Cell Motility and the Cytoskeleton 22:81-91 (1992)

Views and Reviews Dynamics of Neuronal Intermediate Filaments: A Developmental Perspective Ralph A. Nixon and Thomas B. Shea Laboratories for Molecular Neuroscience, McLean Hospital, Haward Medical School, Belmont, Massachusetts

INTRODUCTION

Neuronal intermediate filaments are fast becoming well characterized at the molecular level. What they actually do in neurons and how they do it, however, are less clear. Their restricted cellular localization indicates that certain of their functions are uniquely neuronal. On the other hand, some basic features of neurofilament subunit structure are conserved among the various members of the intermediate filament protein family, suggesting that general principles of intermediate filament organization and dynamics may also apply to neurofilaments. New information from many laboratories now seems to be bearing out both of these predictions. A case in point is the newly discovered ability of certain class I11 intermediate filaments, vimentin and desmin, to assemble and disassemble reversibly and to reorganize rapidly as cells move and change shape [Skalli and Goldman, 19911. These discoveries have been leading some investigators to reconsider whether we may be overlooking a more dynamic side of neurofilaments-structures that have usually been considered to be irreversible polymers. In this review, we focus on what features of class I11 filaments dynamics and regulation apply to neurofilaments. Second, we consider how specializations of polypeptide structure and posttranslational modifications unique to neurofilaments may have evolved to stabilize the filament network and to serve better the purposes of the highly polar, postmitotic neuron. Some aspects of these themes have been reviewed recently [Julien and Grosveld, 1991; Nixon, 1991; Nixon and Sihag, 1991; Shaw, 1991; Skalli and Goldman, 1991; Steinert and Roop, 19881. The issue of neurofilament dynamics can be appreciated best from the perspective of the developing neural cell, which elaborates sequentially a series of transitional filament systems as it matures. At least four intermediate 0 1992 Wiley-Liss, Inc.

filament types, representing several of the five major intermediate filament classes defined by sequence analysis [Steinert and Roop, 19881, have been identified so far in central or peripheral neurons, including the neurofilament triplet (class IV), vimentin (class 111), peripherid57 kDa protein (class 111), a-internexidNF66 (probably class IV), and nestin (an apparent novel “sixth” class [Lendahl et al., 19901) (Fig. 1). Neurons may express certain of these subtypes at distinct stages of cellular differentiation (Fig. 2). Several intermediate filament subtypes may coexist in the same developing neuron, and, in some cases, one of these “transitional” forms may persist in mature neurons together with neurofilaments or may even remain the principal filament system throughout adulthood. The replacement of these transitional forms by neurofilaments and the subsequent modification of neurofilament composition and structure during development coincide with a shift in demand for plastic, intermediate networks to a need for more stable ones to support the highly polar morphologies characteristic of mature neurons. For the purposes of this review, the term neuronal intermediate filament will be used as the general term for intermediate filament systems when they are present in neurons, and neurofilament will refer to assemblies of the triplet proteins, NF-H, NF-M, and NF-L. STRUCTURAL DETERMINANTS OF NEUROFILAMENT DYNAMICS

The N-terminal head and C-terminal tail domains of intermediate filament subunits are considered to be Received December 5 , 1991; accepted January 8, 1992. Address reprint requests to Ralph A. Nixon, Laboratories for Molecular Neuroscience, McLean Hospital, Harvard Medical School, Belmont, MA 02178.

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Ne s tin Coil l a Coil l b

Coil 2

Coil l a Coil 1 b

Coil 2

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Vimentin C+&l f , . ~

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NC E segment

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E and KSP Segment KEP segment

Fig. 1. Domains of neuronal intermediate filaments. Coil 1 and coil 2 regions are conserved a-helical domains containing heptad repeats of hydrophobic amino acids. These regions are separated by rionhelical “links” (represented by separation of boxes) and, within coil 2, by single amino acid breaks in the heptad repeats (represented by vertical lines). Note that NF-M and NF-H possess a unique single, elongated coil I region, which may restrict their ability to form filaments in the absence of a “backbone” subunit such as NF-L. The initial twist of the carboxy-terminus, which constitutes the tail region of vimentin and peripherin, is followed by specialized domains in the other neuronal intermediate filament species as follows. The carboxy-terminus of nestin contains glutamic acid-rich segments (“E”) and -35 repeats of an 11 amino acid sequence. Nestin also possesses a relatively short

N-terminus. NF-66 and neurofilament triplet proteins also each contain one or more glutamic acid-rich regions (“E segments”). NF-66 also contains a region rich in lysine and glutamic acid (“KE segment”). NF-M and NF-H contain repeating regions of lysineserine-proline (“KSP segments”), which represent the sites of extensive phosphorylation of these two species. The extreme termini of NF-M and NF-H possess unique regions rich in lysine and glutamic acid (“ KE segment”) and in lysine, glutamic acid, and proline (“KEP segment”), respectively. The black boxes within the KE region of NF-M represent unique amino acid repeat sequences, which may be involved in binding interactions. (Adapted from Shaw, 1991, with permission of the publisher.)

important determinants of the filament’s potential for plasticity and stability. Recent studies show that the Nterminal head has at least one relatively conserved function related to subunit assembly/disassembly , which, in turn, is regulated by phosphorylation. In the case of vimentin, desmin, and glial fibrillary acidic protein, phosphorylation of the head domain by protein kinase A or protein kinase C in vitro blocks the polymerization of these subunits into filaments and induces assembled filaments to depolymerize [Inagaki et al., 1989, 1990; Kitamura et al., 19891, These effects are reversed by dephosphorylation. More recently it was shown that, as BHK21 cells enter mitosis, disassembly of the vimentin filament network is accompanied by hyperphosphorylation of the subunits at specific N-terminal sites by an

effector-independent kinase [Chou et al., 19901. Similar effects are seen with the type V intermediate filament system making up the nuclear lamina [Peter et al., 19901. The reversibility of type I11 IF subunit assembly in vitro finds an in vivo correlate not only in the cell cycledependent disassembly of filaments that occurs during mitosis [Celis et al., 1983; Evans and Fink, 19821 but also in the ability of soluble subunits to exchange with subunits in the assembled filament during normal cell maintenance and growth (Isaacs et al., 1989; Vikstrom et al., 1989). Which of these behaviors, if any, applies to neurofilaments? So far, comparable information is limited to in vitro studies involving only the core subunit of the low-molecular-weight subunit of neurofilaments, NF-L

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Fig. 2. Generalized developmental profile of intermediate filament species of neurons. The order of appearance of IF species and the accompanying annotations are compiled from hypotheses presented in detail within the text. Not all filament species are necessarily ex-

pressed by all neurons ( e.g ., peripherin and NF-66), and known and potential heteropolymers (e.g., peripherin or NF-66 and the neurofilament triplet proteins) are not represented.

[Angelides et al., 19891. Isolated NF-L subunits readily assemble into homopolymers, and subunits in these filaments can undergo exchange with soluble NF-L subunits in vitro [Angelides et al., 19891. As in the case of vimentin, the phosphorylation of the head domain by protein kinase A prevents NF-L assembly and induces the disassembly of NF-L filaments in vitro [Ando et al., 1989; Hisanaga et al., 1990; Nakamura et al., 19901. The major protein kinase A site on NF-L is localized on the head domain at serine 55 [Sihag and Nixon, 19911a site within a region of the head domain that DNA transfection experiments indicate may be essential for early steps in filament assembly [Gill et al., 19901. This site can incorporate phosphate in vivo and displays relatively rapid phosphate turnover soon after synthesis,

consistent with its having a role in an early behavior of neurofilament subunits [Sihag and Nixon, 19911. The presence of two arginine residues preceding serine 55 further supports the relevance of this site for assembly, since arginine residues on the N-terminal end of intermediate filaments play essential roles in assembly [Traub and Vorgias, 19891. The fact that vimentin, desmin, and NF-L all have protein kinase A-phosphorylated sites and arginine residues within the same 40-60 amino acid residue sequence [Geisler and Weber, 1988; Inagaki et al., 19891 suggests that the function served by this site is conserved. The relevance of the in vitro phenomena involving NF-L to the behavior of neurofilaments in the cell, however, is not easily interpreted. Although it has never been

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demonstrated directly, most neurofilaments in the mature neuron are presumed to be in the triplet subunit form [Willard and Simon, 1981; Hirokawa et al., 1984; Trojanowski et al., 19861. Indeed, the reversible assembly of class 111 intermediate filament subunits and NF-L homopolymers in vitro [Angelides et al., 19891 contrasts with the apparent in vitro and in vivo stability of the neurofilaments composed of all three subunits. For example, under the same hyperphosphorylation conditions that disassemble NF-L homopolymers, filaments containing the triplet proteins resist disassembly (Dr. Ram K. Sihag, personal communication). Moreover, subunit exchange has not yet been demonstrated for triplet protein polymers. The irreversible polymerization of most neurofilaments, at least in mature axons, is also suggested by the low percentage of neurofilaments subunits found in the soluble state in axon preparations [Morris and Lasek, 1982; Nixon et al., 19891. Supporting the notion that neurofilaments are transported mainly as assembled polymers is the fact that, although newly synthesized neurofilament proteins are Triton-soluble, >99% of this pool enters the axon in a Triton-insoluble form [Nixon et al., 19891 and in the same subunit stoichiometry as that found in bulk isolated axonal neurofilaments [Nixon and Lewis, 19861. On the other hand, greater amounts of soluble neurofilament subunits exist in cell bodies [Shea et al., 1988a, 1990, 1991al. The possibility therefore exists that reversible assembly or exchange of subunits is important in the perikaryon or dendrite, where the presence of NF-L homopolymers or other filaments with altered subunit stoichiometry has not been excluded. Based on the phosphate turnover kinetics and changes in solubility states of newly synthesized NF-L subunits, it has been suggested that reversible assembly of NF-L and subunit exchange may be needed early after synthesis to facilitate the proper docking and integration of NF-M and NF-H subunits during assembly of the triplet [Nixon and Sihag, 19911. The developing neuron is another case where the need for neurofilament plasticity is apparent, as is discussed below. The presence of two other high-molecular-weight subunits in the mature neurofilament is the most obvious feature that distinguishes the stable polymer from its reversibly polymerized class 111 relatives. How these subunits might stabilize the assembled states, if they do, is not clear. NF-M and NF-H do not form filaments by themselves [Liem and Hutchinson, 19821 even though both have conserved a-helical rod domains; however, NF-M and NF-H are unique among the neuronal intermediate filament proteins in lacking the “stagger” in their heptad structure in the coiled-coil region nearest the N terminus (Fig. 1) [Lees et al., 1988; Myers et al., 19871 and in having instead a single, long coiled coil.

Perhaps the unique rigidity of this area restricts the ability of NF-M and NF-H to form homopolymers and necessitates copolymerization of these subunits with a “backbone” filament subunit (e.g., NF-L). In addition to this difference, the head and tail domains of these subunits are highly specialized and under complex control by several kinase systems [Nixon and Sihag, 19911. At least six distinct phosphorylation sites have been identifed on the head domain of NF-M, most of which appear to be regulated by second messenger-dependent protein kinases [Sihag and Nixon, 19901. Certain of these sites completely turn over their phosphates in vivo without hours after synthesis, before neurofilaments have moved a considerable distance into the axon [Sihag and Nixon, 1989, 19911. It seems likely that some head domain sites are binding sites for other proteins, although none has yet been reported, and, remarkably, most of the binding sites for cytoskeletal proteins have so far been found on NF-L [Frappier et al., 1987, 1991; Heimann et al., 1985; Miyata et al., 19861. Another possible function of NF-M head domain sites regulated by second messenger-dependent kinases may be to stabilize the assembled state of the neurofilament and to reduce its sensitivity to phosphorylation-induced disassembly [Nixon and Sihag, 19911-a property that may be disadvantageous to the long-lived filaments that support the axon. A different type of neurofilament dynamics is governed in part by the phosphorylation state of the long, highly charged C-terminal tail regions of NF-M and NFH, which are peripherally situated on the filament and may correspond to the sidearms of neurofilaments [Pant et al., 1978; Julien and Mushynski, 1983; Sharp et al., 1982; Leterrier et al., 1982; Hirokawa et al., 19841. The phosphorylation of many sites on the tail domains, which is mediated by one or more neuron-specific cytoskeletonassociated kinases [Julien et al., 1983; Runge et al., 1981; Shecket and Lasek, 19821, is delayed until after neurofilaments have entered the axon and continues as neurofilaments are transported [Glicksman et al., 1987; Oblinger, 1987; Nixon et al., 19871. The gradual Cterminal tail phosphorylation that occurs as neurofilaments are transported is associated with the slowing of neurofilament transport [Watson et al., 1989, 19911 and with the integration of filaments into an essentially stationary, slowly turning-over cytoskeletal network in the axon [Lewis and Nixon, 1988; Nixon and Logvinenko, 19861. The phosphorylation process generates families of extensively phosphorylated isoforms of NF-H and NFM. The observation that the most extensively phosphorylated form of each subunit is associated with stationary neurofilaments [Lewis and Nixon, 19881 provided initial support for the idea that C-terminal tail phosphorylation regulates the relative affinity of neurofilament proteins for the transport mechanisms and for stationary struc-

Dynamics of Neuronal Intermediate Filaments

tures in the axon and therefore is one of the factors that controls the transport rate of neurofilament proteins [Nixon and Sihag, 1991; Watson et al., 19911. This process may be facilitated by extension of the C-terminal domains radially from the neurofilament core [Julien and Mushynski, 1983; Sharp et al., 1982; Leterrier et al., 1982; Hirokawa et al., 1984; Carden et al., 19871 or rigidification of this extended conformation by the addition of numerous charged groups to this region [Sternberger and Sternberger, 1983; Hagestedt et al., 19891. This process would be expected to hinder neurofilament movement sterically , possibly to reduce attachment to the transport vector, and to increase accessibility to new binding sites for cross-linking proteins all along the filament [Nixon and Sihag, 19911. Supporting this hypothesis are studies that neurofilament transport rates vary in the predicted ways when the phosphorylation state of the C-terminal domain of NF-H changes during axonal maturation and in neurotoxic states [Watson et al., 1989, 19911. Presumably, stationary neurofilaments are interconnected with microtubules and other structures via microtubule-associated proteins and membrane cytoskeleta1 proteins [Nixon, 1987; Nixon et al., 19901, since binding sites for these are present on neurofilament subunits [Frappier et al., 1987; Miyata et al., 1986; Heimann et al., 19851. The existence of multiple phosphorylated isoforms of NF-M and NF-H, some of which are preferentially associated with transported neurofilaments and others with stationary neurofilaments, raises the possibility that neurofilament networks may be capable of undergoing dynamic rearrangement by altering the strength and varieties of interactions with other cytoskeleton proteins by means of phosphateinduced charge alterations and conformational changes that expose particular binding sites [Eyer and Leterrier, 1988; Hisanaga and Hirokawa, 1989, 19901. The rearrangements of axonal cytoskeleton that accompany nerve stretching [Ochs et al., 19891 and the marked nonuniformity of neurofilament distribution at nodes of Ranvier and internodal regions [Reles and Friede, 19911 or at other specialized axonal regions imply a dynamic local regulation of the size and density of neurofilament networks even in the mature axon. Indeed, NF-H subunits appear to be less phosphorylated in neurofilaments at nodes of Ranvier than at those of internodal regions [Mata et al., 19921, in accordance with the suspected role of phosphorylation in regulating numbers of neurofilaments in axons [Nixon, 1992; Nixon and Sihag, 19911. During development, when an axon may need to stabilize a given trajectory toward its target, a shift from independent, moving neurofilaments to interconnected, stationary neurofilaments may also be quite important.

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TRANSITIONAL INTERMEDIATE FILAMENT NETWORKS IN NEURONS

The sequential appearance and disappearance of different subtypes of neuronal intermediate filament proteins during development (Fig. 2) can be partly understood by considering the changing demands for plasticity and stability of cell shape as neurons migrate, elaborate neurites, and establish a permanent fiber trajectory. Vimentin filaments, which are present in many mesenchyma1 cell types, are also abundant in most central nervous system neurons before they differentiate. These earliest stages of development are associated with the events of cell division, which involve nuclear rearrangements and changes in cell shape. The reversible assembly of vimentin [Chou et al., 19901 and the ability of its tail domain to attach to the nuclear envelope protein lamin B and to cytoskeleton and membrane proteins via linkages apparently regulated by phosphorylation [Foisner et a]. , 19911 are possible molecular correlates of this behavior. Vimentin is gradually replaced by neurofilament proteins shortly before neuronal precursor cells stop dividing [Cochard and Paulin, 1984; Tapscott et al., 19811, and, for a time, both filament protein types may coexist [Cochard and Paulin, 1984; Jacobs et al., 1982; Shea et al., 1988b; Shea and Nixon, 1988; Shea, 1990; Tapscott et al., 19811. A transient increase in vimentin is observed within developing neurites in neuroblastoma cells [Shea, 19901. Furthermore, treatment of these cells with vimentin antisense oligonucleotides and intracellular delivery of anti-vimentin antibodies, but not anti-NF-L, inhibits the initiation of neurite outgrowth [Shea et al., 1991b1, indicating an additional, unique role for vimentin in the initial stages of neuritogenesis. During this period, at least, the majority of vimentin is assembled into homopolymers and is not coassembled with neurofilament subunits [Shea and Nixon, 19881. By contrast, transfection studies of nonneuronal cells demonstrate that all three neurofilament subunits readily coassemble with vimentin [Chin and Liem, 1989, 1990; Monteiro and Cleveland, 1989; Soifer et al., 19911. These findings may indicate an ability of developing neuronal cells to segregate the assembly of different intermediate filament systems. A newly described intermediate filament protein, nestin, is also expressed early in embryonic development in neuroectoderm cells (neuronal stem cells and muscle cells) [Lendahl et a]. , 19901. Morphogenetic activity during this period is intense and includes the folding of the neural plate and the formation of highly elongated radial glia, upon which young neurons migrate. Although nestin retains the conserved core domain structure of other intermediate filament proteins, its intron

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placement and certain unusual features, including a large C-terminal tail, suggest that it defines a novel sixth class of intermediate filament proteins [Lendahl et a]. , 19901. The long C-terminal domain recalls a similar structural feature of neurofilament subunits that is believed to serve as a filament sidearm that increases the space between neurofilaments and enhances their ability to fill space and interconnect other cytoskeletal elements [Julien et al., 1988; Shaw, 19911. Nestin C-terminal side arms, by analogy, may help to maintain the long processes of the radial glia. Although these two intermediate filament systems are easily distinguished from neurofilament networks by their restricted appearance during development, the term neurofilament takes on broader meaning with the recent discovery of additional intermediate filament proteins that, as in the case of the neurofilament triplet, reside exclusively in neurons and may persist in some of these cells as a major component throughout life. One of these, peripherin (57 kDa protein) [Parysek et al., 1988; Portier et al., 19841, appears in some neurons, particularly those of the peripheral nervous system, after their final division and after migrating cells have reached their destinations [Escurat et al., 1990; Gorham et al., 19901. Peripherin expression declines in most of these neurons as they mature, but, unlike vimentin, it persists at some level throughout life in many of these same cells [Troy et a]., 19901. Like vimentin, it increases after axotomy, while expression of the neurofilament triplet decreases [Oblinger et al., 19891. These observations suggest a principal role of peripherin in facilitating axonal outgrowth. The domain structure of peripherin may indicate a potential for the kind of plasticity tentatively ascribed to type I11 intermediate filament systems. The rod domain and N-terminal helix domain show highest homology (65-75%) with vimentin and desmin. The N-terminal head domain contains high numbers of serine residues, suggesting the potential regulation of this region by phosphorylation, in analogy to NF-L, with which it shows about 28% homology [Leonard et al., 19881. Sites in this region are phosphorylated in vivo [Huc et al., 19891 and may be under regulation by extrinsic signals such as nerve growth factor (NGF) in some cells [Aletta et al., 19891. Significantly, peripherin lacks the highly acidic C-terminal domain characteristic of neurofilament proteins and instead shows a fairly high degree of homology with the C-terminal domains of vimentin and desmin (40%). Through this domain, peripherin, like vimentin and desmin, binds to nuclear lamin B [Djabali et al., 19911, which may be important within the period of intense protein synthesis during axonal outgrowth. One curious feature of most peripherin-containing neurons is their tendency to have long axons that project, at

least in part, outside of the central nervous system (CNS). Understanding what special problems an axon encounters when growing outside of the CNS should help to uncover peripherin’s special function. One intriguing association has been made between the presence of peripherin and the regenerative potential of peripheral axons [Wong and Oblinger, 19901. One might speculate that, in the absence of a long C-terminal tail, peripherin filaments are less capable of forming stationary networks. This is consistent with a diminishing role for peripherin as axons destined to be large-caliber fibers begin their radial growth spurt. If, in addition, they retain an ability to reversibly assemble and disassemble, peripherin filaments might provide a degree of structural support with minimal impedance to the transport of organelles along the axon and preserve the ability of axons to change directions or retract as they seek their targets. The advantages of these properties diminish as the axon becomes established, but, in long peripheral axons, facilitation of transport may require axonal domains in which the filament network is relatively plastic. Yet another neuronal intermediate filament protein, NF-66 (60-66 kDa molecular mass), has been recently identified by Chiu et al. [1989] and confirmed by sequencing data [Fliegner et al., 19901 to be identical to a previously described cytoskeletal protein, a-internexin [Pachter and Liem, 19851. This intermediate filament system persists in many neurons in the adult brain, however, and predominates in some cell types. Although it is widely distributed in the CNS, its distribution does not entirely overlap with that of neurofilament triplet proteins. NF66 has enough structural homology to the neurofilament proteins for it to qualify as a class IV intermediate filament protein [Chiu et al., 19901. Moreover, it has the ability to self-assemble like NF-L and has properties characteristic of NF-M, including a highly homologous ll-residue sequence (35-45) in the Nterminal head; a C-terminal tail rich in Glu, Lys, Ser and Thr residues; and high sequence homology in the extreme C-terminal area [Fliegner et al., 19901. These properties, and a close similarity to neurofilaments in its axonal transport kinetics and posttranslational modifications during transport [Kaplan et al., 1990; Nixon et a]. , 19901, raise the possibility that NF66 subunits may copolymerize with the neurofilament triplet protein. Differences in the ratio of NF-L and NF66 subunits along optic axons, however, imply that, if copolymerization occurs, the subunit stoichiometry is highly variable or that pools of NF66 filaments and NF66-triplet protein hybrid filaments exist in varying proportions along axons. In view of the reciprocal changes in levels of expression of NF-L and NF66 during development [Fliegner et a]., 19901, copolymerization would imply a considerable degree of neurofilament plasticity, includ-

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ing presumably subunit exchange. These possibilities have not yet been investigated. Apart from this issue, the NF66 subunits, which have the properties of both NF-L and NF-M, are suitable filaments as homopolymers to perform certain roles in small-caliber axons analogous to those of neurofilaments. DYNAMICS OF NEUROFILAMENTS DURING DEVELOPMENT

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uble NF-H subunits, including isoforms that are extensively phosphorylated [Shea et al., 1988b], and have been shown in pulse-chase radio-labeling studies to incorporate into axonal neurofilaments [Shea et al., 19901. Interestingly, substantial pools of soluble, extensively phosphorylated NF-H subunits were also detected in brains of young mice, and these pools decreased to low levels only in fully mature animals [Shea et al., 1991al. The initial appearance of NF-H is accompanied by up-regulation of NF-L and NF-M expression during the final phase of neuronal maturation. Intracellular delivery of anti-NF-H antisera into neuroblastoma had indicated that NF-H subunits are critical to the development of colchicine resistance of neurites. Thus, NF-H subunits mediate the establishment of a stable supporting structure that prevents the rapid retraction of the neurite [Shea et al., 1991bl. Increased neurofilament gene expression is followed by a gradual and complex series of phosphate additions to the C-terminal tails of NF-H and NF-M, which begin during the period when axons expand radially to their full size and myelination is proceeding [Foster et al., 1987; Dahl et al., 1986; Bignami and Dahl, 1987; Carden et al., 19871. These phosphorylation events appear to be intimately involved in the formation of the stationary network of neurofilaments and other cytoskeletal proteins that predominates in central axons [Nixon, 1987; Nixon and Logvinenko, 19861 and likely contributes to stabilization of axon structure [Dahl, 1988; Bignami and Dahl, 19871 and, together with increased neurofilament synthesis and export [Hoffman et al., 1984, 19871, to the radial growth of the axon. It has recently been observed, for example, that the axonal region of optic axons where C-terminal phosphorylation begins to be extensive marks a significant transition in the morphology of the axon and its supporting neurofilament network. Beginning at the level of the optic axon where extensively phosphory Iated isoforms of NF-H and NF-M first appear, the number of neurofilaments abruptly increases threefold, and the average distance between them becomes greater, resulting in a marked expansion of axon caliber [Nixon et al., 19911.

Neurofilaments first appear at embryonic stages when neurites have extended but have not reached their target. Most if not all central neurons first express an incomplete set of neurofilament proteins (NF-L and NFM) at relatively low levels before going on to express NF-H. The tightly coordinated expression of the NF-L and NF-M genes in different cells with varying schedules of development has suggested that these two subunits are assembled together into an “immature” filament [Carden et al., 1987; Scott et al., 19851. Although the subsequent conversion to a three-subunit filament is fairly generalized [Trojanowski et al., 19861, two subunit assemblies may persist in some neurons [Shaw et al., 1981; Shaw and Weber, 19821, possibly reflecting a continued need for some greater plasticity of the filament network in these axons [Carden et al., 19871. These early-appearing neurofilament proteins might form relatively unstable structures, since neurofilaments are not commonly seen ultrastructurally until well after all three subunits are detected immunochemically [Pachter and Liem, 19841. Oligomeric ‘‘assembly intermediates” have in fact been observed in several neuronal systems [Weisenberg et al., 1987; Shea et al., 19901. The addition of NF-H is a gradual process beginning only after the axon is fairly well established and has likely formed synaptic contacts [Carden et al., 19871. It is still an open question, when NF-subunits appear, whether they are recruited into preexisting “immature” filaments. Also unanswered is whether NF-H subunits exchange between soluble and filamentous pools once the triplet is assembled. Some evidence, particularly from the immature neuron, supports this idea [Hirokawa et al., 1984; Scott et al., 19851. The ratio of NF-H subunits to NF-L and NF-M increases during development. SUMMARY: FROM PLASTIC TO STABLE The number of binding sites for this subunit is smaller INTERMEDIATE FILAMENT NETWORKS than the actual number of NF-H subunits bound as deFrom these studies we see that a succession of neutermined from in vitro assembly studies of isolated filaments. This holds true even in adults [Scott et al., 19851. ronal intermediate filament systems facilitates the tranThe stoichiometry of NF-H relative to the other subunits sitions of the developing neuron from a highly plastic, varies somewhat in different species [Scott et al., 19851, mitotic, and sometimes motile cell to a postmitotic, in different neuronal cell lines [Lee and Page, 1984; Lee, highly polar cell with relatively fixed morphology. The 1986; Lee et al., 1991; Shea et al., 1988a1, and in dif- earliest phases of neuroblast multiplication and migration ferent regions of the neuron [Hirokawa et al., 19841. involve class I11 intermediate filament systems endowed Mouse neuroblastoma cells contain a large pool of sol- with properties of subunit exchange and reversible as-

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ment-11. Immunofluorescence study of rat spinal cord ,and cerebellum with axon-specific neurofilament antibodies. Int. J. Dev. Neurosci. 5:29-37. Carden, M. J., Trojanowski, J.Q., Schlaepfer, W. W., and Lee, V.M.Y. (1987): Two-stage expression of neurofilament polypeptides during rat neurogenesis with early establishment of adult phosphorylation patterns. J. Neurosci. 7:3489-3504. Celis, J.E., Larsen, P.M., Fey, S.J., and Celis, A. (1983): Phosphorylation of keratin and vimentin polypeptides in normal and transformed mitotic human epithelial amino cells: Behavior of keratin and vimentin filaments during mitosis. J. Cell Biol. 97: 1429-1 434. Chin, S.S.M., and Liem, R.K.H. (1989) Expression of rat neurofilament proteins NF-L and NF-M n transfected non-neuronal cells. Eur. J . Cell Biol. 50:475-490. Chin, S.S.M., and Liem, R.K.H. (1990): Transfected rat high POlecular weight neurofilament (NF-H) co-assembles with vimentin in a predominantly non-phosphorylated form. J. Neurosci. 10:3714-3726. Chiu, F.-C., Barnes, E.A., Das, K., Haley, J., Socolow, P., Macaluso, F.P., and Fant, J. (1989): Characterization of a novel 66 kd subunit of mammalian neurofilaments. Neuron 2:14351445. Chou, C.-H., Rosevear, E., and Goldman, R.D. (1990): Phosphorylation and disassembly of intermediate filaments in mitotic cells. Roc. Natl. Acad. Sci. USA 86:1885-1889. Cochard, P., and Paulin, D. (1984): Initial expression of neurofilaments and vimentin in the central and peripheral nervous system of the mouse embryo in vivo. J . Neurosci. 4:2080-2094. Dahl, D. (1988): Early and late appearance of neurofilament phosphorylated epitopes in rat nervous system development: In vivo and in vitro study with monoclonal antibodies. J . Neurosci. Res. 20:431-441. Dahl, D., Crosby, C.J., Gardner, E.E., and Bignami, A. (1986): Delayed phosphorylation of the largest neurofilament protein in rat optic nerve development. J. Neurosci. Res. 15:513-519. Djabali, K., Portier, M., Gors, R., Blobel, G., and Georgatos, S.D. (1991): Network antibodies identify nuclear lamin B as a physiological attachment site for peripherin intermediate filaments. Cell 64:109-121. ACKNOWLEDGMENTS Escurat, M., Djabali, K., Gumpel, M., Gros, F., and Portier, M.M. (1990): Differential expression of two neuronal intermediateWe thank Drs. F.-C. Chui, I. Fischer, and R.K. filament proteins, peripherin and the low-molecular mass neuSihag for helpful discussions. We are also grateful to J. rofilament protein (NF-L), during the development of the rat. J . Khan and C. Jefferson for help in preparing the manuNeurosci. 10:769-784. script and to John Gawrn for assistance with computer Evans, R.M., and Fink, L.M. (1982): An alteration in the phosphorgraphics. The authors’ work was supported by grants ylation of vimentin-type intermediate filaments is associated with mitosis in cultured mammalian cells. Cell 29:43-52. from NIA, the NSF, and the Alzheimer’s Disease and Eyer, J., and Letemer, J-F. (1988): Influence of the phosphorylation Related Disorders Association. state of neurofilament proteins on the interactions between purified filaments in vitro. J. Biochem. 252:655-660. Fliegner, K.H., Ching, G.Y., and Liem, R.K.H. (1990): The preREFERENCES dicted amino acid sequence of u-internexin is that of a novel neuronal intermediate filament protein. EMBO J. 9:749-755. Aletta, J.M., Shelanski, M.L., and Greene, L.A. (1989): Phosphorylation of the peripherin 58-kDa neuronal intermediate filament Foisner, R., Traub, P., and Wiche, G. (1991): Protein kinase A- and protein. J . Biol. Chem. 264:4619-4627. protein kinase C-regulated interaction of plectin with lamin B and vimentin. Proc. Natl. Acad. Sci. USA 88:3812-3816. Ando, S . , Tanabe, K., Gonda, Y., Sato, C., and Inagaki, M. (1989): Domain- and sequence-specific phosphorylation of vimentin Foster, G.A., Dahl, D., and Lee, V.M.-Y. (1987): Temporal and induces disassembly of the filament structure. Biochemistry topographic relationships between the phosphorylated and non2812974-2979, phosphorylated epitopes of the 200 kDa neurofilament protein Angelides, K.J., Smith, K.E., and Takeda, M. (1989): Assembly and during development in vitro. J. Neurosci. 7:2651-2663. exchange of intermediate filament proteins of neurons: Neuro- Frappier, T., Regnouf, F., and Pradel, L.A. (1987): Binding of brain filaments are dynamic structures. J. Cell Biol. 108:1495-1506. spectrin to the 70 kDa neurofilament subunit protein. Eur. J. Bignami, A , , and Dahl, D. (1987): Axonal maturation in developBiochem. 109:651-657.

sembly . As differentiation begins and neurite outgrowth is initiated, cell morphology is still highly plastic since neurites constantly advance, retract, and change direction as they seek their targets. New dynamics are required for the support of extreme polar morphologies and to transport filaments or their subunits considerable distances from their site of synthesis. For this, transitional filament networks are invoked, some retaining mostly class I11 features (e.g., peripherin), vimentin, and others evolving C-terminal specializations of class IV filaments (e.g., NF66). Still unclear is whether these transitional networks also have the ability to assemble and disassemble reversibly and to rearrange with the same facility as vimentin networks. In completely differentiated neurons, where the fully grown and established axon may constitute >99% of the cytoplasmic volume, the neurofilament network must also assume space-occupying and permanent structural support roles. Although “immature” forms of the neurofilament may retain some plasticity, there is little evidence so far that triplet protein assemblies are reversibly polymerized, or need to be, to perform their functions. The possibility cannot be excluded, however, on the basis of existing data. Cross-linking of neurofilaments to each other, or to other stationary cytoskeletal structures [Shaw and Hou, 19901, could further enhance network stability. Reversible cross links between neurofilaments and other axonal structures may be a different level of dynamic neurofilament behavior that allows for regional specializations of the cytoskeleton at nodes of Ranvier and other local domains of the neuron.

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