Molecular Biology of the Cell
Vol. 14, 1468–1478, April 2003
Nestin Promotes the Phosphorylation-dependent
Disassembly of Vimentin Intermediate Filaments
Ying-Hao Chou*, Satya Khuon*, Harald Herrmann†, and
Robert D. Goldman*‡
Department of Cell and Molecular Biology, Northwestern University, 303 East Chicago Avenue,
Chicago, IL 60611, USA. Department for Cell Biology, German, Cancer Research Center, D-69120,
Submitted August 29, 2002; Revised November 24, 2002; Accepted December 9, 2003
Monitoring Editor: Paul T. Matsudaira
The expression of the intermediate filament (IF) protein nestin is closely associated with rapidly
proliferating progenitor cells during neurogenesis and myogenesis, but little is known about its
function. In this study, we examine the effects of nestin expression on the assembly state of
vimentin IFs in nestin-free cells. Nestin is introduced by transient transfection and is positively
correlated with the disassembly of vimentin IFs into nonfilamentous aggregates or particles in
mitotic but not interphase cells. This nestin-mediated disassembly of IFs is dependent on the
phosphorylation of vimentin by the maturation/M-phase–promoting factor at ser-55 in the
amino-terminal head domain. In addition, the disassembly of vimentin IFs during mitosis appears
to be a unique feature of nestin-expressing cell types. Furthermore, when the expression of nestin
is downregulated by the nestin-specific small interfering RNA in nestin-expressing cells, vimentin
IFs remain assembled throughout all stages of mitosis. Previous studies suggest that nonfilamen-
tous vimentin particles are IF precursors and can be transported rapidly between different
cytoplasmic compartments along microtubule tracks. On the basis of these observations, we
speculate that nestin may play a role in the trafficking and distribution of IF proteins and
potentially other cellular factors to daughter cells during progenitor cell division.
Cell division marks a period in the cell cycle during which
both the cytoplasmic and nuclear compartments are disas-
sembled, reorganized, and partitioned into daughter cells. In
vertebrate cells, this process is orchestrated by the three
major cytoskeletal systems: intermediate filaments (IFs), mi-
crotubules, and microfilaments. Although the changes in
organizational states of microtubules and microfilaments are
highly conserved during both the assembly of the mitotic
spindle and the formation of the contractile ring in cytoki-
nesis, the structural changes in cytoplasmic IF networks
appear to be cell- and IF-type specific (Chou et al., 1996). For
example, in mitotic BHK-21 cells, the interphase IF network,
which is composed primarily of vimentin and desmin, is
completely disassembled from 10-nm IFs into nonfilamen-
tous particles in late prophase (Rosevear et al., 1990). In
PtK-2 epithelial cells, both vimentin and keratin IF networks
remain intact during mitosis (Aubin et al., 1980). In HeLa
cells, which also possess keratin and vimentin networks, the
keratin network is disassembled into spheroid bodies,
whereas vimentin remains filamentous (Franke et al., 1982;
Jones et al., 1985). The various organizational fates of cyto-
plasmic IFs in different cell types undergoing mitosis sug-
gest that the biochemical factors regulating their restructur-
ing during mitosis are not identical.
Although the factors involved in regulating the structural
changes of IFs in vivo are not completely understood, pro-
tein phosphorylation is known to play an essential role in
determining the assembly states (Inagaki et al., 1997). In
dividing BHK-21 cells, it has been shown that the disassem-
bly of vimentin IFs is correlated with an elevated phosphor-
ylation of vimentin mediated by two mitotic protein kinases,
maturation/M-phase promoting factor (MPF; p34cdc2/cyclin
B) and p37 kinase (Chou et al., 1990; Tsujimura et al., 1994;
Chou et al., 1996). Whereas MPF phosphorylates vimentin at
Article published online ahead of print. Mol. Biol. Cell 10.1091/
mbc.E02–08–0545. Article and publication date are at www.molbi-
‡Corresponding author. E-mail address: r-goldman@northwestern.
Abbreviations used: IF, intermediate filament; MPF, matura-
tion/M-phase promoting factor.
1468© 2003 by The American Society for Cell Biology
ser-55 and plays an essential role in the disassembly of
vimentin IFs, p37 kinase phosphorylates vimentin at thr-457
and ser-458 and has no apparent impact on the disassembly
of vimentin IFs in mitotic BHK-21 cells (Chou et al., 1996).
The phosphorylation of vimentin by MPF is a universal
feature of mitotic cells expressing vimentin. Yet the break-
down of vimentin networks during mitosis has been re-
ported only in MDBK (Franke et al., 1982), BHK-21 (Rosevear
et al., 1990), and ST15A (Sahlgren et al., 2001) cells. Therefore,
additional factors, unique to these three cell lines, are re-
quired for the disassembly of IFs.
Clues regarding the identity of these cell type–specific
factors come from our recent studies of the high-molecular-
weight proteins present in purified IF preparations of
BHK-21 cells (Steinert et al., 1999). One of these has been
identified as nestin, a protein known to be expressed in
neuroepithelial cells and developing muscle cells (Lendahl et
al., 1990; Sejersen and Lendahl, 1993; Kachinsky et al., 1995;
Vaittinen et al., 1999). Nestin cannot form filaments on its
own, but it can readily form copolymer IFs when combined
with type III IF proteins such as vimentin both in vitro and
in vivo (Marvin et al., 1998; Eliasson et al., 1999; Steinert et al.,
1999). The inability of nestin to form IFs is most likely
because of its very short N-terminus, a domain known to be
essential for IF 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-
dependent manner (Steinert et al., 1999). These observations
have led us to investigate the possible role of nestin in
regulating the structural dynamics of vimentin IFs in vivo.
MATERIALS AND METHODS
Cell Culture and Transfection
Hamster BHK-21 cells were cultured as described previously
(Prahlad et al., 1998). Mouse 3T3 cells were grown in DMEM sup-
plemented with 10% calf serum. Chinese hamster ovary (CHO) and
bovine MDBK cells were grown in Ham’s F12 medium with 10%
fetal calf serum. African green monkey CV-1 cells were grown in
MEM containing 10% fetal calf serum. Rat embryonic fibroblasts
(REFs) were obtained and cultured as described (Goldman, 1998).
Human MCF-7 and rat C6–2 glioma cells were cultured in DMEM
with 10% fetal calf serum. Penicillin and streptomycin (100 U/ml)
were added to all culture media. Rat C6 glioma cells express various
levels of vimentin as determined by immunofluorescence, but the
expression of vimentin and nestin is always coincidental. The C6–2
glioma cells used in this study were derived from a subclone that
expresses both vimentin and nestin.
Transient transfection was performed by electroporation as de-
scribed previously (Prahlad et al., 1998) in OPTI-MEM (R) medium
(Life Technologies/Invitrogen, San Diego, CA) at 0.27 V/960 ?F
(Gene Pulser II, Bio-Rad Laboratories, Hercules, CA). Transfected
cells were allowed to grow for 48–72 h before fixation and staining.
Under our experimental conditions, transfection rates of ?30–50%
and ?10–25%, respectively, were obtained in single and double
transfection assays. In the case of doubly transfected cells, ?3–5%
were mitotic. The numbers in Figure 3M were pooled from three to
Cells were fixed and processed for indirect immunofluorescence as
previously described (Helfand et al., 2002). Images were acquired
using a Zeiss LSM 510 confocal microscope (Carl Zeiss, Inc.,
Oberkochen, Germany). Fluorescein, rhodamine, and toto-3 images
were visualized using excitation at 488, 543, 633 nm and emission at
505–530, 585–615, and ? 670 nm, respectively. Mitotic cells were
identified by both phase contrast and the fluorescence patterns of
condensed chromosomes stained with toto-3 iodide (Molecular
Probes, Eugene, OR). To visualize vimentin, two polyclonal anti-
bodies (Prahlad et al., 1998; Helfand et al., 2002) and one monoclonal
antibody V9 (Sigma Chemical Co., St. Louis, MO) were used. For
following the fate of nestin and immunoblotting, a polyclonal anti-
body (No. 268) raised against purified hamster nestin (Steinert et al.,
1999) was used. This antibody was affinity-purified using Affi-gel-10
beads (Bio-Rad) coupled with two glutathione S-transferase–nestin-
tail fusion proteins described below. Two monoclonal antibodies,
411C for hamster nestin (Yang et al., 1992) and 401 for rat nestin (BD
PharMingen, San Diego, CA), were also used.
Cloning and Expression of Nestin
The complete rat nestin cDNA was amplified by RT-PCR with total
C6 glioma cell RNA as template using the Thermoscript RT-PCR
and Elongase systems (Invitrogen). Sense and antisense primers
were made corresponding to the starting and the ending sequences
of the open reading frame of the published rat nestin cDNA (acces-
sion No. m34384). Primers were designed to create two different sets
of unique restriction sites at the 5? and the 3? ends of the amplified
cDNA to facilitate the subsequent cloning into either mammalian or
bacterial expression vectors. For expression in bacteria, the nestin
cDNA was cloned into the pET-24 vector (Novagen, Madison, WI)
between the NdeI and EcoRI sites. For expression in cultured mam-
malian cells, the nestin cDNA was cloned into the pCMV-myc
vector (Clontech Laboratories, Cambridge, UK) between the SalI
and NotI sites. The nestin cDNA (accession No. AF538924) amplified
from rat C6 glioma cells is longer than the published sequence and
is predicted to encode a protein of 1893 amino acids, which is 88
amino acids longer than those predicted from the published rat
sequence (Lendahl et al., 1990). This additional sequence is located
in the 11-amino-acid repeat region of the C-terminal domain. The
size of the C6 nestin cDNA does not appear to be the result of
mispriming during RT-PCR amplification or rearrangements during
cloning. PCR reactions using genomic C6 cell DNA as template and
two different pairs of primers corresponding to the flanking se-
quences of the repeat region produced the expected size of the
repeat region (i.e., 49 ? 33 base pairs; data not shown).
The C-terminal 1350 amino acids of hamster nestin (accession
No. af110498) were cloned as two separate nestin tail fragments
(NT334–949and NT950–1683) into pGEX vector (Pharmacia/Amer-
sham Biosciences, Arlington Heights, IL) between the EcoRI and
SalI sites and expressed as glutathione fusion proteins. These
fusion proteins were expressed in BL-21 bacterial cells (Strat-
Sepharose 4B column according to the manufacturer’s protocol
(Pharmacia). These purified fusion proteins were used for anti-
body purification mentioned above.
Small Interfering RNA Studies
A 21 nucleotide sequence (AAG AUG UCC CUU AGU CUG GAG)
which is conserved in rat and hamster nestin (residues 299–319,
accession No. af110498) was selected for synthesizing the small
interfering RNA (siRNA) duplex with two 2?-deoxythymidine over-
hangs at the 3? ends. This nestin siRNA duplex as well as the
negative control luciferase–GL2 siRNA duplex (Harborth et al.,
2001) were purchased from Dharmacon (Lafayette, CO). Transient
transfection of siRNA-nestin into BHK-21 and C6–2 cells was per-
formed exactly as described previously (Harborth et al., 2001), and
the effects of siRNAs were examined 60 h after transfection.
Function of Nestin in Mitosis
Vol. 14, April 20031469
Immunoblotting and Two-Dimensional Gel
IF-enriched cytoskeletal fractions were prepared according to estab-
lished procedures (Prahlad et al., 1998; Steinert et al., 1999). Then 2.5
?g of each sample was used for immunoblotting analyses. The same
amount of the BL-21 cell lysates expressing the full-length rat nestin
was used as a positive control. Because of the strong reactivity of the
antibody toward the hamster nestin, the amount of the BHK-21 IF
sample was reduced to 0.5 ?g. For two-dimensional gel electro-
phoresis studies, 5 ?g of each of the IF-enriched samples was used,
and 0.5 ?g of bacterially expressed tailless vimentin (Correia et al.,
1999) (a gift of Brian Helfand) was included in samples and served
as a reference mobility marker for unphosphorylated vimentin and
its acidic variants. Isoelectric focusing and SDS-PAGE were per-
formed using a published protocol (O’Farrell, 1975). Mitotic cells
were enriched by treating untransfected or transfected CHO cells
with 2 ?M of nocodazole for 4 h and then collected by mechanical
shake-off as described previously (Chou et al., 1989).
Nestin Expression Affects the Assembly State of
Vimentin in Mitotic Cells
To begin to determine whether nestin plays a role in the
organization of IF networks, CHO cells that express vimen-
tin but not nestin (see Figure 1, A and B, and 4A) were used
to study the effects of nestin expression. CHO cells were
transiently transfected with an expression vector carrying
the rat nestin cDNA. As is shown in Figure 1, C–E, ectopi-
cally expressed nestin colocalized with vimentin in a fila-
mentous pattern, suggesting that it was incorporated into
the vimentin IF network in interphase CHO cells. The ex-
pression of nestin did not appear to alter the overall distri-
bution or the assembly state of endogenous IFs. In contrast,
nestin expression caused significant changes in the organi-
zation of the vimentin IF network in mitotic cells. In untrans-
fected mitotic cells, vimentin remained filamentous, and it
formed a cage-like structure surrounding the mitotic appa-
ratus during all stages of mitosis (Aubin et al., 1980; Zieve et
al., 1980). Polymerized IFs persisted through mid to late
cytokinesis (Figure 2D). In contrast, vimentin IFs in mitotic
cells expressing nestin were disassembled, as indicated by
the transformation from a filamentous into a punctate and
diffuse pattern of fluorescence (Figure 2, E and F). In addi-
tion, the majority of the vimentin and nestin patterns in
these cells appeared to be coincident (Figure 2G). This dis-
assembled vimentin pattern persisted throughout all mitotic
stages until the completion of cytokinesis, when cells began
to reassemble their typical interphase IF networks (data not
shown; see Figure 1, A and C, for examples of interphase
Nestin-Mediated Disassembly of Vimentin IFs
Depends on the MPF-specific Phosphorylation of the
N-Terminal Domain of Vimentin at ser-55
Because an elevated phosphorylation level is the major bio-
chemical modification of vimentin that has been character-
ized during the interphase–mitosis transition, studies were
performed to determine whether vimentin phosphorylation
is required for the nestin-mediated disassembly of IFs dur-
ing mitosis. To approach this, we performed transient trans-
fection assays in MCF-7 cells, a cell line that expresses ker-
atin IFs but is devoid of vimentin. This cell type allowed us
to assemble a vimentin network solely from wild-type (WT)
or either one of the two mitotic phosphorylation mutant
vimentins (Chou et al., 1996). When these proteins were
expressed individually, each formed filamentous IF net-
works in interphase MCF-7 cells that were indistinguishable
from each other (for example, see Figure 3A, and data not
terphase CHO cells. In untrans-
fected interphase cells, vimentin
(VIM, red) is seen in a typical fila-
mentous pattern (A). These cells
express no detectable nestin (NES,
green) after staining with nestin
antibody (B). When nestin is ex-
pressed in CHO cells, it is incorpo-
rated into the endogenous vimen-
tin IF network with no apparent
impact on its assembly state or or-
ganization (C–E). Bar, 10 ?m.
Nestin expression in in-
Y.-H. Chou et al.
Molecular Biology of the Cell 1470
shown). During mitosis, in each case, dense arrays of vimen-
tin IFs surrounded the mitotic apparatus (see Figure 3, D–F).
The fraction of cells that gave filamentous patterns was
?84% (n ? 51) for WT-vimentin, ?90% (n ? 48) for ser-55:
ala-vimentin, and ?94% (n ? 33) for thr-457:ala/ser-458:ala
vimentin. These observations are consistent with the idea
that elevated phosphorylation alone is not sufficient to cause
the disassembly of vimentin IFs during mitosis.
When nestin was coexpressed with either the WT or one
of the two vimentin mutants in MCF-7 cells, nestin and
vimentin colocalized in indistinguishable filamentous net-
works in interphase cells (for example, see Figure 3, B and
C). In mitotic cells, however, the structural changes associ-
ated with the nestin/WT-vimentin and the nestin/thr-457:
ala/ser-458:ala-vimentin networks were significantly differ-
ent from those associated with the nestin/ser-55:ala-
vimentin networks (Figure 3, G–L). The filamentous
networks of the nestin/WT-vimentin (Figure 3, G and J)
were disassembled in the majority (?79%, n ? 43) of doubly
transfected cells. A similar result was observed in mitotic
cells expressing both nestin and thr-457:ala/ser-458:ala-vi-
mentin (Figure 3, I and L; ?97%, n ? 35). In contrast, IF
networks formed from the nestin and ser-55:ala-vimentin
remained largely intact (Figure 3, H and K; ?90%, n ? 42).
Taken together, these observations suggest that the disas-
sembly of vimentin IFs during mitosis requires both the
presence of nestin and phosphorylation of vimentin at the
MPF-specific site, ser-55.
IF Breakdown During Mitosis Is a General Feature
of Cells Expressing Nestin
The conversion of vimentin IFs into nonfilamentous struc-
tures during mitosis has been reported in BHK-21, ST15A,
and MDBK cells. Furthermore, it has been demonstrated
that nestin is expressed in BHK-21 (Steinert et al., 1999) and
ST15A (Sahlgren et al., 2001) cells. This led us to examine
whether MDBK cells also express nestin and whether there
is a correlation between the expression of nestin and the
disassembly of mitotic vimentin networks in other cultured
cell types. We first screened, by immunoblotting with an
affinity-purified polyclonal nestin antibody (No. 268), a
number of commonly used cell lines. Bacterially expressed
full-length rat nestin was used as a positive control. Among
the seven cell lines screened, the strongest nestin reaction
was detected in IF-enriched preparations obtained from
BHK-21 cells (Figure 4A) (note: the amount of the BHK IF
protein sample loaded for this blotting assay was only 20%
of that used for other samples). C6–2 glioma cell IF prepa-
rations gave the second strongest reaction, whereas a rela-
tively weaker signal was obtained in IF samples prepared
from MDBK cells. A very faint band in the REF sample could
also be detected after longer exposure. In all positive immu-
noblots, the antibodies recognized primarily a doublet in the
240- to 280-kDa range, which is consistent with the sizes of
nestin predicted from published cDNA sequences (Dahl-
strand et al., 1992; Steinert et al., 1999; Yang et al., 2001) (also
see Materials and Methods).
the mitotic spindle. The relationship between vimentin IFs (red) and spindle tubulin microtubules (green) in an untransfected mitotic cell is
shown in three consecutive optical sections (A–C). The vimentin IFs persist into mid to late cytokinesis (D). In mitotic cells expressing nestin,
the endogenous vimentin IFs are disassembled and vimentin (red) and nestin (green) are extensively colocalized in punctate and diffuse
structures (E–G). Mitotic cells were identified by the presence of condensed chromosomes (blue). Bar, 5 ?m.
Nestin expression in mitotic CHO cells. During mitosis, vimentin IFs remain intact and form a filamentous network surrounding
Function of Nestin in Mitosis
Vol. 14, April 2003 1471
The cell lines expressing nestin were also examined by
immunofluorescence. Double labeling of C6–2 glioma and
BHK-21 cells showed that nestin expression could be de-
tected in the majority (?95%) of the cells and that there
was extensive coincidence of the vimentin and nestin
staining patterns during interphase. During mitosis, the
majority of these filamentous networks were disassem-
bled into nonfilamentous structures that in C6–2 cells
appeared as punctate structures surrounded by more dif-
fuse staining (Figure 4, B and C). This latter morpholog-
ical feature is very similar to the one seen in ST15A cells,
which express a similar level of nestin (Sahlgren et al.,
2001). In mitotic BHK-21 cells, the disassembled vimentin
displayed a distinct punctate pattern (Figure 6, G–I).
When the fate of IFs in mitotic REF, 3T3, CV-1, and CHO
cells was examined, the majority of them appeared to be
tin at ser-55 is required for the nestin-
mediated disassembly of IFs during
mitosis. Wild-type vimentin (WT) or
either the ser-55:ala (S55A) or thr-457:
ala/ser-458:ala (S458A) mutant vi-
mentins were transfected individually
or together with nestin in MCF-7 cells.
Subsequently, the assembly state of
vimentin was examined by indirect
immunofluorescence with a rabbit an-
tibody directed against vimentin (red)
and a monoclonal antibody directed
against rat nestin (NES, green). In in-
terphase cells, typical vimentin IF net-
works assembled from WT vimentin
alone (A) or from WT vimentin and
nestin as demonstrated by double-la-
bel immunofluorescence (B and C).
During mitosis, all three forms of vi-
mentin, when expressed individually,
retained their filamentous networks
(D–F). However, when vimentin was
coexpressed with nestin, IFs formed
with WT vimentin/nestin (G and J) or
tin/nestin (I and L) were disassem-
bled into punctate and diffuse struc-
tures, whereas IFs assembled with
intact (H and K). Images G and J, H
and K, and I and L are pairs of the
same cells using double immunofluo-
rescence. Mitotic cells were identified
by the presence of condensed chromo-
somes (blue). (M) Summary table of
the quantitative results of singly and
doubly transfected mitotic MCF-7
cells. Bar in interphase (A–C) and mi-
totic (D–L) cells, 10 ?m.
Phosphorylation of vimen-
Y.-H. Chou et al.
Molecular Biology of the Cell1472
filamentous (see Figure 2, A–C, and Figure 4, D–G, for
examples of CHO, 3T3, and CV-1 cells).
When MDBK cells were examined by immunofluores-
cence, the nestin signals detected in these cell lines were
different from those of BHK-21 and C6–2 cells. Fewer cells
(?63%, n ? 553) were fluorescent (Figure 5, A and B), and
the intensity of the fluorescence was variable among the
nestin-positive cells. The organizational fate of vimentin IF
networks in mitotic MDBK cells also appeared to be heter-
ogeneous. In all of the mitotic cells that lacked nestin and in
almost half of the mitotic cells that appeared to express low
levels of nestin, the vimentin networks remained filamen-
tous (Figure 5, E and F). However, in ?30% (n ? 58) of the
mitotic cells, vimentin IF networks appeared to be frag-
mented, with some punctate vimentin structures (Figure 5,
C and D). In general, this latter phenotype was correlated
with cells expressing higher levels of nestin, as indicated by
the intensity of fluorescence. Together, these results suggest
that the various filamentous states of vimentin seen during
mitosis can be correlated qualitatively with different expres-
sion levels of nestin in different cells. These results provide
further support for the idea that nestin expression is causally
linked to the mechanism regulating the extent of vimentin IF
disassembly during mitosis.
blotting analysis of IF-enriched cytoskeletal samples from seven
cultured cell lines with an affinity-purified polyclonal nestin anti-
body (see Materials and Methods). In cell types such as C6–2,
vimentin (VIM, red) and nestin (NES, green) are present in a non-
filamentous punctate pattern during mitosis (B and C). The mitotic
state (metaphase) of this cell is indicated by the presence of con-
densed chromosomes (blue). In cell types such as 3T3 (D and E) and
CV-1 (F and G) that do not express nestin, the vimentin IFs remain
filamentous, as shown in an anaphase 3T3 cell (D and E) and a
metaphase/early anaphase CV-1 cell (F and G). Bar for B–E, F, and
G, 5 ?m.
Nestin expression in cultured cell types. (A) Immuno-
MDBK cells. (A and B) All cells display a filamentous vimentin
pattern (VIM, red), but fewer are positive for nestin (NES, green), as
determined by double indirect immunofluorescence. During mito-
sis, cells exhibiting brighter nestin fluorescence contain a rather
punctate vimentin and nestin pattern (C and D), whereas cells
expressing no or low levels of nestin have a filamentous vimentin
network (E and F). Mitotic chromosomes are stained with toto-3
(blue). Bar, 10 ?m.
Nestin expression and the assembly state of vimentin in
Function of Nestin in Mitosis
Vol. 14, April 2003 1473
Downregulation of Nestin Expression Blocks
Vimentin IF Network Breakdown during Mitosis
It was recently demonstrated that double-stranded and se-
quence-specific siRNAs could effectively downregulate the ex-
pression of specific genes in cultured cells (Elbashir et al., 2001;
Harborth et al., 2001). We therefore designed one 21-nucleo-
tide-long RNA duplex corresponding to sequences shared by
both rat and hamster, and the siRNA was then introduced into
BHK-21 or C6–2 cells to study the effects on the assembly state
of vimentin IFs. As shown in Figure 6, D–F, this treatment
resulted in a significant loss of nestin signal from ? 50% of the
BHK-21 cells. The specificity of these reagents was shown by
immunoblotting analysis of siRNA-treated cells. Although the
expression level of nestin was significantly reduced in cells
treated with nestin siRNA, its expression was not affected in
the luciferase GL2 siRNA-treated cells (Figure 6L). Further-
more, the expression levels of vimentin under these conditions
appeared to be the same. By immunofluorescence, the partial
or complete loss of nestin did not appear to change the orga-
nization or the assembly state of vimentin IFs during inter-
phase (compare Figure 6, A–C and D–F). In contrast, during
mitosis, the reduction of nestin by siRNA was accompanied by
a concurrent change in vimentin IF organization from a punc-
tate to a filamentous pattern (compare Figure 6, G–I and J and
K). Similar results were obtained in siRNA-treated C6–2 gli-
oma cells (data not shown).
vents the disassembly of vimentin IFs
in mitotic BHK-21 cells. Interphase
cells display filamentous vimentin
(VIM, red) and nestin (NES, green)
networks that are very similar to each
other (A–C, double-label and merged
images). After cells were treated with
nestin siRNA, nestin fluorescence was
greatly reduced or nondetectable in
many cells. Reduction of nestin ex-
pression had no detectable effect on
the expression or the organization of
endogenous vimentin (D–F, double-
label and merged images). Untreated
mitotic cells had both vimentin and
nestin in a punctate pattern (G–I). In
mitotic cells, in which the nestin sig-
nal was greatly reduced by nestin
siRNA, the vimentin remained in a
filamentous pattern (J and K). The mi-
totic state of the cells was identified
by condensed chromosomes in blue.
(L) Immunoblotting analysis of vi-
mentin and nestin in cell lysates de-
rived from cells untreated (?) or
siRNA (GL2) or nestin siRNA (NES).
Bar, 10 ?m.
Nestin-specific siRNA pre-
Y.-H. Chou et al.
Molecular Biology of the Cell1474
Ectopic Expression of Nestin Has No Effect on the
Basal Phosphorylation Level of Vimentin
Because the incorporation of nestin into vimentin IFs could
potentially change the structural properties of IFs and
thereby modulate vimentin phosphorylation, we performed
two-dimensional gel electrophoresis analyses to determine
whether the basal level of vimentin phosphorylation was
affected by the expression of nestin. This approach has been
used previously in evaluating the phosphorylation state of
vimentin (Evans and Fink, 1982; Celis et al., 1983). As shown
in Figure 7A, vimentin derived from untransfected inter-
phase cells migrated as two distinct spots, a major/unphos-
phorylated vimentin (V, open circle) and a minor acidic/
phosphorylated variant (closed circle). Conversely, vimentin
prepared from untransfected mitotic cells was resolved into
three distinct spots, a minor/unphosphorylated vimentin
(Figure 7B, open circle) and two more prominent acidic/
phosphorylated variants (Figure 7B, closed circles). When
samples from nestin-transfected cells were compared with
those derived from untransfected cells, the relative abun-
dance of the unphosphorylated vimentin and its acidic vari-
ants was not obviously altered (compare Figure 7, A and B,
with C and D). We also performed similar two-dimensional
gel analyses with samples prepared from untreated and
nestin siRNA–treated BHK-21 cells. No detectable difference
in phosphorylation levels was observed between these two
sets of samples (data not shown).
These results are consistent with our previous observation
that vimentin is only slightly phosphorylated at ?0.3 mol
Pi/mol protein during interphase, and the level of phos-
phorylation is elevated approximately sixfold to ?1.9 mol
Pi/mol protein when cells enter mitosis (Chou et al., 1989).
The single acidic vimentin variant associated with the inter-
phase sample is probably a result of the partial phosphory-
lation of one of many previously identified interphase-spe-
cific sites (Inagaki et al., 1997). The two acidic vimentin
variants seen in mitotic samples can be accounted for by the
two major phosphorylation sites at ser-55 and ser-458 (Chou
et al., 1996). Together, our results suggest that the presence
or absence of nestin does not significantly alter the phos-
phorylation state of vimentin. However, the transition from
interphase to mitosis appears to be the major contributing
factor in elevating the phosphorylation state of vimentin.
Although it has been known for many years that there are
significant differences in the assembly states of vimentin IFs
during mitosis in different cell types, the mechanisms re-
sponsible for these variations are not understood. The phos-
phorylation of vimentin at ser-55 by the ubiquitous mitotic
kinase MPF has been shown to be essential for the disassem-
bly of vimentin IFs during mitosis (Chou et al., 1996). Fur-
thermore, this site has been conserved during vertebrate
evolution (Herrmann et al., 1996b). However, the persistence
of vimentin IFs in many other cell types (Aubin et al., 1980;
Jones et al., 1985) during mitosis suggests that the phosphor-
ylation of vimentin by MPF alone is insufficient to disassem-
ble IF networks. The results of this study identify nestin as a
modulator of IF structure during mitosis. Furthermore, the
results show that nestin works in concert with MPF to
induce the disassembly of vimentin IFs during mitosis.
Several lines of evidence support the synergistic roles of
nestin and the specific phosphorylation of vimentin by MPF
to achieve an extensive disassembly of IFs during mitosis.
For example, the ectopic expression of nestin in vimentin?/
nestin? cell types, such as CHO cells, promotes the disas-
sembly of vimentin IFs during mitosis, whereas there are no
apparent effects on the organization of interphase IF net-
works. In addition, this nestin-mediated disassembly of IF
networks is dependent on vimentin phosphorylation at ser-
55, the MPF-specific site. Furthermore, the nestin-facilitated
disassembly of IFs in mitosis is blocked by the siRNA-
mediated downregulation of nestin expression. Finally, we
show that the nestin-mediated disassembly of vimentin IFs
during mitosis is a general feature of cell types expressing
sufficient amounts of nestin.
The molecular basis of how nestin and vimentin phos-
phorylation by MPF work synergistically to disassemble the
copolymers of vimentin and nestin is unknown. Previous
studies indicate that nestin and vimentin can form het-
erodimers and heterotetramers in vitro. It has also been
demonstrated in vitro that nestin-vimentin heterodimers
and heterotetramers are less stable than similar oligomers
formed by vimentin alone when subjected to increasing
concentrations of urea (Steinert et al., 1999). This latter ob-
servation may be partly because nestin has only a short
amino-terminal head domain. In the case of vimentin ho-
mopolymer IFs, it is known that the head domain plays an
lation state of endogenous vimentin. IF-enriched preparations from
untransfected (A and B) and nestin-transfected (38% transfection
rate; C and D) CHO cells were analyzed by two-dimensional gel
electrophoresis, and the phosphorylation states of vimentin were
determined by the relative abundance of unphosphorylated and
phosphorylated (more acidic) electrophoretic variants. In interphase
samples (A and C), vimentin is largely unphosphorylated (marked
as V, open circle), and only a small fraction of it exits as a more
acidic variant (closed circle). However, when cells enter mitosis, the
phosphorylation state of vimentin changes significantly (B and D),
with the majority of the vimentin present as two more acidic vari-
ants (closed circles), and only a small fraction of it remains unphos-
phorylated (open circle). The positions of the endogenous actin (A),
which is frequently associated with crude IF preparations, and the
exogenously added ?C-vimentin (?C) were used as mobility refer-
The expression of nestin does not affect the phosphory-
Function of Nestin in Mitosis
Vol. 14, April 20031475
important role in dimer-dimer interaction (Herrmann et al.,
1996a). Therefore, the presence of heterodimers containing
only one full-length head (i.e., that of vimentin) may lead to
the formation of less stable though still long IFs. As a con-
sequence, it is conceivable that the phosphorylation of the
vimentin head domain in a vimentin/nestin heteropolymer
would generate a dimer or tetramer unable to sustain inter-
actions essential for maintaining the structural integrity of
IFs, thereby leading to the fragmentation of filaments and
Vimentin is phosphorylated throughout the cell cycle, but
the phosphorylation level is significantly higher during mi-
tosis. The apparently normal IF networks seen during inter-
phase in nestin-expressing cells suggest that a higher level of
phosphorylation is required for nestin to exert its effect on
vimentin structure, and this level of phosphorylation may be
achieved only when cells enter mitosis (Chou et al., 1989).
Consistent with this idea is the observation that interphase
vimentin IFs in BHK-21 cells are rapidly disassembled when
cells are exposed to low doses of the phosphatase inhibitor
calyculin A, which elevates the phosphorylation level of
vimentin (Eriksson et al., 1992). Furthermore, phosphoryla-
tion of nestin by MPF during mitosis has also been corre-
lated with the reorganization of vimentin IFs in ST15A cells
(Sahlgren et al., 2001). Although it has not been explored in
this study, the phosphorylation of nestin may also play a
role in the extent of disassembly of vimentin IFs during
mitosis. Finally, the persistence of vimentin IFs during mi-
tosis in some of the nestin? MDBK cells described in this
study suggests that a critical ratio of nestin/vimentin is
required for nestin to exert the above-described effects on
vimentin IF assembly states. This latter suggestion is sup-
ported by the dose-dependent effects of nestin on the disas-
sembly of vimentin filaments in vitro (Steinert et al., 1999).
Like those of vimentin, the phosphorylation levels of the
keratins are elevated in mitosis, and the organizational fates
of keratin IF networks vary from one cell type to another
(Horwitz et al., 1981; Franke et al., 1982; Lane et al., 1982;
Celis et al., 1983; Ku and Omary, 1994). It is therefore likely
that variations in the disassembly of keratin IFs observed in
different types of epithelial cells are also regulated by keratin
phosphorylation, along with the expression of unique kera-
tin-associated proteins (Fuchs and Karakesisoglou, 2001;
Leung et al., 2002). Because nestin does not appear to coas-
semble with keratin IFs in epithelial cell types such as those
used in this study (MCF-7, MDBK), proteins other than
nestin are most likely responsive for their disassembly. A
number of other IF proteins, such as synemin (Bellin et al.,
1999), paranemin (Hemken et al., 1997), and syncoilin
(Newey et al., 2001), exhibit some properties similar to those
of nestin, because they cannot assemble into IFs on their
own. Each of these proteins requires a type III IF protein for
its assembly into IFs (Schweitzer et al., 2001). Therefore, they
could potentially function like nestin, regulating the dy-
namic properties or assembly states of IF networks in dif-
ferent cells and tissues as well as in different stages of
The disassembly of vimentin IFs during mitosis is obvi-
ously not required for mitosis per se, because many cell
types do not express nestin. Furthermore, phosphorylation
at ser-55 is not essential for the distribution of vimentin IFs
to daughter cells (Yasui et al., 2001). In mitotic cells in which
there is no obvious disassembly of vimentin IFs, the parti-
tioning of IFs into daughter cells is facilitated by a highly
localized phosphorylation and disassembly of IFs restricted
to the cleavage furrow in late cytokinesis. This involves
phosphorylation of vimentin at multiple specific sites by
C-kinase, rho-kinase, and an unidentified protein kinase.
Mutation of these sites produces an abnormally long IF-
enriched bridge between daughter cells (Goto et al., 2000;
Yasui et al., 2001).
The potential benefit of the mitotic disassembly of vimen-
tin IFs for cells expressing nestin remains an open question.
However, nonfilamentous vimentin in the form of particles
has also been observed in spreading interphase cells. These
particles move at high speeds along microtubules because of
their association with the molecular motors such as kinesin
and dynein (Prahlad et al., 1998; Helfand et al., 2002). Motile
and nonfilamentous keratin structures have also been re-
ported in mitotic epithelial cells (Windoffer and Leube,
1999). One of the suggested functions for the fast-moving
nonfilamentous IF structures is to provide a rapid transit
system to move IF precursors between various cytoplasmic
compartments. In light of the unusually long C-terminus
(?1200 amino acids) of nestin, which is likely to interact
with other cellular factors, nonfilamentous IF particles could
potentially carry other “cargoes” in their journey. Therefore,
the expression of nestin may be associated with an increase
in cytoplasmic trafficking required for progenitor cells un-
dergoing rapid rounds of division, interspersed with active
interphase migration. These activities are hallmarks of the
nestin-expressing cells found in early developing nerve and
muscle systems (Lendahl et al., 1990; Sejersen and Lendahl,
1993; Kachinsky et al., 1995; Vaittinen et al., 1999) and in cells
responding to the injury and regeneration of adult tissues
(Frisen et al., 1995; Vaittinen et al., 1999).
From the developmental perspective, the nestin-express-
ing progenitor or stem cells of the early developing neural
tube are organized in a polarized manner between the inner
ventricular edge and the outer pial surface. As the cell
number increases, proliferation is confined primarily to the
ventricular zone, whereas postmitotic differentiating neu-
rons migrate toward the pial surface (Frederiksen and
McKay, 1988; Rakic, 1988). The polarized distribution of the
dividing and differentiating cells within the neuroepithe-
lium may be caused by the uneven partitioning of key
cellular components during cell divisions of the progenitor
cells. In this regard, the nestin-mediated disassembly of IFs
and the motility of vimentin particles during mitosis could
also take part in the asymmetric allocation of cytoskeletal
and other cellular factors to daughter cells.
This study was supported by a MERIT grant from the National
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