Regulation: How is the Spir-Capu complex regulated?

Cell polarity is commonly established in response to external signals which lead to rearrangement of the actin cytoskeleton. Given its role in actin nucleation, the Spir-Capu complex is a very likely target of regulation. We already know that Spir is removed from the oocyte cortex in a temporally controlled manner (Fig. 3). The Spir-Capu complex or Spir alone may be degraded or physically swept away at the onset of streaming. Based on existing data, candidate regulators of the Spir-Capu complex include Jun- N-terminal kinase, Rab11 and Rho. We will combine standard biochemical approaches and genetic screens to find proteins that interact with and regulate the Spir-Capu complex. A FRET assay that reports Spir-Capu interaction will facilitate rapid screening of potential regulators. We will combine the FRET assay and pyrene-actin polymerization assays to test regulation of the complex and its affect on nucleation activity

Immunofluorescence image of stage 9 and 10 Drosophila egg chambers. Spir (green) colocalizes with filamentous actin (red) at the oocyte cortex (arrow) only before stage 10 (arrowhead).

 Interaction between Spir and Capu affects actin nucleation. (A) The Spir-KIND domain inhibits actin nucleation by Capu-family formins. We induced polymerization by mixing pyrene-labeled actin with Mg2+, K+ and Capu-FH1FH2. Addition of KIND to Capu-FH1FH2 before mixing with actin potently inhibited nucleation activity in a dose dependent manner. Protein concentrations: Actin – 4 mM (5% pyrene labeled), Capu-FH1FH2 – 10 nM, KIND – as indicated. (B) Capu enhances actin nucleation by Spir.  We mixed several concentrations of a nucleation incompetent mutant of Capu-FH1FH2 (Capu-FH1FH2(I706A)) with the N-terminal half of Spir (NTSpir), which contains the KIND domain and four WH2 domains (only three concentrations are shown for clarity). Nucleation activity of NTSpir was increased by Capu-FH1FH2(I706A) until the proteins were approximately equimolar and then activity decreased. Inset. Capu-FH1FH2(I706A) does not enhance the activity of the WH2 cluster alone, indicating that an interaction between the KIND domain and the formin is necessary. Protein concentrations: Actin – 4 mM (5% pyrene labeled), NTSpir and WH2 – 250 nM, Capu-FH1FH2(I706A) –as indicated.