Intermediate filaments as dynamic structures

Cancer and Metastasis Reviews 15: 417428, 1996. © 1996 Kluwer Academic Publishers. Printed in the Netherlands.

Intermediate filaments as dynamic structures

Michael W. Klymkowsky Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, Boulder, Colorado 80309-0347, USA

Introduction

In the last decade, our view of intermediate filaments (IFs) has changed dramatically. Once thought of a static structures, with ill-defined functions, it is now clear that IFs are quite dynamic and undergo dramatic reorganizations in response to cell cycle signals, cellular differentiation, and pathogenic events. Moreover, IFs have been shown to play important architectural roles in epidermis, neurons and muscle. At the same time, the roles of IFs in a number of other cell types, e.g. glia, fibroblasts, simple epithelia, remains much more enigmatic. The more general questions of how IF protein expression and network formation influence a cell's oncogenic potential and behavior (the subject of this volume) remains largely unclear. As a general introduction to this volume I will briefly describe the known IF proteins and their intracellular organization.

Vertebrate IF proteins IF proteins are united by their ability to assemble into 8-10 nm filaments. Assembly can occur in vitro from denatured subunit proteins, in the absence of nucleotide hydrolysis or other co-factors [for more extensive reviews of IF structure and assembly see 1-4]. The known vertebrate IF proteins share a number of similar features: these include a characteristic 'central rod domain' of"310 amino acids in length, characterized by stretches of 'heptad repeat', interrupted by non-heptad domains. The heptad repeat regions drive the formation of parallel, unstaggered a-helical coiled-coiled dimers,

which go on to form higher order oligomers (Figure 1). The N- and C-terminal 'helix initiation' and 'helix termination' regions of this 'rod' domain are particularly well conserved among the known IF proteins; it is in these regions in particular that mutations associated with severe keratin-based diseases of the skin are most commonly found [5]. The monoclonal antibody anti-IFA (available through the American Type Culture Collection), reacts with most IF proteins [6], and binds to a sequence at the C-terminus of the rod domain [7]. The N-terminal 'head' and C-terminal 'tail' domains of IF proteins, in contrast, differ rather dramatically both in size and composition. Head domains appear essential for IF assembly [8, 9] and behavior; 'swapping' the heads of IF proteins changes their behavior within the cell [10]. The roles of the tail domains are more enigmatic. There are a number of examples in which deletion of the tail domain has little noticeable effect on the ability of the truncated protein to assemble into IFs [11-13]. At the same time, tails domains have been shown to be involved in the organization of IF networks [14-16]. The IF proteins of vertebrates fall into four major groups, based on their genomic structure, polymerization properties, and patterns of expression: the vimentin-like proteins, the neurofilament proteins, the keratins, and the lens IF proteins [17]. The vimentin-like proteins, i.e., vimentin, desmin, peripherin, and glial fibrillary acidic protein (GFAP), form homopolymeric IFs both in vitro and in vivo. They readily co-polymerize with one another and with the proteins of the neurofilament protein group, i.e. the three neurofilament proteins (NF-L, NF-M and NF-H), ~-internexin, and nestin. When expressed in the same cell, vimentin-like and neuro-

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Figurel. A cartoon that illustrates the steps in IF protein assembly and the difference between lamin and IF structure.

filament proteins form a single hybrid system. Recently, characterization of a cDNA encoding the IFassociated protein synemin indicates that it is a bona fide IF protein [18]; based on the co-localization of vimentin, desmin and synemin [19, 20], it seems likely that these proteins co-polymerize. The major feature that distinguishes the vimentin-like proteins from the neurofilament group proteins is the pattern of introns and exons in the genomic sequence of the genes [21-23]. In addition, the neurofilament group proteins, only ct-internexin has yet been shown to form an IF network of its own in vitro [24]; the other neurofilament proteins require the presence of other IF proteins to form an extended IF network [24, 25]. During the course of normal development, neurofilament proteins are initially expressed in the presence of vimentin or

ct-internexin [26] which can support NF assembly. Other IF proteins, e.g. plasticin [27], XNIF [28], gefiltin [29], and tannabin [30] have been described in amphibians and fish, but whether they represent novel IF proteins or are homologs of specific mammalian IF proteins remains to be determined. In the case of the NF-M and NF-H proteins, the C-terminal tail domains extend outward from the central filament core [14]. In the neuron, NF-M and NF-H form cross-bridges between neurofilaments, and together with MAP2 form cross-bridges with microtubules [31]; these neurofilament tail interaction are critical for the normal maintenance of axonal diameter [16]. In addition, the NF-M tail domain appears to play an important role in the normal elongation of neurofilaments [32]. The structure of the NF-M and NF-H tail domains is regulated by phosphorylation [33]. In myelinated neurons, where axonal caliber changes dramatically between nodal and non-nodal (myelinated) segments, changes in neurofilament protein phosphorylation (involving Schwann cell/neuron interactions) are associated with changes in neurofilament organization [34-36]. The keratins are the largest group of IF proteins. They are divided into the type I keratins (K9 through K20) and the type II keratins (K1 through K8) based of their overall charge and molecular weights. In addition to the 'cytokeratins', which are expressed in epithelial cells, there are the 'hard' keratins which are found in hair, nail, and scale [37]. In contrast to the vimentin-like and neurofilament proteins, a single keratin cannot form an IF and will not co-assemble with non-keratin IF proteins; rather keratin filaments are obligate heteropolymers of type I and type II proteins in a 1:1 stoichiometry [38]. When keratins and non-keratins are co-expressed in the same cell they form distinct, although often interacting, networks [39, 40]. A number of different keratins can co-assemble into a single filament. There is growing evidence that the composition of a keratin network effects its organization, and presumably function [41, 42]. Since dramatic changes in the composition of keratin accompany normal differentiation, response to injury, or other pathogenic states [43], changes in keratin expression can

419 be expected to generate changes in the nature of the cell's keratin filament network. For example, upon wounding of the epidermis, the keratins K6 and K16 are induced. These keratins differ in their assembly properties from other epidermal keratins and have been proposed to facilitate the reorganization of keratin filament organization associated with wound healing [44]. It seems likely that changes in keratin composition associated with cancer could also lead to physiologically significant alterations in cellular behavior. The most recently recognized group of IF proteins, filensin and CP49 (also known as phakinin), are found in lens [45]. Filensin (Mr q00 kDa), which behaves very much like a peripheral membrane protein [46, 47], has a 'truncated' central rod domain that is missing "29 amino acids (including the epitope recognized by the anti-IFA antibody) [48]. CP49/phakinin has a complete (310 amino acid) central domain but lacks a C-terminal tail; sequence comparison indicates that it is most closely related to type I keratins. Filensin and phakinin co-assemble with one another to form the 'beaded filaments' characteristic of lens [49, 50]. Neither protein coassembles with vimentin, which is also expressed in the lens [51].

The evolutionary history and functions of IF proteins Ten years ago, the surprising discovery was made that the major structural proteins of the nuclear lamina, the lamins, are closely related to the IF proteins [52, 53]. Like IF proteins, the lamins contain a central helical rod domain; the lamin rod domain, however, is approximately 6 heptad repeats longer than that found in the vertebrate IF proteins. Interestingly, sequence analysis of invertebrate IF proteins, revealed that they also have a 'lamin-like' rod domain [54-57]. Of vertebrate IF proteins, only the recent described splice variant of the vertebrate IF protein CP49/phakinin, CP49ins, contains such a lamin-like rod domain [58]. The relationship between lamins and IF proteins suggests a number of possible evolutionary scenarios. If we could find 'lower eukaryotes' with either

only lamins or only IF proteins, we might be able to define a family tree. However, the search for lamins/IF proteins in lower organisms has not been particularly fruitful [17]. In fact, there is no convincing evidence that lamins or IF proteins are present in any unicellular eukaryote. With the recent determination of the entire genomic sequence of the yeast Saccharomyces ceriviseae, it has become possible to search directly for genes that may encode lamins/IF proteins. A preliminary search failed to reveal any obvious lamin/IF protein genes (M. Winey and M.W. Klymkowsky, unpub, obs.). Similarly, studies on the nuclear lamina of Amoeba proteus [59] suggest that it is structurally distinct from the nuclear lamina of higher eukaryotes. We therefore are left without a phyllogenetic clue as to the origin of lamins/IF proteins. In any case, it is now abundantly clear, from a number of experimental systems, that lamins and IFs are not defining elements of eukaryotic cells. Moreover, the characterization of spontaneously occurring 'IF-free' cells [60, 61], and most recently, gene knock-out studies of keratin K8 [63], vimentin [64] and GFAP [65, 66] indicate that IFs are not involved in a number of simple cellular functions. At the same time lamins and IFs are found in essentially all metazoans and are highly conserved, suggesting that they increase organismic fitness. The functions of lamins/IFs are now being elucidated. In the case of the lamins, antibody-depletion studies [67, 68] and co-localization studies [69] suggest that lamins are involved in DNA replication and nuclear organization in vertebrate cells [70, 71]. Based on the results of various experimental manipulations [72-75], spontaneous mutations [76] and 'knock-out' mutagenesis [77-81], it is clear that IFs play important roles in epithelia, muscle and nerve.

IF structure and assembly Since IF assembly does not involve nucleotide hydrolysis, it must be a simple equilibrium reaction. Structural studies indicate that both lamins and IF proteins first dimerize in a parallel, unstaggered manner (Figure 1). In the case of the lamins, there

420 appears to be strong tendency to form 'head-to-tail' dimer polymers [82]. With IF proteins, in contrast, the dimers rapidly associated in a lengthwise, antiparallel manner to form tetramers; tetramers associated into higher order oligomers and protofilaments [83]. Between 6 to 8 protofilaments associate to form an IF [84]. The exact structure of either lamin or intermediate filaments, however, remains poorly determined. When the typical cell is extracted with detergent, under mild ionic conditions, the bulk of the IF proteins are found to be insoluble, presumably in the form of filaments; what soluble form exists apears, when analyzed by sucrose velocity sedimentation analysis, to be tetrameric [85-87 and references therein]. Our own studies in Xenopus oocytes [87] indicate that the soluble form of keratin is in fact a larger oligomer, but that this form is unstable under conditions of sucrose gradient analysis. Pulse-case studies of IF assembly suggest that the on-off rate relatively fast [87 and references therein]. A similar conclusion emerges from 'fluorescence recovery after photobleaching' studies [88, 89] and the time-course of subunit addition into pre-existing IF networks [90, 91]. Based on immunofluorescence studies, it appears that there are discrete sites of new subunit addition along the length of pre-existing filaments [92, 93]. Whether these are, in fact, protofilament ends has yet to be determined. In any case, it is clear that there are sites for subunit addition scattered throughout the IF network. One advantage to such an assembly pathway is that it preserves the structural integrity of the IF network, even as the IF is replaced.

1F network organization Lamin fibers are found associated with the inner surface of the nuclear envelope, where they form a fibrous mesh. In most cells, the organization of the lamina appears rather diffuse, and discrete filaments are rarely described [94]. A striking exception is the orthogonal array of lamin filaments found in the nucleus (germinal vesicle) of Xenopus oocytes [95]. During interphase, the nucleus, and the nuclear lamin, grows in size and lamins are as-

sumed to enter the nucleus and incorporate, more or less uniformly, into the lamina [71]. Upon entry into M-phase, the nuclear lamina undergoes a controlled depolymerization [96], which is mediated by phosphorylation of lamins by the cyclin-dependent kinase p34 cdc2[97-99]. During M-phase, the 'B-type' lamins remain associated with the membranous remnants of the nuclear envelope, while the 'A/Ctype' lamins are found in a soluble form. At the end of M-phase, the disassembly process is reversed and a new nuclear lamina is established [71, 100]. IFs also undergo reorganization during M-phase in some cells, although only rarely as extensively as seen for the nuclear lamins (see below). In most somatic cell types, the onset of M-phase is associated with an increase in the level of IF protein phosphorylation [101,102]. Typically, IF networks are seen to undergo a 'collapse' and can form a 'cage' that surrounds, or lies to one side of the spindle [39, 103]. The collapse of IF organization is particularly dramatic in cells that express vimentin. In cells that express keratin, the keratin filament network, while recognized, generally remains largely extended [39]. There are, however, examples in which the filamentous structure of both keratin [1-4-106] and vimentin/desmin [107] IF networks is transformed into 'amorphous' insoluble aggregates, which are found scattered throughout the cytoplasm. At the end of M-phase, these aggregates reorganize again to reform the cell's IF network [106]. The most dramatic example of IF reorganization so far described occurs during oocyte maturation in Xenopus [108]. In the prophase oocyte, there are three keratins (the homologs of the simple epithelial keratins K8, K18 and K19) arranged in cortical network [109]. During oocyte maturation, this keratin network disassembles completely into soluble oligomers [87, 110]. Size exclusion chromatography indicates that these oligomers are larger than tetrainer, with an apparent molecular weight of -750 kDa; a similar oligomeric form of keratin is seen in the soluble fraction of prophase oocytes and in in vitro translation reactions synthesizing both type I and type II keratins (but not either type alone) [87]. The disassembly of keratin filaments is initiated by maturation promoting factor MPF or cdc2 kinase), but unlike the direct effects of MPF on lamin orga-

421 nization, the disassembly of keratin filaments requires ongoing protein synthesis [111]. Keratin disassembly is associated with the hyperphosphorylation of the K8-1ike keratin of the oocyte [110], whether this is the true cause of keratin filament disassembly remains to be established unambiguously. Many IF proteins have been shown to be phosphorylateable by MPF/cdc2 kinase [112-116]. That phosphorylation by MPF/cdc2 kinase is, by itself, not sufficient to induce IF disassembly is suggested by the observation that when vimentin is expressed in the Xenopus oocyte and that oocyte is matured, the occyte's vimentin filament network does not disassemble, but rather becomes much more robust [117], while at the same time, the oocyte's keratin network is disassembling completely (see above). Using antibodies specific for various phosphorylated regions of IF proteins, Inagaki and colleagues have shown that i) cdc2-mediated phosphorylation of vimentin occurs throughout the cytoplasmic system but vimentin network reorganization occurs only in cell lines with high levels of cdc2 kinase activity [116]; and a different kinase acts in the region of the contractile ring [118,119], presumably leading the localized disassembly of IFs, thereby facilitating cytokinesis. In addition, to phosphorylation by cdc2 kinase, IF proteins have also been found to be phosphorylated by, and associated with a number of other kinases [120-124 and references therein], as well as modified by glycosylation [125-127]. The functional significance of many of these modifications remains unclear, although it seems likely that they modify interactions, either between IFs themselves, or between IFs and other cytoplasmic factors, e.g. 14-3-3 proteins [128]. The best understood interactions between IFs and other cellular components involve IF-microtubule/microfilament interactions and IF-desmosomal and hemi-desmosomal interactions. In cells that express vimentin-like or neuronal IF proteins, the organization of the IF network depends upon interactions with microtubules (MTs). Disruption of the microtubule system generally leads to a centripetal 'collapse' of IFs to a region near the cell center. The collapse of IF network organization appears to require energy and is microfilament-de-

pendent [129, 130]. The current view would be that IFs are 'dragged' outward via interactions with MTs, and 'pulled' inward via interactions with the microfilament system. The same basic type of mechanism is thought to act in neurons, where neurofilaments are arrayed in a more or less parallel manner within the axon. Neurofilament proteins move along the axon at the same rate as tubulin (and only slightly slower than aetin) [131]. Numerous connections between IFs and MTs are observed [31, 32]. It should be noted that the interaction between IFs and MTs appears to be a particularly sensitive one, in that any of a number of noxious treatments lead to the reorganization (collapse) of IF organization [133]. A similar 'metabolic' defect appears to underlie the reorganization of IFs seen in at least a subpopulation of patients with the genetic disease Giant Axonal Neuropathy [134, 135]. Disruption of normal IF organization is associated with a number of myopathic and cardiomyopathic [136, 137], neuropathie [138-140], renal diseases [141]. In this light it is interesting to note that disruption of IF organization by mutated forms of desmin can lead to cell death [142], suggesting that disrupted IF networks, while not the direct 'locus' of the disease, may exacerbate symptoms. That there are molecules within the cell that can mediate interactions between IFs, MTs and MFs was graphically suggested by observation of 2 nm 'linker' elements connecting all three cytoskeletal systems [132,143]. In the case of the IF-MT interaction, kinesin [144], a 210 kDa microtubule-associated protein [145], and plectin [146] have been implicated. Plectin, a membrane of the desmoplakin/ bullous pemphigoid antigen (BPAG) family of proteins [147], has also been observed to interact with actin (MF)-containing structures [148]. In epithelial cells, which express keratin-type IFs, interactions between IFs-MTs-MFs also play an important role in the determination of IF network organization, since anti-microtubule or microfilament drugs can generate dramatic changes in IF network organization [149, 150; see also 109]. However, keratin network organization appears to be determined in large measure by interactions with hemidesmosomes and desmosomes, cell-matrix

422 and cell-cell adherence junctions [151, 152], Hemidesmosomes are integrin-based structures to which IFs are attached. 'Knock-out' mutagenesis of the bullous pemphigoid antigen-1 (BPAG-1) gene [153] indicates the BPAG-1 plays a role in linking IFs to the hemidesmosome (interestingly, the presence of defects in sensory neurons in BPAG-1 null mice, which do not contain hemidesmosomes suggests multiple functions of the BPAG-1 gene product). In the case of desmosomes, IFs are attached to cadherin-type cell-cell adherence molecules through a set of accessory proteins, which include desmoplakin [147], plakophilin (band 6 protein) [154] and perhaps IFAP300 [155]. During development, desmosomes appear to form first and to then connect to IFs, which are often seen initially as 'tuffs' emerging from the desmosomal plaque [156159]. Disruption of desmosomes, brought on by the expression of the tail domain of the desmosomal cadherin desmoglein [160] leads to a disruption of normal keratin network organization. Interestingly, a similar construct introduced into mice leads to a wide range of defects [161]. Some of these are likely to be due to keratin filament disorganization, as seen in diseases of keratins [162,163], whereas other aspects of the observed phenotype are probably due to effects on transcellular signaling [164]. For example, disrupting desmosomes is like to disrupt the distribution of desmosome-associated proteins. One of these, plakoglobin, has been shown to be able to alter cellular differentiation [165] and to suppress tumorigenicity in transformed cells [166]. It should be noted that disruption of keratin filament organization, mediated by either antibody injection [167], toxic compounds [168], the expression of mutant IF proteins [169] or in keratin gene 'knock-out' mice [78] does not appear to significantly alter desmosome formation or maintenance.

IF proteins as determinants of IF network organization Finally, there are dramatic differences between different types of IF proteins and the types of networks they form within cells; these differences appear to be intrinsic to the IF proteins themselves.

For example keratins tend to form bundles, also known as 'tonofilaments', whereas bundle formation is much less common among then non-keratins. The extent of keratin filament bundling differs between different cell types and may be a direct function of the keratins expressed in these tissues [44 for a discussion]. Antibodies against certain keratins lead to an apparent 'fraying' of tonofilaments [167, 170], perhaps by directly blocking keratin-keratin or keratin-'bundling protein' interactions. Among the non-keratins, there is also clear evidence that IF composition dramatically influences organization. In neurons, changing the ration of NF-L, NF-M and NF-H leads to changes in neurofilament organization and axonal diameter [15, 16]. Similarly, in muscle we have found that the muscle specific IF protein desmon behaves quite differently from vimentin (see 10,171). In contrast to the longitudinal IF network formed by vimentin, desmin is preferentially associated with the sarcolemma, where it appears to act to strengthen the attachment of myofibrils [74, 80, 81]. Based on the differences in organization in vimentin and desmin-type IFs, we predict that the re-introduction of vimentin into desmin 'null' mice will fail to completely rescue the desmin null phenotype. The significance of differences in IF proteins, and the networks they form, for cellular behavior, particularly in the oncogenic state is the focus of this issue and will be dealt with in greater detail by the other authors.

Acknowledgements Our work has been supported by grants from the National Science Foundation, the American Cancer Society, the National Institutes of Health, the Muscular Dystrophy Association, and the Colorado Chapter of the American Heart Association.

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Address for offprints: M.W. Klymkowsky, Molecular, Cellular & Developmental Biology, University of Colorado, Boulder, Boulder, Colorado 80309-0347, USA