Funktionelle Charakterisierung von Adenylatzyklasen der Honigbiene Apis mellifera

Adenylyl cyclases (ACs) are enzymes that synthesize the intracellular messenger adenosine 3',5'-cyclic monophosphate (cAMP). The physiological impact of ACs has been intensively investigated in the mammalian brain and in the fruitfly Drosophila melanogaster. Some of the AC-enzymes are involved in processes underlying learning and memory. An organism, which is well suited for learning studies, is the honeybee Apis mellifera. The bee provides a rich behavioral repertoire that can be experimentally adressed. Within the last decades, several tests to study visual, olfactory, and tactile learning skills of the honeybee have been established. However, the cellular mechanisms and molecular components controlling the bees' behavior are largely unknown. In the beginning of this study, the molecular and biochemical properties of adenylyl cyclases in the bee had not been uncovered. I have cloned three genes (Amac2, Amac3, and Amac8) that encode membrane-bound ACs from honeybee brain. The amino-acid sequences have striking similarities to ACs from mammals and Drosophila. Heterologously expressed AmAC2t and AmAC3 proteins are activated by forskolin as well as by $\alpha$-subunits of stimulatory G-proteins (Gs$\alpha$). The Amac2-gene encodes an N-terminally truncated protein (AmAC2t). Notably, AmAC2t is the first truncated AC-enzyme that could be functionally expressed. The expression profile of the Amac3 gene was analyzed by in situ hybridization of braintissue sections. The Amac3 mRNA is predominatly expressed in the mushroom bodies, the optic lobes, and the deutocerebrum. To analyze the in vivo function of AmAC3, the expression of the Amac3 gene should be suppressed in the brain. To pursue such analyses, RNA interference (RNAi) is a promising technique. When Amac3-specific, double-stranded (ds) RNA is introduced into the honeybee brain, the endogenous Amac3 mRNA-level should be reduced. Bees that were injected with Amac3-dsRNA exhibited a higher responsiveness to sugar compared to controls. These results suggest that AmAC3 modulates the gustatory sensitivity of the animal. Interestingly, the injected bees had a higher amount of Amac3 mRNA than control bees. This unexpected finding evoked an alternative interpretation of the RNAi-effect. Further experiments are necessary, however, to uncover how the RNAi mechanism in the honeybee really works and to establish this method as a reliable tool to study gene function in this insect

SpermQ–A Simple Analysis Software to Comprehensively Study Flagellar Beating and Sperm Steering

Motile cilia, also called flagella, are found across a broad range of species; some cilia propel prokaryotes and eukaryotic cells like sperm, while cilia on epithelial surfaces create complex fluid patterns e.g., in the brain or lung. For sperm, the picture has emerged that the flagellum is not only a motor but also a sensor that detects stimuli from the environment, computing the beat pattern according to the sensory input. Thereby, the flagellum navigates sperm through the complex environment in the female genital tract. However, we know very little about how environmental signals change the flagellar beat and, thereby, the swimming behavior of sperm. It has been proposed that distinct signaling domains in the flagellum control the flagellar beat. However, a detailed analysis has been mainly hampered by the fact that current comprehensive analysis approaches rely on complex microscopy and analysis systems. Thus, knowledge on sperm signaling regulating the flagellar beat is based on custom quantification approaches that are limited to only a few aspects of the beat pattern, do not resolve the kinetics of the entire flagellum, rely on manual, qualitative descriptions, and are only a little comparable among each other. Here, we present SpermQ, a ready-to-use and comprehensive analysis software to quantify sperm motility. SpermQ provides a detailed quantification of the flagellar beat based on common time-lapse images acquired by dark-field or epi-fluorescence microscopy, making SpermQ widely applicable. We envision SpermQ becoming a standard tool in flagellar and motile cilia research that allows to readily link studies on individual signaling components in sperm and distinct flagellar beat patterns

Biochemical properties of heterologously expressed and native adenylyl cyclases from the honeybee brain (Apis mellifera L.)

Fuss N, Mujagic S, Erber J, Wachten S, Baumann A. Biochemical properties of heterologously expressed and native adenylyl cyclases from the honeybee brain (Apis mellifera L.). Insect Biochem.Mol.Biol. 2010;40(8):573-580.Cyclic AMP is an important intracellular signaling molecule participating e.g. in sensory signal transduction, cardiac myocyte regulation, learning and memory. The formation of cAMP is catalyzed by adenylyl cyclases. A variety of factors can modulate the properties of these enzymes and lead to dynamic changes of the intracellular cAMP concentration. Here we determined the tissue distribution of a recently cloned adenylyl cyclase (AmAC3) in honeybee brain. The protein is present in all neuropils. Intensive immunoreactivity was found in parts of the proto- and deutocerebrum and in the suboesophageal ganglion. Biochemical and pharmacological properties of AmAC3 and of native adenylyl cyclases in subregions of the honeybee brain were examined. Values for half-maximal activation with NKH477 were in the low micromolar range with 10.2 mu M for AmAC3 and 3.6-8.1 mu M for native enzymes. Biosynthesis of cAMP was specifically blocked by P-site inhibitors. Adenylyl cyclases in antennal lobes and AmAC3 share the inhibitory profile with 2',5'dd3'ATP > 3'AMP > 2'deoxyadenosine. In addition to P-site inhibitors AmAC3 activity was impaired by Ca2+/calmodulin. The results suggest that AmAC3 is a likely candidate to fulfill an integrative role in sensory, motor and higher-order information processing in the honeybee brain. (C) 2010 Elsevier Ltd. All rights reserved

Direct demonstration of discrete Ca2+ microdomains associated with different isoforms of adenylyl cyclase

Ca2+-sensitive adenylyl cyclases (ACs) orchestrate dynamic interplay between Ca2+ and cAMP that is a crucial feature of cellular homeostasis. Significantly, these ACs are highly selective for capacitative Ca2+ entry (CCE) over other modes of Ca2+ increase. To directly address the possibility that these ACs reside in discrete Ca2+ microdomains, we tethered a Ca2+ sensor, GCaMP2, to the N-terminus of Ca2+-stimulated AC8. GCaMP2-AC8 measurements were compared with global, plasma membrane (PM)-targeted or Ca2+-insensitive AC2-targeted GCaMP2. In intact cells, GCaMP2-AC8 responded rapidly to CCE, but was largely unresponsive to other types of Ca2+ rise. The global GCaMP2, PM-targeted GCaMP2 and GCaMP2-AC2 sensors reported large Ca2+ fluxes during Ca2+ mobilization and non-specific Ca2+ entry, but were less responsive to CCE than GCaMP2-AC8. Our data reveal that different AC isoforms localize to distinct Ca2+-microdomains within the plasma membrane. AC2, which is regulated via protein kinase C, resides in a microdomain that is exposed to a range of widespread Ca2+ signals seen throughout the cytosol. By contrast, a unique Ca2+ microdomain surrounds AC8 that promotes selectivity for Ca2+ signals arising from CCE, and optimizes CCE-mediated cAMP synthesis. This direct demonstration of discrete compartmentalized Ca2+ signals associated with specific signalling proteins provides a remarkable insight into the functional organization of signalling microdomains

Functional characterization of transmembrane adenylyl cyclases from the honeybee brain

Balfanz S, Ehling P, Wachten S, et al. Functional characterization of transmembrane adenylyl cyclases from the honeybee brain. Insect Biochemistry and Molecular Biology. 2012;42(6):435-445.The second messenger cAMP has a pivotal role in animals physiology and behavior. Intracellular concentrations of cAMP are balanced by cAMP-synthesizing adenylyl cyclases (ACs) and cAMP-cleaving phosphodiesterases. Knowledge about ACs in the honeybee (Apis mellifera) is rather limited and only an ortholog of the vertebrate AC3 isoform has been functionally characterized, so far. Employing bioinformatics and functional expression we characterized two additional honeybee genes encoding membrane-bound (tm)ACs. The proteins were designated AmAC2t and AmAC8. Unlike the common structure of tmACs, AmAC2t lacks the first transmembrane domain. Despite this unusual topography, AmAC2t-activity could be stimulated by norepinephrine and NKH477 with EC50s of 0.07 mu M and 3 mu M. Both ligands stimulated AmAC8 with EC50s, of 0.24 mu M and 3.1 mu M. In brain cryosections, intensive staining of mushroom bodies was observed with specific antibodies against AmAC8, an expression pattern highly reminiscent of the Drosophila rutabaga AC. In a current release of the honeybee genome database we identified three additional tmAC- and one soluble AC-encoding gene. These results suggest that (1) the AC-gene family in honeybees is comparably large as in other species, and (2) based on the restricted expression of AmAC8 in mushroom bodies, this enzyme might serve important functions in honeybee behavior. (C) 2012 Elsevier Ltd. All rights reserved

Insights into the residence in lipid rafts of adenylyl cyclase AC8 and its regulation by capacitative calcium entry

Adenylyl cyclases (ACs) are a family of critically important signaling molecules that are regulated by multiple pathways. Adenylyl cyclase 8 (AC8) is a Ca2+ stimulated isoform that displays a selective regulation by capacitative Ca2+ entry (CCE), the process whereby the entry of Ca2+ into cells is triggered by the emptying of intracellular stores. This selectivity was believed to be achieved through the localization of AC8 in lipid raft microdomains, along with components of the CCE apparatus. In the present study, we show that an intact leucine zipper motif is required for the efficient N-linked glycosylation of AC8, and that this N-linked glycosylation is important to target AC8 into lipid rafts. Disruption of the leucine zipper by site-directed mutagenesis results in the elimination of N-glycosylated forms and their exclusion from lipid rafts. Mutants of AC8 that cannot be N-glycosylated are not demonstrably associated with rafts, although they can still be regulated by CCE; however, raft integrity is required for the regulation of these mutants. These findings suggest that raft localized proteins in addition to AC8 are needed to mediate its regulation by CCE

Capacitative Ca2+ Entry via Orai1 and Stromal Interacting Molecule 1 (STIM1) Regulates Adenylyl Cyclase Type 8

Capacitative Ca2+ entry (CCE), which occurs through the plasma membrane as a result of Ca2+ store depletion, is mediated by stromal interacting molecule 1 (STIM1), a sensor of intracellular Ca2+ store content, and the pore-forming component Orai1. However, additional factors, such as C-type transient receptor potential (TRPC) channels, may also participate in the CCE apparatus. To explore whether the store-dependent Ca2+ entry reconstituted by coexpression of Orai1 and STIM1 has the functional properties of CCE, we used the Ca2+-calmodulin stimulated adenylyl cyclase type 8 (AC8), which responds selectively to CCE, whereas other modes of Ca2+ entry, including those activated by arachidonate and the ionophore ionomycin, are ineffective. In addition, the Ca2+ entry mediated by previous CCE candidates, diacylglycerol-activated TRPC channels, does not activate AC8. Here, we expressed Orai1 and STIM1 in HEK293 cells and saw a robust increment in CCE, and a proportional increase in CCE-stimulated AC8 activity. Inhibitors of the CCE assembly process ablated the effects on cyclase activity in both AC8-overexpressing HEK293 cells and insulin-secreting MIN6 cells endogenously expressing Ca2+-sensitive AC isoforms. AC8 is believed to be closely associated with the source of CCE; indeed, not only were AC8, Orai1, and STIM1 colocalized at the plasma membrane but also all three proteins occurred in lipid rafts. Together, our data indicate that Orai1 and STIM1 can be integral components of the cAMP and CCE microdomain associated with adenylyl cyclase type 8