giant axon. II. Morphological identification of microtubule- and microfilament-associated domains of axolemma

In the preceding paper (Kobayashi, T., S. Tsukita, S. Tsukita, Y. Yamamoto, and G. Matsumoto, 1986, J. Cell Biol., 102:1710-1725), we demonstrated biochemically that the subaxolemmal cytoskeleton of the squid giant axon was highly specialized and mainly composed of tubulin, actin, axolinin, and a 255-kD protein. In this paper, we analyzed morphologically the molecular organization of the subaxolemmal cytoskeleton in situ. For thin section electron microscopy, the subaxolemmal cytoskeleton was chemically fixed by the intraaxonal perfusion of the fixative containing tannic acid. With this fixation method, the ultrastructural integrity was well preserved. For freeze-etch replica electron microscopy, the intraaxonally perfused axon was opened and rapidly frozen by touching its inner surface against a cooled copper block (4 degrees K), thus permitting the direct stereoscopic observation of the cytoplasmic surface of the axolemma. Using these techniques, it became clear that the major constituents of the subaxolemmal cytoskeleton were microfilaments and microtubules. The microfilaments were observed to be associated with the axolemma through a specialized meshwork of thin strands, forming spot-like clusters just beneath the axolemma. These filaments were decorated with heavy meromyosin showing a characteristic arrowhead appearance. The microtubules were seen to run parallel to the axolemma and embedded in the fine three-dimensional meshwork of thin strands. In vitro observations of the aggregates of axolinin and immunoelectron microscopic analysis showed that this fine meshwork around microtubules mainly consisted of axolinin. Some microtubules grazed along the axolemma and associated laterally with it through slender strands. Therefore, we were led to conclude that the axolemma of the squid giant axon was specialized into two domains (microtubule- and microfilament-associated domains) by its underlying cytoskeletons.     

Membrane-Associated Cytoskeletal Proteins in Squid Giant Axons

Abstract: Cytoskeletal proteins (e.g., tubulin, actin, and neurofilament proteins) in the squid giant axon are separable into KF-soluble and -insoluble forms. The KF-insoluble cytoskeletal components appear to constitute the major proteins in the subaxolemmal fibrous network on the inner surface of the axon. These cytoskeletal proteins and the subaxolemmal network are both highly soluble in KI solutions. Whereas giant axons tolerate prolonged perfusions in KF solutions with no loss of excitable properties, a relatively short perfusion with KI solution completely eliminates the excitability of the axon. The loss of this excitability correlates with the simultaneous dissolution of the subaxolemmal network of cytoskeletal proteins and the release of its proteins into the perfusate. These data support the hypothesis that cytoskeletal proteins associated with the inner surface of the axolemma are involved in the regulation of axonal excitability.     

The effects of nitroprusside and putative agonists on guanylate cyclase activity in squid giant axons

cGMP content of axoplasm from the giant axon of Loligo forbesi was investigated after subjecting the axon to various treatments. Repetitive electrical stimulation or depolarisation by high K+ caused no change in cGMP content. Glutamate and serotonin were also without effect. The nicotinic agonist carbachol (100 microM) increased cGMP levels by 90% (n = 5). A large transient elevation of cGMP content was evoked by external nitroprusside (10 nM-20 microM in intact axons. Nitroprusside injected into both extruded axoplasm and intact axons also increased cGMP content, the stimulation being considerably higher in intact axons where the axolemma was also present. Nitroprusside was also active in axons where the soluble cytoplasmic components were washed out by internal perfusion.     

Organization of the Cortical Endoplasmic Reticulum in the Squid Giant Axon

The organization of the cortical endoplasmic reticulum in the squid giant axon was investigated by rapid freeze and freeze-substitution electron microscopy, thereby eliminating the effects of fixatives on this potentially labile structure. Juvenile squid, which have thinner Schwann sheaths, were used in order to achieve freezing deep enough to include the entire axonal cortex. The smooth endoplasmic reticulum is composed of subaxolemmal and deeper cisternae, tubules, tethers and vesicles. The subaxolemmal cisternae make junctional contacts with the axolemma which are characterized by filamentous-granular bridging structures approximately 3 nm in diameter. The subaxolemmal junctions with the axolemma resemble the coupling junctions between the sarcoplasmic reticulum and the T-tubules in muscle. Reconstruction of short series of sections showed that a number of the elements of the endoplasmic reticulum were continuous but numerous separate vesicles were present as well. The morphology of endoplasmic reticulum as described here suggests that it is a highly dynamic entity as well as a Ca²⁺ sequestering organelle.     

Macromolecular structure of reassembled neurofilaments as revealed by the quick-freeze deep-etch mica method: difference between NF-M and NF-H subunits in their ability to form cross-bridges.

Neurofilament (NF) structure and ability to form cross-bridges were examined by quick-freeze deep-etch mica and low-angle rotary-shadow electron microscopy in NFs purified from bovine spinal cord and reassembled in various combinations of NF subunits. When NFs were reassembled from triplet proteins, NF-L, NF-M and NF-H, they were oriented randomly and often fragmented, but their elongated filaments (12-15 nm wide) and the cross-bridges (4-5 nm wide) connecting them were similar in appearance to those of isolated bovine NFs or in vivo rat NFs. Projections extended from the wall of the core filament in almost the same pattern as the cross-bridges and were the same in width and interval (minimum interval, 20-25 nm) as the cross-bridges. Projections were more conspicuous when core filaments were separated by 60 to 80 nm or more, while cross-bridges were more conspicuous when core filaments were close to each other. Projections or cross-bridges extended bilaterally at intervals of 20 to 25 nm where core filaments expanded and formed a network between filaments which were far from one another. When NFs were reconstructed from NF-L alone, only core filaments appeared, the same width as the filaments of triplet NFs. The core filaments were occasionally in almost direct contact with each other, with no projection or cross-bridge. When NFs were reassembled from NF-M alone or NF-L + NF-M, although NF-M core filaments were shorter and slightly thinner than NF-L + NF-M core filaments, both had projections, and both had cross-bridges, but cross-bridges were less evident. Cross-bridges were almost the same in width as those of triplet NFs, but significantly shorter and much less frequent although the minimum interval was the same, and core filaments were not attached to each other. In contrast, when NFs were reconstituted from NF-H alone or NF-L + NF-H, both had conspicuous projections and cross-bridges, similar to those of triplet NFs. Thus, when NFs contained NF-H, they formed frequent cross-bridges and long projections with extensive peripheral branching. When NFs contained NF-M but no NF-H, they tended to form cross-bridges, and to form projections that were shorter and straighter and without peripheral branching. That is, there appears to be a significant difference between NF-M and NF-H in ability to form cross-bridges and thus in interaction with adjacent NFs.     

Subaxolemmal filamentous network in the giant nerve fiber of the squid (Loligo pealei L.) and its possible role in excitability

A new technique utilizing the squid giant nerve fiber has been developed which permits direct examination of the inner face of the axolemma by scanning electron microscopy. The axoplasm was removed sequentially in a 15-mm long segment of the fiber by intracellular perfusion with a solution of KF, KCl, Ca++-containing seawater, or with pronase. The action potential of the fibers was monitored during these treatments. After brief prefixation in 1% paraformaldehyde and 1% glutaraldehyde, the perfused segment was opened by a lne could be related to information on the detailed morphology of the cytoplasmic face of the axolemma and the ectoplasm. The results obtained by scanning electron microscopy were further substantiated by transmission electron microscopy of thin sections. In addition, living axons were studied with polarized light during axoplasm removal, and the identification of actin by heavy meromyosin labeling and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis was accomplished. These observations demonstrate that a three-dimensional network of interwoven filaments, consisting partly of an actinlike protein, is firmly attached to the axolemma. The axoplasmic face of fibers in which the filaments have been removed partially after perfusion with pronase displays smooth membranous blebs and large profiles which sppose the axolemma. In fibers where the excitability has been suppressed by pronase perfusion, approximately one-third of the inner face of the axolemma in the perfusion zone is free of filaments. It is hypothesized that the attachment of axoplasm filaments to the axolemma may have a role in the maintenance of the normal morphology of the axolemma, and, thus, in some aspect of excitability.     

Microtubules and actin in giant nerve fibers of the spiny lobster, Panulirus argus

Giant axons of the spiny lobster, Panulirus argus, are filled with microtubules that are decorated with fine, irregular filaments. Mitochondria and membrane-limited clear vesicles are the only other distinguishable elements in the axoplasm and are located around the periphery of the axon near the axolemma. Neither 100 A neurofilaments nor 70 A microfilaments are evident in fixed, intact axons or in negatively stained axoplasm. Actin-like microfilaments are a prominent constituent of the glial cells that closely ensheathe the axons, and gel electrophoresis studies suggest that most of the actin in the nerve fibers is located in the glia rather than in the axons. Studies of isolated axoplasm indicate that microtubules are the primary elements stabilizing the axoplasm. The microtubules in the isolated axoplasm are disrupted by Ca2+ in the medium in the presence of protease inhibitors.     

Antisense Morpholino Oligonucleotides Reduce Neurofilament Synthesis and Inhibit Axon Regeneration in Lamprey Reticulospinal Neurons

The sea lamprey has been used as a model for the study of axonal regeneration after spinal cord injury. Previous studies have suggested that, unlike developing axons in mammal, the tips of regenerating axons in lamprey spinal cord are simple in shape, packed with neurofilaments (NFs), and contain very little F-actin. Thus it has been proposed that regeneration of axons in the central nervous system of mature vertebrates is not based on the canonical actin-dependent pulling mechanism of growth cones, but involves an internal protrusive force, perhaps generated by the transport or assembly of NFs in the distal axon. In order to assess this hypothesis, expression of NFs was manipulated by antisense morpholino oligonucleotides (MO). A standard, company-supplied MO was used as control. Axon retraction and regeneration were assessed at 2, 4 and 9 weeks after MOs were applied to a spinal cord transection (TX) site. Antisense MO inhibited NF180 expression compared to control MO. The effect of inhibiting NF expression on axon retraction and regeneration was studied by measuring the distance of axon tips from the TX site at 2 and 4 weeks post-TX, and counting the number of reticulospinal neurons (RNs) retrogradely labeled by fluorescently-tagged dextran injected caudal to the injury at 9 weeks post-TX. There was no statistically significant effect of MO on axon retraction at 2 weeks post-TX. However, at both 4 and 9 weeks post-TX, inhibition of NF expression inhibited axon regeneration.     

Injury-induced vesiculation and membrane redistribution in squid giant axon

Injury of isolated squid giant axons in sea water by cutting or stretching initiates the following unreported processes: (i) vesiculation in the subaxolemmal region extending along the axon several mm from the site of injury, followed by (ii) vesicular fusions that result in the formation of large vesicles (20-50 micron diameter), 'axosomes', and finally (iii) axosomal migration to and accumulation at the injury site. Some axosomes emerge from a cut end, attaining sizes up to 250 microns in diameter. Axosomes did not form after axonal injury unless divalent cations (Ca2+ or Mg2+) were present (10mM) in the external solution. The requirement for Ca2+ and the action of other ions are similar to that for cut-end cytoskeletal constriction in transected squid axons (Gallant, P.E. (1988) J. Neurosci. 8, 1479-1484) and for electrical sealing in transected axons of the cockroach (Yawo, H. and Kuno, M. (1985) J. Neurosci. 5, 1626-1632). Axosomes probably consist of membrane from different sources (e.g., axolemma, organelles and Schwann cells); however, localization of axosomal formation to the inner region of the axolemma and the formation dependence on divalent cations suggest principal involvement of cisternae of endoplasmic reticulum. Patch clamp of excised patches from axosomes liberated spontaneously from cut ends of transected axons showed a 12-pS K+ channel and gave indications of other channel types. Injury-induced vesiculation and membrane redistribution seem to be fundamental processes in the short-term (minutes to hours) that precede axonal degeneration or repair and regeneration. Axosomal formation provides a membrane preparation for the study of ion channels and other membrane processes from inaccessible organelles.     

Ultrastructure of the Squid Axon Membrane as Revealed by Freeze-Fracture Electron Microscopy

The structure of the axolemma of the squid giant axon was studied by freeze-fracture electron microscopy. Three types of preparations were examined: intact axons, axons with their Schwann cell sheaths stripped off prior to freezing, and axons with their Schwann cell sheaths chemically detached but not mechanically removed. Because of a problem of cross-fracturing, the first two types of preparations revealed very few membrane faces of the axolemma. This cross-fracturing problem, however, was eliminated when we used a complementary replication method to fracture the third type of preparation. We found that the E-face of the axon membrane was smooth relative to the P-face, which showed many prominent intramembrane particles (IMP). The diameters of the typical IMP range from 6 to 15 nm. The P-face of the adjacent Schwann cells also showed many large IMP. The sizes and heights of the Schwann-cell IMP, however, appear to be more homogeneous than the P-face axolemma.     

270K microtubule-associated protein cross-reacting with anti-MAP2 IgG in the crayfish peripheral nerve axon

MAPs (microtubule-associated proteins) were isolated from crayfish walking leg nerves. A major MAP was identified as a high molecular weight protein (270K). This protein co-migrated with mammalian MAP2, stimulated the polymerization of rat brain tubulin into microtubules, and was heat resistant. Rotary shadowing revealed that the 270K MAP is a long thin flexible structure. It formed cross-bridges of fine strands, linking microtubules with each other in vitro. These strands resemble the cross-bridges between microtubules observed in the crayfish axon permeabilized with saponin and quick-frozen, deep-etched. Antibodies against mammalian MAP2 cross-reacted with this crayfish MAP and stained the axoplasm of the walking leg nerves. Thus MAPs, especially the 270K MAP, appear to be a major component of the cross-linking strands between microtubules observed in the crayfish axon.     

Electron tomographic analysis of cytoskeletal cross-bridges in the paranodal region of the node of Ranvier in peripheral nerves

The node of Ranvier is a site for ionic conductances along myelinated nerves and governs the saltatory transmission of action potentials. Defects in the cross-bridging and spacing of the cytoskeleton are a prominent pathological feature in diseases of the peripheral nerve. Electron tomography was used to examine cytoskeletal–cytoskeletal, membrane–cytoskeletal, and heterologous cell connections in the paranodal region of the node of Ranvier in peripheral nerves. Focal attachment of cytoskeletal filaments to each other and to the axolemma and paranodal membranes of the Schwann cell via narrow cross-bridges was visualized in both neuronal and glial cytoplasm. A subset of intermediate filaments associates with the cytoplasmic surfaces of supramolecular complexes of transmembrane structures that are presumed to include known and unknown junctional proteins. Mitochondria were linked to both microtubules and neurofilaments in the axoplasm and to neighboring smooth endoplasmic reticulum by narrow cross-bridges. Tubular cisternae in the glial cytoplasm were also linked to the paranodal glial cytoplasmic loop juxtanodal membrane by short cross-bridges. In the extracellular matrix between axon and Schwann cell, junctional bridges formed long cylinders linking the two membranes. Interactions between cytoskeleton, membranes, and extracellular matrix associations in the paranodal region are likely critical not only for scaffolding, but also for intracellular and extracellular communication.     

Cytoskeletal reorganization of human platelets after stimulation revealed by the quick-freeze deep-etch technique

We studied the cytoskeletal reorganization of saponized human platelets after stimulation by using the quick-freeze deep-etch technique, and examined the localization of myosin in thrombin-treated platelets by immunocytochemistry at the electron microscopic level. In unstimulated saponized platelets we observed cross-bridges between: adjoining microtubules, adjoining actin filaments, microtubules and actin filaments, and actin filaments and plasma membranes. After activation with 1 U/ml thrombin for 3 min, massive arrays of actin filaments with mixed polarity were found in the cytoplasm. Two types of cross-bridges between actin filaments were observed: short cross-bridges (11 +/- 2 nm), just like those observed in the resting platelets, and longer ones (22 +/- 3 nm). Actin filaments were linked with the plasma membrane via fine short filaments and sometimes ended on the membrane. Actin filaments and microtubules frequently ran close to the membrane organelles. We also found that actin filaments were associated by end-on attachments with some organelles. Decoration with subfragment 1 of myosin revealed that all the actin filaments associated end-on with the membrane pointed away in their polarity. Immunocytochemical study revealed that myosin was present in the saponin-extracted cytoskeleton after activation and that myosin was localized on the filamentous network. The results suggest that myosin forms a gel with actin filaments in activated platelets. Close associations between actin filaments and organelles in activated platelets suggests that contraction of this actomyosin gel could bring about the observed centralization of organelles.     

Nonelectrolyte Permeability, Sodium Influx, Electrical Potentials, and Axolemma Ultrastructure in Squid Axons of Various Diameters

The penetration of ¹⁴C-labeled ethylene glycol, erythritol, mannitol, and sucrose was measured in giant axons of various diameters isolated from the hindmost stellar nerves of Doryteuthis plei squid. Axon diameter depends mainly on the age of the squid. The influx of ²²Na, some electrical properties, and the ultrastructure of the axolemma were also studied. The results confirm our previous observation that in medium sized axons of D. plei stimulation causes an increase in the permeability to the penetration of erythritol, mannitol, and sucrose. They also demonstrate that the magnitude of the increase in the penetration of these probing molecules diminishes progressively as the axon diameter increases. The diminution in permeability may be due to a reduction in size of the pathways used by nonelectrolytes to enter the axon. No effect of stimulation on the ethylene glycol permeability is observed. The sodium influx and electrical properties are independent of axon size. The ultrastructural study shows that the axolemma thickness increases with axon diameter. The present experiments indicate that the nonelectrolyte permeability of stimulated axons depends on nerve fiber properties related to axon diameter and on the size of the hydrophilic nonelectrolyte probe.     

Requirement of Heavy Neurofilament Subunit in the Development of Axons with Large Calibers

Neurofilaments (NFs) are prominent components of large myelinated axons. Previous studies have suggested that NF number as well as the phosphorylation state of the COOH-terminal tail of the heavy neurofilament (NF-H) subunit are major determinants of axonal caliber. We created NF-H knockout mice to assess the contribution of NF-H to the development of axon size as well as its effect on the amounts of low and mid-sized NF subunits (NF-L and NF-M respectively). Surprisingly, we found that NF-L levels were reduced only slightly whereas NF-M and tubulin proteins were unchanged in NF-H–null mice. However, the calibers of both large and small diameter myelinated axons were diminished in NF-H–null mice despite the fact that these mice showed only a slight decrease in NF density and that filaments in the mutant were most frequently spaced at the same interfilament distance found in control. Significantly, large diameter axons failed to develop in both the central and peripheral nervous systems. These results demonstrate directly that unlike losing the NF-L or NF-M subunits, loss of NF-H has only a slight effect on NF number in axons. Yet NF-H plays a major role in the development of large diameter axons.     

Proteins transported in slow components a and b of axonal transport are distributed differently in the transverse plane of the axon

The distribution of the proteins migrating with the slow components a (SCa) and b (SCb) of axonal transport were studied in cross-sections of axons with electron microscope autoradiography. Radiolabeled amino acids were injected into the hypoglossal nucleus of rabbits and after 15 d, the animals were killed. Hypoglossal nerves were processed either for SDS-polyacrylamide gel electrophoresis fluorography to identify and locate the two components of slow transport, or for quantitative electron microscope autoradiography. Proteins transported in SCa were found to be uniformly distributed within the cross-section of the axon. Labeled SCb proteins were also found throughout the axonal cross-section, but the subaxolemmal region of the axon contained 2.5 times more SCb radioactivity than any comparable area in the remainder of the axon.     

The neurofilament middle molecular mass subunit carboxyl-terminal tail domains is essential for the radial growth and cytoskeletal architecture of axons but not for regulating neurofilament transport rate

The phosphorylated carboxyl-terminal "tail" domains of the neurofilament (NF) subunits, NF heavy (NF-H) and NF medium (NF-M) subunits, have been proposed to regulate axon radial growth, neurofilament spacing, and neurofilament transport rate, but direct in vivo evidence is lacking. Because deletion of the tail domain of NF-H did not alter these axonal properties (Rao, M.V., M.L. Garcia, Y. Miyazaki, T. Gotow, A. Yuan, S. Mattina, C.M. Ward, N.S. Calcutt, Y. Uchiyama, R.A. Nixon, and D.W. Cleveland. 2002. J. Cell Biol. 158:681-693), we investigated possible functions of the NF-M tail domain by constructing NF-M tail-deleted (NF-MtailDelta) mutant mice using an embryonic stem cell-mediated "gene knockin" approach that preserves normal ratios of the three neurofilament subunits. Mutant NF-MtailDelta mice exhibited severely inhibited radial growth of both motor and sensory axons. Caliber reduction was accompanied by reduced spacing between neurofilaments and loss of long cross-bridges with no change in neurofilament protein content. These observations define distinctive functions of the NF-M tail in regulating axon caliber by modulating the organization of the neurofilament network within axons. Surprisingly, the average rate of axonal transport of neurofilaments was unaltered despite these substantial effects on axon morphology. These results demonstrate that NF-M tail-mediated interactions of neurofilaments, independent of NF transport rate, are critical determinants of the size and cytoskeletal architecture of axons, and are mediated, in part, by the highly phosphorylated tail domain of NF-M.     

Arrival, reversal, and departure of neurofilaments at the tips of growing axons

We have investigated the movement of green fluorescent protein-tagged neurofilaments at the distal ends of growing axons by using time-lapse fluorescence imaging. The filaments moved in a rapid, infrequent, and asynchronous manner in either an anterograde or retrograde direction (60% anterograde, 40% retrograde). Most of the anterograde filaments entered the growth cone and most of the retrograde filaments originated in the growth cone. In a small number of cases we were able to observe neurofilaments reverse direction, and all of these reversals occurred in or close to the growth cone. We conclude that neurofilament polymers are delivered rapidly and infrequently to the tips of growing axons and that some of these polymers reverse direction in the growth cone and move back into the axon. We propose that 1) growth cones are a preferential site of neurofilament reversal in distal axons, 2) most retrograde neurofilaments in distal axons originate by reversal of anterograde filaments in the growth cone, 3) those anterograde filaments that do not reverse direction are recruited to form the neurofilament cytoskeleton of the newly forming axon, and 4) the net delivery of neurofilament polymers to growth cones may be controlled by regulating the reversal frequency.     

Two distinct functions of the carboxyl-terminal tail domain of NF-M upon neurofilament assembly: cross-bridge formation and longitudinal elongation of filaments

Neurofilaments are the major cytoskeletal elements in the axon that take highly ordered structures composed of parallel arrays of 10-nm filaments linked to each other with frequent cross-bridges, and they are believed to maintain a highly polarized neuronal cell shape. Here we report the function of rat NF-M in this characteristic neurofilament assembly. Transfection experiments were done in an insect Sf9 cell line lacking endogenous intermediate filaments. NF-L and NF-M coassemble to form bundles of 10-nm filaments packed in a parallel manner with frequent cross-bridges resembling the neurofilament domains in the axon when expressed together in Sf9 cells. Considering the fact that the expression of either NF-L or NF-M alone in these cells results in neither formation of any ordered network of 10-nm filaments nor cross- bridge structures, NF-M plays a crucial role in this parallel filament assembly. In the case of NF-H the carboxyl-tail domain has been shown to constitute the cross-bridge structures. The similarity in molecular architecture between NF-M and NF-H suggests that the carboxyl-terminal tail domain of NF-M also constitutes cross-bridges. To examine this and to further investigate the function of the carboxyl-terminal tail domain of NF-M, we made various deletion mutants that lacked part of their tail domains, and we expressed these with NF-L. From this deletion mutant analysis, we conclude that the carboxyl-terminal tail domain of NF-M has two distinct functions. First, it is the structural component of cross-bridges, and these cross-bridges serve to control the spacing between core filaments. Second, the portion of the carboxyl- terminal tail domain of NF-M that is directly involved in cross-bridge formation affects the core filament assembly by helping them to elongate longitudinally so that they become straight.