genome wide assessment of mrna in astrocyte protrusions by direct rna sequencing reveals mrna...
Post on 07-Apr-2017
213 Views
Preview:
TRANSCRIPT
RESEARCH ARTICLE
Genome Wide Assessment of mRNA inAstrocyte Protrusions by Direct RNA
Sequencing Reveals mRNA Localization forthe Intermediate Filament Protein Nestin
Rune Thomsen,1 Jonatan Pallesen,1,2,3 Tina F. Daugaard,1
Anders D. B�rglum,1,2,3,4 and Anders L. Nielsen1,2,3
Subcellular RNA localization plays an important role in development, cell differentiation, and cell migration. For a comprehen-sive description of the population of protrusion localized mRNAs in astrocytes we separated protrusions from cell bodies in aBoyden chamber and performed high-throughput direct RNA sequencing. The mRNAs with localization in astrocyte protru-sions encode proteins belonging to a variety of functional groups indicating involvement of RNA localization for a palette ofcellular functions. The mRNA encoding the intermediate filament protein Nestin was among the identified mRNAs. By RT-qPCR and RNA FISH analysis we confirmed Nestin mRNA localization in cell protrusions and also protrusion localization ofNestin protein. Nestin mRNA localization was dependent of Fragile X mental retardation syndrome proteins Fmrp and Fxr1,and the Nestin 3’-UTR was sufficient to mediate protrusion mRNA localization. The mRNAs for two other intermediate fila-ment proteins in astrocytes, Gfap and Vimentin, have moderate and no protrusion localization, respectively, showing that indi-vidual intermediate filament components have different localization mechanisms. The correlated localization of Nestin mRNAwith Nestin protein in cell protrusions indicates the presence of a regulatory mechanism at the mRNA localization level forthe Nestin intermediate filament protein with potential importance for astrocyte functions during brain development andmaintenance.
GLIA 2013;61:1922–1937Key words: intermediate filaments, Nestin, Gfap, Vimentin, cytoskeleton, glia
Introduction
Astrocytes constitute the most abundant cell type in the
CNS (Allen and Barres, 2009; Freeman, 2010; Sofro-
niew and Vinters, 2010). Astrocytes typically exhibit a highly
polarized morphology extending multiple pseudopodial pro-
trusions participating in (i) establishing scaffolds for crawling
neurons during CNS development, (ii) establishing a part of
the gliovascular structure and a part of the blood-brain-
barrier, and (iii) mediating interactions with synapses aiding
in optimal neuronal signal transduction (Allen and Barres,
2009; Morest and Silver, 2003; Sofroniew and Vinters,
2010). Many studies have been carried out in order to under-
stand the dynamics of astrocyte protrusions. During cell dif-
ferentiation and migration the astrocyte morphology changes
dramatically by formation of pseudopodial protrusions driven
by coordinated polymerization and depolymerization of the
cytoskeleton (Etienne-Manneville, 2004). The mammalian
cytoskeleton consists of three types of filaments: actin fila-
ments, microtubules, and intermediate filaments (IFs). The
highly diverse family of IF proteins are encoded by �70
genes, and the complexity of the IF family is increased by
generation of multiple protein isoforms (Herrmann et al.,
2009). Proteins of the IF family are subdivided into different
classes due to sequence homology and capability to
View this article online at wileyonlinelibrary.com. DOI: 10.1002/glia.22569
Published online September 5, 2013 in Wiley Online Library (wileyonlinelibrary.com). Received Jan 9, 2013, Accepted for publication Aug 5, 2013.
Address correspondence to Anders Lade Nielsen, Department of Biomedicine, Aarhus University, DK-8000 Aarhus C, Denmark. E-mail: aln@hum-gen.au.dk
From the 1Department of Biomedicine, Aarhus University, Aarhus, Denmark; 2Center for Integrative Sequencing, iSEQ, Department of Biomedicine, Aarhus
University, Aarhus, Denmark; 3Lundbeck Foundation Initiative for Integrative Psychiatric Research, iPSYCH, Department of Biomedicine, Aarhus University, Aarhus,
Denmark; 4Center for Psychiatric Research, Aarhus University Hospital, Aarhus, Denmark.
Additional Supporting Information may be found in the online version of this article.
1922 VC 2013 The Authors. GLIA published by Wiley Periodicals, Inc.This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in anymedium, provided the original work is properly cited.
co-assemble into IFs (Eriksson et al., 2009; Herrmann et al.,
2009). IF proteins have a common overall structure com-
posed of an amino-terminal head domain and a carboxy-
terminal tail domain linked together by a highly coiled
a-helical rod domain. The rod domain is highly conserved
among IF proteins whereas the head and tail domains exhibit
a large degree of variability (Eriksson et al., 2009; Herrmann
et al., 2009; Middeldorp and Hol, 2011). The coiled a-
helical rod domain mediates generation of parallel homo-
dimers, and the homo-dimers form anti-parallel tetramers
that are linked head to tail. Eight tetramers associate into
unit-length filaments by which the mature 7- to 11-nm thick
IFs are comprised (Herrmann et al., 2009). Apart from pro-
viding static mechanical support to the cell structure, IF pro-
teins are also involved in dynamic reorganization of the cell
morphology during growth and migration (Eriksson et al.,
2009; Michalczyk and Ziman, 2005).
Astrocytes of the mammalian brain express the IF pro-
teins Nestin, Vimentin, and Glial fibrillary acidic protein
(Gfap) (Middeldorp and Hol, 2011). Nestin is considered a
marker for undifferentiated progenitor cells, down regulated
in terminally differentiated cells, but become reactivated dur-
ing injury responses in a process termed reactive gliosis char-
acterized by cell hypertrophy and proliferation (Gilyarov,
2008). The Nestin protein has a short amino-terminal head
domain of only six amino acids (Herrmann et al., 2009).
Nestin is unable to form homomeric filaments but coassem-
bles with Vimentin and Gfap (Eliasson et al., 1999; Herr-
mann et al., 2009; Michalczyk and Ziman, 2005). Nestin is
believed to play a pivotal role in cell morphology changes
during mitosis through the dynamic assembly and disassem-
bly of Vimentin including IFs (Chou et al., 2003). Vimentin,
like Nestin, is expressed early during brain development.
Vimentin and Nestin becomes gradually replaced by Gfap in
terminally differentiated astrocytes but Gfap expression has
also been described in neuronal and astrocyte progenitor cells
(Doetsch et al., 1999; Imura et al., 2006; Michalczyk and
Ziman, 2005; Middeldorp and Hol, 2011; Middeldorp et al.,
2010; Zhu and Dahlstrom, 2007). Gfap expression is upregu-
lated along with Nestin and Vimentin during reactive gliosis
after CNS injury (Pekny et al., 2007). Gfap mRNA is alterna-
tive spliced to generate protein isoforms whereas alternative
splicing generating protein isoforms of Nestin and Vimentin
is not yet described, but alternative Vimentin mRNA splicing
exists (Blechingberg et al., 2007a; Condorelli et al., 1999;
Middeldorp and Hol, 2011; Nielsen and Jorgensen, 2004;
Nielsen et al., 2002; Quinlan et al., 2007; Zhou et al.,
2010). Attention is emerging to understand how IFs are regu-
lated during cell growth, migration and morphology changes.
RNA localization in cell protrusions was demonstrated
in a considerable number of cell types including oocytes,
crawling fibroblasts and in axons and dendrites of neurons
(Doyle and Kiebler, 2011; Mili and Macara, 2009). RNA
localization is important for development, cell differentia-
tion and cell functionality (Mili and Macara, 2009). A lim-
ited number of studies have described IF mRNA
localization in astrocytes. One study demonstrated subcellu-
lar localization of the Nestin mRNA in protrusions of radial
glia cells (Dahlstrand et al., 1995). Gfap mRNA localization
was demonstrated in the branch points and distal parts of
astrocyte protrusions (Landry et al., 1994; Medrano and
Steward, 2001). However, detailed studies of the subcellular
mRNA localization patterns in astrocytes remains to be con-
ducted. Comprehensive examinations of RNA molecules
localized in fibroblast, neuronal and cancer cell protrusions
were performed by microarray analysis of RNA purified
from pseudopodial cell protrusions separated from cell
bodies using the Boyden chamber assay (Feltrin et al.,
2012; Mili et al., 2008; Shankar et al., 2010). In this study
we took advantage of a recently presented high throughput
next generation sequencing (NGS) method, termed single
molecule direct RNA sequencing (DRS) (Ozsolak et al.,
2009). DRS analyses were applied on mouse astrocyte RNA
from protrusions and cell bodies isolated using a modified
Boyden chamber assay. We identified numerous mRNAs
with localization to cell protrusions including NestinmRNA. We further analyzed Nestin mRNA and protein
localization in astrocyte protrusions and the presented
results indicate that the function of Nestin in reorganization
of astrocyte morphology can be regulated through coordi-
nated mRNA and protein localization.
Materials and Methods
Primary Cells, Cell Lines, and Brain TissuePrimary astrocytes cultures were prepared as described (Andres-
Barquin et al., 1994; Thomsen and Lade Nielsen, 2011). A pregnant
mouse of the NMRI mouse strain was purchased from Taconic,
Denmark and astrocytes were isolated from the cerebral cortex of
newborn (P0) mice. After 8 days in vitro (DIV) cells showed a
95% confluence and >80% of the cell were staining positive for
the astrocyte marker Gfap. Before experimental procedure cells
were trypsinized using 0.5% trypsin-EDTA (GIBCO). Under these
conditions, neurons, oligodendrocytes and microglia rapidly die or
do not adhere (Imura et al., 2006). The type 2 astrocyte mouse
cell line C8-S, the mouse embryo-derived teratocarcinoma cell line
P19, the mouse neuroblastoma cell line N1E-115, and the mouse
fibroblast cell line NIH/3T3 were purchased from the American
Tissue Culture Collection and cultured in Dulbecco’s Modified
eagle Medium (DMEM) with 10% Fetal Bovine Serum (FBS),
streptomycin, penicillin and glutamine, in a 5% CO2 humidified
atmosphere at 37�C. P19 cells were, if indicated, incubated with
retinoic acid (1 lM, R2625, Sigma) for 24 h. Whole brains were
collected from P0 and 21-month-old (P21m) NMRI mouse.
Thomsen et al.: Nestin mRNA Localization in Mouse Astrocytes
November 2013 1923
Boyden Chamber Isolation of Cell ProtrusionsCell protrusions were isolated using a modified Boyden chamber
assay as previously described (Thomsen and Lade Nielsen, 2011). To
obtain sufficient protein and RNA, six 9.6 cm2 cell culture inserts
(BD Falcon) with a 1-lm pore size polystyrene membrane, and three
75 cm2 growth flasks of cell culture were used for each experiment.
Cell culture inserts were coated with extra cellular matrix (ECM)
protein Collagen type-I (Sigma; C7661) (final concentration 10 lg
mL21 in phosphate buffered saline (PBS)) at 37�C for 2 h. The cell
cultures inserts were afterward directly transferred to a six-well tissue
culture dish containing serum free DMEM. Cells were grown to
90% confluence and medium changed to serum free medium with
antibiotics and glutamine before cells were grown for additional
24 h. Cells were detached using 0.5% Trypsin-EDTA, and the tryp-
sin was inactivated by dissolving cells in DMEM with 10% FBS.
Cells were pelleted and re-dissolved in serum free DMEM, and 2 3
106 cells were seeded for each cell culture insert. Cell protrusions
were growing through the membrane for 24 h at 37�C. Inserts were
washed in PBS and the cells were gently scraped off the membrane.
First the cell protrusion fraction (PF) was made by scraping the bot-
tom side of the membrane. Cells were lysed by carefully washing the
cell scraper in 1 mL TRI-Reagent (Sigma) for RNA or in 1 mL 1x
protein gel loading buffer (Fermentas) for protein isolation. After-
wards, the cell body fraction (CF) was made by scraping the upper
side of the membrane and dissolving the cell material in TRI-
reagent or 1x protein gel loading buffer.
RNA Purification, cDNA Synthesis and Real TimeQuantitative PCR (RT-qPCR)RNA from cells and tissues was purified by standard TRI-reagent
protocol (Sigma) using 1 lg of glycogen for precipitation (Thomsen
and Lade Nielsen, 2011). cDNA was made using an iScript cDNA
synthesis kit (Bio-Rad). Nearly 1 lL of total RNA solution was used
per reaction. RT-qPCR was performed using SYBR Green 480 mas-
ter mix (Roche). RT-qPCR analyses were performed using a Roche
LightcyclerTM 480, with a primer annealing temperature of 58�C.
Primers were designed as intron spanning and PCR amplicons were
verified by gel electrophoresis and melting curve peak analysis.
Primer sequences are shown in Supporting Information Table 1.
Direct RNA Sequencing (DRS), Data Processing andAnalysisA modified Boyden chamber assay was used for isolation of cell pro-
trusions and cell bodies as previously described (Thomsen and Lade
Nielsen, 2011). Single RNA molecules were sequenced by DRS
(Ozsolak et al., 2009) using the Helicos Biosciences platform (Heli-
cos Biosciences, Boston, MA). Sequencing reads were mapped to a 2
kb region surrounding the 30 distal part of 27,131 genes for poly-
adenylated RNA of the mm9 version of the mouse genome. Bioin-
formatics raw data analysis and sequence alignment was made by
Helicos Biosciences, and transcript reads were presented as an excel
spread sheet. Details of the RNA preparation and sequencing can be
found at http://www.helicosbio.com/. Expression values were proc-
essed as RNA transcripts per million reads (tpm). To avoid including
false positives due to stochastic counts in the lower range, transcripts
exhibiting counts of <5 tpm in both PF and CF were excluded. The
RNA enrichment in protrusions was presented as the relative expres-
sion value in PF compared to CF [tpm PF/tpm CF]. A list of the
250 transcripts with the highest ratio were submitted for ordination
of gene name, location, and type, followed by functional annotation
analysis using the IPA ingenuity online platform, URL: http://
www.ingenuity.com. For increased stringency a direct relationship
analysis was performed including pathways described both human
rat and mouse. Results were presented with P values calculated by
the Benjamini-Hochberg method, to control for multiple testing.
RT-qPCR amplifications were made in triplicates for each gene and
Ct values were converted into linear values using the Xo method
(Thomsen et al., 2010). The mRNA localization ratio was calculated
as the ratio between the mean expression level in CF and PF and
afterwards normalized to the determined mRNA localization ratio
for Arpc3. Differences in localization ratios were analyzed by a Stu-
dent’s unpaired two tailed t test. All experiments were repeated three
times.
siRNA TransfectionsFor siRNA experiments 1.000.000 C8-S cells were immediately
before the transfection plated into 10 cm dishes in DMEM with
10% FBS. In 640 lL serum free medium was mixed siRNA to a
final concentration of 2 lM and incubated for 5 min. Nearly 13 lL
Dharmafect was mixed with 1267 lL serum free medium and incu-
bated 5 min. The two solutions were mixed and incubated 20 min,
added to the cells, and incubated 24 h. The medium was changed to
serum free medium and cells incubated for further 24 h. The cells
were used in a standard Boyden chamber assay with 1 lm mem-
branes and RNA purified from three membranes for each transfec-
tion and pooled. siRNA sequences Fmr121063: GGAUCAAGA
UGCAGUGAAA; Fmr12447: GUGAUGAAGUUGAGGUUUA;
Fxr12219: GAGAUGAAGUAGAGGUAUA; Fxr12560: GCAACU
GUGAAGAGAGUAA; Fxr221269: GGAAAGAACGGGAAAG
UGA; Fxr221336: GAGAUAACGACAAGAAGAA; non-specific
control: AGGUAGUGUAAUCGCCUUG.
Cloning of 30-UTR Reporter Constructs andTransfectionsThe 30-UTR sequences were amplified from mouse C8S cDNA
(primer sequences are shown in Supp. Info. Table 1) and purified
bands digested with XmaI and XbaI. The pcDNA5-beta-globin-
6xMS2-SV40-LpA vector was XmaI and XbaI digested and after liga-
tion with UTR inserts and E. coli transformation, positive constructs
were verified by sequencing. To remove the 6xMS2-sites the vectors
were digested with NotI and SmaI and by Klenow polymerase treat-
ment blunt-ended. After ligation and E. coli transformation con-
structs were verified by sequencing. The constructs were pooled and
transfected into NIH/3T3 cells using 2 lg total DNA mixture. In
parallel was transfected the control construct pTAG4 (Blechingberg
et al., 2007b). For each transfection were used 150,000 cells in six-
well plates using 200 lL serum-free medium and 3 lL Xtreme
Gene 9 DNA transfection reagent version 03 (Roche). Cells were
incubated 24 h before a medium shift to serum free medium and a
subsequent incubation for 24 h. The cells were used in a standard
1924 Volume 61, No. 11
Boyden chamber assay with 1 lM membranes and RNA purified
from three membranes for each transfection and pooled.
Western Blot AnalysisProteins were extracted for Western blot analysis by the Boyden
chamber assay. To normalize the protein amount from CF and PF
a-Tubulin and Actb were used as loading controls. Proteins were sep-
arated by SDS-PAGE in a 4–15% gradient polyacryl amide gel (Bio-
Rad). Proteins were transferred to a nitrocellulose membrane and
analyzed with following primary antibodies: rabbit anti a-Tubulin
(Rockland), rabbit anti b-Actin (Actb) (Sigma; A2103), rabbit anti
Histone H3 (Abcam; ab1791), mouse anti Nestin (Millipore;
MAB253), Mouse monoclonal anti Vimentin (Abcam; ab20346),
and goat anti Gfap (Santa Cruz; sc-6170). Nestin antibody was
diluted 1:1,000 and all other antibodies were diluted 1:10,000.
Horse radish peroxidase conjugated anti mouse, anti rabbit and anti
goat secondary antibodies (DAKO) were used for detection.
Single RNA Molecule FISH and ISHSingle molecule RNA fluorescence in situ hybridization (FISH) was
essential done as described (Femino et al., 1998). Probes consisting
of 50-mer single stranded DNA oligonucleotides were synthesized
and labeled with 4–5 Cy3 fluorophores. A total of eight various oli-
gonucleotides were hybridized to each target mRNA. Cells were
seeded onto 0.17-mm-thick coverslips (Marienfeld) either coated
with Collagen type 1 or uncoated and cultured in DMEM with
10% FBS, penicillin, streptomycin, and glutamine. At �60% con-
fluence cells were fixed in 4% paraformaldehyde for 20 min at room
temperature, and washed and stored in phosphate buffered saline
(PBS) at 4�C. Before hybridization, cells were permeabilized using
0.5% triton X-100 in PBS for 10 min at room temperature, washed
in PBS, and then incubated in pre-hybridization solution: (50%
formamide (Sigma; F4761) and 2 3 SSC (Ambion)) for 15 min. at
room temperature. The probes were hybridized in prehybridization
solution supplemented with 2 mg mL21 BSA (Roche), 0.2 mg
mL21 E. coli tRNA (Roche), and 0.2 mg mL21 sheared salmon
sperm DNA (Sigma; D7656) for 3 h at 37�C. About 10 ng DNA
probe was used per coverslip. Cells were washed twice with pre-
hybridization solution for 20 min at 37�C, then 10 min in 23 SSC
at room temperature, and in PBS for 10 min at room temperature.
Cell nuclei were counterstained with DAPI (0.5 mg L21 in PBS).
After a final wash in PBS, coverslips were rinsed in double distilled
water to remove excess salt, dried and mounted using ProLong gold
(InVitrogen). Actb probes were a kind gift from Dr. Robert H.
Singer. Probe sequences for Nestin and Gfap mRNA are shown in
Supporting Information Table 2. SSA4 oligonucleotides were previ-
ously described (Jensen et al., 2001).
Mouse P4 sagittal section mRNA in situ hybridization (ISH) data
for Nestin, Gfap, Actb, CamKIIa and Tubb3 were extracted from Allen
Developing Mouse Brain Atlas (http://developingmouse.brain-map.org)
and used for publication with permission.
ImmunofluorescenceCells were grown on 0.17 mm coverslips until 60% confluence, then
fixed in 4% paraformaldehyde for 20 min at room temperature, and
washed and stored in PBS at 4o C. Cells were permeabilized using
0.5% triton X-100 in PBS for 10 min at room temperature. A
blocking step was made using 1% BSA in PBS for 30 min at room
temperature. Primary antibodies were dissolved in blocking buffer
and incubated for 1 h at room temperature. Cells were washed three
times in PBS and incubating with secondary antibody dissolved in
blocking buffer. After three washes in PBS double immunofluores-
cence was performed as described above with a second treatment of
primary and secondary antibodies. After final secondary antibody
incubation cell were washed two times and cell nuclei were stained
with DAPI and washed once in PBS. Coverslips were rinsed in dou-
ble distilled water to remove salt, dried, and mounted with ProLong
gold. Primary antibodies used: Mouse anti Nestin (Milipore; MAB
253), mouse anti Vimentin (Abcam; ab20346), rabbit anti Gfap
(DAKO), rabbit anti a-Tubulin (Rockland), and rabbit anti Actb
(Sigma; A2103). All primary antibodies were diluted 1:500. Second-
ary antibodies were Alexa 488 conjugated goat anti rabbit IgG (Invi-
trogen A11034) and Alexa 555 conjugated goat anti mouse IgG
(Invitrogen A21127) both diluted 1:2000.
Microscopy and Image ProcessingAll images were made on a Zeiss axiovert 200m microscope, with a
plan apochromatic 633 1.4 NA objective, a HBO 100 W mercury
light source, and a CoolSNAP-HQ camera (Roper Scientific), oper-
ated by the MetaMorphVR software. Filters were from Chroma, Cy3
(41003), FITC (41001) and DAPI (31000). For RNA FISH analy-
ses were used a z-stack of 20 z-sections with 0.2-lm step size and
500 msec exposure. For FISH images z-stacks were collapsed to a
2D maximum intensity projection and for immunofluorescence a
single 2D image was selected from a 20 section z-stack. Images were
processed by background subtraction and normalization using the
open source software Image J (url: rsbweb.nih.gov/ij). To calculate
the mRNA localization ratio FISH images were processed by sub-
tracting the mean background value and the coordinates of the cell
center was determined manually in the DAPI channel, and the coor-
dinates of the apex of the cell protrusion was determined in the Cy3
channel. The intensities of each pixel and their respective coordinates
together with the coordinates of the cell center and protrusion were
imported into a custom made computer program. In this program, a
line was drawn from the center of the cell to the apex of the protru-
sion, and each point was perpendicularly projected to this line. A
pixel was categorized as localized if the position was more than 2/3
of the total length of the line toward the apex of the protrusion. A
localization score was finally calculated by dividing the sum of the
intensities of all localized pixels by the sum of the intensities of all
pixels projected on the line.
Statistical AnalysisRT-qPCR amplifications were made in triplicates for each gene and
Ct values were converted into linear values using the Xo method
(Thomsen et al., 2010). All the relative expression levels were nor-
malized to Arpc3 (for mRNA localization) or Gapdh (for mRNA
expression). Differences in the localization ratios and expression lev-
els were analyzed by a Student’s unpaired two tailed t test. All
experiments were repeated three times.
Thomsen et al.: Nestin mRNA Localization in Mouse Astrocytes
November 2013 1925
Results
Identification of Localized mRNAs in Mouse PrimaryAstrocyte Protrusions by DRSRNA localization has shown to play pivotal roles in cell sig-
naling, morphology, and migration during both embryonic
development, brain maintenance and in cancer metastasis
(Mili and Macara, 2009). Whereas mRNA localization in
neurons is extensively described, RNA localization in astro-
cytes is rather uncharacterized. To identify on a genome
wide scale mRNA species localized in astrocyte protrusions
we took advantage of the Boyden chamber cell fractionation
method to separate cell protrusions from cell bodies
(Thomsen and Lade Nielsen, 2011). Mouse primary astro-
cyte cultures were established from P0 mouse cortices and
grown for 8 DIV. The mRNA expression pattern in the
mouse primary astrocytes and two brain samples was by
RT-qPCR examined for expression of cell type specific
markers for microglia (Aif1), endothelial cells (Pecam1), oli-
godendrocytes (Mbp and Cnp), neurons (Nptx1), and astro-
cytes (Aldh1l1 and Gfap) (Imura et al., 2006; Stahlberg
et al., 2011) (Supp. Info. Fig. 1). The expression analysis
supported an in majority astrocyte lineage content of the
primary astrocyte cultures and this was further substantiated
by immunofluorescence staining showing that more than
80% of the cells were GFAP positive as also previously
described (Imura et al., 2006; Pekny et al., 1998; Stahlberg
et al., 2011; Thomsen and Lade Nielsen, 2011). It should
be emphasized that the heterogeneity of the primary astro-
cyte cultures still could result in identification by the Boy-
den chamber approach of mRNA species with expression
and RNA localization which cannot be confined to astro-
cytes. Compared to Stahlberg et al. (2011) presenting data
for 10–12 DIV primary astrocytes we note relative more
Nestin mRNA expression in our 8 DIV primary astrocyte
cultures (Supp. Info. Fig. 1). In accordance, by RT-qPCR
analysis of primary astrocyte cultures grown further DIV we
observed a decrease in Nestin mRNA expression (Supp.
Info. Fig. 2). We note a decrease in cell culture growth
capability and lack of efficient plating in the Boyden cham-
ber setting by using older DIV primary astrocyte cultures
and selected eight DIV astrocytes for the subsequent
analysis.
By RT-qPCR purification by the Boyden chamber
assay of protrusion and cell body RNA from eight DIV
mouse primary astrocytes was consolidated as described
(Thomsen and Lade Nielsen, 2011). Absence of nuclear
contamination of the PF was further controlled by the lack
of DAPI staining and lack of histone proteins. Reproducible
NGS analysis by single molecule DRS using minute RNA
quantities were recently presented (Ozsolak et al., 2010). In
the DRS procedure cDNA synthesis and amplification are
evaded and NGS analysis can be performed directly on pol-
yadenylated RNA (Ozsolak et al., 2009). RNA samples
from primary mouse astrocyte PFs and CFs isolated by the
Boyden chamber assay were analyzed by DRS. The resulting
output of 36 bases average size sequences were mapped to a
2 kb region surrounding the distal 30 end of 27131 genes of
the mm9 mouse genome. The total number of mapped
sequence counts in primary astrocytes was 5444770 for the
CF and 2147050 counts for the PF. The individual
sequence number for each transcript was normalized to
transcripts per million reads (tpm). The tpm values repre-
sent the relative abundance of a given mRNA in the total
population of mRNA molecules present in the CF or the
PF. In all subsequent analyses we included transcripts with
count numbers �5 tpm in both CF and PF to avoid insig-
nificantly expressed mRNAs and false positives due to even-
tual stochastic fluctuations in the lower range sequence
number. The total number of transcripts having �5 tpm in
both CF and PF was 8894. The localization ratio was calcu-
lated as the ratio between the tpm values from the PF and
CF, and 2298 mRNAs were determined to have a localiza-
tion ratio >1 (Fig. 1A). Complete list is shown in Support-
ing Information Table 3A and the 250 mRNAs with
highest localization ratios are shown in Supporting Informa-
tion Table 4. The localization ratio for a given mRNA will
be a variable depending on cell morphology and experimen-
tal settings, but the hierarchical order for localization ratios
is envisaged to be relative independent of these variable fac-
tors. Moreover we note that a mRNA localization ratio> 1
is not describing that a majority of this mRNA is localized
in protrusions but instead describes the relative enrichment
in protrusions and can thereby represent localization of only
a minor fraction of the total amount of a particular mRNA
within the cell. DRS determined mRNA localization ratios
for Rab13 mRNA (ratio 16.7), p0071/Pkp4 mRNA (ratio
15.7), and Kank2/Ankrd25 mRNA (ratio 10.9) are in
accordance with previous mRNA localization observations
(Mili et al., 2008; Thomsen and Lade Nielsen, 2011).
These results demonstrate that the Boyden chamber assay
combined with single molecule DRS are applicable to iden-
tify localized RNAs on a genome wide scale. Comparing
the 250 most localized mRNAs with mRNA expression data
from in vivo mouse P7 astrocytes (Cahoy et al., 2008) con-
firmed expression of the localized mRNAs in astrocytes invivo (Data not shown). Annotation analysis revealed that
the most localized mRNAs encode a broad variety of pro-
tein families (Supp. Info. Tables 4, 5 and Fig. 3). The most
localized mRNAs encode proteins with a localization pat-
tern strikingly similar to the total population of expressed
mRNAs and in this line we notice that a significant fraction
of protrusion localized mRNAs encode nuclear proteins.
1926 Volume 61, No. 11
mRNA Localization Analysis in the Mouse C8-SAstrocyte Cell LineWe next included the mouse astrocyte like cell line C8-S
that originally was isolated from the cerebellum of a post
natal (P8) mouse and resembles type II astrocyte Bergmann
glia cells (Alliot and Pessac, 1984). C8-S is a homogenous
and non-cancerous cell line with a highly polarized mor-
phology making it a qualified model for RNA localization
studies. We note that the RNA localization analysis in
mouse primary astrocytes represent an analysis of a hetero-
geneous cell population whereas C8-S RNA localization
analysis would be confined to a more homogenous cell type
of astrocyte lineage. To identify mRNAs localized in C8-S
cells, we again separated protrusions from the cell bodies by
the Boyden chamber assay and purified total RNA for
DRS. Sequences were mapped to a 2 kb region of the 30
distal part of 27131 genes from the mm9 mouse genome
sequence. The total counts number for CF was 2269453
and for PF 2090020, and 7697 transcripts had read num-
bers �5 tpm in both PF and CF. The normalized reads
were plotted as the ratio between PF and CF (Fig. 1C). The
number of transcripts with a localization ratio> 1 was
1436. The 250 mRNAs with highest localization ratios are
shown in Supporting Information. Table 6 and the com-
plete list in Supporting Information Table 3B. Annotation
analysis revealed that the most localized mRNAs encode a
FIGURE 1: Transcriptome analysis by DRS. (A) Graphical summary of the Boyden chamber assay for isolation of cell protrusions. ECMindicates coating of the membranes with Collagen type-1 prior to the seeding of cells to stimulate protrusion migration through the1 lm pore size membrane. Protrusions were growing for 24 h before isolation. The protrusions and cell bodies were isolated in two sep-arate fractions before subsequent analysis. (B,C) DRS transcriptome analysis of Boyden chamber purified RNA from mouse primary astro-cyte protrusions (B) and mouse C8-S cell protrusions (C). Bar plots displaying the RNA localization ratios between the normalizednumbers of DRS identified transcripts in protrusions over normalized number of transcripts in cell bodies in a hierarchical order. RNAswith �5 tpm in both protrusions and cell bodies are included. (D) Plot of the RNA localization ratio in primary astrocytes relative to theRNA localization ratio in C8-S cells. For visualization a red line indicates equal localization ratios, a dashed green line indicates the 26relative most localized RNAs in C8-S cells and a dashed yellow line indicating the 28 relative most localized RNAs in primary astrocytes.(E) RT-qPCR analysis of the RNA localization ratio in C8-S cells compared with the RNA localization ratio in primary astrocytes. RNAlocalization ratios are normalized to Arpc3. P values were calculated by an unpaired student’s t test. [Color figure can be viewed in theonline issue, which is available at wileyonlinelibrary.com.]
Thomsen et al.: Nestin mRNA Localization in Mouse Astrocytes
November 2013 1927
broad variety of protein families (Supp. Info. Table 7 and
Fig. 3).
Comparative mRNA Localization Analysis of MousePrimary Astrocytes and C8-S CellsA total of 7119 mRNA species had transcript reads �5 tpm
in PF and CF for both C8-S cells and mouse primary astro-
cytes. To examine whether we could identify cell type specific
localized mRNAs, we plotted the localization ratio of primary
astrocytes against C8-S cells (Fig. 1D). The analyses revealed
that majority of the localized mRNAs in mouse primary
astrocytes also localized in C8-S cells but also identified vari-
ability in localization patterns (Supp. Info. Tables 8 and 9).
Moreover, we note that the annotation analysis of localized
mRNAs also revealed differences for C8-S and primary astro-
cytes (Supp. Info. Fig. 3, Supp. Info. Tables 5 and 7).
To verify observed mRNA localizations in primary
astrocytes and C8-S cells we conducted RT-qPCR analysis of
representative candidates using independent biological RNA
samples from Boyden chambers. We selected Tensin3 (Ten3)
mRNA as it is highly expressed in both primary astrocytes
and C8-S cells but exhibited a larger RNA localization ratio
in primary astrocytes than C8-S cells, sperm flagellar 1
(Spef1) mRNA which is highly expressed in both primary
astrocytes and C8-S cells but with a higher RNA localization
ratio in C8-S cells than primary astrocytes, cytochrome c oxi-
dase subunit IV isoform 1 (Cox4i1) mRNA which is highly
expressed but displays no RNA localization in primary astro-
cytes and C8-S cells, and Rab13 and p0071/Pkp4 mRNAs
which are localized in both cell types. The RNA localization
ratios were normalized to Arpc3, a component of the Arp2/3
complex (Mili et al., 2008). Arpc3 mRNA showed localiza-
tion in neither the DRS analysis and nor in RT-qPCR analy-
ses using NIH/3T3 cells, primary astrocytes and C8-S cells
(Feltrin et al., 2012; Mili et al., 2008; Thomsen and Lade
Nielsen, 2011). RT-qPCR analysis revealed a significant local-
ization of Tensin3 mRNA in primary astrocytes compared to
C8-S cells and a significant localization of Spef1 mRNA in
C8-S cells compared to primary astrocytes (Fig. 1E). Cox4i1mRNA had no RNA localization in primary astrocytes and
C8-S cells, whereas Rab13 and p0071 mRNAs have RNA
localization in both cell types (Fig. 1E). The results of the
RT-qPCR analysis were similar to the DRS analysis, support-
ing that the Boyden chamber method combined with a DRS
analysis also is applicable for identification of specific RNA
localization patterns between different cell types.
Next we searched for candidate mRNAs having protru-
sion localization and in addition being enriched for astrocyte
expression. A comprehensive identification of mRNAs rela-
tively enriched in in vivo mouse astrocytes, mature astrocytes,
and developing astrocytes compared to other brain cell types
was described by Cahoy et al. (Cahoy et al., 2008). The study
also included identification of mRNAs enriched in in vitrogrown astroglia cells compared to in vivo astrocytes (Cahoy
et al., 2008). Of the 250 most localized mRNAs in mouse
primary astrocytes 28 were also identified to be enriched in at
least one of these four defined astrocyte populations (Supp.
Info. Table 10). Twenty two of the mRNAs have enriched
expression in astrocytes in vivo and 15 have enriched expres-
sion in astroglia cells grown in vitro (Supp. Info. Table 10).
We note that all identified mRNAs to be both localized in
astrocyte protrusions and expression enriched in developing
astrocytes in vivo also are relatively enriched in primary astro-
glia cells in vitro [Supp. Info. Table 10 and (Cahoy et al.,
2008)]. Moreover, the comparative analysis pointed that albeit
some mRNAs with localization in astrocyte protrusions have
enriched expression in astrocytes only few were assigned to be
astrocyte specific (Dio2, Ppp1r3c and Gfap). Recently, Feltrin
et al. described 80 mRNAs localized in mouse N1E-115 neu-
roblastoma cells (Feltrin et al., 2012). Of the 25 most protru-
sion localized mRNAs in mouse primary astrocytes and C8-S
cells 13 mRNAs were also identified to be significantly local-
ized in N1E-115 cells and of the 80 most protrusion localized
mRNAs in mouse primary astrocytes and C8-S cells, 28 and
26, respectively, were also significantly localized in N1E-115
cells (Supp. Info. Tables 11 and 12) (Feltrin et al., 2012).
Such mRNAs could represent a group of commonly expressed
and protrusion localized mRNAs and several of these were
also identified to be localized in mouse fibroblast protrusions
(Mili et al., 2008). Of the 80 N1E-115 localized mRNAs 4
were overlapping with the astrocyte enriched and localized
mRNAs (Cyb5r3, Ddr2, Arhgap11a, and Kctd10) (Supp. Info.
Table 10).
mRNA Localization for the IF Protein NestinWe observed that the mRNA for the IF protein Nestin had a
high localization ratio in both mouse primary astrocytes and
in C8-S cells and a preferential expression in developing
astrocytes (and other neural cell type progenitors in vivo)(Fig. 2A, Supp. Info. Tables 4, 6, 10) (Cahoy et al., 2008).
One of our long term research aims is to identify mechanisms
for IF regulation and we accordingly focused the subsequent
studies on Nestin mRNA. In primary astrocytes NestinmRNA had a localization ratio of 42 and was identified as
the relatively most protrusion localized mRNA. In C8-S cells
the localization ratio was 19. The RNA localization ratios for
the two Nestin related IF proteins in astrocytes, Gfap and
Vimentin, were 5 and 0.8 in primary astrocytes and 1 and
0.7 in C8-S cells, respectively. The RNA localization outcome
of the DRS analysis was confirmed by RT-qPCR analyses
using independent biological samples including primers
against Nestin, Vimentin, and Gfap cDNA. As references we
1928 Volume 61, No. 11
included analyses of Actb and Arpc3 cDNA. The RNA local-
ization ratios were normalized to Arpc3 given the value 1
(Fig. 2B). The RT-qPCR results from mouse primary astro-
cytes showed localization of Nestin mRNA in the cell protru-
sions with a localization ratio of 130. Likewise, we noted that
the Gfap mRNA was localized in the protrusions with a 13
times higher ratio than Arpc3. The Vimentin mRNA had no
localization, with a ratio of 0.6, which was concurrent to the
ratios of Arpc3 and Actb mRNAs. In a similar RT-qPCR
experimental setting using RNA material from C8-S cells Nes-tin mRNA showed a significant localization in cell protru-
sions, whereas neither Vimentin nor Gfap mRNAs displayed
localization (Fig. 2C). We note that the Gfap mRNA expres-
sion in C8-S cells was approximately 500-fold lower than for
primary astrocytes. In a time course experiment for C8-S cell
protrusion growth Nestin RNA localization was at maximum
FIGURE 2: Nestin mRNA localization in cell protrusions from mouse primary astrocytes and C8-S cells. (A) Localization ratios determinedby DRS for mRNA for IF proteins and controls. (B,C) RT-qPCR analysis of RNA localization ratios in primary astrocytes (B) and C8-S cells(C). cDNA was made from RNA from the protrusion and the cell body fractions. The ratios are normalized to Arpc3. (D) RT-qPCR analysisof RNA localization ratios in C8-S cells after growth in Boyden chamber assays for the indicated times. The RNA localization ratios arenormalized to Arpc3 and subsequently to the RNA localization ratio after 24 h growth time given the value 100. (E) RT-qPCR analysis ofRNA localization ratios in C8-S cells after growth in Boyden chamber assays with Laminin and Fibronectin membrane coating. (F) RT-qPCR analysis of RNA localization ratios in P19 cells without (2RA) and with (1RA) retinoic acid incubation for 24 h and the localizationratios normalized to Arpc3.
Thomsen et al.: Nestin mRNA Localization in Mouse Astrocytes
November 2013 1929
after 24 h (Fig. 2D). Nestin RNA localization was also
observed in C8-S cells after coating membranes in the Boy-
den chamber with the extracellular matrix proteins Laminin
or Fibronectin (Fig. 2E), whereas absence of coating hindered
quantitative experiments because only few cell protrusions
penetrated the membrane.
To examine if the observed Nestin mRNA localization
was restricted to cells of astrocyte lineage we next examine
mouse embryo-derived teratocarcinoma P19 cell line in the
Boyden chamber setting. P19 cells are pluripotent and can
differentiate into cell types of all three germ layers and in
response to retinoic acid treatment P19 cells first become
neural stem-like cells with Nestin expression and finally dif-
ferentiate to neural cells (neuron, glia, etc.) (Jones-Villeneuve
et al., 1982; Tan et al., 2010). After the Boyden chamberassay P19 cells without incubation with retinoic acid had alow level of Nestin mRNA expression but RT-PCR analyseswere able to determine Nestin mRNA localization to the P19cell protrusions (Fig. 2F). After retinoic acid incubation for24h Nestin mRNA expression was increased �25-fold (datanot shown) and again Nestin mRNA localization to cell pro-trusions was determined (Fig. 2F). We were unable to detectGfap mRNA expression to a significant level in the P19 cellsamples (data not shown). We conclude that the NestinmRNA present in mouse P19 pluripotent embryonic carci-noma cells also have protrusion localization capability.
Cis-elements determining mRNA localization are typi-
cally enclosed in the 3’-UTR (Mili and Macara, 2009). To
determine if the 30-UTR was involved in mediating localiza-
tion of Nestin mRNA to cell protrusions we cloned the 419
bp 30-UTR sequence including the intrinsic poly-adenylation
signal into a modified pcDNA5-beta-globin-6xMS2-SV40-
LpA vector downstream of the spliced b-globin transcription
unit. We also cloned the Rab13 30-UTR for positive localiza-
tion control and the Vimentin 30-UTR for negative control
together with the vector without an UTR insert. We were
unable to transfect mouse primary astrocytes and C8-S suffi-
ciently for quantitative ectopic RNA localization measure-
ments and instead used mouse NIH/3T3 cells which
previously were used as model in mRNA localization studies
(Mili and Macara, 2009). After cell transfection and Boyden
assay purified RNA was examined by RT-qPCR for expression
of the ectopic mRNAs by a forward primer recognizing b-
globin exon 1 included in the chimeric mRNAs and a reverse
primer corresponding to the inserted UTR fragments or aspecific primer for the vector without an UTR insert. cDNAfrom the PF and the CF from cells transfected with the con-
trol vector, pTAG, resulted in no significant RT-qPCR ampli-fication. Insertion of the Nestin 30-UTR resulted in anapproximately fourfold increase in localization ratio comparedto the Vimentin 30-UTR and vector control (Fig. 3A). The
Rab13 30-UTR resulted in a further fourfold increase in
localization ratio (Fig. 3A). From the transfection experimentswe conclude that the Nestin 30-UTR includes sequence deter-minants which alone can mediate RNA localization.
We noted the presence of a high Guanine (G) content
and algorithm predicted putative G-quadruplex motifs in
the Nestin 30-UTR (http://bioinformatics.ramapo.edu/
QGRS). The fragile X mental retardation protein family
composed of Fmrp, Fxr1, and Fxr2 can through the RGG-
box associate with G-quadruplex motifs and mediate trans-
port to neuronal dendrites (Bagni and Greenough, 2005;
Darnell et al., 2001; Mili et al., 2008; Schaeffer et al.,
2001). To determine if these factors are involved in NestinmRNA localization we depleted C8-S cells of these factors
individually or in combinations by transient siRNA treat-
ments and by Boyden chamber following measured NestinmRNA localization. Semi-quantitative measurements
showed that the Fxr2 mRNA expression level in C8-S was
several fold lower than Fxr1 (data not shown). Fmr1mRNA, which encodes the Fmrp protein, was also lower
expressed than Fxr1 (data not shown). Fmr1, Fxr1 and Fxr2
siRNA mediated co-depletion resulted in an approximately
twofold reduction in the Nestin RNA localization ratio (Fig.
3B). For the individual siRNAs we observed that depletion
of Fmr1 and Fxr1 resulted in an approximately twofold
reduction in the Nestin RNA localization ratio supporting
that the proteins, directly or indirectly, are involved in Nes-
tin mRNA localization (Fig. 3B).
FISH Analysis of Nestin mRNATo substantiate the Nestin mRNA localization studies we
examined for Nestin mRNA localization in mouse primary
astrocytes by FISH. For optimal cytoplasmic RNA detection
we took advantage of the single RNA molecule FISH tech-
nique (Femino et al., 1998). We designed probes complemen-
tary to mRNAs of Nestin and Gfap. For the FISH assay we
used 50-mer DNA oligonucleotide probes each labeled with
five covalently coupled Cy3 fluorophores and single mRNA
molecules were detected by hybridizing a pool of eight differ-
ent probes targeting each mRNA species. For control we
included probes against Actb mRNA. Primary astrocytes (8
DIV) plated on Collagen ECM protein coated coverslips
were used for analysis and the results of the FISH assays were
monitored by blinded cell counting. Approximately 50% of
the primary astrocytes were scored positive for Nestin mRNA
expression due to the number of Nestin mRNA FISH signals.
We note that Nestin mRNA FISH signals also could be
detected in cells initially scored negative for Nestin mRNA
expression by the blinded cell counting indicating that the
actual number of Nestin expressing cells is higher than 50%.
In approximately half of the positively scored cells numerous
Nestin mRNA FISH signals could be detected in cell
1930 Volume 61, No. 11
protrusions in accordance with Nestin mRNA protrusion
localization (Fig. 4A). FISH analysis showed that GfapmRNA was more uniformly distributed in the cytoplasm
compared to Nestin mRNA. However, Gfap mRNA could be
observed in the outermost regions of the cytoplasm in a
majority of the Gfap positive cells, whereas Actb mRNA pre-
dominantly was confined to the peri-nuclear part of the cells
(Fig. 4A).
We also examined subcellular RNA localization in C8-S
cells by RNA FISH, using probes against Nestin, Actb and
mRNA for the S. cereviseae heat shock protein SSA4 as nega-
tive control. Gfap mRNA expression is very low in C8-S cells
and accordingly not included in the analysis. C8-S cells were
plated on collagen ECM protein coated coverslips and the
results of the FISH assay were analyzed by blinded cell count-
ing. Most C8-S cells (>99%) were scored positive due to the
number of Nestin mRNA FISH signals. In approximately half
of the positively scored cells numerous Nestin mRNA FISH
signals could be detected in cell protrusions in accordance
with protrusion localization of Nestin mRNA (Fig. 4B, upper
panels). Actb mRNA was predominantly confined to the peri-
nuclear region of the cell body with a low amount of mRNA
also observed in cell protrusions (Fig. 4B, middle panels).
The negative control probe showed no signal (Fig. 4B, lower
panels). To determine if there is an effect of ECM compo-
nents on Nestin mRNA localization we performed a similar
RNA FISH analysis using C8-S cells plated on ECM
uncoated coverslips (Supp. Info. Fig. 4). We observed no
changes in Nestin and Actb mRNA localization patterns
(Supp. Info. Fig. 4). To further examine the difference of
mRNA localization between Nestin and Actb in C8-S cells,
we randomly selected 10 cells with Nestin mRNA signals and
9 cells with Actb signals (Fig. 4C). Signal intensities were
summarized and signals that displayed a relative distance
more than two thirds from the center of the nucleus were
regarded as localized. Localized signal intensities were divided
by the total signal intensities within the cell to obtain a local-
ization ratio. The ratios were statistically analyzed by a stu-
dent’s t test, which showed that the localization of Nestin
FIGURE 3: (Continued)
FIGURE 3: Cis- and trans-factors involved in Nestin mRNA local-ization. (A) The Nestin 30-UTR include cis-sequences for mRNAlocalization to protrusions. The 30-UTR sequences of Nestin,Vimentin and Rab13 were transfected as a pool into mouse NIH/3T3 cells together with the expression vector without insert.RNA fractions from protrusions and cell bodies were purified bythe Boyden chamber assay. By RT-qPCR the RNA localizationratios were determined and normalized to the localization ratiofor the vector without insert (control) given the value 1. (B)Involvement of the FMRP protein family for Nestin mRNA local-ization. In C8-S cells Fmr1, Fxr1, and Fxr2 were either depletedtogether using a pool of siRNA (sifpool, left panels) or by indi-vidually siRNA (right panels). At 24 h after transfection C8-S cellswere used in Boyden chamber assay and Nestin RNA localizationratio determined and normalized to 100 for the control siRNAtransfection. The efficiency of siRNA treatments were measuredby RT-qPCR analysis of Fmr1, Fxr1, and Fxr2 and normalized to1 for the control siRNA transfection.
Thomsen et al.: Nestin mRNA Localization in Mouse Astrocytes
November 2013 1931
mRNA was significantly higher than Actb (Fig. 4D). In con-
clusion, the observations from the FISH assays were in con-
cordance with the biochemical analysis showing localization
of a fraction of the Nestin mRNA in cell protrusions.
Nestin Protein Localization in Astrocyte ProtrusionsWe examined whether the Nestin protein display localization
analogous to the mRNA. In this line we note that it was pre-
viously shown that mouse Nestin protein localizes in growth
cones of P19 derived neurons and cerebellar granule cells
(Yan et al., 2001). We isolated protein extracts from the PF
and CF of mouse primary astrocytes using the Boyden
chamber assay and subsequently performed Western blot anal-
ysis. To normalize the protein content between the PF and
the CF we included analysis of a-Tubulin and Actb represent-
ing controls for uniform protein distribution. Histone H3
was included to control that cell nuclei were confined to the
CF. Western blot analysis showed that Nestin and Vimentin
were relatively more present in the PF whereas Gfap was
equally detected in the CF and the PF (Fig. 5A). Western
blot analysis also showed that in the C8-S cell PF Nestin and
Vimentin proteins were relatively enriched (Fig. 5B). Notably,
the absence in the presented Western blot analysis of detecta-
ble Vimentin and Nestin in the CF fraction shall not be
interpreted as absence of the proteins in this fraction but
solely reflects a relative increased localization in protrusions
compared to a-tubulin and Actb.
To examine subcellular protein distributions in closer
details we performed immunofluorescence analysis. Nestin
and Vimentin were detected using mouse primary antibodies
and an Alexa 555 conjugated secondary antibody and Gfap
was detected using a rabbit primary antibody and an Alexa
488 conjugated secondary antibody. In primary mouse astro-
cytes in the order of 80% of the cells were scored positive for
Gfap, Vimentin and Nestin but we note very heterogeneous
expression levels. Most Gfap positive cells showed filamentous
Gfap distribution throughout the cytoplasm (Fig. 5C,D). The
largest fraction of the Vimentin positive cells has filamentous
staining throughout the cytoplasm (Fig. 5C, upper panels)
but some cells (�30%) have Vimentin accumulation in cell
protrusions with simultaneous Vimentin filaments mostly in
the central part of the cell (Fig. 5C, lower panels). In Vimen-
tin and Gfap positive cells we observed co-localization includ-
ing colocalization in protrusions (Fig. 5C). Most Nestin
positive cells showed filamentous Nestin distribution
FIGURE 4: (Continued)
FIGURE 4: Subcellular detection of Nestin mRNA by single mole-cule RNA FISH. (A) Single molecule RNA FISH analysis of endog-enous Nestin, Actb and Gfap mRNAs in cultured primaryastrocytes isolated from P0 mice (left panels). A region of inter-est (ROI) is highlighted and magnified by a 53 zoom factor (mid-dle panels). Cell nuclei were stained by DAPI (right panels). Scalebar 5 10 lm. (B) Single molecule RNA FISH of Nestin and ActbmRNAs in C8-S cells (left panels). SSA4 served as negative con-trol. Middle panels show a ROI representing the tip of a cell pro-trusion enlarged five times (middle panels). Cell nuclei werestained by DAPI (right panels). Scale bar 5 20 lm. (C) Heat plotdisplaying C8-S Nestin and Actb mRNA localization analysis out-put (right panels) and their respective raw images (left panels).Cell center is marked by a red spot en the length of the cell pro-trusion is marked by a green line. Examples of cells with ActbmRNA signals (upper two panels) and Nestin mRNA signals(lower two panels) are shown. (D) The outcome of a paired twotailed t test for Nestin and Actb mRNA localization according tothe procedure in (C). The cell counting numbers were for Actbn 5 9 and for Nestin n 5 10. [Color figure can be viewed in theonline issue, which is available at wileyonlinelibrary.com.]
1932 Volume 61, No. 11
throughout the cytoplasm including the cell protrusions (Fig.
5D, upper panels). In Nestin and Gfap positive cells we
observed co-localization including colocalization in protru-
sions. Some Nestin positive cells (�20%) showed more pro-
nounced Nestin accumulation in protrusions with Gfap
colocalization and simultaneous Nestin in the central part of
the cell (Fig. 5D, lower panels). In C8-S cells Gfap expression
could not be detected whereas Vimentin and Nestin were
detected in nearly all cells (Fig. 5E). In �50% of the C8-S
cells Nestin accumulation was present throughout the protru-
sions (Fig. 5E). In nearly all C8-S cells Vimentin accumula-
tion was present throughout protrusions (Fig. 5E). The
control proteins a-Tubulin and Actb were uniformly dispersed
throughout the cytoplasm (Fig. 5E). Thus, both in primary
astrocytes and C8-S cells a fraction of the Nestin protein, as
well as the Nestin mRNA, was present in protrusions.
Discussion
The number of mRNAs that exhibit a distinct subcellular
localization pattern has increased dramatically as genome
wide technologies are improved (Lecuyer et al., 2007). More
than 1,000 mRNAs are identified to be relatively localized in
neuronal protrusions (Eberwine et al., 2001; Feltrin et al.,
2012; Taylor et al., 2009; Willis and Twiss, 2010; Willis
et al., 2007). Our presented DRS-based results strongly indi-
cate that numerous polyadenylated RNAs also are localized in
protrusions of primary astrocytes and the astrocyte cell line
C8-S. The heterogeneity of the used primary astrocytes could
in principle result in identification of mRNA localization
which cannot be confined to astrocytes but may represent
expression or localization in a minor cell subpopulation of
different lineage within the cell culture. We note that non-
Gfap positive cells within primary astrocyte cultures most
probably are primarily meningeal cells of fibroblast lineage
(Imura et al., 2006). However, comparing the 250 most local-
ized mRNAs from primary astrocytes with expression data for
in vivo astrocytes (Cahoy et al., 2008) we could verify astro-
cyte expression of the identified localized mRNAs. Annota-
tion analysis revealed that the 250 most localized mRNAs in
primary astrocytes and the C8-S astrocyte cell line encode a
broad variety of protein families. Moreover, both the 50 and
the 250 most localized mRNAs encode proteins which exhibit
a localization pattern similar to the total population of
expressed mRNAs (Supp. Info. Fig. 3). In this line, a signifi-
cant fraction of the protrusion localized mRNAs encode
nuclear proteins, which indicates that RNA localization to
protrusions might not necessarily be predictive for a direct
function of the encoded protein in cellular protrusions or
that such protein has moonlighting capacity. A previous study
showed that a majority (�70%) of all expressed RNAs are
localized in Drosophila embryos (Lecuyer et al., 2007) and
FIGURE 5: Nestin protein localization analyses. (A) Western-blotanalysis of protein isolated from the cell body fraction (CF, Boy-den chamber upper side) and cell protrusion fraction (PF, Boydenchamber lower side) using mouse primary astrocytes. Proteinsare detected by Western blotting using primary antibodiesagainst Nestin, Vimentin, Gfap, a-Tubulin, Actb, and Histone H3.a-Tubulin and Actb served as load controls and Histone H3 tocontrol for the lack of cell body migration through the Boydenchamber membrane. (B) Western blot analysis of protein isolatedfrom CF and PF by the Boyden chamber assay using C8-S cells.(C,D) Double immunofluorescence analysis of Vimentin and Gfap(C) and Nestin and Gfap (D) in mouse primary astrocytes. Pro-teins were detected using primary antibodies against Nestin,Vimentin and Gfap. Nuclei were stained with DAPI and shown inmerged pictures (Gfap, green; Nestin, red; Vimentin, red; DAPI,Blue) of representative cells. Scale bar 5 20 lm. (E) Immunofluo-rescence analysis of C8-S cells showing subcellular protein local-ization of Nestin, Vimentin, Actb and a-Tubulin. Cell nuclei werestained with DAPI (blue). Scale bar 5 20 lm. [Color figure can beviewed in the online issue, which is available atwileyonlinelibrary.com.]
Thomsen et al.: Nestin mRNA Localization in Mouse Astrocytes
November 2013 1933
points toward the notion that RNA localization is a norm
rather than the exception. We determined a higher number of
localized mRNAs in primary astrocytes than in C8-S cells, a
result that could be dependent on the different morphologies
of the cells. To further verify the determined RNA localiza-
tion patterns we conducted RT-qPCR analysis of representa-
tive mRNAs. The RT-qPCR analyses verified that Tensin3
mRNA displays a larger RNA localization ratio in primary
astrocytes than C8-S cells, and Spef1 mRNA shows a higher
RNA localization ratio in C8-S cells than primary astrocytes
(Fig. 1D). The result of the RT-qPCR analysis was similar to
the DRS analysis, further supporting that the Boyden cham-
ber assay combined with a DRS analysis is applicable to com-
pare and identify both commonly and cell type specific
localized RNAs. The Spef1 protein was previously determined
to have a developmental dependent subcellular localization in
mouse sperm cells (Chan et al., 2005). Tensin proteins act as
mediators between the extracellular matrix and the cytoskele-
ton, and studies of human cancer cell lines showed that Ten-
sin3 act as a negative regulator of cell migration
(Martuszewska et al., 2009). The results suggest that the two
examined astrocyte cell types have different intrinsic capacity
for Spef1 and Tensin3 mRNA localization which could be
mediated through nonidentical expression patterns of mRNA
localizing trans-factors or inclusion of different yet unidenti-
fied cis-sequences in the transcripts through alternative RNA
processing.
Directional transport by cytoskeletal motors is the pre-
dominant mechanism for delivering mRNA to the destination
(Bullock, 2011). mRNA and associated protein trans-factors
are cotransported as messenger ribonucleoprotein (mRNP)
particles (Mili and Macara, 2009). The mRNP transport is
facilitated by myosin motor proteins on actin filaments or via
kinesin and dynein motor proteins on microtubules (Shav-Tal
and Singer, 2005). Different motors can be active in sorting
mRNP in the same cell at the same time leading to differen-
tial patterns of mRNA localization (Bullock, 2011). Assembly
of the cytoskeletal filaments used for mRNP transport can be
self-regulated through mRNA localization dependent mecha-
nisms which are well established for actin filaments (Shav-Tal
and Singer, 2005). Actb mRNA is localized to the leading
edge of fibroblasts in an actin filament dependent manner
and disruption of Actb mRNA localization results in slower
cell motility, loss of directionality, delocalization of actin poly-
merization and altered adhesion dynamics (Katz et al., 2012;
Mingle et al., 2005; Shestakova et al., 2001). The actin-
related protein 2/3 (Arp2/3) complex is a crucial actin poly-
merization nucleator and is localized to the leading protru-
sions of migrating fibroblasts. mRNAs for the seven subunits
of the Arp2/3 complex (Arpc1a, Arpc2, Arpc3, Arpc4, Arpc5,
Actr2, and Actr3) are localized to fibroblast protrusions in
both actin filaments and microtubules dependent manners
supporting that the Arp2/3 complex is targeted to the site of
function by mRNA localization (Mingle et al., 2005). In the
Boyden chamber approach we neither observed mRNA local-
ization for Actb mRNA nor the seven mRNAs for the Arp2/3
complex (Supp. Info. Table 3). This observation is in accord-
ance with other reports describing the lack of mRNA localiza-
tion for these actin filament components and may reflect the
use of different experimental settings to detect mRNA local-
ization (Feltrin et al., 2012; Mili et al., 2008).
Only few studies have addressed mRNA localization in
astrocytes in relation to IFs. In this study we have identified
mRNA localization of Nestin. Compared to control Arpc3mRNA, Nestin mRNA exhibited a significant localization in
astrocyte protrusions as well as in protrusions of P19 embryo-
derived teratocarcinoma cells. Gfap mRNA displayed a more
moderate localization. Vimentin mRNA exhibited no signifi-
cant localization, neither by RT-qPCR nor DRS analysis. For
detailed subcellular analysis of Nestin mRNA we performed
single RNA molecule FISH. This revealed that Nestin mRNA
was localized to protrusions in �50% of the primary astro-
cytes and C8-S cells scored positive for Nestin mRNA expres-
sion. Moreover, when we applied a computer program to
analyze FISH images of Nestin mRNA positive cells and com-
pared these with Actb mRNA positive cells, we found that
Nestin mRNA had significantly more localization to cell pro-
trusions than Actb mRNA. The Nestin mRNA localization
results sustain previous results obtained from the developing
mouse brain indicating that Nestin mRNA is localized in
columnar neuroepithelial cells and radial glial cells (Dahl-
strand et al., 1995), and studies made on tissue sections of
chicken brain and cell cultures demonstrating mRNA localiza-
tion for Transitin, a Nestin like IF protein, in developing
radial glia cells from chickens (Lee and Cole, 2000). To
examine if Nestin mRNA localization also could be detected
in vivo we analyzed mouse P4 brain sagittal section mRNA in
situ hybridization (ISH) data for Nestin, Gfap, Actb,
CamKIIa, and Tubb3 (extracted from Allen Developing
Mouse Brain Atlas) (Supp. Info. Figs. 5 and 6). Nestin ISH
signals were sparsely detected and based on the data we were
not able to conclusively determine if Nestin mRNA in vivo is
localized in astrocyte protrusions.
The presented Nestin mRNA localization observations by
DRS, RT-qPCR and FISH in astrocyte cells are concordant
with the results showing presence of Nestin protein in the cell
protrusions (Fig. 5). Together the data supports that Nestin
mRNA can be localized and most likely also locally translated
in astrocyte protrusions. Although local proteins synthesis was
not shown directly, our results suggest that Nestin protein is
subcellular localized, at least partly, as a consequence of local
mRNA translation. Changes of cell morphology depend on
1934 Volume 61, No. 11
coordinated assembly and disassembly of cytoskeleton proteins.
Notably, it has been demonstrated that Nestin can inhibit
Vimentin filament formation in a concentration dependent
manner (Steinert et al., 1999). Thus, localization and local
translation of the Nestin mRNA can be used by the cell to cre-
ate the necessary local environment to provide optimal condi-
tions for IF modulation in the early onset of protrusion
formation. The notion that local assembly of Nestin and
Vimentin could participate in modulation of astrocyte mor-
phology is further indicated by the results showing significant
Vimentin protein localization in protrusions. Interestingly, we
showed no significant Vimentin mRNA localization and
Vimentin is accordingly most likely localized by protein trans-
port mechanisms. This is supported by numerous studies dem-
onstrating mictrotubule and dynein dependent transport of
non-filamentous IF protein particles (Perlson et al., 2005; Prah-
lad et al., 1998; Yoon et al., 1998). Moreover, studies of
migrating endothelial cells have shown Vimentin localization to
focal adhesions sites (Tsuruta and Jones, 2003). Gfap expression
is graduate up-regulated as Nestin and Vimentin expression
decreases and it is proposed that Vimentin and Nestin filaments
are scaffolds for the establishment of long term Gfap filaments
(Dahlstrand et al., 1995). Our data indicates that different
localization of Vimentin, Gfap, and Nestin mRNAs and the
resulting proteins altogether participates in the control of IF
dynamics in cell protrusions.
Several studies have demonstrated that mRNA localiza-
tion depends on cis-elements that typically, but not exclu-
sively, are enclosed in the 30-UTR and associated RNA
binding trans-factors (Mili and Macara, 2009). In a reporter
assay we determined that the Nestin mRNA 30-UTR was suf-
ficient to mediate localization to cell protrusions whereas the
Vimentin mRNA 30-UTR lacked this functionality. Fmrp and
the autosomal paralogues, Fxr1 and Fxr2, compose a family
of functional homologous RNA-binding proteins including
two ribonucleoprotein K homology domains and a cluster of
arginine and glycine residues in the RGG box (Bassell and
Warren, 2008; Tan et al., 2009). These domains are impor-
tant for RNA binding and polyribosome association. The
FMRP-family has an important role in translation control,
both in vivo and in vitro, and FMRP regulates protein syn-
thesis at sites where mRNAs are locally translated (Kindler
and Kreienkamp, 2012). The FMRP-family shuttles between
the nucleus and the cytoplasm and after mRNA association
in the nucleus forms a ribonucleoprotein complex transported
to dendrites and spines (Kim et al., 2009; Kindler and
Kreienkamp, 2012). An mRNA target sequence for the
FMRP-family consists of a G-quadruplex motif recognized by
the RGG box (Melko and Bardoni, 2010). Isolation of
FMRP containing ribonucleoprotein complexes from mouse
brains identified 432 mRNA species whereof 70% included
putative G-quadruplex motifs (Brown et al., 2001). Moreover,
30% of an examined group of mRNA localized in neuronal
dendrites, such as Psd95 and CamkIIa, contain G-quadruplex
motifs in the 30-UTR (Subramanian et al., 2011). We showed
that Fmrp and Fxr1, either directly or indirectly, are involved
in Nestin mRNA localization. The Nestin mRNA contains
several putative G-quadruplex motifs in the 30-UTR which
could indicate that these elements in association with Fmrp
and Fxr1 participates in Nestin mRNA localization. In
genome wide identification analysis of mRNAs associating
with Fmrp the Nestin mRNA does not appear as a highly sig-
nificant Fmrp target (Ascano et al., 2012; Brown et al., 2001;
Darnell et al., 2011). It should be emphasized that NestinmRNA expression is relatively low in the examined cell lines
and mouse brain samples which could be hindering identifi-
cation of Fmrp and Nestin mRNA interactions. In this line
Darnell et al. found interactions between Fmrp and Nestin
mRNA but the interaction was not scored significantly posi-
tive (Darnell et al., 2011).
In summary, we identified that IFs potentially can be
regulated at the level of mRNA localization mechanisms. We
demonstrated that Nestin mRNA and Nestin protein can have
localization in cell protrusions proposing that at least some
Nestin protein can be localized as a consequence of local
translation of the Nestin mRNA. The Vimentin mRNA dis-
played no significant protrusion localization whereas the
Vimentin protein is present. Finally, Gfap mRNA is moder-
ately localized in protrusions whereas the Gfap protein is dis-
tributed relatively evenly in the cytoplasm. The identified
mRNA localization patterns might reflect that IF proteins use
different sets of localization mechanisms with potential to reg-
ulate astrocyte morphology and migration during brain devel-
opment and maintenance.
Acknowledgment
Grant sponsors: The Lundbeck Foundation; Fonden til Læge-
videnskabens Fremme; NANONET—COST Action
BM1002; The Health Faculty, Aarhus University, Denmark.
The authors thank Robert H. Singer, Albert Einstein College
of Medicine, Bronx, New York, USA, for introducing them to
the FISH technique and the donation of FISH control probes.
The grant funders had no role in study design, data collection
and analysis, decision to publish or preparation of the manu-
script. The authors declare no conflicts of interests.
References
Allen NJ, Barres BA. 2009. Neuroscience: Glia—More than just brain glue.Nature 457:675–677.
Alliot F, Pessac B. 1984. Astrocytic cell clones derived from established cul-tures of 8-day postnatal mouse cerebella. Brain Res 306:283–291.
Thomsen et al.: Nestin mRNA Localization in Mouse Astrocytes
November 2013 1935
Andres-Barquin PJ, Fages C, Le Prince G, Rolland B, Tardy M. 1994. Thyroidhormones influence the astroglial plasticity: Changes in the expression of glialfibrillary acidic protein (GFAP) and of its encoding message. Neurochem Res19:65–69.
Ascano M Jr, Mukherjee N, Bandaru P, Miller JB, Nusbaum JD, Corcoran DL,Langlois C, Munschauer M, Dewell S, Hafner M, et al. 2012. FMRP targetsdistinct mRNA sequence elements to regulate protein expression. Nature492:382–386.
Bagni C, Greenough WT. 2005. From mRNP trafficking to spine dysmorpho-genesis: The roots of fragile X syndrome. Nat Rev Neurosci 6:376–387.
Bassell GJ, Warren ST. 2008. Fragile X syndrome: Loss of local mRNA regula-tion alters synaptic development and function. Neuron 60:201–214.
Blechingberg J, Holm IE, Nielsen KB, Jensen TH, Jorgensen AL, Nielsen AL.2007a. Identification and characterization of GFAPkappa, a novel glial fibril-lary acidic protein isoform. Glia 55:497–507.
Blechingberg J, Lykke-Andersen S, Jensen TH, Jorgensen AL, Nielsen AL.2007b. Regulatory mechanisms for 3’-end alternative splicing and polyadenyl-ation of the glial fibrillary acidic protein, GFAP, transcript. Nucleic Acids Res35:7636–7650.
Brown V, Jin P, Ceman S, Darnell JC, O’Donnell WT, Tenenbaum SA, Jin X,Feng Y, Wilkinson KD, Keene JD, et al. 2001. Microarray identification ofFMRP-associated brain mRNAs and altered mRNA translational profiles infragile X syndrome. Cell 107:477–487.
Bullock SL. 2011. Messengers, motors and mysteries: Sorting of eukaryoticmRNAs by cytoskeletal transport. Biochem Soc Trans 39:1161–1165.
Cahoy JD, Emery B, Kaushal A, Foo LC, Zamanian JL, Christopherson KS,Xing Y, Lubischer JL, Krieg PA, Krupenko SA, et al. 2008. A transcriptomedatabase for astrocytes, neurons, and oligodendrocytes: A new resourcefor understanding brain development and function. J Neurosci 28:264–278.
Chan SW, Fowler KJ, Choo KH, Kalitsis P. 2005. Spef1, a conserved noveltestis protein found in mouse sperm flagella. Gene 353:189–199.
Chou YH, Khuon S, Herrmann H, Goldman RD. 2003. Nestin promotes thephosphorylation-dependent disassembly of Vimentin intermediate filamentsduring mitosis. Mol Biol Cell 14:1468–1478.
Condorelli DF, Nicoletti VG, Barresi V, Conticello SG, Caruso A, Tendi EA,Giuffrida Stella AM. 1999. Structural features of the rat GFAP gene and iden-tification of a novel alternative transcript. J Neurosci Res 56:219–228.
Dahlstrand J, Lardelli M, Lendahl U. 1995. Nestin mRNA expression corre-lates with the central nervous system progenitor cell state in many, but notall, regions of developing central nervous system. Brain Res Dev Brain Res84:109–129.
Darnell JC, Jensen KB, Jin P, Brown V, Warren ST, Darnell RB. 2001. FragileX mental retardation protein targets G quartet mRNAs important for neuronalfunction. Cell 107:489–499.
Darnell JC, Van Driesche SJ, Zhang C, Hung KY, Mele A, Fraser CE, StoneEF, Chen C, Fak JJ, Chi SW, et al. 2011. FMRP stalls ribosomal translocationon mRNAs linked to synaptic function and autism. Cell 146:247–261.
Doetsch F, Caille I, Lim DA, Garcia-Verdugo JM, Alvarez-Buylla A. 1999. Sub-ventricular zone astrocytes are neural stem cells in the adult mammalianbrain. Cell 97:703–716.
Doyle M, Kiebler MA. 2011. Mechanisms of dendritic mRNA transport and itsrole in synaptic tagging. EMBO J 30:3540–3552.
Eberwine J, Miyashiro K, Kacharmina JE, Job C. 2001. Local translation ofclasses of mRNAs that are targeted to neuronal dendrites. Proc Natl Acad SciUSA 98:7080–7085.
Eliasson C, Sahlgren C, Berthold CH, Stakeberg J, Celis JE, Betsholtz C,Eriksson JE, Pekny M. 1999. Intermediate filament protein partnership inastrocytes. J Biol Chem 274:23996–24006.
Eriksson JE, Dechat T, Grin B, Helfand B, Mendez M, Pallari HM, GoldmanRD. 2009. Introducing intermediate filaments: From discovery to disease. JClin Invest 119:1763–1771.
Etienne-Manneville S. 2004. Actin and microtubules in cell motility: Whichone is in control? Traffic 5:470–477.
Feltrin D, Fusco L, Witte H, Moretti F, Martin K, Letzelter M, Fluri E,Scheiffele P, Pertz O. 2012. Growth cone MKK7 mRNA targeting regulatesMAP1b-dependent microtubule bundling to control neurite elongation. PLoSBiol 10:e1001439.
Femino AM, Fay FS, Fogarty K, Singer RH. 1998. Visualization of single RNAtranscripts in situ. Science 280:585–590.
Freeman MR. 2010. Specification and morphogenesis of astrocytes. Science330:774–778.
Gilyarov AV. 2008. Nestin in central nervous system cells. Neurosci BehavPhysiol 38:165–169.
Herrmann H, Strelkov SV, Burkhard P, Aebi U. 2009. Intermediate filaments:Primary determinants of cell architecture and plasticity. J Clin Invest 119:1772–1783.
Imura T, Nakano I, Kornblum HI, Sofroniew MV. 2006. Phenotypic and func-tional heterogeneity of GFAP-expressing cells in vitro: Differential expressionof LeX/CD15 by GFAP-expressing multipotent neural stem cells and non-neurogenic astrocytes. Glia 53:277–293.
Jensen TH, Patricio K, McCarthy T, Rosbash M. 2001. A block to mRNAnuclear export in S. cerevisiae leads to hyperadenylation of transcripts thataccumulate at the site of transcription. Mol Cell 7:887–898.
Jones-Villeneuve EM, McBurney MW, Rogers KA, Kalnins VI. 1982. Retinoicacid induces embryonal carcinoma cells to differentiate into neurons and glialcells. J Cell Biol 94:253–262.
Katz ZB, Wells AL, Park HY, Wu B, Shenoy SM, Singer RH. 2012. beta-ActinmRNA compartmentalization enhances focal adhesion stability and directscell migration. Genes Dev 26:1885–1890.
Kim M, Bellini M, Ceman S. 2009. Fragile X mental retardation protein FMRPbinds mRNAs in the nucleus. Mol Cell Biol 29:214–228.
Kindler S, Kreienkamp HJ. 2012. Dendritic mRNA targeting and translation.Adv Exp Med Biol 970:285–305.
Landry CF, Watson JB, Kashima T, Campagnoni AT. 1994. Cellular influenceson RNA sorting in neurons and glia: An in situ hybridization histochemicalstudy. Brain Res Mol Brain Res 27:1–11.
Lecuyer E, Yoshida H, Parthasarathy N, Alm C, Babak T, Cerovina T, HughesTR, Tomancak P, Krause HM. 2007. Global analysis of mRNA localizationreveals a prominent role in organizing cellular architecture and function. Cell131:174–187.
Lee JA, Cole GJ. 2000. Localization of transitin mRNA, a Nestin-like interme-diate filament family member, in chicken radial glia processes. J Comp Neu-rol 418:473–483.
Martuszewska D, Ljungberg B, Johansson M, Landberg G, Oslakovic C,Dahlback B, Hafizi S. 2009. Tensin3 is a negative regulator of cell migrationand all four Tensin family members are downregulated in human kidney can-cer. PLoS One 4:e4350.
Medrano S, Steward O. 2001. Differential mRNA localization in astroglial cellsin culture. J Comp Neurol 430:56–71.
Melko M, Bardoni B. 2010. The role of G-quadruplex in RNA metabolism:Involvement of FMRP and FMR2P. Biochimie 92:919–926.
Michalczyk K, Ziman M. 2005. Nestin structure and predicted function in cel-lular cytoskeletal organisation. Histol Histopathol 20:665–671.
Middeldorp J, Boer K, Sluijs JA, De Filippis L, Encha-Razavi F, Vescovi AL, SwaabDF, Aronica E, Hol EM. 2010. GFAPdelta in radial glia and subventricular zoneprogenitors in the developing human cortex. Development 137:313–321.
Middeldorp J, Hol EM. 2011. GFAP in health and disease. Prog Neurobiol93:421–443.
Mili S, Macara IG. 2009. RNA localization and polarity: From A(PC) to Z(BP).Trends Cell Biol 19:156–164.
Mili S, Moissoglu K, Macara IG. 2008. Genome-wide screen reveals APC-associated RNAs enriched in cell protrusions. Nature 453:115–119.
1936 Volume 61, No. 11
Mingle LA, Okuhama NN, Shi J, Singer RH, Condeelis J, Liu G. 2005. Localiza-tion of all seven messenger RNAs for the actin-polymerization nucleator Arp2/3complex in the protrusions of fibroblasts. J Cell Sci 118 (Part 11):2425–2433.
Morest DK, Silver J. 2003. Precursors of neurons, neuroglia, and ependymalcells in the CNS: What are they? Where are they from? How do they getwhere they are going? Glia 43:6–18.
Nielsen AL, Holm IE, Johansen M, Bonven B, Jorgensen P, Jorgensen AL.2002. A new splice variant of glial fibrillary acidic protein, GFAP epsilon,interacts with the presenilin proteins. J Biol Chem 277:29983–29991.
Nielsen AL, Jorgensen AL. 2004. Self-assembly of the cytoskeletal glial fibril-lary acidic protein is inhibited by an isoform-specific C terminus. J Biol Chem279:41537–41545.
Ozsolak F, Platt AR, Jones DR, Reifenberger JG, Sass LE, McInerney P,Thompson JF, Bowers J, Jarosz M, Milos PM. 2009. Direct RNA sequencing.Nature 461:814–818.
Ozsolak F, Ting DT, Wittner BS, Brannigan BW, Paul S, Bardeesy N,Ramaswamy S, Milos PM, Haber DA. 2010. Amplification-free digital geneexpression profiling from minute cell quantities. Nat Methods 7:619–621.
Pekny M, Eliasson C, Chien CL, Kindblom LG, Liem R, Hamberger A,Betsholtz C. 1998. GFAP-deficient astrocytes are capable of stellation in vitrowhen cocultured with neurons and exhibit a reduced amount of intermediatefilaments and an increased cell saturation density. Exp Cell Res 239:332–343.
Pekny M, Wilhelmsson U, Bogestal YR, Pekna M. 2007. The role of astrocytesand complement system in neural plasticity. Int Rev Neurobiol 82:95–111.
Perlson E, Hanz S, Ben-Yaakov K, Segal-Ruder Y, Seger R, Fainzilber M. 2005.Vimentin-dependent spatial translocation of an activated MAP kinase ininjured nerve. Neuron 45:715–726.
Prahlad V, Yoon M, Moir RD, Vale RD, Goldman RD. 1998. Rapid movementsof Vimentin on microtubule tracks: Kinesin-dependent assembly of intermedi-ate filament networks. J Cell Biol 143:159–170.
Quinlan RA, Brenner M, Goldman JE, Messing A. 2007. GFAP and its role inAlexander disease. Exp Cell Res 313:2077–2087.
Schaeffer C, Bardoni B, Mandel JL, Ehresmann B, Ehresmann C, Moine H.2001. The fragile X mental retardation protein binds specifically to its mRNAvia a purine quartet motif. EMBO J 20:4803–4813.
Shankar J, Messenberg A, Chan J, Underhill TM, Foster LJ, Nabi IR. 2010.Pseudopodial actin dynamics control epithelial-mesenchymal transition inmetastatic cancer cells. Cancer Res 70:3780–3790.
Shav-Tal Y, Singer RH. 2005. RNA localization. J Cell Sci 118 (Part 18):4077–4081.
Shestakova EA, Singer RH, Condeelis J. 2001. The physiological significanceof beta-actin mRNA localization in determining cell polarity and directionalmotility. Proc Natl Acad Sci USA 98:7045–7050.
Sofroniew MV, Vinters HV. 2010. Astrocytes: Biology and pathology. ActaNeuropathol 119:7–35.
Stahlberg A, Andersson D, Aurelius J, Faiz M, Pekna M, Kubista M, Pekny M.2011. Defining cell populations with single-cell gene expression profiling:Correlations and identification of astrocyte subpopulations. Nucleic Acids Res39:e24.
Steinert PM, Chou YH, Prahlad V, Parry DA, Marekov LN, Wu KC, Jang SI,Goldman RD. 1999. A high molecular weight intermediate filament-associated protein in BHK-21 cells is Nestin, a type VI intermediate filamentprotein. Limited co-assembly in vitro to form heteropolymers with type IIIVimentin and type IV alpha-internexin. J Biol Chem 274:9881–9890.
Subramanian M, Rage F, Tabet R, Flatter E, Mandel JL, Moine H. 2011.G-quadruplex RNA structure as a signal for neurite mRNA targeting.EMBO Rep 12:697–704.
Tan H, Li H, Jin P. 2009. RNA-mediated pathogenesis in fragile X-associateddisorders. Neurosci Lett 466:103–108.
Tan Y, Xie Z, Ding M, Wang Z, Yu Q, Meng L, Zhu H, Huang X, Yu L, MengX, et al. 2010. Increased levels of FoxA1 transcription factor in pluripotentP19 embryonal carcinoma cells stimulate neural differentiation. Stem CellsDev 19:1365–1374.
Taylor AM, Berchtold NC, Perreau VM, Tu CH, Li Jeon N, Cotman CW. 2009.Axonal mRNA in uninjured and regenerating cortical mammalian axons. JNeurosci 29:4697–4707.
Thomsen R, Lade Nielsen A. 2011. A Boyden chamber-based method forcharacterization of astrocyte protrusion localized RNA and protein. Glia 59:1782–1792.
Thomsen R, Solvsten CA, Linnet TE, Blechingberg J, Nielsen AL. 2010. Analy-sis of qPCR data by converting exponentially related Ct values into linearlyrelated X0 values. J Bioinform Comput Biol 8:885–900.
Tsuruta D, Jones JC. 2003. The Vimentin cytoskeleton regulates focal contactsize and adhesion of endothelial cells subjected to shear stress. J Cell Sci116 (Part 24):4977–4984.
Willis DE, Twiss JL. 2010. Regulation of protein levels in subcellular domainsthrough mRNA transport and localized translation. Mol Cell Proteom 9:952–962.
Willis DE, van Niekerk EA, Sasaki Y, Mesngon M, Merianda TT, Williams GG,Kendall M, Smith DS, Bassell GJ, Twiss JL. 2007. Extracellular stimuli specifi-cally regulate localized levels of individual neuronal mRNAs. J Cell Biol 178:965–980.
Yan Y, Yang J, Bian W, Jing N. 2001. Mouse Nestin protein localizes ingrowth cones of P19 neurons and cerebellar granule cells. Neurosci Lett 302:89–92.
Yoon M, Moir RD, Prahlad V, Goldman RD. 1998. Motile properties of Vimen-tin intermediate filament networks in living cells. J Cell Biol 143:147–157.
Zhou Z, Kahns S, Nielsen AL. 2010. Identification of a novel Vimentin pro-moter and mRNA isoform. Mol Biol Rep 37:2407–2413.
Zhu H, Dahlstrom A. 2007. Glial fibrillary acidic protein-expressing cells in theneurogenic regions in normal and injured adult brains. J Neurosci Res 85:2783–2792.
Thomsen et al.: Nestin mRNA Localization in Mouse Astrocytes
November 2013 1937
top related