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XIII - Wachten

Analyzing the physiological function of cAMP in primary cilia using optogenetics


1) Dagmar Wachten (PI; caesar Bonn), coworker: Jan Jikeli


Analyzing the physiological function of cAMP in primary cilia using optogenetics

Cilia are membrane protrusions emanating from the cell surface and come in two different flavors: immotile, primary cilia and motile cilia, which are also called flagella. Motile cilia and flagella are involved in cell movement or the generation of fluid flow. Primary cilia are subcellular compartments that protrude from the surface of almost every mammalian cell. Their physiological role is less clear, but primary cilia dysfunction has been implicated in many human diseases, which are collectively termed ciliopathies.

Primary cilia are considered as sensory organelles that receive extracellular signals and mediate a downstream cellular response. Cyclic-nucleotide signaling has been proposed to be an evolutionary conserved signaling pathway, which acts not only in specialized sensory cilia but also in other cilia. Whereas in motile cilia or flagella, e.g. of Paramecium, Chlamydomonas, airway epithelial cells, or mammalian sperm, cAMP regulates ciliary beating, the role of cAMP signaling in non-motile primary cilia is much less understood. Ciliary G-protein coupled receptors (GPCRs) are believed to act as extrasynaptic signaling devices, which sense the local environment. Upon stimulation, ciliary GPCRs have been proposed to locally change the cAMP concentration in the primary cilium, thereby, controlling cellular functions like gene expression.

So far, attempts to study cAMP signaling in primary cilia have been hindered by the lack of suitable tools to manipulate and measure cAMP levels in this small compartment with high spatial and temporal resolution. Our approach to tackle this is to use optogenetics. We have already generated mouse models that express the photoactivated adenylate cyclase bPAC or the light-activated phosphodiesterase (LAPD) exclusively in sperm. Using these mouse models, we have demonstrated the strength of optogenetics in controlling flagellar signaling and function.

We now want to apply these tools to primary cilia. Our main goal is to investigate cAMP signaling in primary cilia using optogenetics and genetically-encoded biosensors. We will design ciliary-specific target approaches to localize our tools exclusively to cilia. We will measure endogenous cAMP dynamics in primary cilia using our novel genetically-encoded FRET (Förster resonance energy transfer)-based cAMP biosensor. This will allow to reveal whether primary cilia utilize cAMP signaling downstream of different environmental stimuli e.g. mechanical stimulation or hormones. Furthermore, we will manipulate cAMP signaling in primary cilia using our optogenetic tools and analyze the downstream response. In particular, we will focus on changes in gene expression. This requires single-cell gene expression analysis.

In combination, our approach will open up new avenues for optogenetics and contribute to answering a fundamental biological question - what is the physiological function of primary cilia.


Relevant Publications:

Mukherjee S*, Jansen V*, Jikeli J*, Hamzeh H, Alvarez L, Dombrowski M, Balbach M, Strünker T, Seifert R, Kaupp UB, Wachten D. A novel biosensor to study cAMP dynamics in cilia and flagella. eLife, 2016 in press.

Jansen V, Alvarez L, Balbach M, Strünker T, Hegemann P, Kaupp UB, Wachten D. Controlling Fertilization and cAMP signaling in sperm flagella by optogenetics. eLife, 2015, e05161.

Raju D, Schonauer S, Hamzeh H, Flynn KC, Bradke F, vom Dorp K, Dörmann P, Yildiz Y, Trötschel C, Poetsch A, Breiden B, Sandhoff K, Körschen HG, Wachten D. Accumulation of glucosylceramide in the absence of the beta-glucosidase GBA2 alters cytoskeletal dynamics. PLoS Genetics, 2015, e1005063, doi:10.1371/journal.pgen.1005063.

Schiffer C, Müller A, Egeberg DL, Alvarez L, Brenker C, Rehfeld A, Frederiksen H, Wäschle B, Kaupp UB, Balbach M, Wachten D, Skakkebaek NE, Almstrup K, Strünker T. Direct action of endocrine disrupting chemicals on human sperm. EMBO Rep., 2014, 15(7): p. 758-65.

Krähling M, Alvarez L, Debowski K, Van Q, Gunkel M, Irsen S, Al-Amoudi A, Strünker T, Kremmer E, Krause E, Voigt I, Wörtge S, Waisman I, Weyand I, Seifert R, Kaupp UB, Wachten D. CRIS – a novel cAMP-binding protein controlling spermiogenesis and the development of the flagellar bending. PLOS Genetics, 2013, e1003960, doi: 10.1371/journal.pgen.1003960.