Current insights into the formation and
breakdown of hemidesmosomes
Sandy H.M. Litjens1, Jose ´ M. de Pereda2and Arnoud Sonnenberg1
1Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands
2Instituto de Biologia Molecular y Celular del Cancer, Centro de Investigacion del Cancer, University of Salamanca - CSIC,
E-37007 Salamanca, Spain
complexes that promote epithelial stromal attachment
in stratified and complex epithelia. Modulation of their
function is of crucial importance in a variety of biological
processes, such as differentiation and migration of
keratinocytes during wound healing and carcinoma
invasion, in which cells become detached from the
substrate and acquire a motile phenotype. Although
much is known about the signaling potential of the a6b4
integrin in carcinoma cells, the events that coordinate
the disassembly of hemidesmosomes during differen-
tiation and wound healing remain unclear. The binding
of a6b4 to plectin has a central role in hemidesmosome
assembly and it is becoming clear that disrupting this
interaction is a crucial event in hemidesmosome
disassembly. In addition, further insight into the
functional interplay between a3b1 and a6b4 has
contributed to our understanding of hemidesmosome
disassembly and cell migration.
The keratinocytes of the basal layer of the skin are firmly
attached to the underlying basement membrane through
protein complexes called hemidesmosomes(Box1 provides
further details of skin structure). Hemidesmosomes were
first defined at the ultrastructural level as small electron-
dense domains in the plasma membrane, connecting the
extracellular basement membrane to the intracellular
intermediate filament system [1,2]. The hemidesmosomal
protein complex provides stable adhesion of the epidermis
to the underlying dermis and ensures resistance to
mechanical stress when applied to the skin. In the skin,
hemidesmosomes contain the integrin a6b4, the type XVII
collagen BP180, the tetraspanin CD151 and the two
plakin family members plectin and BP230 [1–3]. It is the
a6b4 integrin that connects the cells to a major component
of the basement membrane, laminin-5 (now also referred
to as laminin-332 ), whereas the cytoplasmic proteins
plectin and BP230 connect to the keratin filaments,
thereby creating a stable anchoring complex. Although
the binding of BP180 to laminin-332 has been shown
in vitro, this interaction by itself is not sufficiently strong
to mediate adhesion of cells to laminin-332 in the absence
of a6b4 . Stable attachment of basal keratinocytes to the
basement membrane through hemidesmosomes is of
fundamental importance for maintaining skin integrity;
inherited or acquired diseases in which either of the
hemidesmosome components are affected lead to a variety
of skin blistering disorders [6–8].
The a6b4 integrin has been associated not only with
stable adhesion, but also with cell migration [3,9,10]. In all
studies investigating keratinocyte migration or carcinoma
cell invasion, hemidesmosomes are disassembled, their
components are redistributed and a6b4 is no longer
exclusively concentrated at the basal cell surface [9,10].
Here, we focus on recent findings on the structural
properties of hemidesmosomes and their constituents,
and the regulation of their assembly and disassembly.
Hemidesmosome structure and assembly
The absence of a6b4 in genetically modified mice results in
the loss of hemidesmosomes, and hence of stable
epidermal adhesion [11–13]. This is also observed in
patients carrying mutations in either the a6 or the b4
subunit of the integrin, which can result in devastating
diseases characterized by skin fragility and blistering .
In affected patients, hemidesmosomes are less robust or
completely absent. Notably, two mutations identified in
the b4 gene (ITGB4) of patients with a nonlethal form of
junctional epidermolysis bullosa disrupt the binding to
plectin, indicating that the interaction of b4 with plectin is
crucial for the formation of stable hemidesmosomes
[14–16]. These two proteins interact at two levels; the
primary interaction occurs between the plectin actin-
binding domain (ABD) and a region on b4 comprising the
first pair of fibronectin type III (FnIII) domains and a
small region of the adjacent connecting segment, and the
secondary between the plakin domain of plectin and a
more C-terminal part of the connecting segment of b4, and
the ultimate C-tail (i.e. the region following the fourth
FnIII domain) [14,17–20] (Figure 1). Consistent with the
idea that the a6b4–plectin connection is crucial for the
mechanical stability of hemidesmosomes, plectin-deficient
mice show extensive epithelial detachment and die
2–3 days after birth . Patients with mutations in the
PLEC gene also suffer from skin fragility, although the
symptoms are less severe than those observed in plectin-
deficient mice. Most of these mutations are located within
the rod domain (Figure 1), which is not present in
Corresponding author: Sonnenberg, A. (firstname.lastname@example.org).
Available online 6 June 2006
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a smaller splice variant that is expressed at low levels in
cells [17,22]. It is conceivable that this variant can
partially compensate for the loss of the canonical plectin
molecule in humans. Interestingly, neither BP180 nor
BP230 are efficiently recruited into hemidesmosomes
when b4 cannot bind to plectin [15,17,23]. In addition,
simple epithelia (e.g. that of the intestine) contain
adhesion complexes consisting of only a6b4 and plectin,
described as type II hemidesmosomes [24,25]. This
suggests that the a6b4–plectin interaction is one of the
first steps in the formation of hemidesmosomes and occurs
before the recruitment of BP180 and BP230 .
CD151 is also an early constituent of the hemidesmo-
somal adhesion structures. It is strongly associated with
another laminin-binding integrin on keratinocytes, a3b1,
with which it forms ‘pre-hemidesmosomal’ clusters at the
basal cell surface of human keratinocytes . These
clusters might serve as a ‘nucleation site’ for the assembly
of hemidesmosomes by the integrin a6b4. In contrast to
CD151, which becomes a component of mature hemi-
desmosomes (via binding to a6), a3b1 is recruited into
focal contacts or redistributed to cell–cell contacts after
hemidesmosomes have been assembled and cell–cell
contacts established. Although a3b1 does not appear
directly to participate in hemidesmosome assembly, it
might, together with other b1-containing integrins,
contribute to the efficacy of their formation, possibly by
affecting the localization of a6b4 at the basal membrane.
Indeed, in b1-deficient mice, the number of hemidesmo-
somes is reduced, which can partially explain the skin
defects that are observed in these mice [27,28]. However, if
only a3b1 is absent, as in a3-null mice, the number and
Box 1. Structure of the skin
The skin, the largest organ of our body, is crucial for protecting the
underlying tissues and organs from the external environment. It is
composed of three layers : the epidermis (keratinocytes in
various stages of differentiation and melanocytes), the dermis
(blood vessels, nerve endings, sebaceous glands and hair follicles,
surrounded by connective tissue and collagen fibers) and the
subcutaneous layer (connective tissue and fat). The basal layer of
cells in the epidermis contains the least differentiated keratinocytes,
and among these are the stem cells, which ensure the constant
renewal of the skin by their unlimited dividing capacity. Stem cells
produce transit-amplifying cells that are destined to withdraw from
the cell cycle and terminally differentiate after a few rounds of
division [58,59]. During terminal differentiation, the keratinocytes
detach from the basement membrane, migrate upward in the
epidermis, gradually flatten, start producing more keratins, lose
their nucleus and ultimately die and flake off the skin . The basal
keratinocytes are attached to the basement membrane, a meshwork
of extracellular macromolecules separating the dermis from the
epidermis, through specialized adhesion structures called hemi-
desmosomes (Figure 1).
Integrin α α6β β4
Plakin domain ABDCoiled-coil rod domainPlakin repeat domains
B1 B2B3B4 B5C
FnIII-1 FnIII-2FnIII-3 FnIII-4
Subbasal dense plate
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Figure 1. Components and structure of hemidesmosomes. (a) A schematic drawing of the hemidesmosome, showing its six components (a6b4 heterodimer, CD151, BP180,
BP230 and plectin) together with its ligand laminin-332 and intracellular intermediate filament partners keratin-5 and -14 (IF). (b) The structures of a6b4 and plectin. The
various domains in the proteins are indicated, as well as the residues in the b4 cytoplasmic domain that are important for plectin binding and regulation of this interaction. (c)
An electron micrograph of several hemidesmosomes. (d) An enlargement of the indicated area in the middle panel. Abbreviations: BM, basement membrane; CH, calponin
homology domain; HD, hemidesmosome; IP, inner plaque; LD, lamina densa; LL, lamina lucida; OP, outer plaque.
TRENDS in Cell BiologyVol.16 No.7 July 2006377
size of hemidesmosomes appears to be normal .
Similarly, no deficiencies in hemidesmosomes were
observed in CD151 null mice, and these mice also have
no signs of skin fragility . Moreover, in cells expressing
b4-chimeras that are unable to associate with a6 and thus
CD151, hemidesmosome-like structures, containing plec-
tin, BP180 and BP230, are still formed [31,32]. Thus, in
mice and in human cultured keratinocytes, CD151 is not
required for the formation of hemidesmosomes, and its
precise function with a3b1 in the ‘pre-hemidesmosomal’
clusters remains unknown.
Interestingly, wound healing was defective in CD151-
deficient mice; this was found to be the result of an
impaired deposition of laminin-332 into the wound bed
and a failure to upregulate a6b4 . Impaired organiz-
ation of the basement membrane has also been described
in a3-null mice, which hints at an important function of
the complexes of a3b1 and CD151 in this process .
Intriguingly, although the absence of CD151 in mice has
no effect on epidermal integrity, a nonsense mutation in
the CD151 gene in a patient, resulting in a truncated
protein, and loss of the integrin binding site, leads to skin
blistering, albeit less severe and more localized than in
patients with mutations in either a6 or b4 . Moreover,
it has been shown that the tetraspanin TSP-15, which is
similar to mammalian CD151, is required for epidermal
integrity in Caenorhabditis elegans . It is possible that
the truncated CD151 molecule acts in a dominant-
negative fashion, with its presence being more deleterious
for tissue integrity than is the total lack of CD151.
Alternatively, the absence of a functional CD151 molecule
in mice, but not in humans, might be compensated by
another tetraspanin . Together, these findings indicate
that the binding of b4 to plectin represents the first step in
the formation of hemidesmosomes, and that complexes of
a3b1 and CD151 might facilitate this step by providing a
platform at the basal surface of keratinocytes through
Role of laminin-332 in the formation of
In several reports, data have been presented suggesting
that the processing of laminin-332 is important for the
a3 chain of laminin-332 is cleaved, securing the formation
of stable a6b4-containing hemidesmosomes; however,
when keratinocytes need to migrate [e.g. following skin
wounding (Box 2)], unprocessed laminin-332 is secreted at
the leading edge, which stimulates migration through the
a3b1 integrin [36,37]. It is generally assumed that when
the a3 chain of laminin-322 has not been processed,
keratinocytes switch from a6b4-mediated adhesion to
one mediated by a3b1, because the latter binds more
efficiently to unprocessed laminin-332. Conversely, a6b4
might bind less efficiently to this unprocessed laminin-322
than to the cleaved form, which might contribute to a
higher turnover of hemidesmosomes. In addition, there is
evidence that matrix metalloproteinase 19-dependent
processing of the laminin-g2 chain can effectuate the
above switch, leading to increased migration of keratino-
cytes . Therefore, whether laminin-332 is processed or
not determines which of the two laminin-binding integrins
mentioned above becomes engaged, and consequently the
turnover of hemidesmosomes. Interestingly, in a recent
report it was shown that unprocessed laminin-332 (a3
chain) does indeed prevent the formation of mature
hemidesmosomes but not the nucleation of hemidesmoso-
mal plaques . Furthermore, it showed that keratino-
cytes migrate equally well on both processed and
unprocessed laminin-332, whereas mature hemidesmo-
somes can only be formed after the a3 chain of laminin-332
is processed. Nevertheless, mainly unprocessed laminin-
332 is present underneath the leading edge of keratino-
cytes within a skin wound. It is possible that in the case of
excessive production of laminin-332 following wounding,
the efficiency of processing will be reduced and the
formation of mature hemidesmosomes inhibited, favoring
a migratory phenotype.
Box 2. The role of epidermal integrins in wound healing
Injury to the skin causes the transition of quiescent adherent
keratinocytes on laminin-332 into migrating cells on the newly
formed provisional matrix containing fibrin, fibronectin and vitro-
nectin. During this process, various growth factors and cytokines are
secreted to induce healing of the wound . Transforming growth
factor b is one of these and contributes to re-epithelialization of skin
wounds by modulating integrin expression, thereby stimulating
keratinocyte migration towards the provisional wound matrix. As
described in this review, stable adhesion of keratinocytes by a6b4 to
the basement membrane is lost and the integrin is redistributed
from hemidesmosomes to lamellipodia, which might facilitate their
stabilization. In addition, the expression of the integrins a2b1 and
a3b1, mostly restricted to the lateral membranes of basal keratino-
cytes in normal epidermis, becomes extended throughout the entire
epidermis and their concentration at the basal membrane of basal
keratinocytes increases [61–63]. After binding of keratinocytes by
a3b1 to newly deposited laminin-332 at the wound edge, the T
lymphoma invasion and metastasis 1 (TIAM1)–Rac1 signaling
pathway is activated to promote lamellipodia formation and
subsequent spreading [64,65]. The initial spreading induced by
a3b1 is followed by the activation of RhoA, which in in vitro
experiments was found to be required for the adhesion of leading
keratinocytes to collagen by a2b1  However, more recent studies
have questioned the importance of a2b1 in wound healing because
wound closure is normal in a2-deficient mice [67,68]. The
fibronectin- and vitronectin-binding integrins a5b1, avb5 and avb6
probably have a more important role in wound healing because
these integrins are receptors for the various components of the
provisional matrix and their expression is induced or strongly
increased. Of these integrins, only avb5 is expressed at low levels on
proliferating keratinocytes, whereas a5b1 and avb6 are normally
absent [69,70]. (Increased) expression of these three integrins results
in a more migratory phenotype of the keratinocytes. Interestingly,
upregulation of avb6 causes a fibronectin-dependent upregulation of
matrix metalloproteinase 9, which might partly be responsible for
the increased migration . Although not well documented, a role
of the fibronectin- and tenascin-binding integrin a9b1 in epidermal
wound repair has been proposed [72,73].
TRENDS in Cell BiologyVol.16 No.7 July 2006378
Although the above studies suggest an important
function of laminin-332 in the formation and stabilization
of hemidesmosomes, studies from several laboratories
have indicated that the formation of hemidesmosomes can
be driven entirely from within the cell through interaction
of the cytoplasmic domain of b4 with plectin [31,40–42].
Keratinocytes expressing a b4 subunit that is unable to
interact with its ligand form structures that contain all of
the components of type I hemidesmosomes. Although
these data came as a surprise at the time, they do not
imply that laminin-332 does not have a role in nucleating
hemidesmosomes. Indeed, hemidesmosomes can also be
initiated through an interaction of a6b4 with clustered
laminin-332 that has been deposited by the cells
themselves. Furthermore, ligation of the integrin clearly
affects the organization and density of the formed clusters
[32,36,38]. When a6b4 is not bound to laminin-332, plectin
clusters the integrin into tight complexes. However, upon
ligation to laminin-332 in the matrix, the dynamics of
a6b4 are reduced and, therefore, the degree of intra-
cellular clustering by plectin is restricted . Thus, the
formation of hemidesmosomes is driven both from within
the cells through an interaction of the cytoplasmic domain
of b4 with plectin and from outside the cell through
binding of a6b4 to laminin-332.
Basolateral targeting of hemidesmosomal components
in polarized epithelia
Evidence is emerging that hemidesmosomes are also
affected by mechanisms that control polarized vesicular
transport in cells. In polarized epithelia, where tight
junctions prevent the free diffusion of plasma membrane
proteins between the apical and basolateral membranes,
membrane trafficking pathways are used to transport
proteins to their respective domains. These trafficking
mechanisms are not only important for maintaining, but
also for establishing epithelial cell polarity. One of the
proteins that has been implicated in directing protein
trafficking to the basolateral membrane is the cytoskeletal
protein lethal giant larvae 2 (Lgl2). Indeed, this protein is
crucial for correct hemidesmosome formation in zebrafish
. Lgl2 functions in polarized exocytosis by regulating
the function of target membrane soluble N-ethylma-
leimide attachment protein receptors (t-SNAREs) at the
basolateral plasma membrane [44,45]. At the subapical
region, its activity is inhibited as result of phosphorylation
by atypical protein kinase C (PKC), which is a member of
the Par protein complex [46,47].
Another protein that might have a role in the
basolateral targeting of hemidesmosomal proteins is
Erbin, which binds to both b4 and BP230 . Erbin was
originally identified as an ErbB2-associated protein
involved in the targeting of this receptor to the basolateral
membrane. It is a member of the LAP [leucine-rich repeats
(LRR) and postsynaptic density protein-95, Discs-large
and zonula occludens-1 (PDZ)] family of proteins, impli-
cated in establishing epithelial cell polarity [49,50]. In
addition to the hemidesmosomal proteins b4 and BP230,
Erbin interacts with components of adherens junctions
and desmosomes, both of which are cell–cell adhesion
structures at the lateral membrane. These observations
suggest that Erbin might have a general role in the
establishment and maintenance of cell–cell and cell–
basement membrane adhesions.
Although nothing is known about the dynamics of
hemidesmosomes in the epidermis, in cultured keratino-
cytes these structures appear continuously to renew
themselves [32,51]. This dynamic state is thought to be a
prerequisite to enable hemidesmosomes to be quickly
disassembled when required – for example, for cell
migration or differentiation, when cells detach from the
underlying basement membrane. Although the role of b4
in the migration of keratinocytes, and particularly of
carcinoma cells, has been studied extensively, only a few
models for hemidesmosome disassembly have been pro-
posed [52,53]. The regulatory requirements for each
assembly and disassembly step are not yet clear. It is
known that the interaction between plectin and b4 is
regulated by serine phosphorylation of residues 1356,
1360 and 1364 in the connecting segment of b4.
Phosphorylation of these residues by PKCa, and possibly
other enzymes, results in partial hemidesmosome disas-
sembly owing to loss of interaction with plectin . Most
likely, activation of a phosphatase is required to induce
binding of plectin and reassembly of hemidesmosomes.
This putative phosphatase might also be activated upon
ligation of a6b4 to processed laminin-332.
The cytoplasmic domain of b4 can adopt a folded
conformation by binding of the C-tail to the connecting
segment  (Box 3). Interestingly, these same sequences
in b4 are involved in binding to the plectin plakin domain
[17,20,54]. Therefore, the intramolecular folding of the b4
cytoplasmic domain might also regulate the interaction
with plectin. Posttranslational modifications, such as
phosphorylation, might determine the conformation of
the b4 cytoplasmic domain and thereby affect its affinity
for other hemidesmosome components. Based on the
limited data available, several mechanisms for phos-
phorylation-induced hemidesmosome disassembly are
conceivable (Figure 2).
First, phosphorylation of the connecting segment might
disrupt the interaction between b4 and the plakin domain
of plectin and/or the intramolecular interaction, thereby
destabilizing the interaction with plectin (Figure 2a).
Second, phosphorylation of b4 might increase its affinity
for a third protein, which subsequently competes with
plectin for binding to the connecting segment and/or
interferes with binding of plectin with the first pair of
FnIII domains in the integrin C-tail (Figure 2b,c). Indeed,
macrophage-stimulating protein-induced serine phos-
phorylation of the b4 connecting segment by PKC-a,
leads to hemidesmosome disassembly and relocation of
a6b4 to lamellipodia at the leading edge of migrating
keratinocytes via binding of 14-3-3 proteins to the
phosphorylated connecting segment . Interestingly,
macrophage-stimulating protein is one of the growth
factors secreted in the wound fluid and might, by inducing
the disassembly of hemidesmosomes, contribute to wound
repair [55,56] (Box 2). Third, phosphorylation of the
TRENDS in Cell BiologyVol.16 No.7 July 2006379
conformational change of the b4 cytoplasmic domain that
would distort the binding sites for the plectin ABD and/or
plakin domain (Figure 2d). In the first two models, binding
of the plectin plakin domain is lost, whereas binding of the
plectin ABD to b4 is still possible. In the latter two models,
the binding of the plectin ABD is prevented and the
affinity of the plakin domain of plectin for b4 is not strong
enough to induce binding. In these models, the interaction
between plectin and b4 has a key role. Obviously, other
hemidesmosomal interactions might be subject to
Box 3. Structure of hemidesmosomal components
Knowledge of the structure of the protein components of hemi-
desmosomes has contributed to our understanding of the structure,
assembly and disassembly of these adhesion complexes. The
cytoplasmic domain of b4 is w1000 residues long and contains
four FnIII domains arranged in two tandem pairs separated by a
connecting segment (Figure I). The knowledge of the structure of b4
is limited to the crystal structure of the first pair of FnIII domains
. This region and the first 35 residues of the adjacent connecting
segment (1321–1355) constitute the primary plectin binding site in
b4, a region essential for targeting plectin to hemidesmosomes
[14,17,18]. Residues important for the interaction with plectin map
onto a continuous area of b4 extending over the first and second
FnIII domains and define the binding interface (Figure I). Plectin
binds to the first pair of FnIII domains of b4 via its N-terminal ABD.
The ABD is formed by two CH domains arranged in a closed
conformation, as shown in the crystal structure [75,76]. Residues
crucial for binding to b4 are clustered on the surface of the N-
terminal CH domain, and overlap partially with one of the actin-
binding sequences essential for binding to F-actin, providing a
structural basis for the competitive nature of plectin binding to b4
and F-actin [14,77,78]. The structure of the complex of the first pair
of FnIII domains of b4 and the ABD of plectin is not known.
However, a validated model of this binary complex has been
proposed in which the N-terminal CH domain contacts b4 at the
inter-FnIII domain area .
The PLEC gene contains many alternative first exons, resulting in 11
plectin splice variants differing in their N-terminal sequence .
Although it has been shown that both plectin-1A and plectin-1C can
interact with b4 in vitro, only plectin-1A is expressed in basal
keratinocytes and is localized in hemidesmosomes [14,77,79,80]. The
N-terminal sequence in plectin-1A might be important for regulating
the affinity of the ABD for b4 and F-actin .
The cytoplasmic domain of b4 might adopt a closed conformation
owing to the interaction between its connecting segment and its C-
terminal tail, which might affect the affinity for plectin . For the
folded state of b4, a strong bending of the second pair of FnIII domains
is probably required and might be favored by the 20-residue linker
connecting the third and fourth FnIII domains. The b4 regions involved
in the intramolecular interaction contain binding sites for the plakin
domain of plectin located adjacent to the ABD [17,54]. Similarly to b4,
plectin contains a binding site for BP180, and together these proteins
recruit BP180 into hemidesmosomes . Ultimately, BP230 becomes
recruited into hemidesmosomes by binding to both b4 and BP180, and
thus type I hemidesmosomes are formed.
TRENDS in Cell Biology
FigureI. Structure of thebinding interfaces of integrin b4 and plectin. Schematic representation of a model of the complex betweenthe first pairof FnIII domains of b4 and
the plectin ABD obtained by computational docking methods . The Ca atoms of residues whose mutation affects binding are shown as red spheres. The b4 residues
that are important for binding to plectin are located in the loop EF of the first FnIII domain and in loops BC, EC0and the region preceding strand A of the second FnIII
domain. The initial residues of the connecting segment are necessary for plectin binding but are not present in the crystal structure, yet they are likely to be orientated
towards the plectin ABD in the model (dashed line). Residues in the plectin ABD relevant for binding to b4 are located in a continuous area of the first calponin homology
(CH) domain (orange) and include the loop preceding the a-helix C and the a-helices E and F. The second CH domain (pink) does not contain particular residues crucial for
binding but its presence is essential for the interaction. Abbreviation: N-term, amino terminus.
TRENDS in Cell BiologyVol.16 No.7 July 2006380
regulation, and their posttranslational modifications
might contribute to the balance between hemidesmosome
formation and disassembly.
Although research on carcinoma cell lines has informed us
about possible pathways for hemidesmosome disassembly,
it should be kept in mind that cancer cells have developed
mechanisms enabling them to escape pathways that
normally regulate proliferation and migration. Therefore,
in physiological conditions in the skin, the pathways
leading to the disassembly of hemidesmosomes might be
entirely different. It is clear that serine phosphorylation of
the b4 cytoplasmic domain is involved in the disassembly
of hemidesmosomes in keratinocytes to initiate migration.
However, it is not yet known which signals induce
of other hemidesmosome components, and whether
(de-)phosphorylation of the b4 cytoplasmic domain is
required for plectin binding. Furthermore, it is not clear
whether serine phosphorylation of b4 is sufficient to
induce hemidesmosome disassembly. The answers to
these questions will help to understand the switch in
function of the a6b4 integrin between the formation of
stable adhesion structures and induction of migration.
We would like to thank Drs K. Wilhelmsen, E. Roos and L. Borradori for
critical reading of the manuscript. The research of Arnoud Sonnenberg is
supported by grants from the Dutch Cancer Society and Dystrophic
Epidermolysis Bullosa Research Association (DEBRA, Crowthorne, UK).
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TRENDS in Cell Biology
Figure 2. Four putative models for phosphorylation-induced hemidesmosome disassembly.Upon phosphorylationof the b4 connecting segment, the interaction with plectin
is lost. (a,b) Binding of plectin might disappear by interfering with binding of the plakin domain, thereby destabilizing the interaction between plectin and b4. Binding of the
plakin domain can be (a) prevented directly by phosphorylation, or (b) prevented indirectly by the binding of a third protein to the phosphorylated connecting segment. (c,d)
Alternatively, binding of the plectin ABD might be lost owing to phosphorylation of b4. Phosphorylation might (c) indirectly lead to loss of plectin ABD binding through
competition with a third protein, or (d) directly lead to loss of binding by inducing a large conformational change. Only the cytoplasmic domain of b4 is shown for simplicity.
Abbreviations: CS, connecting segment; PD, plakin domain.
TRENDS in Cell Biology Vol.16 No.7 July 2006381
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TRENDS in Cell BiologyVol.16 No.7 July 2006383
KASH-domain proteins in nuclear migration, anchorage and
Kevin Wilhelmsen, Mirjam Ketema, Hoa Truong and Arnoud Sonnenberg*
Division of Cell Biology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX
Amsterdam, The Netherlands.
*E-mail address for the corresponding author:
Short title: Nuclear Migration and Anchorage
Key words: nuclear envelope, outer nuclear membrane, KASH domain, nesprin
The nucleus in eukaryotic cells can move within the cytoplasm, and its position is critical
for many cellular events, including migration and differentiation. Nuclear anchorage and
movement can be achieved through association of outer nuclear membrane (ONM)
proteins with the three cytoskeletal systems. Two decades ago studies described C.
elegans mutants with defects in such events, but only recently it has been shown that the
strategies for nuclear positioning are indeed conserved in C. elegans, as well as in
Drosophila, mammals and potentially all eukaryotes. The integral ONM proteins
implicated in these processes thus far all contain a conserved Klarsicht/ANC-1/Syne
homology domain at their C-terminus that can associate with Sad1p/UNC-84-domain
proteins of the inner nuclear membrane within the periplasmic space of the nuclear
envelope (NE). The complex thus formed is responsible, not only for association with
cytoplasmic elements, but also for the integrity of the NE itself.
The contents of the nucleus are enclosed by two lipid bilayers, the inner (INM) and the
outer (ONM) nuclear membrane, which together form the nuclear envelope (NE). The
INM and the ONM fuse at the nuclear pore complexes (NPCs) and the lumen region
between these membranes is called the periplasmic (or perinuclear) space (PS) (Gerace
and Burke, 1988). Beneath the INM is the nuclear lamina, a web of intermediate filament
(IF) proteins composed of A- and B-type lamins. These proteins give shape and stability
to the NE, in addition to associating with proteins bound to chromatin (Gruenbaum et al.,
2005). The ONM, by contrast, is continuous with the endoplasmic reticulum and, in fact,
these two structures share a set of proteins as well as ribosomes (Gerace and Burke,
The nucleus and other organelles can move within the cytoplasm. The position of the
nucleus is important for processes such as mitosis, meiosis, cell migration and
polarization, and nuclear positioning occurs in the cells of most eukaryotes (Morris,
2000). Interactions with the three cytoskeletal filament systems (i.e. F-actin, IFs and
microtubules (MTs)) anchor the nucleus or allow it to move in the cytoplasm. The
different filament systems, in turn, are connected to each other through members of the
plakin family of cytoskeletal cross-linkers (Fuchs and Karakesisoglou, 2001; Leung et al.,
2002). Although defects in nuclear positioning have been observed for a relatively long
time, the mechanisms responsible are only now becoming evident.
Nuclear Positioning in Caenorhabditis elegans
Nuclear migration and anchorage were initially studied in C. elegans. The positions of
the nuclei within the developing embryo can be mapped in great detail because C.
elegans has a transparent body (Sulston and Horvitz, 1977; Sulston et al., 1983). Nuclear
migration has been extensively studied in hyp7 precursor and P cells within the embryo
and larvae, respectively. Prior to the cell fusion events that form the large, multi-
nucleated hypodermal syncytium (the hyp7 syncytium), the hyp7 precursor cells go
through a series of elongation and nuclear migration events, whereby the nuclei move
past the dorsal midline to the opposing lateral side (Sulston et al., 1983; Williams-
Masson et al., 1998). Similarly, the P cells, which ultimately develop into motor neurons
and epithelial cells, have nuclei that migrate from the lateral sides of the newly hatched
larva to form a single row in the ventral cord (Sulston, 1976; Sulston and Horvitz, 1977).
The first worm strains shown to have defects in nuclear migration and anchorage in these
two cell types, which result in uncoordinated movement, were unc-83 and unc-84
(Horvitz and Sulston, 1980; Sulston and Horvitz, 1981; Malone et al., 1999). The unc-83,
and some unc-84, worms also display defective nuclear migration in cells of the intestinal
primordium (Starr et al., 2001). In the majority of the unc-84 mutants, the nuclei move
slowly and do not migrate past the dorsal midline in the hyp7 precursor cells and float
freely within the hyp7 syncytium. However, some unc-84 worms only display the
migratory defects. In P cells, the nuclei fail to migrate to the ventral cord and the cells
ultimately apoptose (Malone et al., 1999). The unc-83 worms have the same phenotype,
except that the nuclei have never been observed to float freely within the cytoplasm of
the hyp7 syncytium (Starr et al., 2001) (Fig. 1A).
The unc-83 and unc-84 genes encode an ONM protein, UNC-83, and an INM protein,
UNC-84, respectively. UNC-83 has a conserved C-terminal Klarsicht/ANC-1/Syne-1
homology (KASH) domain, which contains a transmembrane region and residues that lie
within the PS, and a cytoplasmic N-terminus that does not show sequence similarity to
any known protein (McGee et al., 2006). UNC-84 contains several potential
transmembrane domains and a C-terminal region that is homologous to the yeast protein
Sad1p and, accordingly, is called the Sad1p, UNC-84 (SUN) domain (Malone et al.,
1999; McGee et al., 2006). The localization of UNC-84 to the INM is not dependent upon
its SUN domain but rather on the presence of nuclear lamins, which probably interact
with its N-terminus (Lee et al., 2002). Deletion of UNC-84, mutations in the UNC-84
SUN domain or mutations within the UNC-83 KASH domain can prevent the localization
of UNC-83 at the ONM (McGee et al., 2006; Starr et al., 2001), presumably because of a
loss of direct interaction between the UNC-83 KASH and UNC-84 SUN domains within
the PS (McGee et al., 2006) (Fig. 2). This explains why the nuclear migration defects in
unc-83 and unc-84 mutant worms are very similar (Malone et al., 1999). Curiously,
UNC-83 is present at the NE in a limited number of cell types (including P, hyp7,
intestinal, pharyngeal and uterine cells), unlike UNC-84, which is localized at the NE in
nearly all cells (Starr et al., 2001).
UNC-83 and -84 were originally proposed to tether the nucleus to centrosomes, which
was hypothesized to drive nuclear migration (Malone et al., 1999; Reinsch and Gonczy,
1998); however, later studies showed a normal association between the centrosomes and
nuclei in unc-83 and unc-84 mutant cells whose nuclei fail to migrate (Lee et al., 2002;
Starr et al., 2001). The proteins that interact with the N-terminus of UNC-83 to facilitate
nuclear migration have not been identified. Recently, another ONM protein, ZYG-12,
was shown to mediate an association between the centrosomes and the nucleus in C.
elegans. In zygote defective (zyg)-12 mutant worms, the centrosome detachment defect in
the developing embryo results in death as a consequence of chromosome segregation
defects (Fig. 1B). ZYG-12 is a member of the Hook family and is localized at both the
centrosomes, which is MT-dependent, and the NE. Interestingly, its recruitment to the NE
is dependent upon the expression of the only other SUN-domain-containing protein in C.
elegans, Matefin/SUN-1 (Fridkin et al., 2004; Malone et al., 2003). This is consistent
with the results of the experiments showing that in unc-84 null cells the centrosomes are
correctly localized at the NE (Lee et al., 2002). Unlike for UNC-84, nuclear lamins are
not involved in the retention of Matefin/SUN-1 at the INM (Fridkin et al., 2004). There
are three splice variants of ZYG-12 (A, B and C), two of which contain a KASH domain
(B and C). Localization of these two variants at the ONM probably depends upon an
association between their C-termini and that of Matefin/SUN-1 (Fig. 2).
Defects in nuclear anchorage were first detected in the hyp7 syncytium. Five mutant
alleles of the anchorage defective (anc)-1 gene were isolated from mutant worms in
which unanchored nuclei float freely in the cytoplasm of the syncytium (Hedgecock and
Thomson, 1982). In these worms, mitochondria also appear unanchored, which suggests
that the same mechanism is responsible for the anchorage of the nuclei and mitochondria.
The ANC-1 protein contains two N-terminal calponin homology (CH) motifs, which
together comprise an actin-binding domain (ABD), and a C-terminal KASH domain
(Starr and Han, 2002). The presence of a cytoplasmic ABD indicated that the defects in
nuclear and mitochondrial anchorage might be due to a loss of interaction with the actin
cytoskeleton. Indeed, a direct association with F-actin has been demonstrated in vitro and
an N-terminal fragment of ANC-1, containing the ABD, specifically decorates F-actin in
cells (Starr and Han, 2002). ANC-1 is an enormous molecule of ~950 kDa. This size is
due to a region of repetitive structural elements between the ABD and KASH domains.
Therefore, it seems that a relatively large distance needs to be maintained between the
nucleus and F-actin. The KASH domain of ANC-1 is necessary for its localization at the
ONM and mutations within the SUN domain of UNC-84 prevent the proper localization
of ANC-1 to the NE (Starr and Han, 2002). Additionally, overexpression of a construct
containing the KASH domain alone results in nuclear anchorage defects in the
hypodermal syncytium, probably due to a competition with endogenous ANC-1 for a
limited number of UNC-84 binding sites (Starr and Han, 2002). Interestingly, an unc-84
null mutation has no effect on mitochondrial anchorage in muscle cells (Starr and Han,
2002). The SUN domain of UNC-84 is thus also responsible for the recruitment of ANC-
1 to the ONM through its KASH domain, although note that in this case a direct
interaction has not been detected (Starr and Han, 2002) (Fig. 2).
Worms with mutations in both anc-1 and unc-83 have a compound phenotype. The nuclei
within the syncytium are misplaced as well as unanchored because they fail to migrate
prior to fusion and are unable to interact with the actin cytoskeleton (Hedgecock and
Thomson, 1982). Most of the unc-84 mutant alleles similarly produce nuclear migration
and anchorage defects, but some only lead to defects in nuclear migration (Fig. 1A). The
worms that show both defects either lack UNC-84 or have mutations in regions in or near
the SUN domain; those that only display defects in migration have mutations within the
nucleoplasmic N-terminus of UNC-84 (Malone et al., 1999), indicating that anchorage
may not only depend upon an interaction with the nuclear lamins.
The data thus support a model in which UNC-84 becomes localized at the INM by
binding to the nuclear lamins with its N-terminus, which leaves its periplasmic SUN
domain available for making associations with the KASH domain of either ANC-1 or
UNC-83 to retain these proteins at the ONM. ANC-1 is responsible for nuclear anchorage
through interactions with the actin cytoskeleton, whereas UNC-83 is necessary for
nuclear migration, although how this is achieved is currently unknown (Fig. 2).
Nuclear Positioning in Drosophila melanogaster
Two KASH-domain proteins have been identified in Drosophila, Klarsicht and Msp-
300/nesprin. Klarsicht, originally called Marbles, is important for the development of the
compound eye in Drosophila. Mutation of the klarsicht gene results in the failure of the
nuclei in photoreceptors to migrate to the apex of the developing eye imaginal disc and,
therefore, most nuclei remain at the basal side, which results in oddly shaped
photoreceptors (Fischer-Vize and Mosley, 1994). Importantly, this nuclear migration
defect coincides with the detachment of the centrosome from the nucleus (Patterson et al.,
2004) (Fig. 3A). Klarsicht is also involved in the transport of lipid droplets in Drosophila
embryos (Welte et al., 1998), but this involves a different C-terminal splice variant
(Klarsicht-β, rather than Klarsicht-α and -γ) (Guo et al., 2005). Interestingly, klarsicht-
null mutants are viable and fertile and only display major defects in eye morphology
(Mosley-Bishop et al., 1999).
Klarsicht-α is ~251 kDa, whereas Klarsicht-γ is ~62 kDa. Both contain a C-terminal
KASH domain and are localized at the NE in several cell types (Klarsicht-β does not
have a KASH domain) (Guo et al., 2005). Klarsicht also associates with MTs in
photoreceptors (Fischer et al., 2004; Patterson et al., 2004); the N-terminal region of
Klarsicht, which is not present in Klarsicht-γ and shows no sequence similarity to other
known proteins, is responsible (Fischer et al., 2004). Analyses of fly phenotypes
associated with mutations in the Drosophila B-type lamin Dm0 indicate that Klarsicht
and lamin Dm0 are part of the same pathway (Patterson et al., 2004). Lamin Dm0 may
therefore be required for the localization of an UNC-84-like protein to the INM, and the
consequent retention of Klarsicht at the ONM. The Drosophila genome encodes two
uncharacterized SUN-domain proteins, although these are not predicted to have
transmembrane regions (Malone et al., 2003; Starr and Han, 2003) (Fig. 2). Moreover,
overexpression of the Klarsicht KASH domain in photoreceptors does not result in a
mutant phenotype (Fischer et al., 2004). This suggests that they do not compete with the
endogenous protein for a limited number of binding sites at the NE, in contrast to the
ANC-1 KASH domain (Starr and Han, 2002).
Although Klarsicht and ZYG-12 are both necessary for the association of the centrosome
with the nucleus, these two proteins are not related except in the KASH domain. In
Drosophila, only two proteins show some sequence similarity to ZYG-12. One is a
member of the Hook family, dHk (Kramer and Phistry, 1996; Kramer and Phistry, 1999),
and the other is a recently identified Hook-related protein, dHkRP (Simpson et al., 2005).
Both contain a putative N-terminal MT-binding domain, but do not contain a KASH
domain or are localized at the NE. dHk is cytosolic and reported to play a role in
endocytic trafficking (Kramer and Phistry, 1996; Kramer and Phistry, 1999). The Hook
proteins and ZYG-12 therefore do not appear to have similar functions in the two species,
although, Klarsicht and ZYG-12 seem to have acquired analogous functions in some
cells, given their retention at the NE by a KASH domain and their ability to tether the
nucleus to the centrosome.
Msp300/nesprin is another KASH-domain protein present in Drosophila. Msp-300
(muscle-specific protein 300 kDa) was originally identified in a search for muscle-
specific genes expressed during embryonic myotube migration and attachment (Volk,
1992). Given its localization to actin-rich muscle-attachment sites, Z-lines and the
leading edge of migrating myotubes, Msp-300 was hypothesized to be critical for muscle
morphogenesis in the developing Drosophila embryo (Volk, 1992). Indeed, an embryonic
lethal mutation in Msp-300 leads to defects in some of these processes. In the Msp-300SZ-
75 mutant flies, the myotubes cannot extend towards to the epidermal attachment sites
and, as a result, the ability of the embryonic somatic muscle cells to contract is severely
compromised (Rosenberg-Hasson et al., 1996).
Ten years after Msp-300 was characterized, a gene immediately downstream of Msp-300,
provisionally called nesprin (nuclear envelope spectrin-repeat), was shown to encode a
KASH domain (Zhang et al., 2002). Careful analysis revealed that Msp-300 and nesprin
are in fact part of the same enormous gene spanning about 80 kb. Msp-300 encodes the
N-terminal portion of the full-length protein and nesprin encodes the C-terminal region.
A repetitive coding region lies between the two and, therefore, the complete gene can
potentially encode a gigantic molecule of ~1300 kDa: Msp-300/nesprin (Zhang et al.,
2002). Like ANC-1, Msp-300/nesprin contains a C-terminal KASH domain and an N-
terminal ABD, which binds directly to F-actin in vitro and decorates these filaments in
vivo (Rosenberg-Hasson et al., 1996; Volk, 1992). The region in between comprises a
series of spectrin-repeats (SRs) (Fig. 2) - three-helix bundles that can give proteins length
and elasticity and mediate protein interactions, including homodimerization (Djinovic-
Carugo et al., 2002; Mislow et al., 2002a). Interestingly, the repetitive regions in ANC-1
are not related to SRs, although they apparently have an analogous function. Msp-
300/nesprin appears to be localized predominantly at the NE in nurse cells and the
oocyte, but some also appears to be present in the cytoplasm (Yu et al., 2006). Curiously,
expression of a GFP-KASH domain fusion protein did not have any obvious detrimental
defects on the position of the nuclei in these cells. This suggests that the docking sites for
Msp-300/nesprin, like those of Klarsicht, are not limited on the NE.
During oogenesis in Drosophila, one of the cells in the egg chamber becomes the oocyte;
while the other 15 become the supporting polyploid nurse cells. Eventually, the nurse
cells “dump” their cytoplasmic contents into the oocyte through ring canals, and then
undergo apoptosis (Spradling, 1993). The positions of the nuclei are thought to be critical
during this process because if they would become detached they would block the canals
(Yu et al., 2006). Since actin structures are important for nuclear anchorage in the nurse
cells during dumping (Guild et al., 1997), Yu et al. investigated whether Msp-300/nesprin
plays a role in this process by generating flies carrying the Msp-300SZ-75 allele only in the
germ line (these flies are viable but do not contribute any wild-type Msp-300/nesprin to
the developing Msp-300SZ-75 egg chamber) (Yu et al., 2006). Indeed, the egg chambers in
these flies exhibit defects in cytoplasmic dumping and nuclear positioning (Yu et al.,
2006) (Fig. 3B). Interestingly, they also have defective actin structures, which suggests
that Msp-300/nesprin also has a role in actin organization (i.e. bundling and/or cross-
linking), a role attributed to other ABD-containing proteins (Winder and Ayscough,
Interestingly, the Msp-300SZ-75 mutation is lethal, unlike the deletion of ANC-1 in C.
elegans. Larvae die because they do not hatch from the chorion owing to the previously
mentioned defects in muscle attachment and contraction and not of nuclear anchoring
defects (Rosenberg-Hasson et al., 1996). In contrast, ANC-1 seems not to have critical
functions besides those associated with nuclear and mitochondrial anchorage.
The Mammalian Nesprins
The mammalian nesprins were originally identified in yeast two-hybrid screens for
binding partners of a tyrosine kinase of the postsynaptic membrane in muscle (MuSK)
(which yielded nesprin-1) (Apel et al., 2000) and for the cytoskeletal cross-linker protein
plectin (which yielded nesprin-3) (Wilhelmsen et al., 2005). Nesprin-1, originally named
synaptic nuclear envelope (Syne)-1 because of its presence at postsynaptic nuclei of
neuromuscular junctions (Apel et al., 2000), is actually localized at the ONM of various
cell types (Wilhelmsen et al., 2005; Zhang et al., 2001; Zhang et al., 2002). It is also
referred to as myocyte nuclear envelope (Myne)-1 (Mislow et al., 2002b) or Enaptin
(Padmakumar et al., 2004). Nesprin-2 was identified in database searches for sequences
related to nesprin-1 or the α-actinin ABD (Apel et al., 2000; Zhen et al., 2002). It is also
known as Syne-2 (Apel et al., 2000) or NUANCE (Zhen et al., 2002).
Nesprin-1 and nesprin-2 are giant proteins of ~976 kDa and ~764 kDa, respectively
(Zhang et al., 2002), and each contains an N-terminal ABD (Padmakumar et al., 2004;
Zhen et al., 2002). Nesprin-3 is much smaller, ~110 kDa, and instead binds to plectin at
its N-terminus to connect it to IFs (Wiche, 1998; Wilhelmsen et al., 2005). All three
contain a series of homologous SRs and a C-terminal KASH domain (Fig. 4). In vitro
binding assays and localization studies with the isolated N-terminal regions indicate that
the nesprins can indeed interact with actin filaments and IFs (Fischer et al., 2004;
Padmakumar et al., 2004; Starr and Han, 2002; Wilhelmsen et al., 2005; Zhen et al.,
2002). Interestingly, plectin is ~500 kDa and, when bound to nesprin-3, generates a
bridge to IFs similar in size as the one that nesprin-1 and -2 provide for F-actin. This
suggests that it is important that there is a significant distance between the nucleus and
both the IF and F-actin systems (Fig. 2).
In transgenic mice overexpressing the nesprin-1 KASH domain in muscle cells, the
nuclei do not aggregate beneath the postsynaptic membrane because endogenous nesprin-
1 is displaced from the NE (Grady et al., 2005). This implies that the number of KASH
domain binding sites at the NE is limited (as is the case for ANC-1). How nesprin-1
tethers nuclei to the postsynaptic membrane is not known, although an association with
actin structures or MuSK is probably required (Apel et al., 2000; Grady et al., 2005).
Surprisingly, although the nuclei are not properly localized, the neuromuscular junction
Nesprin-1 and nesprin-2 exhibit a variety of isoforms produced through the use of
different translational initiation and stop sites and alternative splicing (Cottrell et al.,
2004; Mislow et al., 2002b; Padmakumar et al., 2004; Zhang et al., 2001; Zhang et al.,
2005). Interestingly, some of these do not contain an ABD and/or a KASH domain.
Several isoforms are localized and function at areas of the cell other than the ONM, such
as the INM. For example, nesprin-1α might localize at the INM because it can associate
with the INM protein emerin and lamin-A/C, although this has not been confirmed
(Mislow et al., 2002a; Mislow et al., 2002b). Nesprin-1 is also found within the nucleus,
where it is associated with heterochromatin (Zhang et al., 2001). Additionally, expression
of lamin A/C can influence the localization of certain nesprin-2 isoforms at the NE and in
vitro experiments suggested that these bind directly to lamin A/C and emerin within the
nucleus. Indeed, ultrastructural analysis suggested that they are present at the INM
(Libotte et al., 2005; Zhang et al., 2005). Notably, no splice variants of nesprin-3 have
been detected at the INM (Wilhelmsen et al., 2005). The function of the nesprins at the
INM is currently not known, although it is possible that they help to organize the nuclear
Membrane Trafficking and Organization
Nesprin-1 might also be involved in protein trafficking and it has been detected at the
Golgi (Gough et al., 2003). Moreover, expression of certain SR fragments of nesprin-1 in
cells results in the collapse of the Golgi apparatus, prevents the recycling of protein
disulfide isomerase and alters the localization of both the KDEL ER-retrieval receptor
and β-COP (part of the COPI coat complex involved in ER-Golgi transport and NE
breakdown (Liu et al., 2003; Nickel et al., 2002)) (Gough and Beck, 2004). The nesprin-1
splice variant Golgi-localized SR-containing protein (GSRP)-56 contains two SRs
present in the central region of the full-length protein and lacks both the ABD and the
KASH domain. GSRP-56 is localized at the Golgi apparatus and interestingly, its
overexpression results in expansion of the Golgi apparatus (Kobayashi et al., 2006). The
brain-specific splice variant CPG2 is also derived from the SR-rich region (Cottrell et al.,
2004; Padmakumar et al., 2004). Detected in a screen for plasticity-related genes
upregulated after neuronal excitation in rat brains (Nedivi et al., 1993), it is localized at
the postsynaptic endocytic zones of excitatory synapses in neurons. CPG2 regulates the
constitutive internalization of glutamate receptors and the activity-induced internalization
of α-amino-3-hydroxy-5-methyl-isoxazole-4-proprionic acid (AMPA) receptors (Cottrell
et al., 2004), and is thereby proposed to modify synaptic strength. Variants of nesprin-1
might thus function in protein trafficking, control of Golgi morphology and, possibly, NE
Several other roles of nesprin-1 isoforms have been described. For instance, nesprin-1α
can recruit muscle A-kinase anchoring protein (mAKAP) to the NE in striated myocytes
through an association between their SRs (Pare et al., 2005). mAKAP is a scaffold
protein that forms signaling complexes containing protein kinase A (PKA), the ryanodine
receptor (RyR) calcium release channel, protein phosphatase 2A and phosphodiesterase
type 4D3 (Kapiloff et al., 2001). Phosphorylation of the RyR by PKA augments the
release of calcium from the sarcoplasmic reticulum and other luminal areas associated
with the NE (Bers and Perez-Reyes, 1999). Nesprin-1α might thus serve as a receptor on
Nesprin-1 has also been shown to be involved in cytokinesis. A non-SR central fragment
of nesprin-1 interacts with the KIF3B subunit of the kinesin II motor. Overexpression of
this fragment or the C-terminus of KIF3B prevents cytokinesis. Furthermore, the proteins
are co-localized at the central spindle and midbody during cytokinesis. Their association
might allow nesprin-1 to attach vesicles to the kinesin motor protein, bringing extra
membrane to the cleavage site to increase the surface area available for the formation of
the two daughter cells (Fan and Beck, 2004).
Finally, two splice variants of nesprin-3 have been characterized: nesprin-3α and -3β.
Interestingly, nesprin-3β is unable to associate with the plakin family members
(Wilhelmsen et al., 2005). This suggests that nesprin-3, like the smaller isoforms of
nesprin-1 and -2, has functions in the cell other than tethering the nucleus to the
Links with Microtubules
Like the Drosophila proteins dHk and dHkRP, the mammalian Hook and Hook-related
(HkRP) family members share regions of similarity with ZYG-12, but lack a conserved
KASH domain (Malone et al., 2003; Simpson et al., 2005; Walenta et al., 2001) and do
not appear to be responsible for the association of the centrosome with the nucleus.
Nesprin-3, however, has been shown to associate with the ABDs of the plakin family
members MACF and BPAG1. These proteins interact with MTs (Wilhelmsen et al.,
2005) and, thus, Nesprin-3 might provide the necessary link between the nucleus and this
cytoskeletal system. However, an interaction between these proteins has not yet been
demonstrated in cells.
The Mammalian SUN-domain Proteins
Two mammalian UNC-84 orthologues were discovered through an EST database search
for sequences that share similarity with unc-84: SUN1 (UNC84A) and SUN2 (UNC84B)
(Malone et al., 1999). SUN1 was independently discovered in a proteomic analysis of
neuronal NE components (Dreger et al., 2001). Both SUN1 and SUN2 are INM
components. Two other mammalian proteins containing a SUN domain have been
identified, SUN3 and SPAG4, but they were instead primarily detected at the ER
membranes (Crisp et al., 2006; Hasan et al., 2006). In common with matefin/SUN-1, but
not UNC-84, SUN1 does not require lamin proteins for its localization at the INM,
although it does bind specifically to lamin A (Crisp et al., 2006; Haque et al., 2006;
Hasan et al., 2006; Padmakumar et al., 2005). Lamin A is, however, partially responsible
for the localization of SUN2. Interestingly, both SUN1 and SUN2 preferentially bind to
the unprocessed (i.e. non-cleaved) form of lamin A in vitro and in vivo (Crisp et al.,
2006), which suggests that they play a role in the recruitment and organization of these
proteins at the nuclear lamina.
SUN1 and SUN2 each contain a conserved SUN domain that extends into the PS and
interacts with the KASH domains of nesprin-1, -2 and -3 (Crisp et al., 2006; Haque et al.,
2006; Ketema and Sonnenberg, unpublished; Padmakumar et al., 2005). However, a
region just upstream of the SUN domain may mediate high-affinity binding to the KASH
domain (Padmakumar et al., 2005). The importance of this interaction is highlighted in
transgenic mice overexpressing the KASH domain of nesprin-1, which prevents the
localization of endogenous nesprin-1 at the ONM in muscle cell nuclei at the
neuromuscular junction (Grady et al., 2005). Additionally, RNAi-induced depletion of
SUN1 and SUN2 proteins in HeLa cells results in the mislocalization of nesprin-2γ away
from the ONM. Importantly, this study also showed that the interactions of the nesprins
with the SUN-domain proteins is critical for maintenance of the NE because the absence
of SUN1 and SUN2 leads to an expansion of the PS (Crisp et al., 2006). The
evolutionarily conserved interactions that take place between the conserved SUN and
KASH domains within the PS are thus critical not only for associations between the
nucleus and cytoplasmic proteins, but also for the integrity of the NE in mammalian cells.
It is now clear that SUN-domain proteins of the INM retain the KASH-domain proteins at
the ONM and these proteins probably directly interact within the PS. This generates a
continuous protein scaffold that physically links the nucleoskeleton to the cytoskeleton:
the so-called LINC complex (Crisp et al., 2006) (Figs 2, 5). However, other proteins
present in the PS might influence these interactions.
It is intriguing that the ONM proteins in C. elegans, although unrelated to those of
Drosophila and mammals except for their KASH domain, have evolved similar
mechanisms for nuclear anchorage and migration. ANC-1 has a function analogous to
that of Msp-300/nesprin and the mammalian nesprins in nuclear anchorage, whereas
ZYG-12 has a function analogous to that of Klarsicht in the attachment of the centrosome
to the nucleus. Although no proteins related to UNC-83 have been identified, its role in
migration is likely to be similarly conserved in higher organisms.
For several reasons, few studies have examined the importance of the mammalian
nesprins in nuclear positioning. First, the genes and their mRNAs are very large (Zhang
et al., 2002), which makes molecular, biochemical and cellular studies difficult.
Secondly, there are many splice variants, some of which are cell-type specific, which
complicates the interpretation of the results. Thirdly, the three nesprins may be able to
compensate for one another if one is absent or disfunctional. Studies in which the
function of all three nesprins is disrupted may ultimately prove their importance in
nuclear positioning, but such studies are complicated by the fact that the nesprins have
other critical functions.
The only study to demonstrate that the mammalian nesprins are essential in nuclear
positioning depended on the overexpression of the nesprin-1 KASH domain in muscle
cells (Grady et al., 2005) and was based on similar experiments using the C. elegans
ANC-1 KASH domain (Starr and Han, 2002). Both studies suggest that the number of
SUN-domain binding sites in the PS is limited. However, overexpressed KASH domains
might displace other KASH-domain proteins from the ONM, such as nesprin-3 in
mammals, which is also expressed in muscle cells (Wilhelmsen et al., 2005).
Furthermore, overexpression of the Klarsicht and Msp-300/nesprin KASH domains in
Drosophila does not affect nuclear positioning, or produce other phenotypic defects, in
photoreceptors or the egg chamber, respectively (Fischer et al., 2004; Yu et al., 2006).
Therefore one should be cautious when interpreting results from studies using KASH
domain overexpression in higher organisms.
An intriguing theory called cellular tensegrity proposes that eukaryotic cells are pre-
stressed through the concerted action of all three cytoskeletons (Ingber, 2003a; Ingber,
2003b). Importantly, it would explain how simultaneous changes in cytoskeletal, nuclear
and other cellular structures can occur in response to localized force in the absence of any
biochemical changes (Maniotis et al., 1997). We have known for a relatively long time
that integrins can mediate cross-talk between the ECM and the different cytoskeletal
systems (Geiger et al., 2001), and that integral INM proteins can mediate the interactions
of the nuclear lamina with heterochromatin (Gruenbaum et al., 2005). The well-studied
NPCs are known to bridge the nucleoskeleton and the cytoskeleton and have been
suggested to mediate the mechanotransduction of signals from the cytoskeleton to the
nuclear environment (Ingber, 2006). KASH-domain proteins may provide an alternative
mechanism to direct forces into the nucleus (Fig. 5). There is no doubt that future studies
on the architecture and positioning of the nucleus and cellular mechanotransduction will
reveal many novel, exciting functions for this family of proteins.
We would like to thank Drs. Rik Korswagen, Yosef Gruenbaum and Nick Brown for
critical reading of the manuscript. Kevin Wilhelmsen and Mirjam Ketema are supported
by grants from the Dutch Cancer Society (KWF) and the Netherlands Science
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Phenotypes of the C. elegans mutants anc-1, unc-83, unc-84 and zyg-12. A. In C.
elegans, hyp7 precursor cells go through a series of elongation and alternating
intercalation events on the dorsal side of the developing embryo. In the wild-type
embryo, the nuclei (grey and black circles) then move to the opposite side of the embryo
(S3). After fusion of the hyp7 precursors into the large multi-nucleated hyp7 syncytium,
the nuclei are evenly anchored near the dorsal midline (DM) (S4). Unc-83 and some unc-
84 mutant alleles result in failure of the nuclei to migrate past the DM (S3); these are
therefore mispositioned in the syncytium, although they appear to be anchored (S4). In
the anc-1 worms, the nuclei migrate normally (S3) but are unanchored and typically form
clumps in the syncytium (S4). The majority of the unc-84 mutant alleles, and all the unc-
83 anc-1 double mutants, result in incorrect positioning and anchorage of the nuclei in
the syncytium (S4). Only a few of the hyp7 precursor cells are depicted for simplicity. B.
In the single-cell embryo of C. elegans, MTs associated with the two centrosomes pull
the two pronuclei (black ovals) together; these then fuse and the nuclear envelope breaks
down. At the start of the first mitosis, MTs associated with the centrosomes pull the
paired chromosomes apart. In the zyg-12 worms, the centrosomes are detached from the
pronuclei. As a result, the nuclei are not pulled together and do not fuse properly. The
nuclear envelopes still break down and the chromosomes from each pronucleus still
associate with MTs. Cytokinesis occurs but results in daughter cells that contain
abnormal numbers of chromosomes.
Proposed protein complexes spanning the NE in C. elegans, Drosophila and
mammals. The nuclear lamins of several species are proposed to interact with the N-
terminal regions of the SUN-domain proteins of the INM. Within the PS, the SUN-
domains (or regions immediately upstream of the SUN-domains) associate with the
KASH domains of ONM proteins. Within the cytoplasm, the N-terminal regions of these
proteins associate with the microtubule organizing center (MTOC), MTs, F-actin or
plectin (which can directly bind to the IFs). Thus, there is a continuous protein scaffold
from the nuclear lamina, through the NE, to the different cytoskeletal systems in several
different species. The SUN-domain proteins are shown as dimers. SUN1 crosses the INM
three times, while the other SUN-domain proteins cross it at least once. The two
Drosophila SUN-domain proteins identified in database searches (labeled Dm-SUN in
this depiction) do not contain any recognizable membrane spanning regions, although
these may not represent full-length sequences.
Phenotypes of the Drosophila mutants klarsicht and Msp-300SZ-75. A. In the wild-type
eye imaginal disc, passage of the morphogenetic furrow (MF) initiates the differentiation
of photoreceptors. Normally, the nuclei (black circles) are pulled up to the apical side
through an association with the centrosome and MTs. In the klarsicht mutant flies, the
centrosomes are no longer attached to the nuclei. As a result, the nuclei remain at the
basal side. B. At the start of stage 10B in wild-type Drosophila ovaries, the nurse cells
(NCs) start to dump their cytoplasmic contents into the oocyte through the ring canals
(RCs). By stage 13, most NCs, and their nuclei (large black ovals), have disappeared and
the dorsal appendages are extruded at the anterior end. In Msp-300SZ-75 flies, the NC
nuclei become detached once cytoplasmic dumping occurs and either form multi-
nucleated cells, enter the oocyte or become mispositioned within individual cells. The
germinal vesicle (GV) (the oocyte nucleus) (grey oval) is also mispositioned. For
simplicity, only some of the NCs are depicted. A-anterior; P-posterior.
Alignment of KASH domains from various proteins. The amino acid residues present
in the predicted transmembrane regions are shown in bold. The Zyg-12B KASH domain
sequence is used because the Zyg-12C KASH domain sequence in the database lacks 16
residues encompassing part of the neck and transmembrane regions. Dark green
highlighting indicates conserved amino acids and light green highlighting indicates amino
acids with similar chemical properties in more than half of the KASH domains. Mm-Mus
musculus; Hs-Homo sapiens.
In mammals, a continuous protein scaffold connects the extracellular matrix to the
nuclear lamina via IFs and F-actin. Integrin α6β4, present in hemidesmosomes (HDs),
and β1/β3 integrins, present in focal contacts (FCs), connect the extracellular matrix
(ECM) to cytoplasmic IFs (through plectin) and F-actin (through talin), respectively.
These two cytoskeletal systems are interconnected to each other via the plakins (orange
circles) or to themselves via filamin or α-actinin (red circles) and the plakins (grey
circles). F-actin associates with the ABDs of nesprin-1 and –2; IFs associate with plectin
dimers bound to nesprin-3. In turn, the ONM nesprins associate with either SUN1 or -2
within the PS. The INM SUN-domain proteins associate with lamin A through their N-
termini within the nucleus. ER-endoplasmic reticulum; INM-inner nuclear membrane;
ONM-outer nuclear membrane; PM-plasma membrane; PS-periplasmic space.
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