Analyzing the role of cAMP in controlling the flagellar beat using optogenetics

• Applicant: Professorin Dr. Dagmar Wachten, since 10/2018
• Subject Area: Cell Biology ; Biophysics
• Term: from 2017 to 2022
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 365450677

Many eukaryotic cells, such as sperm or green algae, deploy flagella, lash-like appendages protruding from the cell surface. Flagella serve as propeller that moves the cell forward and as antennae that gather sensory cues. Receptors on the flagellar surface register and transduce stimuli into cellular signals. Finally, these signals modulate the undulatory beating of the flagellum and result in the swimming path. Cyclic nucleotide signaling represents an evolutionary conserved signaling pathway that regulates flagella beating e.g. of Paramecium, Chlamydomonas, airway epithelial cells, or mammalian sperm. In mammalian sperm, cAMP is essential for motility: sperm lacking cAMP are immotile and cannot fertilize the egg. However, the spatial organization of cAMP dynamics and how these changes are translated into a change in the flagella beat pattern is unknown. Preliminary data indicate that the beat pattern is not homogeneous along the flagellum: the beat pattern of the last third of the flagellum features a higher mode beat pattern in comparison to the main part of the flagellum. We have recently generated two mouse models expressing either the photoactivated adenylate cyclase bPAC or the light-activated phosphodiesterase LAPD exclusively in sperm flagella. These optogenetic tools in combination with a novel micro mirror illumination system will allow us to increase or decrease the intracellular cAMP level with high spatial-temporal precision along the beating flagellum and allow to answer the question: Is a compartmentalized beat pattern related to the intracellular cAMP concentration? To measure cAMP dynamics, we have generated a transgenic mouse line, expressing a novel genetically-encoded FRET (Förster resonance energy transfer)-based cAMP biosensor in sperm flagella. Our main goal is to correlate cAMP dynamics in sperm flagella with the beat pattern. This will only be possible using our unique approach of combining optogenetics with genetically-encoded biosensor and thereby address a fundamental biophysical question: How is the flagellar beat regulated by cAMP?

• DFG Programme: Priority Programmes
• Subproject of: SPP 1726: Microswimmers - From Single Particle Motion to Collective Behaviour
• Ehemaliger Antragsteller: Dr. Jan Fritz Jikeli, until 10/2018