accumulation of synaptic vesicle proteins and cytoskeletal specializations at the peripheral node of...

MICROSCOPY RESEARCH AND TECHNIQUE 34~462-473 (1996)

Accumulation of Synaptic Vesicle Proteins and Cytoskeletal Specializations at the Peripheral Node of Ranvier HERBERT ZIMMERMANN Biozentrum der J.W. Goethe-Uniuersitat, AK Neurochemie, Zoologisches Znstitut, 0-60439 Frankfurt am Main, Germany

KEY WORDS SV2, Synaptophysin, Neurofilament, rab3, Membrane turnover, Axonal trans- Port

ABSTRACT Nodes of Ranvier of peripheral nerve fibres represent repetitive physiological axon constrictions. The nodal attenuation of the axon cylinder is expected to facilitate eliciting axon potentials. But as revealed by immunocytochemical analysis of synaptic vesicle proteins such as SV2 and synaptophysin, nodes are also sites of accumulation of the synaptic vesicle membrane compartment. Results from our studies and other laboratories suggest that the local nodal retar- dation of the axonally transported synaptic vesicle membrane compartment serves membrane processing and/or turnover. Nodes of Ranvier as well as incisures of Schmidt-Lanterman are rich in filamentous actin and can easily be depicted by fluoresceinated phalloidin. At the node and paranode phalloidin fluorescence appears to be mainly associated with the Schwann cell compartment. Immunofluorescence demonstrates that this compartment also contains myosin and spectrin. The nodal contents in actin and myosin may be effective in actively constricting the axon cylinder at both the node of Ranvier and the Schmidt-Lanterman incisures. This hypothesis is discussed in the light of the nodal cytoskeletal specializations of the axon cylinder and the ensheathing Schwann cell. 8 1996 Wiley-Liss, Inc.

INTRODUCTION The major physiological function of the node of Ran-

vier is considered to be in the accelerated propagation of action potentials. Although often neglected, one of the most significant morphological characteristics of the node of peripheral axons is the constriction of the axon cylinder. This constriction occurs both at central and peripheral nodes of Ranvier and is particularly prominent in large myelinated axons (Berthold, 1978; Hildebrand et al. 1993). Depending on fibre size the cross-sectional area of the node of peripheral axons is reduced to approximately 30 to 15% of the internodal area. Generally, the constriction is most pronounced at the two paranodal regions where the pockets of the Schwann cell lamellae indent the surface of the axon (compare Figs. 1, 23). In the nodal region that is covered by interdigitating processes extending from the outer cytoplasmic layers of the Schwann cells, the axon often bulges outward. The biological function of the nodal axon constriction is unknown. It may be of rele- vance for axonal conduction of action potentials. It may, in addition, serve the accumulation and controlled interaction of axonally transported organelles. The article provides evidence for the nodal accumulation of the synaptic vesicle membrane compartment and discusses its possible function. This is followed by a detailed analysis of the nodal cytoskeletal specializations and the mechanisms that might effect the constriction of the axon cylinder.

CONSTRICTION FACILITATES ELICITING ACTION POTENTIALS

The node carries dense populations of sodium and potassium channels. The voltage-dependent opening of

the sodium channels is a precondition for the successful propagation of action potentials from node to node. The constricted nodal axon cylinder has a considerably reduced plasma membrane area. A reduction of the axon diameter by 50% results in a reduction of the crossec- tional area of the axon and of the volume of the axon cylinder by 75% and of the axonal surface area by 50%. This means that the membrane capacitance at the node of Ranvier is twofold decreased as compared to a corresponding segment with internodal axon diameter. Eliciting action potentials should therefore be facili- tated at the node. The nodal constriction may thus represent a safety margin or even a necessary condition to allow eliciting action potential in axons with large axon diameters and long internodal segments. Model- ling of the physical properties of the node of Ranvier has revealed that the nodal-paranodal constriction pro- motes higher conduction velocities (Halter and Clark, 1993).

CONSTRICTION FUNCTIONS IN THE ACCUMULATION OF

AXONALLY TRANSPORTED MEMBRANE COMPARTMENTS

The axon is the pipeline for metabolic support, of the far distant synaptic region with fast anterograde and retrograde and also slow axonal transport continuously

Received February 13, 1995; accepted in revised form March 20, 1995. Address reprint requests to Dr. Herbert Zimmermann, Biozentrum der J.W.

Goethe-Universitit, AK Neurochemie, Zoologisches Institut, Marie-Curie-Str. 9, D-60439 Frankfurt am Main, Germany.

6 1996 WILEY-LISS, INC.

VESICLE ACCUMULATION AND NODAL CYTOSKELETON 463

taking place. The nodal constriction may be deleterious to and greatly hamper axoplasmic transport. If the bulk flow of axoplasm throughout the axon was to be maintained, the transport velocity through the constricted nodal segment would need to be increased up to 40-fold (Berthold, 1978). There is broad evidence that membrane compartments that are transported by fast axonal flow are locally arrested and accumulate at nodes of Ranvier (Berthold et al., 1993; Fabricius et al., 1993). This accumulation can be observed even in Tolu- idine blue stained sections (see, for example, Raine et al., 1983; Berthold et al., 1993). In accordance with this, part of intraaxonal organelles in isolated and liv- ing myelinated nerve fibres from the African claw frog Xenopus laeuis were found to be temporarily arrested at nodes (Cooper and Smith, 1974). After injection of [3Hlfucose into the rat spinal cord, axoplasmic accumulation of radiolabelled glycoproteins was demonstrated at nodes of Ranvier in the sciatic nerve (Armstrong et al., 1987). Horseradish peroxidase injected into the cat gastrocnemius muscle is retrogradely transported within membrane compartments and accumulates a t consecutive nodes of Ranvier (Berthold and Mellstrom, 1986; Berthold et al., 1986, 1988). Even 30 nm fluorescent latex beads injected into a crushed nerve are accumulated at nodes (Persson and Gatzinsky, 1993). It is also of interest that under conditions of anterograde transport the proximal paranodal segment tends to be labelled more intensively (Armstrong et al., 1987) whereas it is the distal paranodal segments in retrograde transport experiments (Berthold and Mellstrom, 1986; Persson and Gatzinsky, 1993). Also acid hydro- lase positive organelles belonging to the lysosomal compartment are accumulated at peripheral nodes of Ranvier (Holtzman and Novikoff, 1965; Gatzinsky et al., 1988,1991a). Finally, the membrane compartment of synaptic vesicles is equally found to be accumulated (Janetzko et al., 1989; Zimmermann and Vogt, 1989; Okajima et al. 1993). All these data suggest that the temporary arrest at the node of Ranvier of anterogradely and retrogradely transported organelles is not simply an accident or unavoidable consequence of the nodal constriction. It may rather represent a physiological mechanism that serves the local turnover of axonal membrane compartments at consecutive nodes.

SYNAPTIC VESICLE MEMBRANE COMPARTMENT IS ACCUMULATED

AT THE NODE In mature nerve cells the major membrane compart-

ment axonally transported to and from nerve terminals is presumably the synaptic vesicle compartment. The origin of synaptic vesicles is presently not completely understood. Vesicles may either originate directly from the Golgi-apparatus and become transported into the nerve terminals for release (Zimmermann et al., 19931, or they may be formed first as vesicles of the constitu- tive exocytotic pathway and-after a sorting step within the nerve terminal-become transformed into release competent synaptic vesicles (Regnier-Vigour- oux and Huttner, 1993). Finally, the vesicular membrane may be transported as part of the endoplasmic reticulum and originate by budding from this intraax-

onal membrane system only in the nerve terminal (Ka- dota et al. 1991). In electron micrographs vesicular structures are frequently observed at the node of Ran- vier (Tsukita and Ishikawa, 1976; Fabricius et al. 1993).

Identification of Vesicle Proteins by Indirect Immunocytochemistry

In immunocytochemical studies the synaptic vesicle membrane compartment can be labelled by antibodies against specific membrane integral proteins such as synaptic vesicle protein 2 (SV2), or synaptophysin or against vesicle surface associated proteins such as the small GTP-binding protein rab3 (Volknandt et al., 1993) or synapsin I. The function of SV2 is unknown but as deduced from its membrane topography it might function as a transporter (Buckley, 1994). Synapto- physin is a ubiquitous vesicle protein possibly function- ing as a pore in the vesicle membrane (Thomas et al., 1988). Other proteins are associated with the synaptic vesicle surface. The GTP-binding protein rab3 is thought to target synaptic vesicles to sites of docking and exocytosis within the nerve terminal (see references in Ferronovick and Novick, 1993). The synapsins, on the other hand, stabilize synaptic vesicles within the actin meshwork of the axon terminal (Val- torta et al. 1992). We performed a detailed analysis of the axonal distribution of individual vesicle proteins. The varying association of the proteins with membrane compartments on their passage through the axon was expected to provide further insights into the life cycle of the vesicular membrane compartment. This led to the discovery of an intense nodal accumulation of the vesicular membrane compartment and a further char- acterization of the underlying mechanisms.

Membrane Integral Proteins The synaptic vesicle membrane compartment is best

studied in a neural system that has a high production rate and turnover of the organelle. The electromotor system of the electric ray Torpedo is a well-character- ized neural system and contains cholinergic nerve terminals; axons and perikarya corresponding to the identical type of neuron are easily accessible. Each axon has impressive arborizations within the electric organ that require intensive support of vesicular membrane compartments. The cylinders of the axons have a mean diameter of about 7 prn with the traverse area reduced by about 80% at the paranodes (Zimmermann and Vogt, 1989) (Fig. 1).

As revealed by indirect fluorescein isothiocyanate (FITC) immunof luorescence using a monoclonal antibody against the vesicle protein SV2 vesicular membrane proteins are accumulated within the nodal and paranodal segments (Zimmermann and Vogt, 1989) (Fig 4). This can also be observed by electron microscopical immunocytochemistry or by light microscopical immunocytochemistry using peroxidase-labelled second antibodies (Janetzko et al., 1989). Outside the nodes immunof luorescence is weak revealing a punctuate pattern. Interestingly, nodal accumulation is first observed at the CNS-PNS borderline. The diameter of the electromotor axons is considerably reduced

464 H. ZIMMERMA”

Figs. 1-10.


within the CNS and the density of synaptic vesicle protein is enriched in this initial axon segment.

The nature of the axonal membrane compartments carrying the vesicular protein could be further charac- terized by immunoelectron microscopy (Janetzko et al., 1989). If the antigen is directed to the vesicle surface, the colloidal gold technique can be applied using a preembedding protocol. Since SV2 is an integral membrane protein it can serve as a marker of the synaptic vesicle membrane compartment even if this is a con- stituent of another organelle (such as an endosome). Within the nerve terminals in the electric organ synaptic vesicles (and the very rare multivesicular bodies) are the only labelled membrane compartments. Within the non-terminal axon, typical synaptic vesicle profiles are considerably rarer. Instead, there are numerous additional membrane-bound organelles such as vesic- ulo-tubular structures, lamellar bodies, and multivesicular bodies. With the exception of the axoplasmic reticulum and mitochondria all these organelles are labelled by the anti-SV2 antibody. This means that they share at least part of their membrane proteins with synaptic vesicles and that they may interact at

Figs. 1-10. Structural aspects of the node of Ranvier in the axons of the electromotor nerve of the electric ray Torpedo murmomta.

Fig. 1. Longitudinal semithin section through an axon at the node of Ranvier (glutaraldehyde and osmium fixation; methylene-blue staining).

Fig. 2. Structural detail of nodal binding of TRITC-labelled phalloidin (longitudinal cryoatat section, fluorescence). In both Figures 1 and 2 the nodal segment (long arrow) and the paranodal segments (short arrows) can clearly be distinguished.

Fig. 3. Section across an axon bundle in the electromotor nerve and application of TRITC-labelled phalloidin. Only nodes of Ranvier are labelled (arrowheads) whereas internodal axon profiles remain unlabelled (thin arrow).

Fig. 4. Localization of binding sites for an antibody against the synaptic vesicle membrane protein SV2 using indirect FITC-immunofluorescence and longitudinal sections. Punctuate distribution in the axon and accumulation at nodes (arrows).

Fig. 5. Identical image as in Figure 4. Double-labelling of nodes of Ranvier (arrows) with TRITC-phalloidin. Thin arrows depict labelling of Schwann-cell cytoplasm.

Fig. 6. Localization of binding sites for an antibody against the synaptic vesicle associated GTP-binding protein o-rab3 (indirect FITC-immunofluorescence). As compared to the protein SV2 the punctuate distribution in the axon and accumulation at nodes (arrows) is less intense.

Fig. 7. Binding sites for an antibody against actin (indirect FITC- immunofluorescence, longitudinal section) at nodes of Ranvier (arrows) and the Schwann-cell cytoplasm (thin arrows).

Fig. 8. A similar image as in Figure 7 is obtained when an antibody against myosin is applied.

Fig, 9. Nodal enhancement of tubulin (arrow) as revealed by binding of an antibody against p-tubulin (FITC-immunofluorescence and longitudinal sections).

Fig. 10. Identical image aa in Figure 9, double labelling of node of Ranvier (arrow) with TRITC-phalloidin. Bars represent 10 pm.

some stage with the vesicle membrane during its life cycle. All these axonal organelles are found to accumulate at consecutive nodes of Ranvier of the peripheral electromotor axons.

Both SV2- and synaptophysin-containing membrane organelles are also observed at nodes of the myelinated axons of the rat sciatic nerve (Zimmermann and Vogt, 1989). Using FITC-labelled second antibodies, the im- munoreaction in the rat is considerably weaker than in Torpedo electromotor axons, presumably due to reduced membrane traffic in the mammalian motor ax- 0118 with their few terminal ramifications. Further- more, immunoreactivity was restricted to the paranodes. However, when the peroxidase technique is applied, the nodal accumulation in rat sciatic axons can be impressively demonstrated (Okajima et al. 1993) (Fig. 14) In this case reaction product is found in both the paranodal and nodal section. When the axon is crushed the nodal accumulation of vesicle proteins is enhanced near the crush (Fig. 15). A strong nodal accumulation of the synaptic vesicle membrane compartment could also be observed when an antiserum against the total of synaptic vesicle proteins is applied. In the crushed nerve the accumulation is particularly prominent at the proximal paranodal segment (Fig. 16) (see also Biiiij et al. 1986; Dahlstriim et al. 1992). This is presumably the result of a blockade of the axonal export of vesicular membrane constituents. A similar observation is made when the accumulation of membrane compartments containing the enzyme acetylcholinesterase is analyzed (Fig. 17). At present it is not clear whether acetylcholinesterase and protein components of the synaptic vesicle membrane reside in identical or functionally different membrane compartments when transported into the nerve terminal region.

Membrane Associated Proteins Torpedo electromotor nerves reveal also a nodal ac-

cumulation of o-rab3, an electric ray homologue of the mammalian small Gprotein rab3A (Fig. 6). This is of interest since the protein is associated with the vesicle surface by polyisoprenylation and it has been suggested that the association of rab3-proteins with vesicles is reversible (see references in Femnovick and Novick, 1993). Our result suggests that o-rab 3 is associated with the synaptic vesicle compartment at an early state of its biogenesis. On the other hand, no nodal accumulation of the vesicle surface associated synapsin I could be detected in normal electromotor axons. The interaction of synapsin I with the vesicle surface is reversible and phosphorylation controlled and may be restricted to the synaptic terminal. How- ever, in rat sciatic nerve and after a crush and bulk accumulation of transported synaptic vesicle material, synapsin I reveals also a nodal accumulation (Fig. 18) (B6oj et al. 1986).

Possible hrnctional Significance Obviously, membrane bound vesicular organelles

are not able to pass nodes of Ranvier unhindered. Nodes of Ranvier thus represent physiological axon constrictions and sites of repetitive (about every mm)

466 H. ZIMMERMA”

physiological accumulations of intraaxonal membrane compartments including that of synaptic vesicles. Analysis of nodal membrane structure and interactions is therefore expected to yield more reliable insights into axonal organelle structure and turnover than experimental ligation of the axon. Since the vesicular membrane compartments are accumulated at the proximal and distal paranode, both anterograde and retrograde membrane transport (Dahlstrom et al. 1992) must be involved in the accumulation. The labelled anterograde organelles either represent synaptic vesicles themselves or a membrane compartment from which vesicles will finally emerge. The retrograde material is expected to be more heterogeneous and to be- long to a large extent to the endosomal compartment. When an extracellular volume marker such as horseradish peroxidase is applied to the nerve terminals it is taken up into endocytotic vesicles and finally transported to the soma. As shown by Berthold and Mell- strom (1982, 1986) in the cat peripheral nerve, this results in a distal accumulation of horseradish peroxidase at consecutive nodes of Ranvier. The endocytosed enzyme once in the axonal compartment was detected in a variety of membrane-bounded organelles such as lamellar bodies, multivesicular bodies and vesiculotu- bular structures. These are the same types of organelles that were labelled by antibodies against the vesicle membrane protein SV2 in the axons of the electric ray (Janetzko et al., 1989). This indicates a functional connectivity of these organelles with the synaptic vesicle membrane compartment. Similar organelles were also found to contain acid phosphatase in alpha- motor and dorsal root ganglion cells of the cat (Gatzin- sky et al., 1988, 1991b). They were located mainly at nodes of Ranvier where they were more frequent distal than proximal to the nodal midlevel. The accumulation of the organelles a t the nodes may facilitate fusion of or exchange between the various intraaxonal organelles. On retrograde transport synaptic vesicles may enter prelysosomal compartments (Holtzman and Novikoff, 1965; Krikorian et al. 1980; Gatzinsky et al. 1988, 1991a; Holtzman, 1992) of increasing maturity by step- wise fusions from node to node. They may become degraded together with material that has been taken up by endocytosis from the terminal axon compartment. This is supported by the observation that the protein SV2 is found intact in the nerve terminal compartment but partially degraded in the axonal compartment (Jan- etzko et al., 1989). The exchange between membrane compartments is presumably regulated. The mechanism is unknown but activity-dependent influx of calcium into myelinated axons has been suggested (Lev- Ram and Grinvald, 1987). Within a 10 cm long nerve, the retrogradely transported synaptic vesicle membrane compartment would face nodal accumulation and membrane processing about 100 times before reaching the cell body. This applies also to the anterogradely transported vesicle membrane compartment. Little is known concerning a potential processing of this anterograde compartment. The small G-protein 0-rab3 might have a function in this. In non-myelinated neurons these processes may take place throughout the length of the axon. The well-insulated myelinated fibres may

have to rely on their nodal compartments for regulated membrane turnover.

At present it is not known whether synaptic vesicle protein containing membrane compartments can fuse with the nodal plasma membrane. It is, however, note- worthy, that in the CNS synaptic vesicle filled presyn- aptic compartments can originate directly fi-om the nodes where they form en passant synapses (see, for example, Nicol and Walmsley, 1991). Furthermore, axonal sprouting of lesioned peripheral nerves originates from nodes of Ranvier, demonstrating the dynamic potential of the nodal membrane compartment (Kato and Ide, 1994).

CONTRACTILE PROTEINS ARE CONCENTRATED AT NODES OF RANVIER The mechanisms that maintain the paranodal and

nodal constriction of the axon cylinder and control nodal width are not known. One would expect that the constriction needs to be actively maintained against a considerable hydrostatic pressure developed by the transported contents of the axon tube. Indeed, contrac-

Figs. 11-18. Structural aspects of nodes of Ranvier and of

Fig. 11. Longitudinal cryostat section with TRITC-phalloidin binding to a node of Ranvier (large arrow) and to Schmidt-Lanterman incisures (open arrows, with only select examples depicted). Thin arrows indicate labelling of Schwann cell cytoplasm.

Fig. 12. Longitudinal section as for Figure 11 with detail of a node of Ranvier showing “spiny bracelet of Nageotte” (arrow). Schmidt- Lanterman incisures are indicated by open arrows.

Fig. 13. Cross-section with TRITC-phalloidin binding to Schmidt- Lanterman incisures.

Fig. 14. Rat sciatic nerve immunostained with an antibody against the synaptic vesicle membrane protein synaptophysin (cryostat sections, indirect immunoperoxidase). Nodal and paranodal staining is depicted (arrows).

Fig. 15. As for Figure 14 but after ligation for 24 hours (2 mm proximal to the site of ligation). There is increased immunoreactivity in the axons with intense accumulation at individual nodes (arrows). Figures 14 and 15 have been kindly provided by Seiichiro Okajima (Kobe) and are reproduced from Okajima et a]., 1993, with permission of Elsevier Science Publishers.

Fig. 16. Ventral root crushed 3 hours before dissection. The fat arrow points towards the nerve terminal and is situated 1.5 mm proximal of the crush. Accumulation of synaptic vesicle membranes is particularly intense proximal of the nodes (arrows). It is depicted by indirect FITC-immunofluorescence using an antibody against the total of synaptic vesicle proteins isolated from the electric ray Narcine.

Fig. 17. As for Figure 16 but 6 hours after the crush and 2-3 mm proximal of the crush. Staining for acetylcholinesterase (enzyme his- tochemistry) is intense at the proximal side of the nodes (arrows) and very similar to that observed with the synaptic vesicle proteins (Figs. 16, 18).

Fig. 18. As for Figure 17 but application of an antibody against the surface associated synaptic vesicle protein synapsin I and with nodes (arrows) situated 3 mm proximal to the crush. Figures 16-18 have kindly been provided by Annica Dahlstram (Goteborg). Bars represent 10 p m (11-13), 20 pm (14,15), or 30 pm (16-18).

Schmidt-Lanterman incisures in axons of the rat sciatic nerve.


Figs. 11-18.

468 H. ZIMMERMANN

tile elements such as filamentous actin and myosin are rich at the nodes of Ranvier.

Phalloidin Labels Nodes of Ranvier and Schmidt-Lanterman Incisures

When studying the axonal cytoskeleton we found that application of tetraethylrhodamine isothiocyanate (TRJTC)-conjugated phalloidin that selectively binds to filamentous actin results in intense labelling of nodes of Ranvier. This provides an easy means of identifying nodes by fluorescence microscopy. In double labelling experiments using an additional fluorophor phalloidin can be successfully applied together with immunocytochemistry (Zimmermann and Vogt, 1989). In the electromotor nerve of Torpedo nodal phalloidin labelling expresses a typical pattern (Figs. 2,3). In longitudinal sections there is a strongly labelled central band at the nodal segment that displays discrete parallel subunits. In the paranodal segments there are several thinner bands surrounding the axon cylinder without interrup- tion. This paranodal structure o h n is symmetrical but can also be distorted and less symmetrical in other cases (Figs. 5,lO). In all cross-sectional views a ring of intense fluorescence becomes apparent at the nodal segment. Comparison with phase contrast images suggests that phalloidin-labelling corresponds mostly to extra-axonal, Schwann cell derived cellular elements. But it is possible that the cortical layer of the axolemma contributes to the labelling. This typical fluorescent pattern is only observed in the peripheral axon and is lost in the initial axon segment myelinated by oligodendroglial cells.

In rat sciatic axons, TRITC-phalloidin labels two prominent structures: nodes of Ranvier and Schmidt- Lanterman incisures (Figs. 11-13). Less intensely fluorescent structures include circumferential and longitudinal bands at the Schwann cell surface. Fluores- cence at the node of Ranvier often displays a cross-like structure (cross of Ranvier) with an intensely fluorescent band marking the nodal segment. There is a weaker and banded labelling at the paranodal segments (Fig. 12). This banded labelling corresponds to the previously described “spiny bracelet of Nageotte” (Nageotte 1922). Other nodes display only a central band within the nodal segment corresponding to the “cementing disc” (Ranvier, 1878; Hess and Young, 1952). It thus appears that the silver stains of the early investigators depicting the structure of nodes and incisures were mainly based on the distribution of filamentous actin in nerve fibres.

Labelling with FlTC-phalloidin of the sciatic nerve of the African claw frog (Xenopus Zaevis) yields nodal structures and incisures very similar to that of the rat sciatic nerve (Fig. 19). Also the nodes of Ranvier of the giant axons that innervate the electric organ of the electric catfish (MuZuptemrus electricus) reveal a band of intense phalloidin fluorescence (Fig. 20). These axons have a diameter of up to 50 pm (Janetzko et al., 1987). Schmidt-Lanterman incisures are apparent at regular intervals (Fig. 21).

The distinct pattern of phalloidin fluorescence at the nodes of Ranvier includes cytoplasmic specializations of the Schwann cell. In myelinated fibres of the periph-

eral nervous system the nodal segment is covered by cytoplasmic processes of the Schwann cell which may form a dense microvilli containing sheath (compare Fig. 23). This is particularly prominent at mammalian nodes (Berthold and Rydmark, 1983) but can also be observed at the nodes of Torpedo electromotor nerves (Janetzko et al., 1989). These microvillus-like Schwann cell processes are presumably rich in filamentous actin and provide the structural basis for the nodal phalloidin labelling. The fluorescent paranodal rings might then represent the actin-filament content in the periaxonal rings of Schwann cell pockets. Both Schwann cell pockets and incisures of Schmidt and Lanterman have previously been shown to contain circumferen- tially running microtubules (Hall and Williams, 1970). Owing to their contents in filamentous actin they now also have to be considered as potentially contractile elements of the myelin sheath.

Bulk of Actin and Myosin Whereas phalloidin labels only filamentous actin,

the total of axonal actin and myosin can be labelled by the use of antibodies. Both actin and myosin have been visualized by indirect immunofluorescence and are contained throughout the axoplasm of peripheral and central nerve fibres (Unsicker et al., 1978a,b; Drenck- hahn and Kaiser, 1983; Walker et al. 1985). A closer look revealed the presence of bulk actin and myosin also at the nodes of Ranvier (Zimmermann and Vogt, 1989). In Torpedo electromotor nerves, the fluorescence for both actin and myosin is enhanced at the nodes and paranodes but as compared to phalloidin labelling less structural detail is revealed (Figs. 7,8). The axon cylinder and the surface layer of the Schwann cell sheath in the internodal segments are also labelled. The same applies to the labelling of nodes of Ranvier at rat sciatic 8x0118 (Zimmermann and Vogt, 1989:~ There is increased fluorescence at the nodal segment. Axon cylinders and the peripheral layer of the Schwann cell sheath are more intensely labelled with the antibody than with phalloidin. Neither antibodies to actin nor to myosin label the incisures of Schmidt and Lanterman. Possibly, phalloidin can penetrate the myelin more easily than the larger antibodies.

INTRAAXONAL TUBULIN AND NEUROFILAMENTS

It is obvious that the constriction of the axon cylinder must have a strong impact on the distribution of lon- gitudinally extended cytoskeletal elements such as neurofilaments and microtubules. The constriction would result in a tremendous concentration of the filamentous structures and further impair axonal transport through the node. Indeed, progression of fluid injected into the axon is hindered at the node. That has previously led to the suggestion that there was a trans- verse septum in the nodal axoplasm (see references in Berthold, 1978).

The distribution of tubulin and neurofilaments as major filamentous elements of the nodal axoplasm has previously been investigated by electron microscopy. The number of microtubules in nodes of peripheral axons was found to stay constant (Tsukita and Ishikawa,


Figs. 19-21. Aspects of TRITC-labelled phalloidin labelling of

Fig. 19. Detection of nodes of Ranvier (arrows) and Schmidt-Lan- terman incisures (open arrows) by TRITC-labelled phalloidin in the sciatic nerve of the African clawfrog, Xenopus lueuis (longitudinal cryostat section, fluorescence).

Fig. 20. Nodal binding (arrow) in a collateral of the giant axon of the electromotoneuron of the electric catfish, Mulupterurus electricus (longitudinal cryostat section). The axon is surrounded by numerous sheaths of connective tissue (Janetzko et al., 1987).

Fig. 21. Incisures of Schmidt-Lanterman (thin arrows) in a collateral of the electric catfish giant electromotor axon depicted by TRITC- labelled phalloidin (longitudinal cryostat section, fluorescence). Bars represent 10 pm (19,20) or 20 pm (21).

nodes of Ranvier and of Schmidt-Lanterman incisures.

1981, mouse saphenous nerve) or to be even increased (Berthold, 1982; Reles and Friede, 1991; feline peripheral nerve and mouse sciatic nerve, respectively) re- sulting in considerable tubule packaging. Immunoflu- orescence using antibodies against P-tubulin revealed an increased immunofluorescence at nodes of Ranvier in Torpedo electromotor axons (Figs. 9,101 but not a t nodes of the rat sciatic nerve (Zimmermann and Vogt, 1989). The latter corresponds to the immunoelectron- microscopical analyses by Mata et al. (1992) and Hsieh et al. (1994). Neurofilaments are markedly decreased in number at nodes of Ranvier of mouse peripheral nerves indicating that most of them were discontinu- ous at the nodal region (Tsukita and Ishikawa, 1981; Reles and Friede, 1991; saphenous and sciatic nerve, respectively). Similarly, neurofilament numbers are reduced in avian peripheral nerves (Price et al., 1990, 1993). In the oculomotor nerve nodal neurofilament numbers were only 14-41% of that at the adjacent compact myelinated regions (Price et al., 1990). Nev- ertheless, neurofilament densities were still somewhat higher a t the nodal region (6-42%). Recently, Hsieh et al. (1994) reported that nodes of Ranvier at primary sensory neurons of the rat had three times less neurofilaments and 2 0 4 0 % less nearest neighbour spacing than internodes.

MEMBRANE-CYTOSKELETAL INTERACTIONS AT THE NODE

Membrane-cytoskeletal interactions at the node may serve two distinct functions. On the one hand they may be involved in arranging and preserving the supramo- lecular structure of the node including the concentration of molecular constituents essential for propagation of action potentials. Among these are the voltage-dependent sodium and potassium channels and the Na+/ K' -ATPase (see references in Baines, 1990; Ichimura and Ellisman, 1991). On the other hand, node and paranodes are sites of intense contact and presumably also functional interaction between the axon cylinder and cytoplasmic extensions of the Schwann cell. This axon Schwann cell network may also be important for gross axon-Schwann cell transfers, particularly during pathological conditions (Gatzinsky et al., 1988, 1991~). Subaxolemmal densities have long been identified as a peculiarity of the nodal region. By fine structural analysis a complex array of filamentous structures can be revealed at the paranodal axon-glia junction (Fig. 22) (Ichimura and Ellisman, 1991, rat sciatic nerve). This array provides a direct structural connection between the plasma membrane and microtubules and interme- diate filaments in both the axon and Schwann cell. In addition, a system of transcellular filaments links axon

470 H. ZIMMERMANN

Fig. 22. Diagram representing the membrane-cytoskeletal interaction at the node of Ranvier. Both the cytoplasm of the glial loops and the axon reveal focal attachment of cytoskeletal filaments to the nodal and paranodal membranes. Cytoskeletal filaments include microtubules, neuro and glial filaments, and finer filaments of various sizes. A subset of these filaments anchors to the cytoplasmic end of transmembrane structures. Nodal gap filaments in the node and the

bridging structures in the paranode link the axolemma to the membranes of the surrounding glia. Paranodal cisternae that may be involved in fluctuations of intracellular Ca2+-levels are also linked to the axolemma by protein filaments. (Reproduced from Ichimura and Ellisman, 1991, with permission of Chapman and Hall Ltd. Figure kindly provided by Marc H. Ellisman, San Diego).

and Schwann cell a t the node and paranode. Both, extracellular gap-crossing filaments and membrane-cytoskeletal linkers are joined to prominent membrane particles of the nodal axolemma. The arrangement of filamentous contacts is more complex at the paranode than at the node.

Less is known concerning the molecular identity of the various filamentous constituents observed. Specific adhesion molecules are essential for establishing and possibly maintaining the Schwann cell-axon contact. Interestingly, the extracellular matrix protein cytotactin that is involved in neuron-glia interaction is concentrated at nodes of Ranvier (Rieger et al., 1986). This also applies to the cell adhesion molecules Ng-CAM (neuron-glia cell adhesion molecule), N-CAM (neural cell adhesion molecule), and N-cadherin (a member of the calcium-dependent cell adhesion molecule family) (Rieger et al., 1986, sciatic nerves of adult chicken and mice; Cifuentes-Diaz et al., 1994, chick sciatic nerve). Similarly, myelin associated glycoprotein (MAG, a

member of the immunoglobulin gene superfamily of cell adhesion molecules) is localized in the node-paranode region (Martini and Schachner, 1986; Trapp et al., 1989; Martini, 1994). Members of the integrin family of adhesion receptors that mediate cell-cell and cell-extracellular matrix contact are enriched both at Ranvier paranodal areas and at Schmidt-Lanterman in cisures (Niessen et al., 1994, human peripheral nerve).

Proteins that represent linkers to the cytoskeleton and are candidates for controlling neuronal plasma membrane topography are spectrin and ankyrin. At the nodes of the Torpedo electromotor nerves a poly- clonal antibody against chicken spectrin reveals increased immunofluorescence. A similar accumulation is observed at nodes of axons from rabbit ventral roots (Koenig and Repasky, 1985). There spectrin colocalizes with F-actin. Spectrin can bind to actin filaments, microtubules, or neurofilaments and thus could link any of them to the plasma membrane. It may interact either directly with membrane proteins or indirectly via

VESICLE ACCUMULATION AND NODAL CYTOSKELETON 47 1

the intermediary protein ankyrin. An isoform of ankyrin was found to be selectively associated with the nodal region of rat sciatic nerves (Kordeli and Bennet, 1991). Ankyrin could link to axolemmal proteins such as voltage-gated sodium channels or the Na+/K+- ATPase and may connect them to filamentous proteins such as microtubules and neurofilaments (Baines, 1990). Could these protein components also be involved in the nodal constriction of the axon cylinder?

POTENTIAL MECHANISMS OF NODAL AXON CONSTRICTION

Two principal mechanisms have been considered. One assumes that there is an active constriction with participation of filamentous actin and myosin of the nodal Schwann cell compartment or possibly also the subcortical axolemma (Zimmermann and Vogt, 1989). The other emphasizes the functional interaction between Schwann cell and axon and postulates an intraaxonal mechanism that determines cylinder volume (Raine, 1982; de Waegh et al., 1992).

Contraction Model Our finding that filamentous actin and also myosin

are associated with the paranodal circumferential collars of the Schwann cell pockets and possibly also the cortical layer of the axoplasm suggests that these may exert an active contractile force. This would lead to a constriction of the axon cylinder preferentially at the paranodes (Fig. 23). Continued maintenance of the constriction against the moving contents of the axon cylinder is probably an energy consuming process. The very high concentrations of mitochondria in the paranodal cytoplasmic dilations of the Schwann cell (Lan- don and Hall, 1976; Berthold, 1978) could provide the necessary energy. Individual microtubules that were shown to follow the extension of the periaxonal cytoplasmic rings (see references in Landon and Hall, 1976) may contribute to stabilize the constriction. But the actin-myosin contractile system may not only be responsible for the constriction of the axon cylinder. Lon- gitudinal nodal contractions might also occur that control the width of the nodal gap. This could be mediated by the actin-rich finger- and brushborder-like nodal processes (Raine, 1982) of the mutually attached oppos- ing Schwann cells. Thus, nodal constriction and shape could be a variable of the metabolic state or other cellular conditions of the Schwann cell and possibly also the axon.

Also incisures of Schmidt and Lanterman are sites of local-albeit less pronounced-constrictions of the axon cylinder (Zimmermann and Vogt, 1989). Similar to the nodal segment there is an increased packing density of neurofilaments (Price et al., 1990, 1993). Furthermore, like nodes of Ranvier, Schmidt-Lanter- man incisures were shown to reduce transport of [3H]fucose labelled glycoproteins (Armstrong et al., 1987). Although this does not exclude other functions of the incisures, their high contents in filamentous actin suggest that they may be involved in actively constricting the axon cylinder a t these sites. The possibil- ity that axonal constrictions might be mediated by external compressive forces has also been suggested by

I / ,

Fig. 23. Schematic representation of the nodal axon constriction and of the “contraction model.” Filamentous actin and also myosin associated with the paranodal circumferential collars of the Schwann cell pockets may exert an active contractile force. Also longitudinal nodal contractions might occur (tMi) that control the width of the nodal gap. M1, cytoplasmic pockets of the Schwann cell formed by dilation of the myelin lamellae; My, compact myelin; Nc, nodal collar with Schwann cell microvilli.

Price et al. (1990,1993) based on their studies on axon diameter and neurofilament densities.

Schwann Cell Axon Interaction-Neurofilament Model

It has been noticed that an axon divested of its myelin sheath collapses to about 40% of its original diameter (Raine et al., 1969; Raine, 1982). On remyelination the internodal diameter of the axon cylinder increases slightly but it never returns to its original dimensions. From this it has been concluded that the presence of the myelin sheath permits the internodal axon to maintain a diameter greater than at the node. More recently the relation between neurofilament density, neurofilament phosphorylation, and axonal caliber was studied in detail in the dysmyelinating mutant Trem- bler mouse (de Waegh et al., 1992). Sciatic nerve axons of Trembler mice had a reduced average axonal caliber, a twofold increased density of neurofilaments, a decreased neurofilament phosphorylation, and reduced slow axonal transport velocities. Neurofilaments are generally homogeneously distributed within the axon and neurofilament numbers increase with axon caliber (Muma and Hoffman, 1993). Increased neurofilament spacing has been observed at internodes (Hsieh et al., 1994). Neurofilaments have characteristic sidearms and it has been suggested that the degree of extension of the sidearms away fiom the neurofilament core is increased by phosphorylation (see references in Nixon and Sihag, 1991; Muma and Hoffman, 1993). In turn, dephosphorylation would reduce spacing between neurofilaments. It has been hypothesized (de Waegh et al., 1992) that Schwann cell axon interactions act to mod- ulate a kinase-phosphatase cycle that is responsible for maintaining the phosphorylation state and thus the spacing in normal myelinated axons. A disruption of the myelin sheath would thus produce a net reduction in kinase to phosphatase activity, increase neurofilament density, and make the axon shrink. Such a mechanism might also be effective at the node of Ranvier where myelinization is interrupted at sites of contacts between two Schwann cells. Indeed, using an immunocytochemical approach, a reduction in the degree of phosphorylation of the neurofilament subunits was found at

472 H. ZIMMERMANN

nodes of Ranvier (Mata et al. 1992, rat sciatic nerves; Hsieh et al., 1994, rat primary sensory neurons). This demonstrates that neurofilaments within the nodal region are subject to posttranslational modification.

Open Questions Remain What mechanisms would regulate the interaction be-

tween filamentous actin and myelin at the node and possibly also at the incisures of Schmidt-Lanterman? How would neurofilaments be able to alter axonal volume? What force would transmit the alteration in neurofilament structure to a constriction of the axon cylinder that presumably exerts hydrostatic pressure? The major quantitative alteration in nodal neurofilaments is presumably not their increase in density but rather their considerable reduction in numbers (Tsu- kita and Ishikawa, 1981; Price et al. 1990; Reles and Friede, 1991; Hsieh et al. 1994). Thus, a large propor- tion of the neurofilaments appears to be actually dis- continued at the node. The myelin sheath is not interrupted at the incisures of Schmidt Lanterman where the axon cylinder is also indented. The paranodes as sites of the most intense axonal constriction are never- theless densely covered by Schwann cell collars. In contrast, the node that displays less intense contact with the Schwann cell generally is bulged rather than further constricted. But the relation between neurofilament phosphorylation and axon caliber further under- lines the dynamic potential of the Node of Ranvier. Both neurofilament structure and active constrictive forces may interact in shaping the structure of the node.

ACKNOWLEDGMENTS The support by the Deutsche Forschungsgemein-

schaft (SFB 169/A10) is gratefully acknowledged. I am greatly indebted to Annica Dahlstrom (Goteborg), Marc H. Ellisman (San Diego), and Seiichiro Okajima (Kobe) for their supply of micrographs and to Norbert Braun, Angela Hausinger, Kai Schafer, and Manfred Vogt (all Frankfurt) for their supply of unpublished material from their theses.

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