Localized inhibition in the Drosophila mushroom body

Many neurons show compartmentalized activity, in which activity does not spread readily across the cell, allowing input and output to occur locally. However, the functional implications of compartmentalized activity for the wider neural circuit are often unclear. We addressed this problem in the Drosophila mushroom body, whose principal neurons, Kenyon cells, receive feedback inhibition from a non-spiking interneuron called the anterior paired lateral (APL) neuron. We used local stimulation and volumetric calcium imaging to show that APL inhibits Kenyon cells’ dendrites and axons, and that both activity in APL and APL’s inhibitory effect on Kenyon cells are spatially localized (the latter somewhat less so), allowing APL to differentially inhibit different mushroom body compartments. Applying these results to the Drosophila hemibrain connectome predicts that individual Kenyon cells inhibit themselves via APL more strongly than they inhibit other individual Kenyon cells. These findings reveal how cellular physiology and detailed network anatomy can combine to influence circuit function

Export of functional Streptomyces coelicolor alditol oxidase to the periplasm or cell surface of Escherichia coli and its application in whole-cell biocatalysis

Streptomyces coelicolor A3(2) alditol oxidase (AldO) is a soluble monomeric flavoprotein in which the flavin cofactor is covalently linked to the polypeptide chain. AldO displays high reactivity towards different polyols such as xylitol and sorbitol. These characteristics make AldO industrially relevant, but full biotechnological exploitation of this enzyme is at present restricted by laborious and costly purification steps. To eliminate the need for enzyme purification, this study describes a whole-cell AldO biocatalyst system. To this end, we have directed AldO to the periplasm or cell surface of Escherichia coli. For periplasmic export, AldO was fused to endogenous E. coli signal sequences known to direct their passenger proteins into the SecB, signal recognition particle (SRP), or Twin-arginine translocation (Tat) pathway. In addition, AldO was fused to an ice nucleation protein (INP)-based anchoring motif for surface display. The results show that Tat-exported AldO and INP-surface-displayed AldO are active. The Tat-based system was successfully employed in converting xylitol by whole cells, whereas the use of the INP-based system was most likely restricted by lipopolysaccharide LPS in wild-type cells. It is anticipated that these whole-cell systems will be a valuable tool for further biological and industrial exploitation of AldO and other cofactor-containing enzymes.

Fras1, a basement membrane-associated protein mutated in Fraser syndrome, mediates both the initiation of the mammalian kidney and the integrity of renal glomeruli

FRAS1 is mutated in some individuals with Fraser syndrome (FS) and the encoded protein is expressed in embryonic epidermal cells, localizing in their basement membrane (BM). Syndactyly and cryptophthalmos in FS are sequelae of skin fragility but the bases for associated kidney malformations are unclear. We demonstrate that Fras1 is expressed in the branching ureteric bud (UB), and that renal agenesis occurs in homozygous Fras1 null mutant blebbed (bl) mice on a C57BL6J background. In vivo, the bl/bl bud fails to invade metanephric mesenchyme which undergoes involution, events replicated in organ culture. The expression of glial cell line-derived neurotrophic factor and growth-differentiation factor 11 was defective in bl/bl renal primordia in vivo, whereas, in culture, the addition of either growth factor restored bud invasion into the mesenchyme. Mutant primordia also showed deficient expression of Hoxd11 and Six2 transcription factors, whereas the activity of bone morphogenetic protein 4, an anti-branching molecule, was upregulated. In wild types, Fras1 was also expressed by nascent nephrons. Foetal glomerular podocytes expressed Fras1 transcripts and Fras1 immunolocalized in a glomerular BM-like pattern. On a mixed background, bl mutants, and also compound mutants for bl and my, another bleb strain, sometimes survive into adulthood. These mice have two kidneys, which contain subsets of glomeruli with perturbed nephrin, podocin, integrin α3 and fibronectin expression. Thus, Fras1 protein coats branching UB epithelia and is strikingly upregulated in the nephron lineage after mesenchymal/epithelial transition. Fras1 deficiency causes defective interactions between the bud and mesenchyme, correlating with disturbed expression of key nephrogenic molecules. Furthermore, Fras1 may also be required for the formation of normal glomeruli

Expression of Fraser syndrome genes in normal and polycystic murine kidneys

BACKGROUND: Fraser syndrome (FS) features renal agenesis and cystic kidneys. Mutations of FRAS1 (Fraser syndrome 1)and FREM2 (FRAS1-related extracellular matrix protein 2)cause FS. They code for basement membrane proteins expressed in metanephric epithelia where they mediate epithelial/mesenchymal signalling. Little is known about whether and where these molecules are expressed in more mature kidneys. METHODS: In healthy and congenital polycystic kidney (cpk)mouse kidneys we sought Frem2 expression using a LacZ reporter gene and quantified Fras family transcripts. Fras1 immunohistochemistry was undertaken in cystic kidneys from cpk mice and PCK (Pkhd1 mutant) rats (models of autosomal recessive polycystic kidney disease) and in wildtype metanephroi rendered cystic by dexamethasone. RESULTS: Nascent nephrons transiently expressed Frem2 in both tubule and podocyte epithelia. Maturing and adult collecting ducts also expressed Frem2. Frem2 was expressed in cpk cystic epithelia although Frem2 haploinsufficiency did not significantly modify cystogenesis in vivo. Fras1 transcripts were significantly upregulated, and Frem3 downregulated, in polycystic kidneys versus the non-cystic kidneys of littermates. Fras1 was immunodetected in cpk, PCK and dexamethasone-induced cystepithelia. CONCLUSIONS: These descriptive results are consistent with the hypothesis that Fras family molecules play diverse roles in kidney epithelia. In future, this should be tested by conditional deletion of FS genes in nephron segments and collecting ducts

The small RNA repertoire of Dictyostelium discoideum and its regulation by components of the RNAi pathway

Small RNAs play crucial roles in regulation of gene expression in many eukaryotes. Here, we report the cloning and characterization of 18–26 nt RNAs in the social amoeba Dictyostelium discoideum. This survey uncovered developmentally regulated microRNA candidates whose biogenesis, at least in one case, is dependent on a Dicer homolog, DrnB. Furthermore, we identified a large number of 21 nt RNAs originating from the DIRS-1 retrotransposon, clusters of which have been suggested to constitute centromeres. Small RNAs from another retrotransposon, Skipper, were significantly up-regulated in strains depleted of the second Dicer-like protein, DrnA, and a putative RNA-dependent RNA polymerase, RrpC. In contrast, the expression of DIRS-1 small RNAs was not altered in any of the analyzed strains. This suggests the presence of multiple RNAi pathways in D. discoideum. In addition, we isolated several small RNAs with antisense complementarity to mRNAs. Three of these mRNAs are developmentally regulated. Interestingly, all three corresponding genes express longer antisense RNAs from which the small RNAs may originate. In at least one case, the longer antisense RNA is complementary to the spliced but not the unspliced pre-mRNA, indicating synthesis by an RNA-dependent RNA polymerase

Hierarchical chemosensory regulation of male-male social interactions in Drosophila

Pheromones regulate male social behaviors in Drosophila, but the identities and behavioral role(s) of these chemosensory signals, and how they interact, are incompletely understood. We found that (z)-7-tricosene, a male-enriched cuticular hydrocarbon that was previously shown to inhibit male-male courtship, was essential for normal levels of aggression. The mechanisms by which (z)-7-tricosene induced aggression and suppressed courtship were independent, but both required the gustatory receptor Gr32a. Sensitivity to (z)-7-tricosene was required for the aggression-promoting effect of 11-cis-vaccenyl acetate (cVA), an olfactory pheromone, but (z)-7-tricosene sensitivity was independent of cVA. (z)-7-tricosene and cVA therefore regulate aggression in a hierarchical manner. Furthermore, the increased courtship caused by depletion of male cuticular hydrocarbons was suppressed by a mutation in the olfactory receptor Or47b. Thus, male social behaviors are controlled by gustatory pheromones that promote aggression and suppress courtship, and whose influences are dominant to olfactory pheromones that enhance these behaviors

SecA, a remarkable nanomachine

Biological cells harbor a variety of molecular machines that carry out mechanical work at the nanoscale. One of these nanomachines is the bacterial motor protein SecA which translocates secretory proteins through the protein-conducting membrane channel SecYEG. SecA converts chemically stored energy in the form of ATP into a mechanical force to drive polypeptide transport through SecYEG and across the cytoplasmic membrane. In order to accommodate a translocating polypeptide chain and to release transmembrane segments of membrane proteins into the lipid bilayer, SecYEG needs to open its central channel and the lateral gate. Recent crystal structures provide a detailed insight into the rearrangements required for channel opening. Here, we review our current understanding of the mode of operation of the SecA motor protein in concert with the dynamic SecYEG channel. We conclude with a new model for SecA-mediated protein translocation that unifies previous conflicting data

Turning Males On: Activation of Male Courtship Behavior in Drosophila melanogaster

The innate sexual behaviors of Drosophila melanogaster males are an attractive system for elucidating how complex behavior patterns are generated. The potential for male sexual behavior in D. melanogaster is specified by the fruitless (fru) and doublesex (dsx) sex regulatory genes. We used the temperature-sensitive activator dTRPA1 to probe the roles of fruM- and dsx-expressing neurons in male courtship behaviors. Almost all steps of courtship, from courtship song to ejaculation, can be induced at very high levels through activation of either all fruM or all dsx neurons in solitary males. Detailed characterizations reveal different roles for fruM and dsx in male courtship. Surprisingly, the system for mate discrimination still works well when all dsx neurons are activated, but is impaired when all fruM neurons are activated. Most strikingly, we provide evidence for a fruM-independent courtship pathway that is primarily vision dependent

Internal representations of smell in the Drosophila brain

Recent advances in sensory neuroscience using Drosophila olfaction as a model system have revealed brain maps representing the external world. Once we understand how the brain's built-in capability generates the internal olfactory maps, we can then elaborate how the brain computes and makes decision to elicit complex behaviors. Here, we review current progress in mapping Drosophila olfactory circuits and discuss their relationships with innate olfactory behaviors

Co- and post-translational translocation through the protein-conducting channel:analogous mechanisms at work?

Many proteins are translocated across, or integrated into, membranes. Both functions are fulfilled by the 'translocon/translocase', which contains a membrane-embedded proteinconducting channel (PCC) and associated soluble factors that drive translocation and insertion reactions using nucleotide triphosphates as fuel. This perspective focuses on reinterpreting existing experimental data in light of a recently proposed PCC model comprising a front-to-front dimer of SecY or Sec61 heterotrimeric complexes. In this new framework, we propose (i) a revised model for SRP-SR-mediated docking of the ribosome-nascent polypeptide to the PCC; (ii) that the dynamic interplay between protein substrate, soluble factors and PCC controls the opening and closing of a transmembrane channel across, and/or a lateral gate into, the membrane; and (iii) that co-and post-translational translocation, involving the ribosome and SecA, respectively, not only converge at the PCC but also use analogous mechanisms for coordinating protein translocation