Intermediate filaments and their associated proteins: multiple dynamic personalities Megan K Houseweart and Don W Cleveland

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1 93 Intermediate filaments and their associated proteins: multiple dynamic personalities Megan K Houseweart and Don W Cleveland A fusion of mouse and human genetics has now proven that intermediate filaments form a flexible scaffold essential for structuring cytoplasm in a variety of cell contexts. In some cases, the formation of this scaffold is achieved through a newly identified family of intermediate-filament-associated proteins that form cross-bridges between intermediate filaments and other cytoskeletal elements, including actin and microtubules. Addresses Ludwig Institute for Cancer Research and Division of Cellular and Molecular Medicine, University of California at San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA Ludwig Institute for Cancer Research, Division of Cellular and Molecular Medicine, and Department of Medicine and Neuroscience, University of California at San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA; dcleveland@ucsd.edu Current Opinion in Cell Biology 1998, 10: Current Biology Ltd ISSN Abbreviations ALS amyotrophic lateral sclerosis BPAG bullous pemphigoid antigen GFAP glial fibrillary acidic protein IF intermediate filament IFAP IF-associated protein MD-EBS muscular dystrophy with epidermolysis bullosa NF neurofilament NLS nuclear localization signal SOD1 superoxide dismutase 1 Introduction Intermediate filaments (IFs) have long been thought of as fixed structural bystanders around whom the lively activity of the cell is distributed. More recently, however, IFs and their associated proteins have been firmly established as constituents of deformable cellular latticeworks, imparting integrity and strength to tissues throughout the body. Long known to extend throughout the cytoplasm, possibly positioning the nucleus within the cell, IF networks have been shown to reversibly link the plasma membrane to other cytoskeletal components to modulate cell shape and confer resistance to mechanical stress. As dynamic components of the cytoplasmic and nuclear cytoskeletons, IFs are thought to contribute to cellular structural rearrangements that occur during cell division. Evidence for pools of soluble IF subunits that can exchange along the entire length of assembled filaments has grown and now helps to explain the basis for the rapid changes in IF organization that observed in response to such stimuli as heat shock and application of growth factors. As many examples of IF dynamics appear to be regulated by phosphorylation, the recent identification of several kinases involved strengthens the argument that these dynamics are true in vivo events. The growing list of intermediate-filament-associated proteins (IFAPs) surely expands the repertoire of possible interactions permitted to IFs and may also add another layer of regulatory complexity. Indeed, the discovery of several human and mouse diseases caused by mutations in the IFAP proteins plectin and neuronal bullous pemphigoid antigen (BPAG1n) illustrates the importance of these molecules in providing links between components of the cellular cytoskeleton. Most would agree that intermediate filaments can no longer be thought of as the least dynamic components of the cell cytoskeleton. This review focuses on the recent advances in understanding IFs and their associated proteins. A growing family of vital intermediate filament cross-linkers Perhaps the most exciting new development in the IF field has been the recognition that IFs have binding partners and that these partners that have important functions in structuring the three-dimensional cytoplasm. The IF-associated proteins, or IFAPs as they have come to be called, are steadily gaining attention as more diseases in humans and mice are shown to arise from mutations in these proteins. The ability of IFAPs to link various components of the cytoskeleton in many different cell types suggests several potential functions as dynamic regulators of cytoskeletal assembly and maintainers of IF network integrity. Support for this notion came recently from studies that utilized peptides corresponding to a conserved helix region of IF proteins that, when injected into fibroblast cells, disrupted the IF, microtubule, and microfilament networks [1 ]. The authors proposed that the introduced peptides effectively competed for IFAPs that would normally link the filament networks together and in this way caused the rapid disassembly of the cytoskeleton. Currently, the list of IFAPs includes members that interact with IFs from all five IF subtypes, but as new IFs (including the lens-specific beaded filaments [2 ]) are identified, the discovery of new linking partners will surely follow. Plectin: an essential linker between intermediate filaments, microtubules, myosin, and actin One of the most thoroughly characterized IFAPs is plectin, an abundant and extremely large (>500 kda) cytoskeletal cross-linker. Plectin is expressed in many cell types and was initially shown by solid phase binding analysis to interact with a wide variety of other proteins such as vimentin, glial fibrillary acidic protein, keratins, lamin B,

2 94 Cytoskeleton microtubule-associated proteins, α-spectrin, and all three neurofilament proteins [3,4]. More recently, immunoelectron microscopy studies [5 ] to evaluate plectin-binding interactions revealed that a) plectin forms cross-bridges between IFs and microtubules, b) vimentin filaments are decorated with plectin projections, c) plectin links IFs and actin filament bundles, and d) plectin associates with myosin filaments in cultured cells. The extraordinarily clear images of plectin cross-bridges between various filament networks supports many of the previously postulated plectin interactions and is further corroborated by biochemical and domain sequence data. Inspection of the rat and human plectin sequences has demonstrated the existence of a putative amino-terminal actin-binding domain [6]. Sequence inspection and mutagenesis studies have mapped a nuclear localization signal (NLS) within plectin s carboxyterminal vimentin binding domain motif [7 ]. Beyond its role as a versatile cross-linker in interphase, the presence of a plectin NLS [6], the fact that plectin binds lamin B in vitro [4], and the established pattern of plectin disassociation from vimentin upon entry into mitosis [8 ] suggest several possible roles for plectin during cell division. One scenario predicts that separating from cytoplasmic IFs would free plectin and collapse the IF network into the typical cagelike IF structure seen during mitosis. Thus freed, the uncovered plectin NLS could sequester NLS binding proteins. Alternatively, plectin s association with lamin B may act to disassemble the nuclear matrix during nuclear envelope breakdown, or perhaps the interaction really functions in the opposite manner, that is, to promote nuclear reassembly (as argued in detail [9]). Although these ideas are as yet only speculative, the protein kinase p34 cdc2 has been shown to phosphorylate plectin and cause its dissociation from vimentin during mitosis [8 ]. Similarly, phosphorylation of plectin by protein kinase A or C can inhibit the binding of plectin to lamin B [4], providing the means by which plectin could accomplish the mitosis-specific roles proposed above. Comparison of plectin s structural domains with those of other cytoskeletal components has yielded intriguing similarities. Plectin shares a high degree of sequence homology and similar domain organization with the neuronal protein BPAG1n/dystonin [6], the epidermal isoforms of BPAG [10], macf7 [11 ], and the desmosomal proteins envoplakin, desmoplakin I, and desmoplakin II. Because of these similarities, this group of proteins has recently been dubbed the plakin family [12]. Plectin, the desmoplakins and cadherins are linker components of desmosomes in cells that experience mechanical stress and function to link keratin IF networks to the plasma membrane, thereby imparting mechanical strength to the individual cell and to the entire tissue. Similarly, plectin, BPAG2e, BPAG1e, and the α6β4 integrin link the components of hemidesmosomes and mediate the attachment of epidermal cells to the basement membrane. That the strength provided by plectin is an essential feature of cellular architecture has been demonstrated by the recent discovery of mutations in the gene for plectin as a cause of the human disease muscular dystrophy with epidermolysis bullosa (MD-EBS) [13 15 ]. This inherited disease is characterized by muscle degeneration and skin blistering due to a failure to anchor the cellular IF network to the plasma membrane via hemidesmosomes. Three of the four known mutations, an eight base pair insertion [13 ], an eight base pair deletion [14 ], and a single nucleotide deletion [15 ], occur within the same region of plectin and result in premature termination approximately one third of the way through the >500 kda polypeptide. The fourth known mutation, a nine nucleotide deletion [15 ], removes three amino acids from within a 23 amino acid stretch that was shown to be identical between human and rat plectin. Tissues from patients harboring the three truncation mutations are typically devoid of plectin, whereas the fourth mutation apparently encodes an otherwise full length protein and results in reduced plectin levels. Patients with any of the four mutations typically display reduced levels of BPAG1e, muscle fiber abnormalities, and hemidesmosomes without an inner attachment plate [13 15 ]. In order to determine more directly which aspects of plectin cross-linking activities are essential in vivo, transgenic mice were engineered that lack the plectin protein [16 ]. These plectin-null mice die two to three days after birth and display pathological features typical of MD-EBS, with a few notable differences. For example, the keratinocytes of MD-EBS skin rupture at the basal hemidesmosome level, whereas the keratinocytes of plectin-null mice rupture at all levels and contain ultrastructurally normal hemidesmosomes and desmosomes. Plectin-deficient skeletal muscle cells in mice were often necrotic and disrupted sarcomeres were prevalent, whereas cardiomyocytes displayed abnormally arranged sarcomeres and disintegrating intercalated discs. Additionally, the distribution and expression levels of selected cytoskeletal components were shown to be altered in the mice lacking plectin, suggesting the involvement of plectin in some aspect of their function. The existence of muscle and skin tissue in these newborn mice indicate that plectin is not necessary for formation and assembly of IFs in these tissues, but is required to provide the stability to withstand subsequent mechanical stresses during life. The loss of plectin protein expression resulting from the previously described mutations in humans and in plectin-null mice accounts for the skin fragility, muscle degeneration and neurodegeneration typically seen in MD-EBS. One important question that remains unaddressed concerns the aberrant muscle phenotype: is it the loss of plectin s linkage between the IF network and the

3 Intermediate filaments and their associated proteins Houseweart and Cleveland 95 plasma membrane, or is it the loss of plectin s binding to actin fibers, that accounts for the fragile muscle cells of MD-EBS patients and plectin-deficient mice? Overall, it appears safe to conclude that plectin is a true linker of multiple cytoskeletal components, providing flexible tensile strength to the three-dimensional cytoplasm within many different cell types. BPAG1n/dystonin: an essential component of sensory neurons As mentioned earlier, the epidermal forms of bullous pemphigoid antigen and the neuronal form, BPAG1n, are members of a sequence-related family of cytoskeletal-linking proteins that also includes plectin, envoplakin, the desmoplakins, macf7 [11 ], and a 450 kda plectin-like antigen [17]. Like the other members of this family, the BPAG members are large proteins with a central α-helical coiled-coil flanked by a globular head and repeating tail segments. On the basis of sequence analysis and its position within epithelial hemidesmosomes, it was initially proposed that BPAG1e would help to form the connections between keratin IF networks and the basement membrane. To address this possibility, mice bearing disruptions in the BPAG1e gene were developed [18]. The basal epidermal cells of BPAG1e-null mice contained hemidesmosomes that were disengaged from the keratin network and cytolysis occurred at this level upon mechanical stress, demonstrating the importance of BPAG1e in maintaining IF contacts with the hemidesmosome. Surprisingly, these mice also displayed severe neurologic defects characteristic of the dystonia musculorum (dt/dt) mouse mutant [18]. The explanation for this additional neuronal phenotype was unclear until positional cloning studies revealed that the locus targeted in the BPAG1e-null mouse also codes for an alternatively spliced isoform, BPAG1n/dystonin [19]. Subsequent characterization of the previously unknown neuronal isoform BPAG1n/dystonin demonstrated that it contains a carboxy-terminal neurofilament-binding domain, found in common with the epidermal BPAG isoforms, and an amino-terminal actin-binding domain that is found only in the neuronal isoform. Upon expression in tissue culture cells lacking IFs, BPAG1n/dystonin was shown to co-align neurofilaments with actin filaments, suggesting that such a linking property is likely to be physiologically important in neurons [7 ]. The appearance of abnormal neurofilamentous networks in degenerating BPAG1n-deficient neurons supports this possibility [20 ]. One problem with this scenario is the finding that, although the expression pattern of BPAG1n/dystonin generally corresponds to the affected areas of the dt/dt and BPAG-null nervous system, there are instances where this is not the case [20,21]. For example, some neurons that would normally express BPAG1n/dystonin do not degenerate in the dt/dt and BPAG-null mice, questioning the universal requirement for such a linking protein in axons. In addition, it is unclear why the primary sensory neurons in the BPAG1n-deficient mice are the most severely affected of all the neurons when it is likely that motor neurons should have similar needs for linker proteins [20,21]. One explanation for this apparent paradox is that some other protein can perform the necessary linking function when BPAG1n/dystonin is missing or not expressed in a particular neuronal cell type. One such candidate protein is the most recently discovered plakin family member, macf7 [11 ]. Although the analysis of the macf7 gene is at an early stage, this protein shows significant homology to BPAG1n/dystonin, contains an actin-binding domain, and is expressed at appreciable levels in the nervous system, making it an attractive potential linker protein. Neuronal intermediate filaments: understanding normal function and role in disease Neurofilaments (NFs) are the predominant type of intermediate filament in most adult neurons of both the central and the peripheral nervous system. The NFs of mature myelinated axons are composed of an NF-L core, with NF-H and NF-M subunits incorporated into the fiber allowing their tails to extend laterally (reviewed in depth in [22]). Several observations have led to the idea that NFs control the increases in axonal diameter that occur following synapse formation. As neurons mature, expression of NFs is increased and accumulation of NFs corresponds directly with increased diameter of developing axons, a key determinant of conduction velocity. NF investment into axons was initially shown to be essential for the establishment of proper axon diameter by the study of a quail mutant that lacks NFs [23]. This finding has been confirmed both in transgenic mice expressing an NF-H gene that blocks filament transport into axons [24], and, more recently, in mice lacking NF-L [25 ]. These latter mice lack axonal NFs (or NF-L, NF-M, and NF-H) and display decreased axonal outgrowth, delayed regeneration after nerve injury, and have a 15 20% loss of motory and sensory axons at 2 months of age. Studies using transgenic mice engineered to overexpress different combinations of NF subunits have proven that NF-dependent radial growth of axons requires the presence of NF-L to drive filament assembly as well as properties provided by NF-M and NF-H [26,27 ]. The latter two are thought to organize axoplasm in a three-dimensional cross-linked array that is capable of supporting axonal expansion. However, a separate increase in either NF-M or NF-H levels reduces the number of axonal filaments (by trapping NFs in the cell body), while increasing the levels of either NF-M or NF-H in the presence of increased NF-L stimulates radial growth without changing the nearest neighbor spacing of neurofilaments [26,27 ]. Challenging this finding is the demonstration of increased filament number and closer filament spacing in the smaller central nervous system

4 96 Cytoskeleton axons of mice overexpressing the human NF-M gene [28]. Here, the elevated levels of NF-M that provoke increased accumulations of NF-L and reduced levels of phosphorylated NF-H may have reduced the amount of NF-H subunits available to form filaments with NF-L. Both studies seem to agree that increased radial growth requires a carefully balanced ratio of NF-L subunits and of the cross-bridge-forming components NF-M and NF-H, but do not fully resolve the question of how an NF array specifies axon diameter. It is clear that, in the largest caliber axons of mice, when very different ratios of the three subunits are expressed and very different amounts of axonal NF are made, the nearest neighbor spacing of these filaments is unchanged. This must indicate the existence of attractive forces between adjacent NFs (these forces are quite apparent in mice with few axonal NFs), but also that the cross-linkers crucial for radial growth must provide longer-range interactions either between NFs that are not nearest neighbors or between NFs and other axonal components. These NF transgenic mice have also been used to show that expression of NF-H at four times the endogenous level selectively slows filament transport through axons [26 ]. It has been suggested that this overabundance of NF-H leads to the assembly of more NFs than can be effectively transported, resulting in the NF accumulation typically seen in cell bodies and swollen axons of these mice. Despite the dual insults of slowed transport and widespread NF accumulations, these mice with high levels of wild-type mouse NF-H do not display phenotypic abnormalities or a loss of neurons. This is in sharp contrast to mice expressing lower amounts of wild-type human NF-H, which develop symptoms characteristic of the disease amyotrophic lateral sclerosis (ALS), such as neurologic defects, muscle atrophy, and neuronal NF accumulations amid a general slowing of slow axonal transport [29]. The finding strongly indicates that the human NF-H protein acts as a mutant protein in mice [26 ]. The discovery that neuronal accumulations of NFs are a common pathological hallmark of several human neurodegenerative diseases, including ALS, has sparked a great deal of effort aimed at understanding whether NFs are themselves the cause of neuron dysfunction, or, at the other extreme, whether they are innocent bystanders. ALS is a late-onset neurodegenerative disease in which the selective loss of motor neurons in the brain and spinal cord leads to progressive muscle weakness, followed by paralysis and death. Several transgenic mouse lines expressing either mutant NFs or high levels of NFs have been created that faithfully mimic the symptoms of ALS. Elevation of wild-type mouse NF-L levels [30] or human NF-H levels [31] to approximately four times the normal amounts resulted in NF accumulations in the cell bodies and axons of motor neurons, muscle atrophy, and axonal degeneration. Even more compelling, the introduction of a single point mutation into the mouse NF-L gene (modeled after keratin mutations frequently found in epidermolysis bullosa simplex) produces mice with selective spinal motor neuron degeneration and death, NF accumulations, and skeletal muscle atrophy [32]. The ability of these varied NF alterations to selectively cause neurodegenerative disease in mice provoked a search for mutations in NFs as the primary cause of ALS in humans. These efforts revealed no mutations in any NF gene from 100 individuals with familial ALS [33], and none in the NF-H KSP region of 117 familial ALS patients [34]. Nevertheless, a previous effort identified either of two small deletions within a KSP-repeating domain of the NF-H gene in 5 of 356 patients with sporadic ALS [35]. Despite these disappointing figures, 15% of familial ALS patients have been shown to bear mutations in the antioxidant metalloenzyme superoxide dismutase 1 (SOD1) [36]. Although the exact mechanism by which mutant SOD1 causes ALS is unknown, a series of experiments has shown that the SOD1 defects are not caused by a loss of SOD1 activity, but instead may result from a gained toxic property. This proposed toxic activity may cause the most damage to long-lived and abundant neuronal components such as NFs. Strong evidence for this proposal comes from the recent discovery of neurofilamentous inclusions in individuals with SOD1 mutations [37,38]. In summary, the data available to date clearly demonstrate that NFs themselves can be the primary cause of ALS-like motor neuron disease in mice, but whether the same is true for humans remains to be proven. Desmin: an essential role in cardiac, skeletal, and smooth muscle The muscle specific IF desmin is expressed in all three muscle tissue types: skeletal; cardiac; and smooth muscle. In developing mammalian muscles, desmin is initially co-expressed with vimentin, but upon terminal differentiation vimentin is downregulated and desmin accumulates around the Z discs of the maturing cells. This localization led to suggestions that desmin functions to maintain the adult contractile apparatus by aligning striated myofibrils laterally via their Z discs and also by linking myofibrils to the nucleus, T tubules, mitochondria, and sarcolemma. Another possible function of desmin that has received more attention lately is a potential role in muscle differentiation and morphogenesis. To test these predictions, two groups used gene disruption to engineer mice lacking desmin. Both obtained viable, fertile animals with strikingly similar phenotypes [39,40 ]. Specifically, skeletal, cardiac, and smooth muscle developed normally, but displayed widespread cell architecture defects such as misaligned muscle fibers, abnormal sarcomeres, swollen mitochondria, and calcium deposits in cardiac muscle tissue. The desmin-deficient

5 Intermediate filaments and their associated proteins Houseweart and Cleveland 97 mice developed weaker skeletal muscles with less endurance and force generation capabilities than normal mice [41 ]. Moreover, although all early stages of muscle differentiation and cell fusion occurred normally, it was only after birth that myofibers were ruptured during contraction and underwent an aberrant repair process that led to the final pathological state of the muscle [41 ]. The severe morphological and functional abnormalities observed in the most active muscle types of both desminnull animals mentioned above underscores the importance of desmin in maintaining the structural integrity of muscle cells, and lends support to the structural model of desmin function. These findings prove that desmin itself is not required for muscle commitment or differentiation and, when combined with the lack of evidence for any compensatory increase in vimentin levels during muscle development [39,40,41 ] and regeneration in desmin-null mice [41 ], or muscle formation in a double mutant mouse lacking both desmin and vimentin [41 ], it is apparent that IFs are not required early in muscle development. Although no human myopathies or cardiomyopathies have been attributed to a complete lack of desmin, there are several accounts of myopathies with excess desmin in the form of granular and filamentous aggregates [39 ]. Glial fibrillary acidic protein: deletion in mice produces subtle, but measurable, defects The IF family member glial fibrillary acidic protein (GFAP) is expressed in astrocytes of the central nervous system, the enteric glia, and in myelin-forming Schwann cells of the peripheral nervous system. Most developing astrocytes initially express vimentin, but later switch to express their final adult IF type, GFAP, as they mature. Astrocytes are thought to modulate neuronal function, help form the blood brain barrier, provide structural and nutritional support for adult neurons, and maintain glial processes as paths for migrating neurons during development. An early study removed GFAP from an astrocyte cell line using antisense transfection and demonstrated an inhibition of glial process extension in the presence of neurons, implying that GFAP is necessary for this important developmental event [42]. The in vivo validity of this result has been diminished in light of the recent findings from a series of GFAP-null mice [43 45,46 ]. All four independently derived GFAP-deficient mice exhibit normal behavior, motor activity, growth, reproduction, and life span. This does not mean that GFAP is completely dispensable, however. Despite the absence of gross structural aberrations, missing astrocyte populations and a loss of blood brain barrier integrity [43 45,46 ], are examples of more subtle phenotypes that have emerged as more sophisticated functional tests have been performed and older mice have been examined. For example, one group has reported important late-onset abnormalities in myelination by oligodendrocytes and a loss of white matter in aged mice [46 ]. In addition, although various electrophysiological parameters such as basic synaptic transmission were found to be normal in the GFAP-null mice, enhancement of hippocampal LTP (long-term potentiation) [45] and deficits in cerebellar LTD (long-term depression) were also demonstrated [47 ]. The possibility that another IF protein such as vimentin may partially compensate for the absence of GFAP seems plausible given the discovery of GFAP filament assembly defects in mice without vimentin [48 ]. It seems likely that a cross between a GFAP-null mouse and the vimentin-null mouse [49] would definitively settle the question of whether vimentin compensates for a lack of GFAP. Nuclear lamins: emerging dynamic functions Although related structurally to the cytoplasmic IFs, nuclear lamin IFs are found exclusively in the nucleus and are the major constituents of the nuclear lamina. The nuclear lamina underlies the inner nuclear membrane and is thought not only to provide mechanical support to the nucleus, but also to aid in nuclear membrane reassembly following mitosis [50]. A recent method used to test the potential roles of lamins and other putative cell cycle proteins involves a nuclear assembly system made from Xenopus laevis egg extracts which, upon entry into an interphase state, assembles many physiological features of the mitotic nucleus [51 ]. Addition of a dominant truncated human lamin A protein to disrupt the endogenous nuclear lamin structure blocked the formation of a normal nuclear lamina and resulted in increased fragility of the nuclei, aggregation of endogenous and mutant lamin A into nucleoplasmic spheriods, a decreased ability to replicate DNA, and the redistribution of chain elongation factors from chromatin to the abnormal lamin aggregates [51 ]. These results lend support to arguments that nuclear lamins are integral to nuclear function and contribute to the formation of some aspects of the DNA replication machinery. Keratins: more telling mutations Keratin proteins constitute the largest and most complex class of intermediate filaments. They are expressed in epidermal cells throughout the body where they form a structural network that spans the cell cytoplasm, linking the plasma membrane, nucleus, and other cytoskeletal components. Keratin deletions in both humans and mice have proven that the keratin network is crucial for maintaining the physical integrity and diverse connections required of various epithelial tissues [52]. Keratin filaments are obligate heteropolymers, meaning that they naturally consist of a 1:1 ratio of type I to type II keratin monomers. The 12 type I keratins and 8 type II keratins all share the typical IF family structure, which is characterized by an amino-terminal, non-helical, head domain followed by a central α-helical rod domain and a non-helical tail domain at the carboxyl terminus. Certain defined combinations of keratin monomer pairing occur

6 98 Cytoskeleton in a tissue-specific and developmentally regulated fashion, thereby expanding the properties of keratin filaments to suit the requirements of various epithelial cell types (reviewed in detail in [52]). In the past few years, rapid progress has resulted from investigators ability to link a great number of skin disorders to specific keratin mutations, thereby gaining a better understanding of both the etiology of the particular disease and the function of individual keratins. One unifying theme emerging from the wealth of data on keratin mutations is that mutations within the keratin genes generally cluster at the ends of the keratin rod domain in severe forms of dominantly inherited disease, but milder forms of disease result from more tolerated changes in the less conserved head and tail regions [52]. Important conclusions about requirements for filament alignment and higher-order assembly can be drawn from these observations. As expected, the number of skin disease causing keratin mutations has continued to grow and has led to the identification of other non-skin diseases caused by keratin mutations. Previous studies had shown that all three major subtypes of the skin blistering disease epidermolysis bullosa simplex (EBS) can result from mutations in keratin 14 K14 or its binding partner keratin 5 (K5). Recently, a rare subtype of EBS, EBS with mottled pigmentation, was also shown to be caused by a point mutation in the head domain of K5 [53 ]. Many mutations that cause the severest forms of epidermolytic hyperkeratosis (EH) had traditionally been found in the rod domains of the K1 and K10 genes, with less severe forms of the disease caused by head domain mutations, but a recent report was able to show that a severe form of EH can result from a head domain mutation as well [54]. To date, only about half of the epidermolytic palmoplantar keratoderma (EPPK) patients examined have mutations in the K9 gene, raising the possibility that other genes will soon be found that may contribute to this disorder. The genetic basis for icthyosis bullosa of Siemens (IBS) had previously been ascribed to mutations within the rod domain of Ke2; the oral/esophageal mucosal disorder white sponge nevus (WSN) has been documented to arise from K4 and K13 rod domain mutations; and pachyonychia congenita (PC) results from mutations in K17, K16 and K6a (other diseases caused by mutations in keratins are reviewed in [52,55]). Most recently, a mutation in K18 has been found in one patient with a liver cirrhosis of unknown etiology, raising speculation that similar keratin mutations may either cause or predispose individuals to liver disease [56 ]. In addition to the numerous mutations of epithelial keratins found to underlie many serious skin disorders, novel mutations in the cornea-specific keratins of the eye and the hard α-keratins of the hair and nail have come forth. Specifically, K3 and K12 missense mutations were found to cause Meesmann s corneal dystrophy, a late-onset disorder characterized by fragility of the corneal epithelium [57]. Also, the description of a point mutation in the type II hair cortex keratin, hhb6, in two unrelated families with the disease monilethrix is the first to provide direct evidence for involvement of keratins in inherited hair disease [58 ]. Like many mutations found in epidermal keratin forms, both the cornea-specific and the hair-specific keratin mutations found to date were confined to the ends of the central rod domain, demonstrating the universal importance of these regions across keratin subtypes. At the last count, 14 of the 20 epithelial keratin genes and one of the ten hard α-keratin genes had been shown to harbor mutations causing human genetic disorders. At the current rate, it seems likely that additional mutations will be discovered in the remaining keratins that may, in turn, help determine what aspect of each abnormal keratin actually induces the cell fragility that is characteristic of these diseases. In order to directly study the in vivo cellular sequence of events that leads to mechanosensitivity due to abnormal keratin, it would be helpful to begin with a normal animal model cell population and be able to induce production of an altered keratin gene of interest into a defined set of cells at any time. Such a system was developed using the human K6a promoter and shows promise both as a tool to study keratin function and as a way to deliver foreign gene products to humans via inducible, transgenic skin grafts [59 ]. This technique makes use of the fact that expression of the K6a gene is spatially restricted and can be induced in response to the topical application of various chemicals. It was shown that the expression of a gene of interest (in this case lac Z or hk6a) could be reproducibly induced in the stratified epithelia and epidermis of mice upon mechanical stress or treatment with retinoic acid or the phorbol ester PMA (phorbol-12-myristate-13-acetate). In a similar fashion, the human K14 promoter was harnessed to overproduce a growth hormone in the skin cells of a donor mouse that were then successfully grafted to a recipient mouse. The recipient mice did display elevated levels of the hormone in the bloodstream, indicating the potential utility of using keratin promoters and skin grafts manipulated in vitro to secrete specific compounds as drug delivery systems [60 ]. Conclusions and prospects The collective findings from the past few years have gone a long way in advancing our understanding of intermediate filaments and their associated proteins. In particular, the widespread use of transgenic and gene deletion mice has helped prove that IFs and their cross-linking proteins, including plectin and BPAG1n, structure the cell cytoplasm by forming flexible, reversible arrays that provide essential resistance to environmental stresses. The pace at which new human disorders are found to be associated with, or directly caused by, aberrant/missing IF proteins and faulty IF connections has revealed an

7 Intermediate filaments and their associated proteins Houseweart and Cleveland 99 efficient way to learn more about the effects of IFs in vivo. The continued identification of novel IFs and their binding partners in different cell types will also lead to a better awareness of what role IFs normally play in cellular dynamics and how these proteins can cause disease in humans. Acknowledgements The authors wish to thank their fellow investigators who sent preprints of work in progress, and apologize to those whose work was not cited because of space considerations. References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest 1. Goldman RD, Khuon S, Chou YH, Opal P, Steinert PM: The function of intermediate filaments in cell shape and cytoskeletal integrity. J Cell Biol 1996, 134: The use of injected intermediate filament (IF) mimetic peptides that rapidly disrupt all three filamentous networks of the cytoskeleton suggests a central role for filaments in maintaining cellular architecture and hints that IF-associated proteins may act as adaptors between the different network types. 2. Georgatos SD, Gounari F, Goulielmos G, Aebi U: To bead or not to bead? Lens specific intermediate filaments revisited. J Cell Sci 1997, 110: This thorough discussion of lens-specific beaded intermediate filaments (IFs) describes the current state of the field and highlights the work that led to our present understanding of this distinct class of IFs. 3. Foisner R, Leichtfried FE, Herrmann H, Small JV, Lawson D, Wiche G: Cytoskeleton-associated plectin: in situ localization, in vitro reconstitution, and binding to immobilized intermediate filament proteins. J Cell Biol 1988, 106: Foisner R, Traub P, Wiche G: Protein kinase A- and protein kinase C-regulated interaction of plectin with lamin B and vimentin. Proc Natl Acad Sci USA 1991, 88: Svitkina TM, Verkhovsky AB, Borisy GG: Plectin sidearms mediate interaction of intermediate filaments with microtubules and other components of the cytoskeleton. J Cell Biol 1996, 135: The immunoelectron microscopy images presented in this work convincingly document the association of plectin with multiple cellular partners (intermediate filaments, microtubules, actin filaments, and myosin), confirming previous biochemical data and establishing plectin as a true organizer of intracellular space. 6. Nikolic B, Mac Nulty E, Mir B, Wiche G: Basic amino acid residue cluster within nuclear targeting sequence motif is essential for cytoplasmic plectin-vimentin network junctions. J Cell Biol 1996, 134: Yang Y, Dowling J, Yu QC, Kouklis P, Cleveland DW, Fuchs E: An essential cytoskeletal linker protein connecting actin microfilaments to intermediate filaments. Cell 1996, 86: The discovery that the neuronal protein BPAG1n/dystonin can co-align neurofilaments with actin filaments in cultured cells strengthens its claim as a cytoskeletal cross-linking protein and provides an explanation for the defects seen in mice disrupted in this locus (see also [18]). 8. Foisner R, Malecz N, Dressel N, Stadler C, Wiche G: M-phasespecific phosphorylation and structural rearrangement of the cytoplasmic cross-linking protein plectin involve p34cdc2 kinase. Mol Biol Cell 1996, 7: The identification of a kinase responsible for the redistribution of the crosslinking protein plectin from an insoluble vimentin-bound state in interphase cells to a more soluble vimentin-independent state during mitosis strongly suggests that plectin acts to modulate cell architecture in many stages of the cell cycle. 9. Foisner R: Dynamic organization of intermediate filaments and associated proteins during the cell cycle. Bioessays 1997, 17: Liu CG, Maercker C, Castanon MJ, Hauptmann R, Wiche G: Human plectin: organization of the gene, sequence analysis, and chromosome localization (8q24). Proc Natl Acad Sci USA 1996, 93: Bernier G, Mathieu M, de Repentigny Y, Vidal SM, Kothary R: Cloning and characterization of mouse ACF7, a novel member of the dystonin subfamily of actin binding proteins. Genomics 1996, 38: This preliminary characterization of the newest plakin family member, macf7, reveals that the three macf7 isoforms have considerable homology to BPAG1n/dystonin and also contain actin binding domain regions. It seems likely that this is the first of many such linking proteins to be found using homology with other proteins known to connect intermediate filaments to other cytoskeletal components. 12. Ruhrberg C, Watt FM: The plakin family: versatile organizers of cytoskeletal architecture. Curr Opin Genet Dev 1997, 7: Smith FJ, Eady RA, Leigh IM, McMillan JR, Rugg EL, Kelsell DP, Bryant SP, Spurr NK, Geddes JF, Kirtschig G et al.: Plectin deficiency results in muscular dystrophy with epidermolysis bullosa. Nat Genet 1996, 13: The demonstration that mutations in a cytoskeletal linker protein such as plectin can cause a human disease characterized by a lack of cell integrity was a clear indication of plectin s essential function in different cell types and argued strongly for the importance of other linker proteins. See also [14 16 ]. 14. McLean WH, Pulkkinen L, Smith FJ, Rugg EL, Lane EB, Bullrich F, Burgeson RE, Amano S, Hudson DL, Owaribe K et al.: Loss of plectin causes epidermolysis bullosa with muscular dystrophy: cdna cloning and genomic organization. Genes Dev 1996, 10: The demonstration that mutations in a cytoskeletal linker protein such as plectin can cause a human disease characterized by a lack of cell integrity was a clear indication of plectin s essential function in different cell types and argued strongly for the importance of other linker proteins. See also [13,15,16 ]. 15. Pulkkinen L, Smith FJ, Shimizu H, Murata S, Yaoita H, Hachisuka H, Nishikawa T, McLean WH, Uitto J: Homozygous deletion mutations in the plectin gene (PLEC1) in patients with epidermolysis bullosa simplex associated with late-onset muscular dystrophy. Hum Mol Genet 1996, 5: The demonstration that mutations in a cytoskeletal linker protein such as plectin can cause a human disease characterized by a lack of cell integrity was a clear indication of plectin s essential function in different cell types and argued strongly for the importance of other linker proteins. See also [13,14,16 ]. 16. Andra K, Lassmann H, Bittner R, Shorny S, Fassler R, Propst F, Wiche G: Targeted inactivation of plectin reveals essential function in maintaining the integrity of skin, muscle, and heart cytoarchitecture. Genes Dev 1997, in press. By disrupting the plectin gene in mice, these authors demonstrate the importance of the abundant cytoskeletal cross-linking protein plectin for reinforcement of several tissues. The defects displayed by these mice are most similar to the human disease muscular dystrophy with epidermolysis bullosa, which was also recently shown (in 14 ]) to result from a loss of plectin. 17. Fujiwara S, Kohno K, Iwamatsu A, Naito I, Shinki H: Identification of a 450kDa autoantigen as a new member of the plectin family. J Invest Dermatol 1996, 106: Guo L, Degenstein L, Dowling J, Yu QC, Wollmann R, Perman B, Fuchs E: Gene targeting of BPAG1: abnormalities in mechanical strength and cell migration in stratified epithelia and neurologic degeneration. Cell 1995, 81: Brown A, Bernier G, Mathieu M, Rossant J, Kothary R: The mouse dystonia musculorum gene is a neural isoform of bullous pemphigoid antigen 1. Nat Genet 1995, 10: Dowling J, Yang Y, Wollmann R, Reichardt J, Fuchs E: Developmental expression of BPAG1-n: insights into the spastic ataxia and gross neurologic degeneration in dystonia musculorum mice. Dev Biol 1997, 187: A more thorough examination of BPAG1n/dystonin expression in normal mice and the pathological changes caused by disrupting the BPAG locus in transgenic mice showed a less restricted expression pattern and distribution of neuronal abnormalities than was previously reported [18]. 21. Bernier G, Brown A, Dalpe G, de Repentigny Y, Mathieu M, Kothary R: Dystonin expression in the developing nervous system predominates in the neurons that degenerate in dystonia musculorum mutant mice. Mol Cell Neurosci 1995, 6: Lee MK, Cleveland DW: Neuronal intermediate filaments. Annu Rev Neurosci 1996, 19:

8 100 Cytoskeleton 23. Yamasaki H, Itakura C, Mizutani M: Hereditary hypotrophic axonopathy with neurofilament deficiency in a mutant strain of the Japanese quail. Acta Neuropathol (Berl) 1991, 82: Eyer J, Peterson A: Neurofilament-deficient axons and perikaryal aggregates in viable transgenic mice expressing a neurofilament-beta-galactosidase fusion protein. Neuron 1994, 12: Zhu Q, Couillard-Despres S, Julien J-P: Delayed maturation of regenerating myelinated axons in mice lacking neurofilaments. Exp Neurol 1997, in press. Disruption of the neurofilament-l gene in mice resulted in the production of axons without neurofilaments, reduced axonal diameter, and axonal regeneration defects. These animals are another example supporting the view that filament number is a key determinant of axonal caliber. 26. Marszalek JR, Williamson TL, Lee MK, Xu Z-S, Crawford TO, Hoffman PN, Cleveland DW: Neurofilament subunit NF- H modulates axonal diameter by affecting the rate or neurofilament transport. J Cell Biol 1996, 135: With a series of transgenic mice expressing completely wild-type mouse NF- H at up to four times the normal amount, this work demonstrates a selective slowing of NF transport, but the absence of any neuronal degeneration under these conditions. This indicates that that motor neuron disease developed by mice expressing human NF-H must arise from the human protein acting like a mutant in mice. 27. Xu Z-S, Marszalek JR, Lee MK, Wong PC, Folmer J, Crawford TO, Hsieh S-T, Griffin JW, Cleveland DW: Subunit composition of neurofilaments specifies axonal diameter. J Cell Biol 1996, 133: By mating transgenic mice expressing elevated levels of murine NF-L, NF-M or NF-H, to produce mice with elevated synthesis of any pair of subunits, this work demonstrates that NF-dependent stimulation of growth in axonal diameter requires NF-L to drive assembly and NF-M and NF-H to mediate interactions in the axoplasm. 28. Tu PH, Elder G, Lazzarini RA, Nelson D, Trojanowski JQ, Lee VM: Overexpression of the human NFM subunit in transgenic mice modifies the level of endogenous NFL and the phosphorylation state of NFH subunits. J Cell Biol 1995, 129: Collard JF, Cote F, Julien JP: Defective axonal transport in a transgenic mouse model of amyotrophic lateral sclerosis. Nature 1995, 375: Xu Z, Cork LC, Griffin JW, Cleveland DW: Increased expression of neurofilament subunit NF-L produces morphological alterations that resemble the pathology of human motor neuron disease. Cell 1993, 73: Cote F, Collard JF, Julien JP: Progressive neuropathy in transgenic mice expressing the human neurofilament heavy gene: a mouse model of amyotrophic lateral sclerosis. Cell 1993, 73: Lee MK, Marszalek JR, Cleveland DW: A mutant neurofilament subunit causes massive, selective motor neuron death: implications for the pathogenesis of human motor neuron disease. Neuron 1994, 13: Vechio JD, Bruijn LI, Xu Z, Brown RH Jr, Cleveland DW: Sequence variants in human neurofilament proteins: absence of linkage to familial amyotrophic lateral sclerosis. Ann Neurol 1996, 40: Rooke K, Figlewicz DA, Han FY, Rouleau GA: Analysis of the KSP repeat of the neurofilament heavy subunit in familiar amyotrophic lateral sclerosis. Neurology 1996, 46: Figlewicz DA, Krizus A, Martinoli MG, Meininger V, Dib M, Rouleau GA, Julien J-P: Variants of the heavy neurofilament subunit are associated with the development of amyotrophic lateral sclerosis. Hum Mol Genet 1994, 3: Rosen DR, Siddique T, Patterson D, Figlewicz DA, Sapp P, Hentati A, Donaldson D, Goto J, O Regan JP, Deng HX: Mutations in Cu/Zn superoxide dismutase gene are associated with familial amyotrophic lateral sclerosis. Nature 1993, 362: Rouleau GA, Clark AW, Rooke K, Pramatarova A, Krizus A, Suchowersky O, Julien JP, Figlewicz D: SOD1 mutation is associated with accumulation of neurofilaments in amyotrophic lateral sclerosis. Ann Neurol 1996, 39: This work demonstrates that neurofilament misaccumulation, itself capable of causing motor neuron disease, is a downstream consequence of a mutation in superoxide dismutase 1 that causes amyotrophic lateral sclerosis. 38. Shibata N, Hirano A, Kobayashi M, Siddique T, Deng HX, Hung WY, Kato T, Asayama K: Intense superoxide dismutase-1 immunoreactivity in intracytoplasmic hyaline inclusions of familial amyotrophic lateral sclerosis with posterior column involvement. J Neuropathol Exp Neurol 1996, 55: Milner DJ, Weitzer G, Tran D, Bradley A, Capetanaki Y: Disruption of muscle architecture and myocardial degeneration in mice lacking desmin. J Cell Biol 1996, 134: This work showed directly that desmin imparts the strength and structural integrity that are necessary for all three muscle types. A lack of desmin in mice does not preclude muscle formation, but does result in mitochondrial disorganization, myofiber misalignment, and degeneration. 40. Li Z, Colucci-Guyon E, Pincon-Raymond M, Mericskay M, Pournin S, Paulin D, Babinet C: Cardiovascular lesions and skeletal myopathy in mice lacking desmin. Dev Biol 1996, 175: Together with [39,41 ], this work with desmin-deficient mice demonstrates the importance of desmin in maintaining the structural integrity of highly used muscle types and proves that desmin is not necessary for the formation of muscle during development. 41. Li Z, Mericskay M, Agbulut O, Butler-Borwne G, Carlsson L, Thronell L-E, Babinet C, Paulin D: Desmin is essential for the tensile strength and integrity of myofibrils but not for myogenic commitment, differentiation, and fusion of skeletal muscle. J Cell Biol 1997, 139:1-16. Together with [39,40 ], this work with desmin-deficient mice demonstrates the importance of desmin in maintaining the structural integrity of highly used muscle types and proves that desmin is not necessary for the formation of muscle during development. 42. Weinstein DE, Shelanski ML, Liem RK: Suppression by antisense mrna demonstrates a requirement for the glial fibrillary acidic protein in the formation of stable astrocytic processes in response to neurons. J Cell Biol 1991, 112: Gomi H, Yokoyama T, Fujimoto K, Ikeda T, Katoh A, Itoh T, Itohara S: Mice devoid of the glial fibrillary acidic protein develop normally and are susceptible to scrapie prions. Neuron 1995, 14: Pekny M, Leveen P, Pekna M, Eliasson C, Berthold C-H, Westermark B, Betsholtz C: Mice lacking glial fibrillary acidic protein display astrocytes devoid of intermediate filaments but develop and reproduce normally. EMBO J 1995, 14: McCall MA, Gregg RG, Behringer RR, Brenner M, Delaney CL, Galbreath EJ, Zhang CL, Pearce RA, Chiu SY, Messing A: Targeted deletion in astrocyte intermediate filament (GFAP) alters neuronal physiology. Proc Natl Acad Sci USA 1996, 93: Liedtke W, Edelmann W, Bieri PL, Chiu F-C, Cowan NJ, Kucherlapati R, Raine CS: GFAP is necessary for the integrity of CNS white matter architecture and long-term maintenance of myelination. Neuron 1996, 17: More detailed examination of older mice lacking GFAP revealed defects in axon myelination and white matter abnormalities that were not apparent in earlier efforts [43,44]. With [47 ], it demonstrates the surprisingly subtle effects of removing the primary intermediate filament constituent, GFAP, from support cells of the CNS. 47. Shibuki K, Gomi H, Chen L, Bao S, Kim JJ, Wakasuki H, Fujisaki T, Fujimoto K, Katoh A, Ikeda T et al.: Deficient cerebellar long term depression, impaired eyeblink conditioning, and normal motor coordination in glial fibrillary acidic protein mutant mice. Neuron 1996, 16: Mice devoid of glial fibrillary acidic protein were found to be deficient in a form of motor learning that is thought to be mediated by long-term depression, supporting the view that astrocytes can modulate higher neuronal functions. 48. Galou M, Colucci-Guyon E, Ensergueix D, Ridet JL, Gimenez Y, Ribotta M, Privat A, Babinet C, Dupouey P: Disrupted glial fibrillary acidic protein network in astrocytes from vimentin knockout mice. J Cell Biol 1996, 133: This report demonstrated that mature astrocytes normally co-expressing vimentin and glial fibrillary acidic protein (GFAP) require the presence of vimentin for normal assembly of a GFAP network, whereas other astrocytes that only expressed vimentin during development form GFAP structures in the absence of vimentin. This represents the first step in understanding the differential IF requirements of even highly related cell types. 49. Colucci-Guyon E, Portier MM, Dunia I, Paulin D, Pournin S, Babinet C: Mice lacking vimentin develop and reproduce without an obvious phenotype. Cell 1994, 79: Moir RD, Spann TP, Goldmann RD: The dynamic properties and possible functions of nuclear lamins. Int Rev Cytol 1995, 162B: Spann TP, Moir RD, Goldman AE, Stick R, Goldman RD: Disruption of nuclear lamin organization alters the distribution