G-protein-coupled receptor (GPCR) activity gradients evoke important cell behavior but there is a dearth of methods to induce such asymmetric signaling in a cell. Here we achieved reversible, rapidly switchable patterns of spatiotemporally restricted GPCR activity in a single cell. We recruited properties of nonrhodopsin opsins-rapid deactivation, distinct spectral tuning, and resistance to bleachingto activate native Gi, Gq, or Gs signaling in selected regions of a cell. Optical inputs were designed to spatiotemporally control levels of second messengers, IP3, phosphatidylinositol (3,4,5)-triphosphate, and cAMP in a cell. Spectrally selective imaging was accomplished to simultaneously monitor optically evoked molecular and cellular response dynamics. We show that localized optical activation of an opsin-based trigger can induce neurite initiation, phosphatidylinositol (3,4,5)-triphosphate increase, and actin remodeling. Serial optical inputs to neurite tips can refashion early neuron differentiation. Methods here can be widely applied to program GPCRmediated cell behaviors.optogenetics | cell polarity G -protein-coupled receptors (GPCRs) initiate most of the signaling in metazoans and regulate a wide variety of cellular responses that include differentiation, migration, secretion, and contraction. Asymmetric activation of GPCR signaling activity in a cell is thought to play a critical role in varied processes such as cell polarization (1) and modulation of neuron function (2). There is still limited information about activation of signaling that is restricted in space and time across a single cell. An impediment is the lack of methods to continuously vary signal input to a single cell with high time resolution and precision to quantitate second messenger and cellular output from the same cell. A method that provides reversible, temporal control over GPCR activity in restricted regions of a single cell may help govern cell behavior and probe the cellular and molecular basis of single-cell responses.Here we used a set of light-triggered GPCRs, human color opsins, and related nonrhodopsin opsins, to achieve such confined GPCR activation in a single cell. In contrast to molecular gradients, an optical signal provides higher spatiotemporal control and it can be switched on or off or relocated almost instantaneously. We recruited the properties of color opsins to develop optical triggers that spatiotemporally confine signaling. These opsins deactivate rapidly and demonstrate relatively low sensitivity to light (3, 4). The deactivation characteristics curtail the diffusion of activated receptors across a cell and help localize receptor activation to selected regions of a cell. Low susceptibility to bleaching allows continuous reproducible activation. In addition, nonrhodopsin opsins selectively activate different G-protein types, suggesting that they can be individually used to regulate distinct second messengers (4, 5). Rhodopsin or its chimeric forms have been valuable for globally activating G proteins (6-8). Howev...
Retinopathy of prematurity (ROP) is a neurovascular complication in preterm babies, leading to severe visual impairment, but the underlying mechanisms are yet unclear. The present study aimed at unraveling the molecular mechanisms underlying the pathogenesis of ROP. A comprehensive screening of candidate genes in preterms with ROP (n = 189) and no-ROP (n = 167) was undertaken to identify variants conferring disease susceptibility. Allele and genotype frequencies, linkage disequilibrium and haplotypes were analyzed to identify the ROP-associated variants. Variants in CFH (p = 2.94 × 10−7), CFB (p = 1.71 × 10−5), FBLN5 (p = 9.2 × 10−4), CETP (p = 2.99 × 10−5), and CXCR4 (p = 1.32 × 10−8) genes exhibited significant associations with ROP. Further, a quantitative assessment of 27 candidate proteins and cytokines in the vitreous and tear samples of babies with severe ROP (n = 30) and congenital cataract (n = 30) was undertaken by multiplex bead arrays and further validated by western blotting and zymography. Significant elevation and activation of MMP9 (p = 0.038), CFH (p = 2.24 × 10−5), C3 (p = 0.05), C4 (p = 0.001), IL-1ra (p = 0.0019), vascular endothelial growth factor (VEGF) (p = 0.0027), and G-CSF (p = 0.0099) proteins were observed in the vitreous of ROP babies suggesting an increased inflammation under hypoxic condition. Along with inflammatory markers, activated macrophage/microglia were also detected in the vitreous of ROP babies that secreted complement component C3, VEGF, IL-1ra, and MMP-9 under hypoxic stress in a cell culture model. Increased expression of the inflammatory markers like the IL-1ra (p = 0.014), MMP2 (p = 0.0085), and MMP-9 (p = 0.03) in the tears of babies at different stages of ROP further demonstrated their potential role in disease progression. Based on these findings, we conclude that increased complement activation in the retina/vitreous in turn activated microglia leading to increased inflammation. A quantitative assessment of inflammatory markers in tears could help in early prediction of ROP progression and facilitate effective management of the disease, thereby preventing visual impairment.
There is a dearth of approaches to experimentally direct cell migration by continuously varying signal input to a single cell, evoking all possible migratory responses and quantitatively monitoring the cellular and molecular response dynamics. Here we used a visual blue opsin to recruit the endogenous G-protein network that mediates immune cell migration. Specific optical inputs to this optical trigger of signaling helped steer migration in all possible directions with precision. Spectrally selective imaging was used to monitor cellwide phosphatidylinositol (3,4,5)-triphosphate (PIP3), cytoskeletal, and cellular dynamics. A switch-like PIP3 increase at the cell front and a decrease at the back were identified, underlying the decisive migratory response. Migration was initiated at the rapidly increasing switch stage of PIP3 dynamics. This result explains how a migratory cell filters background fluctuations in the intensity of an extracellular signal but responds by initiating directionally sensitive migration to a persistent signal gradient across the cell. A twocompartment computational model incorporating a localized activator that is antagonistic to a diffusible inhibitor was able to simulate the switch-like PIP3 response. It was also able simulate the slow dissipation of PIP3 on signal termination. The ability to independently apply similar signaling inputs to single cells detected two cell populations with distinct thresholds for migration initiation. Overall the optical approach here can be applied to understand G-proteincoupled receptor network control of other cell behaviors.optogenetics | ultrasensitivity A variety of cells sense gradients of chemoattractants and respond by migrating toward increasing concentrations. Migration is central to immune cell function, morphogenesis, cancer cell metastasis, and the life cycle of the social amoeba, Dictyostelium discoideum (1). Cell migration is made up of a characteristic sequence of identifiable cellular events that are governed by G-protein-coupled receptor (GPCR)-driven signaling networks. Although considerable information exists about molecules involved in migration, the challenge is in translating a static map of these molecules into a spatially and temporally dynamic network that orchestrates migratory behavior. Effective methods to probe the basis of network control of migration need to be able to faithfully evoke migratory behavior experimentally and quantitatively monitor response dynamics at the cellular and molecular level. Microfluidic devices and electrical fields have been used to regulate migration and provide insights into the process (2-6). However, there are limitations at present in the ability to direct a series of signaling inputs to a single cell in spatially and temporally complex patterns. Such inputs are essential to continually choreograph the events that constitute the migratory response: initiation, translocation, directional changes, and adaptation. A light-sensitive domain of a plant protein has been inserted into Rac1, a downstrea...
G-protein βγ subunits translocate reversibly from the plasma membrane to internal membranes on receptor activation. Translocation rates differ depending on the γ subunit type. There is limited understanding of the role of the differential rates of Gβγ translocation in modulating signaling dynamics in a cell. Bifurcation analysis of the calcium oscillatory network structure predicts that the translocation rate of a signaling protein can regulate the damping of system oscillation. Here, we examined whether the Gβγ translocation rate regulates calcium oscillations induced by G-protein-coupled receptor activation. Oscillations in HeLa cells expressing γ subunit types with different translocation rates were imaged and quantitated. The results show that differential Gβγ translocation rates can underlie the diversity in damping characteristics of calcium oscillations among cells. Mathematical modeling shows that a translocation embedded motif regulates damping of G-protein-mediated calcium oscillations consistent with experimental data. The current study indicates that such a motif may act as a tuning mechanism to design oscillations with varying damping patterns by using intracellular translocation of a signaling component.
The rising family of two-dimensional (2D) materials, MXene, has stirred great scientific interest due to their unique physicochemical properties and potential applications. Much research has focused on the creative strategy and rational development of MXene-derived quantum dots (MXQDs). They contribute to various applications such as sensing, biomedicine, catalysis, optoelectronic devices, energy storage and conversion, and so forth due to their unique properties compared to their 2D-MXene counterparts. Research has focused on the efficient synthetic strategies and potential applications of MXQDs, particularly toward energy conversion and energy storage applications. Therefore, it is highly useful to corroborate the existing data which could provide a decisive track for the booming research on electrocatalysts utilizing MXQDs as a new scaffold. Exploiting the platform, we have reviewed the various synthetic approaches employed to prepare MXQDs and their intriguing applications to develop challenging and fascinating energy conversion and energy storage devices. Furthermore, we have critically highlighted the prospects as well as the challenges. We hope this review will enlighten the path toward preparing more and more promising MXQDs-based materials with captivating properties and potential applications.
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