G protein-coupled receptors (GPCRs) are increasingly recognized to operate from intracellular membranes as well as the plasma membrane. The β2-adrenergic GPCR can activate Gs-linkedcyclic AMP (cAMP) signaling from endosomes. We show here that the homologous human β1-adrenergic receptor initiates an internal Gs-cAMP signal from the Golgi apparatus. By developing a chemical method to acutely squelch G protein coupling at defined membrane locations, we demonstrate that Golgi activation contributes significantly to the overall cellular cAMP response. Golgi signalling utilizes a pre-existing receptor pool rather than receptors delivered from the cell surface, requiring separate access of extracellular ligands. Epinephrine, a hydrophilic endogenous ligand, accesses the Golgi-localized receptor pool by facilitated transport requiring the organic cation transporter 3 (OCT3) whereas drugs can access the Golgi pool by passive diffusion according to hydrophobicity. We demonstrate marked differences among both agonist and antagonist drugs in Golgi-localized receptor access, and show that β-blocker drugs presently used in the clinic differ markedly in ability to antagonize the Golgi signal. We propose ’location bias’ as a new principle for achieving functional selectivity of GPCR-directed drug action.
Self-complementing split fluorescent proteins (FPs) have been widely used for protein labeling, visualization of subcellular protein localization, and detection of cell–cell contact. To expand this toolset, we have developed a screening strategy for the direct engineering of self-complementing split FPs. Via this strategy, we have generated a yellow–green split-mNeonGreen21–10/11 that improves the ratio of complemented signal to the background of FP1–10-expressing cells compared to the commonly used split GFP1–10/11; as well as a 10-fold brighter red-colored split-sfCherry21–10/11. Based on split sfCherry2, we have engineered a photoactivatable variant that enables single-molecule localization-based super-resolution microscopy. We have demonstrated dual-color endogenous protein tagging with sfCherry211 and GFP11, revealing that endoplasmic reticulum translocon complex Sec61B has reduced abundance in certain peripheral tubules. These new split FPs not only offer multiple colors for imaging interaction networks of endogenous proteins, but also hold the potential to provide orthogonal handles for biochemical isolation of native protein complexes.
We designed an epi-illumination SPIM system which utilizes a single objective and has an identical sample interface as an inverted fluorescence microscope with no additional reflection elements. It achieves subcellular resolution and single-molecule sensitivity and is compatible with common biological sample holders, including multi-well plates. We demonstrated multicolor fast volumetric imaging, single-molecule localization microscopy, parallel imaging of sixteen cell lines and parallel recording of cellular responses to perturbations.
SUMMARY Quorum sensing (QS) is a bacterial communication mechanism in which secreted signaling molecules impact population function and gene expression. QS-like phenomena have been reported in eukaryotes with largely unknown contributing molecules, functions, and mechanisms. We identify Qsp1, a secreted peptide, as a central signaling molecule that regulates virulence in the fungal pathogen Cryptococcus neoformans. QSP1 is a direct target of three transcription factors required for virulence, and qsp1Δ mutants exhibit attenuated infection, slowed tissue accumulation, and greater control by primary macrophages. Qsp1 mediates autoregulatory signaling that modulates secreted protease activity and promotes cell wall function at high cell densities. Peptide production requires release from a secreted precursor, proQsp1, by a cell-associated protease, Pqp1. Qsp1 sensing requires an oligopeptide transporter, Opt1, and remarkably, cytoplasmic expression of mature Qsp1 complements multiple phenotypes of qsp1Δ. Thus, C. neoformans produces an autoregulatory peptide that matures extracellularly but functions intracellularly to regulate virulence.
CRISPR/Cas technologies have transformed our ability to add functionality to the genome by knockin of payload via homology-directed repair (HDR). However, a systematic and quantitative profiling of the knock-in integration landscape is still lacking. Here, we present a framework based on longread sequencing and an integrated computational pipeline (knock-knock) to analyze knock-in repair outcomes across a wide range of experimental parameters. Our data uncover complex repair profiles, with perfect HDR often accounting for a minority of payload integration events, and reveal markedly distinct mis-integration patterns between cell-types or forms of HDR templates used. Our analysis demonstrates that the two sides of a given double-strand break can be repaired by separate pathways and identifies a major role for sequence micro-homology in driving donor mis-integration. Altogether, our comprehensive framework paves the way for investigating repair mechanisms, monitoring accuracy, and optimizing the precision of genome engineering.Recent developments in gene editing technologies have transformed our ability to manipulate genomes. Programmable site-specific nucleases -in particular CRISPR/Cas systems -introduce double-strand breaks (DSBs) at chosen genomic locations, prompting the activation of two separate DNA repair pathways which can be leveraged for genome engineering 1 . On the one hand, nonhomologous end-joining (NHEJ) can introduce insertions or deletions (in-dels) at the DSB site to inactivate gene products or regulatory elements. On the other hand, homology-directed repair (HDR)can use exogenous DNA sequences as templates to integrate (knock-in) new genetic information in the locus of interest 2 . Knock-in strategies have wide applications ranging from correcting diseasecausing mutations or inserting therapeutic payloads in a clinical context 3-5 , to introducing functional reporters for cell biology research 6,7 .Ultimately, the power of genetic engineering will rely on our ability to predictably edit genomes in order to precisely control cellular behaviors. Traditionally, NHEJ and HDR have been set apart by their degree of predictability: NHEJ is often thought to drive random repair outcomes, while HDR is considered to enable precise and templated editing. Recent data is challenging this simple distinction, however. High-throughput sequencing of NHEJ repair has uncovered complex but actionable sequence patterns that control in-del outcomes at DSBs 8-13 . In some cases, predictable NHEJ outcomes can be leveraged to precisely correct pathogenic human mutations, paving the way for template-free therapeutic genome editing 9 . By contrast, while accumulating evidence suggests that the integration of payload in knock-in experiments is not always precise 7,14,15 , a systematic and quantitative profiling of the full spectrum of HDR repair outcomes is still missing.Given its wide range of applications in both clinical and research settings, understanding the parameters that govern the efficiency and precision of knock...
Self-complementing split fluorescent proteins (FPs) have been widely used for protein labeling, visualization of subcellular protein localization, and detection of cell-cell contact.To expand this toolset, we have developed a screening strategy for the direct engineering of self-complementing split FPs. Via this strategy, we have generated a yellow-green split-mNeonGreen2 1-10/11 that improves the ratio of complemented signal to the background of FP 1-10 -expressing cells compared to the commonly used split GFP 1-10/11 ; as well as a 10-fold brighter red-colored split-sfCherry2 1-10/11 . Based on split sfCherry2, we have engineered a photoactivatable variant that enables single-molecule localization-based super-resolution microscopy. We have demonstrated dual-color endogenous protein tagging with sfCherry2 11 and GFP 11 , revealing that endoplasmic reticulum translocon complex Sec61B has reduced abundance in certain peripheral tubules. These new split FPs not only offer multiple colors for imaging interaction networks of endogenous proteins, but also hold the potential to provide orthogonal handles for biochemical isolation of native protein complexes. 5,6 , identification of cell contacts and synapses 7,8 , as well as scaffolding protein assembly 3, 9, 10 . Recently, they have also enabled the generation of large-scale human cell line libraries with fluorescently tagged endogenous proteins through CRISPR/Cas9-based gene editing 11 .So far, the most commonly used self-complementing split FP was GFP 1-10D7/11M3 OPT (which we refers to as GFP 1-10/11 ), engineered from super-folder GFP (sfGFP) 12 . With the splitting point between the tenth and eleventh β-strands, the resulting GFP 11 fragment is a 16-amino acid (a.a.) short peptide. The corresponding GFP 1-10 fragment remains almost non-fluorescent until complementation, making GFP 1-10/11 well suited for protein labeling by fusing GFP 11 to the target protein and over-expressing GFP 1-10 in the corresponding subcellular compartments. However, there lacks a second, orthogonal split FP system with comparable complementation performance for multicolor imaging and multiplexed scaffolding of protein assembly. Previously, a sfCherry 1-10/11 system 3 was derived from super-folder Cherry, an mCherry variant optimized for folding efficiency 13 . However, its overall fluorescent brightness is substantially weaker than an intact sfCherry fusion, potentially due to its limited complementation efficiency 3 . Although two-color imaging with sfCherry 1-10/11 and GFP 1-10/11 has been done using tandem sfCherry 11 to amplify the sfCherry signal for over-expressed targets, it is still too dim to detect most endogenous proteins.In this paper, we report a screening strategy for the direct engineering of self-complementing split FPs. Using this strategy, we have generated a yellow-green-colored mNeonGreen2 1-10/11 (mNG2) that has an improved ratio of complemented signal to the background of FP 1-10 -expressing cells as compared to GFP 1-10/11 , as well as a red-colored sfCherry2 1-...
GPCR-regulated cAMP production from endosomes can specify signaling to the nucleus by moving the source of cAMP without changing its amount. How this is possible remains unknown because cAMP gradients dissipate over the nanoscale whereas endosomes typically localize microns from the nucleus. We show that the key location-dependent step for endosome-encoded transcriptional control is nuclear entry of cAMP-dependent protein kinase (PKA) catalytic subunits. These are sourced from punctate accumulations of PKA holoenzyme that are densely distributed in the cytoplasm and titrated by global cAMP into a discrete metastable state, in which catalytic subunits are bound but dynamically exchange. Mobile endosomes containing activated receptors collide with the metastable PKA puncta and pause in close contact. We propose that these properties enable cytoplasmic PKA to act collectively like a semiconductor, converting nanoscale cAMP gradients generated from endosomes into microscale elevations of free catalytic subunit to direct downstream signaling.
Labeling proteins with high specificity and efficiency is a fundamental prerequisite for microscopic visualization of subcellular protein structures and interactions. While the comparatively small size of epitope tags makes them less perturbative to fusion proteins, they require the use of large antibodies that often limit probe accessibility and effective resolution. Here we use the covalent SpyTag-SpyCatcher system as an epitope-like tag for fluorescent labeling of intracellular proteins in fixed cells for both conventional and super-resolution microscopy. We have also applied this method to endogenous proteins via gene editing, demonstrating its high labeling efficiency and capability for isoform-specific labeling.
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