To perform quantitative live cell imaging, investigators require fluorescent reporters that accurately report protein localization and levels, while minimally perturbing the cell. Yet, within the ...biochemically distinct environments of cellular organelles, popular fluorescent proteins (FPs), including EGFP, can be unreliable for quantitative imaging, resulting in the underestimation of protein levels and incorrect localization. Specifically, within the secretory pathway, significant populations of FPs misfold and fail to fluoresce due to non-native disulphide bond formation. Furthermore, transmembrane FP-fusion constructs can disrupt organelle architecture due to oligomerizing tendencies of numerous common FPs. Here, we describe a powerful set of bright and inert FPs optimized for use in multiple cellular compartments, especially oxidizing environments and biological membranes. Also, we provide new insights into the use of red FPs in the secretory pathway. Our monomeric 'oxFPs' finally resolve long-standing, underappreciated and important problems of cell biology and should be useful for a number of applications.
Lipolysis is a critical metabolic pathway contributing to energy homeostasis through degradation of triacylglycerides stored in lipid droplets (LDs), releasing fatty acids. Neutral lipid lipases act ...at the oil/water interface. In mammalian cells, LD surfaces are coated with one or more members of the perilipin protein family, which serve important functions in regulating lipolysis. We investigated mechanisms by which three perilipin proteins control lipolysis by adipocyte triglyceride lipase (ATGL), a key lipase in adipocytes and non-adipose cells. Using a cell culture model, we examined interactions of ATGL and its co-lipase CGI-58 with perilipin 1 (perilipin A), perilipin 2 (adipose differentiation-related protein), and perilipin 5 (LSDP5) using multiple techniques as follows: anisotropy Forster resonance energy transfer, co-immunoprecipitation, 32Porthophosphate radiolabeling, and measurement of lipolysis. The results show that ATGL interacts with CGI-58 and perilipin 5; the latter is selectively expressed in oxidative tissues. Both proteins independently recruited ATGL to the LD surface, but with opposite effects; interaction of ATGL with CGI-58 increased lipolysis, whereas interaction of ATGL with perilipin 5 decreased lipolysis. In contrast, neither perilipin 1 nor 2 interacted directly with ATGL. Activation of protein kinase A (PKA) increased 32Porthophosphate incorporation into perilipin 5 by 2-fold, whereas neither ATGL nor CGI-58 was labeled under the incubation conditions. Cells expressing both ectopic perilipin 5 and ATGL showed a 3-fold increase in lipolysis following activation of PKA. Our studies establish perilipin 5 as a novel ATGL partner and provide evidence that the protein composition of perilipins at the LD surface regulates lipolytic activity of ATGL.
Lipolysis is an important metabolic pathway controlling energy homeostasis through degradation of triglycerides stored in lipid droplets and release of fatty acids. Lipid droplets of mammalian cells ...are coated with one or more members of the PAT protein family, which serve important functions in regulating lipolysis. In this study, we investigate the mechanisms by which PAT family members, perilipin A, adipose differentiation-related protein (ADFP), and LSDP5, control lipolysis catalyzed by hormone-sensitive lipase (HSL), a major lipase in adipocytes and several non-adipose cells. We applied fluorescence microscopic tools to analyze proteins in situ in cultured Chinese hamster ovary cells using fluorescence recovery after photobleaching and anisotropy Forster resonance energy transfer. Fluorescence recovery after photobleaching data show that ADFP and LSDP5 exchange between lipid droplet and cytoplasmic pools, whereas perilipin A does not. Differences in protein mobility do not correlate with PAT protein-mediated control of lipolysis catalyzed by HSL or endogenous lipases. Forster resonance energy transfer and co-immunoprecipitation experiments reveal that each of the three PAT proteins bind HSL through interaction of the lipase with amino acids within the highly conserved amino-terminal PAT-1 domain. ADFP and LSDP5 bind HSL under basal conditions, whereas phosphorylation of serine residues within three amino-terminal protein kinase A consensus sequences of perilipin A is required for HSL binding and maximal lipolysis. Finally, protein kinase A-mediated phosphorylation of HSL increases lipolysis in cells expressing ADFP or LSDP5; in contrast, phosphorylation of perilipin A exerts the major control over HSL-mediated lipolysis when perilipin is the main lipid droplet protein.
Collective cell migration (CCM) plays an important role in embryogenesis, vascular sprouting, and wound healing, but is also a significant driver of cancer metastasis. Ras homolog family member A ...(RhoA) facilitates CCM by modulating contraction of the actomyosin cytoskeleton. Given the precise spatiotemporal regulation of RhoA during CCM, we wanted to investigate the activity of downstream effector Rho‐associated kinase (ROCK) in collectively migrating fibroblasts. Using a new FRET‐based ROCK biosensor, we have observed calcium‐dependent activation of ROCK. We have previously demonstrated blunting of CCM in response to pharmacological inhibition of ROCK or depletion of intracellular calcium with 100 µM EGTA. To study the role of gap junction signaling in calcium‐dependent activation of ROCK during CCM, carbenoxolone, a gap junction blocker, was applied to collectively migrating cells following scratch wounding. Carbenoxolone treatment blunted CCM, as well as decreased ROCK activity and intracellular calcium levels at corresponding time points. Treatment with PQ7, a gap junction activator, caused modest increases in CCM. These results indicate that calcium signaling through gap junctions drives ROCK activity in the context of collective cell migration.
Cyan fluorescent proteins (CFPs), such as Cerulean, are widely used as donor fluorophores in Förster resonance energy transfer (FRET) experiments. Nonetheless, the most widely used variants suffer ...from drawbacks that include low quantum yields and unstable flurorescence. To improve the fluorescence properties of Cerulean, we used the X-ray structure to rationally target specific amino acids for optimization by site-directed mutagenesis. Optimization of residues in strands 7 and 8 of the β-barrel improved the quantum yield of Cerulean from 0.48 to 0.60. Further optimization by incorporating the wild-type T65S mutation in the chromophore improved the quantum yield to 0.87. This variant, mCerulean3, is 20% brighter and shows greatly reduced fluorescence photoswitching behavior compared to the recently described mTurquoise fluorescent protein in vitro and in living cells. The fluorescence lifetime of mCerulean3 also fits to a single exponential time constant, making mCerulean3 a suitable choice for fluorescence lifetime microscopy experiments. Furthermore, inclusion of mCerulean3 in a fusion protein with mVenus produced FRET ratios with less variance than mTurquoise-containing fusions in living cells. Thus, mCerulean3 is a bright, photostable cyan fluorescent protein which possesses several characteristics that are highly desirable for FRET experiments.
Pulses of insulin released from pancreatic β-cells maintain blood glucose in a narrow range, although the source of these pulses is unclear. We and others have proposed that positive feedback ...mediated by the glycolytic enzyme phosphofructokinase-1 (PFK1) enables β-cells to generate metabolic oscillations via autocatalytic activation by its product fructose 1,6-bisphosphate (FBP). Although much indirect evidence has accumulated in favor of this hypothesis, a direct measurement of oscillating glycolytic intermediates has been lacking. To probe glycolysis directly, we engineered a family of inter- and intramolecular FRET biosensors based on the glycolytic enzyme pyruvate kinase M2 (PKAR; pyruvate kinase activity reporter), which multimerizes and is activated upon binding FBP. When introduced into Min6 β-cells, PKAR FRET efficiency increased rapidly in response to glucose. Importantly, however, metabolites entering downstream of PFK1 (glyceraldehyde, pyruvate, and ketoisocaproate) failed to activate PKAR, consistent with sensor activation by FBP; the dependence of PKAR on FBP was further confirmed using purified sensor in vitro. Using a novel imaging modality for monitoring mitochondrial flavin fluorescence in mouse islets, we show that slow oscillations in mitochondrial redox potential stimulated by 10 mm glucose are in phase with glycolytic efflux through PKM2, measured simultaneously from neighboring islet β-cells expressing PKAR. These results indicate that PKM2 activity in β-cells is oscillatory and are consistent with pulsatile PFK1 being the mediator of slow glycolytic oscillations.
Background: Pyruvate kinase M2 controls glycolytic efflux.
Results: Novel FRET sensors report PKM2 structural changes in response to activation by metabolites and phosphorylation events in pancreatic β-cells.
Conclusion: PKM2 activity is oscillatory and synchronized between islet β-cells.
Significance: This approach is broadly applicable for measuring pyruvate kinase M2 activity in living cells.
Glucokinase (GCK) controls the rate of glucose metabolism in pancreatic β cells, and its activity is rate-limiting for insulin secretion. Posttranslational GCK activation can be stimulated through ...either G protein-coupled receptors or receptor tyrosine kinase signaling pathways, suggesting a common mechanism. Here we show that inhibiting Ca2+ release from the endoplasmic reticulum (ER) decouples GCK activation from receptor stimulation. Furthermore, pharmacological release of ER Ca2+ stimulates activation of a GCK optical biosensor and potentiates glucose metabolism, implicating rises in cytoplasmic Ca2+ as a critical regulatory mechanism. To explore the potential for glucose-stimulated GCK activation, the GCK biosensor was optimized using circularly permuted mCerulean3 proteins. This new sensor sensitively reports activation in response to insulin, glucagon-like peptide 1, and agents that raise cAMP levels. Transient, glucose-stimulated GCK activation was observed in βTC3 and MIN6 cells. An ER-localized channelrhodopsin was used to manipulate the cytoplasmic Ca2+ concentration in cells expressing the optimized FRET-GCK sensor. This permitted quantification of the relationship between cytoplasmic Ca2+ concentrations and GCK activation. Half-maximal activation of the FRET-GCK sensor was estimated to occur at ∼400 nm Ca2+. When expressed in islets, fluctuations in GCK activation were observed in response to glucose, and we estimated that posttranslational activation of GCK enhances glucose metabolism by ∼35%. These results suggest a mechanism for integrative control over GCK activation and, therefore, glucose metabolism and insulin secretion through regulation of cytoplasmic Ca2+ levels.
Basal‐like breast cancers typically correspond with increased enrichment of EpCAM‐/CD49f‐ cancer stem cells (CSC) and a propensity toward metastasis. However, the molecular mechanisms underlying ...these general characteristics are not well understood. To provide further insight concerning CSCs and their intrinsic metastatic mechanisms, we compared the 450K DNA methylation profile of EpCAM‐/CD49f‐ poor breast cancer cell lines to that of EpCAM‐/CD49f‐ enriched breast cancer cell lines. From our results, we were able to determine and highlight IL32 as a gene whose promoter is hypomethylated in EpCAM‐/CD49f‐ enriched cell lines. The hypomethylated IL32 promoter corresponded with increased IL32 expression in both cell lines and basal‐like breast cancer patients from The Cancer Genome Atlas (TCGA) database. Interestingly, increased IL32 expression preferentially occurred for the IL32‐beta transcript and corresponds with previous reports demonstrating that IL32‐beta is not secreted from the cell like other canonical interleukins and preferentially localizes to the mitochondria in breast cancer cells. Additionally, expression of the beta‐transcript could be suppressed when CSC‐enriched cells were treated with the BET‐bromodomain inhibitor JQ1. Because of this phenomenon, we sought to determine the effects of suppressing IL32 in the EpCAM‐/CD49f‐ enriched cell line SUM159PT via siRNA‐mediated knockdown and subsequent RNAseq differential expression analysis. From our results, we determined that transcripts involved in extracellular matrix (ECM) organization and collagen/integrin interaction were preferentially affected by IL32 silencing. Additionally, IL32 suppression decreased the invasiveness of SUM159PT based on an ECM‐matrix cell invasion assay. Collectively, our results reflect the notion that differential IL32 expression by promoter hypomethylation in breast CSCs plays a role in ECM remodeling for purposes of breast cancer cell invasion and metastasis.
Optogenetic effectors and sensors provide a novel real-time window into complex physiological processes, enabling determination of molecular signaling processes within functioning cellular networks. ...However, the combination of these optical tools in mice is made practical by construction of genetic lines that are optically compatible and genetically tractable. We present a new toolbox of 21 mouse lines with lineage-specific expression of optogenetic effectors and sensors for direct biallelic combination, avoiding the multiallelic requirement of Cre recombinase -mediated DNA recombination, focusing on models relevant for cardiovascular biology. Optogenetic effectors (11 lines) or Ca
sensors (10 lines) were selectively expressed in cardiac pacemaker cells, cardiomyocytes, vascular endothelial and smooth muscle cells, alveolar epithelial cells, lymphocytes, glia, and other cell types. Optogenetic effector and sensor function was demonstrated in numerous tissues. Arterial/arteriolar tone was modulated by optical activation of the second messengers InsP
(optoα1AR) and cAMP (optoß2AR), or Ca
-permeant membrane channels (CatCh2) in smooth muscle (
) and endothelium (
). Cardiac activation was separately controlled through activation of nodal/conducting cells or cardiac myocytes. We demonstrate combined effector and sensor function in biallelic mouse crosses: optical cardiac pacing and simultaneous cardiomyocyte Ca
imaging in
-CatCh2/
-GCaMP8 crosses. These experiments highlight the potential of these mice to explore cellular signaling in vivo, in complex tissue networks.
Classically, exit from the endoplasmic reticulum (ER) is rate-limiting for secretory protein trafficking because protein folding/assembly occurs there. In this study, we have exploited ...“hPro-CpepSfGFP,” a human proinsulin bearing “superfolder” green fluorescent C-peptide expressed in pancreatic β cells where it is processed to human insulin and CpepSfGFP. Remarkably, steady-state accumulation of hPro-CpepSfGFP and endogenous proinsulin is in the Golgi region, as if final stages of protein folding/assembly were occurring there. The Golgi regional distribution of proinsulin is dynamic, influenced by fasting/refeeding, and increased with β cell zinc deficiency. However, coexpression of ER-entrapped mutant proinsulin-C(A7)Y shifts the steady-state distribution of wild-type proinsulin to the ER. Endogenous proinsulin coprecipitates with hPro-CpepSfGFP and even more so with hProC(A7)Y-CpepSfGFP. Using Cerulean and Venus-tagged proinsulins, we find that both WT-WT and WT-mutant proinsulin pairs exhibit FRET. The data demonstrate that wild-type proinsulin dimerizes within the ER but accumulates at a poorly recognized slow step within the Golgi region, reflecting either slow kinetics of proinsulin hexamerization, steps in formation of nascent secretory granules, or other unknown molecular events. However, in the presence of ongoing misfolding of a subpopulation of proinsulin in β cells, the rate-limiting step in transport of the remaining proinsulin shifts to the ER.
Background: Proinsulin assembly is linked to its intracellular transport.
Results: Proinsulin self-associates in the endoplasmic reticulum but, surprisingly, accumulates at a rate-limiting transport step in the Golgi region.
Conclusion: Proinsulin transport is a dynamic process, and its perturbation may be measured under steady-state conditions.
Significance: Proinsulin distribution may be a useful tool to characterize proinsulin trafficking in disease states.
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