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.     

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).     

Release of proteins from the inner surface of squid axon membrane labeled with tritiated N-ethylmaleimide

Proteins in the inner surface of the squid axon membrane were labeled by intracellular perfusion of [3H]N-ethylmaleimide (NEM), which forms covalent bonds with free sulfhydryl groups. The excitability of the axon was unaffected by the [3H]NEM perfusion. After washout of the unbound label, the perfusate was monitored for the release of labeled proteins. Labeled proteins were released from the inner membrane surface by potassium depolarization of the axon only in the presence of external calcium ions. Replacement of the fluoride ion in the perfusion medium by various anions also caused labeled protein release. The order of effectiveness was SCN- greater than Br- greater than Cl- greater than F-. The extent of labeled protein release by the various anions was correlated with their effects on axonal excitability. The significance of these results is discussed.     

Are axoplasmic microtubules necessary for membrane excitation?

Summary The excitability of the squid giant axon was studied as a function of transmembrane hydrostatic pressure differences, the latter being altered by the technique of intracellular perfusion. When a KF solution was used as the internal medium, a pressure difference of about 15 cm water had very little effect on either the membrane potential or excitability. However, within a few minutes after introducing either a KCl-containing, a KBr-containing, or a colchicine-containing solution as the internal medium, with the same pressure difference across the membrane, the axon excitability was suppressed. In these cases, removal of the pressure difference restored the excitability, indicating that the structure of membrane was not irreversibly damaged. Electron-microscopic observations of these axons revealed that the perfusion with a KF solution or colchicine-containing solution preserves the submembranous cytoskeletal layer, whereas perfusion with a KCl or KBr solution dissolves it. These results suggest that the submembranous cytoskeletons including microtubules provide an important mechanical support to the excitable membrane but are not essential elements in channel activities.     

Tetrodotoxin affects submembranous cytoskeletal proteins in perfused souid giant axons

Application of tetrodotoxin or saxitoxin to intracellularly perfused squid giant axons caused the release of protein from the cytoskeletal network underlying the axolemma into the stream of perfusing solution. This protein was analyzed by one-dimensional polyacrylamide gel electrophoresis and found to be composed of the following major components: tubulin, actin and proteins having molecular weights of 96, 69 and 38 K-daltons. This observation is consistent with the hypothesis (1,2,8) that the integral membrane proteins controlling excitability in the axolemma (channel proteins) interact with the underlying cytoskeleton to maintain the stability of the excitable membrane.     

Squid (Loligo pealei) giant fiber system: a model for studying neurodegeneration and dementia?

In many neurodegenerative disorders that lead to memory loss and dementia, the brain pathology responsible for neuronal loss is marked by accumulations of proteins in the form of extracellular plaques and intracellular filamentous tangles, containing hyperphosphorylated cytoskeletal proteins. These are assumed to arise as a consequence of deregulation of a normal pattern of topographic phosphorylation-that is, an abnormal shift of cytoskeletal protein phosphorylation from the normal axonal compartment to cell bodies. Although decades of studies have been directed to this problem, biochemical approaches in mammalian systems are limited: neurons are too small to permit separation of cell body and axon compartments. Since the pioneering studies of Hodgkin and Huxley on the giant fiber system of the squid, however, the stellate ganglion and its giant axons have been the focus of a large literature on the physiology and biochemistry of neuron function. This review concentrates on a host of studies in our laboratory and others on the factors regulating compartment-specific patterns of cytoskeletal protein phosphorylation (primarily neurofilaments) in an effort to establish a normal baseline of information for further studies on neurodegeneration. On the basis of these data, a model of topographic regulation is proposed that offers several possibilities for further studies on potential sites of deregulation that may lead to pathologies resembling those seen in mammalian and human brains showing neurodegeneration, dementia, and neuronal cell death.     

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.     

Cell-to-cell transfer of glial proteins to the squid giant axon: The glia- neuron protein transfer hypothesis

The hypothesis that glial cells synthesize proteins which are transferred to adjacent neurons was evaluated in the giant fiber of the squid (Loligo pealei). When giant fibers are separated from their neuron cell bodies and incubated in the presence of radioactive amino acids, labeled proteins appear in the glial cells and axoplasm. Labeled axonal proteins were detected by three methods: extrusion of the axoplasm from the giant fiber, autoradiography, and perfusion of the giant fiber. This protein synthesis is completely inhibited by puromycin but is not affected by chloramphenicol. The following evidence indicates that the labeled axonal proteins are not synthesized within the axon itself. (a) The axon does not contain a significant amount of ribosomes or ribosomal RNA. (b) Isolated axoplasm did not incorporate [(3)H]leucine into proteins. (c) Injection of Rnase into the giant axon did not reduce the appearance of newly synthesized proteins in the axoplasm of the giant fiber. These findings, coupled with other evidence, have led us to conclude that the adaxonal glial cells synthesize a class of proteins which are transferred to the giant axon. Analysis of the kinetics of this phenomenon indicates that some proteins are transferred to the axon within minutes of their synthesis in the glial cells. One or more of the steps in the transfer process appear to involve Ca++, since replacement of extracellular Ca++ by either Mg++ or Co++ significantly reduces the appearance of labeled proteins in the axon. A substantial fraction of newly synthesized glial proteins, possibly as much as 40 percent, are transferred to the giant axon. These proteins are heterogeneous and range in size from 12,000 to greater than 200,000 daltons. Comparisons of the amount of amino acid incorporation in glia cells and neuron cell bodies raise the possibility that the adaxonal glial cells may provide an important source of axonal proteins which is supplemental to that provided by axonal transport from the cell body. These findings are discussed with reference to a possible trophic effect of glia on neurons and metabolic cooperation between adaxonal glia and the axon.     

Squid Giant Axon Contains Neurofilament Protein mRNA but does not Synthesize Neurofilament Proteins

When isolated squid giant axons are incubated in radioactive amino acids, abundant newly synthesized proteins are found in the axoplasm. These proteins are translated in the adaxonal Schwann cells and subsequently transferred into the giant axon. The question as to whether any de novo protein synthesis occurs in the giant axon itself is difficult to resolve because the small contribution of the proteins possibly synthesized intra-axonally is not easily distinguished from the large amounts of the proteins being supplied from the Schwann cells. In this paper, we reexamine this issue by studying the synthesis of endogenous neurofilament (NF) proteins in the axon. Our laboratory previously showed that NF mRNA and protein are present in the squid giant axon, but not in the surrounding adaxonal glia. Therefore, if the isolated squid axon could be shown to contain newly synthesized NF protein de novo, it could not arise from the adaxonal glia. The results of experiments in this paper show that abundant 3H-labeled NF protein is synthesized in the squid giant fiber lobe containing the giant axon’s neuronal cell bodies, but despite the presence of NF mRNA in the giant axon no labeled NF protein is detected in the giant axon. This lends support to the glia–axon protein transfer hypothesis which posits that the squid giant axon obtains newly synthesized protein by Schwann cell transfer and not through intra-axonal protein synthesis, and further suggests that the NF mRNA in the axon is in a translationally repressed state.     

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.     

Topographic regulation of phosphorylation in giant neurons of the squid, Loligo pealei: role of phosphatases

In previous studies of phosphorylation in squid stellate ganglion neurons, we demonstrated that a specific multimeric phosphorylation complex characterized each cellular compartment. Although the endogenous protein profile of cell body extracts (giant fiber lobe, GFL), as determined by Coomassie staining, was similar to that of axoplasm from the giant axon, in this study we show that the protein phosphorylation profiles are qualitatively different. Whereas many axoplasm proteins were phosphorylated, including most cytoskeletal proteins, virtually all phosphorylation in perikarya was confined to low molecular weight compounds (<6 kDa). Because phosphorylation of exogenous substrates, histone and casein, was equally active in extracts from both compartments, failure to detect endogenous protein phosphorylation in cell bodies was attributed to the presence of more active phosphatases. To further explore the role of phosphatases in these neurons, we studied phosphorylation in the presence of serine/threonine and protein tyrosine phosphatase (PTP) inhibitors. We found that phosphorylation of axonal cytoskeletal proteins was modulated by okadaic acid-sensitive ser/thr phosphatases, whereas cell body phosphorylation was more sensitive to an inhibitor of protein tyrosine phosphatases, such as vanadate. Inhibition of PTPs by vanadate stimulated endogenous phosphorylation of GFL proteins, including cytoskeletal proteins. Protein tyrosine kinase activity was equally stimulated by vanadate in cell body and axonal whole homogenates and Triton X-100 free soluble extracts, but only the Triton X soluble fraction (membrane bound proteins) of the GFL exhibited significant activation in the presence of vanadate, suggesting higher PTP activities in this fraction than in the axon. The data are consistent with the hypothesis that neuronal protein phosphorylation in axons and cell bodies is modulated by different phosphatases associated with compartment-specific multimeric complexes.     

The functional organization and assembly of the axon initial segment

Action potential initiation, modulation, and duration in neurons depend on a variety of Na+ and K+ channels that are highly enriched at the axon initial segment (AIS). The AIS also has high densities of cell adhesion molecules (CAMs), modulatory proteins, and a unique extracellular matrix (ECM). In contrast to other functional domains of axons (e.g. the nodes of Ranvier and axon terminals) whose development depends on the interactions with different cells (e.g. myelinating glia and postsynaptic cells), the recruitment and retention of AIS proteins is intrinsically specified through axonal cytoskeletal and scaffolding proteins. We speculate that the AIS has previously unappreciated forms of plasticity that influence neuronal excitability, and that AIS plasticity is regulated by the developmental or activity-dependent modulation of scaffolding protein levels rather than directly altering ion channel expression.     

Restoration of the excitability of squid giant axon by tubulin-tyrosine ligase and microtubule proteins.

Membrane excitability of the squid giant axon was destroyed by perfusing with a medium containing 0.2 mM Ca ions that depolymerize microtubules. Restoration of the membrane excitability was achieved by perfusing the axon with a medium that contained purified tubulin-tyrosine ligase, tubulin, ATP, Mg2+, K+, cyclic AMP, and 300 K Protein.     

Changes in cell-substratum adhesion and nuclear-cytoskeletal anchorage during cytodifferentiation of BeWo choriocarcinoma cells

Changes in the substratum anchorage of cells and nuclei were examined during methotrexate (MTX)-induced cytodifferentiation of BeWo human choriocarcinoma cells. During this process cytotrophoblast-like cells (CTLs) transform into giant mono- and multinucleated syncytiotrophoblast-like cells (STLs). Cells treated with MTX for 24 h exhibited significantly faster rates of substratum detachment by EDTA, trypsin-EDTA, EDTA-glycine, and DMSO than did uninduced controls. The decrease in cell-substratum adhesiveness occurred prior to the onset of morphological transformation. By 48 h, when morphological transformation was first observed, there had occurred a marked change in nuclear-cytoskeletal anchorage to the substratum, as evidenced by a difference in sensitivity of Triton-extracted STL and CTL monolayers to detachment by KI. STL monolayers were completely detached within 5 min of exposure to 0.3 M KI, while CTL monolayers remained firmly attached to the substratum for at least 3 h. KI-extracted residues were examined by electron microscopy and found to consist of nuclear shells attached to intermediate filaments. When cytoskeletal residues and KI-extracted proteins of STL and CTL cells were compared by two-dimensional polyacrylamide gel electrophoresis (PAGE), qualitative and quantitative differences were seen in a number of minor components. Thus the sensitivity of STL nuclear-cytoskeletal monolayers to removal by KI, an effective actin depolymerizing agent, may involve changes in the organization, stability, or interactions of actin with other components of the cytoskeletal framework.     

Regulation of axonal outgrowth and pathfinding by integrin-ECM interactions

Developing neurons use a combination of guidance cues to assemble a functional neural network. A variety of proteins immobilized within the extracellular matrix (ECM) provide specific binding sites for integrin receptors on neurons. Integrin receptors on growth cones associate with a number of cytosolic adaptor and signaling proteins that regulate cytoskeletal dynamics and cell adhesion. Recent evidence suggests that soluble growth factors and classic axon guidance cues may direct axon pathfinding by controlling integrin-based adhesion. Moreover, because classic axon guidance cues themselves are immobilized within the ECM and integrins modulate cellular responses to many axon guidance cues, interactions between activated receptors modulate cell signals and adhesion. Ultimately, growth cones control axon outgrowth and pathfinding behaviors by integrating distinct biochemical signals to promote the proper assembly of the nervous system. In this review, we discuss our current understanding how ECM proteins and their associated integrin receptors control neural network formation.     

The Drosophila lethal(2)giant larvae tumor suppressor protein forms homo-oligomers and is associated with nonmuscle myosin II heavy chain

Inactivation of the Drosophila lethal(2)giant larvae (l(2)gl) gene causes malignant tumors in the brain and the imaginal discs and produces developmental abnormalities in other tissues, including the germline, the ring gland and the salivary glands. Our investigations into the l(2)gl function have revealed that the gene product, or p127 protein, acts as a cytoskeletal protein distributed in both the cytoplasm and on the inner face of lateral cell membranes in a number of tissues throughout development. To determine whether p127 can form oligomers or can stably interact with other proteins we have analyzed the structure of the cytosolic form of p127. Using gel filtration and immunoaffinity chromatography we found that p127 is consistently recovered as high molecular weight complexes that contain predominantly p127 and at least ten additional proteins. Blot overlay assays indicated that p127 can form homo-oligomers and the use of a series of chimaeric proteins made of segments of p127 fused to protein A, which alone behaves as a monomer, showed that p127 contains at least three distinct domains contributing to its homo-oligomerization. Among the proteins separated from the immuno-purified p127 complexes or isolated by virtue of their affinity to p127, we identified one of the proteins by microsequencing as nonmuscle myosin II heavy chain. Further blot overlay assay showed that p127 can directly interact with nonmuscle myosin II. These findings confirm that p127 is a component of a cytoskeletal network including myosin and suggest that the neoplastic transformation resulting from l(2)gl gene inactivation may be caused by the partial disruption of this network.     

Morphology and Electrophysiological Properties of Squid Giant Axons Perfused Intracellularly with Protease Solution

Squid giant axons were perfused intracellularly with solutions containing various kinds of proteases (1 mg/ml). Except for a 10 µ layer inside the axolemma the axoplasm was removed by a 5 min perfusion with Bacillus protease, strain N' (BPN'). The resting and action potentials were unchanged and the axon maintained its excitability for more than 4 hr on subsequent enzyme-free perfusion. After perfusion with protease solution for 30 min the axoplasm was almost completely removed. The excitability was maintained, but the action potential became prolonged and rapidly developed a plateau of several hundred milliseconds. The change was not reversible even when the enzyme was removed from the perfusing fluid. Two other enzymes, prozyme and bromelin, also removed the protoplasm without blocking conduction. Trypsin suppressed within 3 min the excitability of the axon. It is suggested that the proteases alter macromolecules in the excitable membrane and thus affect the shape of the action potential.     

Protein Transport in Intact and Severed (Anucleate) Crayfish Giant Axons

Abstract: Using video-enhanced microscopy and a pulse-radiolabeling paradigm, we show that proteins synthesized in the medial giant axon cell body of the crayfish ( Procambarus clarkii ) are delivered to the axon via fast (∼62 mm/day) and slow (∼0.8 mm/day) transport components. These data confirm that the medial giant axon cell body provides protein to the axon in a manner similar to that reported for mammalian axons. Unlike mammalian axons, the distal (anucleate) portion of a medial giant axon remains intact and functional for >7 months after severance. This axonal viability persists long after fast transport has ceased and after the slow wave front of radiolabeled protein has reached the terminals. These data are consistent with the hypothesis that another source (i.e., local glial cells) provides a significant amount of protein to supplement that delivered to the medial giant axon by its cell body.     

Restoration of membrane excitability of squid giant axons by reagents activating tyrosine-tubulin ligase

Summary Using squid giant axon, an experimental survey was performed on restoration of the membrane excitability which had been partially suppressed. Among reagents examined, a combination of 400mm KF, 50 m tyrosine, 1mm ATP, 1mm Mg ions and 5 m cAMP was found to induce the restoration of the excitability to a large extent. Further addition of a small amount of either porcine brain microtubule proteins or the squid axoplasm was found to support complete restoration. The experiments suggest that tubulin-tyrosine ligase contained in the porcine brain microtubule protein fraction or the squid axoplasm maintains the coupling between cytoskeletal structures and the plasma membrane.