Faculty Bibliography

The odour of acids has a distinct quality that is perceived as sharp, pungent and often irritating. How acidity is sensed and translated into an appropriate behavioural response is poorly understood. Here we describe a functionally segregated population of olfactory sensory neurons in the fruitfly, Drosophila melanogaster, that are highly selective for acidity. These olfactory sensory neurons express IR64a, a member of the recently identified ionotropic receptor (IR) family of putative olfactory receptors. In vivo calcium imaging showed that IR64a+ neurons projecting to the DC4 glomerulus in the antennal lobe are specifically activated by acids. Flies in which the function of IR64a+ neurons or the IR64a gene is disrupted had defects in acid-evoked physiological and behavioural responses, but their responses to non-acidic odorants remained unaffected. Furthermore, artificial stimulation of IR64a+ neurons elicited avoidance responses. Taken together, these results identify cellular and molecular substrates for acid detection in the Drosophila olfactory system and support a labelled-line mode of acidity coding at the periphery

Neural systems controlling the vital functions of sleep and feeding in mammals are tightly interconnected: sleep deprivation promotes feeding, whereas starvation suppresses sleep. Here we show that starvation in Drosophila potently suppresses sleep, suggesting that these two homeostatically regulated behaviors are also integrated in flies. The sleep-suppressing effect of starvation is independent of the mushroom bodies, a previously identified sleep locus in the fly brain, and therefore is regulated by distinct neural circuitry. The circadian clock genes Clock (Clk) and cycle (cyc) are critical for proper sleep suppression during starvation. However, the sleep suppression is independent of light cues and of circadian rhythms as shown by the fact that starved period mutants sleep like wild-type flies. By selectively targeting subpopulations of Clk-expressing neurons, we localize the observed sleep phenotype to the dorsally located circadian neurons. These findings show that Clk and cyc act during starvation to modulate the conflict of whether flies sleep or search for food.

Nuclear factor IA (NFIA) is a transcription factor that specifies glial cell identity and promotes astrocyte differentiation during embryonic development. Its expression and function in gliomas are not known. Here, we examined NFIA protein expression in gliomas and its association with clinical outcome in pediatric malignant astrocytomas. We analyzed expression of NFIA by immunohistochemistry in 88 existing glioma specimens from Childrens Hospital Los Angeles and the University of Southern California. Association between NFIA expression and progression-free survival (PFS) was examined in high-grade astrocytomas for which clinical data were available (n = 23, all children). NFIA was highly expressed in astrocytomas of all grades, but only in a minority of cells in oligodendroglial tumors. NFIA was expressed on a higher percentage of tumor cells in low-grade astrocytomas (91 +/- 5% and 77 +/- 14% in World Health Organization [WHO] I and II, respectively) compared with high-grade astrocytomas (48 +/- 18% and 37 +/- 16% in WHO III and IV, respectively; P < .001, low- vs high-grade astrocytomas). There was a significant association between NFIA expression and PFS in children with astrocytoma WHO grade III or IV (Cox regression P = .019; logrank trend test for NFIA tertiles P = .0040 and NFIA quartiles P = .014). The association was not consistently significant in this small series of patients after adjustment was made for WHO grade III or IV. This is the first study to demonstrate expression of NFIA protein in astrocytomas and its association with grades of astrocytoma and PFS, suggesting that NFIA may play a role in astrocytoma biology

Carbon dioxide (CO2) elicits different olfactory behaviors across species. In Drosophila, neurons that detect CO2 are located in the antenna, form connections in a ventral glomerulus in the antennal lobe, and mediate avoidance. By contrast, in the mosquito these neurons are in the maxillary palps (MPs), connect to medial sites, and promote attraction. We found in Drosophila that loss of a microRNA, miR-279, leads to formation of CO2 neurons in the MPs. miR-279 acts through down-regulation of the transcription factor Nerfin-1. The ectopic neurons are hybrid cells. They express CO2 receptors and form connections characteristic of CO2 neurons, while exhibiting wiring and receptor characteristics of MP olfactory receptor neurons (ORNs). We propose that this hybrid ORN reveals a cellular intermediate in the evolution of species-specific behaviors elicited by CO2

How specific sensory stimuli evoke specific behaviors is a fundamental problem in neurobiology. In Drosophila, most odorants elicit attraction or avoidance depending on their concentration, as well as their identity [1]. Such odorants, moreover, typically activate combinations of glomeruli in the antennal lobe of the brain [2-4], complicating the dissection of the circuits translating odor recognition into behavior. Carbon dioxide (CO2), in contrast, elicits avoidance over a wide range of concentrations [5, 6] and activates only a single glomerulus, V [5]. The V glomerulus receives projections from olfactory receptor neurons (ORNs) that coexpress two GPCRs, Gr21a and Gr63a, that together comprise a CO2 receptor [7-9]. These CO2-sensitive ORNs, located in the ab1 sensilla of the antenna, are called ab1c neurons [10]. Genetic silencing of ab1c neurons indicates that they are necessary for CO2-avoidance behavior [5]. Whether activation of these neurons alone is sufficient to elicit this behavior, or whether CO2 avoidance requires additional inputs (e.g., from the respiratory system), remains unclear. Here, we show that artificial stimulation of ab1c neurons with light (normally attractive to flies) elicits the avoidance behavior typical of CO2. Thus, avoidance behavior appears hardwired into the olfactory circuitry that detects CO2 in Drosophila

All animals exhibit innate behaviours in response to specific sensory stimuli that are likely to result from the activation of developmentally programmed neural circuits. Here we observe that Drosophila exhibit robust avoidance to odours released by stressed flies. Gas chromatography and mass spectrometry identifies one component of this 'Drosophila stress odorant (dSO)' as CO2. CO2 elicits avoidance behaviour, at levels as low as 0.1%. We used two-photon imaging with the Ca2+-sensitive fluorescent protein G-CaMP to map the primary sensory neurons governing avoidance to CO2. CO2 activates only a single glomerulus in the antennal lobe, the V glomerulus; moreover, this glomerulus is not activated by any of 26 other odorants tested. Inhibition of synaptic transmission in sensory neurons that innervate the V glomerulus, using a temperature-sensitive Shibire gene (Shi(ts)), blocks the avoidance response to CO2. Inhibition of synaptic release in the vast majority of other olfactory receptor neurons has no effect on this behaviour. These data demonstrate that the activation of a single population of sensory neurons innervating one glomerulus is responsible for an innate avoidance behaviour in Drosophila

COP9 signalosome (CSN) cleaves the ubiquitin-like protein Nedd8 from the Cul1 subunit of SCF ubiquitin ligases. The Jab1/MPN domain metalloenzyme (JAMM) motif in the Jab1/Csn5 subunit was found to underlie CSN's Nedd8 isopeptidase activity. JAMM is found in proteins from archaea, bacteria, and eukaryotes, including the Rpn11 subunit of the 26S proteasome. Metal chelators and point mutations within JAMM abolished CSN-dependent cleavage of Nedd8 from Cul1, yet had little effect on CSN complex assembly. Optimal SCF activity in yeast and both viability and proper photoreceptor cell (R cell) development in Drosophila melanogaster required an intact Csn5 JAMM domain. We propose that JAMM isopeptidases play important roles in a variety of physiological pathways

Different classes of photoreceptor neurons (R cells) in the Drosophila compound eye form connections in different optic ganglia. The R1-R6 subclass connects to the first optic ganglion, the lamina, and relies upon glial cells as intermediate targets. Conversely, R cells promote glial cell development including migration of glial cells into the target region. Here, we show that the JAB1/CSN5 subunit of the COP9 signalosome complex is expressed in R cells, accumulates in the developing optic lobe neuropil, and through the analysis of a unique set of missense mutations, is required in R cells to induce lamina glial cell migration. In these CSN5 alleles, R1-R6 targeting is disrupted. Genetic analysis of protein null alleles further revealed that the COP9 signalosome is required at an earlier stage of development for R cell differentiation

Accumulation of tetracycline in Escherichia coli was studied to determine its permeation pathway and to provide a basis for understanding efflux-mediated resistance. Passage of tetracycline across the outer membrane appeared to occur preferentially via the porin OmpF, with tetracycline in its magnesium-bound form. Rapid efflux of magnesium-chelated tetracycline from the periplasm was observed. In E. coli cells that do not contain exogenous tetracycline resistance genes, the steady-state level of tetracycline accumulation was decreased when porins were absent or when the fraction of Mg(2+)-chelated tetracycline was small. This is best explained by assuming the presence of a low-level endogenous active efflux system that bypasses the outer membrane barrier. When influx of tetracycline is slowed, this efflux is able to reduce the accumulation of tetracycline in the cytoplasm. In contrast, we found no evidence of a special outer membrane bypass mechanism for high-level efflux via the Tet protein, which is an inner membrane efflux pump coded for by exogenous tetA genes. Fractionation and equilibrium density gradient centrifugation experiments showed that the Tet protein is not localized to regions of inner and outer membrane adhesion. Furthermore, a high concentration of tetracycline was found in the compartment that rapidly equilibrated with the medium, most probably the periplasm, of Tet-containing E. coli cells, and the level of tetracycline accumulation in Tet-containing cells was not diminished by the mutational loss of the OmpF porin. These results suggest that the Tet protein, in contrast to the endogenous efflux system(s), pumps magnesium-chelated tetracycline into the periplasm. A quantitative model of tetracycline fluxes in E. coli cells of various types is presented