Optogenetics for transcriptional programming and genetic engineering.

Optogenetics combines genetics and biophotonics to enable noninvasive control of biological processes with high spatiotemporal precision. When engineered into protein machineries that govern the cellular information flow as depicted in the central dogma, multiple genetically encoded non-opsin photosensory modules have been harnessed to modulate gene transcription, DNA or RNA modifications, DNA recombination, and genome engineering by utilizing photons emitting in the wide range of 200-1000 nm. We present herein generally applicable modular strategies for optogenetic engineering and highlight latest advances in the broad applications of opsin-free optogenetics to program transcriptional outputs and precisely manipulate the mammalian genome, epigenome, and epitranscriptome. We also discuss current challenges and future trends in opsin-free optogenetics, which has been rapidly evolving to meet the growing needs in synthetic biology and genetics research.

Probing the Substrate Requirements of the In†Vitro Geranylation Activity of Selenouridine Synthase (SelU).

Natural RNA modifications diversify the structures and functions of existing nucleic acid building blocks. Geranyl is one of the most hydrophobic groups recently identified in bacterial tRNAs. Selenouridine synthase (SelU, also called mnmH) is an enzyme with a dual activity which catalyzes selenation and geranylation in tRNAs containing 2-thiouridine using selenophosphate or geranyl-pyrophosphate as cofactors. In this study, we explored the in vitro geranylation process of tRNA anticodon stem loops (ASL) mediated by SelU and showed that the geranylation activity was abolished when U35 was mutated to A35 (ASL-tRNALys (s2U)UU to ASL-tRNAIle (s2U)AU ). By examining the SelU cofactor geranyl-pyrophosphate (gePP) and its analogues, we found that only the geranyl group, but not dimethylallyl- and farnesyl-pyrophosphate with either shorter or longer terpene chains, could be incorporated into ASL. The degree of tRNA geranylation in the end-point analysis for SelU follows the order of ASLLys (s2UUU) ≃ ASLGln (s2UUG) >ASLGlu (s2UUC) . These findings suggest a putative mechanism for substrate discrimination by SelU and reveal key factors that might influence its enzymatic activity. Given that SelU plays an important role in bacterial translation systems, inhibiting this enzyme and targeting its geranylation and selenation pathways could be exploited as a promising strategy to develop SelU-based antibiotics.

Lateral diffusion contributes to FRET from lanthanide-tagged membrane proteins.

Diffusion can enhance Förster resonance energy transfer (FRET) when donors or acceptors diffuse distances that are similar to the distances separating them during the donor's excited state lifetime. Lanthanide donors remain in the excited state for milliseconds, which makes them useful for time-resolved FRET applications but also allows time for diffusion to enhance energy transfer. Here we show that diffusion dramatically enhances FRET between membrane proteins labeled with lanthanide donors. This phenomenon complicates interpretation of experiments that use long-lived donors to infer association or proximity of mobile membrane proteins, but also offers a method of monitoring diffusion in membrane domains in real time in living cells.

Sensitive and high resolution localization and tracking of membrane proteins in live cells with BRET.

Peripheral and integral membrane proteins can be located in several different subcellular compartments, and it is often necessary to determine the location of such proteins or to track their movement in living cells. Image-based colocalization of labeled membrane proteins and compartment markers is frequently used for this purpose, but this method is limited in terms of throughput and resolution. Here we show that bioluminescence resonance energy transfer (BRET) between membrane proteins of interest and compartment-targeted BRET partners can report subcellular location and movement of membrane proteins in live cells. The sensitivity of the method is sufficient to localize a few hundred protein copies per cell. The spatial resolution can be sufficient to determine membrane topology, and the temporal resolution is sufficient to track changes that occur in less than 1 second. BRET requires little user intervention, and is thus amenable to large-scale experimental designs with standard instruments.

Segregation of family A G protein-coupled receptor protomers in the plasma membrane.

G protein-coupled receptors (GPCRs) transduce many important physiological signals and are targets for a large fraction of therapeutic drugs. Members of the largest family of GPCRs (family A) are thought to self-associate as dimers and higher-order oligomers, although the significance of such quaternary structures for signaling or receptor trafficking is known for only a few examples. One outstanding question is the physical stability of family A oligomers in cell membranes. Stable oligomers would be expected to move through cellular compartments and membrane domains as intact groups of protomers. Here, we test this prediction by recruiting subsets of affinity-tagged family A protomers into artificial microdomains on the surface of living cells and asking if untagged protomers move into these domains (are corecruited) at the same time. We find that tagged ß2 adrenergic and µ-opioid protomers are unable to corecruit untagged protomers into microdomains. In contrast, tagged metabotropic glutamate receptor protomers do corecruit untagged protomers into such microdomains, which is consistent with the known covalent mechanism whereby these family C receptors dimerize. These observations suggest that interactions between these family A protomers are too weak to directly influence subcellular location, and that mechanisms that move these receptors between subcellular compartments and domains must operate on individual protomers.

Rab26 modulates the cell surface transport of α2-adrenergic receptors from the Golgi.

The molecular mechanisms underlying the transport from the Golgi to the cell surface of G protein-coupled receptors remain poorly elucidated. Here we determined the role of Rab26, a Ras-like small GTPase involved in vesicle-mediated secretion, in the cell surface export of α(2)-adrenergic receptors. We found that transient expression of Rab26 mutants and siRNA-mediated depletion of Rab26 significantly attenuated the cell surface numbers of α(2A)-AR and α(2B)-AR, as well as ERK1/2 activation by α(2B)-AR. Furthermore, the receptors were extensively arrested in the Golgi by Rab26 mutants and siRNA. Moreover, Rab26 directly and activation-dependently interacted with α(2B)-AR, specifically the third intracellular loop. These data demonstrate that the small GTPase Rab26 regulates the Golgi to cell surface traffic of α(2)-adrenergic receptors, likely through a physical interaction. These data also provide the first evidence implicating an important function of Rab26 in coordinating plasma membrane protein transport.

Caffeine-Operated Synthetic Modules for Chemogenetic Control of Protein Activities by Life Style.

A genetically encoded caffeine-operated synthetic module (COSMO) is introduced herein as a robust chemically induced dimerization (CID) system. COSMO enables chemogenetic manipulation of biological processes by caffeine and its metabolites, as well as caffeinated beverages, including coffee, tea, soda, and energy drinks. This CID tool, evolved from an anti-caffeine nanobody via cell-based high-throughput screening, permits caffeine-inducible gating of calcium channels, tumor killing via necroptosis, growth factors-independent activation of tyrosine receptor kinase signaling, and enhancement of nanobody-mediated antigen recognition for the severe acute respiratory distress coronavirus 2 (SARS-CoV-2) spike protein. Further rationalized engineering of COSMO leads to 34-217-fold enhancement in caffeine sensitivity (EC50 = 16.9 nanomolar), which makes it among the most potent CID systems like the FK506 binding protein (FKBP)-FKBP rapamycin binding domain (FRB)-rapamycin complex. Furthermore, bivalent COSMO (biCOMSO) connected with a long linker favors intramolecular dimerization and acts as a versatile precision switch when inserted in host proteins to achieve tailored function. Given the modularity and high transferability of COMSO and biCOSMO, these chemical biology tools are anticipated to greatly accelerate the development of therapeutic cells and biologics that can be switched on and off by caffeinated beverages commonly consumed in the daily life.

Third-party bioluminescence resonance energy transfer indicates constitutive association of membrane proteins: application to class a g-protein-coupled receptors and g-proteins.

Many of the molecules that mediate G-protein signaling are thought to constitutively associate with each other in variably stable signaling complexes. Much of the evidence for signaling complexes has come from Förster resonance energy transfer and bioluminescence resonance energy transfer (BRET) studies. However, detection of constitutive protein association with these methods is hampered by nonspecific energy transfer that occurs when donor and acceptor molecules are in close proximity by chance. We show that chemically-induced recruitment of local third-party BRET donors or acceptors reliably separates nonspecific and specific BRET. We use this method to reexamine the constitutive association of class A G-protein-coupled receptors (GPCRs) with other GPCRs and with heterotrimeric G-proteins. We find that beta2 adrenoreceptors constitutively associate with each other and with several other class A GPCRs. In contrast, GPCRs and G-proteins are unlikely to exist in stable constitutive preassembled complexes.

Targeting plasma membrane phosphatidylserine content to inhibit oncogenic KRAS function.

The small GTPase KRAS, which is frequently mutated in human cancers, must be localized to the plasma membrane (PM) for biological activity. We recently showed that the KRAS C-terminal membrane anchor exhibits exquisite lipid-binding specificity for select species of phosphatidylserine (PtdSer). We, therefore, investigated whether reducing PM PtdSer content is sufficient to abrogate KRAS oncogenesis. Oxysterol-related binding proteins ORP5 and ORP8 exchange PtdSer synthesized in the ER for phosphatidyl-4-phosphate synthesized in the PM. We show that depletion of ORP5 or ORP8 reduced PM PtdSer levels, resulting in extensive mislocalization of KRAS from the PM. Concordantly, ORP5 or ORP8 depletion significantly reduced proliferation and anchorage-independent growth of multiple KRAS-dependent cancer cell lines, and attenuated KRAS signaling in vivo. Similarly, functionally inhibiting ORP5 and ORP8 by inhibiting PI4KIIIα-mediated synthesis of phosphatidyl-4-phosphate at the PM selectively inhibited the growth of KRAS-dependent cancer cell lines over normal cells. Inhibiting KRAS function through regulating PM lipid PtdSer content may represent a viable strategy for KRAS-driven cancers.

BRET evidence that ß2 adrenergic receptors do not oligomerize in cells.

Bioluminescence resonance energy transfer (BRET) is often used to study association of membrane proteins, and in particular oligomerization of G protein-coupled receptors (GPCRs). Oligomerization of class A GPCRs is controversial, in part because the methods used to study this question are not completely understood. Here we reconsider oligomerization of the class A ß2 adrenergic receptor (ß2AR), and reevaluate BRET titration as a method to study membrane protein association. Using inducible expression of the energy acceptor at multiple levels of donor expression we find that BRET between ß2AR protomers is directly proportional to the density of the acceptor up to ~3,000 acceptors µm(-2), and does not depend on the density of the donor or on the acceptor:donor (A:D) stoichiometry. In contrast, BRET between tightly-associating control proteins does not depend on the density of the acceptor, but does depend on the density of the donor and on the A:D ratio. We also find that the standard frameworks used to interpret BRET titration experiments rely on simplifying assumptions that are frequently invalid. These results suggest that ß2ARs do not oligomerize in cells, and demonstrate a reliable method of assessing membrane protein association with BRET.

Internalization dissociates ß2-adrenergic receptors.

G protein-coupled receptors (GPCRs) self-associate as dimers or higher-order oligomers in living cells. The stability of associated GPCRs has not been extensively studied, but it is generally thought that these receptors move between the plasma membrane and intracellular compartments as intact dimers or oligomers. Here we show that ß(2)-adrenergic receptors (ß(2)ARs) that self-associate at the plasma membrane can dissociate during agonist-induced internalization. We use bioluminescence-resonance energy transfer (BRET) to monitor movement of ß(2)ARs between subcellular compartments. BRET between ß(2)ARs and plasma membrane markers decreases in response to agonist activation, while at the same time BRET between ß(2)ARs and endosome markers increases. Energy transfer between ß(2)ARs is decreased in a similar manner if either the donor- or acceptor-labeled receptor is mutated to impair agonist binding and internalization. These changes take place over the course of 30 minutes, persist after agonist is removed, and are sensitive to several inhibitors of arrestin- and clathrin-mediated endocytosis. The magnitude of the decrease in BRET between donor- and acceptor-labeled ß(2)ARs suggests that at least half of the receptors that contribute to the BRET signal are physically segregated by internalization. These results are consistent with the possibility that ß(2)ARs associate transiently with each other in the plasma membrane, or that ß(2)AR dimers or oligomers are actively disrupted during internalization.

A draft sequence of the rice genome (Oryza sativa L. ssp. japonica).

The genome of the japonica subspecies of rice, an important cereal and model monocot, was sequenced and assembled by whole-genome shotgun sequencing. The assembled sequence covers 93% of the 420-megabase genome. Gene predictions on the assembled sequence suggest that the genome contains 32,000 to 50,000 genes. Homologs of 98% of the known maize, wheat, and barley proteins are found in rice. Synteny and gene homology between rice and the other cereal genomes are extensive, whereas synteny with Arabidopsis is limited. Assignment of candidate rice orthologs to Arabidopsis genes is possible in many cases. The rice genome sequence provides a foundation for the improvement of cereals, our most important crops.