Fuchs 1996

October 24, 1996 9:37 Annual Reviews CHAPTER9.
TXT AR21-09
Annu. Rev. Genet. 1996. 30:197–231

Copyright c 1996 by Annual Reviews Inc. All rights reserved
THE CYTOSKELETON AND DISEASE:

Genetic Disorders of Intermediate
Filaments
Annu. Rev. Genet. 1996.30:197-231. Downloaded from www.annualreviews.org
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Elaine Fuchs
Howard Hughes Medical Institute and Department of Molecular Genetics and Cell
Biology, The University of Chicago, Chicago, Illinois 60637
KEY WORDS: intermediate filaments, cytoskeleton, protein structure, genetic disease, multi-
gene family, protein function
ABSTRACT
Specialized cytoskeletons play many fascinating roles, including mechanical in-
tegrity and wound-healing in epidermal cells, cell polarity in simple epithelia, con-
traction in muscle cells, hearing and balance in the inner ear cells, axonal transport
in neurons, and neuromuscular junction formation between muscle cells and mo-
tor neurons. These varied functions are dependent upon cytoplasmic networks of
actin microfilaments (6 nm), intermediate filaments (10 nm) and microtubules (23
nm), and their many associated proteins. In this chapter, I review what is known
about the cytoskeletons of intermediate filaments and their associated proteins. I
focus largely on epidermal cells, which devote most of their protein-synthesizing
machinery to producing an extensive intermediate filament network composed of
keratin. Recent studies have shown that many of the devastating human disorders
that arise from degeneration of this cell type have as their underlying basis either
defects in the genes encoding keratins or abnormalities in keratin IF networks.
I discuss what we know about the functions of IFs, and how the link to genetic
disease has enhanced this understanding.
CLASSES OF INTERMEDIATE FILAMENT PROTEINS

Members of the IF superfamily are α-helical polypeptides that intertwine in a
coiled-coil fashion to form the dimer subunit structure of 10 nm filaments (54).
IF polypeptides can be subdivided on the basis of their amino acid and cDNA
sequence similarities. Type I and type II IF proteins are the keratins, which are
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198 FUCHS
broadly expressed in epithelial cells (53, 167, 181). There are approximately
30 different keratin genes in the human genome that are differentially expressed
in pairs of type I (K9-K20 and Ha1-Ha4) and type II (K1-K8 and Hb1-Hb4)
proteins ranging from 40 to 67 kDa (112). Among keratins of a single type, the
α-helical domains share 50–99% sequence identity, while keratins of opposite
type display only ∼ 30% homology in these regions (65, 66, 162).
Type III IF proteins share only ∼ 25–40% sequence identity with type I and
type II keratins. The most common of type III IF proteins include: (a) vimentin,
produced by mesenchymal cells (123); (b) desmin, concentrated at the Z discs of
smooth, skeletal and cardiac muscle cells (92); (c) glial fibrillary acidic protein
(GFAP), expressed in neuroglial cells (59); and (d) peripherin, contributing to

the IF network in some neurons of the dorsal root ganglia, sympathetic ganglia,
cranial nerves, and ventral portion of the spinal cord (129). A distant relative of
the type III IF family is nestin, a protein expressed in proliferating stem cells of
the developing central nervous system and to a lesser extent in skeletal muscle
(94).
Neurofilament (NF) proteins, including NF-L, NF-M, NF-H and α-internexin,
have been classified as type IV IF proteins, sharing ∼ 50% sequence identity
with their closest type III relative (24, 58, 98, 99). NF-L, NF-M and NF-H form
the extensive array of 10 nm IFs in the axons of motor and sensory neurons,
whereas α-internexin is restricted to embryonic neurons (124).
The nuclear lamina is composed of a 10 nm filament meshwork of type V IF
proteins, referred to as lamins (1, 49, 107). Lamins interconnect to nuclear pore
complexes on the inner surface of the nuclear membrane (77; for review, see 60).
This structure is universal to higher eukaryotic nuclei and provides a framework
for the nucleus. It has also been implicated in chromatin organization (169).
Lamins differ from other IF protein types by virtue of an insertion of 42 amino
acid residues within the polypeptide chain. Otherwise, their sequence relation
to other IF types is similar. All lamins possess sequences which signal their
import to the nucleus (102).
Several recently sequenced cDNAs encode proteins that are clearly members
of the IF superfamily, but which are atypical and do not fall into any of the
designated sequence types. Two of these, filensin and phakinin, form beaded
heteropolymeric filaments in the lens of the eye (62, 110). Analyses of cloned
cDNA sequences reveal that filensin is a member of the IF superfamily, sharing
the greatest similarities with type III and type IV IF proteins. This said, the
sequence identities shared between filensin and any of these IF proteins are
considerably less than 50%. Another protein, restin, was recently cloned and
shown to be an IF-associated protein of the IF superfamily (12). Restin is found
at high levels in the cells diagnostic for Hodgkin’s disease (41).
THE CYTOSKELETON AND DISEASE 199
Finally, a few proteins have been cloned and share some very distant homol-
ogy with IF proteins. Among the most interesting is the MDM1 protein cloned
by Yaffe and colleagues (105). This yeast protein can assemble into filaments
in vitro, but the filaments have not yet been analyzed by more sophisticated
physicochemical means to ascertain the extent to which they resemble bona
fide 10 nm IFs. The protein is interesting in that its gene was identified in a
genetic screen for a mutant unable to segregate mitochondria appropriately.
IF STRUCTURE AND ASSEMBLY

Cytoplasmic IFs can assemble in vitro in the absence of auxiliary proteins or

factors, suggesting that the information necessary to form a 10 nm filament is

intrinsic, contained within the primary sequence of the IF polypeptide (158).
Given the remarkable sequence heterogeneity among IF proteins, the ability of
proteins to assemble into IFs must rely upon common secondary and tertiary
structures.
The type I-IV IF proteins can be aligned perfectly without introduction of
gaps or insertions (56, 65, 66). The most prominent feature of IF proteins is
a central α-helical domain, the rod, flanked by nonhelical head (amino end)
and tail (carboxy end) domains (Figure 1). The IF α-helical rod is subdivided
into helices 1A, 1B, 2A and 2B, which are interrupted by three short nonhelical
linker segments, linkers 1, 1-2 and 2 (65, 66). The rods of two polypeptide
chains intertwine in a coiled-coil, driven by the existence of heptad repeats
of hydrophobic amino acids, and yielding a hydrophobic seal on the helical
surface of each rod. The coiling of two right-handed α-helical IF rods about
a common axis in a left-handed fashion occurs in register and in parallel, i.e.
with the amino terminal ends of the two rods aligned (1, 56, 126, 133, 180).
In most cases, this interaction results in the formation of homodimers (133 and
references therein). In contrast, keratin IFs appear to be formed largely if not
solely from heterodimers of one type I and one type II polypeptide (37, 68; see
also 156).
Dimers of cytoplasmic IF proteins associate in an antiparallel fashion to form
stable tetramers (132, 180). In register and staggered alignments of dimers have
been described (37, 55, 130, 157, 165), and recent nearest neighbor analyses of
crosslinking data suggest that both forms are likely to exist within the filament
(57, 160, 161). Based on the relative amounts of different cross-linked species,
and on data from paracrystals of IF proteins, the major IF building block is
predicted to be a half-staggered tetramer, and these subunits are linked in a
head-to-tail fashion to yield linear chains, or protofilaments (57, 111, 160, 161,
165). Ultrastructural analyses and scanning transmission electron microscopy
has led to the postulate that groups of two protofilaments intertwine to form
200 FUCHS
Figure 1 Model of intermediate filament assembly. Top: Stick figures depict secondary structures
of IF proteins (65, 66). Boxes denote the coiled-coil rod segment typical of IF dimers; arrow
indicates direction of polypeptides, from base (N-terminus) to tip (C-terminus). Large boxes
encompass the α-helical rod domain, interrupted by short nonhelical linker segments. Hatched
boxes denote highly conserved ends of the rod. Thinner bars denote nonhelical head and tail
domains, with the H1 domain unique to type II keratins (161) shown as solid bar. Bottom: Putative
arrangement of dimer subunits in the IF. In the diagram, the conserved rod ends are overlapping by
∼ 10 amino acid residues, as suggested by Steinert et al (160, 161). Note: in this model, unstaggered
antiparallel alignments of dimers arise at the level of protofibril-protofibril associations (73). Small
black bar in each arrow denotes the sequence in the L1-2 linker region, which according to the
model is directly opposite the putative rod end-overlap in an adjacent dimer (20). Model is adapted
from that previously described [51, 54 (with permission from the Journal of Cell Biology); see also
54, 73, 161].
protofibrils, and groups of four protofibrils intertwine to produce the 10 nm

filament (2, 46, 164). At one of these higher ordered levels of interactions,
unstaggered alignments are predicted (73). Figure 1 provides a model that is
consistent with the data at hand (54). Additional studies will be necessary to
assess the extent to which these models accurately predict the relative align-
ments of IF subunits within the 10 nm filament.
DIFFERENTIAL EXPRESSION OF KERATIN GENES

IN EPITHELIAL TISSUES
A. Expression of Keratins in the Epidermis and Hair Follicles
The epidermis is a stratified squamous epithelium, the outermost layer of which
is the skin surface (Figure 2A). In neonatal skin, only the innermost or basal
layer is mitotically active. As cells withdraw from the cell cycle and begin to
move outward they undergo a series of morphological and biochemical changes
that culminate in the production of dead, flattened squames, which are then
sloughed from the skin surface. For most body regions, it takes two to four
weeks for cells to complete terminal differentiation.

The epidermis and its appendages, such as hair and nail, serve a protective
function in the body, keeping microorganisms out and important bodily fluids
in. These tissues are composed of keratinocytes, which produce an extensive
network of 10 nm keratin filaments. The keratin cytoskeleton is likely to play
an important role in manifesting the protective functions of keratinocytes, and
the complexity in the patterns of keratin expression suggests that the keratin
cytoskeleton is specifically tailored to suit the particular structural needs of
these cells in the skin.
Cells in the mitotically active layer have a dynamic keratin cytoskeleton
composed of filaments that stretch from the nuclear envelope to either the
hemidesmosomes that attach the keratinocyte to the basement membrane, or
the desmosomes that interconnect keratinocytes to one another. Keratins K14
(type I) and K5 (type II) constitute the keratin network in basal cells, and they
account for greater than 10% of the total cell protein in vivo. As cells exit the
basal layer they switch off the transcription of K5 and K14 genes and induce
expression of K1 (type II) and K10 (type I) genes (53, 140). In the upper spinous
layers, K2e (type II) is also produced (29). These suprabasal keratins are typical
of nearly all epidermal regions. In contrast, K9 is a suprabasal keratin unique
to palmar and plantar skin, and to calluses (53, 91). The synthesis of K1, K10,
K2e and K9 in the suprabasal layers is significantly greater than the synthesis of
K5 and K14 in basal cells. The suprabasal keratins can account for up to 85%
of the total protein of fully differentiated cells. In vitro, suprabasal keratins
assemble into 10 nm filaments that aggregate, a feature which correlates with
the progressive bundling of filaments that occurs in vivo as cells move into the
spinous layers.
When epidermal cells reach the granular layer, they produce filaggrin, which
in vitro, has been shown to bundle 10 nm filaments (103). This feature may
relate to the further bundling of keratin filaments into large macrofibrillar struc-
tures as cells move into the stratum corneum layers. The granular layer is the
202 FUCHS
last stage of terminal differentiation where the transcriptional machinery is still

active. As cells become permeable, transglutaminase is activated by calcium,
and a cornified envelope forms from γ -glutamyl -lysine crosslinking of pro-
teins deposited beneath the plasma membrane (137). Recent studies show that
keratin macrofibrils form specific crosslinks to this envelope, thereby produc-
ing a highly insoluble sac of macrofibrils (159). The suprastructure is resistant
to the massive destructive phase which then ensues, and it provides protection
to the skin surface.
The hair follicle has an equally elaborate program of keratin gene expres-
sion (Figure 2B). The matrix cells form a bulb of relatively undifferentiated,
A B
Figure 2 The patterns of keratin expression in epidermis and hair follicle. (A) Epidermis. Epider-
mal cells in the innermost, or basal, layer express keratins 5 and 14, hallmarks of mitotically active
cells of stratified squamous epithelia. As basal cells withdraw from the cell cycle and commit to
terminally differentiate, they switch off the expression of K5 and K14, and begin their journey
towards the skin surface. As they enter the spinous layer, cells induce the expression of K1 and
K10, and in palmo and plantar skin, K9 is also induced. As epidermal cells continue to move
outward, they express K2e, which is the last keratin to join the program of terminal differentiation.
(B) Hair follicle. In the growth phase of the hair cycle, cells from dermal papilla interact with
proliferating matrix cells, which are relatively undifferentiated and express little if any IF proteins.
Under an as yet unidentified trigger, matrix cells cease to divide, begin to migrate upward, and
commit to concentric rings of differentiated states: cortex, which express the Ha and Hb keratins,
i.e. those that are specific to hair; inner root sheath (IRS), expressing keratins 1 and 10; outer root
sheath (ORS), and expressing keratins 5 and 14. The nomenclature of the keratins is according to
Moll et al (112).
mitotically active cells that surround the dermal papilla cells at the base of the
follicle. These cells differentiate in upward cylindrical columns. Cells at the
very center become cortex cells as they leave the matrix. The cortical cells
express the Ha and Hb keratins that constitute the 10 nm filaments forming
the bulk of the differentiated medulla or hair shaft. The concentric cylinder of
cells surrounding the medulla is the hair cuticle which may also express these
hair-specific keratins. The hair cuticle remains with the medulla when it breaks
the skin surface and emerges as a hair.
The hair is also surrounded by two sheaths. The inner root sheath (IRS) con-
sists of three concentric cylinders of cells. A layer of inner root sheath cuticle
cells keeps contact with the hair shaft. Circling the cuticle shaft is Henle’s and
then Huxley’s layers of cells. Both of these layers express K1 and K10 and in
many respects, these cells resemble differentiating spinous cells. The IRS is
surrounded by an outer root sheath (ORS). The ORS is contiguous with the
epidermis. In its upper third, the outermost layer of ORS cells transcribes the
K5 and K14 genes and is likely to be derived from the epidermal basal layer.
These ORS cells differ from epidermal basal cells in that switch on expression
of K6 and K16 rather than K1 and K10 as they differentiate. In the epider-
mis, K6 and K16 are induced only transiently during wound healing (86, 104,
167). They are also expressed suprabasally in the epidermis of proliferative skin
disorders (167). This said, there is no direct link between K6 and K16 expres-
sion and proliferation, since factors such as TGFβ, which inhibits keratinocyte
proliferation, can also induce these genes (26).
Below the isthmus of the hair follicle, the ORS is not derived from the
epidermis, but rather from cells at the base of the follicle. In contrast to other
cells that originate from the matrix, a population of ORS cells seem to remain
proliferative and appear to be self-generating (139). These cells express very
low levels of K5 and K14, but as they move upward and inward, they increase
the expression of these keratins (40).
By electron microscopy, the outermost layer of ORS cells below the isthmus
are considerably less differentiated and contain many fewer keratin filaments
than the basal layer of the epidermis. The matrix cells of the follicle contain
almost no discernable keratin filaments (40). There is still controversy as to
whether matrix cells are the true stem cells of the hair follicle or whether they
are derived from a population of putative stem cells residing in a region called
the bulge up near the sebaceous glands (35) or in the lower portion of the
ORS (139). The extent to which these controversies arise from species-specific
differences is unclear, and must await the development of biochemical markers
to more intricately dissect out this pathway. However, as judged by keratin
expression alone, matrix cells are the least differentiated population of cells
within the skin.
204 FUCHS
B. Keratins Expressed in Other Epithelial Tissues

K5 and K14 are not restricted to the skin, but are broadly expressed in nearly
all stratified squamous epithelia (119). Their expression is most abundant
in surface epithelia, which in addition to the epidermis and ORS of the hair
follicles includes the limbal cells of the cornea, which induce expression of
K3 and K12 as they terminally differentiate (15, 119, 167). Oral epithelia,
including the tongue, and anal and vaginal epithelia are also a rich source of
K14 and K5 (166, 177). As these cells differentiate, they express K6 and K16,
again setting them apart from many other stratified tissues. Basal cells of more
internal stratified tissues, such as the esophagus, express K5 and K14, but their
keratin network is more disperse and less abundant than the network of external
basal cells. A closer inspection of these differences reveals that another keratin,
K15, is actually the predominant type I keratin in neonatal esophagus, and a
keratin equal in amounts to K14 in adult esophagus (101 and references therein).
Conversely, while K15 is found in the basal layers of external stratified tissues,
including the epidermis, it is a minor component of these tissues. Thus, while
K14 and K5 are the major basal keratins of external stratified tissues, K15 and
K5 may be the major basal keratins of more internal tissues.
Finally, K5 and K14 are expressed in a number of nonstratified squamous
epithelia, including the myoepithelial cells of the mammary glands, salivary
glands and sweat glands, in the reticular cells and Hassel’s corpuscles of the
thymus, and in the parathyroid gland. Only in the Hassel’s corpuscles are there
differentiation-specific keratins, in this case, K1 and K10.
In contrast to stratified squamous epithelia, nearly all simple epithelial tissues
express K8 and K18, and in some situations, they also express K7 and K19
(181). K7 is a marker of mesothelium, while K19 has been tauted as a keratin
expressed often in transitional epithelia (100, 155). Surprisingly, despite the
comprehensive studies of the 1980s, a new keratin, K20, has recently been
cloned (113).
MOLECULAR MUTAGENESIS EXPERIMENTS ON

INTERMEDIATE FILAMENT PROTEINS:
PLANTING THE SEEDS OF A REVERSE GENETIC
APPROACH TO ELUCIDATE GENETIC DISORDERS
OF INTERMEDIATE FILAMENT GENES
The sequence identity among all IF proteins is especially high at the start of
coil 1A and near the end of coil 2B (Figure 1). Molecular mutagenesis exper-
iments revealed that these sequences are particularly important for IF network
formation and filament assembly. The first studies of this sort involved deletion
mutations of human keratin 14 (3, 4). Cultured human epidermal cells were en-
gineered to express various truncated versions of an epitope-tagged K14 cDNA.
Mutants missing the nonhelical head or tail domains of K14 integrated into
the endogenous K14-K5 network without perturbation. In contrast, mutants
missing one or the other of the highly conserved rod ends elicited dramatic per-
turbations in the endogenous network, and in some cases, resulted in punctate
aggregates of keratin throughout the cell cytoplasm (3, 4). When these mutants
were engineered in bacterial expression systems and then purified and subjected
to in vitro dimerization assays, these mutants behaved in a true dominant nega-
tive fashion to produce mutant heterodimers with their wild-type partners (36).
In filament assembly assays, these mutant dimers assembled into filamentous

structures that were considerably shorter than wild-type 10 nm filaments (36).
Subsequent transfection and filament assembly studies with other truncated IF
proteins further underscored the importance of the rod end segments in filament
network formation and filament elongation (3, 4, 102, 134).
As the relevance of the rod end segments became clearer, site-directed muta-
genesis was utilized to explore the significance of specific residues within these
domains (70, 96, 102). Even conservative amino acid substitutions in the amino
end of helix 1A or the carboxy end of 2B resulted in marked perturbations in
10 nm filament structure. Surprisingly, proline substitutions more centrally
within the rod often less severely affected IF assembly than did subtle mu-
tations at the rod ends (96). Further support for the importance of the rod
ends came from the use of synthetic peptides to these sequences (and certain
other conserved regions): At a large (50 molar) excess, these peptides led to IF
disassembly (25, 71, 88).
In contrast to the rod ends, the nonhelical tail domains of IF proteins have
often been dispensable in filament assembly assays. Thus, for example, pro-
teolytic removal of a desmin tail segment (84), or deletion of the complete
vimentin tail (44, 106), has only very minor effects on IF assembly in vitro.
Similarly, in vitro, tailless K14 and K5 can still assemble into filaments that
look surprisingly normal (69, 178, but see also 8). Even for the type IV and V
IF proteins, the removal of the tail segment does not seem to block intrafilament
associations from occurring (but see 73). However, at least for type III IF pro-
teins (106) and keratins (69, 178), filaments formed from tailless proteins have
a tendency to unravel under conditions where wild-type IFs do not, suggesting
a possible role for the tails in stabilizing protofibrillar interactions and filament
diameter.
In most cases, IF proteins that are missing their nonhelical head domain are
more severely compromised in filament-forming efficiency than those miss-
ing the tail segment. Thus, neither headless desmin nor headless vimentin are
206 FUCHS
competent to form IFs on their own (67, 84, 134, 172). Headless lamins form
dimers, but they do not associate in a head-to-tail fashion. In contrast, headless
NF-Ls form linear arrays of tetramers, but these protofilaments do not assemble
further unless 1–2 mM calcium is added (73). Even under these conditions,
IFs composed of headless NF-L possess fewer protofibrils than normal (73).
Collectively, these observations suggest that the head domain is involved in
lateral associations and/or in end-to-end stabilization of subunits.
The only IF head thus far scoring as dispensable for in vitro filament assembly
is the K14 head (36). While part of the explanation for the functionality of
headless K14 is likely to reside in the obligatory heteropolymeric nature of
keratins, it is worth noting that conversely, headless K5 does not assemble into
bona fide IFs with wild-type K14 (178). This suggests a dichotomy of the type
II and type I head domains, a feature also indicated by computer modeling (54;
see also 161).
Very few studies have been conducted on the role of the nonhelical linker
segments that interrupt the α-helical rod domain. Two indicators suggest that
these sequences are important for the assembly process: (a) While not con-
served in sequence, these segments always contain helix-breaking residues and
are situated within the same proximity relative to the rod ends in all IF proteins
(65, 66), and (b) the removal of proline residues within these linker segments
elicits filament aggregation in vitro (96).
THE GENETIC BASIS OF EPIDERMOLYSIS BULLOSA

SIMPLEX: THE DISCOVERY OF A HUMAN GENETIC
DISORDER OF AN INTERMEDIATE FILAMENT PROTEIN
Two major cell types of the body, keratinocytes and neurons, devote most of
their protein-synthesizing machinery to developing an elaborate network of
10 nm intermediate filaments. Given that keratin filaments and neurofilaments,
respectively, are the major structural components of these cells, it seemed that
there should be genetic disorders involving the genes encoding these IF proteins.
If this premise was correct, then the dominant negative behavior of most IF
mutants predicted that IF diseases should be largely autosomal dominant in
nature.
Transgenic Mice Engineered to Express Mutant K14 Genes

Provided Important Clues to the Genetic Basis
of Epidermolysis Bullosa Simplex
The first study that directly tested this hypothesis was conducted by Vassar
et al (175), who expressed a truncated human K14 gene in transgenic animals.
The mutant had already been shown by transfection and filament assembly
studies to be severely disrupting to IF structure (3, 36). At birth, transgenic
mice displayed signs of skin blistering upon mild physical trauma (Figure 3A).
Sectioning and ultrastructural analysis revealed that the blistering arose from
cytolysis within the basal epidermal layer (Figure 3B, C). In some cells, clumps
or aggregates of keratin filaments were present that labeled with antibodies
against K5 and K14, as well as the epitope-tagged K14 transgene product
(Figure 3C). Oral involvement was also detected, consistent with the known
pattern of transgene expression (175, 176). When the phenotype and pathology
of the mice was compared with that of known human genetic skin disorders,
a striking resemblance was noted to the human skin disorder, Dowling-Meara

epidermolysis bullosa simplex (D-M EBS) (175).
Human Epidermolysis Bullosa Simplex
Epidermolysis Bullosa Simplex (EBS) constitutes a group of autosomal dom-
inant genetic skin diseases affecting 1 in 40,000 in the population (48). All
EBS disorders are typified by skin blistering as a consequence of cell cytolysis
within the basal epidermal layer. In all cases, the blisters are induced by me-
chanical stress, and they heal without scarring. EBS is further classified into
three major subgroups according to clinical severity. Dowling-Meara (D-M)
EBS is the most severe form in which blistering is present at birth and occurs
over the entire body surface (Figure 4A). Occasional oral and nail involvement
have also been described. Ultrastructurally, clumps or aggregates of keratin
filaments are seen in the basal layer (Figure 4B) and these label with antibodies
against K5 or K14 (Figure 4C).
Koebner EBS (K-EBS) is intermediate in severity, with generalized but less
painful blistering and with abnormalities, but no keratin clumping, in the basal
layer (48). Oral involvement is not seen in this milder subtype of EBS. Even
milder is the Weber-Cockayne (W-C) EBS subtype (Figure 5). In this form,
blistering is confined to the hands and feet. Unlike the other forms of EBS,
symptoms in W-C EBS are often not apparent at birth, and sometimes not
detected until adult life.
Interestingly, transgenic mice engineered to express more mildly disrupting
mutant K14 proteins displayed basal cell rupturing in paw skin within ∼ 2–5
days after birth (Figure 5A; 39). The delayed onset and restricted location of
blistering resembles that of W-C EBS in humans (Figure 5B). Ultrastructurally,
W-C EBS basal cells exhibited few if any aberrations in keratin filament archi-
tecture, but nevertheless, signs of intraepidermal cell cleavage are still present
(arrowheads in Figure 5C; 64). Similarly, epidermis from transgenic mice ex-
pressing mild disrupters of basal keratin filament assembly displayed little or
no discernible abnormalities in basal keratin networks (39).
208 FUCHS
Figure 3 Transgenic mice expressing a K14 mutant gene display characteristics of Dowling-
Meara EBS. (A) Phenotype of mice. Mice engineered to express a severely disrupting K14 mutant
exhibit gross blistering over their entire body trunk. The blistering arises from mild mechanical
stress, in this case simply moving through the birth canal (175). (B) Histopathology of the skin of
K14 mutant transgenic animals. Skin section from animal expressing a mutant K14 gene reveals
that the blistering (arrows) arises from degeneration in the basal epidermal layer [reprinted with
permission from the Journal of Cell Biology; 39]. Immunoelectron microscopy of basal epidermal
cells from mice expressing the severely disrupting K14 mutant. Transgenic basal epidermal cells,
such as the one shown here, exhibit clear cytoplasm, indicative of cell cytolysis. The keratin
network is in clumps or aggregates that label with antibodies to the transgene (shown) as well as
antibodies against K5 and K14 (not shown). These features are typical of basal cells from patients
with Dowling-Meara EBS (39, 175). BL, basal lamina; asterisks denote cell cytolysis; KC, keratin
clump. Bar in B represents 100 µm in B and 0.5 µm in C.
At first, despite the remarkable similarities between mutant K14-expressing

transgenic mice and EBS, it seemed quite unlikely that EBS was a keratin
defect: two different reports had mapped the defect to chromosome 1 (79, 81,
115), whereas the keratin 14 gene had been mapped to chromosome 17 (143)
and the keratin 5 gene had been mapped to chromosome 12 (142). Moreover, a
number of biochemical studies had focused on the possibility that the cytolysis
in EBS might be due to an underlying enzymatic or glycosylation defect (121,
151, 153). Biochemical studies on EBS keratins led to one conclusion that EBS
was likely not a keratin disorder (170) and another conclusion that EBS might
be an abnormality in transcriptional regulation of keratin gene expression (82).
Thus, despite elegant early electron microscopy studies raising the possibility
that EBS might be a keratin defect (6) and despite noted similarities between
cultured EBS cells and keratinocytes transfected with mutant keratin genes (85),
few researchers in the field had given much credence to the notion that a defect
in keratin could give rise to the massive cell cytolysis seen in EBS.
The ability to genetically engineer an EBS mouse by expressing a mutant
K14 gene made it likely that at least some cases of human EBS would also
have as their basis defects in the K14, or partner K5, genes (175). Within less
than a year, two groups demonstrated that human EBS is indeed an autosomal
Figure 4 Human Dowling-Meara EBS. (A) Leg of patient with Dowling-Meara EBS. Note pres-
ence of blistering over the body surface. In D-M EBS, blistering is often in clusters, and it arises as
a consequence of mild physical trauma (courtesy of AS Paller). (B) Electron microscopy of basal
layer of epidermis from patient with Dowling-Meara EBS. Note presence of clumps or aggregates
of keratin in the basal cell cytoplasm. Note that not all keratin is in clumps; some keratin appears
to be in relatively normal IF bundles. The appearance of these IFs misled early researchers into
thinking that the disease was not rooted in a keratin mutation. kf, keratin filaments; kc, keratin
clumps; N, nucleus; bm, basement membrane; he, hemidesmosome; arrowheads denote desmo-
somes [reprinted with permission from Cell; 38] (C) Immunoelectron microscopy of keratin clump
from basal cell of patient with Dowling-Meara EBS. Cells were labeled with anti-K14 antibodies.
Note the specific labeling over the protein aggregates [reprinted with permission from Cell; 38].
Bars represent 1 µm.
October 24, 1996
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dominant disorder of keratins 5 and 14 (13, 38). In one study, two unrelated
patients with Dowling-Meara EBS were shown to harbor point mutations in the
same arginine residue located within the highly conserved amino end of the K14
rod domain (38). One individual had an R125C substitution, while the other one
had an R125H mutation. This C to T transition at a CpG dinucleotide is a hotspot
for methylation/deamination (33) and is now known to account for nearly half
of all of the D-M EBS patients thus far analyzed (22, 38, 163; E Hutton, P
Rice & E Fuchs, unpublished data). The arginine residue is located within the
first heptad of helix 1A, and is conserved even in the invertebrate IF proteins
(Figure 6; see also Figure 1). Functional studies have now demonstrated that
either R125 mutations is sufficient to cause dominant negative disruption of the

endogenous keratin network and perturbations in filament elongation (23, 38,
72, 97).
In the other study on human EBS, classical genetics was used to map the
defects in two EBS families (13). Affected members of a family suffering from
Koebner EBS were found to have a defect that mapped to chromosome 17q12-
q21, near or at the K14 locus, and subsequent sequence analysis revealed an
L383P mutation in helix 2B of the rod domain (13). Affected members of a
family with the mildest form, Weber-Cockayne EBS, mapped to chromosome
12q11-13, near or at the K5 locus. While this study did not report the mutation
involved, within a few years, it was confirmed by mutational analyses that
W-C EBS is also a disorder of keratin, most frequently harboring mutations in
K5 (20, 21, 150). These reports had higher lod scores than most earlier EBS
mapping reports, and now provided genetic mapping data confirming that EBS
is indeed a disorder of keratin.
The location of known keratin mutations relative to IF secondary structure
is summarized in Figure 6. The growing catalogue of EBS keratin mutations
underscores the existence of genetic hotspots, first unveiled with the initial
publication on the genetic basis of human EBS (38). As predicted from random
point mutagenesis (96), nearly all keratin mutations causing the most severe
form of EBS reside in the amino (1A) or carboxy (2B) ends of the central
←−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−
Figure 5 Weber-Cockayne EBS in mice and men. (A) Transgenic mouse typical of those express-
ing mild-disrupting K14 mutations. Note blistering confined to the paws (39). (B) Feet from human
patient with Weber-Cockayne EBS. In this form of EBS, patients have mild blistering, generally
restricted to the palmo and plantar skin (courtesy of AS Paller). (C) Electron microscopy of basal
layer from human patient with Weber-Cockayne EBS. Note the rupturing of the basal cells beneath
the nucleus and above the hemidesmosomes (arrowheads). This region of the cell appears to be
more fragile and prone to breakage (21, 39, 64). Note the absence of cytolysis and the relatively
normal-looking keratin filament network.
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coiled-coil rod domain of the polypeptides (19, 22, 38, 90, 163, 183). In
contrast, Koebner mutations are frequently proline substitutions more centrally
located within the rod segment (13, 42). Again, this finding agrees well with
random mutagenesis studies showing that proline mutations more centrally in
the rod domain are less deleterious to IF assembly than those at the rod ends (96).
Finally, W-C mutations are often nonproline substitutions that reside either in
a nonhelical linker (L12) segment within the rod, or in the head domain of K5
(20, 21, 45, 150). Although the precise location of W-C mutations had not been
anticipated from random mutagenesis studies, the importance of the K5 head
domain and the L12 domain to filament assembly had already been noted (96,
178).
Further verification that the location of EBS mutations correlates with disease
severity stems from engineering and testing the precise EBS mutations found
in patients (20, 21, 38, 97). Interestingly, while D-M mutations predominantly
affect the filament elongation process, W-C mutations seem to influence lateral
associations within the filament (Figure 7).
Insights as to how these mutations elicit the responses that they do stem
from recent chemical crosslinking studies (57, 160, 161). Crosslinks were
detected between the beginning of helix 1A of one keratin polypeptide and
the end of helix 2B of another. According to alignment models arising from
crosslinking studies, a 1.6 nm (10–11 residue) overlap is predicted between the
conserved rod ends of two head-to-tail oriented dimers (for review, see 54). This
fascinating observation provides an explanation for why the rod ends are highly
evolutionarily conserved and why D-M EBS point mutations in the first heptad
of helix 1A and the last heptad of helix 2B might affect filament elongation.
Such associations may involve ionic associations, given that the beginning of
helix 1A and the end of helix 2B are highly basic and acidic, respectively. This
slight overlap between rod ends also explains why in rotary shadowed IFs, the
axial pitch is only 21–22 nm, rather than an exact half (23 nm) of the rod length
(see 54 and references therein). Thus, through genetic analyses of severe EBS
←−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−
Figure 6 EBS Mutations relative to the secondary structure of keratin. Summary of mutations
found in patients with EBS and correlation between the location of mutation and disease severity
of EBS. Stick figure depicts secondary structure of human keratins. Large boxes encompass the
central α-helical rod domain. Hatched boxes denote the highly conserved end domains of the
rod. Lines denote the nonhelical linker segments. Solid bars denote the nonhelical head and tail
domains, often conserved only for a single IF type. Note that D-M EBS mutations are clustered
within the highly conserved rod ends whereas K-EBS mutations are more internal. In contrast,
W-C EBS mutations are in the nonhelical regions of keratin, particularly in the L1-2 linker segment
separating helix 1B from helix 2A [reprinted with permission from Blackwell Publishers; Chan &
Fuchs, 1996].
214 FUCHS
Figure 7 In vitro filament assembly studies of D-M EBS and W-C EBS mutants. Human K5
and K14 were expressed in bacteria, purified by FPLC, and assembled into filaments in vitro.
(A) Filaments formed from normal K5 and K14; (B) filaments formed from normal K5 and D-M
EBS mutant (K14 R125C) giving rise to severely shortened filaments; (C) filaments formed from
normal K14 and W-C EBS mutant (K5 M327T) showing minor perturbation on IF structure, mostly
unraveling of the filaments. [For methods and additional examples, see References 20, 38, 96].
Bar, 100 nm.
cases, new hypotheses have emerged that have led to a refinement of 10 nm

filament structure.
Why do most of the W-C EBS mutations reside in the carboxy half of the
L1-2 linker? In a model of linear arrays of dimers, where the helix 1A ends of
one dimer have been postulated to overlap with the helix 2B ends of the next
dimer in line, the carboxy ends of the L1-2 linker segments within one chain of
dimers might be nearly opposite to the end overlap segments within an adjacent
row of dimers (see Figure 1; 20). If this model is correct, then this portion of
the L1-2 linker is in a position to play a role in promoting appropriate staggered
lateral associations between the linear arrays of alternating dimer chains. This
segment could also participate in further stabilizing the end-overlap interactions
(Figure 1). The fact that mutations in the carboxyl end of the linker L1-2 region
cause unraveling of filaments assembled in vitro is in agreement with this notion
(20).
Taken together, it appears that keratin mutations giving rise to greater clinical
severity cause perturbations in filament elongation, whereas mutations causing
milder phenotypes often affect lateral associations within the filament. While
the future may yet provide us with additional and important refinements to this
trend, a knowledge of keratin structure has given us a better understanding of
how the genetic defects give rise to the clinical manifestations of the disease.
There are several rare subtypes of EBS, and recently it has been discovered
that at least one of these, EBS with Mottled Pigmentation (MP), is also a genetic
disorder of keratins 5 and 14 (173). Patients with MP-EBS not only have gener-
alized blistering due to basal cell cytolysis, but also hyper- and hypopigmented
spots over the body surface. Affected members of two seemingly unrelated
families with MP-EBS have a P24L mutation in one of two K5 alleles, and
linkage analyses confirmed a genetic defect on chromosome 12q11-q13 (173).
Only conserved between K5 and K6, and not among any of the other type II ker-
atins, 24P is in the nonhelical head domain of K5, and only mildly perturbs the
length of 10 nm keratin filaments assembled in vitro. However, this part of the
K5 head domain is likely to protrude on the filament surface, perhaps leading
to additional aberrations in IF architecture and/or in melanosome distribution
that are seen ultrastructurally in patients with the mutation.

Finally, it should be noted that although most EBS cases examined to date
carry point mutations in the coding region of either one of the two basal keratin
genes, it is still possible that in some cases of EBS, the genetic defect may
reside outside the K5 or K14 genes. In this regard, it was recently shown that
defects in the proteins involved in connecting keratin IFs to hemidesmosomes
can lead to basal cell cytolysis, similar to EBS (63). It will be of interest in
the future to assess whether there are subtypes of human EBS disorders that
involve mutations in one or more of the hemidesmosomal genes.
ADDITIONAL DISORDERS OF KERATIN

The discovery of the genetic basis of EBS set the paradigm for a keratin disor-
der. A severe filament-disrupting defect in a keratin gene would be predicted
to cause mechanical stress-induced degeneration in the cells where the gene is
expressed, and ultrastructurally, there should be aggregates or clumps of ker-
atin IFs in these cells. There are more than 30 different keratin genes in the
human genome, and different pairs of keratin genes are differently expressed
in different epithelial cells and at various stages of differentiation and develop-
ment. Therefore, when transgenic mouse studies demonstrated that EBS could
be generated from K5/K14 mutations, additional candidate disorders of keratin
surfaced immediately.
Epidermolytic Hyperkeratosis
Epidermolytic hyperkeratosis (EH) is an autosomal dominant disease, which
shares certain striking mirror-image similarities to EBS. The histopathology of
EH reveals a normal basal epidermal layer, but cytolysis in the suprabasal layers
(for review, see 6). As in EBS, clumps of keratin filaments and perinuclear
shells of keratin aggregates can be found in suprabasal cells. These parallels
between EBS and EH were noted many years ago (7), and resurfaced when
comparisons were made between mutant K14 transgenic mice and EBS (175).
216 FUCHS
Given that epidermal cells switch from K5/K14 to K1/K10 expression as they
leave the basal layer and move outward towards the skin surface (53, 140), it
was predicted that EH would be a disorder involving mutations in K1 and K10
(175).
Soon thereafter, it was demonstrated that mice engineered to express a trun-
cated human keratin 10 gene display the pathobiological and biochemical char-
acteristics of EH (52). Genetic mapping of an EH family revealed linkage
to the keratin cluster on chromosome 12 (32), and then point mutations were
identified in the K1 and K10 genes of patients with human EH (23, 25, 144).
In less than two years after the paradigm for the first keratin disorder was set,
the genetic basis of EH was solved.

Remarkably, three of the first EH cases evaluated had 156R:H/C mutations
in K10 at an arginine residue equivalent to the hotspot residue so often mutated
in the K14 gene of D-M EBS patients (23, 144). Thus the same mutation in
a highly conserved residue of two genes can give rise to two distinct genetic
diseases by virtue of the differential expression of the genes. Many additional
cases of EH have now been sequenced, and overall, the K1 and K10 mutations
fall into patterns similar to those seen for EBS (145, 168, 184).
The first case analyzed of extremely mild EH, sometimes referred to as
Ichthyosis Bullosa of Siemens, had a mutation in helix 2B of the K10 gene,
outside the 10 amino acids of the putative rod overlap (168). Intriguingly,
additional mild cases have mutations in the rod end domains of K2e (89, 108
and references therein). Presumably, severely disrupting mutations in K2e
cause milder cases of EH, because cytolysis can only take place in the upper
spinous layers, where K2e is expressed (29).
Epidermal Nevi of the EH Type
Many cutaneous disorders manifest clinically in a mosaic pattern, often as alter-
nating stripes of affected and unaffected skin along the body surface. Referred
to as lines of Blaschko, these stripes do not track along vascular, neural, or
lymphatic structures of skin. Although never tested, this patterning has been
attributed to clonal proliferation of two genetically distinct cell populations
arising from a postzygotic mutation during embryo genesis. Epidermal nevi
of the epidermolytic hyperkeratosis type is one such disease that tends to fol-
low Blaschko’s lines of clinical mosaicism. These patients sometimes have
offspring with generalized EH. Recently, it was shown that not only do the
EH offspring of three EN patients have mutations in their K10 genes, but in
addition, the patients are genetically mosaic for the keratin mutation, with the
mutation existing in lesional, but not nonlesional skin (125).
Given the parallels between EH and EBS, it is interesting that no mosaic
disorder analogous to epidermal nevi is known for EBS. It could be that genetic
mosaicism in basal keratin genes does not manifest itself clinically in a mosaic
fashion. It seems likely that within a mosaic layer of epidermal basal cells,
healthy cells divide and move laterally into sites vacated by mutant, degenerat-
ing cells. The layer is thus disproportionately populated with wild-type cells,
generating clinically normal skin despite the genetic abnormality. An exception
might occur when the majority of epidermal cells carry the K5 or K14 mutation,
but in this case, a diagnosis of EBS would be probable and the minor mosaicism
might go undetected. In contrast to basal cells, suprabasal cells terminally dif-
ferentiate in upward columns of cells. Hence, when a somatic mutation exists
in a suprabasally expressed gene, lateral migration in the suprabasal layer does
not occur, and clinical mosaicism is observed.

Palmoplantar Keratoderma
Epidermolytic palmoplantar keratoderma (EPPK) is another autosomal domi-
nant disorder that fits the paradigm for a keratin disorder. EPPK patients display
palmoplantar skin blistering due to keratin filament clumping and cytolysis in
suprabasal layers. K9 is a keratin previously shown to be expressed specifically
only in the suprabasal layers of palmo and plantar skin (53), and thus EPPK is
a candidate for K9 gene defects. Indeed, K9 mutations have been discovered
in the rod end segments of K9 in a number of different EPPK patients (75, 135,
147, 171). Based on parallels between W-C EBS and EPPK, some patients
might also be expected to have mild defects in their K1 or K10 genes.
Intriguingly, there are noncytolytic forms of PPK, and one of these was
recently shown to have a point mutation in a lysine residue located in the head
domain of K1, at a considerable distance from the rod segment (31). In the 10
nm filament, this portion of the head domain is thought to protrude along the
filament surface (162). This residue is also quite highly conserved, and in K5,
this residue is in a domain that participates in the direct association between
desmoplakin and keratin (87).
Might some forms of PPK involve subtle perturbations in desmosomes and/or
their IF connections? Although it is still too early to say, it is relevant that a
family with PPK has a genetic defect that was recently mapped to the region of
chromosome 18 where the desmosomal cadherin genes reside (74). Moreover,
transgenic mice engineered to express a mutant desmosomal cadherin were
recently shown to display a number of phenotypic similarities to PPK (5).
Pachyonychya Congenita
Pachyonychya congenita (PC) is an autosomal dominant, degenerative disorder
involving a thickening of the skin surrounding hair follicles and palmoplantar
skin, accompanied by defects in the nails and oral tissues (50). Based on the
additional ultrastructural abnormalities in keratin filaments, it has traditionally
218 FUCHS
been classified clinically as a keratinization disorder, and often discussed in

the context of EBS, EH, and PPK. Given the keratin filament abnormalities
and the location of the keratinocytes that exhibit signs of degeneration in PC,
PC fits the paradigm to be a disorder of K6, K16, and K17. Indeed, in 1995,
PC was genetically mapped to the type I and type II keratin gene clusters on
chromsome 17 and 12, respectively (116), and several laboratories have now
found mutations in each of these three genes in different PC families (14, 109).
White Sponge Nevus: An Oral and Esophageal Disorder
White sponge nevus (WSN) is a rare autosomal dominant disorder typified
by white, thick plaques in the oral (primarily buccal) mucosa, occasionally

accompanied by esophageal, genitalial, and/or rectal involvement (50). Thick-

ening of the epithelium, degeneration of the suprabasal cells, and clumps or
aggregates of keratin filaments signify this disorder as a candidate for a keratin
defect. Based on the distribution of clinical abnormalities, and the particular
cells involved, defects in K4 and K13 are expected (112). Recently, K4 and
K13 mutations in WSN patients were identified by several laboratories (138,
148). This analysis now extends keratin disorders to organs and tissues that go
beyond the skin.
Additional Disorders of Keratin
Several possible candidate hair diseases share features that fit the paradigm
for keratin disorders (6, 51). In some of these cases, abnormalities in two-
dimensional gel patterns and amino acid compositions of hair keratins from
patients with hair diseases have been reported (see for example 61). It may
also be relevant that several autosomal dominant mouse mutants, Re, Bsk, and
Reden , map in close proximity to the type I epidermal keratin genes (118 and
references therein): Rex mice have curly whiskers and bent hair shafts, and
denuded mutant mice and bare skin mice undergo hair loss after completion of
the first hair cycle.
ARE THERE DISORDERS OF OTHER INTERMEDIATE

FILAMENT MEMBERS?
Given that virtually all higher eukaryotic cells express IF genes, the question
arises as to whether there might be genetic disorders involving other types of
IF proteins. Of these, disorders of the type IV neurofilament proteins might be
most likely, given their prominence in large myelinated axons of motor neurons,
where they are the major structural components of these cells. Several neurolog-
ical disorders fit the paradigm for a genetic disorder of IFs. Amyotrophic lateral
sclerosis (ALS or Lou Gehrig’s disease), infantile spinal muscular atrophy, and
hereditary sensory motor neuropathy are all clinically characterized by progres-

sive muscle denervation and atrophy arising from motor neuron degeneration.
Most importantly, aggregates of neurofilament proteins and aberrant accumula-
tion of NFs in motor neuron cell bodies are associated with these disorders. A
major question, which was also applicable to EBS in the 1980s, is whether the
aggegations of NFs are cause or consequence of these diseases. In this regard,
it is interesting that mice engineered to express a mutant NF transgene ex-
hibit pathological features of ALS, including axonal and perikaryal swellings,
slowed axonal transport, and selective degeneration of spinal motor neurons,
resulting in neurogenic atrophy of the skeletal muscle (28, 34, 93, 182). Ad-
ditionally, several mutations in NF-H have been detected in individuals with

sporadic ALS, and these have not been detected in the general population (47
but see 141). A difficulty in assessing if NF mutations are causative in degen-
erative disorders of the nervous system has been the late onset of the disorders,
and this and other factors have made the problem considerably more difficult
than elucidating the genetic basis of EBS and other keratin disorders.
Another candidate disorder of IFs includes forms of congenital myopathies
typified not only by cell degeneration, but also by aberrations in their desmin
IF networks. Perturbations in the desmin IF network have been observed in
a number of distal myopathies (16, 78, 117, 174). Although no linkage has
yet been found between any form of cardiomyopathies and the desmin locus
on chromosome 2, as more studies are conducted on degenerative cytoskeletal
disorders such as cardiomyopathies, the list of human genetic diseases whose
pathology is based on defects in IF networks is likely to grow.
FUNCTIONS OF IFs
Prior to the discovery of keratin disorders, the functions of IFs remained elusive.
It is now clear that a major function of the stratified squamous epithelial keratins,
and perhaps other IF proteins as well, is to impart mechanical integrity to cells.
Without the underlying framework of keratins, epithelial cells become fragile
and prone to rupture upon physical stress. This function was first revealed
upon analyses of two independent incidences of human recessive EBS (18,
149). These rare cases have homozygous premature stop codon mutations in
both of their K14 alleles, yielding nondetectable levels of K14 in the basal
epidermal layer. In the absence of K14, K5 is unstable, and its turnover time
changes from days to less than 30 min (R Lersch & E Fuchs, unpublished
data; see 95). Thus, ablation of one of the two major basal keratins results
in the loss of the other (18, 149). Although a residual network of K5-K15
keratins is still detected, this network is not sufficiently extensive and/or robust
to maintain mechanical strength in the basal epidermal layer. Interestingly,
220 FUCHS
whether recessive or autosomal dominant in nature, EBS cells rupture in a

defined zone, beneath the nucleus and above the hemidesmosomes. This is the
longest portion of the columnar basal cell, and the zone expected to be most
fragile when filament elongation is compromised.
Recent gene targeting, in which the keratin 14 gene has been removed from
the mouse genome, has extended functional analyses of keratins (101). In this
case, mechanical fragility is in the keratinocytes of not only the epidermis,
but also the hair follicles, cornea, oral epithelia, and esophagus (101). In
addition, mice probably lacking the K10 gene display similar aberrations in
their suprabasal cells (128). Thus, while the autosomal dominant disorders of
keratin might have displayed mechanical fragility due to a gain-of-function,

rather than a loss-of-function manner, the recessive K14 and K10 cases in mice
and humans leave little doubt that keratin networks serve a mechanical role in
cell survival. Although additional knockout studies will be necessary to test this
function in other epithelial cells and tissues, it seems likely that this function
will be one that is characteristic of many keratins.
Several lines of evidence suggest that at least some other IFs also play a role as
mechanical integrators of space. Rheologic studies show that wild-type type III
IFs harden and resist breakage under stresses where other cytoskeletal networks
will rupture (83). Newport and colleagues (120) discovered that a Xenopus
oocyte extract, depleted of its single lamin (LIII ), encapsulates chromatin, fuses
membrane vesicles and assembles nuclear pores, but the resulting nuclei are
fragile. Transgenic mice overexpressing a wild-type hair keratin gene have
cortical cells that are fragile, leading to brittleness and hair breakage (131).
Finally, when the keratin 8 gene, normally expressed in liver, is ablated in mice
by embryonic stem cell technology, embryos often do not survive past a critical
stage in development when the liver becomes vascularized, i.e. when there is
potentially substantial mechanical stress exerted to this tissue (10, 11).
By the above criteria, at least some additional members of the IF superfamily
seem to function by imparting mechanical integrity to cells. However, a mutant
vimentin causing disruption of endogenous vimentin assembly is without effect
when present during early embryogenesis in the frog (27). Additionally, some
mice lacking keratin K8 altogether can escape hemorrhaging in the liver and
develop to adulthood without any apparent complications (10). Finally, recent
knockouts of vimentin (30) and GFAP (127) in mice have not revealed any signs
of tissue-specific degeneration expected if mechanical integrity were compro-
mised. Thus, despite the documented function for some IFs in resisting stress
(83, 101), this function may be more important in certain cells than in others.
Are there other functions for IFs? An additional potential function for keratin
IFs is in the organization of mitochondria and other organelles within the
cytoplasm. Thus, in K14 knockout patients and mice, mitochondria tend to

cluster within the cytoplasm (18). This finding is intriguing in light of the
phenotype of the yeast mutant MDM1 IF-like protein, which when defective
leads to improper mitochondrial segregation (105). In addition, some studies
have pointed to the possibility that the cytoplasmic IF network may function
in providing external mechanical support to a nucleus inside a cell. Thus, for
example, a fibroblast cell line that appears to lack cytoplasmic IFs altogether
exhibits abnormalities in nuclear shape (152). While the possible role of IFs in
organelle and/or nuclear organization is by no means unequivocal, the number
of circumstantial associations continues to grow, and warrants further inves-
tigation. However, the finding that some cytoplasmic IFs can apparently be
dispensed with in vivo without compensation would argue against their being
any universal or essential role for IFs in all cell types.
Indeed, mounting evidence has strengthened the view that many functions for
IFs are tissue specific. Although NFs are not abundant during neurite extension,
their density increases greatly at a time when neurites expand radially, leading
to the hypothesis that these IFs may be involved in establishing neuronal caliber
(76 and references therein). Moreover, when axons are severed, NF synthesis
is downregulated, with a subsequent decrease in axonal NFs and a shrinkage
in neuronal caliber. In addition, when NF-H, but not NF-L, is overexpressed
in transgenic mice, radial growth of the axons is increased (34, 114, 182).
Conversely, when NF-M is overexpressed, radial growth is decreased (179).
Finally, a nonsense mutation in the quail NF-L gene leads to an absence of NFs
and a concomitant decrease in axonal calibers (122). Collectively, these data
convincingly demonstrate that NFs are intrinsic determinants of axonal caliber.
Even though variation in IF density can influence axon diameter, overexpres-
sion of up to 10× the physiological levels of wild-type vimentin leads to no
detectable morphological abnormalities during the early stages of Xenopus em-
bryogenesis, suggesting that cells in early development can tolerate substantial
changes in IF density (27). Similarly, the creation of a dense NF network in
transgenic mouse kidney (normally NF−− ) is without apparent consequence.
However, too many cytoplasmic IFs can be deleterious: Overexpression of
type III or type IV IFs in lens epithelium of transgenic mice leads to cataract
formation (17, 43).
The localization of desmin at Z discs has led to the hypothesis that desmin
might be involved in either laterally linking Z-bands of adjacent myofibrils to
one another and to the sarcolemma or in longitudinally connecting successive
Z-bands in elongating myofibrils. However, when a truncated desmin is ex-
pressed transiently in postmitotic myoblasts and multinucleated myotubes, the
preexisting IF networks are completely dismantled, and yet striated myofibrils
222 FUCHS
are still assembled and laterally aligned, and contraction proceeds apparently
unaffected (154). Thus, the specialized role of desmin in muscle cells is at
present unclear, and awaits knockout studies.
In summary, in addition to their potential role in providing mechanical in-
tegrity to cells, IFs also serve more specialized roles, ranging from stabilizers of
DNA replication complexes to tough, resilient survivors of terminal differentia-
tion. As additional studies are conducted, their roles in determining the intricate
architecture and physiology of different cell types and tissues should become
increasingly apparent. Other gene products may also contribute to IF function.
This notion is underscored by the recent observation that K8 gene disruption
causes colorectal hyperplasia in some but not other strains of mice (9). This
marked effect of genetic background on an IF (−/−) phenotype opens the door
for the future discovery of additional genetic modifiers of as yet unexplored IF
functions.
CONCLUSIONS AND PERSPECTIVES

IF proteins constitute a superfamily whose members share marked conservation
in secondary structure. This enables these proteins to assemble into coiled-coil
dimers, approximately 20,000 of which then assemble into the superstructure
of a single 10-nm filament. The use of transgenic mice as a stepping stone to
elucidate the genetic basis of Epidermolysis Bullosa Simplex provided the first
example of how reverse genetics can be used to solve the basis for human dis-
eases. This approach led to the first in vivo demonstration of a function for IFs
and set the paradigm for the subsequent elucidation of a growing list of IF dis-
orders. Although specialized functions are still to be defined for many IFs, the
diverse patterns of IF expression provide strong evidence that their 10-nm net-
works play roles that extend beyond mechanical integrity. By manipulating IF
gene sequences and expressing them or ablating them in cultured cells, in bac-
teria and in mice, powerful experimental systems have been generated by which
to dissect the complex issues of IF structure, function, and disease. Combined
approaches involving gene knockout and replacement technologies should con-
tinue to illuminate the functions of these proteins and the significance of their
multiplicity of sequences. The list of keratin disorders is likely to grow, although
the relation between aberrancies of other IFs and genetic diseases remains un-
clear. It seems increasingly less likely that ALS will be a disorder of NF gene
mutations, but transgenic studies nevertheless underscore the importance of NF
aggregations in the manifestations similar to an ALS phenotype. The possible
role of desmin mutations in certain types of cardiomyopathies remains largely
unexplored, and further insights await the generation of desmin knockout mice.
Finally, although the absence of apparent phenotype in some recent knockouts,
namely vimentin and GFAP, has been disappointing to the IF field, subtle, as
yet unidentified changes in mice might translate into significant abnormalities
in humans. Moreover, judging from results with epidermal keratins, dominant
negative mutations in IF genes may lead to more severe abnormalities than null
mutations. While there are many additional experiments yet to be done, research
in the 1990s has placed IFs squarely on the map of interesting cytoskeletal pro-
teins with essential and important functions in cells. A central and relatively
little explored issue for the future will be to understand IFs in the context of the
molecules that they associate with and the networks that they form.
ACKNOWLEDGMENTS
I thank past and current members of my laboratory, who over the years have
contributed to many of the different findings regarding the epidermal keratins
and their relation to human disease: Pierre Coulombe, Kathryn Albers, Douglas
Marchuk, Angela Tyner, Rafi Kopan, Robert Vassar, Anthony Letai, Yiu-mo
Chan, Jian Cheng, Mary Beth McCormick, Andrew S Syder, M Elizabeth
Hutton, Linda Degenstein, and Qian Chun Yu. I thank also the many dermatol-
ogists who have collaborated with us over the years: Amy S Paller and J-David
Fine, Jouni Uitto, Tobias Gedde-Dahl, and Ingrun Anton-Lamprecht. Finally,
I thank many of my colleagues who have contributed so heavily to the field of
intermediate filaments.
Visit the Annual Reviews home page at
http://www.annurev.org.
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October 24, 1996 9:37 Annual Reviews CHAPTER9.

Annu. Rev. Genet. 1996. 30:197–231

THE CYTOSKELETON AND DISEASE:

CLASSES OF INTERMEDIATE FILAMENT PROTEINS

(GFAP), expressed in neuroglial cells (59); and (d) peripherin, contributing to

THE CYTOSKELETON AND DISEASE 199

IF STRUCTURE AND ASSEMBLY

Cytoplasmic IFs can assemble in vitro in the absence of auxiliary proteins or

factors, suggesting that the information necessary to form a 10 nm filament is

protofibrils, and groups of four protofibrils intertwine to produce the 10 nm

THE CYTOSKELETON AND DISEASE 201

DIFFERENTIAL EXPRESSION OF KERATIN GENES

weeks for cells to complete terminal differentiation.

last stage of terminal differentiation where the transcriptional machinery is still

THE CYTOSKELETON AND DISEASE 203

B. Keratins Expressed in Other Epithelial Tissues

MOLECULAR MUTAGENESIS EXPERIMENTS ON

THE CYTOSKELETON AND DISEASE 205

In filament assembly assays, these mutant dimers assembled into filamentous

THE GENETIC BASIS OF EPIDERMOLYSIS BULLOSA

Transgenic Mice Engineered to Express Mutant K14 Genes

THE CYTOSKELETON AND DISEASE 207

a striking resemblance was noted to the human skin disorder, Dowling-Meara

THE CYTOSKELETON AND DISEASE 209

At first, despite the remarkable similarities between mutant K14-expressing

THE CYTOSKELETON AND DISEASE 211

either R125 mutations is sufficient to cause dominant negative disruption of the

THE CYTOSKELETON AND DISEASE 213

cases, new hypotheses have emerged that have led to a refinement of 10 nm

THE CYTOSKELETON AND DISEASE 215

that are seen ultrastructurally in patients with the mutation.

ADDITIONAL DISORDERS OF KERATIN

the genetic basis of EH was solved.

THE CYTOSKELETON AND DISEASE 217

not occur, and clinical mosaicism is observed.

been classified clinically as a keratinization disorder, and often discussed in

by white, thick plaques in the oral (primarily buccal) mucosa, occasionally

accompanied by esophageal, genitalial, and/or rectal involvement (50). Thick-

ARE THERE DISORDERS OF OTHER INTERMEDIATE

THE CYTOSKELETON AND DISEASE 219

hereditary sensory motor neuropathy are all clinically characterized by progres-

ditionally, several mutations in NF-H have been detected in individuals with

whether recessive or autosomal dominant in nature, EBS cells rupture in a

keratin might have displayed mechanical fragility due to a gain-of-function,

THE CYTOSKELETON AND DISEASE 221

cytoplasm. Thus, in K14 knockout patients and mice, mitochondria tend to

CONCLUSIONS AND PERSPECTIVES

THE CYTOSKELETON AND DISEASE 223

Heiner M. 1994. Colorectal hyperplasia 105:629–32

1991. Epidermolysis bullosa simplex: ev- keratosis. Cell 70:821–28

THE CYTOSKELETON AND DISEASE 225

patients: genetic and functional analyses. structural homology to interemediate fila-

Cell 66:1301–11 ment proteins. Proc. Natl. Acad. Sci. USA

Hall/University Press Cleveland DW, Aebi U. 1993. The rod do-

62. Gounari F, Merdes A, Quinlan R, Hess main of NF-L determines neurofilament

THE CYTOSKELETON AND DISEASE 227

THE CYTOSKELETON AND DISEASE 229

expressing an intermediate filament gene. sion. Mol. Cell. Biol. 8:722–36

of a vimentin-type intermediate filament 163. Stephens K, Sybert VP, Wijsman EM,

genesis. J. Cell Biol. 114:953–66

THE CYTOSKELETON AND DISEASE 231

Anton-Lamprecht I, Yu Q-C, et al. 1996. ing neurofilament subunit NF-M expres-

A, Fuchs E. 1989. Tissue-specific and DW. 1993. Increased expression of neu-

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