, 2000; Figure 1A). The organization of the hts locus is shown

in a schematic that includes the position of three molecularly defined mutations and a deficiency that we use in this study ( Figure 1A; Petrella et al., 2007). In addition, two antibodies, Hts1B1 and Hts-M, are available that recognize unique epitopes within Drosophila Hts ( Petrella et al., 2007 and Zaccai PLX4032 molecular weight and Lipshitz, 1996; Figure 1A). These antibodies were used to confirm and define the molecular nature of our mutant hts alleles and to analyze the presence of the Hts-M isoform at the neuromuscular junction (NMJ). Both antibodies clearly label the Drosophila neuromuscular junction. Staining is present in the presynaptic motor axons, throughout the postsynaptic muscle, and is also concentrated within the postsynaptic muscle membrane folds, termed the subsynaptic reticulum (SSR), that surround the

NMJ ( Figure 1C). The P element insertion hts1103 completely eliminates immunostaining assayed in situ and on western blots of larval brains, demonstrating the specificity of these antibodies ( Figures 1B and 1C). The htsW532X mutation results in a premature Fasudil order stop codon and only a small amount of residual staining is detectable in the motor nerve when using the Hts1B1 antibody ( Figure 1C); however, no protein can be detected on the western blot ( Figure 1B). The htsΔG mutation results in a truncated but stable protein lacking the MARCKS domain since staining with the Hts-M antibody is eliminated, while staining with the Hts1B1 antibody is retained. These results are in accordance with prior analysis of the hts mutations in oocytes ( Petrella et al., 2007). The integrity of the NMJ and of individual synapses within the NMJ can be analyzed by immunolabeling synaptic markers that reside pre- and postsynaptically (Eaton et al., 2002, Koch et al., 2008, Massaro et al., 2009, Pielage et al., 2005, Pielage et al., 2006 and Pielage et al., 2008). At the wild-type NMJ, the active-zone associated protein Bruchpilot resides in precise apposition to clusters of postsynaptic glutamate receptors throughout the NMJ (Figure 2A).

In addition, the presynaptic vesicle marker Synapsin and over the presynaptic membrane marker anti-HRP are opposed throughout the NMJ by the postsynaptic marker Dlg (Budnik et al., 1996), which labels the SSR (Figure 2A and 2E). In hts mutant animals, by contrast, we observe large regions of NMJs where postsynaptic antigens are no longer opposed by presynaptic markers. Specifically, within a single NMJ, regions of the presynaptic nerve terminal are devoid of presynaptic Brp but retain postsynaptic glutamate receptor clusters. The regions of the presynaptic nerve terminal that lack Brp have a discontinuous, fragmented presynaptic membrane, whereas regions of the same NMJ that contain Brp have a normal, continuous presynaptic membrane ( Figures 2A–2D).