2010 Colinet & Hoffmann IBMB .pdf
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Title: Gene and protein expression of Drosophila Starvin during cold stress and recovery from chill coma
Author: HervE Colinet; Ary Hoffmann
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Insect Biochemistry and Molecular Biology 40 (2010) 425e428
Contents lists available at ScienceDirect
Insect Biochemistry and Molecular Biology
journal homepage: www.elsevier.com/locate/ibmb
Gene and protein expression of Drosophila Starvin during cold stress
and recovery from chill coma
Hervé Colinet a, b, *, Ary Hoffmann b
Earth and Life Institute, Biodiversity Research Centre, Université catholique de Louvain, 1348 Louvain-la-Neuve, Belgium
Centre for Environmental Stress and Adaptation Research, Department of Genetics, Bio21 Institute, The University of Melbourne, Parkville, Victoria 3010, Australia
a r t i c l e i n f o
a b s t r a c t
Received 1 February 2010
Received in revised form
11 March 2010
Accepted 15 March 2010
In Drosophila melanogaster, the sole member of the Bcl-2-associated anthanogene (BAG)-family proteins,
called Starvin (Stv), has only been recently described. BAG proteins regulate a large range of physiological
processes including life/death cell balance and stress response. The role of Stv has been poorly studied in
the context of abiotic stress and particularly during and after cold stress. In this study we investigated the
temporal expression of Stv gene and protein in adult ﬂies during both the cold stress (up to 9 h at 0 C)
and the subsequent recovery phase (up to 8 h at 25 C). Because BAG proteins can regulate positively and
negatively the function of Hsp70/Hsc70, we also checked whether Stv expression was related to Hsp70
and Hsc70. Stv mRNA and Stv protein both showed a similar expression pattern: no modulation during
the cold period followed by a signiﬁcant up-regulation during the recovery period. A coordinated
response of Stv and Hsp70 mRNA was observed, but not for Hsc70. Our ﬁndings indicate that Stv
expression is part of a stress-induced program in D. melanogaster. It probably acts as a co-chaperone
modulating the activity of Hsp70 chaperone machinery during recovery from cold stress. Finally our
results support the suggestion that Stv and human BAG3 may be functional homologs.
Ó 2010 Elsevier Ltd. All rights reserved.
Bcl-2-associated anthanogene (BAG)-family proteins are regulators involved in a range of processes (Doong et al., 2002; Takayama
and Reed, 2001). The ﬁrst BAG gene was discovered in a search for
proteins interacting with the anti-apoptotic protein (Bcl-2), and was
shown to synergistically enhance cell survival with Bcl-2 (Takayama
et al., 1995). BAG proteins are found throughout evolution, with
several homologs in vertebrates, insects, nematodes, yeast, and
plants (Takayama et al., 1999; Takayama and Reed, 2001). These
proteins contain different N-terminal sequences but all share
a conserved zone of roughly 50 amino acids near the C-terminal end
called the BAG domain (Briknarová et al., 2002). The BAG domain
binds to the ATPase domain of Hsp70/Hsc70 allowing a positive and
negative regulation of these two chaperones (Doong et al., 2002). In
addition to the conserved BAG domain, BAG proteins also contain
a range of additional domains allowing them to interact with
various speciﬁc target proteins (Takayama and Reed, 2001). BAG
proteins can thus form complexes with a range of transcription
* Corresponding author at: Earth and Life Institute, Biodiversity Research Centre,
Université catholique de Louvain, 1348 Louvain-la-Neuve, Belgium. Tel.: þ32 (0)
E-mail address: firstname.lastname@example.org (H. Colinet).
0965-1748/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved.
factors modulating various physiological processes including
tumorigenesis, cell cycle, neuronal differentiation, apoptosis and
stress response (Takayama and Reed, 2001; Doong et al., 2002;
Kabbage and Dickman, 2008).
Six BAG family members have been identiﬁed in humans
(Takayama and Reed, 2001; Kabbage and Dickman, 2008). In
Drosophila melanogaster, the sole member of BAG family, a 69-kDA
protein called Starvin (Stv), was ﬁrst characterized by Coulson et al.
(2005) who showed that Stv was essential for viability and had an
undeﬁned role in food uptake. More recently, this functional role
has been further investigated. Stv-deﬁcient mutant larvae had
reduced locomotion abilities, suggesting an important role of Stv in
somatic muscles (Coulson et al., 2005; Arndt et al., 2010). Moreover
Stv and BAG3 were suggested to be functional homologs that deﬁne
a conserved chaperone pathway essential for muscle maintenance
through the principle ‘degrade to maintain’ (Arndt et al., 2010).
The silkworm Bombyx mori has a gene called Samui encoding
a homolog of Stv (Moribe et al., 2001). The BAG domains of the
D. melanogaster and Bombyx proteins show sequence identity of
67% and a similarity of 92%. Samui was ﬁrst isolated from
diapausing eggs incubated at 5 C (Moribe et al., 2001). Its
expression was also shown to be modulated in cold-exposed nondiapausing eggs. It was suggested that Samui may play a role in
transmitting a signal that protects non-diapause eggs against cold
H. Colinet, A. Hoffmann / Insect Biochemistry and Molecular Biology 40 (2010) 425e428
injury (Moribe et al., 2001, 2002). So far Samui is the only member
of BAG family reported to be up-regulated by a cold treatment. The
function of the D. melanogaster BAG gene and protein has not yet
been explored in the context of cold stress.
The molecular mechanisms behind recovery from cold stress are
complex and it seems that more genes/proteins are activated
during the recovery phases than during the period of the cold stress
itself (Clark and Worland, 2008). It is thus necessary to differentiate
between the cold and the recovery phases and this aspect has been
emphasized in previous work on alternating temperatures (Colinet
et al., 2007a,b; Lalouette et al., 2007). In the present study we
investigated the time-course expression of Stv gene and protein in
adult D. melanogaster during both the cold stress and recovery
phases. We also checked whether the expression of Stv is related to
Hsp70 and Hsc70.
using the Primer3 module in BioManager (http://www.angis.org.au)
(forward: 50 -TCATCAATGCCCACAAGGAGATAC-30 and reverse: 50 GTTTAGTGGCGTCGGTCTGTTG-30 , amplicon size 314 bp). Hsp70 and
Hsc70 primers were the same as described in Colinet et al. (2010).
Real time PCRs were performed on the LightCyclerÒ 480 system
(Roche Diagnostics, Australia) following the method described in
Colinet et al. (2010). Brieﬂy, after 10 min at 95 C, the cycling
conditions were as follows: 60 cycles at 95 C for 10 s, 60 C for 15 s
and 72 C for 15 s. A post ampliﬁcation melting curve analysis was
performed to validate the speciﬁcity of ampliﬁcation. Relative
expression ratios (i.e., fold change) were calculated using the 2DDCt
method (Livak and Schmittgen, 2001). The ratio of the target gene
was expressed in treated samples versus matched controls (calibrators) and normalized using the housekeeping reference gene, RpS20
(see Colinet et al., 2010 for more details).
2. Materials and methods
2.4. Western blot
2.1. Fly culture
Protein samples for western blots were prepared from 25 adult
males homogenized in a lysis buffer containing 2% SDS, 6.5 mM
TriseHCl (pH 6.8), 10 mM DDT, and a protease inhibitor cocktail
added (Roche Diagnostics, Germany). Proteins were run on NuPAGE
4e12% Bis-Tris gels (Invitrogen, Australia), transferred to nitrocellulose membranes and probed with polyclonal rabbit anti-Starvin
antibody (dilution 1:1000) kindly provided by Robert Saint and
Michelle Coulson (see Coulson et al., 2005). Polyclonal goat antirabbit horseradish peroxidase was used as secondary antibody
(dilution 1:7000) (Dako, Denmark). Monoclonal mouse anti-atubulin was used as a loading control (dilution 1:10 000, Sigma,
T6074). Immunoblots were developed with the LAS-3000 Image
Analyzer (Fujiﬁlm, Australia). Densitometry was performed to
quantify results using FujiFilm Multi Gauge V2.3 computer software
and ratios for protein levels were calculated relative to a-tubulin
We conducted our experiments on a mass-bred D. melanogaster
population derived from about 50 females collected in Innisfail
(a tropical location along the Australian east coast, latitude 17 330 S)
in May 2008. Flies were maintained in 250 ml bottles at 19 C and
70% relative humidity under continuous light on a standard ﬂy
medium contained yeast (3.2%), agar (3.2%), sugar (1.6%), potato
(1.6%) as previously described (Hoffmann and Shirriffs, 2002). Flies
had been transferred at 25 C for 3 generations at the time of
2.2. Cold stress and recovery conditions
Tests were performed using synchronized 4-day old males,
visually sexed without CO2 (using an aspirator). To measure Stv
mRNA expression during the cold stress and during the recovery
period, we used the same method described in Colinet et al. (2010).
Brieﬂy, groups of 20 males were placed in empty glass vials
immersed in a glycol solution cooled to 0 C to induce chill coma.
Flies were then sampled after 0.25, 3, 6 and 9 h of cold stress
(denoted S025, S3, S6 and S9 respectively). After 9 h of cold stress,
ﬂies were returned to 25 C to recover and Stv mRNA expression
was measured after 0.5, 2, 4, and 8 h of recovery (denoted R05, R2,
R4 and R8 respectively). For every sampling time there was a corresponding control, consisting of males kept in food vials at 25 C
for the same duration. To ensure that gene expression was not
confounded by a nutritional effect during recovery (i.e., ﬂies refeeding after cold-induced starvation), mRNA expression was
compared in ﬂies recovering with or without food (as described in
Colinet et al., 2010). Because there was no difference between the
two treatments, mRNA expression is only shown for ﬂies recovering
with food. Modulation of Stv mRNA expression was veriﬁed at the
protein level by measuring expression of Stv protein in control ﬂies
kept at 25 C (Co), in ﬂies cold stressed at 0 C for 9 h (S9) and in
ﬂies recovering at 25 C for 0.5, 2, 4, 6 or 8 h (R05, R2, R6, R4 and R8)
in food vials. Four or two biological replicates were used for mRNA
and protein expression respectively.
3. Results and discussion
The mRNA level of Stv was affected by treatments (ANOVA:
F ¼ 24.49; df ¼ 7,19; P < 0.001) and was signiﬁcantly up-regulated
after cold exposure (Fig. 1). There was no modulation relative to
controls during the cold stress period, then Stv transcripts accumulated signiﬁcantly during the recovery phase. A marked peak of
expression occurred after 2 h of recovery with an average fold
change relative to control reaching 7.8 (Fig. 1). At the protein level,
2.3. RNA extraction and real time PCR
RNA extraction was performed using RNeasy RNA extraction kit
and RNase-Free DNase Set (Qiagen, Australia) as described in Colinet
et al. (2010). Three hundred nanograms of total RNA was used in the
reverse transcription to complementary DNA with the Superscript III
First-Strand Synthesis System for qRT-PCR (Invitrogen, Australia),
according to manufacturer’s instructions. Stv primers were designed
Fig. 1. Change in expression of Stv mRNA, relative to control and normalized against
Rps20 (mean values are expressed as fold change þ SE, n ¼ 4). At values of 1, the
expression is similar to the control value. (*) indicates when a mean value is signiﬁcantly different from 1. (S ¼ cold stressed for 0.25e9 h, R ¼ recovered for 0.5e8 h).
H. Colinet, A. Hoffmann / Insect Biochemistry and Molecular Biology 40 (2010) 425e428
expression of Stv was also signiﬁcantly affected by treatments
(ANOVA: F ¼ 8.792; df ¼ 6,7; P ¼ 0.0056) (Fig. 2A and B). Expression
of Stv protein did not show any modulation during the cold period,
and then it was up-regulated from 2 h of recovery (Fig. 2A and B). At
the mRNA level, signiﬁcant up-regulation was already detected
after 0.5 h, however modulation of protein expression was not yet
detected at this time point. This is not surprising as the processes of
transcription and translation are separated both spatially and in
The function of Stv protein has only recently been investigated
and it appears that it acts as a central proteostasis factor essential
for muscle maintenance in both larvae and adults under nonstressful conditions (Coulson et al., 2005; Arndt et al., 2010). It is not
known whether this function is involved in responding to stress.
However muscle maintenance is likely to be important during
recovery from cold stress since (i) chill coma results from perturbations in muscle resting potentials (Hosler et al., 2000) and (ii)
chilling provokes neuromuscular damage (Kelty et al., 1996).
Stv has been poorly studied, especially in the context of abiotic
stress. As far as we know this is the ﬁrst time that Stv is reported to
be cold-responsive. This result corroborates data obtained with
Samui, the Stv homolog in Bombyx (Moribe et al., 2001). Stv protein
contains a BAG domain which is known to bind to the ATPase
domain of Hsp70/Hsc70 and modulates positively or negatively the
activity of these molecular chaperones (Briknarová et al., 2002;
Doong et al., 2002; Kabbage and Dickman, 2008). There is competition between Hsp70 and other protein partners for binding to the
BAG domain (Takayama and Reed, 2001; Doong et al., 2002).
Therefore, BAG proteins have been suggested to work as a molecular
switch in signalling pathways: they have diverse cellular functions
under normal conditions but regulate Hsp70/Hsc70 which accumulate under stressful conditions (Takayama and Reed, 2001;
Doong et al., 2002). In Drosophila species Hsp70 is known to be
strongly up-regulated in response a large array of stressors
(Hoffmann et al., 2003), including cold stress (Goto and Kimura,
1998; Sinclair et al., 2007). In D. melanogaster males submitted to
the same cold treatments as in the present study, Hsp70 mRNAs
Fig. 2. (A) Western blot of D. melanogaster male whole body extracts using polyclonal
anti-Starvin and monoclonal anti-a-tubulin as internal control (Co ¼ control, S9 ¼ cold
stressed for 9 h, R ¼ recovered for 0.5e8 h). (B) Densitometric measurements of
Starvin protein (mean values þ SE, n ¼ 2). Values are normalized against a-tubulin
values. (*) indicates when a value is signiﬁcantly different from the control value.
(Co ¼ control, S9 ¼ cold stressed for 9 h, R ¼ recovered for 0.5e8 h).
display a temporal pattern of expression very similar to Stv: no
regulation during cold stress followed by a signiﬁcant up-regulation
during the recovery phase with a peak after 2 h (Colinet et al., 2010).
In contrast, the constitutively expressed gene Hsc70 does not show
any signiﬁcant modulation during both cold stress and recovery
periods in D. melanogaster (Colinet et al., 2010). When the expression of Stv, Hsp70 and Hsc70 genes was simultaneously measured in
a sample and across all conditions, a positive relationship between
Stv and Hsp70 was observed (Fig. 3; regression: F ¼ 103.2; df ¼ 1,49;
P < 0.001; r2 ¼ 0.678). However, there was no relation between Stv
and Hsc70 (Fig. 4; regression: F ¼ 0.743; df ¼ 1,49; P ¼ 0.392;
r2 ¼ 0.014). The fact that Stv displays a similar temporal mRNA
accumulation to Hsp70 suggests an association between the two
partners. Experimental tests (e.g., in vitro and in vitro binding
assays) might be carried out to conﬁrm this association, although
the interaction of BAG proteins with Hsc70/Hsp70 has previously
been established using protein binding assays (e.g., Takayama et al.,
1999; Moribe et al., 2001).
Stv protein shares sequence similarity with human BAG3
(Coulson et al., 2005) and these two proteins have recently been
shown to be functional homologs (Arndt et al., 2010). Among
proteins of the BAG family, human BAG3 is the only one reportedly
inducible by stressors (Rosati et al., 2007). Moreover, it has been
observed that human BAG3 and Hsp70 mRNAs also display a coordinated temporal pattern of expression following heat stress
(Pagliuca et al., 2003). Our results thus support the notion that Stv
may have functional similarities with human BAG3.
BAG proteins exert an important role in regulating the life/death
cell balance (Rosati et al., 2007). They are known to inhibit
apoptosis through different mechanisms, either via their interactions with Hsc70/Hsp70 and/or via binding to the anti-apoptotic
protein Bcl-2. The interaction with Hsc70/Hsp70 indirectly regulates the chaperone-mediated protein refolding or protein degradation process (Doong et al., 2002). Yi et al. (2007) have shown that
the apoptotic inhibitor Bcl-2 was down-regulated in cold-shocked
ﬂies and also shown that a freezing treatment induced apoptosis in
D. melanogaster cells. In this experiment an acute cold stress (5 C)
was used whereas in present experiment ﬂies were exposed to
non-freezing chronic cold stress (0 C for 9 h). Therefore we do not
know whether the apoptosis machinery may be modulated during
non-freezing cold stress and/or during recovery. Our preliminary
tests on expression of the D. melanogaster death executioner Bcl-2
homolog (debcl) did not show any modulation during either cold
stress or recovery (data not shown).
Fig. 3. Positive relationship between expression of Hsp70 and Stv genes. Data are
normalized against Rps20 as reference. Data are expressed as Log (2Ct targetCt reference).
Each dot represents the level of expression of Hsp70 and Stv measured in the same
cDNA sample. The line indicates the linear regression.
H. Colinet, A. Hoffmann / Insect Biochemistry and Molecular Biology 40 (2010) 425e428
Fig. 4. Absence of relationship between expression of Hsc70 and Stv genes. Data are
normalized against Rps20 as reference. Data are expressed Log (2Ct targetCt reference).
Each dot represents the level of expression of Hsc70 and Stv measured in the same
Taken together, our ﬁndings indicate that Stv expression is part
of a stress response to cold exposure in D. melanogaster and this
was observed at both the mRNA and protein levels. The coordinated
response of Stv and Hsp70 points to an interaction between the two
genes. Our results support the suggestion that Stv and human BAG3
may be functional homologs (Arndt et al., 2010). The precise role of
Stv during stress response to cold is not yet known. However like
human BAG proteins, it probably functions as a co-chaperone
modulating the activity of Hsp70 chaperone machinery during
recovery from cold stress.
We are grateful to Robert Saint and Michelle Coulson for
providing Starvin antibodies and Adam Southon for assistance with
protein work. This study was supported by « Fonds de la Recherche
Scientiﬁque e FNRS » and the Australian Reseach Council and the
Commonwealth Environmental Research Fund. This paper is
number BRC 168 of the Biodiversity Research Centre (BDIV).
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