Spire is a maternal effect locus that affects both the dorsal- ventral and anterior-posterior axes of the Drosophila egg and embryo. It is required for localization of determinants within the developing oocyte to the posterior pole and to the dorsal anterior corner. During mid-oogenesis, spire mutants display premature microtubule-dependent cytoplasmic streaming, a phenotype that can be mimicked by pharmacological disruption of the actin cytoskeleton with cytochalasin D. Spire has been cloned by transposon tagging and is related to posterior end mark-5, a gene from sea squirts that encodes a posteriorly localized mRNA. Spire mRNA is not, however, localized to the posterior pole. SPIRE also contains two domains with similarity to the actin monomer-binding WH2 domain, and we demonstrate that SPIRE binds to actin in the interaction trap system and in vitro. In addition, SPIRE interacts with the rho family GTPases RHOA, RAC1 and CDC42 in the interaction trap system. Thus, our evidence supports the model that SPIRE links rho family signaling to the actin cytoskeleton.

Spire (spir) is a maternal effect locus that affects both the dorsal-ventral and anterior-posterior axes of the Drosophila egg and embryo (Manseau and Schüpbach, 1989). In the dorsal-ventral axis, spir mutant eggs are dorsalized. In the anterior-posterior axis, spir is a member of the posterior group, with mutants lacking abdominal segments, pole cells and polar granules. Spir functions in the germline to help establish both of these axes (Manseau and Schüpbach, 1989). It is required for localization of all known posteriorly localized determinants (Bardsley et al., 1993; Ephrussi, et al., 1991; Kim-Ha et al., 1991; Lasko and Ashburner, 1990; Manseau and Schüpbach, 1989; Newmark et al., 1997; St Johnston et al., 1991; Wang and Lehmann, 1991). In the dorsal-ventral axis, gurken mRNA is not localized correctly to the dorsal-anterior corner of the oocyte (Neuman-Silberberg and Schüpbach, 1993).

Mutations in spir and other genes and pharmacological treatments of developing eggs have revealed links between the actin and microtubule networks during oogenesis. In wild-type stage-10 egg chambers, the microtubules bundle at the cortex of the oocyte and the cytoplasm of the oocyte begins microtubule- dependent streaming (Gutzeit, 1986; Theurkauf et al., 1992). In spir egg chambers, both of these microtubule behaviors happen prematurely, at stage 8 of oogenesis (Theurkauf, 1994). These microtubule-based phenotypes can also be induced upon disruption of the actin cytoskeleton with cytochalasin D (Emmons et al., 1995) and are seen in profilin (chickadee) mutants (Manseau et al., 1996), suggesting that the actin cytoskeleton represses these microtubule behaviors. Cappuccino (capu) shares these phenotypes (Emmons et al., 1995; Theurkauf, 1994) and it and related proteins bind to profilin (Chang et al., 1997; Evangelista et al., 1997; Imamura et al., 1997; Manseau et al., 1996; Wasserman, 1998; Watanabe et al., 1997). These observed links between the actin cytoskeleton and the microtubule cytoskeleton suggest that the relationship of spir to the actin cytoskeleton should be directly examined.

A number of observations link the actin cytoskeleton to patterning of the Drosophila oocyte. Mutations in two key regulators of the actin cytoskeleton, profilin and tropomyosin, disrupt the localization of oskar mRNA to the posterior pole (Erdelyi et al., 1995; Manseau et al., 1996). Profilin mutants also exhibit dorsal-ventral egg shell defects (Manseau et al., 1996). Capu and the formin family of proteins have been linked to regulation of the actin cytoskeleton through binding to rho family GTPases and profilin (J. C. and L. M., unpublished; Chang et al., 1997; Evangelista et al., 1997; Imamura et al., 1997; Manseau et al., 1996; Wasserman, 1998; Watanabe et al., 1997). Capu mutant chambers, similar to those from spir, fail to localize determinants to the posterior pole and exhibit mislocalization of gurken mRNA and protein to the dorsal- anterior corner of the oocyte (Bardsley et al., 1993; Ephrussi et al., 1991; Kim-Ha et al., 1991; Lasko and Ashburner, 1990; Manseau and Schüpbach, 1989; Neuman-Silberberg and Schüpbach, 1993; Newmark et al., 1997; St Johnston et al., 1991; Wang and Lehmann, 1991).

Spir’s relationship to the rho family of GTPases is of specific interest because certain members of this family of signaling proteins are known to link extracellular signaling to regulation of the actin cytoskeleton (Ridley, 1996; Tapon and Hall, 1997). Roles in Drosophila oogenesis have been defined for the rho family GTPases cdc42, rac1 and rhoL (Murphy and Montell, 1996). They have been implicated in actin cytoskeleton processes, including nurse cell dumping, nurse cell integrity, border cell migration and maintaining nurse cell-follicle cell contacts.

To further our understanding of how spir functions during development, we have cloned spir. Here, we report on our sequence analysis of spir and experiments that indicate that there is an interaction between SPIR and actin and rho family GTPases.

Spire cloning

Plasmid rescue was conducted as described in Ashburner (1989). Genomic DNA from homozygous P8 flies was cut with XbaI, recircularized by ligation, and transformed into E. coli. The isolated plasmid, pP8, contains approximately 7 kb of genomic DNA flanking the P element sequences. To determine whether this P element is in the spir genomic region, sequences adjacent to the P element were used to probe a collection of genomic DNA clones from the Drosophila genome project. A region of P1 DS04178 was found to hybridize, confirming that the P element is in the spir genomic region of 38C. cDNA clones were isolated from the Tolias ovary cDNA library (Stroumbakis et al., 1994) using the 7 kb region flanking the P element as a probe. The two largest cDNA clones, 4C and 8D, were chosen for subsequent analysis. The 8D cDNA clone is 2.7 kb long and represents the 2.7 kb mRNA including: (1) 300 bp of the 5′ non-coding region, (2) the ORF and (3) the complete 3′ non-coding region. The 4C cDNA clone is 3.4 kb long and contains the 5′ non-coding region and 2.8 kb of the ORF of the 4.5 kb mRNA. A cDNA clone, BB18-1, containing the entire 3′ end of the 4.5 kb mRNA was found in an ovarian cDNA library made by Jörg Grosshans. It contains 448 bp of overlap with the 4C cDNA.

Whole-mount tissue in situ hybridization

Whole-mount tissue in situ hybridizations were performed as in Tautz and Pfeifle (1989) with DIG-labeled RNA probes. Ovaries were fixed in 4% paraformaldehyde with 0.1% DMSO for 1 hour. An RNA probe specific for the short form of spir was made from the spir 8D cDNA clone cut with Tth111I and a probe specific for the long form of spir was made from the spir 4C cDNA clone cut with NcoI. A probe that would detect both the short and long forms of the spir transcript was made from the 8D cDNA clone cut with SalI.

Interaction trap analysis

The procedures, the yeast strain EGY48, and the vectors used for interaction trap analyses (pJG4-5, pEG202, pJK202, pJK103 and pSH18-34) are as described in Golemis et al. (1999). Yeast transformations were done using the high efficiency method of Gietz et al. (1992). Rho family lexA baits were constructed by in vivo recombination in yeast (Mulrad et al., 1992) or bacteria (Oliner et al., 1993). Each mutant and wild-type rho (templates provided by Liqun Luo and Denise Montel) was amplified by PCR with four primers. Two of the primers were specific to the rho family member. One introduced a C/S change in the CAAX-box isoprenylation site and both added some sequence identity with the interaction trap bait vector pEG202 on each side. These two primers were used at one-tenth the normal PCR primer concentration. The other two primers, used at normal concentration during the PCR reaction, added more sequence identity to vector pEG202 so that the final products had 30 bp of identity with the vector on each side. These PCR products were then co-transformed with pEG202 linearized by SalI and NotI digestion into yeast strain EGY48 or bacterial strain JC8679 (Oliner et al., 1993). The plasmids were recovered and sequenced. The rho-specific primers used were: for RHOA, ggtcgactcgagcggGAGCAAAAGGCtTCTGGTCTTCTTCC- TC and aattcccggggatccgtATGACGACGATTCGCaag; for RHOL, aattcccggggatccgtATGACGGCGAACATAACG and ggtcgactcgag- cggCAGTATTTTGCtCGATTGCTTGGACGTCG; for CDC42, aattcccggggatccgtATGCAAACCATCAAGTGC and ggtcgactcgag- cggTAAGAATTTGCtCTTCCTTTTCTTTGTGGG; for RAC1, aatt-cccggggatccgtATGCAGGCGATCAAGTG and ggtcgactcgagcgg- GAGCAGGGCGCtCTTGCGCTTGGACTTGG. The general primers used in all the reactions were TCGCAACGGCGACTGGctggaa- ttcccggggatccgt and TCGCCCGGAATTAGCTTGGCTgcaggtcgactc- gagcgg.

The interaction trap construct for the long version of SPIR was made by fusing the 4C and BB18-1 cDNA clones. A primer (ACACGCgtcgACACATAATGACGGAGCAC), containing an in- frame SalI site and a T7 primer, were used to amplify from the 4C cDNA clone. The PCR product was cut with SalI and NotI and cloned into pJK202 partially cut with SalI and completely cut with NotI. The 3′ end was added to this clone by cutting BB18-1 out of pJG4-5 with NotI and cloning it into pJK202 4C cut with NotI. Note that the 4C cDNA clone differs from the BB18-1 at position 3149 (4C GCG (Ala); BB18-1 GTG (Val)). The interaction trap construct contains GTG (Val).

The mutated WH2 construct was generated by amplifying from the construct containing aa 296-585 in two pieces. The first half of the mutant construct was made using primers TAGAattCATAATCG- AGACCTGCAGA and CCagatctTATGGACTCCATGAGCTGC. The second half was made using primers TAagatctGGGAAGGAGCa- CgAGCAGATCACCCCG and GTGCTcGAGTGCCTCCTCAAC-CG. The first half was digested with EcoRI and BglII, the second half with BglII and XhoI. They were ligated to pJK202 cut with EcoRI and XhoI. The entire construct was sequenced and contains only the three mutations within the second WH2 domain.

Other constructs tested in the interaction trap system were made using standard molecular methods.

In vitro binding assay

G-actin isolated from chicken breast muscle was generously provided by Bruce Patterson (University of Arizona). G-actin [100 μl of 10 μg/ml in TBS (25 mM Tris-HCl, 137 mM NaCl, 2.7 mM KCl, pH 8.0) per well] was incubated at 4°C overnight in Immulon II HB (Dynex Technologies) microtiter plates. G-actin coated and uncoated wells were washed three times with TBS then blocked for 4 hours with 0.5% bovine serum albumin, 0.05% Tween 20-TBS at room temperature. 1 μl in vitro-translated [35S]methionine-labeled SPIR protein (Ambion’s Retic Lysate IVT kit; Austin, Texas) in 100 μl 0.01% BSA, 0.001% Tween 20-TBS was added to each well and incubated at 4°C overnight. Each well was then washed three times with 0.01% BSA, 0.001% Tween 20-TBS, and bound proteins were removed by washing wells with SDS-protein sample buffer (PSB) at 100°C. Bound labeled SPIR protein was then analyzed by SDS-PAGE and exposure to a phosporimager cassette for 12 hours.

Observation of the cytoskeleton

Visualization of the microtubule cytoskeleton utilized a GFP-tau expressing gene (Gonzalez-Reyes et al., 1995). For actin staining, ovaries were dissected in PBS, fixed in 8% formaldehyde for 1 hour and then washed with PBS. 10 μl of BODIPY-phalloidin (Molecular Probes) was vacuum dried and resuspended in 200 μl PBST (PBS, 0.1% Triton X-100) and incubated with the ovaries for 4 hours. The ovaries were then washed with PBS, dissected and mounted in Aquapolymount (Polysciences, Inc.). Confocal images were taken on a Leica Confocal Microscope.

Spire cloning

Spir has previously been shown to be required for dorsal- ventral and anterior-posterior patterning of A the Drosophila egg and embryo (Manseau and Schüpbach, 1989). It is required for localization of gurken mRNA to the dorsal-anterior corner and STAUFEN and oskar mRNA to the posterior pole of the oocyte (Ephrussi et al., 1991; Kim-Ha et al., 1991; Neuman-Silberberg and Schüpbach, 1993). During mid-oogenesis in spir mutant oocytes, the microtubules prematurely bundle at the cortex of the oocyte, and premature microtubule- dependent cytoplasmic streaming occurs (Theurkauf, 1994). Cytochalasin D treatment mimics these microtubule phenotypes (Manseau et al., 1996), suggesting that the actin cytoskeleton is regulating the microtubule cytoskeleton and that spir might be affecting both the actin and microtubule cytoskeletons.

To begin to address the molecular function of SPIR, we have cloned spir. A P element- induced allele of spir, line P8 (C. Bai and P. Tolias, unpublished), was used to clone spir by plasmid rescue. cDNA clones were isolated from the Tolias ovary cDNA library (Stroumbakis et al., 1994) using the region flanking the P element as a probe. Using a probe from the 5′ end of one of the cDNAs (probe A, Fig. 1), three different RNAs (4.5 kb, 2.7 kb and 1.2 kb) were detected in ovarian RNA by northern analysis (data not shown). Northern analysis with strand-specific probes indicated that the 1.2 kb RNA is transcribed towards the P element (to the left in Fig. 1) while the 4.5 kb and 2.7 kb mRNAs are transcribed away from the P element (to the right in Fig. 1). Lesions identified in P element- and EMS-induced alleles suggest that the 4.5 and 2.7 kb mRNAs encode spir (Fig. 1, see later). We have been unable to identify cDNAs that represent the 1.2 kb mRNA, although we screened with probes that detect the 1.2 kb mRNA on northern blots.

Fig. 1.

Genomic organization of the spir region. Open boxes indicate the regions found in the mature spir transcripts. The positions of the molecular lesions in the P-induced alleles spirMM36 and spirP8 and the roo- induced allele spirI83 are indicated by triangles, while the positions of the molecular lesions in spir2F, spirRP, spirQF and spirPJ are indicated by asterisks. Both spirMM36and spirP8 have been reverted to wild-type by mobilizing the P element. Probe A detects the 1.2 kb mRNA from the opposite strand from spir.

Fig. 1.

Genomic organization of the spir region. Open boxes indicate the regions found in the mature spir transcripts. The positions of the molecular lesions in the P-induced alleles spirMM36 and spirP8 and the roo- induced allele spirI83 are indicated by triangles, while the positions of the molecular lesions in spir2F, spirRP, spirQF and spirPJ are indicated by asterisks. Both spirMM36and spirP8 have been reverted to wild-type by mobilizing the P element. Probe A detects the 1.2 kb mRNA from the opposite strand from spir.

Spir encodes a protein with similarity to PEM-5 and to the actin-binding WH2 domain

cDNAs representing the 2.7 kb mRNA and the 4.5 kb mRNA were sequenced. The DNA sequence and predicted proteins have been deposited in GenBank (accession numbers AF184975, AF184976). The spir genomic region has been completely sequenced (clone # DS05187; accession number AC002503, Berkeley Drosophila Genome Project, unpublished) and comparison of the cDNA sequence with the genomic sequence predicts the genomic organization shown in Fig. 1. Analysis of protein-coding sequences revealed that spir encodes two different proteins of 585 amino acids (aa) and 990 aa. Predicted coiled-coil regions (Lupas et al., 1991) are found at aa 281-294 and aa 932-947, and putative nuclear localization signals at aa 423, 424 and 579.

Database searches revealed that spir is related to pem-5, a gene of unknown function in the ascidian Ciona savignyi (Satou and Satoh, 1997). The proteins contain three regions of high similarity, which we have named SPEM1-3 (SPIR and PEM; Fig. 2). Pem-5 mRNA is localized to the posterior pole of the sea squirt. Since spir has a posterior group phenotype, it is of particular interest to know whether spir mRNA localizes to the posterior pole of the oocyte. In situ hybridization using probes specific for the 4.5 kb and 2.7 kb mRNAs indicates that both spir mRNAs are present in the follicle cells and nurse cells from region 2 of the germarium, but are not localized to the posterior pole of the oocyte (Fig. 3). Spir is also related to human ESTs found in a variety of tissue types (Fig. 2 legend).

Fig. 2.

Sequence alignment of SPIR with PEM-5. (A) Stick diagram showing the SPEM1, SPEM2 and SPEM3 regions with their percentage identity and similarity. Alignments of the SPEM1 region (B), SPEM2 region (C) and SPEM3 region (D) are also shown. Identical amino acids are shaded in black while similar amino acids are shaded in grey. In addition to pem-5, there are human EST sequences related to spir found in neurorendocrine lung carcinoid cells (AA928857, 1e-30), activated T-cells (EST95010, 8e-13), testis (AA383439, 7e-09), HeLa cells (AA085502, 1e-07), fetal liver/spleen (R06205, 7e-05) and mouse total fetus (W54692, 8e-15), hypothalamus (AA982706, 2e-08) and brain (AU035598, 2e-04).

Fig. 2.

Sequence alignment of SPIR with PEM-5. (A) Stick diagram showing the SPEM1, SPEM2 and SPEM3 regions with their percentage identity and similarity. Alignments of the SPEM1 region (B), SPEM2 region (C) and SPEM3 region (D) are also shown. Identical amino acids are shaded in black while similar amino acids are shaded in grey. In addition to pem-5, there are human EST sequences related to spir found in neurorendocrine lung carcinoid cells (AA928857, 1e-30), activated T-cells (EST95010, 8e-13), testis (AA383439, 7e-09), HeLa cells (AA085502, 1e-07), fetal liver/spleen (R06205, 7e-05) and mouse total fetus (W54692, 8e-15), hypothalamus (AA982706, 2e-08) and brain (AU035598, 2e-04).

Fig. 3.

In situ hybridization of spir in ovaries. (A) The earliest detectable spir transcript is in region 2 of the germarium. (B) Spir transcript is present in the nurse cells and follicle cells through early and mid-oogenesis. (C) Spir transcript is present in a stage 10 oocyte in the follicle cells and nurse cells. Similar results were obtained with probes specific for the 2.7 kb and 4.5 kb mRNA as well as with probes that detect both mRNAs.

Fig. 3.

In situ hybridization of spir in ovaries. (A) The earliest detectable spir transcript is in region 2 of the germarium. (B) Spir transcript is present in the nurse cells and follicle cells through early and mid-oogenesis. (C) Spir transcript is present in a stage 10 oocyte in the follicle cells and nurse cells. Similar results were obtained with probes specific for the 2.7 kb and 4.5 kb mRNA as well as with probes that detect both mRNAs.

Both the Simple Modular Architecture Research Tool (SMART) (Schultz et al., 1998) and BLAST searches (Altschul et al., 1990) revealed two regions in SPIR that have a low level of sequence similarity to the WH2 or VPH regions of bovine N-WASP, a protein related to the Wiskott Aldrich Syndrome protein (Miki and Takenawa, 1998; Symons et al., 1996). The sequence alignment of the SPIR WH2 regions with that of N-WASP, WASP, verprolin and Scar1, and additional WH2-containing sequences of unknown function are shown in Fig. 4A. The WH2 region of N-WASP and the WASP-related protein Scar1 have been shown to bind actin monomers in vitro (Machesky and Insall, 1998; Miki and Takenawa, 1998).

Fig. 4.

The WH2 domains of SPIR. (A)Sequence alignment of the WH2 domain of SPIR with other WH2 domains. CLUSTALW was used to generate the WH2 alignment shown and then adjustments to the alignment were made by hand. Black shading indicates positions at which 80% of the sequences are similar, dark grey shading indicates positions at which 60% of the sequences are similar, and light grey shading indicates positions at which 40% of the sequences are similar. Note that N-WASP, like SPIR, contains two, closely spaced WH2 domains, which have been shown to bind directly to actin (Miki and Takenawa, 1998). A mutant version of the second WH2 domain of SPIR that failed to interact with actin is shown at the bottom. Sequences shown are from a C. elegans ORF (GENBANK: locus CELR144, accession U23515), S. pombe YAV1 (accession Q10172), a Xenopus proline rich protein (PIR: S31719), an SH3 binding protein from R. norvegicus (accession U31159), S. cerevisae verprolin (Donnelly et - al., 1993; Munn et al., 1995), Entamoeba actobindin (accession BAA21997), a human ORF kiaa0429 (accession BAA24859), human WASP (Derry et al., 1994), Bos taurus N- WASP (Miki et al., 1996), Dictyostelium Scar1 (Machesky and Insall, 1998) and the two WH2 regions of SPIR. (B) Interaction between SPIR and Actin 5C. The position of the WH2 domains in the short form of SPIR are indicated on the wide bar at the top. The constructs of SPIR tested with actin are shown below. * indicates the second WH2 domain has been mutated, as described above. + indicates the presence of growth on galactose plates lacking leucine. An interaction between SPIR and actin 5C was observed with both the short and long forms of SPIR. β-galactosidase expression was not seen when SPIR and actin 5C were co- expressed, nor did we observe β-galactosidase expression in the positive control of profilin with actin 5C. No interaction between SPIR and actin was detected when the DNA binding and transcriptional activation fusions were switched so that actin was fused to the DNA binding domain and SPIR was fused to the transcriptional activation domain. (C) In vitro binding of SPIR to G-actin. G-actin from chicken muscle was bound to wells of microtiter plates. Wells with (+) and without (−) G-actin were then blocked and incubated overnight at 4°C with in vitro-translated [35S]methionine-labeled SPIR protein. Plates were washed and bound proteins were removed by washing with hot SDS-protein sample buffer and separated by gel electrophoresis. Each lane represents 6 wells. The prominent smaller molecular mass band is a SPIR breakdown product that binds G-actin. (D) A Coomassie-stained SDS-PAGE gel of 2 μg of the actin preparation used to demonstrate in vitro binding with SPIR (lane A); lane M, molecular mass markers.

Fig. 4.

The WH2 domains of SPIR. (A)Sequence alignment of the WH2 domain of SPIR with other WH2 domains. CLUSTALW was used to generate the WH2 alignment shown and then adjustments to the alignment were made by hand. Black shading indicates positions at which 80% of the sequences are similar, dark grey shading indicates positions at which 60% of the sequences are similar, and light grey shading indicates positions at which 40% of the sequences are similar. Note that N-WASP, like SPIR, contains two, closely spaced WH2 domains, which have been shown to bind directly to actin (Miki and Takenawa, 1998). A mutant version of the second WH2 domain of SPIR that failed to interact with actin is shown at the bottom. Sequences shown are from a C. elegans ORF (GENBANK: locus CELR144, accession U23515), S. pombe YAV1 (accession Q10172), a Xenopus proline rich protein (PIR: S31719), an SH3 binding protein from R. norvegicus (accession U31159), S. cerevisae verprolin (Donnelly et - al., 1993; Munn et al., 1995), Entamoeba actobindin (accession BAA21997), a human ORF kiaa0429 (accession BAA24859), human WASP (Derry et al., 1994), Bos taurus N- WASP (Miki et al., 1996), Dictyostelium Scar1 (Machesky and Insall, 1998) and the two WH2 regions of SPIR. (B) Interaction between SPIR and Actin 5C. The position of the WH2 domains in the short form of SPIR are indicated on the wide bar at the top. The constructs of SPIR tested with actin are shown below. * indicates the second WH2 domain has been mutated, as described above. + indicates the presence of growth on galactose plates lacking leucine. An interaction between SPIR and actin 5C was observed with both the short and long forms of SPIR. β-galactosidase expression was not seen when SPIR and actin 5C were co- expressed, nor did we observe β-galactosidase expression in the positive control of profilin with actin 5C. No interaction between SPIR and actin was detected when the DNA binding and transcriptional activation fusions were switched so that actin was fused to the DNA binding domain and SPIR was fused to the transcriptional activation domain. (C) In vitro binding of SPIR to G-actin. G-actin from chicken muscle was bound to wells of microtiter plates. Wells with (+) and without (−) G-actin were then blocked and incubated overnight at 4°C with in vitro-translated [35S]methionine-labeled SPIR protein. Plates were washed and bound proteins were removed by washing with hot SDS-protein sample buffer and separated by gel electrophoresis. Each lane represents 6 wells. The prominent smaller molecular mass band is a SPIR breakdown product that binds G-actin. (D) A Coomassie-stained SDS-PAGE gel of 2 μg of the actin preparation used to demonstrate in vitro binding with SPIR (lane A); lane M, molecular mass markers.

Identification of lesions in mutant alleles

To determine the molecular nature of the mutant lesions in spir, we used reverse transcription-PCR to identify the molecular lesions. The lesions in spir2F, spirRP and spirPJ are nonsense mutations, creating premature termination codons, while the lesion in spirQF alters a splice junction, leading to premature termination within the intron (Fig. 1). SpirI83, a hybrid dysgenesis-induced allele of spir, contains an insertion of a roo element in the region specific to the long form of spir.

SPIR binds to actin

Since SPIR contains sequence similarity to WH2 domains that bind directly to actin in vitro (Machesky and Insall, 1998; Miki and Takenawa, 1998), we tested SPIR for its ability to bind actin in the yeast interaction trap system (Finley and Brent, 1995). Co-expression of SPIR::lexA with Drosophila actin 5C fused to a transcriptional activation domain resulted in growth on galactose medium lacking leucine (Fig. 4B). This indicates that SPIR interacts with actin to stimulate expression of the leucine reporter gene. To identify the region of SPIR responsible for the interaction with actin, we tested smaller fragments of SPIR (Fig. 4B). The actin binding region is contained in a fragment of SPIR (aa 296-585), which contains both WH2 domains (aa 398-416 and aa 462- 479). Smaller fragments within this region of SPIR have only weak or no interactions with actin. A construct of the actin binding region of SPIR containing mutations in the second WH2 domain failed to interact with actin (Fig. 4A,B), confirming that the WH2 domains are responsible for SPIR’s actin binding capability. In vitro binding assays between in vitro translated SPIR and G-actin isolated from chicken muscle demonstrated that SPIR binds directly to purified G- actin (Fig. 4C).

The actin-binding WH2 domains of WASP and SCAR1 are followed by domains that interact with the p21 Arc of the Arp2/3 complex. Together these domains affect polymerization of actin (Machesky and Insall, 1998; Machesky et al., 1999; Yarar et al., 1999). SPIR does not appear to contain these domains and we failed to detect an interaction between SPIR and p21 Arc in the interaction trap system.

SPIR interacts with rho family GTPases

spir has similar phenotypes to capu (Manseau and Schüpbach, 1989; Theurkauf, 1994) and CAPU binds to rho family GTPases (J. Calley and L. Manseau, unpublished). For these reasons, we tested whether SPIR interacts with rho family GTPases in the interaction trap system. SPIR interacts with wild-type and dominant negative mutants of RHOA, RAC1 and CDC42, but not with RHOL (Table 1). We also attempted to test the constitutively active forms of the rho family members, but found that they self-activate the reporters, making it difficult to assess whether SPIR interacts with the constitutively active forms. Deletion analysis localized the rho binding domain of SPIR to the first 100 amino acids (Fig. 5).

Table 1.

Interaction of SPIR with rho family GTPases

Interaction of SPIR with rho family GTPases
Interaction of SPIR with rho family GTPases
Fig. 5.

Mapping the rho interaction domain in SPIR. The constructs of SPIR tested with RHOA are shown. + indicates the presence of growth on galactose plates lacking leucine. The interaction was observed with the short form of SPIR fused to a transcriptional activation domain and the rho family GTPase as a LexA fusion. The long form of SPIR has not been tested, but the region that interacts is common to the short and long forms of SPIR.

Fig. 5.

Mapping the rho interaction domain in SPIR. The constructs of SPIR tested with RHOA are shown. + indicates the presence of growth on galactose plates lacking leucine. The interaction was observed with the short form of SPIR fused to a transcriptional activation domain and the rho family GTPase as a LexA fusion. The long form of SPIR has not been tested, but the region that interacts is common to the short and long forms of SPIR.

Spir phenotypes

As our findings point to a role for SPIR in the actin cytoskeleton, we analyzed the actin cytoskeleton in spir mutants. Rhodamine-phalloidin staining of the actin cytoskeleton in spirEC and spir2F appeared normal (data not shown).

Previous work has shown that in spir mutants, the microtubules bundle at the cortex prematurely, during stage 8 and this bundling of the microtubules is accompanied by rapid, microtubule-dependent swirling of the cytoplasm (Theurkauf, 1994) (Fig. 6D,E). Both the bundling of microtubules and cytoplasmic streaming are normally seen later in stage 10 wild-type oocytes (Theurkauf et al., 1992).

Fig. 6.

Cytoskeletal and patterning phenotypes of spir. (A-E) Microtubule phenotypes of spir visualized with GFP-tau. During stage 6 of oogenesis in wild type (A), the microtubules are organized from the anterior cortex of the oocyte. In spir mutant egg chambers during stage 6 (B,C; spirQF/spirEC), we sometimes observe strong staining of microtubules at both the anterior and posterior cortex. During stage 8 of oogenesis in wild type (D), the microtubules appear diffuse with higher levels in the anterior end of the oocyte. In spir mutant chambers (E; spir242/spirEC), the microtubules are bundled at the cortex. (F,G) Similar expression of BB127, an enhancer trap that is expressed in anterior follicle cells in wild type (F) and in spirEC/spirEC (G). Note that posterior follicle cells do not stain, indicating that they have not undergone an anterior cell fate transformation. (H-K) In situ hybridization showing oskar mRNA distribution in wild type (H,J) and spirRP/spirPJ (I,K). In contrast to the report of Kim-Ha et al. (1991), no central dot of oskar mRNA was ever observed. Similar results were obtained with spirRP/spir242, spirRP/spirEC, spirEC/spir242, spirEC/spirED and spirEC/spirQF. (L-M) In situ hybridization showing similar bicoid mRNA distributions in wild-type (L) and spirRP/spirEC mutant (M) egg chambers.

Fig. 6.

Cytoskeletal and patterning phenotypes of spir. (A-E) Microtubule phenotypes of spir visualized with GFP-tau. During stage 6 of oogenesis in wild type (A), the microtubules are organized from the anterior cortex of the oocyte. In spir mutant egg chambers during stage 6 (B,C; spirQF/spirEC), we sometimes observe strong staining of microtubules at both the anterior and posterior cortex. During stage 8 of oogenesis in wild type (D), the microtubules appear diffuse with higher levels in the anterior end of the oocyte. In spir mutant chambers (E; spir242/spirEC), the microtubules are bundled at the cortex. (F,G) Similar expression of BB127, an enhancer trap that is expressed in anterior follicle cells in wild type (F) and in spirEC/spirEC (G). Note that posterior follicle cells do not stain, indicating that they have not undergone an anterior cell fate transformation. (H-K) In situ hybridization showing oskar mRNA distribution in wild type (H,J) and spirRP/spirPJ (I,K). In contrast to the report of Kim-Ha et al. (1991), no central dot of oskar mRNA was ever observed. Similar results were obtained with spirRP/spir242, spirRP/spirEC, spirEC/spir242, spirEC/spirED and spirEC/spirQF. (L-M) In situ hybridization showing similar bicoid mRNA distributions in wild-type (L) and spirRP/spirEC mutant (M) egg chambers.

We have confirmed these reports (Fig. 6D,E, data not shown).

A bi-directional signaling process occurs between the oocyte and the posterior follicle cells to establish the posterior pole of the egg (Gonzalez-Reyes et al., 1995; Roth et al., 1995). Phenotypes indicative of a defect in this signaling process include transformation of the posterior follicle cells into an anterior follicle cell fate, misorganization of the microtubules at stage 6, localization of oskar mRNA to the center of the oocyte, localization of bicoid mRNA to the posterior pole, and premature cytoplasmic streaming (Gonzalez-Reyes et al., 1995; Roth et al., 1995; Clegg et al., 1997). Similar to the observation by Gonzalez-Reyes et al. (1995), we have found that the posterior follicle cell fates are established correctly in spir (Fig. 6F,G). In contrast to a previous report (Kim-Ha et al., 1991), we have not observed a central spot of oskar mRNA staining (Fig. 6H-K). Finally, bicoid mRNA localization appears relatively normal in spir (Fig. 6L,M). These results suggest that signaling between the posterior follicle cells and the oocyte is not abnormal in spir mutants. We have found that in spir mutant oocytes microtubules sometimes show abnormal distributions during stage 6 (Fig. 6A-C), but we believe that this probably reflects an earlier manifestation of the known spir microtubule defect.

We report here the cloning and molecular analysis of the Drosophila maternal effect gene spir. SPIR is highly similar to the protein encoded by sea squirt pem-5 and contains two WH2 domains. We have shown that SPIR binds to actin through its WH2 domains and that SPIR interacts with rho family GTPases. This suggests that SPIR links rho family GTPase signaling to the actin cytoskeleton.

WH2 domains, like those found in SPIR, have been found in the Wiskott-Aldrich syndrome protein (WASP), verprolin, Scar-1 and a number of other proteins of unknown function (Machesky and Insall, 1998; Symons et al., 1996; Vaduva et al., 1997). The WH2 domains of N-WASP and of Scar1 have been shown to bind directly to G-actin in vitro (Machesky and Insall, 1998; Miki and Takenawa, 1998). In addition, we have shown that SPIR binds to unpolymerized actin in vitro.

Although SPIR is capable of binding to actin monomers through its WH2 domain, we have not observed defects in the actin cytoskeleton in spir mutants, suggesting a number of possibilities. The defects may be in actin structures that are difficult to observe, such as the cell cortex. In fact, we have shown that spir phenotypes can be mimicked by treatment with cytochalasin D, a drug that affects the polymerization state of actin, and we have been unable to observe defects in the actin cytoskeleton in cytochalasin D-treated oocytes. Alternatively, spir may act downstream of the actin cytoskeleton and, thus, not change it. Finally, in vitro experiments have shown that, in the absence of the neighboring cofilin homology and acidic domains, the two WH2 domains of N-WASP have either no effect on or slowly depolymerize filamentous actin (Miki and Takenawa, 1998). Since SPIR is lacking the cofilin homology and acidic domains it, similarly, may have only minor or no effect on filamentous actin.

It is becoming more apparent that a relationship exists between the actin cytoskeleton and premature microtubule- dependent cytoplasmic streaming. We have shown that the premature cytoplasmic streaming phenotype of spir can be mimicked by addition of cytochalasin D (Emmons et al., 1995), a drug which depolymerizes actin filaments. Additional evidence that the actin cytoskeleton is involved in repressing microtubule bundling and streaming comes from analysis of mutant phenotypes of genes linked to the actin cytoskeleton. We have shown that SPIR binds to actin. In addition to spir, mutants in chickadee, which encodes profilin and capu, which is thought to bind to profilin, also exhibit these microtubule behaviors (Manseau et al., 1996).

We have also shown that SPIR is capable of interacting with rho family GTPases, specifically with RHOA, CDC42 and RAC1. While phenotypes for cdc42 and rac1 have been described during oogenesis, their effects on patterning during oogenesis are unknown (Murphy and Montell, 1996). The finding that SPIR interacts with rho family GTPases suggests that at least one of the rho family GTPases is functioning in patterning. Further genetic and biochemical studies will be required to determine the nature of SPIR’s interaction with rho family GTPases in vivo.

Our analysis of spir suggests that rho family GTPAses and actin function with SPIR in patterning the Drosophila oocyte. Further studies on spir should elucidate the role of rho family GTPases and the actin cytoskeleton in patterning during oogenesis.

We would like to thank Martin Muller, Daniel St Johnston, Denise Montell and Marek Mlodzik for providing us with fly stocks, Denise Montell and Liqun Luo for providing us with mutant and wild-type rho family GTPase clones, and Bruce Patterson for providing us with purified chicken G-actin. We thank Eugen Kerkhoff for discussing unpublished results. We are grateful to Trudi Schüpbach, in whose laboratory the initial efforts to clone spire were made. Also, we appreciate the help of other members of the Manseau laboratory, Tom Bunch and Will Staatz throughout the course of this work, and Tom Bunch for careful reading of the manuscript.

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