Nestin structure and predicted function in cellular cytoskeletal organisation.
ABSTRACT Nestin is an intermediate filament protein expressed in dividing cells during the early stages of development in the CNS, PNS and in myogenic and other tissues. Upon differentiation, nestin becomes downregulated and is replaced by tissue-specific intermediate filament proteins. Interestingly, nestin expression is reinduced in the adult during pathological situations, such as the formation of the glial scar after CNS injury and during regeneration of injured muscle tissue. Although it is utilised as a marker of proliferating and migrating cells very little is known about its functions or regulation. In depth studies on the distribution and expression of nestin in mitotically active cells indicate a complex role in regulation of the assembly and disassembly of intermediate filaments which together with other structural proteins, participate in remodeling of the cell. The role of nestin in dynamic cells, particularly structural organisation of the cell, appears strictly regulated by phosphorylation, especially its integration into heterogeneous intermediate filaments together with vimentin or alpha-internexin.
- SourceAvailable from: Ahmet Sukru UmurKafkas Üniversitesi Veteriner Fakültesi Dergisi 01/2014; · 0.29 Impact Factor
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ABSTRACT: There is an urgent need for new in vitro strategies to identify neurotoxic agents with speed, reliability and respect for animal welfare. Cell models should include distinct brain cell types and represent brain microenvironment to attain higher relevance. The main goal of this study was to develop and validate a human 3D neural model containing both neurons and glial cells, applicable for toxicity testing in high-throughput platforms. To achieve this, a scalable bioprocess for neural differentiation of human NTera2/cl.D1 cells in stirred culture systems was developed. Endpoints based on neuronal- and astrocytic-specific gene expression and functionality in 3D were implemented in multi-well format and used for toxicity assessment. The prototypical neurotoxicant acrylamide affected primarily neurons, impairing synaptic function; our results suggest that gene expression of the presynaptic marker synaptophysin can be used as sensitive endpoint. Chloramphenicol, described as neurotoxicant affected both cell types, with cytoskeleton markers’ expression significantly reduced, particularly in astrocytes. In conclusion, a scalable and reproducible process for production of differentiated neurospheres enriched in mature neurons and functional astrocytes was obtained. This3D approach allowed efficient production of large numbers of human differentiated neurospheres, which in combination with gene expression and functional endpoints are a powerful cell model to evaluate human neuronal and astrocytic toxicity.Journal of Biotechnology. 01/2015;
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ABSTRACT: Neurogenesis is an important process in the regulation of brain function and behaviour, highly active in early development and continuing throughout life. Recent studies have shown that neurogenesis is modulated by inflammatory cytokines in response to an activated immune system. To disentangle the effects of the different cytokines on neurogenesis, here we summarise and discuss in vitro studies on individual cytokines. We show that inflammatory cytokines have both a positive and negative role on proliferation and neuronal differentiation. Hence, this strengthens the notion that inflammation is involved in molecular and cellular mechanisms associated with complex cognitive processes and, therefore, that alterations in brain-immune communication are relevant to the development of neuropsychiatric disorders. Copyright © 2014 Elsevier Ltd. All rights reserved.Trends in Neurosciences 01/2015; · 12.90 Impact Factor
Summary. Nestin is an intermediate filament protein
expressed in dividing cells during the early stages of
development in the CNS, PNS and in myogenic and
other tissues. Upon differentiation, nestin becomes
downregulated and is replaced by tissue-specific
intermediate filament proteins. Interestingly, nestin
expression is reinduced in the adult during pathological
situations, such as the formation of the glial scar after
CNS injury and during regeneration of injured muscle
tissue. Although it is utilised as a marker of proliferating
and migrating cells very little is known about its
functions or regulation. In depth studies on the
distribution and expression of nestin in mitotically active
cells indicate a complex role in regulation of the
assembly and disassembly of intermediate filaments
which together with other structural proteins, participate
in remodeling of the cell. The role of nestin in dynamic
cells, particularly structural organisation of the cell,
appears strictly regulated by phosphorylation, especially
its integration into heterogeneous intermediate filaments
together with vimentin or α-internexin.
Key words: Nestin, Intermediate filaments,
Nestin is an intermediate filament protein expressed
predominantly in rapidly dividing progenitor cells of
developing and regenerating tissues. Cell division
requires that cytoplasmic and nuclear compartments be
disassembled, reorganized and partitioned into daughter
cells. These processes of extensive remodeling are
orchestrated by components of the cytoskeleton, a
composite of microtubules (20 nm in diameter),
intermediate filaments (8-12 nm in diameter) and actin
microfilaments (6 nm in diameter) (Geisler et al., 1989;
Klymkowsky, 1996; Ku et al., 1996; Fuchs and
Cleveland, 1998; Goldman et al., 1999).
Intermediate filaments, of which nestin is a member,
comprise more than forty individual proteins that can be
divided into six main classes (I-VI) based on their
molecular structure (Lendahl et al., 1990; Steinert and
Liem, 1990). Class I and class II are basic and acidic
keratins of epithelial cells; class III proteins include
desmin, GFAP, peripherin and vimentin; class IV
consists of neurofilaments and α–internexin and class V
are nuclear lamins. Nestin comprises a novel class VI
intermediate filament protein (Lendahl et al., 1990).
Intermediate filament proteins are differentially
expressed in tissues and depending on the cell type may
comprise from 1 to 85% of total protein, where they are
arranged as homogenous or heterogeneous polymers
(Zehner, 1991; Goldman, 2001).
Changes within the spatial and temporal expression
of intermediate filament proteins regulate remodeling of
the cell cytoskeleton during development. This is
particularly striking in the CNS where intermediate
filaments exhibit sequential expression; preimplantation
embryos express cytokeratins (Classes I and II);
following neurulation, multipotent CNS cells express
nestin (class VI) and vimentin (class III). Finally
terminal differentiation involves down-regulation of
nestin and induction of neurofilaments (class IV) in
neurons or GFAP (class III) in astrocytes (Steinert and
Identified in 1985 (Hockfield and McKay, 1985),
nestin is expressed in the majority of mitotically active
CNS and PNS progenitors that give rise to both neurons
and glia (Cattaneo and McKay, 1990; Lendahl et al.,
1990; Lendahl, 1997; Mujtaba et al., 1998). Nestin is
also found in myogenic precursors of skeletal muscle
and heart (Lendahl et al., 1990; Sejersen and Lendhal,
1993; Kachinsky et al., 1994, 1995), as well as in the
developing tooth bud (Terling et al., 1995), testis
(Fröjdman et al., 1997) and hair follicle sheath
progenitor cells of the skin (Li et al., 2003).
Nestin is downregulated in all cells upon
differentiation (Zimmerman et al., 1994; Lothian and
Lendahl, 1997), but reappears transiently after injury to
muscle or the CNS where it has been found in reactive
Nestin structure and predicted
function in cellular cytoskeletal organisation
K. Michalczyk and M. Ziman
School of Biomedical and Sports Science, Edith Cowan University, Joondalup, Western Australia, Australia
Histol Histopathol (2005) 20: 665-671
Offprint requests to: Dr. Mel Ziman, School of Biomedical and Sports
Science, Edith Cowan University, 100 Joondalup Drive, Joondalup,
Western Australia, Australia 6027. e-mail: email@example.com
Cellular and Molecular Biology
astroglia of the brain and in ependymal cells of the rat
spinal cord after injury (Lendahl, 1997; Krum and
Rosenstein, 1999; Namiki and Tator, 1999; Pekny et al.,
1999; Vaittinen et al., 2001). Moreover, adult tissues
such as CNS and skin contain small populations of
nestin positive stem/progenitor cells (Johansson et al.,
2002; Li et al., 2003). In fact, nestin is now wildly used
as a marker for stem cells that characteristically display
features such as multipotency, self renewal and
regeneration, yet little is known about nestin function. In
this review the basic biological properties of nestin are
described and possible functional roles in cell
remodeling during mitosis are explored.
The nestin gene and its evolution
The grouping and nomenclature of nestin as a
distinct intermediate filament protein is based on the
exon/intron structure of the gene; the positions and
lengths of its introns are highly conserved (Fig. 1).
Moreover two of the introns are in identical positions to
those of neurofilament genes (Fig. 2). Taken together
with their similarity in sequence (20% in regions
encoding the conserved protein rod domains) (Fig. 3), it
is reasonable to argue that nestin and the three
neurofilament genes arose from a common ancestor by
gene duplication (Dahlstrand et al., 1992) (Fig. 2).
The promoter region of the nestin gene controls
spatial and temporal expression
Expression of the nestin gene is driven by a minimal
promoter, present between residues -11 and +183 in the
5’-non-coding region. The promoter contains two
adjacent Sp1-binding sites necessary for promoter
activity, but lacks a functional TATA box (Cheng et al.,
2004). Enhancer elements that specifically regulate
expression in myogenic and neural precursors are in the
first and second intron respectively (Zimmerman et al.,
Within the second intron of nestin reside two
separate enhancer elements; a 204-bp midbrain specific
enhancer between bases 1068 and 1271, and a 206-bp
pan-CNS enhancer between bases 1272 and 1477
(Lothian and Lendahl, 1997; Lothian et al., 1999). These
two enhancers function independently of each other and
contain at least two distinct regulatory sites. In the
midbrain enhancer, there is a midbrain-specifying
element, between bases 1068 and 1199 and a general
transcriptional potentiator element, between bases 1200
and 1255. The midbrain element reproducibly restricts
expression to the ventral midbrain yet in its absence,
gene expression is nonspecific (Lothian et al., 1999;
Kappen and Yaworsky, 2003). Similarly, the CNS
enhancer contains two critical sites between bases 1272
and 1400 and between bases 1401 and 1477. These sites
interact to enhance activity throughout the developing
nervous system (Yaworsky and Kappen, 1999; Lothian
et al., 1999). Such multiple regulatory elements are
presumably required to ensure nestin expression in CNS
Nestin structure and function
Fig. 1. A diagram depicting the exon/intron structure of the nestin gene;
the intron pattern is conserved between the human, rat and mouse
nestin genes. The first three introns are in identical positions however
the fourth intron has not been reported for the human gene. The regions
encoding the α-helices and the 11 amino acid repeats have been
Fig. 2. A schematic representation of intron positions in the genes
encoding the six different classes of intermediate filament proteins.
Filled triangles indicate intronic positions. Identical intronic positions in
NF and nestin are indicated by larger triangles. Keratins (Ichinose et al.,
1988; Krauss and Franke, 1990); GFAP (Balcarek and Cowan, 1985);
NFM (Myers et al., 1987); Lamin (Döring and Stick, 1990); nestin
(Lendahl et al., 1990). Note: the positions of the first two introns of
nestin are perfectly conserved relative to those of neurofilament. The
introns, situated in the fourth coil of the a-helical region, are spliced at
exactly the same nucleotide position (From Dahlstrand et al., 1992).
Fig. 3. A diagram depicting intermediate filament structure, showing
conserved sequences and rod subdomains. Coils 1A and 1B are
separated by linker L1, coils 1B and 2A are separated by L12, and coils
2A and 2B are separated by L2. Nestin has a short N terminal region
and no linker L2 region. N and C designate amino and carboxyl termini,
progenitor cells at specific locations along the anterior-
posterior and dorsal-ventral axes of the embryo.
The CNS and midbrain specific enhancer elements
are thought to be regulated by nuclear hormone receptors
(TRs, RXR, RAR, COUP-TF) and POU-domain
transcription factors. These transcription factors regulate
early embryonic patterning, cell migration and
proliferation (Lothian and Lendahl, 1997; Jaworsky and
Kappen, 1999; Lothian et al., 1999).
Remarkably, in the adult CNS, nestin expression is
regulated by upstream specific enhancer elements which
differ from elements that direct expression in embryos
and cultured adult CNS cells (Johansson et al., 2002).
Detailed characterization of nestin enhancers may be
important for future studies aimed at repairing the adult
The nestin protein
Nestin is a large protein (>1600 amino acids),
structurally similar to other intermediate filaments, with
a highly conserved α-helical core domain of 300-330
amino-acids flanked by amino- and carboxy-terminal
domains. The common core, referred to as the rod
domain, is composed of several α-helical coils, coils 1A
and 1B separated by linker L1, coils 1B and 2A
separated by L12, and coils 2A and 2B (Fig. 3). The
coils are essential for the creation of coiled-coil dimers
that associate in an antiparallel fashion to form tetramers
and protofilaments, which then combine laterally to form
filaments (Fuchs and Weber, 1994; Marvin et al., 1998).
The C-terminal region of nestin contains 1306
amino-acids and a conserved heptad repeat unit. By
contrast, the amino terminus of nestin is much shorter
than that of other intermediate filament proteins
(Dahlstrand et al., 1992). Variation in the amino and
carboxy terminal ends of intermediate filament proteins
allows for complex binding to an array of structural
The deduced human nestin protein is shorter than the
rat and mouse proteins by 187 and 203 amino acids
respectively (Dahlstrand et al., 1992; Yang et al., 2001).
The difference in length is due to variation in the number
of C-terminal heptad repeat units; human nestin has 18
repeats of the heptad S/PLEK/EEN/DQES/PLR, whereas
there are 41 repeats of an almost identical motif
(SLEK/EENQEXLR) in rat and mouse. While the
carboxy-terminal region is only 55% conserved, the rod
α-helical region is highly conserved (82%) between
man, rat and mouse (Dahlstrand et al., 1992; Yang et al.,
2001). Thus the carboxy-terminal region and the α-
helical domain of human nestin have evolved at quite
different rates which highlights the importance of the
helical rod domain in copolymer formation. Similarly,
other filament proteins, including neurofilament, have
conserved rod domains while the lengths of their C-
terminal repeat units vary (Julien et al., 1988; Lees et al.,
1988) (Table 1, Fig. 3).
Mutational analysis has identified conserved
sequences within the rod domain specifically within
coils 1A and 2B that are essential for normal
intermediate filament assembly (Hatzfeld and Weber,
1992; Kouklis et al., 1992; Fuchs and Weber, 1994).
Proteins containing mutations in rod-end sequences were
found to disrupt assembly of normal intermediate
filament subunits in vivo and in vitro and to result in
accumulation of nestin aggregates in the cytoplasm of
CNS precursors and radial glia in vivo (Letai et al., 1992;
Marvin et al., 1998).
Nestin assembles into polymers with other
Nestin preferentially forms intermediate filaments
by assembly with a variety of intermediate filament
proteins, particularly type III vimentin and type IV α-
internexin (Marvin et al., 1998; Eliasson et al., 1999;
Steinert et al., 1999). The formation of filaments
composed of heterodimers and heterotetramers rather
than homodimers is presumably because nestin contains
a very short N-terminus, a domain known to be essential
for filament protein assembly (Fuchs and Weber, 1994;
Herrmann and Aebi, 2000). This possibility is supported
by in vitro studies of nestin-vimentin coassembly, which
demonstrate that nestin inhibits filament formation in a
concentration dependant manner when present at
concentrations greater than 50% (Steinert et al., 1999).
Moreover, composites of nestin-vimentin and nestin-α-
internexin heterodimers are more stable than nestin
homodimers but less stable than vimentin and α-
internexin homopolymers when subjected to increasing
concentrations of urea in vitro (Steinert et al., 1999).
Nestin structure and function
Table 1. Rates of conservation of intermediate filament protein domains.
The percentage of amino acid conservation between man and mouse
(for nestin, man and rat) for the α-helical domain and the carboxy
terminus of several intermediate filament proteins. Values denote amino
acid conservation for GFAP (Balcarek and Cowan, 1985; Reeves et al.,
1989), NFH (Julien et al., 1988; Lees et al., 1988), NFM (Levy et al.,
1987; Myers et al., 1987), NFL (Lewis and Cowan, 1986; Julien et al.,
1987), lamin C (Fisher et al.,1986; Riedel and Werner, 1989; Dahlstrand
et al., 1992), nestin (Lendahl et al., 1990; Dahlstrand et al., 1992).
*Note: For most intermediate filament proteins the α-helical region is
more highly conserved than the C-terminal region, indicating different
rates of conservation (Julien et al., 1988; Lees et al., 1988).
**Note: Nestin is the least conserved particularly at the carboxy-terminal
end. The amino acid sequence in the N terminal α-helical region is 82%
conserved whereas the carboxy-terminal region is only 55% conserved
between man and rat.
To explain the multiple roles of nestin in regulation
of cellular structure, a model has been proposed in which
nestin-vimentin heteropolymers would attach to a core
of vimentin homopolymers (Fig. 4). The long carboxy-
terminal of nestin would protrude from this filament
body and could function as a linker or cross-bridge
between intermediate filaments, microfilaments and
microtubules (Hirokawa et al., 1984; Hisanaga and
Hirokawa, 1988). Thus nestin may play a role in
connecting the three components of the cytoskeleton and
coordinate changes in cell dynamics (Herrmann and
Aebi, 2000) (Fig. 5).
The prescribed role for nestin in the organization of
intermediate filaments during mitosis is supported by in
vitro studies showing that, nestin transfected into
cultured cells colocalises with vimentin consistently
throughout the cell cycle (Chou et al., 2003; Sahlgren et
al., 2003). During interphase, filamentous networks
consisting of nestin and vimentin polymers were
observed extending from the perinuclear region to the
cell surface; during the transition from late prophase to
metaphase, nestin and vimentin copolymer networks
simultaneously reorganized into cage-like structures
surrounding the nucleus; during telophase, nestin and
vimentin networks were disassembled and vimentin and
nestin were extensively colocalised in punctate and
diffuse structures (Chou et al., 2003; Sahlgren et al.,
Phosphorylation of nestin is associated with
disassembly of filaments
To bring about the changes in morphology required
within rapidly dividing and migrating cells, structural
proteins need to be assembled and disassembled in a
strictly regulated spatial and temporal manner. Several
lines of evidence indicate that nestin is involved in this
process during mitosis; nestin forms stable polymers
with several intermediate filament proteins. Moreover,
paradoxically, its expression is also associated with
requisite intermediate filament breakdown. The
mechanisms that control this diversity in function are
still not fully understood, but phosphorylation is thought
to be an important factor (Eriksson et al., 1992; Skalli et
al., 1992; Ku et al., 1996; Inagaki et al., 1997).
Low levels of phosphorylation are associated with
filament assembly. By contrast, during mitosis, a three-
fold increase in the phosphorylation of nestin and a six-
fold increase in the phosphorylation of vimentin
coincide with dramatic reorganisation of filament
networks (Sahlgren et al., 2001). In particular,
phosphorylation of nestin at Threonine316by cdc2 kinase
causes partial disassembly of nestin-containing
intermediate filaments (Sahlgren et al., 2001). Notably,
Threonine316is located in the highly conserved carboxyl
terminus near the end of the rod domain, a region
important for filament assembly. Furthermore, mutations
in this region are associated with intermediate filament-
related diseases, including severe cases of epidermolysis
bullosa simplex (Letai et al., 1992; Zimmerman et al.,
1994; Sahlgren et al., 2001).
Phosphorylation of nestin may regulate not only its
polymerisation with type III and type IV intermediate
filament proteins, but may alter also, its links with other
cytoskeletal components (Steinert et al., 1999; Herrmann
and Aebi, 2000; Sahlgren et al., 2001). The long C
terminal repeats contain serine residues that are
predicted to serve as additional phosphorylation sites.
Their phosphorylation would change the configuration
of the side arms and affect the formation of cross-bridges
between nestin intermediate filaments and other
cytoskeletal components. In summary, phosphorylation
Nestin structure and function
Fig. 4. Cross-sectional representation of individual proteins in an
intermediate filament. a. In the homopolymeric situation, tetramers are
integrated within a symmetrical filament with minimal hollow core
(Steinert et al., 1999). b. Mixed polymers of vimentin-nestin or α-
internexin-nestin may form such that the nestin (shaded subunits) reside
on the filament periphery (adapted from Herrman and Aebit, 2000).
Fig. 5. Schematic representation of the interaction between
microfilaments (MF), microtubules (MT) and intermediate filaments (IF).
The intermediate filament protein consists of a vimentin (yellow)/nestin
(blue) heterodimer. Nestin has a long non-α-helical carboxy-terminal
end that may stably link it to MTs and MFs. (adapted from Herrman and
and dephosphorylation of nestin may modulate
respectively, disassembly and assembly of intermediate
filaments within supramolecular structures and thus
control dynamic changes in cell ultastructure (Hirokawa
et al., 1984; Steinert et al., 1999).
Similarly, phosphorylation has been shown to
regulate the spatial organization of intermediate filament
proteins such as vimentin (Chou et al., 1989, 1990). In
fact, during mitosis, the disassembly of vimentin
intermediate filaments requires the presence and
phosphorylation of both nestin and vimentin (Chou et
al., 2003). To induce the disassembly of vimentin
polymers during mitosis, nestin works in concert with
the ubiquitous mitotic kinase, maturation/M-phase-
promoting factor, MPF, which phosphorylates vimentin
at Ser-55 in the amino-terminal head domain, the region
required for dimerisation (Aubin et al., 1980; Jones et
al., 1985; Chou et al., 1996, 2003; Sahlgren et al., 2003).
The role of nestin in vimentin disassembly was recently
confirmed by downregulation of nestin with specific
siRNAs in vitro, which blocked the disassembly of
vimentin intermediate filaments in mitotic cells (Chou et
The disassembly of vimentin intermediate filaments
is not a feature of all cells – in fact, many cell types do
not express nestin. In dividing cells in which nestin is
absent and there is no apparent disassembly of vimentin
filaments, there is alternately, a restricted local
disassembly of intermediate filaments at the cleavage
furrow in late cytokinesis (Yasui et al., 2001).
Nestin association with cytoplasmic trafficking in
rapidly dividing progenitor cells
The advantage of mitotic disassembly of vimentin
filaments for cells expressing nestin remains unknown.
Mitotic and spreading interphase cells, containing
nonfilamentous keratin or vimentin, are able to move
protein particles at high speeds along microtubules with
molecular motors kinesin and dynein (Prahlad et al.,
1998; Windoffer and Leube, 1999; Helfand et al., 2002).
It is possible that this ensures the rapid transport of
precursor molecules between various cytoplasmic
compartments. Therefore, nestin expression may be
associated with increased cytoplasmic trafficking in
progenitor cells undergoing rapid rounds of division,
interspersed with active interphase migration. Such
characteristics are common features of cells in early
developing nerve and muscle tissues (Lendahl et al.,
1990; Sejersen and Lendahl, 1993; Kachinsky et al.,
1995; Vaittinen et al., 1999) and in regenerating adult
tissues (Frisen et al., 1995; Vaittinen et al., 1999).
Nestin may also play a role in the asymmetric
allocation of cytoskeletal and other cellular factors to
daughter cells. For example, within the developing
neural tube, ventricular cells continue to proliferate
whereas differentiated cells migrate towards the pial
surface. Polarized distribution of material between
dividing and differentiating daughter cells within the
neuroepithelium may be caused by nestin-mediated
disassembly and uneven partitioning of motile vimentin
particles during mitosis (Frederiksen and McKay, 1988;
Rakic, 1988; Chou et al., 2003).
Intermediate filaments represent the least understood
part of the cytoskeleton. Although many parameters are
known, the reasons for the existing diversity of
intermediate proteins as well as their individual
functions remain unknown. Because nestin is expressed
in the majority of mitotically active CNS and PNS
progenitors, it is currently widely used as a marker for
neural stem cells yet its apparent diversity of roles in
heterogeneous cells is still not completely understood.
Several researchers have performed complex
experiments and detailed analyses to ascertain its
functions. While these have aided in the understanding
of the complexity of cell dynamics, the intricate role of
nestin in cellular proliferation during development and
regeneration remains to be conclusively defined.
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Accepted January 5, 2004
Nestin structure and function