A new method to characterize function of the Drosophila heart by means of optical flow.

The minuteness of Drosophila poses a challenge to quantify performance of its tubular heart and computer-aided analysis of its beating heart has evolved as a resilient compromise between instrumental costs and data robustness. Here, we introduce an optical flow algorithm (OFA) that continuously registers coherent movement within videos of the beating Drosophila heart and uses this information to subscribe the time course of observation with characteristic phases of cardiac contraction or relaxation. We report that the OFA combines high discriminatory power with robustness to characterize the performance of the Drosophila tubular heart using indicators from human cardiology. We provide proof of this concept using the test bed of established cardiac conditions that include the effects of ageing, knockdown of the slow repolarizing potassium channel subunit KCNQ and ras-mediated hypertrophy of the heart tube. Together, this establishes the analysis of coherent movement as a suitable indicator of qualitative changes of the heart's beating characteristics, which improves the usefulness of Drosophila as a model of cardiac diseases.

Separate roles of PKA and EPAC in renal function unraveled by the optogenetic control of cAMP levels in vivo.

Cyclic AMP (cAMP) is a ubiquitous second messenger that regulates a variety of essential processes in diverse cell types, functioning via cAMP-dependent effectors such as protein kinase A (PKA) and/or exchange proteins directly activated by cAMP (EPAC). In an intact tissue it is difficult to separate the contribution of each cAMP effector in a particular cell type using genetic or pharmacological approaches alone. We, therefore, utilized optogenetics to overcome the difficulties associated with examining a multicellular tissue. The transgenic photoactive adenylyl cyclase bPAC can be activated to rapidly and reversibly generate cAMP pulses in a cell-type-specific manner. This optogenetic approach to cAMP manipulation was validated in vivo using GAL4-driven UAS-bPAC in a simple epithelium, the Drosophila renal (Malpighian) tubules. As bPAC was expressed under the control of cell-type-specific promoters, each cAMP signal could be directed to either the stellate or principal cells, the two major cell types of the Drosophila renal tubule. By combining the bPAC transgene with genetic and pharmacological manipulation of either PKA or EPAC it was possible to investigate the functional impact of PKA and EPAC independently of each other. The results of this investigation suggest that both PKA and EPAC are involved in cAMP sensing, but are engaged in very different downstream physiological functions in each cell type: PKA is necessary for basal secretion in principal cells only, and for stimulated fluid secretion in stellate cells only. By contrast, EPAC is important in stimulated fluid secretion in both cell types. We propose that such optogenetic control of cellular cAMP levels can be applied to other systems, for example the heart or the central nervous system, to investigate the physiological impact of cAMP-dependent signaling pathways with unprecedented precision.

AKAPS act in a two-step mechanism of memory acquisition.

Defining the molecular and neuronal basis of associative memories is based upon behavioral preparations that yield high performance due to selection of salient stimuli, strong reinforcement, and repeated conditioning trials. One of those preparations is the Drosophila aversive olfactory conditioning procedure where animals initiate multiple memory components after experience of a single cycle training procedure. Here, we explored the analysis of acquisition dynamics as a means to define memory components and revealed strong correlations between particular chronologies of shock impact and number experienced during the associative training situation and subsequent performance of conditioned avoidance. Analyzing acquisition dynamics in Drosophila memory mutants revealed that rutabaga (rut)-dependent cAMP signals couple in a divergent fashion for support of different memory components. In case of anesthesia-sensitive memory (ASM) we identified a characteristic two-step mechanism that links rut-AC1 to A-kinase anchoring proteins (AKAP)-sequestered protein kinase A at the level of Kenyon cells, a recognized center of olfactory learning within the fly brain. We propose that integration of rut-derived cAMP signals at level of AKAPs might serve as counting register that accounts for the two-step mechanism of ASM acquisition.

Consolidated and labile odor memory are separately encoded within the Drosophila brain.

Memories are classified as consolidated (stable) or labile according to whether they withstand amnestic treatment, or not. In contrast to the general prevalence of this classification, its neuronal and molecular basis is poorly understood. Here, we focused on consolidated and labile memories induced after a single cycle training in the Drosophila aversive olfactory conditioning paradigm and we used mutants to define the impact of cAMP signals. At the biochemical level we report that cAMP signals misrelated in either rutabaga (rut) or dunce (dnc) mutants separate between consolidated anesthesia-resistant memory (ARM) and labile anesthesia-sensitive memory (ASM). Those functionally distinct cAMP signals act within different neuronal populations: while rut-dependent cAMP signals act within Kenyon cells (KCs) of the mushroom bodies to support ASM, dnc-sensitive cAMP signals support ARM within antennal lobe local neurons (LNs) and KCs. Collectively, different key positions along the olfactory circuitry seem to get modified during storage of ARM or ASM independently. A precise separation between those functionally distinct cAMP signals seems mandatory to allocate how they support appropriate memories.

Light modulation of cellular cAMP by a small bacterial photoactivated adenylyl cyclase, bPAC, of the soil bacterium Beggiatoa.

The recent success of channelrhodopsin in optogenetics has also caused increasing interest in enzymes that are directly activated by light. We have identified in the genome of the bacterium Beggiatoa a DNA sequence encoding an adenylyl cyclase directly linked to a BLUF (blue light receptor using FAD) type light sensor domain. In Escherichia coli and Xenopus oocytes, this photoactivated adenylyl cyclase (bPAC) showed cyclase activity that is low in darkness but increased 300-fold in the light. This enzymatic activity decays thermally within 20 s in parallel with the red-shifted BLUF photointermediate. bPAC is well expressed in pyramidal neurons and, in combination with cyclic nucleotide gated channels, causes efficient light-induced depolarization. In the Drosophila central nervous system, bPAC mediates light-dependent cAMP increase and behavioral changes in freely moving animals. bPAC seems a perfect optogenetic tool for light modulation of cAMP in neuronal cells and tissues and for studying cAMP-dependent processes in live animals.

Agrobacterium tumefaciens promotes tumor induction by modulating pathogen defense in Arabidopsis thaliana.

Agrobacterium tumefaciens causes crown gall disease by transferring and integrating bacterial DNA (T-DNA) into the plant genome. To examine the physiological changes and adaptations during Agrobacterium-induced tumor development, we compared the profiles of salicylic acid (SA), ethylene (ET), jasmonic acid (JA), and auxin (indole-3-acetic acid [IAA]) with changes in the Arabidopsis thaliana transcriptome. Our data indicate that host responses were much stronger toward the oncogenic strain C58 than to the disarmed strain GV3101 and that auxin acts as a key modulator of the Arabidopsis-Agrobacterium interaction. At initiation of infection, elevated levels of IAA and ET were associated with the induction of host genes involved in IAA, but not ET signaling. After T-DNA integration, SA as well as IAA and ET accumulated, but JA did not. This did not correlate with SA-controlled pathogenesis-related gene expression in the host, although high SA levels in mutant plants prevented tumor development, while low levels promoted it. Our data are consistent with a scenario in which ET and later on SA control virulence of agrobacteria, whereas ET and auxin stimulate neovascularization during tumor formation. We suggest that crosstalk among IAA, ET, and SA balances pathogen defense launched by the host and tumor growth initiated by agrobacteria.

Photoactivatable adenylyl cyclases (PACs) as a tool to study cAMP signaling in vivo: an overview.

Photoactivatable adenylyl cyclases (PACs) are proteins that combine the capacity of a photoreceptor with that of an adenylyl cyclase. When ectopically expressed under the control of specific promoters, these naturally occurring proteins become potent transgenic tools that facilitate the increase of cellular cAMP levels by the use of light. Currently, three different PAC transgenes-the euglenoid euPACα and euPACß, as well as the b eggiatoan bPac-are available. These transgenic tools provide cyclase activity capable of increasing cellular cAMP levels up to a hundredfold with either phasic- or tonic-like kinetic characteristics. Here, we consider the functional features of different cyclases and provide operating guidelines to optimize the use of PACs in vivo.

A central role of abscisic acid in drought stress protection of Agrobacterium-induced tumors on Arabidopsis.

Crown gall tumors induced by Agrobacterium tumefaciens represent a sink that has to be provided with nutrients and water by the host plant. The lack of an intact epidermis or cuticle results in uncontrolled loss of water. However, neither the tumor nor the host plant displays wilting. This phenomenon points to drought adaptation in both tumors and the crown gall host plant. To understand the underlying molecular mechanisms of protection against desiccation the gene expression pattern of Arabidopsis (Arabidopsis thaliana) tumors was integrated with the profile of stress metabolites: Arabidopsis tumors accumulated high amounts of abscisic acid (ABA), the ethylene precursor aminocyclopropyl carboxylic acid, osmoprotectants, and form a suberized periderm-like protective layer. Suberization of the outer tumor cell layers most likely is mediated by ABA since external application of ABA induced suberization of Arabidopsis roots. However, the expression level of the classical marker genes, known to respond to drought stress and/or ABA, was lower in tumors. Instead another set of drought and/or ABA-inducible genes was more highly transcribed. Elevated transcription of several ABA-dependent aquaporin genes might indicate that ABA controls the water balance of the tumor. The retarded tumor growth on abi and aba mutant plants underlined the importance of a tumor-specific ABA signaling pathway. Taken together, we propose that ABA is an important signal for protection of tumors against desiccation and thus supports tumor development.

An integrated view of gene expression and solute profiles of Arabidopsis tumors: a genome-wide approach.

Transformation of plant cells with T-DNA of virulent agrobacteria is one of the most extreme triggers of developmental changes in higher plants. For rapid growth and development of resulting tumors, specific changes in the gene expression profile and metabolic adaptations are required. Increased transport and metabolic fluxes are critical preconditions for growth and tumor development. A functional genomics approach, using the Affymetrix whole genome microarray (approximately 22,800 genes), was applied to measure changes in gene expression. The solute pattern of Arabidopsis thaliana tumors and uninfected plant tissues was compared with the respective gene expression profile. Increased levels of anions, sugars, and amino acids were correlated with changes in the gene expression of specific enzymes and solute transporters. The expression profile of genes pivotal for energy metabolism, such as those involved in photosynthesis, mitochondrial electron transport, and fermentation, suggested that tumors produce C and N compounds heterotrophically and gain energy mainly anaerobically. Thus, understanding of gene-to-metabolite networks in plant tumors promotes the identification of mechanisms that control tumor development.