1.
Nanobody-directed targeting of optogenetic tools to study signaling in the primary cilium.
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Hansen, JN
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Kaiser, F
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Klausen, C
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Stüven, B
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Chong, R
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Bönigk, W
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Mick, DU
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Möglich, A
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Jurisch-Yaksi, N
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Schmidt, FI
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Wachten, D
Abstract:
Compartmentalization of cellular signaling forms the molecular basis of cellular behavior. The primary cilium constitutes a subcellular compartment that orchestrates signal transduction independent from the cell body. Ciliary dysfunction causes severe diseases, termed ciliopathies. Analyzing ciliary signaling has been challenging due to the lack of tools investigate ciliary signaling. Here, we describe a nanobody-based targeting approach for optogenetic tools in mammalian cells and in vivo in zebrafish to specifically analyze ciliary signaling and function. Thereby, we overcome the loss of protein function observed after fusion to ciliary targeting sequences. We functionally localized modifiers of cAMP signaling, the photo-activated adenylate cyclase bPAC and the light-activated phosphodiesterase LAPD, and the cAMP biosensor mlCNBD-FRET to the cilium. Using this approach, we studied the contribution of spatial cAMP signaling in controlling cilia length. Combining optogenetics with nanobody-based targeting will pave the way to the molecular understanding of ciliary function in health and disease.
2.
Potassium channel-based optogenetic silencing.
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Bernal Sierra, YA
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Rost, BR
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Pofahl, M
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Fernandes, AM
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Kopton, RA
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Moser, S
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Holtkamp, D
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Masala, N
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Beed, P
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Tukker, JJ
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Oldani, S
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Bönigk, W
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Kohl, P
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Baier, H
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Schneider-Warme, F
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Hegemann, P
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Beck, H
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Seifert, R
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Schmitz, D
Abstract:
Optogenetics enables manipulation of biological processes with light at high spatio-temporal resolution to control the behavior of cells, networks, or even whole animals. In contrast to the performance of excitatory rhodopsins, the effectiveness of inhibitory optogenetic tools is still insufficient. Here we report a two-component optical silencer system comprising photoactivated adenylyl cyclases (PACs) and the small cyclic nucleotide-gated potassium channel SthK. Activation of this 'PAC-K' silencer by brief pulses of low-intensity blue light causes robust and reversible silencing of cardiomyocyte excitation and neuronal firing. In vivo expression of PAC-K in mouse and zebrafish neurons is well tolerated, where blue light inhibits neuronal activity and blocks motor responses. In combination with red-light absorbing channelrhodopsins, the distinct action spectra of PACs allow independent bimodal control of neuronal activity. PAC-K represents a reliable optogenetic silencer with intrinsic amplification for sustained potassium-mediated hyperpolarization, conferring high operational light sensitivity to the cells of interest.