Over the past 20 years, protein engineering has been extensively used to improve and modify the fundamental properties of fluorescent proteins (FPs) with the goal of adapting them for a fantastic ...range of applications. FPs have been modified by a combination of rational design, structure-based mutagenesis, and countless cycles of directed evolution (gene diversification followed by selection of clones with desired properties) that have collectively pushed the properties to photophysical and biochemical extremes. In this review, we provide both a summary of the progress that has been made during the past two decades, and a broad overview of the current state of FP development and applications in mammalian systems.
Monomeric red and far-red FPs and indicators now perform nearly as well as the best green FPs (and indicators).
Reversible and irreversible photochromism in FPs can be exploited to increase optical resolution and improve contrast compared with traditional fluorescence microscopy.
Infrared FPs (IFPs) are becoming ever more useful as labels for various proteins that allow correct localization and whole-animal imaging. IFPs can serve as an additional fluorescent ‘color’ for simultaneous imaging with visible FP-labeled proteins.
Bacterial phytochrome (BphP)-based IFPs provide a new scaffold for engineering fluorogenic indicators, which are ideal to visualize spatiotemporal dynamics of cell signaling in vivo.
Small ultra-red FP (smURFP) is the brightest far-red nonprototypical FP (comparable with EGFP) and is extremely photostable. smURFP may prove particularly useful as a photostable FP for super-resolution imaging and as a FRET acceptor for biosensing applications.
The engineering of new fluorescent indicators that combine features of prototypical FP-based indicators with photochromic proteins can reveal the cellular maps of biochemical activities in super-resolution.
FPs can be used as optogenetic actuators to manipulate cellular and protein functions through chromophore-assisted light inactivation or light-controlled protein oligomerization.
Metastasis is the major cause of poor prognosis in colorectal cancer (CRC), and increasing evidence supports the contribution of miRNAs to cancer progression. Here, we found that high expression of ...miR-103 and miR-107 (miR-103/107) was associated with metastasis potential of CRC cell lines and poor prognosis in patients with CRC. We showed that miR-103/107 targeted the known metastasis suppressors death-associated protein kinase (DAPK) and Krüppel-like factor 4 (KLF4) in CRC cells, resulting in increased cell motility and cell-matrix adhesion and decreased cell-cell adhesion and epithelial marker expression. miR-103/107 expression was increased in the presence of hypoxia, thereby potentiating DAPK and KLF4 downregulation and hypoxia-induced motility and invasiveness. In mouse models of CRC, miR-103/107 overexpression potentiated local invasion and liver metastasis effects, which were suppressed by reexpression of DAPK or KLF4. miR-103/107-mediated downregulation of DAPK and KLF4 also enabled the colonization of CRC cells at a metastatic site. Clinically, the signature of a miR-103/107 high, DAPK low, and KLF4 low expression profile correlated with the extent of lymph node and distant metastasis in patients with CRC and served as a prognostic marker for metastasis recurrence and poor survival. Our findings therefore indicate that miR-103/107-mediated repression of DAPK and KLF4 promotes metastasis in CRC, and this regulatory circuit may contribute in part to hypoxia-stimulated tumor metastasis. Strategies that disrupt this regulation might be developed to block CRC metastasis.
Channelrhodopsins (ChRs) are light‐activated channels from algae that provide these organisms with fast sensors to visible light for phototaxis. Since its discovery, channelrhodopsin‐2 (ChR2) has ...been used as a research tool to depolarize membranes of excitable cells with light. Subsequent chimeragenesis, mutagenesis and bioinformatic approaches have introduced additional ChR variants, such as channelrhodopsin‐2 with H134R mutation (ChR2/H134R), channelrhodopsin‐2 with E123T mutation (ChETA), Volvox carteri channelrhodopsin‐1 (VChR1), Volvox carteri channelrhodopsin‐2 (VChR2), channelrhodopsin‐2 with C128 or D156A mutations (ChR2/C128X/D156A), chimera D (ChD), chimera EF (ChEF) and chimera EF with I170V mutation (I170V). Each of these ChR variuants has unique features and limitations, but there are few resources summarizing and comparing these ChRs in a systematic manner. In this review, the seven following key properties of ChRs that have significant influences on their effectiveness as research tools are examined: conductance, selectivity, kinetics, desensitization, light sensitivity, spectral response and membrane trafficking. Using this information, valuable qualities and deficits of each ChR variant are summarized. Optimal uses and potential future improvements of ChRs as optogenetic tools are also discussed.
Channelrhodopsins (ChRs) are used to optogenetically depolarize neurons. We engineered a variant of ChR, denoted red-activatable ChR (ReaChR), that is optimally excited with orange to red light (λ ...∼590-630 nm) and offers improved membrane trafficking, higher photocurrents and faster kinetics compared to existing red-shifted ChRs. Red light is less scattered by tissue and is absorbed less by blood than the blue to green wavelengths that are required by other ChR variants. We used ReaChR expressed in the vibrissa motor cortex to drive spiking and vibrissa motion in awake mice when excited with red light through intact skull. Precise vibrissa movements were evoked by expressing ReaChR in the facial motor nucleus in the brainstem and illumination with red light through the external auditory canal. Thus, ReaChR enables transcranial optical activation of neurons in deep brain structures without the need to surgically thin the skull, form a transcranial window or implant optical fibers.
The efficacy of treatment of Helicobacter pylori infection has decreased steadily because of increasing resistance to clarithromycin, metronidazole, and levofloxacin. Resistance to amoxicillin is ...generally low, and high intragastric pH increases the efficacy of amoxicillin, so we investigated whether a combination of a high-dose proton pump inhibitor and amoxicillin (dual therapy) was more effective than standard first-line or rescue therapies in eradicating H pylori.
We performed a large-scale multihospital trial to compare the efficacy of a high-dose dual therapy (HDDT) with that of standard therapies in treatment-naive (n = 450) or treatment-experienced (n = 168) patients with H pylori infection. Treatment-naive patients were randomly assigned to groups given HDDT (rabeprazole 20 mg and amoxicillin 750 mg, 4 times/day for 14 days, group A1), sequential therapy for 10 days (group B1), or clarithromycin-containing triple therapy for 7 days (group C1). Treatment-experienced patients were randomly assigned to groups given HDDT for 14 days (group A2), sequential therapy for 10 days (B2), or levofloxacin-containing triple therapy for 7 days (C2). H pylori infection was detected by using the (13)C-urea breath test. We evaluated factors associated with treatment outcomes.
In the intention-to-treat analysis, H pylori was eradicated in 95.3% of patients in group A1 (95% confidence interval CI, 91.9%-98.8%), 85.3% in B1 (95% CI, 79.6%-91.1%), and 80.7% in group C1 (95% CI, 74.3%-87.1%). Infection was eradicated in 89.3% of patients in group A2 (95% CI, 80.9%-97.6%), 51.8% in group B2 (95% CI, 38.3%-65.3%), and 78.6% (95% CI, 67.5%-89.7%) in group C2. The efficacy of HDDT was significantly higher than that of currently recommended regimens, irrespective of CYP2C19 genotype. Bacterial resistance to drugs was associated with treatment failure. There were no significant differences between groups in adverse events or patient adherence.
HDDT is superior to standard regimens as empirical first-line or rescue therapy for H pylori infection, with similar safety profiles and tolerability. ClinicalTrials.gov number: NCT01163435.
Optogenetic techniques provide effective ways of manipulating the functions of selected neurons with light. In the current study, we engineered an optogenetic technique that directly inhibits ...neurotransmitter release. We used a genetically encoded singlet oxygen generator, miniSOG, to conduct chromophore assisted light inactivation (CALI) of synaptic proteins. Fusions of miniSOG to VAMP2 and synaptophysin enabled disruption of presynaptic vesicular release upon illumination with blue light. In cultured neurons and hippocampal organotypic slices, synaptic release was reduced up to 100%. Such inhibition lasted >1 hr and had minimal effects on membrane electrical properties. When miniSOG-VAMP2 was expressed panneuronally in Caenorhabditis elegans, movement of the worms was reduced after illumination, and paralysis was often observed. The movement of the worms recovered overnight. We name this technique Inhibition of Synapses with CALI (InSynC). InSynC is a powerful way to silence genetically specified synapses with light in a spatially and temporally precise manner.
•MiniSOG mediates CALI of presynaptic release proteins•Synaptic release is inhibited optogenetically with miniSOG fused VAMP2 or SYP1•Light reversibly reduces movement in miniSOG-VAMP2-expressing C. elegans
Lin et al. tether miniSOG to presynaptic proteins allowing optogenetic inhibition of synaptic release. This technique, InSynC, effectively inhibits synapses in cultured neurons, organotypic slice, and behaving C. elegans and is useful for studying the function of specific projections.
Channelrhodopsin 2 (ChR2), a light-activated nonselective cationic channel from Chlamydomonas reinhardtii, has become a useful tool to excite neurons into which it is transfected. The other ChR from ...Chlamydomonas, ChR1, has attracted less attention because of its proton-selective permeability. By making chimeras of the transmembrane domains of ChR1 and ChR2, combined with site-directed mutagenesis, we developed a ChR variant, named ChEF, that exhibits significantly less inactivation during persistent light stimulation. ChEF undergoes only 33% inactivation, compared with 77% for ChR2. Point mutation of Ile170 of ChEF to Val (yielding “ChIEF”) accelerates the rate of channel closure while retaining reduced inactivation, leading to more consistent responses when stimulated above 25 Hz in both HEK293 cells and cultured hippocampal neurons. In addition, these variants have altered spectral responses, light sensitivity, and channel selectivity. ChEF and ChIEF allow more precise temporal control of depolarization, and can induce action potential trains that more closely resemble natural spiking patterns.
Oxidative stress activates endothelial innate immunity and disrupts endothelial functions, including endothelial nitric oxide synthase-derived nitric oxide bioavailability. Here, we postulated that ...oxidative stress induces sterol regulatory element-binding protein 2 (SREBP2) and microRNA-92a (miR-92a), which in turn activate endothelial innate immune response, leading to dysfunctional endothelium.
Using cultured endothelial cells challenged by diverse oxidative stresses, hypercholesterolemic zebrafish, and angiotensin II-infused or aged mice, we demonstrated that SREBP2 transactivation of microRNA-92a (miR-92a) is oxidative stress inducible. The SREBP2-induced miR-92a targets key molecules in endothelial homeostasis, including sirtuin 1, Krüppel-like factor 2, and Krüppel-like factor 4, leading to NOD-like receptor family pyrin domain-containing 3 inflammasome activation and endothelial nitric oxide synthase inhibition. In endothelial cell-specific SREBP2 transgenic mice, locked nucleic acid-modified antisense miR-92a attenuates inflammasome, improves vasodilation, and ameliorates angiotensin II-induced and aging-related atherogenesis. In patients with coronary artery disease, the level of circulating miR-92a is inversely correlated with endothelial cell-dependent, flow-mediated vasodilation and is positively correlated with serum level of interleukin-1β.
Our findings suggest that SREBP2-miR-92a-inflammasome exacerbates endothelial dysfunction during oxidative stress. Identification of this mechanism may help in the diagnosis or treatment of disorders associated with oxidative stress, innate immune activation, and endothelial dysfunction.
Engineering a memory with LTD and LTP Nabavi, Sadegh; Fox, Rocky; Proulx, Christophe D ...
Nature (London),
07/2014, Letnik:
511, Številka:
7509
Journal Article
Recenzirano
Odprti dostop
It has been proposed that memories are encoded by modification of synaptic strengths through cellular mechanisms such as long-term potentiation (LTP) and long-term depression (LTD). However, the ...causal link between these synaptic processes and memory has been difficult to demonstrate. Here we show that fear conditioning, a type of associative memory, can be inactivated and reactivated by LTD and LTP, respectively. We began by conditioning an animal to associate a foot shock with optogenetic stimulation of auditory inputs targeting the amygdala, a brain region known to be essential for fear conditioning. Subsequent optogenetic delivery of LTD conditioning to the auditory input inactivates memory of the shock. Then subsequent optogenetic delivery of LTP conditioning to the auditory input reactivates memory of the shock. Thus, we have engineered inactivation and reactivation of a memory using LTD and LTP, supporting a causal link between these synaptic processes and memory.
Optogenetics allows the manipulation of neural activity in freely moving animals with millisecond precision, but its application in Drosophila melanogaster has been limited. Here we show that a ...recently described red activatable channelrhodopsin (ReaChR) permits control of complex behavior in freely moving adult flies, at wavelengths that are not thought to interfere with normal visual function. This tool affords the opportunity to control neural activity over a broad dynamic range of stimulation intensities. Using time-resolved activation, we show that the neural control of male courtship song can be separated into (i) probabilistic, persistent and (ii) deterministic, command-like components. The former, but not the latter, neurons are subject to functional modulation by social experience, which supports the idea that they constitute a locus of state-dependent influence. This separation is not evident using thermogenetic tools, a result underscoring the importance of temporally precise control of neuronal activation in the functional dissection of neural circuits in Drosophila.
PDF
