Channelrhodopsins (ChRs) are light-gated ion channels extensively applied as optogenetics tools for manipulating neuronal activity. All currently known ChRs comprise a large cytoplasmic domain, whose function is elusive. Here, we report the cation channel properties of KnChR, one of the photoreceptors from a filamentous terrestrial alga Klebsormidium nitens, and demonstrate that the cytoplasmic domain of KnChR modulates the ion channel properties. KnChR is constituted of a 7-transmembrane domain forming a channel pore, followed by a C-terminus moiety encoding a peptidoglycan binding domain (FimV). Notably, the channel closure rate was affected by the C-terminus moiety. Truncation of the moiety to various lengths prolonged the channel open lifetime by more than 10-fold. Two Arginine residues (R287 and R291) are crucial for altering the photocurrent kinetics. We propose that electrostatic interaction between the rhodopsin domain and the C-terminus domain accelerates the channel kinetics. Additionally, maximal sensitivity was exhibited at 430 and 460 nm, the former making KnChR one of the most blue-shifted ChRs characterized thus far, serving as a novel prototype for studying the molecular mechanism of color tuning of the ChRs. Furthermore, KnChR would expand the optogenetics tool kit, especially for dual light applications when short-wavelength excitation is required.

Light-gated ion channel and ion pump rhodopsins are widely used as optogenetic tools and these can control the electrically excitable cells as (1) they are a single-component system i.e., their light sensing and ion-conducting functions are encoded by the 7-transmembrane domains and, (2) they show fast kinetics with small dark-thermal recovery time. In cellular signaling, a signal receptor, modulator, and the effector components are involved in attaining synchronous regulation of signaling. Optical modulation of the multicomponent network requires either receptor to effector encoded in a single ORF or direct modulation of the effector domain through bypassing all upstream players. Recently discovered modular rhodopsins like rhodopsin guanylate cyclase (RhoGC) and rhodopsin phosphodiesterase (RhoPDE) paves the way to establish a proof of concept for utilization of complex rhodopsin (modular rhodopsin) for optogenetic applications. Light sensor coupled modular system could be expressed in any cell type and hence holds great potential in the advancement of optogenetics 2.0 which would enable manipulating the entire relevant cell signaling system. Here, we had identified 50 novel modular rhodopsins with variant domains and their diverse cognate signaling cascades encoded in a single ORF, which are associated with specialized functions in the cells. These novel modular algal rhodopsins have been characterized based on their sequence and structural homology with previously reported rhodopsins. The presented novel modular rhodopsins with various effector domains leverage the potential to expand the optogenetic tool kit to regulate various cellular signaling pathways across the diverse biological model systems.

In bacteria, cyclic adenosine monophosphate (cAMP) signaling plays an essential regulatory role whose modulation via optogenetic tools would provide researchers an immense opportunity to control biological processes simply by illumination. The cAMP signaling in bacteria is a complex network of regulatory pathways, which utilizes distinct proteomic resources under different nutrient environments. We established an optogenetic modulation of cAMP and studied important cellular process of growth, biofilm formation, and virulence in the model bacterium E. coli using a light-gated adenylate cyclase (LgAC) from Beggiatoa sp. Blue light-induced activation of LgAC elevated the cAMP level in a blue light-dependent manner in E. coli. Quantitative proteomics revealed a decrease in the level of certain proteins governing growth (PTS, Adk, AckA, GlnA, and EFP), biofilm formation (IhfA, flagellin, YajQ, YeaG, and HlfC), and virulence (ClpP, YebC, KatE, BtuE, and Zur) in E. coli cells expressing LgAC upon blue light illumination. This optogenetic modulation of cAMP would be useful for deciphering cAMP-associated host–pathogen signaling of bacterial systems. Proteome knowledge established by this research work would also be useful for the scientific community while adapting LgAC-based optogenetic modulation for studying other relevant cAMP-driven bacterial physiology (e.g., energy metabolism). The systematic utilization of the established method and more extensively designed experiments regarding bacterial growth, biofilm, survival, and virulence might provide a road map for the identification of new targets for developing novel antibacterial drugs.

Microbial rhodopsin-based optogenetics has been widely applied to diverse mammalian and plant cell types for controlling membrane potential mediated responses. However, trafficking of optogenetically active protein to the desired subcellular organelle is still a major concern in optogenetic field. This could be resolved by studying the trafficking mechanism of optogenetically active protein in the native system. Current study is focused on the trafficking of two of the microbial rhodopsins named Chalmyopsin5 and Chlamyopsin6 in a green alga, Chlamydomonas reinhardtii. Chlamyopsin5 and Chlamyopsin6 are modular in nature and possess rhodopsin, histidine kinase, response regulator and cyclase domain in tandem. Immunolocalization of Chlamyopsin5 and Chlamyopsin6 in wild strain suggests their different subcellular localization; Chlamyopsin5 in eyespot and Chlamyopsin6 in flagella. Extensive immunocytochemistry of Chlamyopsin5 and Chlamyopsin6 was performed in different intraflagellar transport (IFT) components-defective strains of Chlamydomonas to dissect their trafficking mode to the destined subcellular compartment. Our results indicated the trafficking of Chlamyopsin5 to the eyespot to be independent of IFT machinery while Chlamyopsin6 to the flagella to be IFT dependent. Further, we demonstrate that IFT88 and IFT52 stabilizes Chlamyopsin6 and IFT20 interacts with Chlamyopsin6 in Chlamydomonas. Protein interactome of Chlamyopsin5 and Chlamyopsin6 indicate their role in nitrogen assimilation, gametogenesis and photoprotection in co-ordination with other photoreceptors. Collectively, our study enabled us to understand the targeting of Chlamyopsins to the subcellular compartment (eyespot and flagella). This study is important to expand optogenetic application of microbial type modular rhodopsin with histidine kinase and response regular. Further research in this direction is required to resolve the current challenge of targeting of optogenetic protein to desired subcellular compartment.