Resonance Energy Transfer Imaging Research Articles

Real-Time Analysis of Individual Ebola Virus Glycoproteins Reveals Pre-Fusion, Entry-Relevant Conformational Dynamics.

The Ebola virus (EBOV) envelope glycoprotein (GP) mediates the fusion of the virion membrane with the membrane of susceptible target cells during infection. While proteolytic cleavage of GP by endosomal cathepsins and binding of the cellular receptor Niemann-Pick C1 protein (NPC1) are essential steps for virus entry, the detailed mechanisms by which these events promote membrane fusion remain unknown. Here, we applied single-molecule Förster resonance energy transfer (smFRET) imaging to investigate the structural dynamics of the EBOV GP trimeric ectodomain, and the functional transmembrane protein on the surface of pseudovirions. We show that in both contexts, pre-fusion GP is dynamic and samples multiple conformations. Removal of the glycan cap and NPC1 binding shift the conformational equilibrium, suggesting stabilization of conformations relevant to viral fusion. Furthermore, several neutralizing antibodies enrich alternative conformational states. This suggests that these antibodies neutralize EBOV by restricting access to GP conformations relevant to fusion. This work demonstrates previously unobserved dynamics of pre-fusion EBOV GP and presents a platform with heightened sensitivity to conformational changes for the study of GP function and antibody-mediated neutralization.

Multiplexed non-invasive tumor imaging of glucose metabolism and receptor-ligand engagement using dark quencher FRET acceptor.

Rationale: Following an ever-increased focus on personalized medicine, there is a continuing need to develop preclinical molecular imaging modalities to guide the development and optimization of targeted therapies. Near-Infrared (NIR) Macroscopic Fluorescence Lifetime Förster Resonance Energy Transfer (MFLI-FRET) imaging offers a unique method to robustly quantify receptor-ligand engagement in live intact animals, which is critical to assess the delivery efficacy of therapeutics. However, to date, non-invasive imaging approaches that can simultaneously measure cellular drug delivery efficacy and metabolic response are lacking. A major challenge for the implementation of concurrent optical and MFLI-FRET in vivo whole-body preclinical imaging is the spectral crowding and cross-contamination between fluorescent probes.Methods: We report on a strategy that relies on a dark quencher enabling simultaneous assessment of receptor-ligand engagement and tumor metabolism in intact live mice. Several optical imaging approaches, such as in vitro NIR FLI microscopy (FLIM) and in vivo wide-field MFLI, were used to validate a novel donor-dark quencher FRET pair. IRDye 800CW 2-deoxyglucose (2-DG) imaging was multiplexed with MFLI-FRET of NIR-labeled transferrin FRET pair (Tf-AF700/Tf-QC-1) to monitor tumor metabolism and probe uptake in breast tumor xenografts in intact live nude mice. Immunohistochemistry was used to validate in vivo imaging results.Results: First, we establish that IRDye QC-1 (QC-1) is an effective NIR dark acceptor for the FRET-induced quenching of donor Alexa Fluor 700 (AF700). Second, we report on simultaneous in vivo imaging of the metabolic probe 2-DG and MFLI-FRET imaging of Tf-AF700/Tf-QC-1 uptake in tumors. Such multiplexed imaging revealed an inverse relationship between 2-DG uptake and Tf intracellular delivery, suggesting that 2-DG signal may predict the efficacy of intracellular targeted delivery.Conclusions: Overall, our methodology enables for the first time simultaneous non-invasive monitoring of intracellular drug delivery and metabolic response in preclinical studies.

Fluorometric visualization of mucin 1 glycans on cell surfaces based on rolling-mediated cascade amplification and CdTe quantum dots.

A rolling-mediated cascade (RMC) amplification strategy is described for improved visualization of profiling glycans of mucin 1 (MUC 1) on cell surfaces. CdTe quantum dots (QDs) are used as fluorescent labels. The RMC based amplification allows even distinct glycoforms of MUC1 to be visualized on the surface of MCF-7 cell via an amplified Förster resonance energy transfer (FRET) imaging strategy that works at excitation/emission wavelengths of 345/610nm. This is achieved by utilizing antibody against MUC1 modified with the fluorescent label 7-amino-4-methylcoumarin-3-acetic acid (AMCA) as the energy donor in FRET. The QDs (used to label surface glycans) act as acceptors. N-Azidoacetylgalactosamine-Acetylated (Ac4GalNAz) as a non-natural azido sugar, can be incorporated into the glycans of the cell surface, which can promote further labeling. The method has the advantage of only requiring a small amount of non-natural sugar to be introduced in metabolic glycan labeling since too much of an artificial sugar will interfere with the physiological functions of cells. Graphical abstract Schematic for theDNA rolling-mediated cascade (RMC)-assisted metabolic labeling of cell surface glycans by using CdTe quantum dots as labels and an intramolecular amplified FRET strategy for imaging glycans on a specific glycosylated protein, MUC1.

Lens-free Dual-color Fluorescent CMOS Image Sensor for F?rster Resonance Energy Transfer Imaging

Lens-free Dual-color Fluorescent CMOS Image Sensor for F?rster Resonance Energy Transfer Imaging

Fluorescence microscopy of biophysical protein dynamics in nanoporous hydrogels

Proteins within nanoporous hydrogels have important biotechnological applications in pharmaceutical purification, tissue engineering, water treatment, biosensors, and medical implants. Yet, oftentimes proteins that are functional in solution lose activity when in contact with soft, nanostructured, condensed phase materials due to perturbations in the folded state, conformation, diffusion, and adsorption dynamics of the protein by the material. Fluorescence microscopy experimentally measures the biophysical dynamics of proteins within hydrogels at the nanoscale and can overcome the limitations of conventional ensemble techniques. An explanation of the benefits of fluorescence is provided, and principles of fluorescence microscope instrumentation and analysis are discussed. Then several nanoscale fluorescence microscopies that image nanoscale protein dynamics within hydrogels are introduced. First, location-based super-resolution imaging resolves the adsorption kinetics of proteins to charged ligands within hydrogels used in pharmaceutical separations. Next, correlation-based super-resolution techniques image the heterogeneity of the nanoscale pore size of the hydrogels and the diffusion of analytes within the pores simultaneously. Finally, fluorescence resonance energy transfer imaging combined with temperature jump perturbations determines the folding and stability of a protein within hydrogels. A common finding with all three fluorescence microscopies is that heterogeneous nanoporous hydrogel materials cause variability of protein behavior dependent on gel sterics and/or interfacial electrostatic forces. Overall, in situ observations of proteins in hydrogels using fluorescence microscopies can inform and inspire soft nanomaterial design to improve the performance, shelf life, and cost of biomaterials.

The allosteric mechanism of substrate-specific transport in SLC6 is mediated by a volumetric sensor

Neurotransmitter:sodium symporters (NSSs) in the SLC6 family terminate neurotransmission by coupling the thermodynamically favorable transport of ions to the thermodynamically unfavorable transport of neurotransmitter back into presynaptic neurons. Results from many structural, functional, and computational studies on LeuT, a bacterial NSS homolog, have provided critical insight into the mechanism of sodium-coupled transport, but the mechanism underlying substrate-specific transport rates is still not understood. We present a combination of molecular dynamics simulations, single-molecule fluorescence resonance energy transfer (smFRET) imaging, and measurements of Na+ binding and substrate transport that reveals an allosteric substrate specificity mechanism. In this mechanism, residues F259 and I359 in the substrate binding pocket couple the binding of substrate to Na+ release from the Na2 site by allosterically modulating the stability of a partially open, inward-facing state. We propose a model for transport selectivity in which residues F259 and I359 act as a volumetric sensor that inhibits the transport of bulky amino acids.

Physiology in Perspective: The Dilemma of Muscle Weakness.

Physiology in Perspective: The Dilemma of Muscle Weakness.

Abstract 1305: Identification of signal transduction kinases inhibited by pan-RAF inhibitor belvarafenib using FRET imaging technique

Abstract The mitogen-activated protein kinase (MAPK) pathway plays an important role for the survival and proliferation of tumor cells. For example, the activation of the MAPK pathway due to mutations in BRAF, NRAS and KRAS kinases is known to be the causes of various solid tumors and hematologic malignancies. Accordingly, there is a strong need for effective therapeutic drugs for treating cancers caused by genetic mutation of BRAF, NRAS or KRAS. Belvarafenib (HM95573 or GDC5573), currently in Phase 1 clinical trials, is a selective and orally bioavailable pan-RAF kinase inhibitor. When biochemically assayed for hundreds of kinases, Belvarafenib showed selectivity toward RAF family kinases, including strong inhibition activities for BRAF WT, BRAF mutants and CRAF. Previously, we reported that Belvarafenib strongly inhibits the growth of cancer cell lines with BRAF, NRAS or KRAS mutations and also shows excellent anticancer efficacy in animal models using the corresponding cancer cells. In this study, employing fluorescence resonance energy transfer (FRET) imaging, we examined which protein kinases involved in signal transduction are regulated by Belvarafenib and investigated the mechanism of action for the regulation in typical cancer cells harboring BRAF, NRAS or KRAS mutations such as A375 (melamona), SK-MEL-30 (Kwmelamona), HCT116 (CRC), and Calu-6 (NSCLC). Among the various protein kinases that can be involved in signal transduction, we identified with time-lapse FRET imaging that the phosphorylation of ERK, AKT and S6K are inhibited after the treatment of Belvarafenib in the above cancer cells of BRAF, NRAS or KRAS mutations. Of note, BRAF mutant selective inhibitors, Vemurafenib and Dabrafenib were not active in cancer cells of NRAS or KRAS mutations. These results correlate with the in vitro and in vivo anticancer activity in cancer cells of BRAF, NRAS or KRAS mutations. In conclusion, unlike Vemurafenib and Dabrafenib, the pan-RAF inhibitor Belvarafenib could be effective for treating the cancer caused by NRAS or KRAS mutations, as well as BRAF mutations. Citation Format: Dong-Jun Bae, Sang-Yeob Kim, JooYun Byun, InHwan Bae, YoungGil Ahn, YoungHoon Kim, Kwee Hyun Suh. Identification of signal transduction kinases inhibited by pan-RAF inhibitor belvarafenib using FRET imaging technique [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1305.

Cyclic nucleotide signalling compartmentation by PDEs in cultured vascular smooth muscle cells.

Up-regulation of phosphodiesterases (PDEs) is associated with several vascular diseases, and better understanding of the roles of each PDE isoform in controlling subcellular pools of cyclic nucleotides in vascular cells is needed. We investigated the respective role of PDE1, PDE5, and PDE9 in controlling intracellular cAMP and/or cGMP concentrations ([cAMP]i , [cGMP]i ) in cultured rat aortic smooth muscle cells (RASMCs). We used selective inhibitors of PDE1 (PF-04471141), PDE5 (sildenafil), and PDE9 (PF-04447943) to measure cAMP- and cGMP-PDE activities with a radioenzymatic assay, in RASMC extracts. Real-time [cAMP]i and [cGMP]i were recorded by Förster resonance energy transfer-imaging in single living cells, and cell proliferation was assessed in FBS-stimulated cells. PDE1, PDE5, and PDE9 represented the major cGMP-hydrolyzing activity in RASMCs. Basal PDE1 exerted a functional role in degrading in situ the cGMP produced in response to activation of particulate GC by C-type natriuretic peptide. In high intracellular Ca2+ concentrations, PDE1 also regulated the NO/soluble GC-dependent cGMP response, as well as the β-adrenoceptor-mediated cAMP response. PDE5 exerted a major role in degrading cGMP produced by NO and the natriuretic peptides. PDE9 only regulated the NO-induced [cGMP]i increase. All three PDEs contributed differently to regulate cell proliferation under basal conditions and upon cGMP-elevating stimuli. Our data emphasize the distinct roles of PDE1, PDE5, and PDE9 in local regulation of [cAMP]i and [cGMP]i , in vascular smooth muscle cells, strengthening the concept of PDEs as key actors in the subcellular compartmentation of cyclic nucleotides.

Sulfonylurea Receptor 1, Transient Receptor Potential Cation Channel Subfamily M Member 4, and KIR6.2:Role in Hemorrhagic Progression of Contusion.

In severe traumatic brain injury (TBI), contusions often are worsened by contusion expansion or hemorrhagic progression of contusion (HPC), which may double the original contusion volume and worsen outcome. In humans and rodents with contusion-TBI, sulfonylurea receptor 1 (SUR1) is upregulated in microvessels and astrocytes, and in rodent models, blockade of SUR1 with glibenclamide reduces HPC. SUR1 does not function by itself, but must co-assemble with either KIR6.2 or transient receptor potential cation channel subfamily M member 4 (TRPM4) to form KATP (SUR1-KIR6.2) or SUR1-TRPM4 channels, with the two having opposite effects on membrane potential. Both KIR6.2 and TRPM4 are reportedly upregulated in TBI, especially in astrocytes, but the identity and function of SUR1-regulated channels post-TBI is unknown. Here, we analyzed human and rat brain tissues after contusion-TBI to characterize SUR1, TRPM4, and KIR6.2 expression, and in the rat model, to examine the effects on HPC of inhibiting expression of the three subunits using intravenous antisense oligodeoxynucleotides (AS-ODN). Glial fibrillary acidic protein (GFAP) immunoreactivity was used to operationally define core versus penumbral tissues. In humans and rats, GFAP-negative core tissues contained microvessels that expressed SUR1 and TRPM4, whereas GFAP-positive penumbral tissues contained astrocytes that expressed all three subunits. Förster resonance energy transfer imaging demonstrated SUR1-TRPM4 heteromers in endothelium, and SUR1-TRPM4 and SUR1-KIR6.2 heteromers in astrocytes. In rats, glibenclamide as well as AS-ODN targeting SUR1 and TRPM4, but not KIR6.2, reduced HPC at 24 h post-TBI. Our findings demonstrate upregulation of SUR1-TRPM4 and KATP after contusion-TBI, identify SUR1-TRPM4 as the primary molecular mechanism that accounts for HPC, and indicate that SUR1-TRPM4 is a crucial target of glibenclamide.

Oral Delivery of Puerarin Nanocrystals To Improve Brain Accumulation and Anti-Parkinsonian Efficacy.

Puerarin (PU) has emerged as a promising herb-derived anti-Parkinsonism compound. However, the undesirable water solubility as well as the unwanted bioavailability of PU limit its application. Therefore, this study aimed to develop and characterize PU nanocrystals (PU-NCs) with enhanced oral bioavailability and improved brain accumulation for the treatment of Parkinson's disease (PD). The fabricated PU-NCs were approximately spherical, with a mean size of 83.05 ± 1.96 nm, a PDI of 0.047 ± 0.009, a drug loading of 72.7%, and a rapid dissolution rate in vitro. Molecular dynamics simulation of PU and Pluronic F68 demonstrated the interaction energy and binding energy of -88.1 kJ/mol and -40.201 ± 0.685 kJ/mol, respectively, indicating a spontaneous binding with van der Waals interactions. In addition, the cellular uptake and permeability of PU-NCs were significantly enhanced as compared to PU alone ( p < 0.01). Moreover, PU-NCs exerted a significant neuroprotective effect against the cellular damage induced by the 1-methyl-4-phenylpyridinium ion (MPP+). Besides, PU-NCs demonstrated no obvious toxic effects on zebrafish, as evidenced by the unaltered morphology, hatching, survival rate, body length, and heart rate. Fluorescence resonance energy transfer (FRET) imaging revealed that intact nanocrystals were found in the intestine and brain of adult zebrafish gavaged with DiO/DiI/PU-NCs. Increased values of Cmax and AUC0- t were observed in the plasma of rats following oral administration of PU-NCs compared to PU suspension. Likewise, brain accumulation of PU-NCs was higher than that of PU suspension. Furthermore, PU-NCs attenuated dopamine depletion, ameliorated 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced behavioral deficits, and enhanced the levels of dopamine and its metabolites. Taken altogether, this study provides evidence that PU-NCs could be exploited as a potential oral delivery system to treat PD, by improving the poor bioavailability of PU and enhancing their delivery into the brain.

ERK Activity Imaging During Migration of Living Cells In Vitro and In Vivo.

Extracellular signal-regulated kinase (ERK) is a major downstream factor of the EGFR-RAS-RAF signalling pathway, and thus the role of ERK in cell growth has been widely examined. The development of biosensors based on fluorescent proteins has enabled us to measure ERK activities in living cells, both after growth factor stimulation and in its absence. Long-term imaging unexpectedly revealed the oscillative activation of ERK in an epithelial sheet or a cyst in vitro. Studies using transgenic mice expressing the ERK biosensor have revealed inhomogeneous ERK activities among various cell species. In vivo Förster (or fluorescence) resonance energy transfer (FRET) imaging shed light on a novel role of ERK in cell migration. Neutrophils and epithelial cells in various organs such as intestine, skin, lung and bladder showed spatio-temporally different cell dynamics and ERK activities. Experiments using inhibitors confirmed that ERK activities are required for various pathological responses, including epithelial repair after injuries, inflammation, and niche formation of cancer metastasis. In conclusion, biosensors for ERK will be powerful and valuable tools to investigate the roles of ERK in situ.

DNA Transformer for Visualizing Endogenous RNA Dynamics in Live Cells.

The functions of RNA are tightly regulated via diverse intracellular mechanisms. However, probing the complex dynamics of endogenous RNA in live cells is a challenging task. In the present study, a DNA transformer is designed for visualizing the abundance, distribution, and mobility of endogenous mRNAs in live human cells. The transformable tetrahedral DNA (T-TED) probe has a flexible hinge structure and is programmed to conform into a 3D tetrahedron upon binding with the target mRNA. By incorporating Förster resonance energy transfer (FRET) imaging, super-resolution localization, and single particle tracking, the T-TED biosensor is applied for investigating the dynamics of Delta-like ligand 4 (Dll4) mRNA, which encodes a transmembrane protein, in human pulmonary microvascular endothelial cells. The data reveal unprecedented subpopulations of Dll4 mRNA with distinct mobility organized spatially in association with the endoplasmic reticulum and microtubule networks. The ability to monitor the dynamics of endogenous RNA in live human cells will provide a useful tool for studying the functions and regulation of RNA.

Automated E-FRET microscope for dynamical live-cell FRET imaging.

Acceptor-sensitised 3-cube fluorescence resonance energy transfer (FRET) imaging (also termed as E-FRET imaging) is a popular fluorescence intensity-based FRET quantification method. Here, an automated E-FRET microscope with user-friendly interfaces was set up for dynamical online quantitative live-cell FRET imaging. This microscope reduces the time of a quantitative E-FRET imaging from 12 to 3 s. After locating cells, calibration of the microscope and E-FRET imaging of the cells can be performed automatically by clicking 'Capture' button on interfaces. E-FRET imaging was performed on the microscope for living cells expressing different FRET tandem constructs. Dynamical E-FRET imaging on the microscope for live cells coexpressing CFP-Bax and YFP-Bax treated by staurosporine (STS) revealed three Bax redistribution stages: Bax translocation from cytosol to mitochondria within 10 min, membrane insertion with conformational change on mitochondrial membrane within about 30 min, and subsequent oligomerisation within about 10 min. Because of excellent user-friendly interface and stability, the automated E-FRET microscope is a convenient tool for quantitative FRET imaging of living cell. LAY DESCRIPTION: Acceptor-sensitised 3-cube fluorescence resonance energy transfer (FRET) imaging (also termed as E-FRET) is a popular fluorescence intensity-based FRET quantification methods. E-FRET measurements are currently performed manually, and a complete FRET measurement takes about 12 s. E-FRET measurement necessitates not only a skilled operator and specialised equipment but also expertise in the interpretation of FRET signals, a considerable challenge in the application of FRET technology in living cells. Furthermore, manual E-FRET microscope is hard to perform dynamical quantitative FRET measurement, the ever-increasing applications in mapping the biochemical signal transduction within cells. Here, an automated E-FRET microscope with user-friendly interfaces was set up for dynamical online quantitative live-cell FRET imaging. This microscope reduces the time of a quantitative E-FRET imaging from 12 to 3 s. After locating cells, calibration of the microscope and E-FRET imaging of the cells can be performed automatically by clicking 'Capture' button on interfaces. Because of excellent user-friendly interface and stability, the automated E-FRET microscope is a convenient tool for quantitative FRET imaging of living cell.

Conformational Dynamics Related to Membrane Fusion Observed in Single Viral Envelope Glycoproteins

All enveloped viruses enter cells by fusing the viral membrane with a cellular membrane. This fusion event in promoted by the viral envelope glycoprotein. The current model of class-I viral membrane fusion describes a static “spring-loaded” fusion domain. Upon triggering by acidic pH or interaction with a receptor, the fusion domain undergoes a singular, irreversible conformational rearrangement. Atomic-resolution structural models of several envelope glycoproteins describe the endpoints of this dynamic event. Yet no direct visualization of these conformational dynamics has ever been made, leaving the current model of class-I-mediated membrane fusion unverified. Here, using single-molecule Förster resonance energy transfer (smFRET) imaging we directly visualized the conformational changes of individual envelope glycoprotein trimers on the surface of viral particles. Thus far, we have visualized the dynamics of influenza hemagglutinin (HA), the canonical class-I viral glycoprotein, and Ebola GP, which shares structural similarities with HA. This novel approach was facilitated by introduction of non-canonical amino acids into HA, which were subsequently labeled with fluorophores. In contrast to the current model of class-I-mediated membrane fusion, we observe spontaneous and reversible exchange between pre-fusion and intermediate conformations. Acidification of pH and interaction with receptors shift the dynamic equilibrium, favoring progression toward membrane fusion. These observations are leading to a new understanding of viral membrane fusion, and conserved and divergent features of distinct class-I viral envelope glycoproteins.

LRRK2 impairs PINK1/Parkin-dependent mitophagy via its kinase activity: pathologic insights into Parkinson's disease.

Mutations of LRRK2, encoding leucine-rich repeat kinase 2 (LRRK2), are the leading cause of autosomal dominant Parkinson's disease (PD). The most frequent of these mutations, G2019S substitution, increases kinase activity, but it remains unclear how it causes PD. Recent studies suggest that LRRK2 modulates mitochondrial homeostasis. Mitochondrial dysfunction plays a key role in the pathogenesis of autosomal recessive PD forms linked to PARK2 and PINK1, encoding the cytosolic E3 ubiquitin-protein ligase Parkin and the mitochondrial kinase PINK1, which jointly regulate mitophagy. We explored the role of LRRK2 and its kinase activity in PINK1/Parkin-dependent mitophagy. LRRK2 increased mitochondrial aggregation and attenuated mitochondrial clearance in cells coexpressing Parkin and exposed to the protonophore carbonylcyanide m-chlorophenylhydrazone. Förster resonance energy transfer imaging microscopy showed that LRRK2 impaired the interactions between Parkin and Drp1 and their mitochondrial targets early in mitophagy. The inhibition of LRRK2 kinase activity by a 'kinase-dead' LRRK2 mutation or with a pharmacological inhibitor (LRRK2-IN-1) restored these interactions. The monitoring of mitophagy in human primary fibroblasts with the novel dual-fluorescence mtRosella reporter and a new hypothermic shock paradigm revealed similar defects in PD patients with the G2019S LRRK2 substitution or PARK2 mutations relative to healthy subjects. This defect was restored by LRRK2-IN-1 treatment in LRRK2 patients only. Our results suggest that PD forms due to LRRK2 and PARK2 mutations involve pathogenic mechanisms converging on PINK1/Parkin-dependent mitophagy.

Illuminating the virus life cycle with single-molecule FRET imaging.

Single-molecule Förster resonance energy transfer (smFRET) imaging has emerged as a powerful tool to probe conformational dynamics of viral proteins, identify novel structural intermediates that are hiding in averaging population-based measurements, permit access to the energetics of transitions and as such to the precise molecular mechanisms of viral replication. One strength of smFRET is the capability of characterizing biological molecules in their fully hydrated/native state, which are not necessarily available to other structural methods. Elegant experimental design for physiologically relevant conditions, such as intact virions, has permitted the detection of previously unknown conformational states of viral glycoproteins, revealed asymmetric intermediates, and allowed access to the real-time imaging of conformational changes during viral fusion. As more laboratories are applying smFRET, our understanding of the molecular mechanisms and the dynamic nature of viral proteins throughout the virus life cycle are predicted to improve and assist the development of novel antiviral therapies and vaccine design.

Multifocal multiphoton volumetric imaging approach for high-speed time-resolved Förster resonance energy transfer imaging in vivo.

In this Letter, we will discuss the development of a multifocal multiphoton fluorescent lifetime imaging system where four individual fluorescent intensity and lifetime planes are acquired simultaneously, allowing us to obtain volumetric data without the need for sequential scanning at different axial depths. Using a phase-only spatial light modulator (SLM) with an appropriate algorithm to generate a holographic pattern, we project a beamlet array within a sample volume of a size, which can be preprogrammed by the user. We demonstrate the capabilities of the system to image live-cell interactions. While only four planes are shown, this technique can be rescaled to a large number of focal planes, enabling full 3D acquisition and reconstruction.

Rab5 activation on macropinosomes requires ALS2, and subsequent Rab5 inactivation through ALS2 detachment requires active Rab7.

Macropinocytosis is a nonspecific bulk uptake of extracellular fluid. During endosome maturation, the Rab5-to-Rab7 switch machinery executes the conversion from early to late endosomes. However, how the Rab switch works during macropinosome maturation remains unclear. Here, we elucidate the Rab switch machinery in macropinosome maturation using Förster resonance energy transfer imaging. Rab5 is activated and concurrently recruited to macropinosomes during ruffle closure. ALS2 depletion abolishes transient Rab5 activation on macropinosomes, while ALS2 is recruited to macropinosomes simultaneously with Rab5 activation. Thus, we conclude ALS2 activates Rab5 on macropinosomes. The absence of active Rab7 prolongs ALS2 presence and Rab5 activation on macropinosomes, indicating that active Rab7 is necessary for Rab5 inactivation through ALS2 dissociation and plays key roles in the Rab switch on macropinosomes.

Peak emission wavelength and fluorescence lifetime are coupled in far-red, GFP-like fluorescent proteins.

The discovery and use of fluorescent proteins revolutionized cell biology by allowing the visualization of proteins in living cells. Advances in fluorescent proteins, primarily through genetic engineering, have enabled more advanced analyses, including Förster resonance energy transfer (FRET) and fluorescence lifetime imaging microscopy (FLIM) and the development of genetically encoded fluorescent biosensors. These fluorescence protein-based sensors are highly effective in cells grown in monolayer cultures. However, it is often desirable to use more complex models including tissue explants, organoids, xenografts, and whole animals. These types of samples have poor light penetration owing to high scattering and absorption of light by tissue. Far-red light with a wavelength between 650-900nm is less prone to scatter, and absorption by tissues and can thus penetrate more deeply. Unfortunately, there are few fluorescent proteins in this region of the spectrum, and they have sub-optimal fluorescent properties including low brightness and short fluorescence lifetimes. Understanding the relationships between the amino-acid sequences of far-red fluorescence proteins and their photophysical properties including peak emission wavelengths and fluorescence lifetimes would be useful in the design of new fluorescence proteins for this region of the spectrum. We used both site-directed mutagenesis and gene-shuffling between mScarlet and mCardinal fluorescence proteins to create new variants and assess their properties systematically. We discovered that for far-red, GFP-like proteins the emission maxima and fluorescence lifetime have a strong inverse correlation.