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Neuronal intermediate filaments
Neuronal intermediate filaments R.K.H. Liem Departments of Pathology and Anatomy and Cell Biology, Columbia University College of Physicians and Surgeons, New York, New York, USA Current Opinion in Cell Biology' 1990, 2:86-90
Introduction Neuronal intermediate filaments (IFs) are 8-10 nm filaments found in most neurons of the central nervous system (CNS) and peripheral nervous systems (PNS). Hoffman and Lasek Or Cell Biol 1975, 66:351-366) first inferred, from axonal transport studies, that neurofilaments (NFs) consist of three polypeptides. These were called the NF triplet, and it was found by sodium dodecytsulfatepolyacrylamide gel electrophoresis that they had molecular weights of 200, 145 and 68kD. Biochemical and immunochemical studies later confirmed the classification of these three polypeptides as NF proteins (Liem et al., J Cell Bio11978, 79:637--643; Schlaepfer and Freeman, J Cell Biol 1978, 78:369-378). Further biochemical studies have also shown that, of the three polypeptides, only the smallest (referred to as NF.L, for low molecular weight subunit) was necessary for the formation of an IF in vitro, and that the other two (NF-M and NFH, for middle and high molecular weight subunits) appeared to be accessory proteins (Liem and Hutchison, Bioclaemistry 1982, 21:3221-3226; Geisler and Weber, J MolBiol1981, 151:565-571). Over the past few years, this relatively simple picture of mammalian NFs has become more complicated. Two additional proteins have been purified, cloned and sequenced and subsequently identified as independent IF systems in neurons, with, presumably, different functions. One of them, named peripherin, is found mainly in the PNS (for review, see [ 1 ]), but the second, named cx-internexin, is found mainly in the CNS (Pachter and Hem, J Cell Bio11985, 101:1316-1322) [2]. This review will describe our current knowledge of these ~'e neuronal IF proteins.
in which the 'a' and 'd' residues are frequently apolar, and it is believed that the basic IF dimeric coiled-coil structure is formed by interactions bem'een these apolar residues. All the neuronal IFs have this basic motif. The NF triplet proteins (NF-L, NF-bl and NF-H) have highly charged C-terminal tail regions and a region rich in glutamic acid, but in the case of NF-H, it is considerably shorter than in the other two NF proteins. Both NF-M and NF-H are heavily phosphorylated in their C-terminal tails: 9-26 and 20-100 phosphate groups, respectively, have been reported (Julien and Mushynski, J Biol Cbem 1982, 257:10467-10470; Jones and Williams, J Biol Chem 1982, 257:9902-9905; Carden et al., J Biol Chem 1985, 260:9805-9817). The consensus sequence for phosphorylation has been identified by chemical and immunological means as lys-ser-pro (KSP in the single letter code; Lee et al., Proc Natl Acad Sci USA 1988, 85:1998-2002). In rat NF-M, this sequence appears five times (Napolitano et al., J Neurosci 1987, 7:2590-2599). In human NF-M, there is a direct repeat which includes the KSP sequence and as a result this sequence appears 13 times (Mc3'ers et al., FallBOJ 1987, 6:1617-1626). Human [4] and mouse [5] NF-H hm'e been cloned and sequenced, and the KSP sequence appears an astonishing 50 times. The sequence of peripherin bears more similarity to the non-neuronal IF proteins, vimentin, desmin and glial fibrillary acidic protein than to NF proteins (Leonard et al., J Cell Biol 1988, 106:181-193) and the tail region shows no homology with the NF triplet proteins. Finally, ct-intemexin is more closely related to the NF triplet proteins than to either peripherin or the other IF proteins (Fliegner et al., EMBOJ 1990, in press). Although smaller than the other NF proteins, it also has a C-terminal tail which is rich in glutamic acid. NF-L, peripherin and cx-intemexin do not have the KSP sequence, but they may be phosphorylated on other serine residues.
Primary sequences All IF have a three-domain structure with ~riable N-terminal and C-terminal head and tail regions flanking a conserved cx-helical domain (for review see [3]). The whelica] domain consists of "--310 amino acids and is cornposed of two cx-helical coils separated by a linker region. Within each coil a heptad motif can be discerned
Molecular biology Genomic sequences have now been published for all the neuronal IF proteins except cx-internexin. The intron-exon arrangements of IF protein genes have been
Abbreviations CNS--central nervous system;IF-~intermediatefilament; MAP--microtubule associated protein; NF(L~4,H)--neurofilament(low, medium, high molecular weight); NGF--nerve growth factor; PNSwperipheralnervous system. 86
(~) Current Science Ltd ISSN 0955-0674
Neonatal intermediate filaments Liem 87 used to classify them into different types [3]. V'unentin, desmin and glial fibrillary acidic protein are known as type III IF proteins and their genes all have eight introns in identical positions. Peripherin has the highest sequence homology with these IF proteins and its gene has eight introns in exactly the same positions, demonstrating that it is also a type III IF protein [6]. The NF triple! protein genes, which encode type IV IF proteins, have introns in different positions [4,5]. Two introns are conserved among the genes of all three proteins, but NFL and NF-H genes each have third introns, albeit in different positions. Interestingly, the poSlUOn of this third intron in the NF-H gene is very close to the eighth intron in genes of the type III IFs. Preliminary data from our laboratory suggest that 0c-intemexin is in the same class as the NF triplet proteins (Ching and Hem, "unpublished data), although a final classification will require further sequencing of the gene. One of the more interesting aspects of the different neuronal IF proteins is that their expression is both tissue-specific and developmentally regulated. Peripherin is found mainly in the PNS, 0~-internexin mainly in the CNS and the NF triplet proteins in both the CNS and the PNS. Our data show that 0c-intemexin precedes NF-L and NF-M in the developing CNS (Kaplan et aL, J Cell Bio11989, 109:729) and NF-H is the last neuronal IF to be expressed during mammalian development (Shaw and Weber, Nature 1982, 298:277-279; Pachter and Hem, Dev Biol 1984, 103:2(X)-210). The tissue-specific and temporally regulated expression of these genes suggests that they contain important regulatory elements. Although some limited homologies were reported between the promoter regions of the peripherin gene and those of NF-L and NF-M [6], no obvious common elements can be discerned in the 5° regions of the genes for the neuronal IF proteins. A number of studies, including experiments involving transgenic mice, are underway to investigate the tissue-specificity of the expression of these genes. In one study already published, the human NF-L gene was expressed in transgenic mice, and the human NF-L protein was shown to be co-expressed with mouse NFs (Julien et al., Genes Dev 1987, 1:1085-1095). This study indicates that the human gene encodes c~acting elements that produce tissue specificity. Interestingly, the expression was somewhat delayed. Further studies using segments of the 5' upstream regions of these neuronal IF proteins linked to reporter genes are being performed in a number of laboratories. •
•~'l
Assembly and expression of NFs in fibroblasts As mentioned above, only NF-L is necessary for the formarion of an IF #z vitro and in most cases, neither NF-M, nor NF-H are able to polymerize into IFs in the absence of NF-L It has been shown that peripherin and ¢z-internexin are both able to form into homopolymers (Parysek and Goldman, JNeurosci 1987, 7:781-791) [2]. In attempts to determine how each of the NF triplet proteins alone would behave #l vivo, we (Chin and Hem, E u r J Cell Biol, 1989, 50: 475-490) and blonteiro and Cleve-
land [7] transfected each of the genes or complementary DNAs for these proteins into fibroblasts. In all three cases, the NF triplet proteins copolymerized with the endogenous vimentin cytoskeleton. The time course for these events was studied in transient transfection studies and an apparent organization center was observed during the early stage, from which the filaments radiated later. Examples of these experiments are shown in Fig. 1. A similar organization center was observed for virnentin filaments when biotinylated vimentin was miocroinjected into fibroblasts [8]. An additional point of potential importance should be noted. When blonteiro and Cleveland [7] transfected L cells with a genomic clone for NF-L, they obtained stable cell lines, which expressed large amounts of NF-L protein. However, in similar experiments using a complementary DNA with a larger 5' untranslated region, we obtained stable cell lines which expressed abundant NF-L message, but.relatively little protein. These studies suggest that the 5' untranslated region in the NF-L messenger RNA may have an additional role in regulation of NF-L translation in neurons• NF proteins are synthesized and assembled in the cell body and transported into the axon. The existence of a focal point for the organization of IFs in fibroblasts is consistent with the observation in PC12 cells that peripherin, NF-L and NF-M appear as a juxtanuclear cap in non-process-beating cells• These cells radiate from this cap into the processes when the cells are treated with nerve growth factor (NGF; Lee and Page, JNeurosci 1984, 4:1705-1714; Parysek and Goldman, 1987). It is still not known whether the NFs are dynamic once they are assembled, but recent evidence has shown that NFs can exchange their subunits in vitro, presumably through a wall-exchange between the polymer and a soluble pool [9]. These results suggest that, even in the axon, there may be a local exchange of subunits allowing the NFs to be dynamic.
Phosphorylation Recent studies have shown that phosphorytation of vimentin, desmin and the nuclear lamins is important for the depolymerization of filaments, especially during mitosis [3]. Neurons do not generally divide and so the phosphorytation of neuronal IF proteins probably involves different functions. The phosphorylation of peripherin in PCl2 cells is increased two- to threefold by treating the cells with NGF [10]. The small proportion of cellular peripherin that is soluble is not phosphorylated, which may mean that phosphorylation of peripherin promotes assembly rather than disassembly. Phosphorytation of NF-L dn distinct domains by different protein kinases has also been reported recently [11]. It is likely that NF-H is phosphylated on the KSP sequence by a NF kinase, which has a high specificity for NF-H and which would not, therefore, be able to phosphorylate other phosphoproteins. There has been a report of the purification of a NF kinase which has these characteristics
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Ii
(a)
(b)
(c)
(d)
Fig. 1. Distribution of neurofilament proteins in cells transiently transfected with a neurofilament low molecular weight and medium molecular weight subunit (NF-L and NF-M) construct at different time points. (a) L tk-cel[s stained with a monoclonal antibody against NF-L 24 h post-transfection with a full complementary DNA encoding NF-L cloned behind a Rous sarcoma virus promoter. (b) Similar staining at 48 h post-transfection. (c) Cos 1 cells transiently transfected with a NF-M complementary DNA construct cloned behind an simian virus 40 promoter and stained with a rnonoclonal antibody against NF-M 48 h post-transfection. (d) Cos I cells similarly stained as in (c) 72 h after transfection. Details of these experiments are given in Chin and Liem (£ur / Cell Biol, 1989 50: 475--490). Note the juxtanuclear focus in both cell types at the earlier time points, from which filaments appear to radiate. Data courtesy of 5teven Chin (private communication) and published by permission.
Neonatal intermediatefilaments Liem 89 [12] and a number of other kinases have been reported that phosphorylate NF-M, but phosphorylate NF-H only poorly. Antibodies specific to either the non-phosphorylated or phosphorylated proteins are important tools in the study of the phosphorylation of NFs. Studies using such antibc~lies showed that phosphorytated NF-H and NF-M epitopes were found in axons, while non-phosphorylated epitopes were primarily .in located cell bodies (Stemberger and Sternberger, Proc Natl Acad Sci USA 1983, 80:6126-6130). Further studies have also shown that there multiple phosphorylated variants of NF-M and NFH, both in cultured cells [13] and in the axons of retinal cell neurons [14]. This suggests that there are constant changes in the phosphorylation state of NFs in the axon and that the kinase is transported together with the NFs. Transfection experiments also show that NF-H is phosphylated by ldnases other than those in fibroblasts, since the NF-H proteins that are made in the transfected cells are not recognized by antibodies against phosphorylated NF-H (Chin and Hem, submitted for publication). Interestingly, NF-M in transfected fibroblasts can be recognized by an antibody specific for phosphorytated NFs, suggesting that the KSP sequence on NF-M may be phosphorytated by ldnases present in fibroblasts. It has been suggested previously that phosphorytation of NF-H may be important for the formation of crossbridges between filaments. However. a recent study by Hirokawa's laboratory showed that non-phosphorylated NF-H was still capable of forming cross-bridges between filaments [15]. Carden et al. (J Neurosci 1987, 7:3489-3504) proposed that phosphorylation may, in fact, cause repulsion between between filaments, which may be a way of increasing axonal diameter (see below) during development. Now that a NF kinase has been identiffed, the role of NF-H phosphorytation may be established in the near future.
Neurofilaments and axonal diameter There is a good correlation between the number of NFs and axonal diameter, suggesting that the NF triplet proteins may determine axonal caliber (for review, see Iasek et al., Cold Spring Harb Syrup Quant Biol 1983, 48:731-744). This is a reasonable hypothesis since NFs increase continuously during development, and decrease after axotomy, when the neurons degenerate (Hoffman et aL, J Cell Bio11985, 101:1332-1340). Interestingly, the messenger RNA levels for NF-L have also been shown to decrease after axotomy (Hoffman et at, Proc Natl Acad USA 1987, 84: 3472-3476) [16], demonstrating that the decrease in NFs is not due to a transport blockade but to a decrease in protein ~3mthesis, and that a retrogradely transported factor, which is activated by the axotomy, may be required to block the transcription of NFs. Studies linking axonal diameter and NF content have overlooked the two recently identified neuronal IFs. Is the
abundance of these also correlated with axonal diameter? Several lines of evidence argue against this possibility and suggest that these proteins may be important in process outgrowth. One is the early appearance of a-intemexin during CNS development (Kaplan et al., J Cell Bio11989, 109:729). Another is the fact that peripherin messenger RNA and protein levels increase after axotomy, indicating that peripherin may be important in process outgrowth during regeneration (Shelanski, personal communication; Oblinger et al., J Neurosci 1989, 9: 3766--3775). However, it is still not known how the functions of these two new neuronal IFs differ and why they have different distributions.
Interactions between NFs and microtubules In our discussion so far we have only considered crossbridging between NFs. In fact, there is an extensive system of cross-bridges between various cytoskeletal elements in nerve cells, including microtubule-- neurofilament cross-bridges. Interactions between microtubules and NFs in vitro have been demonstrated in binding studies (Leterrier et al., J Cell Biol 1982, 95:982-986; Heimann et al., J Biol Chem 1985, 260:2160-2166), and in viscosity studies (Aamodt and Williams, Bix.hemt2try 1984, 23:6023-6031). These interactions have been shown to depend on the microtubule associated proteins (MAPs). In a recent paper, Hirokawa and co-workers [17] showed that MAP2 is a component of cross-bridges between microtubules and NFs. In vitro studies have also shown that tau can bind to NFs (Shelanski et al., NRP Res Bull 1981, 19:32-43), and interactions between MAP1 and NFs can be inferred from direct binding studies (Heimann et al.,JBiol ~ m 1985, 260:2160-2166). Thus, NF-microtubule interactions appear to be mediated by various MAPs. Flynn et al. ( Biochem Biopbys Res Comm 1987, 148:1453-1459), in a recent study, showed that the same domain of MAP2 could bind to both microtubules and NFs, suggesting that cross-bridges require more than one MAP molecule. MAPs interact with a 4 kD carboxyterminal fragment of tubulin which is rich in glutamic acid (Serrano et aZ, Bioc.bemistry 1984, 23:4657-4681). It is likely that the presence of glutamic acid-rich regions on NF proteins could also be responsible for these interactions. With the development of molecular biological probes for all of these molecules, and the ability to make truncated forms of these proteins in vitro, we should soon be able to understand the nature of these interactions in more detail.
Acknowledgements I thank the members of my laboratory,especiallySte~en Chin, Gee Ching, Karsten Fliegner, David Green and MichaelKaplan for their help vdth this article and for providing the recent data presented here.
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Annotated references and recommended
reading • ••
Of interest Of outstanding interest
A fluorescence energy transfer assay is used to show that l',q~ subunit exchange is preceded by dissociation of subunits from the filament and the generation o f a kineticalb" active pool of soluble subunits. The authors also suggest, but do not show, that phosphotylation may be im. portant for the ob~namic assembly and disassembb' o f NFs.
1. GREEr~'EI.A: A n e w neuronal intermediate filament protein. • Trends Neurosci 1989, 12:288-230. A reMew describing the history of the discm~et3; rediscox~ery and somewhat roundabout cloning of the 57 kD IF protein, also known as peripherin.
#a.ETrhJM, StIELA.':SKIML, G~Eh'E LA: Phosphorylation of the peripherin 58-kDa neuronal intermediate protein. Regulation by nerve growth factor and other agents. J Biol Cbem 1989, 264:4619--4627. The phosphorylation of peripherin in PC 12 ceils and its regulation by a number of different factors is described. The results suggest that phosphowIation of peripherin may be important for its assembb"
2. •
11. •
Cmu FC, B.~ffO,T_SEA, DAS K, t ~ J, Socotow P, M,,camso FP, FA.,rr J: Characterization of a novel 66 kd subunit of mammalian NFs. Neuron 1989, 2:1435-1445. The isolation o f a 66 kD neuronal IF protein, which can seLf.pobmerize and is found primarily in the CNS, is described. That this protein is the same as et-intemexin, is now substantiated by the fact that the predicted protein sequence o f ~-intemexin, based on its complemental" DNA, contains a peptide sequence reported in this paper. 3. STEL\'ERTPM, ROOP DR: Molecular and cellular biology of •• intermediate filaments. Annu Rev BiodJem 1988, 5Z593-626. An excel/ent and comprehensh'e review on IFs. 4. •
LEES JF, SfL'x'EIDSL~.NPS, SKUN'rZ SF, CARDEN MJ, LAZZARINIRA: The structure and organization of the human heavy NF subunit (NF-H) and the gene encoding it. EMBO J 1988, 7:1947-1956. The complete amino acid sequence for human NF-tt based on genomic clones shog~ the presence of the phosphot3Otion consensus sequence KSP greater than 40 times. From the intron-exon arrangement for the NF-H gene, it is exSdent that the third intron in this gene nearb" matches the position o f one of the conser~x~d introns in the nonneuronal IF genes. 5. •
JUL/ENJP, COTI~ F, BEAUDET L, SIDKY M, FIAVEI£ D, GROS'.XLD F, MUStWNSKIW: Sequence and structure of the mouse gene coding for the largest NF subunit. Gene 1988, 68:307-314. The complete nucleotide sequence of the mouse gene encoding NF-II is described in this paper, ~fffich xras published at the same time as [4]. The findings are similar to the results described in [4]; the KSP sequence is found 51 times. 6. •
THO.~IPSONMA, ZIFF EB: Structure of the gene encoding peripherin, an NGF-regulated neuronal-specific type III intermediate filament protein. Neuron 1989, 2:1043-1053. The structure of the gene encoding peripherin shows that is is a new ronal t319e III IF protein. Limited homologies with other neuronal and NGF-inducible genes are described. 7. 0•
MO.','TEmOMJ, CLEVELANDDW: Expression o f NF-L and NFM in fibroblasts reveals coassembly o f NF and sSrnentin subunits. J Cell Biol 1989, 108:579-594. Transient and stable DNA transfection techniques are used to force the synthesis of NF-L and NF-M in non-neuronal cultured cells. The results show that both proteins are competent to assemble in fibroblasts and appear to copobmerize vdth ~imentin. 8. ••
VIY,STaO,~! KL, BoalsY GG, GOLDMANRD: Dynamic aspects of intermediate filament networks in BItK-21 cells. Proc Natl Acad Sci USA 1989, 86:549-553. By rnicroinjection o f biotinylated vh'nentin into cultured cells, the authors showed that the incorporation of injected vhnentin is initiated in a perinuclear cap. This region eventualb" gives rise to a filamentous network, which is coincident ~ t h the endogenous IF nem'ork.
9. •
ANGELIDESKJ, 8.~m-H KE, TAKEDAM: Assembly and exchange of intermediate filament proteins of neurons: NFs are dynamic structures. J Cell Biol 1989, 108:1495-1506.
10. •
Slrt~G RK, N1XON RA: In vivo phosphorylation o f distinct domains of the 70-kilodalton NF subunit im'olves different protein kinases. J Biol Chern 1989, 264:457--464. IXqT-Lcan be p h o s p h o r ) • t e d by a number o f different kinases h~ vitro and hi t'hv~ There are 3 potential phosphotTlation sites, x~ilich are phosphorylated by different kinases. 12. •
\VmLEBA, SMml KE, A.XGEUDESKJ: Resolution and purification o f a NF-specific kinase. Proc Nail Acad Sci USA 1989, 86:7207-7224. A NF-specific kinase which has the characteristics o f a phosphorylator for NF-tl, presumabb" on the KSP sequence, is identified and purified. BLACK3t',l, LEE VMY: Phosphorylation of NF proteins in intact neurons: demonstration of phosphoD'lation in cell bodies and axons. J Neurosci 1988, 8:3296-3305. The extent of phosphorylation of NF-M in cultured neurons can be differentiated by the use of antibodies sensitive to the degree of NF.M phosphor)Otion. The paper shows that different is•forms of phosphoDOted NF-M are present in the cell body and in the axon. 13. •
14. t
IE~xasSE, NIXON RA: Multiple phosphoD-lated variants of the high molecular mass subunit of NFs in axons of retinal cell neurons: characterization and evidence for their differential association with stationary and moving NFs. J Cell Bio11988, 107:2689-2702. Multiple phosphotylated ~riants o f NF-tt can be discerned in the optic nerve. These ~riants are distributed differently along the optic axon indicating that the phosphor)Orion state of NF-tt changes during axoplasmic transport. 15. •
ttLS&"gAGASI, IIIROKAWAN: The effects o f dephosphoq,-lation on the structure of the projections of NF. J Neurosci 1989, 9:959-966. The effect o f dephosphor)Otion on the structure of the projections of NFs is examined by quick-freeze deep-etch electron microscop): Surprisingly, it was found that the structure and frequency of cross-bridges appeared to be similar in both control and dephosphorylated NFs. OBI./NGERMM, $ZUMIASRA, WONGJ, LIUZTAFJ: Changes in cytoskeletal gene expression affect the composition of regenerating axonal sprouts elaborated by dorsal root ganglion neurons in ,dr•. J Neurosci 1989, 9:2645-2653. The authors present data which sho'a~ that there are few NFs in the regenerating dorsal root ganglion cells and they suggest that a down-regulation of NF protein gene expression may play a role in the eflqdency of axonal regeneration. 16. •
17. •
HIROKAWAN, HISANAGASl, StUOMURAY: MAP2 is a component o f crossbridges b e t w e e n microtubules and NFs in the neuronal cytoskeletal: quick- freeze, deep-etch immunoelectron microscopy and reconstitution studies. J Neurosci 1988, 8:2769-2779. By use of immunogold procedures on rat spinal cord motor neurons and a quick-freeze, deep-etch technique in conjunction xxSthdecoration ~ t h anti-MAP2 antibody and ferritin-labeled second antibody, the authors show that MAP2 is present in cross-bridges between microtubules and NFs.
Introduction Neuronal intermediate filaments (IFs) are 8-10 nm filaments found in most neurons of the central nervous system (CNS) and peripheral nervous systems (PNS). Hoffman and Lasek Or Cell Biol 1975, 66:351-366) first inferred, from axonal transport studies, that neurofilaments (NFs) consist of three polypeptides. These were called the NF triplet, and it was found by sodium dodecytsulfatepolyacrylamide gel electrophoresis that they had molecular weights of 200, 145 and 68kD. Biochemical and immunochemical studies later confirmed the classification of these three polypeptides as NF proteins (Liem et al., J Cell Bio11978, 79:637--643; Schlaepfer and Freeman, J Cell Biol 1978, 78:369-378). Further biochemical studies have also shown that, of the three polypeptides, only the smallest (referred to as NF.L, for low molecular weight subunit) was necessary for the formation of an IF in vitro, and that the other two (NF-M and NFH, for middle and high molecular weight subunits) appeared to be accessory proteins (Liem and Hutchison, Bioclaemistry 1982, 21:3221-3226; Geisler and Weber, J MolBiol1981, 151:565-571). Over the past few years, this relatively simple picture of mammalian NFs has become more complicated. Two additional proteins have been purified, cloned and sequenced and subsequently identified as independent IF systems in neurons, with, presumably, different functions. One of them, named peripherin, is found mainly in the PNS (for review, see [ 1 ]), but the second, named cx-internexin, is found mainly in the CNS (Pachter and Hem, J Cell Bio11985, 101:1316-1322) [2]. This review will describe our current knowledge of these ~'e neuronal IF proteins.
in which the 'a' and 'd' residues are frequently apolar, and it is believed that the basic IF dimeric coiled-coil structure is formed by interactions bem'een these apolar residues. All the neuronal IFs have this basic motif. The NF triplet proteins (NF-L, NF-bl and NF-H) have highly charged C-terminal tail regions and a region rich in glutamic acid, but in the case of NF-H, it is considerably shorter than in the other two NF proteins. Both NF-M and NF-H are heavily phosphorylated in their C-terminal tails: 9-26 and 20-100 phosphate groups, respectively, have been reported (Julien and Mushynski, J Biol Cbem 1982, 257:10467-10470; Jones and Williams, J Biol Chem 1982, 257:9902-9905; Carden et al., J Biol Chem 1985, 260:9805-9817). The consensus sequence for phosphorylation has been identified by chemical and immunological means as lys-ser-pro (KSP in the single letter code; Lee et al., Proc Natl Acad Sci USA 1988, 85:1998-2002). In rat NF-M, this sequence appears five times (Napolitano et al., J Neurosci 1987, 7:2590-2599). In human NF-M, there is a direct repeat which includes the KSP sequence and as a result this sequence appears 13 times (Mc3'ers et al., FallBOJ 1987, 6:1617-1626). Human [4] and mouse [5] NF-H hm'e been cloned and sequenced, and the KSP sequence appears an astonishing 50 times. The sequence of peripherin bears more similarity to the non-neuronal IF proteins, vimentin, desmin and glial fibrillary acidic protein than to NF proteins (Leonard et al., J Cell Biol 1988, 106:181-193) and the tail region shows no homology with the NF triplet proteins. Finally, ct-intemexin is more closely related to the NF triplet proteins than to either peripherin or the other IF proteins (Fliegner et al., EMBOJ 1990, in press). Although smaller than the other NF proteins, it also has a C-terminal tail which is rich in glutamic acid. NF-L, peripherin and cx-intemexin do not have the KSP sequence, but they may be phosphorylated on other serine residues.
Primary sequences All IF have a three-domain structure with ~riable N-terminal and C-terminal head and tail regions flanking a conserved cx-helical domain (for review see [3]). The whelica] domain consists of "--310 amino acids and is cornposed of two cx-helical coils separated by a linker region. Within each coil a heptad motif can be discerned
Molecular biology Genomic sequences have now been published for all the neuronal IF proteins except cx-internexin. The intron-exon arrangements of IF protein genes have been
Abbreviations CNS--central nervous system;IF-~intermediatefilament; MAP--microtubule associated protein; NF(L~4,H)--neurofilament(low, medium, high molecular weight); NGF--nerve growth factor; PNSwperipheralnervous system. 86
(~) Current Science Ltd ISSN 0955-0674
Neonatal intermediate filaments Liem 87 used to classify them into different types [3]. V'unentin, desmin and glial fibrillary acidic protein are known as type III IF proteins and their genes all have eight introns in identical positions. Peripherin has the highest sequence homology with these IF proteins and its gene has eight introns in exactly the same positions, demonstrating that it is also a type III IF protein [6]. The NF triple! protein genes, which encode type IV IF proteins, have introns in different positions [4,5]. Two introns are conserved among the genes of all three proteins, but NFL and NF-H genes each have third introns, albeit in different positions. Interestingly, the poSlUOn of this third intron in the NF-H gene is very close to the eighth intron in genes of the type III IFs. Preliminary data from our laboratory suggest that 0c-intemexin is in the same class as the NF triplet proteins (Ching and Hem, "unpublished data), although a final classification will require further sequencing of the gene. One of the more interesting aspects of the different neuronal IF proteins is that their expression is both tissue-specific and developmentally regulated. Peripherin is found mainly in the PNS, 0~-internexin mainly in the CNS and the NF triplet proteins in both the CNS and the PNS. Our data show that 0c-intemexin precedes NF-L and NF-M in the developing CNS (Kaplan et aL, J Cell Bio11989, 109:729) and NF-H is the last neuronal IF to be expressed during mammalian development (Shaw and Weber, Nature 1982, 298:277-279; Pachter and Hem, Dev Biol 1984, 103:2(X)-210). The tissue-specific and temporally regulated expression of these genes suggests that they contain important regulatory elements. Although some limited homologies were reported between the promoter regions of the peripherin gene and those of NF-L and NF-M [6], no obvious common elements can be discerned in the 5° regions of the genes for the neuronal IF proteins. A number of studies, including experiments involving transgenic mice, are underway to investigate the tissue-specificity of the expression of these genes. In one study already published, the human NF-L gene was expressed in transgenic mice, and the human NF-L protein was shown to be co-expressed with mouse NFs (Julien et al., Genes Dev 1987, 1:1085-1095). This study indicates that the human gene encodes c~acting elements that produce tissue specificity. Interestingly, the expression was somewhat delayed. Further studies using segments of the 5' upstream regions of these neuronal IF proteins linked to reporter genes are being performed in a number of laboratories. •
•~'l
Assembly and expression of NFs in fibroblasts As mentioned above, only NF-L is necessary for the formarion of an IF #z vitro and in most cases, neither NF-M, nor NF-H are able to polymerize into IFs in the absence of NF-L It has been shown that peripherin and ¢z-internexin are both able to form into homopolymers (Parysek and Goldman, JNeurosci 1987, 7:781-791) [2]. In attempts to determine how each of the NF triplet proteins alone would behave #l vivo, we (Chin and Hem, E u r J Cell Biol, 1989, 50: 475-490) and blonteiro and Cleve-
land [7] transfected each of the genes or complementary DNAs for these proteins into fibroblasts. In all three cases, the NF triplet proteins copolymerized with the endogenous vimentin cytoskeleton. The time course for these events was studied in transient transfection studies and an apparent organization center was observed during the early stage, from which the filaments radiated later. Examples of these experiments are shown in Fig. 1. A similar organization center was observed for virnentin filaments when biotinylated vimentin was miocroinjected into fibroblasts [8]. An additional point of potential importance should be noted. When blonteiro and Cleveland [7] transfected L cells with a genomic clone for NF-L, they obtained stable cell lines, which expressed large amounts of NF-L protein. However, in similar experiments using a complementary DNA with a larger 5' untranslated region, we obtained stable cell lines which expressed abundant NF-L message, but.relatively little protein. These studies suggest that the 5' untranslated region in the NF-L messenger RNA may have an additional role in regulation of NF-L translation in neurons• NF proteins are synthesized and assembled in the cell body and transported into the axon. The existence of a focal point for the organization of IFs in fibroblasts is consistent with the observation in PC12 cells that peripherin, NF-L and NF-M appear as a juxtanuclear cap in non-process-beating cells• These cells radiate from this cap into the processes when the cells are treated with nerve growth factor (NGF; Lee and Page, JNeurosci 1984, 4:1705-1714; Parysek and Goldman, 1987). It is still not known whether the NFs are dynamic once they are assembled, but recent evidence has shown that NFs can exchange their subunits in vitro, presumably through a wall-exchange between the polymer and a soluble pool [9]. These results suggest that, even in the axon, there may be a local exchange of subunits allowing the NFs to be dynamic.
Phosphorylation Recent studies have shown that phosphorytation of vimentin, desmin and the nuclear lamins is important for the depolymerization of filaments, especially during mitosis [3]. Neurons do not generally divide and so the phosphorytation of neuronal IF proteins probably involves different functions. The phosphorylation of peripherin in PCl2 cells is increased two- to threefold by treating the cells with NGF [10]. The small proportion of cellular peripherin that is soluble is not phosphorylated, which may mean that phosphorylation of peripherin promotes assembly rather than disassembly. Phosphorytation of NF-L dn distinct domains by different protein kinases has also been reported recently [11]. It is likely that NF-H is phosphylated on the KSP sequence by a NF kinase, which has a high specificity for NF-H and which would not, therefore, be able to phosphorylate other phosphoproteins. There has been a report of the purification of a NF kinase which has these characteristics
88
Cyloplasm and cell m o l i l i l y
Ii
(a)
(b)
(c)
(d)
Fig. 1. Distribution of neurofilament proteins in cells transiently transfected with a neurofilament low molecular weight and medium molecular weight subunit (NF-L and NF-M) construct at different time points. (a) L tk-cel[s stained with a monoclonal antibody against NF-L 24 h post-transfection with a full complementary DNA encoding NF-L cloned behind a Rous sarcoma virus promoter. (b) Similar staining at 48 h post-transfection. (c) Cos 1 cells transiently transfected with a NF-M complementary DNA construct cloned behind an simian virus 40 promoter and stained with a rnonoclonal antibody against NF-M 48 h post-transfection. (d) Cos I cells similarly stained as in (c) 72 h after transfection. Details of these experiments are given in Chin and Liem (£ur / Cell Biol, 1989 50: 475--490). Note the juxtanuclear focus in both cell types at the earlier time points, from which filaments appear to radiate. Data courtesy of 5teven Chin (private communication) and published by permission.
Neonatal intermediatefilaments Liem 89 [12] and a number of other kinases have been reported that phosphorylate NF-M, but phosphorylate NF-H only poorly. Antibodies specific to either the non-phosphorylated or phosphorylated proteins are important tools in the study of the phosphorylation of NFs. Studies using such antibc~lies showed that phosphorytated NF-H and NF-M epitopes were found in axons, while non-phosphorylated epitopes were primarily .in located cell bodies (Stemberger and Sternberger, Proc Natl Acad Sci USA 1983, 80:6126-6130). Further studies have also shown that there multiple phosphorylated variants of NF-M and NFH, both in cultured cells [13] and in the axons of retinal cell neurons [14]. This suggests that there are constant changes in the phosphorylation state of NFs in the axon and that the kinase is transported together with the NFs. Transfection experiments also show that NF-H is phosphylated by ldnases other than those in fibroblasts, since the NF-H proteins that are made in the transfected cells are not recognized by antibodies against phosphorylated NF-H (Chin and Hem, submitted for publication). Interestingly, NF-M in transfected fibroblasts can be recognized by an antibody specific for phosphorytated NFs, suggesting that the KSP sequence on NF-M may be phosphorytated by ldnases present in fibroblasts. It has been suggested previously that phosphorytation of NF-H may be important for the formation of crossbridges between filaments. However. a recent study by Hirokawa's laboratory showed that non-phosphorylated NF-H was still capable of forming cross-bridges between filaments [15]. Carden et al. (J Neurosci 1987, 7:3489-3504) proposed that phosphorylation may, in fact, cause repulsion between between filaments, which may be a way of increasing axonal diameter (see below) during development. Now that a NF kinase has been identiffed, the role of NF-H phosphorytation may be established in the near future.
Neurofilaments and axonal diameter There is a good correlation between the number of NFs and axonal diameter, suggesting that the NF triplet proteins may determine axonal caliber (for review, see Iasek et al., Cold Spring Harb Syrup Quant Biol 1983, 48:731-744). This is a reasonable hypothesis since NFs increase continuously during development, and decrease after axotomy, when the neurons degenerate (Hoffman et aL, J Cell Bio11985, 101:1332-1340). Interestingly, the messenger RNA levels for NF-L have also been shown to decrease after axotomy (Hoffman et at, Proc Natl Acad USA 1987, 84: 3472-3476) [16], demonstrating that the decrease in NFs is not due to a transport blockade but to a decrease in protein ~3mthesis, and that a retrogradely transported factor, which is activated by the axotomy, may be required to block the transcription of NFs. Studies linking axonal diameter and NF content have overlooked the two recently identified neuronal IFs. Is the
abundance of these also correlated with axonal diameter? Several lines of evidence argue against this possibility and suggest that these proteins may be important in process outgrowth. One is the early appearance of a-intemexin during CNS development (Kaplan et al., J Cell Bio11989, 109:729). Another is the fact that peripherin messenger RNA and protein levels increase after axotomy, indicating that peripherin may be important in process outgrowth during regeneration (Shelanski, personal communication; Oblinger et al., J Neurosci 1989, 9: 3766--3775). However, it is still not known how the functions of these two new neuronal IFs differ and why they have different distributions.
Interactions between NFs and microtubules In our discussion so far we have only considered crossbridging between NFs. In fact, there is an extensive system of cross-bridges between various cytoskeletal elements in nerve cells, including microtubule-- neurofilament cross-bridges. Interactions between microtubules and NFs in vitro have been demonstrated in binding studies (Leterrier et al., J Cell Biol 1982, 95:982-986; Heimann et al., J Biol Chem 1985, 260:2160-2166), and in viscosity studies (Aamodt and Williams, Bix.hemt2try 1984, 23:6023-6031). These interactions have been shown to depend on the microtubule associated proteins (MAPs). In a recent paper, Hirokawa and co-workers [17] showed that MAP2 is a component of cross-bridges between microtubules and NFs. In vitro studies have also shown that tau can bind to NFs (Shelanski et al., NRP Res Bull 1981, 19:32-43), and interactions between MAP1 and NFs can be inferred from direct binding studies (Heimann et al.,JBiol ~ m 1985, 260:2160-2166). Thus, NF-microtubule interactions appear to be mediated by various MAPs. Flynn et al. ( Biochem Biopbys Res Comm 1987, 148:1453-1459), in a recent study, showed that the same domain of MAP2 could bind to both microtubules and NFs, suggesting that cross-bridges require more than one MAP molecule. MAPs interact with a 4 kD carboxyterminal fragment of tubulin which is rich in glutamic acid (Serrano et aZ, Bioc.bemistry 1984, 23:4657-4681). It is likely that the presence of glutamic acid-rich regions on NF proteins could also be responsible for these interactions. With the development of molecular biological probes for all of these molecules, and the ability to make truncated forms of these proteins in vitro, we should soon be able to understand the nature of these interactions in more detail.
Acknowledgements I thank the members of my laboratory,especiallySte~en Chin, Gee Ching, Karsten Fliegner, David Green and MichaelKaplan for their help vdth this article and for providing the recent data presented here.
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Cytoplasm and cell motility
Annotated references and recommended
reading • ••
Of interest Of outstanding interest
A fluorescence energy transfer assay is used to show that l',q~ subunit exchange is preceded by dissociation of subunits from the filament and the generation o f a kineticalb" active pool of soluble subunits. The authors also suggest, but do not show, that phosphotylation may be im. portant for the ob~namic assembly and disassembb' o f NFs.
1. GREEr~'EI.A: A n e w neuronal intermediate filament protein. • Trends Neurosci 1989, 12:288-230. A reMew describing the history of the discm~et3; rediscox~ery and somewhat roundabout cloning of the 57 kD IF protein, also known as peripherin.
#a.ETrhJM, StIELA.':SKIML, G~Eh'E LA: Phosphorylation of the peripherin 58-kDa neuronal intermediate protein. Regulation by nerve growth factor and other agents. J Biol Cbem 1989, 264:4619--4627. The phosphorylation of peripherin in PC 12 ceils and its regulation by a number of different factors is described. The results suggest that phosphowIation of peripherin may be important for its assembb"
2. •
11. •
Cmu FC, B.~ffO,T_SEA, DAS K, t ~ J, Socotow P, M,,camso FP, FA.,rr J: Characterization of a novel 66 kd subunit of mammalian NFs. Neuron 1989, 2:1435-1445. The isolation o f a 66 kD neuronal IF protein, which can seLf.pobmerize and is found primarily in the CNS, is described. That this protein is the same as et-intemexin, is now substantiated by the fact that the predicted protein sequence o f ~-intemexin, based on its complemental" DNA, contains a peptide sequence reported in this paper. 3. STEL\'ERTPM, ROOP DR: Molecular and cellular biology of •• intermediate filaments. Annu Rev BiodJem 1988, 5Z593-626. An excel/ent and comprehensh'e review on IFs. 4. •
LEES JF, SfL'x'EIDSL~.NPS, SKUN'rZ SF, CARDEN MJ, LAZZARINIRA: The structure and organization of the human heavy NF subunit (NF-H) and the gene encoding it. EMBO J 1988, 7:1947-1956. The complete amino acid sequence for human NF-tt based on genomic clones shog~ the presence of the phosphot3Otion consensus sequence KSP greater than 40 times. From the intron-exon arrangement for the NF-H gene, it is exSdent that the third intron in this gene nearb" matches the position o f one of the conser~x~d introns in the nonneuronal IF genes. 5. •
JUL/ENJP, COTI~ F, BEAUDET L, SIDKY M, FIAVEI£ D, GROS'.XLD F, MUStWNSKIW: Sequence and structure of the mouse gene coding for the largest NF subunit. Gene 1988, 68:307-314. The complete nucleotide sequence of the mouse gene encoding NF-II is described in this paper, ~fffich xras published at the same time as [4]. The findings are similar to the results described in [4]; the KSP sequence is found 51 times. 6. •
THO.~IPSONMA, ZIFF EB: Structure of the gene encoding peripherin, an NGF-regulated neuronal-specific type III intermediate filament protein. Neuron 1989, 2:1043-1053. The structure of the gene encoding peripherin shows that is is a new ronal t319e III IF protein. Limited homologies with other neuronal and NGF-inducible genes are described. 7. 0•
MO.','TEmOMJ, CLEVELANDDW: Expression o f NF-L and NFM in fibroblasts reveals coassembly o f NF and sSrnentin subunits. J Cell Biol 1989, 108:579-594. Transient and stable DNA transfection techniques are used to force the synthesis of NF-L and NF-M in non-neuronal cultured cells. The results show that both proteins are competent to assemble in fibroblasts and appear to copobmerize vdth ~imentin. 8. ••
VIY,STaO,~! KL, BoalsY GG, GOLDMANRD: Dynamic aspects of intermediate filament networks in BItK-21 cells. Proc Natl Acad Sci USA 1989, 86:549-553. By rnicroinjection o f biotinylated vh'nentin into cultured cells, the authors showed that the incorporation of injected vhnentin is initiated in a perinuclear cap. This region eventualb" gives rise to a filamentous network, which is coincident ~ t h the endogenous IF nem'ork.
9. •
ANGELIDESKJ, 8.~m-H KE, TAKEDAM: Assembly and exchange of intermediate filament proteins of neurons: NFs are dynamic structures. J Cell Biol 1989, 108:1495-1506.
10. •
Slrt~G RK, N1XON RA: In vivo phosphorylation o f distinct domains of the 70-kilodalton NF subunit im'olves different protein kinases. J Biol Chern 1989, 264:457--464. IXqT-Lcan be p h o s p h o r ) • t e d by a number o f different kinases h~ vitro and hi t'hv~ There are 3 potential phosphotTlation sites, x~ilich are phosphorylated by different kinases. 12. •
\VmLEBA, SMml KE, A.XGEUDESKJ: Resolution and purification o f a NF-specific kinase. Proc Nail Acad Sci USA 1989, 86:7207-7224. A NF-specific kinase which has the characteristics o f a phosphorylator for NF-tl, presumabb" on the KSP sequence, is identified and purified. BLACK3t',l, LEE VMY: Phosphorylation of NF proteins in intact neurons: demonstration of phosphoD'lation in cell bodies and axons. J Neurosci 1988, 8:3296-3305. The extent of phosphorylation of NF-M in cultured neurons can be differentiated by the use of antibodies sensitive to the degree of NF.M phosphor)Otion. The paper shows that different is•forms of phosphoDOted NF-M are present in the cell body and in the axon. 13. •
14. t
IE~xasSE, NIXON RA: Multiple phosphoD-lated variants of the high molecular mass subunit of NFs in axons of retinal cell neurons: characterization and evidence for their differential association with stationary and moving NFs. J Cell Bio11988, 107:2689-2702. Multiple phosphotylated ~riants o f NF-tt can be discerned in the optic nerve. These ~riants are distributed differently along the optic axon indicating that the phosphor)Orion state of NF-tt changes during axoplasmic transport. 15. •
ttLS&"gAGASI, IIIROKAWAN: The effects o f dephosphoq,-lation on the structure of the projections of NF. J Neurosci 1989, 9:959-966. The effect o f dephosphor)Otion on the structure of the projections of NFs is examined by quick-freeze deep-etch electron microscop): Surprisingly, it was found that the structure and frequency of cross-bridges appeared to be similar in both control and dephosphorylated NFs. OBI./NGERMM, $ZUMIASRA, WONGJ, LIUZTAFJ: Changes in cytoskeletal gene expression affect the composition of regenerating axonal sprouts elaborated by dorsal root ganglion neurons in ,dr•. J Neurosci 1989, 9:2645-2653. The authors present data which sho'a~ that there are few NFs in the regenerating dorsal root ganglion cells and they suggest that a down-regulation of NF protein gene expression may play a role in the eflqdency of axonal regeneration. 16. •
17. •
HIROKAWAN, HISANAGASl, StUOMURAY: MAP2 is a component o f crossbridges b e t w e e n microtubules and NFs in the neuronal cytoskeletal: quick- freeze, deep-etch immunoelectron microscopy and reconstitution studies. J Neurosci 1988, 8:2769-2779. By use of immunogold procedures on rat spinal cord motor neurons and a quick-freeze, deep-etch technique in conjunction xxSthdecoration ~ t h anti-MAP2 antibody and ferritin-labeled second antibody, the authors show that MAP2 is present in cross-bridges between microtubules and NFs.