spire: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

Gene name - spire

Synonyms -

Cytological map position - 38C

Function - signal transduction

Keywords - oogenesis, dorsal closure, cytoskeleton

Symbol - spir

FlyBase ID: FBgn0003475

Genetic map position - 2-54

Classification - Wiscott-Aldrich syndrome protein (WASP) homology domain 2 (WH2) family

Cellular location - cytoplasmic

NCBI link: Entrez Gene
spir orthologs: Biolitmine

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 contains two domains with similarity to the actin monomer-binding WH2 domain, and 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. This evidence supports the model that Spire links rho family signaling to the actin cytoskeleton (Wellington, 1999).

Previous work has shown that in spire mutants, the microtubules bundle prematurely, during stage 8 at the cortex. This premature microtubule bundling is accompanied by rapid, microtubule-dependent swirling of the cytoplasm (Theurkauf, 1994). Both the bundling of microtubules and cytoplasmic streaming are normally seen later in stage 10 wild-type oocytes (Wellington, 1999).

A bi-directional signaling process occurs between the oocyte and the posterior follicle cells to establish the posterior pole of the egg. 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. The posterior follicle cell fates are established correctly in spire. In contrast to a previous report (Kim-Ha, 1991), a central spot of Oskar mRNA staining was not observed. Bicoid mRNA localization appears relatively normal in spire. These results suggest that signaling between the posterior follicle cells and the oocyte is not abnormal in spire mutants. In spire mutant oocytes microtubules sometimes show abnormal distributions during stage 6, but it is thought that this probably reflects an earlier manifestation of the known spire microtubule defect (Wellington, 1999).

WH2 domains, like those found in Spire, have been found in the Wiskott-Aldrich syndrome protein (WASP), verprolin, Scar-1 (see Drosophila SCAR), and a number of other proteins of unknown function. The WH2 domains of N-WASP and of Scar-1 have been shown to bind directly to G-actin in vitro. In addition, Spir binds to unpolymerized actin in vitro. Although Spir is capable of binding to actin monomers through its WH2 domain, no defects in the actin cytoskeleton have been observed 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, spir phenotypes can be mimicked by treatment with cytochalasin D, a drug that affects the polymerization state of actin; defects in the actin cytoskeleton in cytochalasin D-treated oocytes have not been observed. 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. Since Spir is lacking the cofilin homology and acidic domains, Spir may have no effect on filamentous actin or only a minor one (Wellington, 1999 and references therein).

It is becoming more apparent that a relationship exists between the actin cytoskeleton and premature microtubule-dependent cytoplasmic streaming. The premature cytoplasmic streaming phenotype of spir can be mimicked by the addition of cytochalasin D, a drug that 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. In addition to spir, mutants in chickadee that encode profilin and capu, which is thought to bind to profilin, also exhibit these microtubule behaviors. While phenotypes for cdc42 and rac1 have been described during oogenesis, their effects on patterning during oogenesis are unknown. 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 (Wellington, 1999).

The p150Spir protein provides a link between c-Jun N-terminal kinase function and actin reorganization

A direct link between JNK signaling and actin organization has not been found previously. The Jun N-terminal kinase (JNK, known as Basket in Drosophila) is a downstream effector of Rac and Cdc42 GTPases involved in actin reorganization. Basket plays a role in the regulation of cell shape changes and actin reorganization during the process of dorsal closure. One potential mechanism for induction of cytoskeletal changes by Basket is via transcriptional activation of the decapentaplegic gene, a member of the TGFbeta superfamily. Spire, termed p150-Spire in this study, has a docking site for Basket/JNK, at its carboxy-terminal end. In mouse fibroblasts, p150-Spir colocalizes with F-actin; its overexpression induces clustering of filamentous actin around the nucleus. p150Spir (Otto, 2000) is slightly longer than the long form characterized by Wellington (1999). When coexpressed with p150-Spire in NIH 3T3 cells, JNK translocates to and colocalizes with p150-Spire at discrete spots around the nucleus. Carboxy-termina l sequences of p150-Spir are phosphorylated by JNK both in vitro and in vivo. It is concluded that p150-Spir is a downstream target of JNK function and provides a direct link between JNK and actin organization (Otto, 2000).

p150Spire was identified in a yeast two-hybrid screen to search for novel Basket interaction partners. The cDNA encoding p150Spire contains an open reading frame of 1,020 amino acids. Transient transfection experiments in mouse fibroblasts reveal that the cDNA directs expression of a protein that migrates with an apparent molecular weight of approximately 150 kDa on an SDS-polyacrylamide gel. The spire gene exhibits significant homology to a maternal gene pem-5 isolated from the ascidian Ciona savignyi (phylum Urochordata). Comparison of Drosophila p150Spire sequences to human EST clones reveals a high conservation between the p150Spire sequences of Drosophila and human (Otto, 2000).

The p150Spire protein contains an acidic domain, a cluster of four WH2 domains, a modified FYVE zinc finger domain and a carboxy-terminal DEJL motif (docking site for Erk and JNK containing an LXL motif). WH2 domains bind monomeric actin: WH2 family proteins such as WASP and WAVE are involved in actin reorganization. Sequences encompassing the Spir WH2 domains interact directly with monomeric actin. As has been shown for WASP and WAVE, transient expression of p150Spire in adherent mammalian cells induces clusters of filamentous actin around the nucleus that colocalize with p150Spire in all transfected cells. A modified FYVE zinc-finger motif is located in the central region of the protein. The modified structure lacks a pocket of basic amino acids between cysteines 3 and 4 of the FYVE finger structures that is necessary for the binding of phosphatidylinositol 3-phosphate (Otto, 2000).

In this study, p150Spire was identified in a yeast two-hybrid screen as a DJNK-interacting protein. The smallest fragment that still binds DJNK consists of the carboxy-terminal 53 amino acids. A DEJL motif, characterized by a cluster of basic amino acids amino-terminal to an L/I-X-L/I motif (in the single-letter amino acid code), is located in this carboxy-terminal sequence (amino acids 997-1014 of p150Spire, KQKRSSARNRTIQNLTLD). DEJL domains have been shown to mediate the docking of the Erk and JNK mitogen-activated protein (MAP) kinases to either activating kinases or substrate proteins. To further characterize the interaction between p150Spire and JNK, the localization of the two proteins was determined when transiently expressed in NIH 3T3 cells. p150Spire accumulates in punctate spots located around the nucleus. Expression of a fusion protein between glutathione-S-transferase and JNK (GST-JNK) reveals both a cytoplasmic and nuclear distribution of the kinase. In the presence of p150Spire, GST-JNK is translocated from these areas and accumulates at places where p150Spire is located. The colocalization of the proteins is detected in all cells expressing both proteins but is not found in cells coexpressing GST alone with p150Spire (Otto, 2000).

Erk and JNK MAP kinases are recruited to substrate proteins via docking sites, enabling the kinases to phosphorylate serine or threonine residues adjacent to prolines (S/TP motifs). As p150Spire contains a JNK docking site (the DEJL motif) and several potential S/TP phosphorylation motifs, attempts were made to determine whether p150Spire is a phosphorylation target of JNK. A highly specific, constitutively active Erk can be generated by the fusion of Erk2 to its upstream activator Mek1 (Erk2-Mek1-LA). Similarly, JNK2 (rat) has been fused to its upstream activator MKK7 (mouse) via a linker region. The fusion protein, JNK-MKK7, is a constitutively active Jun N-terminal kinase. JNK-MKK7 phosphorylates amino-terminal c-Jun sequences in vitro and induces an electrophoretic mobility shift of the c-Jun protein when coexpressed in NIH 3T3 cells, indicating an in vivo c-Jun phosphorylation. MKK7 activates JNK by phosphorylating a TPY motif in the central region of JNK. JNK-MKK7 exhibits autophosphorylation and interacts with a phospho-specific antibody that recognizes activated JNK protein. A similar construct (JNKK2-JNK1) has been shown to be a specific, constitutively active c-Jun kinase (Otto, 2000).

An investigation was carried out to see whether p150Spire sequences are phosphorylated by JNK in vitro. Indeed, arsenite-activated JNK1, immunoprecipitated from NIH 3T3 cell lysates, phosphorylates a carboxy-terminal fragment of the p150Spire protein in an in vitro immune-complex kinase assay. Erk and p38 MAP kinases precipitated from the same lysates exhibit a basal phosphorylation activity that could not be increased by arsenite stimulation of the cells. Coexpression of p150Spire with MLK3-activated JNK2 [mixed lineage kinase (MLK3) is an upstream activator of JNKs] or JNK-MKK7 in mouse fibroblasts, induces several slower migrating forms of the p150Spire protein during SDS-PAGE (Otto, 2000).

The electrophoretic mobility shift of p150Spire strongly suggests that it is phosphorylated by JNK on multiple sites. The slower migrating forms are abolished by phosphatase treatment, demonstrating that the mobility shifts are due to phosphorylation of p150Spire protein. Supporting these data, a kinase-inactive mutant of JNK-MKK7 (containing the JNK mutations K55A and K56A and the MKK7 mutation K76E, called JNK-MKK7-KD) does not induce an electrophoretic mobility shift of p150Spire. To analyse the specificity of JNK phosphorylation of p150Spire in vivo, p150Spire was coexpressed with the constitutively active Erk2-Mek1-LA fusion protein. Although expressed to the same levels as JNK-MKK7, Erk2-Mek1-LA induces only a very weak electrophoretic mobility shift of p150Spire, indicating a preferential phosphorylation of p150Spire by JNK (Otto, 2000).

In conclusion, the JNKs are downstream effectors of the Rac and Cdc42 GTPases involved in actin reorganization. Microinjection of activated Rac and Cdc42 proteins into cells induces changes in actin structures within minutes, before de novo protein synthesis. A WH2 family protein p150Spire, involved in actin reorganization, has been identified as a phosphorylation target of JNK activity. Strikingly, amino-terminal sequences of Spir interact with Rho family GTPases in yeast two-hybrid experiments (Wellington, 1999). One possible mechanism of actin regulation by Rho GTPases could therefore be the activation of a MAP kinase, which subsequently phosphorylates an actin regulator such as WAVE or p150Spire, thus regulating a role in either actin nucleation or the localization of actin regulating proteins. Future experiments will illuminate the cellular functions of p150Spire and its regulation by JNK phosphorylation (Otto, 2000).

Regulatory interactions between two actin nucleators, Spire and Cappuccino

Spire and Cappuccino are actin nucleation factors that are required to establish the polarity of Drosophila oocytes. Their mutant phenotypes are nearly identical, and the proteins interact biochemically. This study found that interaction between Spire and Cappuccino family proteins is conserved across metazoan phyla and is mediated by binding of the formin homology 2 (FH2) domain from Cappuccino (or its mammalian homologue formin-2) to the kinase noncatalytic C-lobe domain (KIND) from Spire. In vitro, the KIND domain is a monomeric folded domain. Two KIND monomers bind each FH2 dimer with nanomolar affinity and strongly inhibit actin nucleation by the FH2 domain. In contrast, formation of the Spire-Cappuccino complex enhances actin nucleation by Spire. In Drosophila oocytes, Spire localizes to the cortex early in oogenesis and disappears around stage 10b, coincident with the onset of cytoplasmic streaming (Quinlan, 2007).

The spire and cappuccino genes have been linked since their discovery in a genetic screen 17 yr ago (Manseau, 1989). The KIND domain of Spir binds with high affinity to the Capu-FH2 domain at a stoichiometry of 2:2 (two KIND monomers to one FH2 dimer). The WH2 cluster of Spir interacts with Capu-FH2 but that this interaction is three orders of magnitude weaker than that between the Capu-FH2 and the KIND domain. Although binding is detected between the two domains, the Capu-FH2 domain has no direct effect on actin nucleation by the Spir-WH2 cluster. However, if the KIND domain is present and correctly folded, binding of the FH2 dimer increased nucleation activity of the WH2 cluster. In contrast, the KIND domain potently inhibits actin nucleation by the Capu-FH2 domain. Constructs containing both the KIND and WH2 cluster do not enhance the inhibition of Capu-FH2-mediated actin nucleation or microtubule bundling over that observed for the KIND domain alone. For these reasons, it is proposed that the KIND-FH2 interaction is more physiologically relevant than the WH2-FH2 interaction. Additional structural and functional studies of the KIND domain are required to determine how many KIND domains are required to inhibit actin nucleation and to compete for actin and microtubule binding (Quinlan, 2007).

The KIND module was initially identified as a conserved region in the N-terminal half of Spir proteins (Ciccarelli, 2003), and the region was named based on its sequence similarity to the C-lobe of the protein kinase fold (Ciccarelli, 2003). The KIND domain is found only in metazoa, and its consensus sequence lacks catalytic residues required for kinase activity. Because the substrates of protein kinases interact with α-helical regions in the C-lobe, it was hypothesized that the KIND domain evolved from a functional kinase into a protein-protein interaction domain. The discovery that the Spir KIND domains bind specifically to Capu family FH2 domains supports this hypothesis (Quinlan, 2007).

What role do Spir and Capu play in oogenesis? Spir disappears from the oocyte cortex at stage 10, when rapid streaming normally begins and its absence in spire mutant flies leads to premature streaming. This strongly suggests that Spir plays an inhibitory role in rapid streaming. It is not yet known whether endogenous Capu has the same restricted temporal pattern observed for Spir. This information will be essential to understanding the nature of the Spir-Capu complex and its role during oogenesis. Spir and Capu interact in the oocyte, and Rosales-Nieves (2006) found that GFP fusions of these proteins both exist at the oocyte cortex, placing them in an ideal location to coordinate actin and possibly anchor microtubules. Rapid streaming is, in part, characterized by bundling and movement of microtubules. Capu bundles microtubules, which is an activity regulated by Spir. If Spir is removed at stage 10 but Capu remains, Capu could play a role in reorganizing the microtubule cytoskeleton and possibly coordinating it with the actin cytoskeleton. A complete understanding of how Spir and Capu achieve this coordination depends on knowing when and how the Spir-Capu complex is regulated (Quinlan, 2007).

Capu and other members of the formin family nucleate de novo actin filament assembly and remain associated with elongating barbed ends of newly formed filaments (Pring, 2003; Quinlan, 2005). The activity of most formin family proteins is regulated by an autoinhibitory interaction between an N-terminal sequence (the Diaphanous inhibitory domain [DID]) and a C-terminal sequence (the Diaphanous autoinhibitory domain [DAD]). Small G proteins of the Rho family stimulate nucleation activity by binding to the DID domain and disrupting its interaction with DAD. However, Capu family formins lack both DID and DAD domains (Higgs, 2005). In fact, Rosales-Nieves (2006) did not observe autoinhibition when combining the N terminus of Capu with the FH2 domain, as has been observed for mDia1 (Li 2003). The results argue strongly that Capu activity is regulated in trans by interaction with Spir (Quinlan, 2007).

The mechanism of actin nucleation by Spir is very different from that of formins like Capu. Spir binds four actin monomers using four closely apposed binding sites and then assembles them into a filament nucleus. After nucleation, Spir proteins remain associated with the slow-growing pointed end of the new filament. If Spir and Capu always function together as a single filament-forming complex, it is suggested that their activities might synergize. One intriguing possibility is that Spir nucleates filaments whose free barbed ends are then handed off to Capu. Such a mechanism would enable the independent control of filament nucleation and barbed end binding. The tight binding that was measured suggests that Spir and Capu may not dissociate upon nucleation but that actin and microtubules do bind competitively. This idea begs two important questions: (1) Does the activation of Capu require the complete dissociation of Spir, or can the two proteins function together as a single filament-forming unit? (2) How is the Spir-Capu interaction modulated by upstream signaling systems? Recent data implicate the GTPase Rho as a regulator of Spir-Capu interaction in Drosophila (Rosales-Nieves, 2006). The Spir-Capu interaction is evolutionally conserved, but whether or not this mode of regulation is conserved remains to be tested (Quinlan, 2007).

Histone acetyltransferase Enok regulates oocyte polarization by promoting expression of the actin nucleation factor spire

KAT6 histone acetyltransferases (HATs) are highly conserved in eukaryotes and have been shown to play important roles in transcriptional regulation. This study demonstrates that the Drosophila KAT6 Enok acetylates histone H3 Lys 23 (H3K23) in vitro and in vivo. Mutants lacking functional Enok exhibited defects in the localization of Oskar (Osk) to the posterior end of the oocyte, resulting in loss of germline formation and abdominal segments in the embryo. RNA sequencing (RNA-seq) analysis revealed that spire (spir) and maelstrom (mael), both required for the posterior localization of Osk in the oocyte, were down-regulated in enok mutants. Chromatin immunoprecipitation showed that Enok is localized to and acetylates H3K23 at the spir and mael genes. Furthermore, Gal4-driven expression of spir in the germline can largely rescue the defective Osk localization in enok mutant ovaries. These results suggest that the Enok-mediated H3K23 acetylation (H3K23Ac) promotes the expression of spir, providing a specific mechanism linking oocyte polarization to histone modification (Huang, 2014).

This study reveals a previously unknown transcriptional role for Enok in regulating the polarized localization of Osk during oogenesis through promoting the expression of spir and mael. Spir and Mael are required for the properly polarized MT network in oocytes from stages 8 to 10A. However, protein levels of both decreased at later stages of oogenesis, allowing reorganization of the MT network and fast ooplasmic streaming. The persistent presence of Spir extending into stage 11 led to loss of ooplasmic streaming and resulted in female infertility. These findings suggest that the temporal regulation of spir expression is crucial for oogenesis, and, interestingly, Enok protein levels were also reduced in egg chambers during stages 10-13 compared with stages 1-9. While the stability of Spir or the translation of spir mRNA may also be a target for regulation, the results suggest that Enok is involved in the dynamic modulation of spir transcript. Furthermore, the results demonstrate the importance of Enok for expression of spir and mael in both ovaries and S2 cells, suggesting that Enok may play a similar role in other Spir- or Mael-dependent processes such as heart development (Huang, 2014).

Notably, Mael is also important for the piRNA-mediated silencing of transposons in germline cells. Mutations in genes involved in the piRNA pathway, including aub and armitage (armi), result in axis specification defects in oocytes as well as persistent DNA damage and checkpoint activation in germline cells. The activation of DNA damage signaling is suggested to cause axis specification defects in oocytes, as the disruption of Osk localization in piRNA pathway mutants can be suppressed by mutations in mei-41 or mnk, which encode ATR or checkpoint kinase 2, respectively. However, mutation in mnk cannot suppress the loss of posteriorly localized Osk in the mael mutant oocyte, indicating that the oocyte polarization defect in the mael mutant is independent of DNA damage signaling. Therefore, although the possibility that the piRNA pathway is affected in enok mutants due to down-regulation of mael cannot be excluded, the Osk localization defect in the enok mutant oocyte is likely independent of mei-41 and mnk (Huang, 2014).

In addition to the osk mRNA localization defect, both spir and mael mutants affect dorsal-ventral (D/V) axis formation in oocytes. However, no defects in the D/V patterning were observed in the eggshells of enok mutant germline clone embryos. Interestingly, among the spir mutant alleles that disrupt formation of germ plasm, only strong alleles result in dorsalized eggshells and embryos, while females with weak alleles produce eggs with normal D/V patterning. Since the enok1 and enok2 ovaries still express ~25% of the wild-type levels of spir mRNA, enok mutants may behave like weak spir mutants. Similarly, the ~40% reduction in mael mRNA levels in enok mutants as compared with the wild-type control may not have significant effects on the D/V axis specification (Huang, 2014).

Redundancy in HAT functions has been reported for both Moz and Sas3, the mammalian and yeast homologs of Enok, respectively. In yeast, deletion of either GCN5 (encoding the catalytic subunit of ADA and SAGA HAT complexes) or SAS3 is viable. However, simultaneously deleting GCN5 and SAS3 is lethal due to loss of the HAT activity of the two proteins, suggesting that Gcn5 and Sas3 can compensate for each other in acetylating histone residues. Indeed, while deleting SAS3 alone had no effect on the global levels of H3K9Ac and H3K14Ac, disrupting the HAT activity of Sas3 in the gcn5Δ background greatly reduced the bulk levels of H3K9Ac and H3K14Ac in yeast. Also, mammalian Moz targets H3K9 in vivo and regulates the expression of Hox genes, but the global H3K9Ac levels are not significantly affected in the homozygous Moz mutant, indicating that other HATs have overlapping substrate specificity with Moz. In flies, a previous study had reported that the H3K23Ac levels were reduced 35% in nejire (nej) mutant embryos, which lack functional CBP/p300 . However, knocking down nej by dsRNA in S2 cells severely reduced levels of H3K27Ac but had no obvious effect on global levels of H3K23Ac. This study showed that the global H3K23Ac levels decreased 85% upon enok dsRNA treatment in S2 cells. This study also showed that the H3K23Ac levels are highly dependent on Enok in early and late embryos, larvae, adult follicle cells and nurse cells, and mature oocytes. Therefore, although Nej may also contribute to the acetylation of H3K23, the results indicate that, in contrast to its mammalian and yeast homologs, Enok uniquely functions as the major HAT for establishing the H3K23Ac mark in vivo (Huang, 2014).

The H3K23 residue has been shown to stabilize the interaction between H3K27me3 and the chromodomain of Polycomb. Therefore, acetylation of H3K23 may affect the recognition of H3K27me3 by the Polycomb complex. Another study showed that the plant homeodomain (PHD)-bromodomain of TRIM24, a coactivator for estrogen receptor α in humans, binds to unmodified H3K4 and acetylated H3K23 within the same H3 tail. Also, the levels of H3K23Ac at two ecdysone-inducible genes, Eip74EF and Eip75B, have been shown to correlate with the transcriptional activity of these two genes at the pupal stage, suggesting the involvement of H3K23Ac in ecdysone-induced transcriptional activation. This study further provided evidence for the activating role of the Enok-mediated H3K23Ac mark in transcriptional regulation (Huang, 2014).

In mammals, MOZ functions as a key regulator of hematopoiesis. Interestingly, one of the genes encoding mammalian homologs of Spir, spir-1, is expressed in the fetal liver and adult spleen, indicating the expression of spir-1 in hematopoietic cells. Thus, it will be intriguing to investigate whether the Drosophila Enok-Spir pathway is conserved in mammals and whether Spir-1 functions in hematopoiesis. Taken together, the results demonstrate that Enok functions as an H3K23 acetyltransferase and regulates Osk localization, linking polarization of the oocyte to histone modification (Huang, 2014).


cDNA clone length - 2.7 and 4.5 kb

Bases in 5' UTR - 585 (long form)

Bases in 3' UTR - 1235 (long form)


Amino Acids - 585 and 990 (short and long form respectively; Wellington, 1999) and 1020 (p150-Spir; Otto, 2000)

Structural Domains

spir encodes two different proteins of 585 amino acids (aa) and 990 aa. Predicted coiled-coil regions are found at aa 281-294 and aa 932-947, and putative nuclear localization signals at aa 423, 424 and 579. Database searches reveal that spire is related to pem-5, a gene of unknown function in the ascidian Ciona savignyi (Satou, 1997). The proteins contain three regions of high similarity, and have been named SPEM1-3 (SPIRE and PEM). 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 Spire 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 Spire 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. Spire is also related to human ESTs found in a variety of tissue types (Wellington, 1999).

Both the Simple Modular Architecture Research Tool (SMART) and BLAST searches reveal two regions in Spire 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. The WH2 region of N-WASP and the WASP-related protein Scar1 have been shown to bind actin monomers in vitro (Wellington, 1999 and references therein).

A novel DJNK-interacting protein, p150Spire, belongs to the Wiscott-Aldrich syndrome protein (WASP) homology domain 2 (WH2) family of proteins involved in actin reorganization. It is a multidomain protein with a cluster of four WH2 domains, a modified FYVE zinc-finger motif, and a DEJL motif that serves as a docking site for JNK, at its carboxy-terminal end (Otto, 2000).


The posterior-vegetal cytoplasm of an ascidian egg contains maternal factors required for pattern formation and cell specification of the embryo. The isolation and characterization is reported of cDNA clones for novel maternal genes, posterior end mark 2 (pem-2), pem-4, pem-5 (a homolog of spire), and pem-6. These clones were obtained from a cDNA library of Ciona savignyi fertilized egg mRNAs subtracted with gastrula mRNAs by examining the localization of the corresponding mRNAs of randomly selected clonesusing whole-mount in situ hybridization. As in the case of pem, all of these mRNAs were localized in the posterior-vegetal cytoplasm of the egg, and they later marked the posterior end of early embryos. The predicted amino acid sequence suggests that PEM-2 contains a signal for nuclear localization, a src homology 3 (SH3) domain, and a consensus sequence of the CDC24 family guanine nucleotide dissociation stimulators (GDSs). PEM-4 has a signal for nuclear localization and three C2H2-type zinc finger motifs, while PEM-5 and PEM-6 show no similarity to known proteins. These results provide further evidence that the ascidian egg contains maternal messages that are localized in the posterior-vegetal cytoplasm (Satou, 1997).

The Wiskott-Aldrich homology domain 2 (WH2) family protein Spir and the formin Cappuccino belong to two distinct classes of actin organizers. Despite their functional classification as actin organizers, a major defect of Drosophila spire and cappuccino mutant oocytes is a failure in the orientation of microtubule plus ends towards the posterior pole. Mammalian homologues of spire are the spir-1 and spir-2 genes. The mouse and human formin-1 and formin-2 genes have high similarity to the cappuccino gene. The mouse formin-2 gene has been found to be expressed in the developing nervous system and in neuronal cells of the adult brain. By analyzing the expression of the spir-1 gene it has been shown that spir-1 and formin-2 have a nearly identical expression pattern during mouse embryogenesis and in the adult brain. In mouse embryos both genes are expressed in the developing nervous system. In the adult brain high expression of the genes was found in the Purkinje cells of the cerebellum and in neuronal cells of the hippocampus and dentate gyrus (Schumacker, 2004).

Coordinated recruitment of Spir actin nucleators and myosin V motors to Rab11 vesicle membranes

There is growing evidence for a coupling of actin assembly and myosin motor activity in cells. However, mechanisms for recruitment of actin nucleators and motors on specific membrane compartments remain unclear. This study reports how Spir actin nucleators (see Drosophila Spire) and myosin V (see Drosophila Didum) motors coordinate their specific membrane recruitment. The myosin V globular tail domain (MyoV-GTD) interacts directly with an evolutionarily conserved Spir sequence motif. Crystal structures of MyoVa-GTD bound either to the Spir-2 motif or to Rab11 (see Drosophila Rab11) was determined, and it was shown that a Spir-2:MyoVa:Rab11 complex can form. The ternary complex architecture explains how Rab11 vesicles support coordinated F-actin nucleation and myosin force generation for vesicle transport and tethering. New insights are also provided into how myosin activation can be coupled with the generation of actin tracks. Since MyoV binds several Rab GTPases, synchronized nucleator and motor targeting could provide a common mechanism to control force generation and motility in different cellular processes (Pylypenko, 2016).

spire: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

date revised: 2 June 2004

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