Total Internal Reflection Fluorescence Research Articles

305 Extracellular Vesicular mRNA and miRNA Biomarkers in Patients With Glioblastoma

INTRODUCTION: Glioblastoma (GBM) is a highly lethal primary brain cancer that is resistant to conventional immunotherapies. However, currently, there is a lack of reliable biomarkers for clinical practice. Extracellular vesicles (EVs) present in biofluids are a potential biomarker for GBM diagnosis. METHODS: EVs from cell culture medium, healthy donor and GBM patient serum were purified using tangential flow filtration (TFF). Morphology, size, and numbers of EVs were characterized by TEM and qNano, respectively. EVs from healthy donor and GBM patient serum were compared against other cancer EV samples using bulk mRNA and miRNA sequencing analysis. Selected mRNA and miRNA name are not mentioned due to patents being drafted. mRNA and miRNA expression in EVs were quantified by a fluorescent nucleic acid detection method using a biochip platform at a single-EV level. Fluorescence images were recorded using high-resolution total internal reflection fluorescence (TIRF) microscopy. RESULTS: From bulk mRNA and miRNA sequencing and bioinformatics analysis, three miRNA and two mRNA were selected as potential candidate biomarkers for GBM. Subsequently, EVs from the serum of GBM patients (n = 39) were evaluated through our biochip platform. mRNA and miRNA expression of these biomarkers were positive in single EVs, for which we detected colocalization of protein/RNAs or multiple RNAs in subpopulations of EVs. Our platform was benchmarked with a standard PCR method showing 100X more sensitivity. Moreover, levels of expression of the different RNAs were compared to a cohort of serum from healthy donors (n=20), and showing significant differences between the two groups. CONCLUSIONS: Our non-invasive biochip assay outperformed qRT-PCR for EV RNA detection. Selected mRNAs and miRNAs in subpopulations of EVs could be used as potential GBM biomarkers.

Abstract 193: Melanoma cytoskeletal dynamics, membrane remodeling, and motility are regulated by TRIM9

Abstract Cell shape change and motility are essential in metastasis and involve remodeling of the actin cytoskeleton, cell adhesions, and plasma membrane. How these cytoskeletal and membrane remodeling are regulated and coordinated remains largely unknown. We previously identified TRIM9 as a regulator of actin dynamics and exocytosis in developing neurons. Deletion of murine Trim9 impairs neuronal migration, netrin-induced axon turning, and axonal and dendritic branching, and increased exocytosis and filopodial stability. In addition, we have shown TRIM9 alters dynamics of the actin polymerase VASP at filopodia tips via non-degrative mono-ubiquitination. TRIM9 is expressed in other motile cells, but the non-neuronal role of TRIM9 remains unknown. TRIM9 was identified as a possible prognostic biomarker in melanoma and high expression correlates with low patient survival. Melanomas undergo phenotype switching, where three distinct phenotypes exist that are associated with differences in gene expression. The three phenotypes consist of a melanocytic proliferative state, a neural crest-like invasive state, an intermediate state. Single cell RNAseq (scRNASeq) data from patient-derived melanoma indicate TRIM9 is highly expressed in the proliferative melanocytic state, which correlates with poor prognosis. We hypothesize that TRIM9 coordinates actin dynamics, adhesion, and exocytosis in melanoma. We show that TRIM9 protein is enriched in several human melanoma lines. Here we examine the role of TRIM9 in regulation of focal adhesions, exocytosis, migration, and invasion in two of these lines. Genetic loss of TRIM9 increased random migration velocity but reduced directional persistence. Interestingly, we find that TRIM9 plays a role in promoting bleb-like morphology and inhibits the ability to durotax on shallow gradients on soft substrate. Fluorescence recovery after photobleaching (FRAP), Total internal reflection fluorescence microscopy (TIRF), and widefield microscopy revealed that loss of TRIM9 results in increased focal adhesions, cell size, and altered dynamics of focal adhesion proteins VASP, zyxin, and paxillin dynamics. In addition, TRIM9 knockout cells exhibit reduced filopodial length and density and altered filopodial localization of VASP. In-gel zymography indicates that TRIM9 knockout cells also display an increased degradative capacity. Current studies are investigating how loss of TRIM9 alters parameters of cell contractility, lamellipodia, invadopodia, and exocytosis to define the role of TRIM9 in melanoma motility. Together these findings suggest TRIM9 reduces adhesion and migration, increases proliferation and blebbing and may be an important regulator of phenotype switching in melanoma. Citation Format: Kimberly Lukasik, Jordan Brooks, Sam Redinbo, James Bear, Stephanie Gupton. Melanoma cytoskeletal dynamics, membrane remodeling, and motility are regulated by TRIM9 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 193.

Abstract 513: Intra-tubular damage is targeted by maytansinoids and rescued by NF1: Revisiting mechanism and biomarkers of an established ADC payload

Abstract There is great interest in the identification of biomarkers to guide development of antibody-drug conjugates (ADC). We previously showed that loss of Neurofibromatosis 1 (NF1), a gene frequently mutated across cancers, enhances the activity of DM1, the maytansinoid payload of T-DM1, through a novel function in regulating microtubule (MT) dynamics. Maytansinoids are puzzlingly more effective in cells (in the nanomolar range) vs in vitro (in the micromolar range). Since maytansinoids bind at the interface between tubulin dimers, they are thought to only bind soluble tubulin dimers or MT ends, which would suggest very few binding sites available for pharmacological interaction in vivo, at odds with data. Here we investigated the interaction of DM1 with NF1 and MTs, using cellular and reductionist in vitro systems. To measure in vivo MT dynamics, we transiently transfected the MT end-binding protein EB3-GFP and reconstructed MT trajectories by live-cell imaging. Upon DM1 treatment, KO cells showed a highly significant reduction in MT speed, demonstrating a direct role for NF1 on MT dynamics in cells. In turbidity-based tubulin polymerization assays, recombinant NF1 greatly accelerated polymerization, and completely rescued DM1-induced inhibition. Visual inspection of fluorescent MTs showed that NF1 induced significant MT bundling, a defining feature of many MT-associated proteins, which generates signal indistinguishable from true MT polymerization in turbidity assays. To follow the dynamics of individual microtubules, we applied Total Internal Reflection (TIRF) microscopy on glass-immobilized MTs. As expected, polymerization in the presence of NF1 led to a significant increase in MT dynamics (elongation speed, rescue and catastrophe rate). Expectedly, DM1 led to significant reduction in the fraction of elongating MTs and speed, but these defects were completely or partially rescued by NF1. Importantly, DM1 did not only lead to MT shortening (as proposed by the current model), but also to clear and frequent MT fracturing, indicating that the drug is not only engaging MT ends but also intra-tubular binding sites. This is consistent with recent models of MT formation which incorporate the frequent presence of areas of discontinuity or damage induced by mechanical stress, exposing intra-tubular DM1 binding sites. Interestingly, adding NF1 to DM1-treated MTs generated areas of de novo intra-tubular tubulin insertion, coincident with damaged sites, suggesting an entirely novel role for NF1 in MT repair.In conclusion, we provide evidence for a model in which maytansinoids bind not only to soluble tubulin dimers and MT ends, but also to intra-tubular damaged sites. Thus, the number of binding sites in cells would be proportional to MT damage, suggesting a mechanism for differential efficacy across tumor types and a potential avenue for combinatorial drug development. Citation Format: Eleonora Messuti, Bruno Achutti Duso, Alessia Castiglioni, Giulia Tini, Emanuele Bonetti, Giuseppe Ciossani, Silvia Monzani, Daria Khuntsariya, Zdeněk Lánský, Marcus Braun, Luigi Scietti, Luca Mazzarella. Intra-tubular damage is targeted by maytansinoids and rescued by NF1: Revisiting mechanism and biomarkers of an established ADC payload [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 513.

Freezing-mediated formation of supraproteins using depletion forces

HypothesisLong-acting formulations such as microparticles, injectable depots and implantable devices can realize spatiotemporally controlled delivery of protein drugs to extend their therapeutic in vivo half-lives. To efficiently encapsulate the protein drugs into such drug delivery systems, (sub)micron-sized protein particles are needed. The formation of micronized supraproteins can be induced through the synergistic combination of attractive depletion forces and freezing. The size of the supraproteins can be fine-tuned from submicron to several microns by adjusting the ice crystallization rate through the freeze-quench depth, which is set by the target temperature.MethodsSupraprotein micron structures were prepared from protein solutions under various conditions in the presence and absence of nonadsorbing polyethylene glycol. Scanning electron microscopy and dynamic light scattering were employed to determine the sizes of the supraproteins and real-time total internal reflection fluorescent microscopy was used to follow the supraprotein formation during freezing. The protein secondary structure was measured before and after micronization by circular dichroism. A phase diagram of a protein–polyethylene glycol mixture was theoretically predicted to investigate whether the depletion interaction can elucidate the phase behavior.FindingsMicronized protein supraparticles could be prepared in a controlled manner by rapid freeze-drying of aqueous mixtures of bovine serum albumin, horseradish peroxidase and lysozyme mixed with polyethylene glycol. Upon freezing, the temperature quench initiates a phase separation process which is reminiscent of spinodal decomposition. This demixing is subsequently arrested during droplet phase separation to form protein-rich microstructures. The final size of the generated protein microparticles is determined by a competition between phase separation and cooling rate, which can be controlled by target temperature. The experimental phase diagram of the aqueous protein–polyethylene glycol dispersion aligns with predictions from depletion theory for charged colloids and nonadsorbing polymers.

Arp2/3 complex- and formin-mediated actin cytoskeleton networks facilitate actin binding protein sorting in fission yeast

While it is well-established that F-actin networks with specific organizations and dynamics are tightly regulated by distinct sets of associated actin-binding proteins (ABPs), how ABPs self-sort to particular F-actin networks remains largely unclear. We report that actin assembly factors Arp2/3 complex and formin Cdc12 tune the association of ABPs fimbrin Fim1 and tropomyosin Cdc8 to different F-actin networks in fission yeast. Genetic and pharmacological disruption of F-actin networks revealed that Fim1 is preferentially directed to Arp2/3-complex mediated actin patches, whereas Cdc8 is preferentially targeted to formin Cdc12-mediated filaments in the contractile ring. To investigate the role of Arp2/3 complex- and formin Cdc12-mediated actin assembly, we used four-color TIRF microscopy to observe the in vitro reconstitution of ABP sorting with purified proteins. Fim1 or Cdc8 alone bind similarly well to filaments assembled by either assembly factor. However, in ‘competition’ reactions containing both actin assembly factors and both ABPs, ∼2.0-fold more Fim1 and ∼3.5-fold more Cdc8 accumulates on Arp2/3 complex branch points and formin Cdc12-assembled actin filaments, respectively. These findings indicate that F-actin assembly factors Arp2/3 complex and formin Cdc12 help facilitate the recruitment of specific ABPs, thereby tuning ABP sorting and subsequently establishing the identity of F-actin networks in fission yeast.

An advanced light scattering imaging model for total internal reflection microscopy considering a stratified medium

An advanced light scattering model for Total Internal Reflection Microscopy (TIRM) is presented. The model considers the specific TIRM geometry and deals with the scattering by an axisymmetric particle of arbitrary orientation placed in a stratified medium and the imaging of the scattered field. The scattered field is computed by truncating the scattered and internal field expansions and by using spherical and plane wave expansions for the free-space dyadic Green’s function. While the first expansion is valid outside a sphere enclosing the particle, the second one is valid outside the tangent planes bounding the particle from above and below. We demonstrate that in both cases, the results are the same, and thus, that the restrictive condition according to which the interface should not intersect the particle’s circumscribed sphere is not relevant. The image of the scattered field is computed by using the Debye diffraction integral and fast Fourier transform, while for a better reconstruction of the particle orientation, an image processing step consisting in a contour extraction and ellipse fitting is considered. The numerical simulations dealing with scattering by a prolate spheroid provide evidence of the remarkably sensitivity of the geometric parameters of the image ellipse to the particle orientation angles, as well as, of the integral response of the detector to the distance between the particle and the interface.

Deep Learning Promotes Profiling of Multiple miRNAs in Single Extracellular Vesicles for Cancer Diagnosis.

Extracellular vesicle microRNAs (EV miRNAs) are critical noninvasive biomarkers for early cancer diagnosis. However, accurate cancer diagnosis based on bulk analysis is hindered by the heterogeneity among EVs. Herein, we report an approach for profiling single-EV multi-miRNA signatures by combining total internal reflection fluorescence (TIRF) imaging with a deep learning (DL) algorithm for the first time. This innovative technique allows for the precise characterization of EV miRNAs at the single-vesicle level, overcoming the challenges posed by EV heterogeneity. TIRF with high resolution and a signal-to-noise ratio can simultaneously detect multi-miRNAs in situ in individual EVs. DL algorithm avoids complicated and inaccurate artificial feature extraction, achieving automated high-resolution image analysis. Using this approach, we reveal that the main variation of EVs from 5 cancer cells and normal plasma is the triple-positive EV subpopulation, and the classification accuracy of single triple-positive EVs from 6 sources can reach above 95%. In the clinical cohort, 20 patients (5 lung cancer, 5 breast cancer, 5 cervical cancer, and 5 colon cancer) and 5 healthy controls are predicted with an overall accuracy of 100%. This single-EV strategy provides new opportunities for exploring more specific EV biomarkers to achieve cancer diagnosis and classification.

TRPV4-dependent Ca2+ influx determines cholesterol dynamics at the plasma membrane

The activities of the transient receptor potential vanilloid 4 (TRPV4), a Ca2+-permeable nonselective cation channel, are controlled by its surrounding membrane lipids (e.g., cholesterol, phosphoinositides). The transmembrane region of TRPV4 contains a cholesterol recognition amino acid consensus (CRAC) motif and its inverted (CARC) motif located in the plasmalemmal cytosolic leaflet. TRPV4 localizes in caveolae, a bulb-shaped cholesterol-rich domain at the plasma membrane. Here, we visualized the spatiotemporal interactions between TRPV4 and cholesterol at the plasma membrane in living cells by dual-color single-molecule imaging using total internal reflection fluorescence microscopy. To this aim, we labeled cholesterol at the cytosolic leaflets of the plasma membrane using a cholesterol biosensor, D4H. Our single-molecule tracking analysis showed that the TRPV4 molecules colocalize with D4H-accessible cholesterol molecules mainly in the low fluidity membrane domains in which both molecules are highly clustered. Colocalization of TRPV4 and D4H-accessible cholesterol was observed both inside and outside of caveolae. Agonist-evoked TRPV4 activation remarkably decreased colocalization probability and association rate between TRPV4 and D4H-accessible cholesterol molecules. Interestingly, upon TRPV4 activation, the particle density of D4H-accessible cholesterol molecules was decreased and the D4H-accessible cholesterol molecules in the fast-diffusing state were increased at the plasma membrane. The introduction of skeletal dysplasia-associated R616Q mutation into the CRAC/CARC motif of TRPV4, which reduced the interaction with cholesterol clusters, could not alter the D4H-accessible cholesterol dynamics. Mechanistically, TRPV4-mediated Ca2+ influx and the C-terminal calmodulin-binding site of TRPV4 are essential for modulating the plasmalemmal D4H-accessible cholesterol dynamics. We propose that TRPV4 remodels its surrounding plasmalemmal environment by manipulating cholesterol dynamics through Ca2+ influx.

Comparison of the SMLM technique and the MSSR algorithm in confocal microscopy for super-resolved imaging of cellulose fibres.

Nowadays, the use of super-resolution microscopy (SRM) is increasing globally due to its potential application in several fields of life sciences. However, a detailed and comprehensive guide is necessary for understanding a single-frame image's resolution limit. This study was performed to provide information about the structural organisation of isolated cellulose fibres from garlic and agave wastes through fluorophore-based techniques and image analysis algorithms. Confocal microscopy provided overall information on the cellulose fibres' microstructure, while techniques such as total internal reflection fluorescence microscopy facilitated the study of the plant fibres' surface structures at a sub-micrometric scale. Furthermore, SIM and single-molecule localisation microscopy (SMLM) using the PALM reconstruction wizard can resolve the network of cellulose fibres at the nanometric level. In contrast, the mean shift super-resolution (MSSR) algorithm successfully determined nanometric structures from confocal microscopy images. Atomic force microscopy was used as a microscopy technique for measuring the size of the fibres. Similar fibre sizes to those evaluated with SIM and SMLM were found using the MSSR algorithm and AFM. However, the MSSR algorithm must be cautiously applied because the selection of thresholding parameters still depends on human visual perception. Therefore, this contribution provides a comparative study of SRM techniques and MSSR algorithm using cellulose fibres as reference material to evaluate the performance of a mathematical algorithm for image processing of bioimages at a nanometric scale. In addition, this work could act as a simple guide for improving the lateral resolution of single-frame fluorescence bioimages when SRM facilities are unavailable.

Real-time single-molecule imaging of CaMKII-calmodulin interactions

The binding of calcium/calmodulin (CAM) to calcium/calmodulin-dependent protein kinase II (CaMKII) initiates an ATP-driven cascade that triggers CaMKII autophosphorylation. The autophosphorylation in turn increases the CaMKII affinity for CAM. Here, we studied the ATP dependence of CAM association with the actin-binding CaMKIIβ isoform using single-molecule total internal reflection fluorescence microscopy. Rhodamine-CAM associations/dissociations to surface-immobilized Venus-CaMKIIβ were resolved with 0.5 s resolution from video records, batch-processed with a custom algorithm. CAM occupancy was determined simultaneously with spot-photobleaching measurement of CaMKII holoenzyme stoichiometry. We show the ATP-dependent increase of the CAM association requires dimer formation for both the α and β isoforms. The study of mutant β holoenzymes revealed that the ATP-dependent increase in CAM affinity results in two distinct states. The phosphorylation-defective (T287.306-307A) holoenzyme resides only in the low-affinity state. CAM association is further reduced in the T287A holoenzyme relative to T287.306-307A. In the absence of ATP, the affinity of CAM for the T287.306-307A mutant and the wild-type monomer are comparable. The affinity of the ATP-binding impaired (K43R) mutant is even weaker. In ATP, the K43R holoenzyme resides in the low-affinity state. The phosphomimetic mutant (T287D) resides only in a 1000-fold higher-affinity state, with mean CAM occupancy of more than half of the 14-mer holoenzyme stoichiometry in picomolar CAM. ATP promotes T287D holoenzyme disassembly but does not elevate CAM occupancy. Single Poisson distributions characterized the ATP-dependent CAM occupancy of mutant holoenzymes. In contrast, the CAM occupancy of the wild-type population had a two-state distribution with both low- and high-affinity states represented. The low-affinity state was the dominant state, a result different from published in vitro assays. Differences in assay conditions can alter the balance between activating and inhibitory autophosphorylation. Bound ATP could be sufficient for CaMKII structural function, while antagonistic autophosphorylations may tune CaMKII kinase-regulated action-potential frequency decoding in vivo.

Extracellular-Vesicle Catch-and-Release Isolation System Using a Net-Charge Invertible Curvature-Sensing Peptide.

Extracellular vesicles (EVs) carry various informative components, including signaling proteins, transcriptional regulators, lipids, and nucleic acids. These components are utilized for cell-cell communication between donor and recipient cells. EVs have shown great promise as pharmaceutical-targeting vesicles and have attracted the attention of researchers in the fields of biological and medical science because of their importance as diagnostic and prognostic markers. However, the isolation and purification of EVs from cell-cultured media remain challenging. Ultracentrifugation is the most widely used method, but it requires specialized and expensive equipment. In the present study, we proposed a novel methodology to isolate EVs using a simple and convenient method, i.e., an EV catch-and-release isolation system (EV-CaRiS) using a net-charge invertible curvature-sensing peptide (NIC). Curvature-sensing peptides recognize vesicles by binding to lipid-packing defects on highly curved membranes regardless of the expression levels of biomarkers. NIC was newly designed to reversibly capture and release EVs in a pH-dependent manner. NIC allowed us to achieve reproducible EV isolation from three human cell lines on resin using a batch method and single-particle imaging of EVs containing the ubiquitous exosome markers CD63 and CD81 by total internal reflection fluorescence microscopy (TIRFM). EV-CaRiS was demonstrated as a simple and convenient methodology for EV isolation, and NIC is promising for applications in the single-particle analysis of EVs.

Mosaic quadrivalent influenza vaccine single nanoparticle characterization

Recent work by our laboratory and others indicates that co-display of multiple antigens on protein-based nanoparticles may be key to induce cross-reactive antibodies that provide broad protection against disease. To reach the ultimate goal of a universal vaccine for seasonal influenza, a mosaic influenza nanoparticle vaccine (FluMos-v1) was developed for clinical trial (NCT04896086). FluMos-v1 is unique in that it is designed to co-display four recently circulating haemagglutinin (HA) strains; however, current vaccine analysis techniques are limited to nanoparticle population analysis, thus, are unable to determine the valency of an individual nanoparticle. For the first time, we demonstrate by total internal reflection fluorescence microscopy and supportive physical–chemical methods that the co-display of four antigens is indeed achieved in single nanoparticles. Additionally, we have determined percentages of multivalent (mosaic) nanoparticles with four, three, or two HA proteins. The integrated imaging and physicochemical methods we have developed for single nanoparticle multivalency will serve to further understand immunogenicity data from our current FluMos-v1 clinical trial.

Probing Activation and Conformational Dynamics of the Vesicle-Reconstituted β2 Adrenergic Receptor at the Single-Molecule Level.

G-protein-coupled receptors (GPCRs) are structurally flexible membrane proteins that mediate a host of physiological responses to extracellular ligands like hormones and neurotransmitters. Fine features of their dynamic structural behavior are hypothesized to encode the functional plasticity seen in GPCR activity, where ligands with different efficacies can direct the same receptor toward different signaling phenotypes. Although the number of GPCR crystal structures is increasing, the receptors are characterized by complex and poorly understood conformational landscapes. Therefore, we employed a fluorescence microscopy assay to monitor conformational dynamics of single β2 adrenergic receptors (β2ARs). To increase the biological relevance of our findings, we decided not to reconstitute the receptor in detergent micelles but rather lipid membranes as proteoliposomes. The conformational dynamics were monitored by changes in the intensity of an environmentally sensitive boron-dipyrromethene (BODIPY 493/503) fluorophore conjugated to an endogenous cysteine (located at the cytoplasmic end of the sixth transmembrane helix of the receptor). Using total internal reflection fluorescence microscopy (TIRFM) and a single small unilamellar liposome assay that we previously developed, we followed the real-time dynamic properties of hundreds of single β2ARs reconstituted in a native-like environment─lipid membranes. Our results showed that β2AR-BODIPY fluctuates between several states of different intensity on a time scale of seconds, compared to BODIPY-lipid conjugates that show almost entirely stable fluorescence emission in the absence and presence of the full agonist BI-167107. Agonist stimulation changes the β2AR dynamics, increasing the population of states with higher intensities and prolonging their durations, consistent with bulk experiments. The transition density plot demonstrates that β2AR-BODIPY, in the absence of the full agonist, interconverts between states of low and moderate intensity, while the full agonist renders transitions between moderate and high-intensity states more probable. This redistribution is consistent with a mechanism of conformational selection and is a promising first step toward characterizing the conformational dynamics of GPCRs embedded in a lipid bilayer.

Analysis of Poly(ethylene terephthalate) degradation kinetics of evolved IsPETase variants using a surface crowding model

Poly(ethylene terephthalate) (PET) is a major plastic polymer utilized in the single-use and textile industries. The discovery of PET-degrading enzymes (PETases) has led to an increased interest in the biological recycling of PET in addition to mechanical recycling. IsPETase from Ideonella sakaiensis is a candidate catalyst, but little is understood about its structure-function relationships with regards to PET degradation. To understand the effects of mutations on IsPETase productivity, we develop a directed evolution assay to identify mutations beneficial to PET film degradation at 30ºC. IsPETase also displays enzyme concentration-dependent inhibition effects, and surface crowding has been proposed as a causal phenomenon. Based on total internal reflectance fluorescence microscopy and adsorption experiments, IsPETase is likely experiencing crowded conditions on PET films. Molecular dynamics (MD) simulations of IsPETase variants reveal a decrease in active site flexibility in free enzymes and reduced probability of productive active site formation in substrate-bound enzymes under crowding. Hence, we develop a surface crowding model to analyze the biochemical effects of three hit mutations (T116P, S238N, S290P) that enhanced ambient temperature activity and/or thermostability. We find that T116P decreases susceptibility to crowding, resulting in higher PET degradation product accumulation despite no change in intrinsic catalytic rate. In conclusion, we show that a macromolecular crowding-based biochemical model can be used to analyze the effects of mutations on properties of PETases and that crowding behavior is a major property to be targeted for enzyme engineering for improved PET degradation.

Pre-fusion motion state determines the heterogeneity of membrane fusion dynamics for large dense-core vesicles.

In neuroendocrine cells, large dense-core vesicles (LDCVs) undergo highly regulated pre-fusion processes before releasing hormones via membrane fusion. Significant heterogeneity has been found for LDCV population based on the dynamics of membrane fusion. However, how the pre-fusion status impacts the heterogeneity of LDCVs still remains unclear. Hence, we explored pre-fusion determinants of heterogeneous membrane fusion procedure of LDCV subpopulations. We assessed the pre-fusion motion of two LDCV subpopulations with distinct membrane fusion dynamics individually, using total internal reflection fluorescence microscopy. These two subpopulations were isolated by blocking Rho GTPase-dependent actin reorganization using Clostridium difficile toxin B (ToxB), which selectively targets the fast fusion vesicle pool. We found that the fast fusion subpopulation was in an active motion mode prior to release, termed "active" LDCV pool, while vesicles from the slow fusion subpopulation were also moving but in a significantly more confined status, forming an "inert" pool. The depletion of the active pool by ToxB also eliminated fast fusion vesicles and was not rescued by pre-treatment with phorbol ester. A mild actin reorganization blocker, latrunculin A, that partially disrupted the active pool, only slightly attenuated the fast fusion subpopulation. The pre-fusion motion state of LDCVs also exhibits heterogeneity and dictates the heterogeneous fusion pore dynamics. Rearrangement of F-actin network mediates vesicle pre-fusion motion and subsequently determines the membrane fusion kinetics.

Unveiling PET Hydrolase Surface Dynamics through Fluorescence Microscopy.

PET hydrolases are an emerging class of enzymes that are being heavily researched for their use in bioprocessing polyethylene terephthalate (PET). While work has been done in studying the binding of PET oligomers to the active site of these enzymes, the dynamics of PET hydrolases binding to a bulk PET surface is an unexplored area. Here, methods were developed for total internal reflection fluorescence (TIRF) microscopy and fluorescence recovery after photobleaching (FRAP) microscopy to study the adsorption and desorption dynamics of these proteins onto a PET surface. TIRF microscopy was employed to measure both on and off rates of two of the most commonly studied PET hydrolases, PHL7 and LCC, on a PET surface. It was found that these proteins have a much slower off rates on the order of 10-3 s-1 , comparable to non-productive binding in enzymes such as cellulose. In combination with FRAP microscopy, a dynamic model is proposed in which adsorption and desorption dominates over lateral diffusion over the surface. The results of this study could have implications for the future engineering of PET hydrolases, either to target them to a PET surface or to modulate interaction with their substrate.

Retention of the RNA ends provides the molecular memory for maintaining the activation of the Csm complex.

The type III CRISPR-Cas effector complex Csm functions as a molecular Swiss army knife that provides multilevel defense against foreign nucleic acids. The coordinated action of three catalytic activities of the Csm complex enables simultaneous degradation of the invader's RNA transcripts, destruction of the template DNA and synthesis of signaling molecules (cyclic oligoadenylates cAn) that activate auxiliary proteins to reinforce CRISPR-Cas defense. Here, we employed single-molecule techniques to connect the kinetics of RNA binding, dissociation, and DNA hydrolysis by the Csm complex from Streptococcus thermophilus. Although single-stranded RNA is cleaved rapidly (within seconds), dual-color FCS experiments and single-molecule TIRF microscopy revealed that Csm remains bound to terminal RNA cleavage products with a half-life of over 1 hour while releasing the internal RNA fragments quickly. Using a continuous fluorescent DNA degradation assay, we observed that RNA-regulated single-stranded DNase activity decreases on a similar timescale. These findings suggest that after fast target RNA cleavage the terminal RNA cleavage products stay bound within the Csm complex, keeping the Cas10 subunit activated for DNA destruction. Additionally, we demonstrate that during Cas10 activation, the complex remains capable of RNA turnover, i.e. of ongoing degradation of target RNA.

Antibody surface mobility amplifies FcγR signaling via Arp2/3 during phagocytosis

We report herein that the anti-CD20 therapeutic antibody, rituximab, is rearranged into microclusters within the phagocytic synapse by macrophage Fcγ receptors (FcγR) during antibody-dependent cellular phagocytosis. These microclusters were observed to potently recruit Syk and to undergo rearrangements that were limited by the cytoskeleton of the target cell, with depolymerization of target-cell actin filaments leading to modest increases in phagocytic efficiency. Total internal reflection fluorescence analysis revealed that FcγR total phosphorylation, Syk phosphorylation, and Syk recruitment were enhanced when IgG-FcγR microclustering was enabled on fluid bilayers relative to immobile bilayers in a process that required Arp2/3. We conclude that on fluid surfaces, IgG-FcγR microclustering promotes signaling through Syk that is amplified by Arp2/3-driven actin rearrangements. Thus, the surface mobility of antigens bound by IgG shapes the signaling of FcγR with an unrecognized complexity beyond the zipper and trigger models of phagocytosis.

Modules for teaching and applying single-molecule total internal reflection fluorescence microscopy in an undergraduate lab course

Modules for teaching and applying single-molecule total internal reflection fluorescence microscopy in an undergraduate lab course

Quantifying tau-microtubule interactions with TIRF microscopy and FCS

Quantifying tau-microtubule interactions with TIRF microscopy and FCS