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; e-mail: [email protected]
Current Opinion in Cell Biology 1998, 10:93–101
http://biomednet.com/elecref/0955067401000093
 Current Biology Ltd ISSN 0955-0674
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,
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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 p34cdc2 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
Intermediate filaments and their associated proteins Houseweart and Cleveland
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
95
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
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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
Intermediate filaments and their associated proteins Houseweart and Cleveland
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
97
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
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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
Intermediate filaments and their associated proteins Houseweart and Cleveland
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
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2.
•
Georgatos SD, Gounari F, Goulielmos G, Aebi U: To bead or not
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11.
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Bernier G, Mathieu M, de Repentigny Y, Vidal SM, Kothary R:
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••
Smith FJ, Eady RA, Leigh IM, McMillan JR, Rugg EL, Kelsell
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The demonstration that mutations in a cytoskeletal linker protein such as
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[14••–16••].
14.
••
McLean WH, Pulkkinen L, Smith FJ, Rugg EL, Lane EB, Bullrich
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The demonstration that mutations in a cytoskeletal linker protein such as
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[13••,15••,16••].
15.
••
Pulkkinen L, Smith FJ, Shimizu H, Murata S, Yaoita H, Hachisuka
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The demonstration that mutations in a cytoskeletal linker protein such as
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Svitkina TM, Verkhovsky AB, Borisy GG: Plectin sidearms
mediate interaction of intermediate filaments with
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The immunoelectron microscopy images presented in this work convincingly
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biochemical data and establishing plectin as a true organizer of intracellular
space.
16.
••
6.
17.
Fujiwara S, Kohno K, Iwamatsu A, Naito I, Shinki H: Identification
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18.
Guo L, Degenstein L, Dowling J, Yu QC, Wollmann R, Perman
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19.
Brown A, Bernier G, Mathieu M, Rossant J, Kothary R: The mouse
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5.
••
Nikolic B, Mac Nulty E, Mir B, Wiche G: Basic amino acid
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The discovery that the neuronal protein BPAG1n/dystonin can co-align neurofilaments with actin filaments in cultured cells strengthens its claim as a
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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
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7.
••
8.
•
Foisner R, Malecz N, Dressel N, Stadler C, Wiche G: M-phasespecific phosphorylation and structural rearrangement of
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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
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9.
Foisner R: Dynamic organization of intermediate filaments
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Liu CG, Maercker C, Castanon MJ, Hauptmann R, Wiche G:
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21.
Bernier G, Brown A, Dalpe G, de Repentigny Y, Mathieu M,
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100
Cytoskeleton
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••
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26.
•
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27.
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47.
•
Shibuki K, Gomi H, Chen L, Bao S, Kim JJ, Wakasuki H, Fujisaki
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Galou M, Colucci-Guyon E, Ensergueix D, Ridet JL, Gimenez
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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,
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Spann TP, Moir RD, Goldman AE, Stick R, Goldman RD:
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Albers KM: Keratin biochemistry. Clin Dermatol 1996, 14:309320.
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Uttam J, Hutton E, Coulombe PA, Anton-Lamprecht I, Yu QC,
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whereas most other disease-causing keratin mutations have been shown to
reside in the more conserved ends of the rod domain.
54.
Yang JM, Nam K, Park KB, Kim WS, Moon KC, Koh JK, Steinert
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56.
•
Ku NO, Wright TL, Terrault NA, Gish R, Omary MB: Mutation of
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accordance with previous work using K8-null mice which die during embryogenesis as a result of liver hemorrhage.
57.
101
Irvine AD, Corden LD, Swensson O, Swensson B, Moore JE,
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58.
•
Winter H, Rogers MA, Langbein L, Stevens HP, Leigh IM, Labreze
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monilethrix. Nat Genet 1997, 16:372-374.
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hair disease demonstrates the universal requirement for keratins in the varied
cell types they inhabit.
59.
••
Takahashi K, Coulombe PA: A transgenic mouse model with an
inducible skin blistering disease phenotype. Proc Natl Acad Sci
USA 1996, 93:14776-14781.
This work used the keratin 6 promoter inducibly to express a heterologous
gene product in the skin of mice. The utility of the system was demonstrated
by producing mice that developed a skin blistering disease most similar to
IBS (icthyosis bullosa of Siemens) upon induction of a keratin 6a mutation.
The work sets the stage for delivery of foreign gene products via transgenic
skin grafts. See also [60••].
60.
••
Wang X, Zinkel S, Polonsky K, Fuchs E: Transgenic studies
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prospects for gene therapy. Proc Natl Acad Sci USA 1997,
94:219-226.
The keratin 14 promoter was used to drive expression of a growth hormone
in the skin of mice. Skin grafts from such donor mice could be transferred to
recipient mice to sustain increased levels of the hormone in the blood stream,
demonstrating the possible utility of this system for human gene therapy by
delivering products secreted by grafted skin cells. See also [59••].