The Akt1 kinase governs a number of cellular activities including cell proliferation, cell survival, and cytoskeleton organization [Fig

The Akt1 kinase governs a number of cellular activities including cell proliferation, cell survival, and cytoskeleton organization [Fig. subsynaptic reticulum (SSR). Several lines of evidence indicated that influences SSR assembly by regulation of Gtaxin, a t-SNARE protein (Gorczyca et al., 2007) in a manner independent of the mislocalization of GluRIIA. Our findings show that governs two critical elements of synapse development, neurotransmitter receptor localization, and postsynaptic membrane elaboration. plays critical roles in cell growth and cell survival (Chen et al., 2001). Akt phosphorylation of AS160 influences exocytosis of glucose transporter-containing vesicles, providing an increased capacity for glucose transport across the plasma membrane (Gonzalez and McGraw, 2006; Watson and Pessin, 2006; Grillo et al., 2009). Consistent with a role of Akt in glucose uptake and homeostasis, mice null for peripheral sensory neurons, demonstrating the capacity of this kinase to govern membrane processes that influence synaptic function (Parrish et al., 2009). The central role of Akt in signal integration prompted us to explore its function in the development of the neuromuscular junction. The neuromuscular junction is a powerful model for molecular analysis of synapse development and plasticity. Each muscle of the larval body wall is innervated by identifiable motoneurons, and these peripheral synapses are well described at the molecular, morphological, and physiological levels (Jan and Jan, 1976; Gramates and Budnik, 1999; Ruiz-Canada and Budnik, 2006; Schuster, 2006). The NMJ is a synapse that expands greatly during larval growth, and the dynamic matching of pre- and postsynaptic elements is critical for its assembly. The growth of the NMJ is accompanied by the expansion of a specialized postsynaptic membrane, the subsynaptic reticulum (SSR), as well as the regulated expression of specific glutamate receptor subunits. GluRIIA is critical for the functional strengthening Rabbit Polyclonal to MUC13 and morphological growth of the synapse that accompanies muscle expansion during development (Petersen et al., 1997; Sigrist et al., 2002). We have explored the function of the single gene in is required for the developmentally regulated expansion of the SSR, in addition to regulating glutamate receptor composition. These findings demonstrate that serves a critical role in two fundamental elements of synapse development. MATERIALS AND METHODS Fly Stocks All fly strains were raised in standard cornmeal food at 25C during embryogenesis and 30C during larval development under a 12-h/12-h day/night cycle, unless otherwise stated. strain served as the wild type stock. and were obtained from the Bloomington Stock Center (BDSC). is a is embryonic lethal, but transheterozygotes are semi-viable and some survive to the adult stage. transposon-containing stocks (BDSC) were used for muscle and neuronal-specific expression of (Vienna Drosophila RNAi Center (VDRC) #103703), (VDRC #105113), (from V. Budnick, University of Massachusetts (Gorczyca et al., 2007)), (Kittel et al., 2006), and (BDSC Sildenafil #5137) constructs, respectively. Protein trap line (Flytrap #”type”:”entrez-nucleotide”,”attrs”:”text”:”G00311″,”term_id”:”485169″,”term_text”:”G00311″G00311) directs the expression of GFP-tagged Basigin under the control of its endogenous promoter. was used together with to increase the effectiveness of RNA interference in neurons (Dietzl et al., 2007). The temperature-sensitive GAL80 repressor, under tubulin promoter (line for temporal control of expression in the muscle (Zeidler et al., 2004). Sildenafil GAL80ts suppressed GAL4 function at the permissive temperature (18C). At the restrictive temperature (30C), GAL80ts released GAL4, allowing its binding to the UAS, and inducing the expression of at the early developmental stage, embryos were kept at 30C for 2 Sildenafil days and then raised at 18C until they reached third instar larval stage. In contrast, animals, which was suppressed at the late stage, were raised at 18C until second instar stage and then shifted to 30C for 2 days before immunohistochemistry. The constitutively active forms of ((and constructs were cloned into vector and then integrated into the third chromosome (99F8) by site-specific Ca2+ in 0.1Na-cacodylate, pH 7.4) at 4C overnight. Postfixation was done in 1% osmium tetroxide, and en bloc.