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Copyright � 2007 by the Genetics Society of AmericaDOI:
10.1534/genetics.107.071472
The Molecular Chaperone Hsp90 Is Required for mRNA Localization
inDrosophila melanogaster Embryos
Yan Song,*,1 Lanette Fee,* Tammy H. Lee* and Robin P.
Wharton*,†,2
*Howard Hughes Medical Institute, Department of Molecular
Genetics & Microbiology and †Department of Cell Biology,Duke
University Medical School, Durham, North Carolina, 27710
Manuscript received January 29, 2007Accepted for publication May
25, 2007
ABSTRACT
Localization of maternal nanos mRNA to the posterior pole is
essential for development of both theabdominal segments and
primordial germ cells in the Drosophila embryo. Unlike maternal
mRNAs such asbicoid and oskar that are localized by directed
transport along microtubules, nanos is thought to be trapped asit
swirls past the posterior pole during cytoplasmic streaming.
Anchoring of nanos depends on integrity of theactin cytoskeleton
and the pole plasm; other factors involved specifically in its
localization have not beendescribed to date. Here we use genetic
approaches to show that the Hsp90 chaperone (encoded by Hsp83
inDrosophila) is a localization factor for two mRNAs, nanos and
pgc. Other components of the pole plasm arelocalized normally when
Hsp90 function is partially compromised, suggesting a specific role
for thechaperone in localization of nanos and pgc mRNAs. Although
the mechanism by which Hsp90 acts is unclear,we find that levels of
the LKB1 kinase are reduced in Hsp83 mutant egg chambers and that
localization of pgc(but not nos) is rescued upon overexpression of
LKB1 in such mutants. These observations suggest that LKB1is a
primary Hsp90 target for pgc localization and that other Hsp90
partners mediate localization of nos.
SUBCELLULAR localization of mRNA is an efficientstrategy for
spatially restricting the encoded pro-tein ( Johnstone and Lasko
2001; St. Johnston 2005).This strategy is common in highly
polarized cell typessuch as neurons, as well as during the
development ofoocytes or embryos when gene regulation is limitedto
post-transcriptional mechanisms. Asymmetric RNAlocalization prior
to mitosis also serves to distinguishdaughter cells in
Saccharomyces cerevisiae. In this organ-ism, localization of ASH1
mRNA to buds just prior tocytokinesis currently provides the most
completely un-derstood example of mRNA localization (Niessing et
al.2004; Gonsalvez et al. 2005).
Localization of maternal mRNAs drives much of thepatterning
along the anteroposterior axis of the Dro-sophila embryo. The
anterior determinant, bicoid (bcd)mRNA, is localized during
oogenesis as ribonucleopro-tein (RNP) cargo associated with
molecular motors thattraverse the microtubule cytoskeleton
(Riechmann et al.2002; Schnorrer et al. 2002; Snee et al. 2005;
Weil et al.2006). Posterior patterning is nucleated by the
localiza-tion of oskar (osk) mRNA to the posterior pole of
theoocyte, also via directed movement along microtubules
(Cha et al. 2002; Braat et al. 2004; Huynh et al. 2004;Yano et
al. 2004). Localization of both bcd and osk mRNAsis thought to
occur via complex, multistep pathwayswith many components.
One role of localized Osk is to direct the
subsequentlocalization of a fraction of the nanos (nos) mRNA late
inoogenesis (Bergsten and Gavis 1999). Localized nosmRNA is the
sole source of Nos protein in the earlyembryo (Gavis and Lehmann
1992) where it plays anumber of key roles in development. Nos is
required inthe somatic cytoplasm of the early embryo to
represstranslation of maternal hunchback mRNA, thereby gov-erning
abdominal segmentation (Sonoda and Wharton1999). Nos is also
required in the primordial germ cellsthat form at the posterior
extreme of the embryo to de-lay proliferation, repress
transcription, facilitate mi-gration into the somatic gonad, and
promote survival(Kobayashi et al. 1996; Forbes and Lehmann
1998;Asaoka-Taguchiet al. 1999; Schaner et al. 2003; Hayashiet al.
2004; Kadyrova et al. 2007). The germ line func-tions of Nos appear
to be conserved in many organisms(Subramaniam and Seydoux 1999;
Tsuda et al. 2003).
The role of Osk in nos mRNA localization is indirect;Osk governs
assembly of the pole plasm (specializedcytoplasm that specifies
germ line identity) via anelaborate, genetically defined pathway in
which recruit-ment of nos mRNA is one of the final steps
(Ephrussiand Lehmann 1992; Kim-Ha et al. 1993). Recruitment
isthought to involve trapping of nos RNPs as they swirlpast the
posterior pole during cytoplasmic streaming
1Present address: Department of Pathology, Stanford University
Schoolof Medicine, Stanford, CA.
2Corresponding author: Howard Hughes Medical Institute,
Departmentof Molecular Genetics & Microbiology, Box 3657, Duke
UniversityMedical School, Durham, NC 27710. E-mail:
[email protected]
Genetics 176: 2213–2222 (August 2007)
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(Forrest and Gavis 2003; Serbus et al. 2005), ratherthan the
directed movement that underlies localizationof osk or ASH1 RNPs.
The factors involved specifically inlocalizing nos mRNA have yet to
be identified.
In this report, we describe the results of a geneticscreen for
nos mRNA localization factors that rely onthe abdomen-patterning
role of Nos.
MATERIALS AND METHODS
Isolation and mapping of the 966 mutation: Homozygousw1118 ; e
nosBN males were mutagenized with EMS and mateden masse with w1118
virgin females. Male progeny were individ-ually crossed to generate
P½mini-nos1�, nosBN/e nosBN * females,which were screened for the
ability to give rise to hatchingprogeny. The P½mini-nos1� transgene
is described by Dahanukarand Wharton (1996), where it is named
nos1(DBX). Candi-date mutant chromosomes (*) were recovered from
siblingmales for further testing. Meiotic recombination with a
rucucachromosome placed 966 between ru and h on the left arm ofthe
third chromosome. Fine mapping by P element-inducedmale
recombination further mapped 966 to a 57-kb intervalbetween
P{SUPor-P}KG05210 and P{SUPor-P}KG00982. The966 mutation was
definitively identified by sequencing theHsp90 coding region
amplified from genomic DNA extractedfrom homozygous 966 larvae
(identified by the absence of aGFP-marked balancer chromosome).
Fly strains and reagents: The following strains were fromthe
Bloomington Stock Center: Hsp83 alleles scratch (08445),E317K
(e6D), S529F (e6A), and j5C2A; transformants bearing the17.5
genomic Hsp83 rescue construct; flies with the P{SUPor-P}KG05210,
P{SUPor-P}KG00982, P{SUPor-P}KG03657, andP{SUPor-P}KG07503 elements
used in male recombination.Flies with the maternal tubulin-GAL4
driver as well as the GFP-LKB1 transgene (Huynh et al. 2001; Martin
and St. Johnston2003) were from D. St. Johnston; flies with the
P{GAL4-arm.S}11armadillo-GAL4 driver were from Bloomington.
Antibodiesagainst various proteins were gifts of P. Macdonald (Hb),
A.Nakamura (Nos), D. St. Johnston (Stau), A. Ephrussi (Osk), andK.
Howard (Vas).
Immunohistochemistry: Egg chamber fixation and antigendetection
were performed as described (Palacios and St.Johnston 2002).
Primary antibodies were diluted as follows:chicken anti-Vas 1:2000,
rabbit anti-Osk 1:2000, rabbit anti-Stau 1:2000, rat anti-Hb
(1:500), and rabbit anti-Nos 1:1000.FITC-, rhodamine-, or
Texas-red-conjugated secondary anti-bodies ( Jackson Laboratories)
were used at 1:200. Nuclei were
stained with TOTO-3, oligreen, or TOPRO-3 (MolecularProbes).
Samples were mounted in Vectashield and imagedon a Zeiss LSM510
confocal microscope. For embryo staining,primary antibodies were
diluted as follows: rat anti-Hb (1:500),rabbit anti-Nos 1:1000,
rabbit anti-Osk 1:2000. In situ hybrid-ization was by standard
methods using digoxigenin-labeleddsDNA probes prepared from cDNA
clones. The adducin-like/hts probe was from clone N4 (Ding et al.
1993).
Western and Northern blots: Homozygous 966 mutantlarvae (e.g.,
non-Tubby) were identified shortly after hatchingand grown under
noncrowded conditions. Samples fromwhole larvae homogenized in SDS
sample buffer were ana-lyzed following transfer to Immobilon P by
standard methods.Hsp90 was detected with the 3E6-1.92 monoclonal
antibody,a gift from R. Tanguay (Carbajal et al. 1990), and ECL
Plus(Amersham). The loading control was a-tubulin, detectedwith DM
1A monoclonal antibody (Sigma F-2168) and ECL(Amersham). For
Northern blots, 5-mg samples of total RNAprepared from 0- to 4.5-hr
embryos were analyzed by standardmethods using radio-labeled probes
to detect smaug mRNA(as a loading control) and mini-nos1 mRNA.
Quantitationwas performed on a Typhoon phosphoimager.
RESULTS
A genetic screen for nos localization factors: Toidentify
factors involved in nos mRNA localization, wechemically mutagenized
flies and screened for domi-nant maternal-effect mutations that
(further) compro-mise abdominal segmentation in a sensitized
background.The rationale for the screen is shown in Figure
1.Inefficient localization and translation of a mini-nos1
mRNA generates only sufficient Nos activity to allowdevelopment
of 5–6 abdominal segments if the endog-enous nos genes are mutant
(Dahanukar and Wharton1996). In such a background, we reasoned that
a mu-tation in one of the two alleles encoding a localizationfactor
might compromise Nos activity sufficiently to pre-clude hatching
(either because the allele is dominantnegative or the gene is
haplo-insufficient). A similarrationale has been used extensively
in screens based onchanges in morphology of the Drosophila eye
(e.g., Rebayet al. 2000).
From a pilot screen of�6000 EMS-mutagenized thirdchromosomes, we
isolated a number of mutations that
Figure 1.—A dominant modifier screen for nosmRNA localization
factors. Shown schematicallyare the 39-UTRs of wild-type nos1 mRNA
and amini-nos1 mRNA that lacks nt 185-849. The firstcolumn shows a
qualitative assessment of the rela-tive efficiency of mRNA
localization to the poste-rior pole (see Figure 2), the second
columnshows the typical number of abdominal segmentsin embryos
where the sole source of Nos is the in-dicated mRNA, and the third
column is a drawingof the body plan after 24 hr of embryonic
develop-ment. The rationale for the screen is that an EMS-induced
mutation (*) would dominantly reducesegmentation such that embryos
do not hatch(and thus remain within the vitelline membrane).
2214 Y. Song et al.
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reduce or eliminate abdominal segmentation in thesensitized
background. One mutation proved to be anallele of pumilio and
another an allele of spindle-E; bothgenes are known to play roles
in posterior specification(Lehmann and Nüsslein-Volhard 1987;
Gillespieand Berg 1995; Martin et al. 2003; Cook et al.
2004),validating the premise of the screen. A third mutation,966,
is the subject of this report.
The 966 mutation appears to affect the localizationbut not the
synthesis or stability of mini-nos1 mRNA(Figure 2). Both the
analysis of Northern blots (Figure2A) and examination of the
unlocalized mini-nos1
mRNA in embryos hybridized with digoxigenin-labeledprobes
(Figure 2B) support the idea that mini-nos1
mRNA stability is unaffected by the 966 mutation. Incontrast,
localization of mini-nos1 mRNA is defective
from the beginning of embryonic development (stage1) through
formation of the syncytial blastoderm (stage4) in embryos from
heterozygous 966 mutant females(Figure 2B). Because Nos protein is
generated exclu-sively from translation of localized mRNA, the
embryosfrom 966 heterozygotes apparently have reduced Nosactivity,
because Hunchback (Hb) accumulates in theposterior and they
subsequently fail to develop abdom-inal segments (Figure 2B). The
966 allele is homozygouslethal and germ line clones are
rudimentary, precludinganalysis of embryos derived from homozygous
females.
We examined the distribution of other mRNAs inembryos from
heterozygous 966 mutant females (here-after 966 mutant embryos) to
determine whether thedefects in mini-nos1 mRNA localization are
specific. Asshown in Figure 3, the localization of full-length
nos1
Figure 2.—The 966 mutation dominantly inter-feres with
localization of mini-nos1 mRNA. (A)Northern blot of samples from 0
to 4.5 hr nosBN/P½mini-nos1�, nosBN (lane 1) and 966
nosBN/P½mini-nos1�, nosBN (lane 2) embryos to detectsmaug and
mini-nos1 mRNAs. The mobility of mo-lecular weight markers is
indicated on the left. (B)The distributions of mini-nos1 mRNA
during em-bryonic stages 2 (early cleavage, first row) and
4(syncytial blastoderm, second row), Hb proteinin nuclear cycle 10
(third row), and the
resultingembryonicbodyplan(darkfieldmicrographsof se-creted cuticle
in the fourth row) are shown. Hereand in all subsequent figures,
embryos and eggchambers are oriented anterior to the left and
dor-sal up. Note that the enzymatic reaction used todetect
hybridized digoxigenin-labeled probe wasnearly twice as long for
the embryos in row 2 (vs.row 1) due to the presence of reduced mRNA
lev-els; control experiments suggest that an apprecia-
ble fraction of the signal in the somatic cytoplasm is due to
background at the longer reaction time. In the cuticle
photographs,abdominal segments are marked by the presenceof bands
of large denticles that are white in darkfield; theeighth abdominal
segmentis highlighted with an arrowhead on the left. The maternal
genotypes are indicated above.
Figure 3.—The 966 mutation does not signifi-cantly affect
localization of other mRNAs in theearly embryo. The distributions
of various mRNAs(indicated on the left) are shown in stage 2
em-bryos. Insets on rows 1 and 2 show the posteriorof stage 4
embryos. The maternal genotypes are in-dicated above. Note that
other experiments showthat the distributions of pgc, bcd, and
add-like mRNAsare unaffected by the nos genotype (not shown).
Hsp90 As an mRNA Localization Factor 2215
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mRNA is essentially normal in early 966 mutant em-bryos,
although the pole cells in slightly older embryosappear to retain
somewhat reduced levels of mRNA. Incontrast, localization of pgc
mRNA to the posterior orbicoid and adducin-like mRNAs to the
anterior is in-distinguishable in wild-type and 966 mutant
embryos.
We next wished to determine whether the 966 geneproduct acts
late in the posterior pathway (when nosmRNA is localized) or early,
perhaps acting indirectly togovern oocyte polarity and accumulation
of Osk, forexample. Three observations suggest that the 966
geneproduct acts downstream of Osk. First, Osk accumula-tion is
normal in 966 mutant embryos (Figure 4A). Sec-ond, Osk activity
appears normal in 966 mutant embryosby the criterion that they have
a similar number of polecells as wild-type embryos (data not
shown). The forma-tion of pole cells is known to be a sensitive
indicator ofOsk activity (Ephrussi and Lehmann 1992; Smith et
al.1992). Third, we find that the 966 mutation interfereswith
localization of mini-nos1 mRNA to the anterior ofosk-bcd embryos
(Figure 4B). In osk-bcd embryos, a chi-meric mRNA (bearing the
protein-coding region of oskand the localization signals of bcd)
generates Osk activityat the anterior of the embryo independent of
the up-stream factors that regulate localization and translationof
native osk mRNA. This ectopic Osk directs efficientlocalization and
translation of full-length nos1 mRNA,resulting in the suppression
of head and thoracic seg-ments, which are replaced by a mirror
symmetric dupli-cation of abdominal segments (Ephrussi and
Lehmann1992). We find that mini-nos1 mRNA is inefficiently
lo-calized to the anterior of osk-bcd embryos where it ap-parently
is translated into only enough Nos to suppresshead segmentation
(Figure 4B). If, in addition, the fe-males are heterozygous for
966, then recruitment ofmini-nos1 mRNA to the anterior is almost
eliminatedand the suppression of anterior development by ectopicNos
is largely relieved (Figure 4B).
Taken together, the results described above suggestthat the 966
mutation alters the function of a factor thatis required downstream
of Osk to localize mini-nos1
mRNA.Hsp90 requirement for mRNA localization: We
mapped the 966 mutation (primarily scoring the asso-ciated
homozygous lethality) using deficiencies, meioticrecombination, and
P element-induced male recombi-nation. In the course of these
experiments, we discov-ered that 966 is semilethal in trans to the
scratch (stc)allele (Yue et al. 1999) of Hsp83, which encodes
thehighly conserved Hsp90 chaperone. Two additionallines of
evidence demonstrate that 966 is indeed anallele of Hsp83. First,
the Hsp83 gene on the 966 chro-mosome bears a single nucleotide
substitution thatresults in an alanine to aspartate substitution at
a highlyconserved residue (133) in the N-terminal ATPasedomain
(Figure 5A). Second, two independently iso-lated Hsp83 alleles that
encode missense forms of the
Hsp90 protein (E317K and S592F) (Cutforth andRubin 1994; van der
Straten et al. 1997) suppressabdominal segmentation in the
mini-nos1 backgroundin a manner similar to 966 (Figure 5A). Thus,
Hsp90function is required for normal localization of mini-nos1
mRNA.We next asked whether the A133D allele acts in a
dominant negative or haplo-insufficient manner tomodify
mini-nos1-dependent segmentation. The E317Kand S592F alleles were
identified in screens for domi-nant modifiers of kinase-dependent
eye phenotypes;since they also modify mini-nos1 activity, we
suspectedthat the A133D mutant and the other missense mutantsact as
dominant negatives for mini-nos1 localization.Consistent with such
an idea, a presumptive null alleleof Hsp83, j5C2A, which bears a
P-element in the first(nonprotein-coding) exon, does not dominantly
mod-ify the mini-nos1 segmentation phenotype. Moreover,rescue of
the segmentation phenotype caused by theA133D allele is
inefficient. A single wild-type Hsp83transgene rescues very poorly,
yielding embryos with 1 to2 abdominal segments; two copies of the
wild-type Hsp83transgene are required for full rescue (Figure
5B).Finally, we find that the A133D mutant protein accu-mulates to
wild-type levels in extracts prepared fromlarvae �1 day before they
die (Figure 5B). Takentogether, these results suggest that the
A133D missenseprotein dominantly interferes with wild-type Hsp90
withrespect to the mini-nos1 segmentation phenotype.
To further investigate the role of Hsp90 in localiza-tion of
maternal mRNAs in general and full-length nos1
Figure 4.—The 966 gene product acts downstream of Os-kar in the
posterior pathway to localize mini-nos1 mRNA. (A)The distribution
of Osk in stage 2 embryos, maternal geno-types as indicated. (B)
The distribution of mini-nos1 mRNAin stage 2 embryos and anterior
cuticle (phase-contrast micro-graphs) of mature embryos from
females of the indicated ge-notype. Note that virtually the entire
head skeleton is absentin the embryo on the left, with only a trace
of sclerotized cu-ticle in evidence (arrowhead). With the exception
of minordefects in the dorsal bridge (arrowhead), the head
skeleton(arrowhead) in the embryo on the right is wild type.
2216 Y. Song et al.
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mRNA in particular, it was necessary to bypass therequirement of
Hsp90 function for viability. A compre-hensive study of Hsp83
alleles had shown that severaltrans-heterozygous allelic
combinations are viable andfertile (Yue et al. 1999). We
reinvestigated the issue andfound that Hsp83stc/Hsp83E317K females
are reasonablyhealthy and produce large numbers of eggs, of
which1–2% develop through late embryonic stages. We there-fore used
this genetic background to investigate theconsequences of impairing
maternal Hsp90 functionon mRNA localization.
As shown in Figure 6, localization of full-length nos1
mRNA is defective in 90–95% of embryos when mater-nal Hsp90
function is compromised. The phenotype isheterogeneous and includes
nearly normal localizationof a reduced amount of nos RNA to a
crescent along theposterior cortex, localization of a ‘‘ball’’ of
nos mRNAonly partially in contact with the posterior cortex,diffuse
localization of a cloud near the posterior, andno detectable
localization. These defects in mRNAlocalization have predictable
effects on subsequentdevelopment: essentially all
Hsp83stc/Hsp83E317K embryoshave reduced levels of Nos protein
(Figure 6). Very fewof these embryos cellularize, presumably due to
someother requirement(s) for maternal Hsp90 function. Butthe 1–2%
of embryos that eventually secrete cuticle haveon average 3
abdominal segments rather than thenormal complement of 8 (Figure
6). Thus, maternalHsp90 is critical for posterior localization of
nos1 mRNA.
We next wished to determine whether localization ofother
maternal mRNAs also relies on Hsp90. Accord-
ingly, we examined the distributions of polar granulecomponent
(pgc), germcell-less (gcl), CyclinB (CycB) (Lehnerand O’Farrell
1990; Jongens et al. 1992; Nakamuraet al. 1996), and bcd mRNAs in
Hsp83stc/Hsp83E317K em-bryos. Of these, only pgc mRNA localization
is abnormal(Figure 6 and not shown). Localization of pgc mRNA
isaffected to a greater extent than is localization of nosmRNA.
Because Hsp90 activity is only partially ablated inthe
Hsp83stc/Hsp83E317K egg chambers, we do not knowwhether the
posterior localization of gcl and CycB mRNAsand the anterior
localization of bcd mRNA rely on lowerHsp90 activity or whether
they are Hsp90 independent.However, the significant observation is
that proper de-ployment of only a subset of the late-localizing
posteriormRNAs (nos and pgc) requires normal Hsp90 activity.
Hsp90 is thought to interact with hundreds of pro-teins in most
cells (Millson et al. 2005; Zhao et al. 2005)and Hsp83 mutants are
highly pleiotropic (Yue et al.1999). We therefore considered
whether the defects innos and pgc localization might be quite
indirectly causedby earlier defects in the assembly of pole plasm
com-ponents at the posterior of the egg chamber. To thisend, we
examined the localization of upstream compo-nents of the posterior
pathway in Hsp83stc/Hsp83E317K eggchambers where Hsp90 activity is
compromised.
Reduction of Hsp90 activity has a relatively specificeffect on
the posterior localization of nos and pgc mRNAs.The initial
localization of three key factors that act up-stream of nos is
essentially normal in Hsp83stc/Hsp83E317K
egg chambers (Figure 7). These include: (1) Stau protein½an
effective proxy for osk mRNA (Martin et al. 2003)�,
Figure 5.—966 is a dominantnegative allele of Hsp83. (A) Atthe
top is a schematic representa-tion of the domains of Hsp90and the
positions of missense sub-stitutions encoded by the 966 al-lele and
the two other allelesused in these studies. Abdominalsegmentation
is visualized in dark-field photographs of cuticle se-creted by
embryos from femalesheterozygous for the indicatedHsp83 allele
(except in row 1,which is from an Hsp831/Hsp831
female). The females here and inB are also P½mini-nos1�,
nosBN/nosBN,such that the sole source of Nosin embryos is from the
mini- nos1
transgene. (B) The A133D proteinacts in a dominant negative
fashion.Cuticle of embryos from femalesheterozygous for the
indicatedHsp83 allele, as in A. Embryos inrows 2 and 3 are from
females that
also bear oneand two extra copies of an Hsp831 transgene,
respectively. Note that onecopy of the transgene does not
efficiently rescuethe lethalityofHsp83A133D/Hsp83stc animals,
consistentwith theidea that thisallele,
likeothermissenseallelesofHsp83(CutforthandRubin 1994; van der
Straten et al. 1997), is dominant-negative. Western blot of
extracts (each containing 5 mg of protein) preparedfrom w1118 (lane
1), nosBN/TM3 (lane 2), and 966 nosBN/966 nosBN (lane 3) larvae
72–74 hr after egg laying. Following transfer, themembrane was cut
in half and probed separately with antibodies against Hsp90 and
a-tubulin.
Hsp90 As an mRNA Localization Factor 2217
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(2) Osk protein, and (3) Vasa protein. Since accumula-tion of
Osk is acutely interdependent with localizationof osk mRNA, Vasa,
and the Par-1 kinase (Breitwieseret al. 1996; Riechmann et al.
2002; Benton and St.Johnston 2003; Johnstone and Lasko 2004), the
datapresented in Figure 7 suggest that pole plasm assemblyis
essentially normal until the late recruitment of nos andpgc.
Oogenesis appears grossly normal in the Hsp83stc/Hsp83E317K
background, which suggests that polarizationof the microtubule
cytoskeleton is likely to be normal.This idea is further supported
by the normal initial lo-calization of Stau to the posterior and
the maintenanceof bcd mRNA at the anterior, both dependent
criticallyon microtubule integrity (Pokrywka and Stephenson1991;
Brendza et al. 2000; Weil et al. 2006). Not onlyis the initial
formation of pole plasm normal, but also itsintegrity is maintained
in Hsp83stc/Hsp83E317K embryos,based on the normal posterior
localization of Osk andVasa (Figure 7). Distribution of the latter
protein was
detected in double-staining experiments, which showthat
Hsp83stc/Hsp83E317K embryos with very little or nodetectable Nos
have a normal crescent of Vasa at theposterior pole. Thus, both the
initial formation andmaintenance of the pole plasm appear
unperturbed inHsp83stc/Hsp83E317K flies.
Taken together, the observations outlined above leadus to
conclude that, despite its general pleiotropy,Hsp90 plays a
relatively specific role for the localizationof nos and pgc mRNAs
to the posterior of the embryo.
Rescue of pgc mRNA localization in Hsp83 mutantembryos by
overexpression of LKB1: Hsp90 is a molec-ular chaperone that
activates and stabilizes a wide varietyof client regulatory and
signaling proteins (Pearl andProdromou 2001); a priori, it seemed
unlikely that Hsp90interacts directly with nos and pgc mRNAs.
Therefore, oneapproach to further understanding its role in
mRNAlocalization would be to identify molecules whose activ-ity is
dependent on Hsp90. To date, none of the othergenetic modifiers
identified in the screen that yieldedthe 966 allele of Hsp83
encodes an obvious Hsp90 client.Therefore, we turned to a candidate
gene approach, fo-cusing on protein kinases previously implicated
in vari-ous aspects of posterior patterning. Two such proteins
arePar-1 and LKB1, which are required for polarization ofthe oocyte
microtubule cytoskeleton and the proper de-position of osk mRNA at
the posterior (Shulman et al.2000; Tomancak et al. 2000; Martin and
St. Johnston2003; Doerflinger et al. 2006).
Two lines of evidence suggest that LKB1 is a signifi-cant Hsp90
client for the localization of pgc mRNA. First,the level of
GFP-LKB1 is significantly reduced whenHsp90 activity is compromised
in essentially all Hsp83stc/Hsp83E317K egg chambers (Figure 8A). In
contrast, noconsistent effect of reducing Hsp90 activity is seen
onlevels of GFP-Par-1 (not shown). Second, overexpres-sion of LKB1
in Hsp83stc/Hsp83E317K females significantlyrescues the
localization of pgc mRNA (Figure 8B). Forthis experiment, we used
the armadillo-GAL4 driver toachieve low-level overexpression of
GFP-LKB1 in the ova-ries, as previously described (Martin and St.
Johnston2003). The rescue of pgc localization does not appear tobe
due to an indirect elevation of posterior Osk levels,which are
normal during both oogenesis and early em-bryogenesis (Figure 8B);
these observations are consistentwith the previous finding that up
to 10-fold overexpres-sion of LKB1 has no significant effect on
localization of thepole plasm component Stau (Martin and St.
Johnston2003). In contrast to the rescue of pgc, no significant
res-cue of nos localization was observed (Figure 8B).
Similarnegative results were obtained using nos-GAL4-VP16 todrive
higher level expression of LKB1 in the germ line(Van Doren et al.
1998) (not shown). Taken together,these observations suggest that,
for localization of pgcmRNA, a major function of Hsp90 is to
stabilize LKB1.Presumably other Hsp90 partners or targets mediate
lo-calization of nos mRNA.
Figure 6.—Hsp90 is required for localization of nos and
pgcmRNAs. The distributions of various mRNAs and proteins(except in
row 3, which shows cuticle secreted by mature em-bryos) in stage 2
embryos from wild-type (w1118) and Hsp83stc/Hsp83E317K females.
Many of the Hsp83stc/Hsp83E317K embryoshave terminal defects not
readily visible in the photograph,consistent with previous reports
that Hsp90 is a cofactor ofthe Raf kinase (van der Straten et al.
1997), which actsdownstream of Torso to specify terminal identity
in the em-bryo. The embryos in row 5 were hybridized
simultaneouslywith probes to bcd (the prominent signal at the
anterior)and pgc (the low-level unlocalized signal as well as the
poste-rior cap that is present in wild type but not Hsp83 mutant
em-bryos). Note that many Hsp83 mutant embryos have anabnormal
shape, with elongated anterior ends to which thevitelline membrane
and micropyle frequently remain at-tached (particularly evident in
rows 4 and 5).
2218 Y. Song et al.
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DISCUSSION
The specific role that maternal Hsp90 plays inlocalization of a
subset of mRNAs to the pole plasm issomewhat surprising, given the
number of proteins thatare thought to require the activity of this
chaperone.Although ubiquitously distributed, Hsp90 is enrichedin
the testes and ovaries and the male germ line is par-ticularly
sensitive to a reduction in Hsp90 activity (Yueet al. 1999). The
experiments reported here define a rolefor maternal Hsp90 in the
localization of nos and pgcmRNAs. We do not yet know the mechanism
by whichHsp90 acts. However, the apparent integrity of the
poleplasm in Hsp83stc/Hsp83E317K ovaries and embryos (Fig-ures 6
and 7) and the rescue of pgc localization uponoverexpression of a
single kinase (Figure 8) are consis-tent with the idea that the
defects in pgc (and perhapsalso nos) localization arise from the
reduction in activityof a few discrete Hsp90 clients. Hsp83 mRNA is
itselfconcentrated in the pole plasm (Ding et al. 1993) andHsp90 is
found at particularly high levels in the germline precursors of
other organisms (Vanmuylder et al.2002; Inoue et al. 2003), which
may reflect a conservedrole in mRNA localization.
We do not know whether Hsp90 acts directly orindirectly to
stabilize LKB1. Mammalian LKB1 bindsdirectly to Hsp90 and Cdc37, a
cochaperone for kinaseclients (Boudeau et al. 2003). However, we
have notobserved a direct interaction between Drosophila Hsp90and
LKB1, either by co-immunoprecipitation or in yeast
interaction experiments in which the DNA-bindingdomain was fused
to the Hsp90 C terminus to avoidinterfering with dimerization, as
described (Millsonet al. 2005). We do not currently know whether
Hsp90binding to LKB1 is ephemeral (and thus difficult todetect) or
whether Hsp90 acts indirectly to stabilizeLKB1.
How might LKB1 act to localize pgc mRNA? Despiteits conserved
role in regulation of cellular polarity (seeAlessi et al. 2006 and
references therein), no LKB1substrate that plays a direct role in
mRNA localizationhas been described, to our knowledge. Loss of
LKB1function in germ line clones of presumptive null
allelesprevents the reorganization of the oocyte microtubulenetwork
at stage 7 that is required for posterior lo-calization of osk mRNA
and affects epithelial polarity inthe ovarian follicle cells
(Martin and St. Johnston2003). LKB1 colocalizes with cortical actin
in the oocyte,integrity of which is required for anchoring of
poleplasm components and nos mRNA (Lantz et al. 1999;Forrest and
Gavis 2003). It is therefore attractive tospeculate that LKB1 might
act at the cortex, where actinand microtubule filaments meet,
phosphorylating acurrently unknown substrate to promote the
trappingof pgc-containing RNPs. Our results suggest that thelevel
of LKB1 in Hsp83stc/Hsp83E317K flies is insufficientfor pgc
localization but sufficient for viability as well asproper
polarization of microtubules during oogenesisand localization of
osk. According to this idea, LKB1
Figure 7.—Assembly and maintenance of thepole plasm in Hsp83
mutants. The distributionsof various proteins in stage 10B egg
chambersor stage 2 embryos from wild-type (w1118)
andHsp83stc/Hsp83E317K females. Nuclei in rows 1–3are in blue,
green, and red, respectively. Notethat the initial accumulation of
Osk at the poste-rior is slightly defective in some stage Hsp83
mu-tant egg chambers at stage 7–8; however, by stage9–10, the level
of posterior Osk in all mutant eggchambers is indistinguishable
from that in wildtype. Also note that the level of Osk in manyHsp83
mutant embryos is higher for reasons wedo not understand. As a
result, the protein is de-tectable further toward the anterior than
in wild-type embryos. In row 5, distributions of Vasa(red) and Nos
(green) at the posterior of stage2 embryos are shown, with both
channels overlaidin the third image for each genotype.
Hsp90 As an mRNA Localization Factor 2219
-
hypomorphs might exhibit many of the defects weobserve in flies
with reduced Hsp90 function.
Two other kinases have been implicated in mRNAlocalization in
Drosophila, but as is the case for the roleof LKB1 in pgc
localization, the critical direct sub-strate(s) for each have yet
to be identified. Proteinkinase A (PKA) is required for the
microtubule re-organization described above that leads to
posteriorlocalization of osk mRNA (Lane and Kalderon 1994).Although
PKA has been shown to phosphorylate LKB1at residue 535 in vitro
(Martin and St. Johnston 2003),overexpression of LKB1 bearing a
phosphomimeticS535E substitution does not rescue the microtubule
de-fects in PKA mutant ovaries, suggesting that some otherprotein
is the major target for PKA during microtubulereorganization
(Steinhauer and Kalderon 2005). Asecond kinase, IkB kinase-like2
(Ik2), and its bindingpartner, Spindle-F (Spn-F), have recently
been shown toregulate both microtubule and actin filament
distribu-tions in the female germ line (Abdu et al. 2006;
Shapiroand Anderson 2006). The authors proposed that Ik2/Spn-F
facilitates the connection of a subset of micro-tubules to cortical
actin, although the mechanism of theiraction is unknown. For each
of these kinases, LKB1,PKA, and Ik2, further biochemical and
genetic experi-ments will be required to determine how they act
tolocalize mRNA.
The differential effects we observe on localization ofnos, pgc,
and CycB (Figures 6 and 8) suggest that eachmRNA is localized by a
somewhat different mechanism.CycB mRNA localization is normal in
Hsp83stc/Hsp83E317K
embryos and thus appears to be relatively Hsp90 in-dependent;
pgc mRNA localization requires normallevels of Hsp90 activity,
primarily to stabilize LKB1; andnos mRNA localization requires
normal levels of Hsp90activity, presumably to stabilize or activate
other (cur-rently unknown) factors. Studies of the polar
granulecomponent Tudor support the idea that different mech-anisms
underlie localization of nos, pgc, and gcl mRNAs,each of which is
concentrated at the posterior pole latein oogenesis (Thomson and
Lasko 2004; Arkov et al.2006). Among this class of mRNAs, only nos
has beenstudied in detail (Forrest and Gavis 2003). Similardetailed
studies of pgc, gcl, and CycB localization mightreveal some of the
mechanistic differences.
The genetic screen we employed to identify Hsp90appears to
constitute a promising approach for theidentification of additional
nos mRNA localization factors.One key aspect of the screen was
reliance on the identifi-cation of dominant modifier mutations.
Such mutationsmay be relatively easy to isolate in the case of
Hsp83, asthe encoded protein is a multidomain dimer that formslarge
complexes with other factors, including its cocha-perones (Pearl
and Prodromou 2001). Nevertheless,
Figure 8.—Overexpression ofthe LKB1 kinase rescues pgcmRNA
localization in Hsp83 mu-tant embryos. (A) The distribu-tion of
GFP-LKB1 in sibling wildtype (Hsp83�/1) or Hsp83stc/Hsp83E317K
stage 10B egg cham-bers. (B) The distributions ofpgc mRNA, Osk, and
nos mRNAare shown in stage 2 embryos orstage 10B egg chambers for
threematernal genotypes: wild type(w1118), Hsp83stc/Hsp83E317K,
andarmadillo-GAL4/UAS-GFP-LKB1 ;Hsp83stc/Hsp83E317K .
2220 Y. Song et al.
-
we are optimistic that a scaled-up version of the screenthat
surveys the entire genome and characterization ofresulting mutants
will yield additional components ofthe nos mRNA localization
machinery.
We especially thank Daniel St. Johnston for his generosity
insupplying strains and reagents; Anne Ephrussi, Akira Nakamura,
PaulLasko, Paul Macdonald, and Ken Howard for reagents; Joe
Heitman,Chris Nicchitta, and Danny Lew for comments on the
manuscript andsuggestions; Sandy Curlee and Estelle Tsalik for
assistance in the officeand lab, respectively; and Jian Chen for
media preparation. We are alsoindebted to the Bloomington Stock
Center. Y. S. was a PredoctoralFellow of the HHMI and Robin Wharton
is an Investigator of theHoward Hughes Medical Institute.
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Communicating editor: W. M. Gelbart
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