The glutathione redox couple (GSH/GSSG) and hydrogen peroxide (H(2)O(2)) are central to redox homeostasis and redox signaling, yet their distribution within an organism is difficult to measure. Using genetically encoded redox probes in Drosophila, we establish quantitative in vivo mapping of the glutathione redox potential (E(GSH)) and H(2)O(2) in defined subcellular compartments (cytosol and mitochondria) across the whole animal during development and aging. A chemical strategy to trap the in vivo redox state of the transgenic biosensor during specimen dissection and fixation expands the scope of fluorescence redox imaging to include the deep tissues of the adult fly. We find that development and aging are associated with redox changes that are distinctly redox couple-, subcellular compartment-, and tissue-specific. Midgut enterocytes are identified as prominent sites of age-dependent cytosolic H(2)O(2) accumulation. A longer life span correlated with increased formation of oxidants in the gut, rather than a decrease.

Stages in development with rapid transitions between mitosis and morphogenesis may require specific mechanisms to coordinate cell shape change. Here we describe a novel mitotic inhibitor that acts during Drosophila gastrulation to counteract String/Cdc25, specifically in the cells that invaginate to form the mesoderm. We have identified two genes, frühstart and tribbles, that are required for this ventral inhibition. tribbles encodes a kinase-related protein whose RNA, however, is also present outside of the ventral region. Effective inhibition of mitosis in the cells of the ventral furrow depends on the transcription factor Snail that triggers the ventral cell shape changes. When overexpressed in a microinjection assay, Tribbles directly inhibits mitosis. We propose that Frühstart and Tribbles form a link between the morphogenetic movements and mitotic control.

The RhoA-effector Dia1 controls actin-dependent processes such as cytokinesis, SRF transcriptional activity, and cell motility. Dia1 polymerizes actin through its formin homology (FH) 2 domain. Here we show that Dia1 acts upstream of RhoA independently of its effects on actin assembly. Dia1 binds to the leukemia-associated Rho-GEF (LARG) through RhoA-dependent release of Dia1 autoinhibition. The FH2 domain stimulates the guanine nucleotide exchange activity of LARG in vitro. Our results reveal that Dia1 is necessary for LPA-stimulated Rho/ROCK signaling and bleb-associated cancer cell invasion. Thus, Dia1-dependent RhoA activation constitutes a positive feedback mechanism to modulate cell behavior.Supplemental material is available at http://www.genesdev.org.Received January 11, 2007; revised version accepted May 8, 2007. Diaphanous-related formins (DRFs) are Rho-GTPasebinding proteins that possess conserved functions in actin cytoskeleton dynamics exerted through their formin homology (FH) 2 domains (Goode and Eck 2007). DRFs are involved in essential cellular processes such as cytokinesis, cell movement, and polarity (Faix and Grosse 2006;Gomez et al. 2007), which are frequently deregulated during pathological situations like tumor cell transformation and metastasis (Sahai 2005). The dormant conformation of the DRF Dia1 is maintained by intramolecular association of its regulatory N terminus to the diaphanous autoregulatory domain (DAD), which is relieved through binding of active RhoA (Lammers et al. 2005;Otomo et al. 2005a). The catalytic FH2 domain is believed to become "exposed" by conformational changes in the DRFs to promote barbed end actin polymerization by forming a tethered dimer (Xu et al. 2004;Otomo et al. 2005a). The FH2 domain of Dia1 promotes stress fiber formation and transcriptional activation of the MAL/SRF pathway through its actin-polymerizing activity (Copeland and Treisman 2002;Grosse et al. 2003;Miralles et al. 2003). A Dia1 mutant defective in FH2 dimerization interferes with lysophosphatidic acid (LPA)-induced stress fiber formation and SRF activity (Copeland and Treisman 2002), suggesting that Dia1 is part of LPA signal transduction known to play an important role in cell proliferation and metastasis of a variety of human cancers (Mills and Moolenaar 2003). LPA receptors belong to the group of G-protein-coupled receptors that activate the heterotrimeric G-proteins G 12 and G 13 , which can bind to RGS-containing Rho-GEFs such as leukemia-associated Rho-GEF (LARG), initially isolated from a patient with acute myeloid leukemia (AML) (Kourlas et al. 2000;Vazquez-Prado et al. 2004). Rhodependent mechanisms have emerged as critical processes in tumor progression (Sahai and Marshall 2002;Lozano et al. 2003), and evidence exists that Rho/ROCK function is essential to promote a specific type of rounded bleb-associated mode of cell invasion (Sahai and Marshall 2003;Wyckoff et al. 2006). However, the role of DRFs in tumor cell behavior has not been investigated.In this study, we provide evi...

The physical interaction of the plasma membrane with the associated cortical cytoskeleton is important in many morphogenetic processes during development. At the end of the syncytial blastoderm of Drosophila the plasma membrane begins to fold in and forms the furrow canals in a regular hexagonal pattern. Every furrow canal leads the invagination of membrane between adjacent nuclei. Concomitantly with furrow canal formation, actin filaments are assembled at the furrow canal. It is not known how the regular pattern of membrane invagination and the morphology of the furrow canal is determined and whether actin filaments are important for furrow canal formation. We show that both the guanyl-nucleotide exchange factor RhoGEF2 and the formin Diaphanous (Dia) are required for furrow canal formation. In embryos from RhoGEF2 or dia germline clones, furrow canals do not form at all or are considerably enlarged and contain cytoplasmic blebs. Both Dia and RhoGEF2 proteins are localised at the invagination site prior to formation of the furrow canal. Whereas they localise independently of F-actin, Dia localisation requires RhoGEF2. The amount of F-actin at the furrow canal is reduced in dia and RhoGEF2 mutants, suggesting that RhoGEF2 and Dia are necessary for the correct assembly of actin filaments at the forming furrow canal. Biochemical analysis shows that Rho1 interacts with both RhoGEF2 and Dia, and that Dia nucleates actin filaments. Our results support a model in which RhoGEF2 and dia control position, shape and stability of the forming furrow canal by spatially restricted assembly of actin filaments required for the proper infolding of the plasma membrane.