Resonance Energy Transfer Microscopy Research Articles

Na+ and K+ Ions Differently Affect Nucleosome Structure, Stability, and Interactions with Proteins.

Inorganic ions are essential factors stabilizing nucleosome structure; however, many aspects of their effects on DNA transactions in chromatin remain unknown. Here, differential effects of K+ and Na+ on the nucleosome structure, stability, and interactions with protein complex FACT (FAcilitates Chromatin Transcription), poly(ADP-ribose) polymerase 1, and RNA polymerase II were studied using primarily single-particle Förster resonance energy transfer microscopy. The maximal stabilizing effect of K+ on a nucleosome structure was observed at ca. 80–150 mM, and it decreased slightly at 40 mM and considerably at >300 mM. The stabilizing effect of Na+ is noticeably lower than that of K+ and progressively decreases at ion concentrations higher than 40 mM. At 150 mM, Na+ ions support more efficient reorganization of nucleosome structure by poly(ADP-ribose) polymerase 1 and ATP-independent uncoiling of nucleosomal DNA by FACT as compared with K+ ions. In contrast, transcription through a nucleosome is nearly insensitive to K+ or Na+ environment. Taken together, the data indicate that K+ environment is more preserving for chromatin structure during various nucleosome transactions than Na+ environment.

Exploring the energy landscape of a SAM-I riboswitch

SAM-I riboswitches regulate gene expression through transcription termination upon binding a S-adenosyl-L-methionine (SAM) ligand. In previous work, we characterized the conformational energy landscape of the full-length Bacillus subtilis yitJ SAM-I riboswitch as a function of Mg2+ and SAM ligand concentrations. Here, we have extended this work with measurements on a structurally similar ligand, S-adenosyl-l-homocysteine (SAH), which has, however, a much lower binding affinity. Using single-molecule Förster resonance energy transfer (smFRET) microscopy and hidden Markov modeling (HMM) analysis, we identified major conformations and determined their fractional populations and dynamics. At high Mg2+ concentration, FRET analysis yielded four distinct conformations, which we assigned to two terminator and two antiterminator states. In the same solvent, but with SAM added at saturating concentrations, four states persisted, although their populations, lifetimes and interconversion dynamics changed. In the presence of SAH instead of SAM, HMM revealed again four well-populated states and, in addition, a weakly populated ‘hub’ state that appears to mediate conformational transitions between three of the other states. Our data show pronounced and specific effects of the SAM and SAH ligands on the RNA conformational energy landscape. Interestingly, both SAM and SAH shifted the fractional populations toward terminator folds, but only gradually, so the effect cannot explain the switching action. Instead, we propose that the noticeably accelerated dynamics of interconversion between terminator and antiterminator states upon SAM binding may be essential for control of transcription.

Single-Molecule Kinetic Studies of Nucleic Acids by Förster Resonance Energy Transfer.

Single-molecule microscopy is often used to observe and characterize the conformational dynamics of nucleic acids (NA). Due to the large variety of NA structures and the challenges specific to single-molecule observation techniques, the data recorded in such experiments must be processed via multiple statistical treatments to finally yield a reliable mechanistic view of the NA dynamics. In this chapter, we propose a comprehensive protocol to analyze single-molecule trajectories in the scope of single-molecule Förster resonance energy transfer (FRET) microscopy. The suggested protocol yields the conformational states common to all molecules in the investigated sample, together with the associated conformational transition kinetics. The given model resolves states that are indistinguishable by their observed FRET signals and is estimated with 95% confidence using error calculations on FRET states and transition rate constants. In the end, a step-by-step user guide is given to reproduce the protocol with the Multifunctional Analysis Software to Handle single-molecule FRET data (MASH-FRET).

Optical section structured illumination-based Förster resonance energy transfer imaging.

Förster resonance energy transfer (FRET) microscopy is an important tool suitable for studying molecular interactions in living cells. Optical section structured illumination microscopy (OS-SIM), like confocal microscopy, has about 200 nm spatial resolution. In this report, we performed quantitative 3-cube FRET imaging in OS-SIM mode and widefield microscopy (WF) mode, respectively, for living cells expressing FRET constructs consisting of Cerulean (C, donor) and Venus (V, acceptor). OS-SIM images exhibited higher resolution than WF images. Four spectral crosstalk coefficients measured under OS-SIM mode are consistent with those measured under WF mode. Similarly, the system calibration factors G and k measured under OS-SIM mode were consistent with those measured under WF mode. The measured FRET efficiency (E) values of C32V and C17V as well as C5V constructs, standard FRET plasmids, in living Hela cells were , and , and the measured acceptor-to-donor concentration ratios ( ) were , , and , consistent with the reported values. Collectively, our data demonstrates that OS-SIM can be integrated into FRET microscopy to build an OS-SIM-FRET with confocal microscopy-like resolution.

A topological switch in CFTR modulates channel activity and sensitivity to unfolding.

The cystic fibrosis transmembrane conductance regulator (CFTR) anion channel is essential to maintain fluid homeostasis in key organs. Functional impairment of CFTR due to mutations in the cftr gene leads to cystic fibrosis. Here, we show that the first nucleotide-binding domain (NBD1) of CFTR can spontaneously adopt an alternate conformation that departs from the canonical NBD fold previously observed. Crystallography reveals that this conformation involves a topological reorganization of NBD1. Single-molecule fluorescence resonance energy transfer microscopy shows that the equilibrium between the conformations is regulated by adenosine triphosphate binding. However, under destabilizing conditions, such as the disease-causing mutation F508del, this conformational flexibility enables unfolding of the β-subdomain. Our data indicate that, in wild-type CFTR, this conformational transition of NBD1 regulates channel function, but, in the presence of the F508del mutation, it allows domain misfolding and subsequent protein degradation. Our work provides a framework to design conformation-specific therapeutics to prevent noxious transitions.

Abstract P-9: Excess of Nhp6 over Spt16/Pob3 is Necessary for Efficient Nucleosome Unfolding by yFACT

Background: DNA accessibility in chromatin is important for proper gene expression and is regulated by multiple factors. One of them is histone chaperone FACT, which conducts large-scale ATP-independent nucleosome unfolding that increases the accessibility of nucleosomal DNA. FACT binding results in dramatic DNA uncoiling from nucleosome, occurs without apparent loss of histones, and proceeds via an ‘all-or-none’ mechanism, but the detailed mechanism of this process is still unknown. FACT-dependent nucleosome unfolding modulates the accessibility of nucleosomal DNA, and it is an important function of FACT in the processes of transcription, DNA replication, and repair in vivo. Methods: Nucleosome-positioning DNA sequences containing fluorescent labels (Cy3 and Cy5) at positions 13/91 or 35/112 from the nucleosome boundary were used. Nucleosomes were assembled by transfer of histone octamers from chicken chromatin to nucleosomal DNA during dialysis from 2M NaCl. After dialysis nucleosomes were gel purified and used at a final concentration of 0.5nM for spFRET measurements or at 10nM for EMSA analysis. For complex formation nucleosomes were incubated in the presence of Spt16/Pob3(0.13μM) and Nhp6(1.3μM) for 10 min at 30°C in buffer containing 17mM HEPES, 2mM Tris-HCl, 0.8mM Na3EDTA, 0.11mM 2-merсaptoethanol, 150mM KCl, 11mM NaCl, 1.1%glycerin, 12% sucrose. In spFRET microscopy analysis, the proximity ratio EPR from each nucleosome was calculated based on the intensity of the signals, corrected to the background and plotted as relative frequency distribution. Each plot was fitted as a sum of two Gaussians to describe two conformational states of nucleosomes. The fractions of nucleosomes in different states were estimated by calculating the surface of areas under the corresponding Gaussian peaks as a fraction of the total area of the plot. Results: Here we report the results of our analysis of nucleosome unfolding by yeast FACT (yFACT) at different ratios of Spt16/Pob3 and Nhp6 using single-particle Förster resonance energy transfer (spFRET) microscopy. Our analysis suggests that the optimal ratio of Spt16/Pob3 to Nhp6 for the most efficient FACT-dependent nucleosome unfolding is 1:10. Importantly, a mere increase in the concentration of FACT results in a decrease of the functional activity, suggesting that the formation of a functional complex having a certain stoichiometry of Spt16/Pob3 to Nhp6 is essential for efficient FACT-dependent nucleosome unfolding. Conclusion: We determined that a certain ratio of Spt16/Pob3 to Nhp6 is essential for efficient FACT-dependent nucleosome unfolding, suggesting the formation of a functional FACT: Nhp6 complex.

Role of VDAC Isoforms in Alpha-Synuclein Entry into Mitochondria

Parkinson's disease (PD) patients show loss of neurons, mitochondrial dysfunction, and the presence of Lewy bodies. Lewy bodies are composed of misfolded α-synuclein, which is a risk factor in both familial and sporadic PD. Studies have shown that α-synuclein can cause mitochondrial dysfunction and increase reactive oxygen species production. This has been attributed to the interaction of α-synuclein with complexes of the electron transport chain in the inner mitochondrial membrane, but it is not clear how α-synuclein crosses the outer membrane to enter the mitochondria. In neurons, α-synuclein has been shown to bind Voltage-Dependent Anion Channel (VDAC), an outer mitochondrial membrane protein that shuttles metabolites between the cytosol and the mitochondria. Also, single-channel electrophysiology experiments show that monomeric α-synuclein can transiently block and translocate through all VDAC isoforms. However, the three VDAC isoforms in mammals — VDAC1, VDAC2, and VDAC3 that form channels of almost identical conductance and anion selectivity —show distinctly different interaction with α-synuclein in experiments with reconstituted VDACs. To understand the mechanism of α-synuclein translocation into mitochondria in live cells, we performed Förster Resonance Energy Transfer (FRET) microscopy using Alexa488-α-synuclein and VDAC1, 2 or 3 -GFP. The change in FRET signal as α-synuclein enters VDAC pore and translocates through it was used to visualize α-synuclein entry into the mitochondria. The contribution of each isoform in α-synuclein translocation was studied by knocking down individual VDAC isoforms in live cells. Elucidating the mechanism of α-synuclein entry into mitochondria and, in particular, the role of each VDAC isoform in α-synuclein translocation is crucial for understanding of the impact of α-synuclein in mitochondrial dysfunction and the ways to ameliorate mitochondrial function at the early stages of neurodegeneration.

Direct coupling of oligomerization and oligomerization-driven endocytosis of the dopamine transporter to its conformational mechanics and activity

Dopamine transporter (DAT) mediates the reuptake of synaptically released dopamine, and thus controls the duration and intensity of dopamine neurotransmission. Mammalian DAT has been observed to form oligomers, although the mechanisms of oligomerization and its role in DAT activity and trafficking remain largely unknown. We discovered a series of small molecule compounds that stabilize trimers and induce high-order oligomers of DAT and concomitantly promote its clathrin-independent endocytosis. Using a combination of chemical cross-linking, fluorescence resonance energy transfer microscopy, antibody-uptake endocytosis assay, live-cell lattice light sheet microscopy, ligand binding and substrate transport kinetics analyses, and molecular modeling and simulations, we investigated molecular basis of DAT oligomerization and endocytosis induced by these compounds. Our study showed that small molecule–induced DAT oligomerization and endocytosis are favored by the inward-facing DAT conformation and involve interactions of four hydrophobic residues at the interface between transmembrane (TM) helices TM4 and TM9. Surprisingly, a corresponding quadruple DAT mutant displays altered dopamine transport kinetics and increased cocaine-analog binding. The latter is shown to originate from an increased preference for outward-facing conformation and inward-to-outward transition. Taken together, our results demonstrate a direct coupling between conformational dynamics of DAT, functional activity of the transporter, and its oligomerization leading to endocytosis. The high specificity of such coupling for DAT makes the TM4-9 hub a new target for pharmacological modulation of DAT activity and subcellular localization.

Co-localization and crosstalk between CD44 and RHAMM depend on hyaluronan presentation

CD44 and the receptor for hyaluronic acid-mediated motility (RHAMM) are the main hyaluronan (HA) receptors. They are commonly overexpressed in different cancers activating signaling pathways related to tumor progression, metastasis and chemoresistance. Besides their involvement in signal transduction via interaction with HA, currently, there is a little information about the possible crosstalk between CD44 and RHAMM and the role of HA in this process. In the present work, we used immunocytochemistry combined with Förster resonance energy transfer (FRET) microscopy and co-immunoprecipitation to elucidate the involvement of HA in CD44 and RHAMM expression, co-localization and crosstalk. We studied breast cancer cells lines with different degrees of invasiveness and expression of these receptors in the absence of exogenous HA and compared the data with the results obtained for cultures supplemented with either soluble HA or seeded on substrates with end-on immobilized HA. Our results demonstrated that cells response depends on the HA presentation: CD44/RHAMM complexation was upregulated in all cell lines upon interaction with immobilized HA, but not with its soluble form. Moreover, the results showed that the expression of both CD44 and RHAMM is regulated via interactions with HA indicating cell-specific feedback loop(s) in the signaling cascade.

Age-Dependent Maturation of iPSC-CMs Leads to the Enhanced Compartmentation of β2AR-cAMP Signalling.

The ability to differentiate induced-pluripotent stem cells into cardiomyocytes (iPSC-CMs) has opened up novel avenues for potential cardiac therapies. However, iPSC-CMs exhibit a range of somewhat immature functional properties. This study explored the development of the beta-adrenergic receptor (βAR) pathway, which is crucial in regulating contraction and signifying the health and maturity of myocytes. We explored the compartmentation of β2AR-signalling and phosphodiesterases (PDEs) in caveolae, as functional nanodomains supporting the mature phenotype. Förster Resonance Energy Transfer (FRET) microscopy was used to study the cyclic adenosine monophosphate (cAMP) levels in iPSC-CMs at day 30, 60, and 90 following βAR subtype-specific stimulation. Subsequently, the PDE2, PDE3, and PDE4 activity was investigated using specific inhibitors. Cells were treated with methyl-β-cyclodextrin (MβCD) to remove cholesterol as a method of decompartmentalising β2AR. As iPSC-CMs mature with a prolonged culture time, the caveolae density is increased, leading to a reduction in the overall cytoplasmic cAMP signal stimulated through β2AR (but not β1AR). Pan-phosphodiesterase inhibition or caveolae depletion leads to an increase in the β2AR-stimulated cytoplasmic cAMP. Moreover, with time in culture, the increase in the βAR-dependent cytoplasmic cAMP becomes more sensitive to cholesterol removal. The regulation of the β2AR response by PDE2 and 4 is similarly increased with the time in culture. We conclude that both the β2AR and PDEs are restricted to the caveolae nanodomains, and thereby exhibit a tighter spatial restriction over the cAMP signal in late-stage compared to early iPSC-CMs.

Abstract PR11: Understanding the role of myddosome dynamics in melanoma using live cell imaging

Abstract Melanoma is a complex and devastating disease that impacts patients all over the world. There are many factors that play a role in melanoma, and current studies have suggested TLR signaling involving IRAK4 can be a contributing element. Signal transduction by Toll-like receptors (TLR) was originally studied in the context of the innate immune response by recognizing pathogen-associated molecular patterns and triggering expansion of the immune cell population. Melanocytes express TLRs and can use this pathway to trigger cellular proliferation. In this study we demonstrate the importance of TLR signaling in melanoma using live cell imaging to track the dynamics of the myddosome. Activation of TLRs induces the recruitment of the death domain (DD) adaptor protein MyD88, which assembles into an oligomeric complex called the myddosome containing IRAK4. The myddosome complex mediates NF-κB activation leading to cell growth and survival. The sequential, multicomponent assembly of the myddosome and its transient nature provides a unique opportunity to study myddosome biology using live cell fluorescence resonance energy transfer (FRET) microscopy. Melanoma cells were engineered to stably express fluorescently tagged forms of MyD88 and IRAK4 to interrogate real-time myddosome dynamics using confocal microscopy to resolve individual complexes responding to TLR ligands and pyrrolopyrimidine IRAK4 inhibitors. Cells treated with LPS, a TLR activator, have more assembled myddosomes as read out by the FRET signal, while cells treated with IRAK4 inhibitors have fewer. I have also recapitulated this pattern by mutating MyD88 and found that the activated form of MyD88 leads to an 8-fold increase in the number of myddosomes, while the negative mutant prevents any FRET signal from being detected. Studies have shown that the majority of melanoma tumor samples have elevated levels of phosphorylated IRAK4, and our own findings suggest that inhibition of IRAK4, in combination with a topoisomerase inhibitor, increases the survival of mice after implantation of melanoma cells with a hazard ratio of 0.25. Additionally, human melanoma samples display a subcellular localization of active IRAK4 similar to cells stably expressing an active myddosome complex, while normal skin samples correspond to cells expressing a nonfunctional myddosome. Melanoma can progress rapidly and is known to develop resistance to current therapies; hence there is a great need to develop alternative therapeutic targets. The work presented here hopes to address this need by illuminating the role of the myddosome in melanoma and how targeting this complex, via IRAK4, presents an attractive therapeutic strategy. This abstract is also being presented as Poster B23. Citation Format: Bridget Kreger, Adam Hendricks. Understanding the role of myddosome dynamics in melanoma using live cell imaging [abstract]. In: Proceedings of the AACR Special Conference on Melanoma: From Biology to Target; 2019 Jan 15-18; Houston, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(19 Suppl):Abstract nr PR11.

Single-Chain Lanthanide Luminescence Biosensors for Cell-Based Imaging and Screening of Protein-Protein Interactions.

SummaryLanthanide-based, Förster resonance energy transfer (LRET) biosensors enabled sensitive, time-gated luminescence (TGL) imaging or multiwell plate analysis of protein-protein interactions (PPIs) in living cells. We prepared stable cell lines that expressed polypeptides composed of an alpha helical linker flanked by a Tb(III) complex-binding domain, GFP, and two interacting domains at each terminus. The PPIs examined included those between FKBP12 and the rapamycin-binding domain of m-Tor (FRB) and between p53 (1–92) and HDM2 (1–128). TGL microscopy revealed dramatic differences (>500%) in donor- or acceptor-denominated, Tb(III)-to-GFP LRET ratios between open (unbound) and closed (bound) states of the biosensors. We observed much larger signal changes (>2,500%) and Z′-factors of 0.5 or more when we grew cells in 96- or 384-well plates and analyzed PPI changes using a TGL plate reader. The modular design and exceptional dynamic range of lanthanide-based LRET biosensors will facilitate versatile imaging and cell-based screening of PPIs.

A mechanism for the extension and unfolding of parallel telomeric G-quadruplexes by human telomerase at single-molecule resolution.

Telomeric G-quadruplexes (G4) were long believed to form a protective structure at telomeres, preventing their extension by the ribonucleoprotein telomerase. Contrary to this belief, we have previously demonstrated that parallel-stranded conformations of telomeric G4 can be extended by human and ciliate telomerase. However, a mechanistic understanding of the interaction of telomerase with structured DNA remained elusive. Here, we use single-molecule fluorescence resonance energy transfer (smFRET) microscopy and bulk-phase enzymology to propose a mechanism for the resolution and extension of parallel G4 by telomerase. Binding is initiated by the RNA template of telomerase interacting with the G-quadruplex; nucleotide addition then proceeds to the end of the RNA template. It is only through the large conformational change of translocation following synthesis that the G-quadruplex structure is completely unfolded to a linear product. Surprisingly, parallel G4 stabilization with either small molecule ligands or by chemical modification does not always inhibit G4 unfolding and extension by telomerase. These data reveal that telomerase is a parallel G-quadruplex resolvase.

The type IV pilin PilA couples surface attachment and cell-cycle initiation in Caulobacter crescentus

Understanding how bacteria colonize surfaces and regulate cell-cycle progression in response to cellular adhesion is of fundamental importance. Here, we use transposon sequencing in conjunction with fluorescence resonance energy transfer (FRET) microscopy to uncover the molecular mechanism for how surface sensing drives cell-cycle initiation in Caulobacter crescentus We identify the type IV pilin protein PilA as the primary signaling input that couples surface contact to cell-cycle initiation via the second messenger cyclic di-GMP (c-di-GMP). Upon retraction of pili filaments, the monomeric pilin reservoir in the inner membrane is sensed by the 17-amino acid transmembrane helix of PilA to activate the PleC-PleD two-component signaling system, increase cellular c-di-GMP levels, and signal the onset of the cell cycle. We termed the PilA signaling sequence CIP for "cell-cycle initiating pilin" peptide. Addition of the chemically synthesized CIP peptide initiates cell-cycle progression and simultaneously inhibits surface attachment. The broad conservation of the type IV pili and their importance in pathogens for host colonization suggests that CIP peptide mimetics offer strategies to inhibit surface sensing, prevent biofilm formation and control persistent infections.

Molecular Mechanisms of PARP-1 Inhibitor 7-Methylguanine

7-Methylguanine (7-MG), a natural compound that inhibits DNA repair enzyme poly(ADP-ribose) polymerase 1 (PARP-1), can be considered as a potential anticancer drug candidate. Here we describe a study of 7-MG inhibition mechanism using molecular dynamics, fluorescence anisotropy and single-particle Förster resonance energy transfer (spFRET) microscopy approaches to elucidate intermolecular interactions between 7-MG, PARP-1 and nucleosomal DNA. It is shown that 7-MG competes with substrate NAD+ and its binding in the PARP-1 active site is mediated by hydrogen bonds and nonpolar interactions with the Gly863, Ala898, Ser904, and Tyr907 residues. 7-MG promotes formation of the PARP-1–nucleosome complexes and suppresses DNA-dependent PARP-1 automodification. This results in nonproductive trapping of PARP-1 on nucleosomes and likely prevents the removal of genotoxic DNA lesions.

Intrinsically Disordered Hs Associated Protein X-1 (HAX-1) Alters the Structure of the SERCA2A - Phospholamban Regulatory Complex

The anti-apoptotic protein HAX-1 has been proposed to modulate cardiac calcium handling by interacting with the sarcoendoplasmic reticulum calcium ATPase (SERCA) and its regulatory partner, phospholamban (PLB). HAX-1 is thought to enhance the inhibitory regulation of SERCA by phospholamban, but the mechanism of this functional effect is unknown. Here we used fluorescence resonance energy transfer (FRET) microscopy to quantify the effects of HAX-1 on the structure and affinity of the SERCA-PLB regulatory complex. In parallel experiments, HAX-1 was either coexpressed with Cer-SERCA and YFP-PLB in HEK cells, or recombinant HAX-1 was applied to saponin-permeabilized cells expressing Cer-SERCA and YFP-PLB. We observed that coexpression of HAX-1 significantly increased SERCA and PLB protein expression in HEK cells, consistent with its role as an anti-apoptotic factor. Despite this, we observed a modest decrease in FRET from SERCA to PLB, consistent with a small change in the quaternary structure of the regulatory complex. This effect was dose-dependent, with increasing expression of HAX-1 further decreasing intrinsic FRET efficiency from 31% toward a minimum of 26%. We did not observe a change in the binding affinity of PLB for SERCA, since HAX-1 did not significantly change the apparent dissociation constant (KD) of the regulatory complex. Overall, the data are consistent with previous NMR studies that suggested HAX-1 binds to the PLB cytoplasmic domain and increases its association with the surface of the bilayer membrane.

YWHA (14-3-3) protein isoforms and their interactions with CDC25B phosphatase in mouse oogenesis and oocyte maturation

BackgroundImmature mammalian oocytes are held arrested at prophase I of meiosis by an inhibitory phosphorylation of cyclin-dependent kinase 1 (CDK1). Release from this meiotic arrest and germinal vesicle breakdown is dependent on dephosphorylation of CDK1 by the protein, cell cycle division 25B (CDC25B). Evidence suggests that phosphorylated CDC25B is bound to YWHA (14-3-3) proteins in the cytoplasm of immature oocytes and is thus maintained in an inactive form. The importance of YWHA in meiosis demands additional studies.ResultsMessenger RNA for multiple isoforms of the YWHA protein family was detected in mouse oocytes and eggs. All seven mammalian YWHA isoforms previously reported to be expressed in mouse oocytes, were found to interact with CDC25B as evidenced by in situ proximity ligation assays. Interaction of YWHAH with CDC25B was indicated by Förster Resonance Energy Transfer (FRET) microscopy. Intracytoplasmic microinjection of oocytes with R18, a known, synthetic, non-isoform-specific, YWHA-blocking peptide promoted germinal vesicle breakdown. This suggests that inhibiting the interactions between YWHA proteins and their binding partners releases the oocyte from meiotic arrest. Microinjection of isoform-specific, translation-blocking morpholino oligonucleotides to knockdown or downregulate YWHA protein synthesis in oocytes suggested a role for a specific YWHA isoform in maintaining the meiotic arrest. More definitively however, and in contrast to the knockdown experiments, oocyte-specific and global deletion of two isoforms of YWHA, YWHAH (14-3-3 eta) or YWHAE (14-3-3 epsilon) indicated that the complete absence of either or both isoforms does not alter oocyte development and release from the meiotic prophase I arrest.ConclusionsMultiple isoforms of the YWHA protein are expressed in mouse oocytes and eggs and interact with the cell cycle protein CDC25B, but YWHAH and YWHAE isoforms are not essential for normal mouse oocyte maturation, fertilization and early embryonic development.

FRET Microscopy in Yeast.

Förster resonance energy transfer (FRET) microscopy is a powerful fluorescence microscopy method to study the nanoscale organization of multiprotein assemblies in vivo. Moreover, many biochemical and biophysical processes can be followed by employing sophisticated FRET biosensors directly in living cells. Here, we summarize existing FRET experiments and biosensors applied in yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, two important models of fundamental biomedical research and efficient platforms for analyses of bioactive molecules. We aim to provide a practical guide on suitable FRET techniques, fluorescent proteins, and experimental setups available for successful FRET experiments in yeasts.

Ca2+ Influx Channel Inhibitor SARAF Protects Mice From Acute Pancreatitis

Pancreatitis is characterized by increased influx of Ca2+ into acinar cells, by unknown mechanisms. Inhibitors of Ca2+ influx channels could be effective in treating acute pancreatitis, but these have deleterious side effects that can result in death. We investigated the expression patterns and functions of acinar cell Ca2+ channels and factors that regulate them during development of acute pancreatitis, along with changes in the channel inactivator store-operated calcium entry-associated regulatory factor (SARAF). We investigated whether SARAF is a target for treatment of acute pancreatitis and its status in human with pancreatitis. We generated mice that expressed SARAF tagged with hemagglutinin, using CRISPR/Cas9 gene editing, and isolated acinar cells. We also performed studies with Saraf-/- mice, Sarafzf/zf mice, mice without disruption of Saraf (control mice), and mice that overexpress fluorescently labeled SARAF in acinar cells. We analyzed interactions between stromal interaction molecule 1 (STIM1) and SARAF in HEK cells stimulated with carbachol using fluorescence resonance energy transfer microscopy and immunoprecipitation. Mice were given injections of caerulein or L-arginine to induce pancreatitis. Pancreatic tissues and blood samples were collected and levels of serum amylase, trypsin, tissue damage, inflammatory mediators, and inflammatory cells were measured. We performed quantitative polymerase chain reaction analyses of pancreatic tissues from 6 organ donors without pancreatic disease (controls) and 8 patients with alcohol-associated pancreatitis. Pancreatic levels of Ca2+ influx channels or STIM1 did not differ significantly between acinar cells from mice with vs. without pancreatitis. By contrast, pancreatic levels of Saraf messenger RNA and SARAF protein initially markedly increased but then decreased during cell stimulation or injection of mice with caerulein, resulting in excessive Ca2+ influx. STIM1 interacted stably with SARAF following stimulation of HEK or mouse acinar cells with physiologic levels of carbachol, but only transiently following stimulation with pathologic levels of carbachol, leading to excessive Ca2+ influx. We observed reduced levels of SARAF messenger RNA in pancreatic tissues from patients with pancreatitis, compared with controls. SARAF knockout mice developed more severe pancreatitis than control mice after administration of caerulein or L-arginine, and pancreatic acinar cells from these mice had significant increases in Ca2+ influx. Conversely, overexpression of SARAF in acini reduced Ca2+ influx, eliminated inflammation, and reduced severity of acute pancreatitis. In mice with pancreatitis, SARAF initially increases but is then degraded, resulting in excessive, pathological Ca2+ influx by acinar cells. SARAF knockout mice develop more severe pancreatitis than control mice, whereas mice that express SARAF from a transgene in acinar cells develop less-severe pancreatitis. SARAF therefore appears to prevent pancreatic damage during development of acute pancreatitis. Strategies to stabilize or restore SARAF to acinar cells might be developed for treatment of pancreatitis.

Super-Resolution Optical Lithography with DNA.

We developed an efficient, versatile, and accessible super-resolution microscopy method to construct a nanoparticle assembly at a spatial resolution below the optical diffraction limit. The method utilizes DNA and a photoactivated DNA cross-linker. Super-resolution optical techniques have been used only as a means to make measurements below the light diffraction limit. Furthermore, no optical technique is currently available to construct nanoparticle assemblies with a precisely designed shape and internal structure at a resolution of a few tens of nanometers (nm). Here we demonstrate that we can fulfill this deficiency by utilizing spontaneous structural dynamics of DNA hairpins combined with single-molecule fluorescence resonance energy transfer (smFRET) microscopy and a photoactivated DNA cross-linker. The stochastic fluorescence blinking due to the spontaneous folding and unfolding motions of DNA hairpins enables us to precisely localize a folded hairpin and solidify it only when it is within a predesigned target area whose size is below the diffraction limit. As the method is based on an optical microscope and an easily clickable DNA cross-linking reagent, it will provide an efficient means to create large nanoparticle assemblies with a shape and internal structure at an optical super-resolution, opening a wide window of opportunities toward investigating their photophysical and optoelectronic properties and developing novel devices.