Convergent findings from our behavioral screen for memory mutants and DNA microarray analysis of transcriptional responses during memory formation in normal animals suggest the involvement of the pumilio/staufen pathway in memory. Behavioral experiments confirm a role for this pathway and suggest a molecular mechanism for synapse-specific modification.

How the brain perceives sensory information and generates meaningful behavior depends critically on its underlying circuitry. The protocerebral bridge (PB) is a major part of the insect central complex (CX), a premotor center that may be analogous to the human basal ganglia. Here, by deconstructing hundreds of PB single neurons and reconstructing them into a common three-dimensional framework, we have constructed a comprehensive map of PB circuits with labeled polarity and predicted directions of information flow. Our analysis reveals a highly ordered information processing system that involves directed information flow among CX subunits through 194 distinct PB neuron types. Circuitry properties such as mirroring, convergence, divergence, tiling, reverberation, and parallel signal propagation were observed; their functional and evolutional significance is discussed. This layout of PB neuronal circuitry may provide guidelines for further investigations on transformation of sensory (e.g., visual) input into locomotor commands in fly brains.

Abstract. When nuclear localization sequences (termed NLS) are placed at the N terminus of cytochrome ca, a mitochondrial inner membrane protein, the resulting hybrid proteins do not assemble into mitochondria when synthesized in the yeast Saccharomyces cerevisiae. Cells lacking mitochondrial cytochrome c~, but expressing the hybrid NLScytochrome c, proteins, are unable to grow on glycerol since the hybrid proteins are associated primarily with the nucleus. A similar hybrid protein with a mutant NLS is transported to and assembled into the mitochondria. To identify proteins that might be involved in recognition of nuclear localization signals, we isolated conditional-lethal mutants (npl, for nuclear protein localization) that missorted NLS-cytochrome c, to the mitochondria, allowing growth on glycerol. The gene corresponding to one complementation group (NPLI) encodes a protein with homology to DnaJ, an Escherichia coli heat shock protein, npU-1 is allelic to sec63, a gene that affects transit of nascent secretory proteins across the endoplasmic reticulum. Rothblatt, J. A., R. J. Deshaies, S. L. Sanders, G. Daum, and R. Schekman. 1989. J. Cell Biol. 109:2641-2652. The npU mutants reported here also weakly affect translocation of preprocarboxypeptidaseY across the ER membrane. A normally nuclear hybrid protein containing a NLS fused to invertase and a nucleolar protein are not localized to the nucleus in np11/sec63 cells at the nonpermissive temperature. Thus, NPLI/SEC63 may act at a very early common step in localization of proteins to the nucleus and the ER. Alternatively, by affecting ER and nuclear envelope assembly, npll may indirectly alter assembly of proteins into the nucleus.ACH organelle in a eukaryotic cell has a distinct set of proteins that are necessary for its specific function. Certain peptides can act as signals to localize proteins to particular organelles such as the ER, the mitochondria (Verner and Schatz, 1988) and the nucleus (Silver and Hall, 1988). Several proteins have been identified that mediate the recognition of ER-destined proteins and their subsequent translocation across or assembly into the ER membrane (Walter and Blobel, 1980;Meyer et al., 1982;Tajima et al., 1986; Wiedrnan et al., 1987). Receptors have been proposed for mitochondrial signal peptides (Pfaller and Neupert, 1987;Pfanner et al., 1987) and recently a receptor for protein import into chloroplasts has been identified (Pain et al., 1988). By analogy, similar components may exist for localization of proteins to the nucleus.Nuclear localization sequences (NLS)' are stretches of amino acids that are capable of redirecting nonnuclear pro-1. Abbreviations used in this paper: DAPI, diamidinophenylindole; EMS, ethyl methanesulfonate; NLS, nuclear localization sequences; preproCPY, preprocarboxypeptidase Y.teins such as/$-galactosidase to the nucleus. When the first 74 amino acids of the yeast DNA binding protein GAL4 are joined to/~-galactosidase, the result is a fusion protein that is found exclusively in the yeast nucleus as...

Extensive molecular, genetic, and anatomical analyses have suggested that olfactory memory is stored in the mushroom body (MB), a higher-order olfactory center in the insect brain. The MB comprises three subtypes of neurons with axons that extend into different lobes. A recent functional imaging study has revealed a long-term memory trace manifested as an increase in the Ca 2ϩ activity in an axonal branch of a subtype of MB neurons. However, early memory traces in the MB remain elusive. We report here learning-induced changes in Ca 2ϩ activities during early memory formation in a different subtype of MB neurons. We used three independent in vivo and in vitro preparations, and all of them showed that Ca 2ϩ activities in the axonal branches of ␣Ј/␤Ј neurons in response to a conditioned olfactory stimulus became larger compared with one that was not conditioned. The changes were dependent on proper G-protein signaling in the MB. The importance of these changes in the Ca 2ϩ activity of ␣Ј/␤Ј neurons during early memory formation was further tested behaviorally by disrupting G-protein signaling in these neurons or blocking their synaptic outputs during the learning and memory process. Our results suggest that increased Ca 2ϩ activity in response to a conditioned olfactory stimulus may be a neural correlate of early memory in the MB.

Infection with Helicobacter pylori cagA-positive strains is associated with gastritis, ulcerations, and gastric cancer. CagA is translocated into infected epithelial cells by a type IV secretion system and can be tyrosine phosphorylated, inducing signal transduction and motogenic responses in epithelial cells. Cellular cholesterol, a vital component of the membrane, contributes to membrane dynamics and functions and is important in VacA intoxication and phagocyte evasion during H. pylori infection. In this investigation, we showed that cholesterol extraction by methyl-␤-cyclodextrin reduced the level of CagA translocation and phosphorylation. Confocal microscope visualization revealed that a significant portion of translocated CagA was colocalized with the raft marker GM1 and c-Src during infection. Moreover, GM1 was rapidly recruited into sites of bacterial attachment by live-cell imaging analysis. CagA and VacA were cofractionated with detergent-resistant membranes (DRMs), suggesting that the distribution of CagA and VacA is associated with rafts in infected cells. Upon cholesterol depletion, the distribution shifted to non-DRMs. Accordingly, the CagA-induced hummingbird phenotype and interleukin-8 induction were blocked by cholesterol depletion. Raft-disrupting agents did not influence bacterial adherence but did significantly reduce internalization activity in AGS cells. Together, these results suggest that delivery of CagA into epithelial cells by the bacterial type IV secretion system is mediated in a cholesterol-dependent manner.

One Drosophila mushroom body (MB) is derived from four indistinguishable cell lineages, development of which involves sequential generation of multiple distinct types of neurons. Differential labeling of distinct MB clones reveals that MB dendrites of different clonal origins are well mixed at the larval stage but become restricted to distinct spaces in adults. Interestingly, a small dendritic domain in the adult MB calyx remains as a fourfold structure that, similar to the entire larval calyx, receives dendritic inputs from all four MB clones. Mosaic analysis of single neurons demonstrates that MB neurons, which are born around pupal formation, acquire unique dendritic branching patterns and consistently project their primary dendrites into the fourfold dendritic domain. Distinct dendrite distribution patterns are also observed for other subtypes of MB neurons. In addition,pruning of larval dendrites during metamorphosis allows for establishment of adult-specific dendrite elaboration/distribution patterns. Taken together,subregional differences exist in the adult Drosophila MB calyx, where processing and integration of distinct types of sensory information begin.

Situations requiring rapid decision-making in response to dynamic environmental demands occur repeatedly in natural environments. Neuromodulation can offer important flexibility to the output of neural networks in coping with changing conditions, but the contribution of individual neuromodulatory neurons in social behavior networks remains relatively unknown. Here we manipulate the Drosophila octopaminergic system and assay changes in adult male decision-making in courtship and aggression paradigms. When the functional state of OA neural circuits is enhanced, males exhibit elevated courtship behavior towards other males in both behavioral contexts. Eliminating the expression of the male form of the neural sex determination factor, Fruitless (FruM), in three OA suboesophageal ganglia (SOG) neurons also leads to increased male-male courtship behavior in these same contexts. We analyzed the fine anatomical structure through confocal examination of labeled single neurons to determine the arborization patterns of each of the three FruM-positive OA SOG neurons. These neurons send processes that display mirror symmetric, widely distributed arbors of endings within brain regions including the ventrolateral protocerebra, the SOG and the peri-esophageal complex. The results suggest that a small subset of OA neurons have the potential to provide male selective modulation of behavior at a single neuron level.

Inclusion body myopathy with Paget's disease of bone and frontotemporal dementia (IBMPFD) is caused by mutations in Valosin-containing protein (VCP), a hexameric AAA ATPase that participates in a variety of cellular processes such as protein degradation, organelle biogenesis, and cell-cycle regulation. To understand how VCP mutations cause IBMPFD, we have established a Drosophila model by overexpressing TER94 (the sole Drosophila VCP ortholog) carrying mutations analogous to those implicated in IBMPFD. Expression of these TER94 mutants in muscle and nervous systems causes tissue degeneration, recapitulating the pathogenic phenotypes in IBMPFD patients. TER94-induced neurodegenerative defects are enhanced by elevated expression of wild-type TER94, suggesting that the pathogenic alleles are dominant active mutations. This conclusion is further supported by the observation that TER94-induced neurodegenerative defects require the formation of hexamer complex, a prerequisite for a functional AAA ATPase. Surprisingly, while disruptions of the ubiquitin-proteasome system (UPS) and the ER–associated degradation (ERAD) have been implicated as causes for VCP–induced tissue degeneration, these processes are not significantly affected in our fly model. Instead, the neurodegenerative defect of TER94 mutants seems sensitive to the level of cellular ATP. We show that increasing cellular ATP by independent mechanisms could suppress the phenotypes of TER94 mutants. Conversely, decreasing cellular ATP would enhance the TER94 mutant phenotypes. Taken together, our analyses have defined the nature of IBMPFD–causing VCP mutations and made an unexpected link between cellular ATP level and IBMPFD pathogenesis.