The Spn-F Ik2 and Javelin-like (Jvl) proteins interact to regulate oocyte

The Spn-F Ik2 and Javelin-like (Jvl) proteins interact to regulate oocyte mRNA localization and cytoskeleton organization. is usually disrupted. Expression of Ik2 rescued Spn-FΔC ovarian phenotypes. We found that whereas Spn-F actually interacts with Ik2 and Jvl Spn-FΔC actually interacts with Ik2 but not Eriocitrin with Jvl. Thus expression of Spn-FΔC which lacks the Jvl-interacting domain name probably interferes with conversation of Ik2 and Jvl. In summary our results demonstrate that Spn-F mediates the conversation between Eriocitrin Ik2 and Jvl to control Ik2 activity. INTRODUCTION During development and cell differentiation mRNA localization is usually a crucial step in the regulation of gene expression of many transcripts. Accurate mRNA localization permits precise temporal and spatial regulation of protein production during development in a variety of organisms and cell types. RNA localization has been described in organisms as diverse as yeast and humans and has been observed in many polarized cells such as oocytes fibroblasts or neurons. In spindle-F (Spn-F) IKKε homologue (Ik2) and novel MT-associated protein Javelin-like (Jvl) together produce a complex of proteins that affect both oogenesis and bristle development (1-4). We as well as others have shown that females carrying mutations in these genes produce eggs and embryos with polarity defects that arise due to disruptions in cytoskeleton business and mRNA localization during oocyte development (1 2 4 We moreover have demonstrated that these three proteins actually interact and that their proper cell localization and function are interdependent (3 4 In their physical conversation Ik2 phosphorylates Spn-F although such phosphorylation does not affect the stability of the protein (4). In addition has also Eriocitrin been found to be involved in Eriocitrin other processes including spindle business (5 6 dendrite pruning (7) bristle MT function (8 9 F-actin assembly regulation (10 11 and the shuttling of recycling endosomes during bristle cell elongation (12). Closer examination of and ovarian defects reveals that whereas both mutants share the same defects in terms of cytoskeleton business the mutations differ in their effects on mRNA localization. In the mutants both transport toward the minus end of the MT and the organization of the Grhpr MTs that surround the oocyte nucleus are strongly affected (1 2 The and mutants also present the same defects in terms of mRNA and protein localization. However while over 90% of the embryos produced by mutant females are bicaudal (2) this phenotype is only rarely found in mutant embryos (1). This Eriocitrin Eriocitrin difference could be attributed to the fact that in ovaries and embryos produced by mutant females (mutant ovaries mRNA and protein localization are not affected. The difference seen in mRNA but not in mRNA localization defects between and mutants raises the question as to which molecular mechanisms control the actions of these proteins. To better understand the function of these genes in mRNA localization and cytoskeleton business during development structure-function analysis of Spn-F protein was conducted. We show that this Spn-F protein may act as a mediator between Ik2 and Jvl to regulate Ik2 activity. Thus our results provide a new perspective around the function of these proteins in pattern formation of the egg and embryo demonstrating that Spn-F and Jvl act on the core Ik2 function to augment the activity of this complex. MATERIALS AND METHODS stocks. Oregon-R served as a wild-type control. The following mutants and transgenic flies were used: (1) hybridization. RNA hybridization on ovaries and embryos was carried out as described previously (1 20 β-Galactosidase and antibody staining. β-Galactosidase staining of ovaries was performed as described by Peretz et al. (18) with the exception that the ovaries were incubated in X-Gal (5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside) stock solution at room heat. Antibody staining of ovaries was performed as described previously (4). The following primary antibodies were used: mouse anti-Grk (1:10; clone 1D12) (21) rabbit anti-Oskar (1:3 0 (22) mouse anti-α-tubulin (1:100; Sigma) and rabbit anti-pIKK (10). Goat anti-mouse Cy3- or Cy2-labeled secondary antibodies and goat anti-rabbit Cy3- or Cy2-labeled secondary antibodies (Jackson ImmunoResearch) were used.