Dataset Information

Optogenetic manipulation of neuronal and cardiomyocyte functions in zebrafish using microbial rhodopsins and adenylyl cyclases.

ABSTRACT: Even though microbial photosensitive proteins have been used for optogenetics, their use should be optimized to precisely control cell and tissue functions in vivo. We exploited GtCCR4 and KnChR, cation channelrhodopsins from algae, BeGC1, a guanylyl cyclase rhodopsin from a fungus, and photoactivated adenylyl cyclases (PACs) from cyanobacteria (OaPAC) or bacteria (bPAC), to control cell functions in zebrafish. Optical activation of GtCCR4 and KnChR in the hindbrain reticulospinal V2a neurons, which are involved in locomotion, induced swimming behavior at relatively short latencies, whereas activation of BeGC1 or PACs achieved it at long latencies. Activation of GtCCR4 and KnChR in cardiomyocytes induced cardiac arrest, whereas activation of bPAC gradually induced bradycardia. KnChR activation led to an increase in intracellular Ca²⁺ in the heart, suggesting that depolarization caused cardiac arrest. These data suggest that these optogenetic tools can be used to reveal the function and regulation of zebrafish neurons and cardiomyocytes.

SUBMITTER: Hagio H

PROVIDER: S-EPMC10435232 | biostudies-literature | 2023 Aug

REPOSITORIES: biostudies-literature

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Publications

Optogenetic manipulation of neuronal and cardiomyocyte functions in zebrafish using microbial rhodopsins and adenylyl cyclases.

Hagio Hanako H Koyama Wataru W Hosaka Shiori S Song Aysenur Deniz AD Narantsatsral Janchiv J Matsuda Koji K Shimizu Takashi T Hososhima Shoko S Tsunoda Satoshi P SP Kandori Hideki H Hibi Masahiko M

eLife 20230817

Even though microbial photosensitive proteins have been used for optogenetics, their use should be optimized to precisely control cell and tissue functions in vivo. We exploited GtCCR4 and KnChR, cation channelrhodopsins from algae, BeGC1, a guanylyl cyclase rhodopsin from a fungus, and photoactivated adenylyl cyclases (PACs) from cyanobacteria (OaPAC) or bacteria (bPAC), to control cell functions in zebrafish. Optical activation of GtCCR4 and KnC ...[more]

PMID: 37589546

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Project description:Adenosine 3',5'-cyclic monophosphate (cAMP) is a key second messenger that activates several signal transduction pathways in eukaryotic cells. Alteration of basal levels of cAMP is known to activate protein kinases, regulate phosphodiesterases and modulate the activity of ion channels such as Hyper polarization-activated cyclic nucleotide gated channels (HCN). Recent advances in optogenetics have resulted in the availability of novel genetically encoded molecules with the capability to alter cytoplasmic profiles of cAMP with unprecedented spatial and temporal precision. Using single molecule based super-resolution microscopy and different optogenetic modulators of cellular cAMP in both live and fixed cells, we illustrate a novel paradigm to report alteration in nanoscale confinement of ectopically expressed HCN channels. We characterized the efficacy of cAMP generation using ensemble photoactivation of different optogenetic modulators. Then we demonstrate that local modulation of cAMP alters the exchange of membrane bound HCN channels with its nanoenvironment. Additionally, using high density single particle tracking in combination with both acute and chronic optogenetic elevation of cAMP in the cytoplasm, we show that HCN channels are confined to sub 100 nm sized functional domains on the plasma membrane. The nanoscale properties of these domains along with the exchange kinetics of HCN channels in and out of these molecular zones are altered upon temporal changes in the cytoplasmic cAMP. Using HCN2 point mutants and a truncated construct of HCN2 with altered sensitivity to cAMP, we confirmed these alterations in lateral organization of HCN2 to be specific to cAMP binding. Thus, combining these advanced non-invasive paradigms, we report a cAMP dependent ensemble and single particle behavior of HCN channels mediated by its cyclic nucleotide binding domain, opening innovative ways to dissect biochemical pathways at the nanoscale and real-time in living cells.

Dataset Information

Optogenetic manipulation of neuronal and cardiomyocyte functions in zebrafish using microbial rhodopsins and adenylyl cyclases.

Publications

Optogenetic manipulation of neuronal and cardiomyocyte functions in zebrafish using microbial rhodopsins and adenylyl cyclases.

Similar Datasets

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