Fragile X mental retardation gene (FMR1) encodes an RNA binding protein that acts as a negative translational regulator. We have developed a Drosophila fragile X syndrome model using loss-of-function mutants and overexpression of the FMR1 homolog (dfxr). dfxr nulls display enlarged synaptic terminals, whereas neuronal overexpression results in fewer and larger synaptic boutons. Synaptic structural defects are accompanied by altered neurotransmission, with synapse type-specific regulation in central and peripheral synapses. These phenotypes mimic those observed in mutants of microtubule-associated Futsch. Immunoprecipitation of dFXR shows association with futsch mRNA, and Western analyses demonstrate that dFXR inversely regulates Futsch expression. dfxr futsch double mutants restore normal synaptic structure and function. We propose that dFXR acts as a translational repressor of Futsch to regulate microtubule-dependent synaptic growth and function.

SUMMARY We report a novel mechanism of ribonucleoprotein (RNP) nucleocytoplasmic export by nuclear envelope budding. During development of Drosophila synapses, a fragment of the Wnt-1 receptor, DFrizzled2, is imported into postsynaptic nuclei where it forms prominent foci. We now show these foci to be composed of large RNP granules harboring synaptic protein transcripts. These RNPs exit the nucleus via a budding mechanism akin to nuclear egress of Herpes-type viruses, a process previously thought to be exclusive to these viruses. During this mechanism, RNP granules bud into the space between the inner and the outer nuclear membranes (the perinuclear space), in a manner dependent on Lamin C, a nuclear protein linked to muscular dystrophies. Like herpes virus nuclear egress, this process requires protein kinase C, which is known to disrupt the lamin through phosphorylation. We suggest that nuclear budding is an endogenous nuclear export pathway for large RNP granules.

Previous studies indicated that CAPS (calcium-dependent activator protein for secretion) functions as an essential component for the Ca 2ϩ -dependent exocytosis of dense-core vesicles in neuroendocrine cells. However, recent mouse knock-out studies suggested an alternative role in the vesicular uptake or storage of catecholamines. To genetically assess the functional role of CAPS, we characterized the sole Caenorhabditis elegans CAPS ortholog UNC-31 (uncoordinated family member) and determined its role in dense-core vesiclemediated peptide secretion and in synaptic vesicle recycling. Novel assays for dense-core vesicle exocytosis were developed by expressing a prepro-atrial natriuretic factor-green fluorescent protein fusion protein in C. elegans. unc-31 mutants exhibited reduced peptide release in vivo and lacked evoked peptide release in cultured neurons. In contrast, cultured neurons from unc-31 mutants exhibited normal stimulated synaptic vesicle recycling measured by FM4-64 [N-(3-triethylammoniumpropyl)-4-(6-(4-diethylamino)phenyl) hexatrienyl)pyridinium dibromide] dye uptake. Conversely, UNC-13, which exhibits sequence homology to CAPS/UNC-31, was found to be essential for synaptic vesicle but not dense-core vesicle exocytosis. These findings indicate that CAPS/UNC-31 function is not restricted to catecholaminergic vesicles but is generally required for and specific to dense-core vesicle exocytosis. Our results suggest that CAPS/ UNC-31 and UNC-13 serve parallel and dedicated roles in dense-core vesicle and synaptic vesicle exocytosis, respectively, in the C. elegans nervous system.

Taken together, these studies demonstrate that the UPS functions locally within synaptic boutons to acutely control levels of presynaptic protein and that the rate of UPS-dependent protein degradation is a primary determinant of neurotransmission strength.

Neuronal injury elicits potent cellular responses from glia, but molecular pathways modulating glial activation, phagocytic function, and termination of reactive responses remain poorly defined. Here we show that positive or negative regulation of glial reponses to axon injury are molecularly encoded by unique isoforms of the Drosophila engulfment receptor Draper. Draper-I promotes engulfment of axonal debris through an immunoreceptor tyrosine-based activation motif (ITAM). In contrast, Draper-II, an alternative splice variant, potently inhibits glial engulfment function. Draper-II suppresses Draper-I signaling through a novel immunoreceptor tyrosine-based inhibitory motif (ITIM)-like domain and the tyrosine phosphatase Corkscrew (Csw). Intriguingly, loss of Draper-II/Csw signaling prolongs expression of glial engulfment genes after axotomy and reduces the ability of glia to respond to secondary axotomy. Our work highlights a novel role for Draper-II in inhibiting glial responses to neurodegeneration, and indicates a balance of opposing Draper-I/-II signaling events is essential to maintain glial sensitivity to brain injury.

Synaptic development, function and plasticity are highly regulated processes requiring a precise coordination of pre-and postsynaptic events. Recent studies have begun to highlight Wingless-Int (Wnt) signaling as a key player in synapse differentiation and function. Emerging roles of Wnts include the differentiation of synaptic specializations, microtubule dynamics, architecture of synaptic protein organization, modulation of synaptic efficacy and regulation of gene expression. Wnt transduction pathways: a brief précisExciting roles for embryonic morphogens of the Wingless-Int family (Wnt) beyond those in early pattern formation are beginning to emerge. These include roles in axon guidance, dendrite morphology, synapse formation and plasticity [1,2]. These studies make a clear case for Wnt function in post-mitotic neurons, and highlight the multitude of mechanisms activated by Wnts within the nervous system. The Wnt pathway has pivotal roles in defining positional information in the embryo. Misregulation of this pathway is linked to cancer, and the pathogenesis of Alzheimer's and Huntington disease [3,4]. The best studied Wnt transduction cascade is the canonical pathway, in which Wnt binds to its receptor Frizzled (Fz) in the receiving cell (Figure 1a,b). This binding activates the postsynaptic density-95/Discs-Large (DLG)/zona occludens-1 (PDZ) protein dishevelled (DVL), which disrupts a so-called 'destruction complex' containing glycogen synthase kinase-3β (GSK-3β), Axin and adenomatous polyposis coli (APC). In the absence of Wnt signaling, this complex constitutively phosphorylates β-catenin, leading to its proteasomal degradation. The binding of Wnt to Fz disrupts the destruction complex, resulting in cytoplasmic stabilization of β-catenin and its import into the nucleus [5]. In the nucleus, β-catenin associates with lymphoid enhancer factor (also known as T-cell factor) (LEF/TCF) transcription factors to regulate transcription. Additionally, β-catenin can bind to cadherins [6,7], but this function is not discussed here.Two non-canonical Wnt pathways have a role in development: (i) the planar cell polarity (PCP) pathway, in which Fz acts through Jun N-terminal kinase (JNK) to regulate the cytoskeleton (Figure 1e), and (ii) the Wnt-Ca 2+ signaling pathway, in which Fz activation

SUMMARYThe inner side of the nuclear envelope (NE) is lined with lamins, a meshwork of intermediate filaments that provides structural support for the nucleus and plays roles in many nuclear processes. Lamins, classified as A-or B-types on the basis of biochemical properties, have a conserved globular head, central rod and C-terminal domain that includes an Ig-fold structural motif. In humans, mutations in A-type lamins give rise to diseases that exhibit tissue-specific defects, such as Emery-Dreifuss muscular dystrophy. Drosophila is being used as a model to determine tissue-specific functions of A-type lamins in development, with implications for understanding human disease mechanisms. The GAL4-UAS system was used to express wild-type and mutant forms of Lamin C (the presumed Drosophila A-type lamin), in an otherwise wild-type background. Larval muscle-specific expression of wild type Drosophila Lamin C caused no overt phenotype. By contrast, larval muscle-specific expression of a truncated form of Lamin C lacking the N-terminal head (Lamin C N) caused muscle defects and semi-lethality, with adult 'escapers' possessing malformed legs. The leg defects were due to a lack of larval muscle function and alterations in hormoneregulated gene expression. The consequences of Lamin C association at a gene were tested directly by targeting a Lamin C DNAbinding domain fusion protein upstream of a reporter gene. Association of Lamin C correlated with localization of the reporter gene at the nuclear periphery and gene repression. These data demonstrate connections among the Drosophila A-type lamin, hormone-induced gene expression and muscle function. DEVELOPMENT 3068 lethality when expressed in larval muscle but not in other tissues (Schulze et al., 2009). By contrast, alterations in the rod domain cause abnormal lamin aggregation within the nucleus but not lethality. Collectively, our studies support a role for the N-terminal head and C-terminal globular domain in larval muscle function.To better understand the role of the N-terminal head domain, we generated transgenic flies expressing a mutant form of Lamin C lacking the first 42 amino acids (Lamin C N) (Schulze et al., 2009). This mutant form localizes to the NE and causes semilethality when expressed in muscle but not in non-muscle tissues (Schulze et al., 2009). Here, we demonstrate that lethality is associated with larval body wall muscle defects that include nuclear and cytoplasmic abnormalities. 'Adult escapers' expressing Lamin C N have leg defects that are consistent with a loss of muscle function and disruptions in ecdysone hormone signaling.Collectively, these studies demonstrate a role for the N-terminal head domain of Lamin C in muscle function and gene expression. MATERIALS AND METHODS Drosophila stocks and genetic analysesDrosophila stocks were raised at room temperature on standard sucrose and cornmeal medium (Shaffer et al., 1994). Generation of transgenic stocks expressing wild type and Lamin C N was previously reported (Schulze et al., 2005;Schulze ...

The rolling blackout (rbo) gene encodes an integral plasma membrane lipase required for Drosophila phototransduction. Photoreceptors are enriched for the RBO protein, and temperature-sensitive rbo mutants show reversible elimination of phototransduction within minutes, demonstrating an acute requirement for the protein. The block is activity dependent, indicating that the action of RBO is use dependent. Conditional rbo mutants show activity-dependent depletion of diacylglycerol and concomitant accumulation of phosphatidylinositol phosphate and phosphatidylinositol 4,5-bisphosphate within minutes of induction, suggesting rapid downregulation of phospholipase C (PLC) activity. The RBO requirement identifies an essential regulatory step in G-protein-coupled, PLC-dependent inositol lipid signaling mediating activation of TRP and TRPL channels during phototransduction.