SUBCELLULAR localization of mrna is an efficient

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1 Copyright Ó 2007 by the Genetics Society of America DOI: /genetics The Molecular Chaperone Hsp90 Is Required for mrna Localization in Drosophila 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, Manuscript received January 29, 2007 Accepted for publication May 25, 2007 ABSTRACT Localization of maternal nanos mrna to the posterior pole is essential for development of both the abdominal segments and primordial germ cells in the Drosophila embryo. Unlike maternal mrnas such as bicoid and oskar that are localized by directed transport along microtubules, nanos is thought to be trapped as it swirls past the posterior pole during cytoplasmic streaming. Anchoring of nanos depends on integrity of the actin cytoskeleton and the pole plasm; other factors involved specifically in its localization have not been described to date. Here we use genetic approaches to show that the Hsp90 chaperone (encoded by Hsp83 in Drosophila) is a localization factor for two mrnas, nanos and pgc. Other components of the pole plasm are localized normally when Hsp90 function is partially compromised, suggesting a specific role for the chaperone 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 LKB1 is a primary Hsp90 target for pgc localization and that other Hsp90 partners mediate localization of nos. SUBCELLULAR localization of mrna is an efficient strategy for spatially restricting the encoded protein ( Johnstone and Lasko 2001; St.Johnston 2005). This strategy is common in highly polarized cell types such as neurons, as well as during the development of oocytes or embryos when gene regulation is limited to post-transcriptional mechanisms. Asymmetric RNA localization prior to mitosis also serves to distinguish daughter cells in Saccharomyces cerevisiae. In this organism, localization of ASH1 mrna to buds just prior to cytokinesis currently provides the most completely understood example of mrna localization (Niessing et al. 2004; Gonsalvez et al. 2005). Localization of maternal mrnas drives much of the patterning along the anteroposterior axis of the Drosophila embryo. The anterior determinant, bicoid (bcd) mrna, is localized during oogenesis as ribonucleoprotein (RNP) cargo associated with molecular motors that traverse 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 localization of oskar (osk) mrnatotheposteriorpoleofthe oocyte, also via directed movement along microtubules 1 Present address: Department of Pathology, Stanford University School of Medicine, Stanford, CA. 2 Corresponding author: Howard Hughes Medical Institute, Department of Molecular Genetics & Microbiology, Box 3657, Duke University Medical School, Durham, NC rwharton@duke.edu (Cha et al. 2002; Braat et al. 2004; Huynh et al. 2004; Yanoet al. 2004). Localization of both bcd and osk mrnas is thought to occur via complex, multistep pathways with many components. One role of localized Osk is to direct the subsequent localization of a fraction of the nanos (nos) mrna late in oogenesis (Bergsten and Gavis 1999). Localized nos mrna is the sole source of Nos protein in the early embryo (Gavis and Lehmann 1992) where it plays a number of key roles in development. Nos is required in the somatic cytoplasm of the early embryo to repress translation of maternal hunchback mrna, thereby governing abdominal segmentation (Sonoda and Wharton 1999). Nos is also required in the primordial germ cells that form at the posterior extreme of the embryo to delay proliferation, repress transcription, facilitate migration into the somatic gonad, and promote survival (Kobayashi et al. 1996; Forbes and Lehmann 1998; Asaoka-Taguchiet al. 1999; Schaner et al. 2003; Hayashi et al. 2004; Kadyrova et al. 2007). The germ line functions 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 (specialized cytoplasm that specifies germ line identity) via an elaborate, genetically defined pathway in which recruitment of nos mrna is one of the final steps (Ephrussi and Lehmann 1992; Kim-Ha et al. 1993). Recruitment is thought to involve trapping of nos RNPs as they swirl past the posterior pole during cytoplasmic streaming Genetics 176: (August 2007)

2 2214 Y. Song et al. Figure 1. A dominant modifier screen for nos mrna localization factors. Shown schematically are the 39-UTRs of wild-type nos 1 mrna and a mini-nos 1 mrna that lacks nt The first column shows a qualitative assessment of the relative efficiency of mrna localization to the posterior pole (see Figure 2), the second column shows the typical number of abdominal segments in embryos where the sole source of Nos is the indicated mrna, and the third column is a drawing of the body plan after 24 hr of embryonic development. The rationale for the screen is that an EMSinduced mutation (*) would dominantly reduce segmentation such that embryos do not hatch (and thus remain within the vitelline membrane). (Forrest and Gavis 2003; Serbus et al. 2005), rather than the directed movement that underlies localization of osk or ASH1 RNPs. The factors involved specifically in localizing nos mrna have yet to be identified. In this report, we describe the results of a genetic screen for nos mrna localization factors that rely on the abdomen-patterning role of Nos. MATERIALS AND METHODS Isolation and mapping of the 966 mutation: Homozygous w 1118 ; e nos BN males were mutagenized with EMS and mated en masse with w 1118 virgin females. Male progeny were individually crossed to generate P½mini-nos 1 Š, nos BN /e nos BN * females, which were screened for the ability to give rise to hatching progeny. The P½mini-nos 1 Š transgene is described by Dahanukar and Wharton (1996), where it is named nos 1 (DBX). Candidate mutant chromosomes (*) were recovered from sibling males for further testing. Meiotic recombination with a rucuca chromosome placed 966 between ru and h on the left arm of the third chromosome. Fine mapping by P element-induced male recombination further mapped 966 to a 57-kb interval between P{SUPor-P}KG05210 and P{SUPor-P}KG The 966 mutation was definitively identified by sequencing the Hsp90 coding region amplified from genomic DNA extracted from homozygous 966 larvae (identified by the absence of a GFP-marked balancer chromosome). Fly strains and reagents: The following strains were from the Bloomington Stock Center: Hsp83 alleles scratch (08445), E317K (e 6D ), S529F (e 6A ), and j5c2a; transformants bearing the 17.5 genomic Hsp83 rescue construct; flies with the P{SUPor- P}KG05210, P{SUPor-P}KG00982, P{SUPor-P}KG03657, and P{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. Johnston 2003) were from D. St. Johnston; flies with the P{GAL4-arm.S}11 armadillo-gal4 driver were from Bloomington. Antibodies against various proteins were gifts of P. Macdonald (Hb), A. Nakamura (Nos), D. St. Johnston (Stau), A. Ephrussi (Osk), and K. Howard (Vas). Immunohistochemistry: Egg chamber fixation and antigen detection 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 antibodies ( Jackson Laboratories) were used at 1:200. Nuclei were stained with TOTO-3, oligreen, or TOPRO-3 (Molecular Probes). Samples were mounted in Vectashield and imaged on 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 hybridization was by standard methods using digoxigenin-labeled dsdna probes prepared from cdna clones. The adducin-like/ hts probe was from clone N4 (Ding et al. 1993). Western and Northern blots: Homozygous 966 mutant larvae (e.g., non-tubby) were identified shortly after hatching and grown under noncrowded conditions. Samples from whole larvae homogenized in SDS sample buffer were analyzed following transfer to Immobilon P by standard methods. Hsp90 was detected with the 3E monoclonal antibody, a gift from R. Tanguay (Carbajal et al. 1990), and ECL Plus (Amersham). The loading control was a-tubulin, detected with DM 1A monoclonal antibody (Sigma F-2168) and ECL (Amersham). For Northern blots, 5-mg samples of total RNA prepared from 0- to 4.5-hr embryos were analyzed by standard methods using radio-labeled probes to detect smaug mrna (as a loading control) and mini-nos 1 mrna. Quantitation was performed on a Typhoon phosphoimager. RESULTS A genetic screen for nos localization factors: To identify factors involved in nos mrna localization, we chemically mutagenized flies and screened for dominant maternal-effect mutations that (further) compromise abdominal segmentation in a sensitized background. The rationale for the screen is shown in Figure 1. Inefficient localization and translation of a mini-nos 1 mrna generates only sufficient Nos activity to allow development of 5 6 abdominal segments if the endogenous nos genes are mutant (Dahanukar and Wharton 1996). In such a background, we reasoned that a mutation in one of the two alleles encoding a localization factor might compromise Nos activity sufficiently to preclude hatching (either because the allele is dominant negative or the gene is haplo-insufficient). A similar rationale has been used extensively in screens based on changes in morphology of the Drosophila eye (e.g.,rebay et al. 2000). From a pilot screen of 6000 EMS-mutagenized third chromosomes, we isolated a number of mutations that

3 Hsp90 As an mrna Localization Factor 2215 Figure 2. The 966 mutation dominantly interferes with localization of mini-nos 1 mrna. (A) Northern blot of samples from 0 to 4.5 hr nos BN / P½mini-nos 1 Š, nos BN (lane 1) and 966 nos BN / P½mini-nos 1 Š, nos BN (lane 2) embryos to detect smaug and mini-nos 1 mrnas. The mobility of molecular weight markers is indicated on the left. (B) The distributions of mini-nos 1 mrna during embryonic stages 2 (early cleavage, first row) and 4 (syncytial blastoderm, second row), Hb protein in nuclear cycle 10 (third row), and the resulting embryonic body plan(darkfield micrographs of secreted cuticle in the fourth row) are shown. Here and in all subsequent figures, embryos and egg chambers are oriented anterior to the left and dorsal up. Note that the enzymatic reaction used to detect hybridized digoxigenin-labeled probe was nearly twice as long for the embryos in row 2 (vs. row 1) due to the presence of reduced mrna levels; control experiments suggest that an appreciable 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 presence of bands of large denticles that are white in darkfield; the eighth abdominal segment is highlighted with an arrowhead on the left. The maternal genotypes are indicated above. reduce or eliminate abdominal segmentation in the sensitized background. One mutation proved to be an allele of pumilio and another an allele of spindle-e; both genes are known to play roles in posterior specification (Lehmann and Nüsslein-Volhard 1987; Gillespie and 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 localization but not the synthesis or stability of mini-nos 1 mrna (Figure 2). Both the analysis of Northern blots (Figure 2A) and examination of the unlocalized mini-nos 1 mrna in embryos hybridized with digoxigenin-labeled probes (Figure 2B) support the idea that mini-nos 1 mrna stability is unaffected by the 966 mutation. In contrast, localization of mini-nos 1 mrna is defective from the beginning of embryonic development (stage 1) through formation of the syncytial blastoderm (stage 4) in embryos from heterozygous 966 mutant females (Figure 2B). Because Nos protein is generated exclusively from translation of localized mrna, the embryos from 966 heterozygotes apparently have reduced Nos activity, because Hunchback (Hb) accumulates in the posterior and they subsequently fail to develop abdominal segments (Figure 2B). The 966 allele is homozygous lethal and germ line clones are rudimentary, precluding analysis of embryos derived from homozygous females. We examined the distribution of other mrnas in embryos from heterozygous 966 mutant females (hereafter 966 mutant embryos) to determine whether the defects in mini-nos 1 mrna localization are specific. As shown in Figure 3, the localization of full-length nos 1 Figure 3. The 966 mutation does not significantly affect localization of other mrnas in the early embryo. The distributions of various mrnas (indicated on the left) are shown in stage 2 embryos. Insets on rows 1 and 2 show the posterior of stage 4 embryos. The maternal genotypes are indicated above. Note that other experiments show that the distributions of pgc, bcd, andadd-like mrnas are unaffected by the nos genotype (not shown).

4 2216 Y. Song et al. mrna is essentially normal in early 966 mutant embryos, although the pole cells in slightly older embryos appear to retain somewhat reduced levels of mrna. In contrast, localization of pgc mrna to the posterior or bicoid and adducin-like mrnas to the anterior is indistinguishable in wild-type and 966 mutant embryos. We next wished to determine whether the 966 gene product acts late in the posterior pathway (when nos mrna is localized) or early, perhaps acting indirectly to govern oocyte polarity and accumulation of Osk, for example. Three observations suggest that the 966 gene product acts downstream of Osk. First, Osk accumulation is normal in 966 mutant embryos (Figure 4A). Second, Osk activity appears normal in 966 mutant embryos by the criterion that they have a similar number of pole cells as wild-type embryos (data not shown). The formation of pole cells is known to be a sensitive indicator of Osk activity (Ephrussi and Lehmann 1992; Smith et al. 1992). Third, we find that the 966 mutation interferes with localization of mini-nos 1 mrna to the anterior of osk-bcd embryos (Figure 4B). In osk-bcd embryos, a chimeric mrna (bearing the protein-coding region of osk and the localization signals of bcd) generates Osk activity at the anterior of the embryo independent of the upstream factors that regulate localization and translation of native osk mrna. This ectopic Osk directs efficient localization and translation of full-length nos 1 mrna, resulting in the suppression of head and thoracic segments, which are replaced by a mirror symmetric duplication of abdominal segments (Ephrussi and Lehmann 1992). We find that mini-nos 1 mrna is inefficiently localized to the anterior of osk-bcd embryos where it apparently is translated into only enough Nos to suppress head segmentation (Figure 4B). If, in addition, the females are heterozygous for 966, then recruitment of mini-nos 1 mrna to the anterior is almost eliminated and the suppression of anterior development by ectopic Nos is largely relieved (Figure 4B). Taken together, the results described above suggest that the 966 mutation alters the function of a factor that is required downstream of Osk to localize mini-nos 1 mrna. Hsp90 requirement for mrna localization: We mapped the 966 mutation (primarily scoring the associated homozygous lethality) using deficiencies, meiotic recombination, and P element-induced male recombination. In the course of these experiments, we discovered that 966 is semilethal in trans to the scratch (stc) allele (Yue et al. 1999) of Hsp83, which encodes the highly conserved Hsp90 chaperone. Two additional lines of evidence demonstrate that 966 is indeed an allele of Hsp83. First, the Hsp83 gene on the 966 chromosome bears a single nucleotide substitution that results in an alanine to aspartate substitution at a highly conserved residue (133) in the N-terminal ATPase domain (Figure 5A). Second, two independently isolated Hsp83 alleles that encode missense forms of the Figure 4. The 966 gene product acts downstream of Oskar in the posterior pathway to localize mini-nos 1 mrna. (A) The distribution of Osk in stage 2 embryos, maternal genotypes as indicated. (B) The distribution of mini-nos 1 mrna in stage 2 embryos and anterior cuticle (phase-contrast micrographs) of mature embryos from females of the indicated genotype. Note that virtually the entire head skeleton is absent in the embryo on the left, with only a trace of sclerotized cuticle in evidence (arrowhead). With the exception of minor defects in the dorsal bridge (arrowhead), the head skeleton (arrowhead) in the embryo on the right is wild type. Hsp90 protein (E317K and S592F) (Cutforth and Rubin 1994; van der Straten et al. 1997) suppress abdominal segmentation in the mini-nos 1 background in a manner similar to 966 (Figure 5A). Thus, Hsp90 function is required for normal localization of mini-nos 1 mrna. We next asked whether the A133D allele acts in a dominant negative or haplo-insufficient manner to modify mini-nos 1 -dependent segmentation. The E317K and S592F alleles were identified in screens for dominant modifiers of kinase-dependent eye phenotypes; since they also modify mini-nos 1 activity, we suspected that the A133D mutant and the other missense mutants act as dominant negatives for mini-nos 1 localization. Consistent with such an idea, a presumptive null allele of Hsp83, j5c2a, which bears a P-element in the first (nonprotein-coding) exon, does not dominantly modify the mini-nos 1 segmentation phenotype. Moreover, rescue of the segmentation phenotype caused by the A133D allele is inefficient. A single wild-type Hsp83 transgene rescues very poorly, yielding embryos with 1 to 2 abdominal segments; two copies of the wild-type Hsp83 transgene are required for full rescue (Figure 5B). Finally, we find that the A133D mutant protein accumulates to wild-type levels in extracts prepared from larvae 1 day before they die (Figure 5B). Taken together, these results suggest that the A133D missense protein dominantly interferes with wild-type Hsp90 with respect to the mini-nos 1 segmentation phenotype. To further investigate the role of Hsp90 in localization of maternal mrnas in general and full-length nos 1

5 Hsp90 As an mrna Localization Factor 2217 Figure is a dominant negative allele of Hsp83. (A) At the top is a schematic representation of the domains of Hsp90 and the positions of missense substitutions encoded by the 966 allele and the two other alleles used in these studies. Abdominal segmentation is visualized in darkfield photographs of cuticle secreted by embryos from females heterozygous for the indicated Hsp83 allele (except in row 1, which is from an Hsp83 1 /Hsp83 1 female). The females here and in BarealsoP½mini-nos 1 Š, nos BN /nos BN, such that the sole source of Nos in embryos is from the mini- nos 1 transgene. (B) The A133D protein acts in a dominant negative fashion. Cuticle of embryos from females heterozygous for the indicated Hsp83 allele, as in A. Embryos in rows 2 and 3 are from females that also bear one and two extra copies of an Hsp83 1 transgene, respectively. Note that one copy of the transgene does not efficiently rescue thelethalityofhsp83 A133D /Hsp83 stc animals,consistentwiththeideathat thisallele,likeother missenseallelesofhsp83 (Cutforth and Rubin 1994; van der Straten et al. 1997), is dominant-negative. Western blot of extracts (each containing 5 mg of protein) prepared from w 1118 (lane 1), nos BN /TM3 (lane 2), and 966 nos BN /966 nos BN (lane 3) larvae hr after egg laying. Following transfer, the membrane was cut in half and probed separately with antibodies against Hsp90 and a-tubulin. mrna in particular, it was necessary to bypass the requirement of Hsp90 function for viability. A comprehensive study of Hsp83 alleles had shown that several trans-heterozygous allelic combinations are viable and fertile (Yue et al. 1999). We reinvestigated the issue and found that Hsp83 stc /Hsp83 E317K females are reasonably healthy and produce large numbers of eggs, of which 1 2% develop through late embryonic stages. We therefore used this genetic background to investigate the consequences of impairing maternal Hsp90 function on mrna localization. As shown in Figure 6, localization of full-length nos 1 mrna is defective in 90 95% of embryos when maternal Hsp90 function is compromised. The phenotype is heterogeneous and includes nearly normal localization of a reduced amount of nos RNA to a crescent along the posterior cortex, localization of a ball of nos mrna only partially in contact with the posterior cortex, diffuse localization of a cloud near the posterior, and no detectable localization. These defects in mrna localization have predictable effects on subsequent development: essentially all Hsp83 stc /Hsp83 E317K embryos have reduced levels of Nos protein (Figure 6). Very few of these embryos cellularize, presumably due to some other requirement(s) for maternal Hsp90 function. But the 1 2% of embryos that eventually secrete cuticle have on average 3 abdominal segments rather than the normal complement of 8 (Figure 6). Thus, maternal Hsp90 is critical for posterior localization of nos 1 mrna. We next wished to determine whether localization of other maternal mrnas also relies on Hsp90. Accordingly, we examined the distributions of polar granule component (pgc), germcell-less (gcl), CyclinB (CycB)(Lehner and O Farrell 1990; Jongens et al. 1992; Nakamura et al. 1996), and bcd mrnas in Hsp83 stc /Hsp83 E317K embryos. Of these, only pgc mrna localization is abnormal (Figure 6 and not shown). Localization of pgc mrna is affected to a greater extent than is localization of nos mrna. Because Hsp90 activity is only partially ablated in the Hsp83 stc /Hsp83 E317K egg chambers, we do not know whether the posterior localization of gcl and CycB mrnas and the anterior localization of bcd mrna rely on lower Hsp90 activity or whether they are Hsp90 independent. However, the significant observation is that proper deployment of only a subset of the late-localizing posterior mrnas (nos and pgc) requires normal Hsp90 activity. Hsp90 is thought to interact with hundreds of proteins 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 in nos and pgc localization might be quite indirectly caused by earlier defects in the assembly of pole plasm components at the posterior of the egg chamber. To this end, we examined the localization of upstream components of the posterior pathway in Hsp83 stc /Hsp83 E317K egg chambers where Hsp90 activity is compromised. Reduction of Hsp90 activity has a relatively specific effect on the posterior localization of nos and pgc mrnas. The initial localization of three key factors that act upstream of nos is essentially normal in Hsp83 stc /Hsp83 E317K egg chambers (Figure 7). These include: (1) Stau protein ½an effective proxy for osk mrna (Martin et al. 2003)Š,

6 2218 Y. Song et al. Figure 6. Hsp90 is required for localization of nos and pgc mrnas. The distributions of various mrnas and proteins (except in row 3, which shows cuticle secreted by mature embryos) in stage 2 embryos from wild-type (w 1118 ) and Hsp83 stc / Hsp83 E317K females. Many of the Hsp83 stc /Hsp83 E317K embryos have terminal defects not readily visible in the photograph, consistent with previous reports that Hsp90 is a cofactor of the Raf kinase (van der Straten et al. 1997), which acts downstream of Torso to specify terminal identity in the embryo. The embryos in row 5 were hybridized simultaneously with probes to bcd (the prominent signal at the anterior) and pgc (the low-level unlocalized signal as well as the posterior cap that is present in wild type but not Hsp83 mutant embryos). Note that many Hsp83 mutant embryos have an abnormal shape, with elongated anterior ends to which the vitelline membrane and micropyle frequently remain attached (particularly evident in rows 4 and 5). (2) Osk protein, and (3) Vasa protein. Since accumulation of Osk is acutely interdependent with localization of osk mrna, Vasa, and the Par-1 kinase (Breitwieser et al. 1996; Riechmann et al. 2002; Benton and St. Johnston 2003; Johnstone and Lasko 2004), the data presented in Figure 7 suggest that pole plasm assembly is essentially normal until the late recruitment of nos and pgc. Oogenesis appears grossly normal in the Hsp83 stc / Hsp83 E317K background, which suggests that polarization of the microtubule cytoskeleton is likely to be normal. This idea is further supported by the normal initial localization of Stau to the posterior and the maintenance of bcd mrna at the anterior, both dependent critically on microtubule integrity (Pokrywka and Stephenson 1991; Brendza et al. 2000; Weil et al. 2006). Not only is the initial formation of pole plasm normal, but also its integrity is maintained in Hsp83 stc /Hsp83 E317K embryos, based on the normal posterior localization of Osk and Vasa (Figure 7). Distribution of the latter protein was detected in double-staining experiments, which show that Hsp83 stc /Hsp83 E317K embryos with very little or no detectable Nos have a normal crescent of Vasa at the posterior pole. Thus, both the initial formation and maintenance of the pole plasm appear unperturbed in Hsp83 stc /Hsp83 E317K flies. Taken together, the observations outlined above lead us to conclude that, despite its general pleiotropy, Hsp90 plays a relatively specific role for the localization of nos and pgc mrnas to the posterior of the embryo. Rescue of pgc mrna localization in Hsp83 mutant embryos by overexpression of LKB1: Hsp90 is a molecular chaperone that activates and stabilizes a wide variety of client regulatory and signaling proteins (Pearl and Prodromou 2001); apriori, it seemed unlikely that Hsp90 interacts directly with nos and pgc mrnas. Therefore, one approach to further understanding its role in mrna localization would be to identify molecules whose activity is dependent on Hsp90. To date, none of the other genetic modifiers identified in the screen that yielded the 966 allele of Hsp83 encodes an obvious Hsp90 client. Therefore, we turned to a candidate gene approach, focusing on protein kinases previously implicated in various aspects of posterior patterning. Two such proteins are Par-1 and LKB1, which are required for polarization of the oocyte microtubule cytoskeleton and the proper deposition of osk mrna at the posterior (Shulman et al. 2000; Tomancak et al. 2000; Martin and St.Johnston 2003; Doerflinger et al. 2006). Two lines of evidence suggest that LKB1 is a significant Hsp90 client for the localization of pgc mrna. First, the level of GFP-LKB1 is significantly reduced when Hsp90 activity is compromised in essentially all Hsp83 stc / Hsp83 E317K egg chambers (Figure 8A). In contrast, no consistent effect of reducing Hsp90 activity is seen on levels of GFP-Par-1 (not shown). Second, overexpression of LKB1 in Hsp83 stc /Hsp83 E317K females significantly rescues the localization of pgc mrna (Figure 8B). For this experiment, we used the armadillo-gal4 driver to achieve low-level overexpression of GFP-LKB1 in the ovaries, as previously described (Martin and St.Johnston 2003). The rescue of pgc localization does not appear to be due to an indirect elevation of posterior Osk levels, which are normal during both oogenesis and early embryogenesis (Figure 8B); these observations are consistent with the previous finding that up to 10-fold overexpression of LKB1 has no significant effect on localization of the pole plasm component Stau (Martin and St.Johnston 2003). In contrast to the rescue of pgc, no significant rescue of nos localization was observed (Figure 8B). Similar negative results were obtained using nos-gal4-vp16 to drive 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 pgc mrna, a major function of Hsp90 is to stabilize LKB1. Presumably other Hsp90 partners or targets mediate localization of nos mrna.

7 Hsp90 As an mrna Localization Factor 2219 Figure 7. Assembly and maintenance of the pole plasm in Hsp83 mutants. The distributions of various proteins in stage 10B egg chambers or stage 2 embryos from wild-type (w 1118 ) and Hsp83 stc /Hsp83 E317K females. Nuclei in rows 1 3 are in blue, green, and red, respectively. Note that the initial accumulation of Osk at the posterior is slightly defective in some stage Hsp83 mutant egg chambers at stage 7 8; however, by stage 9 10, the level of posterior Osk in all mutant egg chambers is indistinguishable from that in wild type. Also note that the level of Osk in many Hsp83 mutant embryos is higher for reasons we do not understand. As a result, the protein is detectable further toward the anterior than in wildtype embryos. In row 5, distributions of Vasa (red) and Nos (green) at the posterior of stage 2 embryos are shown, with both channels overlaid in the third image for each genotype. DISCUSSION The specific role that maternal Hsp90 plays in localization of a subset of mrnas to the pole plasm is somewhat surprising, given the number of proteins that are thought to require the activity of this chaperone. Although ubiquitously distributed, Hsp90 is enriched in the testes and ovaries and the male germ line is particularly sensitive to a reduction in Hsp90 activity (Yue et al. 1999). The experiments reported here define a role for maternal Hsp90 in the localization of nos and pgc mrnas. We do not yet know the mechanism by which Hsp90 acts. However, the apparent integrity of the pole plasm in Hsp83 stc /Hsp83 E317K ovaries and embryos (Figures 6 and 7) and the rescue of pgc localization upon overexpression of a single kinase (Figure 8) are consistent with the idea that the defects in pgc (and perhaps also nos) localization arise from the reduction in activity of a few discrete Hsp90 clients. Hsp83 mrna is itself concentrated in the pole plasm (Ding et al. 1993) and Hsp90 is found at particularly high levels in the germ line precursors of other organisms (Vanmuylder et al. 2002; Inoue et al. 2003), which may reflect a conserved role in mrna localization. We do not know whether Hsp90 acts directly or indirectly to stabilize LKB1. Mammalian LKB1 binds directly to Hsp90 and Cdc37, a cochaperone for kinase clients (Boudeau et al. 2003). However, we have not observed a direct interaction between Drosophila Hsp90 and LKB1, either by co-immunoprecipitation or in yeast interaction experiments in which the DNA-binding domain was fused to the Hsp90 C terminus to avoid interfering with dimerization, as described (Millson et al. 2005). We do not currently know whether Hsp90 binding to LKB1 is ephemeral (and thus difficult to detect) or whether Hsp90 acts indirectly to stabilize LKB1. How might LKB1 act to localize pgc mrna? Despite its conserved role in regulation of cellular polarity (see Alessi et al and references therein), no LKB1 substrate that plays a direct role in mrna localization has been described, to our knowledge. Loss of LKB1 function in germ line clones of presumptive null alleles prevents the reorganization of the oocyte microtubule network at stage 7 that is required for posterior localization of osk mrna and affects epithelial polarity in the ovarian follicle cells (Martin and St. Johnston 2003). LKB1 colocalizes with cortical actin in the oocyte, integrity of which is required for anchoring of pole plasm components and nos mrna (Lantz et al. 1999; Forrest and Gavis 2003). It is therefore attractive to speculate that LKB1 might act at the cortex, where actin and microtubule filaments meet, phosphorylating a currently unknown substrate to promote the trapping of pgc-containing RNPs. Our results suggest that the level of LKB1 in Hsp83 stc /Hsp83 E317K flies is insufficient for pgc localization but sufficient for viability as well as proper polarization of microtubules during oogenesis and localization of osk. According to this idea, LKB1

8 2220 Y. Song et al. Figure 8. Overexpression of the LKB1 kinase rescues pgc mrna localization in Hsp83 mutant embryos. (A) The distribution of GFP-LKB1 in sibling wild type (Hsp83 /1) or Hsp83 stc / Hsp83 E317K stage 10B egg chambers. (B) The distributions of pgc mrna, Osk, and nos mrna are shown in stage 2 embryos or stage 10B egg chambers for three maternal genotypes: wild type (w 1118 ), Hsp83 stc /Hsp83 E317K, and armadillo-gal4/uas-gfp-lkb1 ; Hsp83 stc /Hsp83 E317K. hypomorphs might exhibit many of the defects we observe in flies with reduced Hsp90 function. Two other kinases have been implicated in mrna localization in Drosophila, but as is the case for the role of LKB1 in pgc localization, the critical direct substrate(s) for each have yet to be identified. Protein kinase A (PKA) is required for the microtubule reorganization described above that leads to posterior localization of osk mrna (Lane and Kalderon 1994). Although PKA has been shown to phosphorylate LKB1 at residue 535 in vitro (Martin and St.Johnston 2003), overexpression of LKB1 bearing a phosphomimetic S535E substitution does not rescue the microtubule defects in PKA mutant ovaries, suggesting that some other protein is the major target for PKA during microtubule reorganization (Steinhauer and Kalderon 2005). A second kinase, IkB kinase-like2 (Ik2), and its binding partner, Spindle-F (Spn-F), have recently been shown to regulate both microtubule and actin filament distributions in the female germ line (Abdu et al. 2006; Shapiro and Anderson 2006). The authors proposed that Ik2/ Spn-F facilitates the connection of a subset of microtubules to cortical actin, although the mechanism of their action is unknown. For each of these kinases, LKB1, PKA, and Ik2, further biochemical and genetic experiments will be required to determine how they act to localize mrna. The differential effects we observe on localization of nos, pgc, and CycB (Figures 6 and 8) suggest that each mrna is localized by a somewhat different mechanism. CycB mrna localization is normal in Hsp83 stc /Hsp83 E317K embryos and thus appears to be relatively Hsp90 independent; pgc mrna localization requires normal levels of Hsp90 activity, primarily to stabilize LKB1; and nos mrna localization requires normal levels of Hsp90 activity, presumably to stabilize or activate other (currently unknown) factors. Studies of the polar granule component Tudor support the idea that different mechanisms underlie localization of nos, pgc, and gcl mrnas, each of which is concentrated at the posterior pole late in oogenesis (Thomson and Lasko 2004; Arkov et al. 2006). Among this class of mrnas, only nos has been studied in detail (Forrest and Gavis 2003). Similar detailed studies of pgc, gcl, and CycB localization might reveal some of the mechanistic differences. The genetic screen we employed to identify Hsp90 appears to constitute a promising approach for the identification of additional nos mrna localization factors. One key aspect of the screen was reliance on the identification of dominant modifier mutations. Such mutations may be relatively easy to isolate in the case of Hsp83, as the encoded protein is a multidomain dimer that forms large complexes with other factors, including its cochaperones (Pearl and Prodromou 2001). Nevertheless,

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