Total Internal Reflection Fluorescence Research Articles

Imbalance of APOB Lipoproteins and Large HDL in Type 1 Diabetes Drives Atherosclerosis.

Individuals with type 1 diabetes (T1D) generally have normal or even higher HDL (high-density lipoprotein)-cholesterol levels than people without diabetes yet are at increased risk for atherosclerotic cardiovascular disease (CVD). Human HDL is a complex mixture of particles that can vary in cholesterol content by >2-fold. To investigate if specific HDL subspecies contribute to the increased atherosclerosis associated with T1D, we created mouse models of T1D that exhibit human-like HDL subspecies. We also measured HDL subspecies and their association with incident CVD in a cohort of people with T1D. We generated LDL receptor-deficient (Ldlr-/-) mouse models of T1D expressing human APOA1 (apolipoprotein A1). Ldlr-/-APOA1Tg mice exhibited the main human HDL subspecies. We also generated Ldlr-/-APOA1Tg T1D mice expressing CETP (cholesteryl ester transfer protein), which had lower concentrations of large HDL subspecies versus mice not expressing CETP. HDL particle concentrations and sizes and proteins involved in lipoprotein metabolism were measured by calibrated differential ion mobility analysis and targeted mass spectrometry in the mouse models of T1D and in a cohort of individuals with T1D. Endothelial transcytosis was analyzed by total internal reflection fluorescence microscopy. Diabetic Ldlr-/-APOA1Tg mice were severely hyperglycemic and hyperlipidemic and had markedly elevated plasma APOB levels versus nondiabetic littermates but were protected from the proatherogenic effects of diabetes. Diabetic Ldlr-/-APOA1Tg mice expressing CETP lost the atheroprotective effect and had increased lesion necrotic core areas and APOB accumulation, despite having lower plasma APOB levels. The detrimental effects of low concentrations of larger HDL particles in diabetic mice expressing CETP were not explained by reduced cholesterol efflux. Instead, large HDL was more effective than small HDL in preventing endothelial transcytosis of LDL mediated by scavenger receptor class B type 1. Finally, in humans with T1D, increased concentrations of larger HDL particles relative to APOB100 negatively predicted incident CVD independently of HDL-cholesterol levels. Our results suggest that the balance between APOB lipoproteins and the larger HDL subspecies contributes to atherosclerosis progression and incident CVD in the setting of T1D and that larger HDLs exert atheroprotective effects on endothelial cells rather than by promoting macrophage cholesterol efflux.

Quantification of membrane fluidity in bacteria using TIR-FCS

Plasma membrane fluidity is an important phenotypic feature that regulates the diffusion, function, and folding of transmembrane and membrane-associated proteins. In bacterial cells, variations in membrane fluidity are known to affect respiration, transport, and antibiotic resistance. Membrane fluidity must therefore be tightly regulated to adapt to environmental variations and stresses such as temperature fluctuations or osmotic shocks. Quantitative investigation of bacterial membrane fluidity has been, however, limited due to the lack of available tools, primarily due to the small size and membrane curvature of bacteria that preclude most conventional analysis methods used in eukaryotes. Here, we develop an assay based on total internal reflection-fluorescence correlation spectroscopy (TIR-FCS) to directly measure membrane fluidity in live bacteria via the diffusivity of fluorescent membrane markers. With simulations validated by experiments, we could determine how the small size, high curvature, and geometry of bacteria affect diffusion measurements and correct subsequent measurements for unbiased diffusion coefficient estimation. We used this assay to quantify the fluidity of the cytoplasmic membranes of the Gram-positive bacteria Bacillus subtilis (rod-shaped) and Staphylococcus aureus (coccus) at high (37°C) and low (20°C) temperatures in a steady state and in response to a cold shock, caused by a shift from high to low temperature. The steady-state fluidity was lower at 20°C than at 37°C, yet differed between B. subtilis and S. aureus at 37°C. Upon cold shock, the membrane fluidity decreased further below the steady-state fluidity at 20°C and recovered within 30 min in both bacterial species. Our minimally invasive assay opens up exciting perspectives for the study of a wide range of phenomena affecting the bacterial membrane, from disruption by chemicals or antibiotics to viral infection or change in nutrient availability.

Propafenone-induced connexin-43 protein accumulation and intracellular dynamics

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): China Scholarship Council Background Connexin-43 (Cx43) plays a pivotal role in intercellular communication through gap junctions. It is instrumental in propagating electrical signals and allowing ions to flow between neighboring cardiomyocytes. This synchronized electrical activity allows the heart to work as a functional syncytium. Any disruption or malfunction of Cx43 can lead to arrhythmias and other cardiac issues. Understanding the functions and regulation of Cx43 is vital in both basic research and clinical contexts. Propafenone, a class Ic antiarrhythmic drug, has shown promise in rhythm control; however, its precise impact on cardiac cellular physiology, particularly regarding Cx43, remains incompletely understood. Purpose To investigate propafenone’s effect on Cx43 expression, physiology, and underlying mechanisms in cell systems. Methods Used cell lines include human embryonic kidney HEK293 cells transfected with Cx43; differentiated murine embryonic carcinoma EPI7 cells with an epithelioid morphology and visceral endoderm-like END2 cells, endogenously expressing high amounts of functional Cx43. Cx43 protein expression levels were determined by Western blot analysis, whereas immunofluorescence (IF) imaged by a Total Internal Reflection Fluorescence (TIRF) microscope was used to assess the subcellular localization of Cx43 proteins. Dye injections were used to gain insight into the effects of propafenone on Cx43 metabolic coupling. Results and Conclusion Full length Cx43 protein levels were increased after treatment with propafenone and IF microscopy showed an intracellular accumulation of Cx43 protein, both on ectopically and endogenously expressed Cx43. Propafenone did not alter Cx43 half-life, in contrast to the lysosomal inhibitor chloroquine. Finally, gap-junctional coupling was decreased by chronic propafenone treatment. We conclude that the class 1c antiarrhythmic drug propanone increases Cx43 protein expression, resulting in its intracellular accumulation, as a side effect.

Zero-shot learning enables instant denoising and super-resolution in optical fluorescence microscopy.

Computational super-resolution methods, including conventional analytical algorithms and deep learning models, have substantially improved optical microscopy. Among them, supervised deep neural networks have demonstrated outstanding performance, however, demanding abundant high-quality training data, which are laborious and even impractical to acquire due to the high dynamics of living cells. Here, we develop zero-shot deconvolution networks (ZS-DeconvNet) that instantly enhance the resolution of microscope images by more than 1.5-fold over the diffraction limit with 10-fold lower fluorescence than ordinary super-resolution imaging conditions, in an unsupervised manner without the need for either ground truths or additional data acquisition. We demonstrate the versatile applicability of ZS-DeconvNet on multiple imaging modalities, including total internal reflection fluorescence microscopy, three-dimensional wide-field microscopy, confocal microscopy, two-photon microscopy, lattice light-sheet microscopy, and multimodal structured illumination microscopy, which enables multi-color, long-term, super-resolution 2D/3D imaging of subcellular bioprocesses from mitotic single cells to multicellular embryos of mouse and C. elegans.

Serotonin Promotes Vesicular Association and Fusion by Modifying Lipid Bilayers.

The primary event in chemical neurotransmission involves the fusion of a membrane-limited vesicle at the plasma membrane and the subsequent release of its chemical neurotransmitter cargo. The cargo itself is not known to have any effect on the fusion event. However, amphiphilic monoamine neurotransmitters (e.g., serotonin and dopamine) are known to strongly interact with lipid bilayers and to affect their mechanical properties, which can in principle impact membrane-mediated processes. Here, we probe whether serotonin can enhance the association and fusion of artificial lipid vesicles in vitro. We employ fluorescence correlation spectroscopy and total internal reflection fluorescence microscopy to measure the attachment and fusion of vesicles whose lipid compositions mimic the major lipid components of synaptic vesicles. We find that the association between vesicles and supported lipid bilayers is strongly enhanced in a serotonin dose-dependent manner, and this drives an increase in the rate of spontaneous fusion. Molecular dynamics simulations and fluorescence spectroscopy data show that serotonin insertion increases the water content of the hydrophobic part of the bilayer. This suggests that the enhanced membrane association is likely driven by an energetically favorable drying transition. Other monoamines, such as dopamine and norepinephrine, but not other related species, such as tryptophan, show similar effects on membrane association. Our results reveal a lipid bilayer-mediated mechanism by which monoamines can themselves modulate vesicle fusion, potentially adding to the control toolbox for the tightly regulated process of neurotransmission in vivo.

Monocytes release cystatin F dimer to associate with Aβ and aggravate amyloid pathology and cognitive deficits in Alzheimer’s disease

BackgroundUnderstanding the molecular mechanisms of Alzheimer’s disease (AD) has important clinical implications for guiding therapy. Impaired amyloid beta (Aβ) clearance is critical in the pathogenesis of sporadic AD, and blood monocytes play an important role in Aβ clearance in the periphery. However, the mechanism underlying the defective phagocytosis of Aβ by monocytes in AD remains unclear.MethodsInitially, we collected whole blood samples from sporadic AD patients and isolated the monocytes for RNA sequencing analysis. By establishing APP/PS1 transgenic model mice with monocyte-specific cystatin F overexpression, we assessed the influence of monocyte-derived cystatin F on AD development. We further used a nondenaturing gel to identify the structure of the secreted cystatin F in plasma. Flow cytometry, enzyme-linked immunosorbent assays and laser scanning confocal microscopy were used to analyse the internalization of Aβ by monocytes. Pull down assays, bimolecular fluorescence complementation assays and total internal reflection fluorescence microscopy were used to determine the interactions and potential interactional amino acids between the cystatin F protein and Aβ. Finally, the cystatin F protein was purified and injected via the tail vein into 5XFAD mice to assess AD pathology.ResultsOur results demonstrated that the expression of the cystatin F protein was specifically increased in the monocytes of AD patients. Monocyte-derived cystatin F increased Aβ deposition and exacerbated cognitive deficits in APP/PS1 mice. Furthermore, secreted cystatin F in the plasma of AD patients has a dimeric structure that is closely related to clinical signs of AD. Moreover, we noted that the cystatin F dimer blocks the phagocytosis of Aβ by monocytes. Mechanistically, the cystatin F dimer physically interacts with Aβ to inhibit its recognition and internalization by monocytes through certain amino acid interactions between the cystatin F dimer and Aβ. We found that high levels of the cystatin F dimer protein in blood contributed to amyloid pathology and cognitive deficits as a risk factor in 5XFAD mice.ConclusionsOur findings highlight that the cystatin F dimer plays a crucial role in regulating Aβ metabolism via its peripheral clearance pathway, providing us with a potential biomarker for diagnosis and potential target for therapeutic intervention.

Impact of temporal resolution in single particle tracking analysis

Temporal resolution is a key parameter in the observation of dynamic processes, as in the case of single molecules motions visualized in real time in two-dimensions by wide field (fluorescence) microscopy, but a systematic investigation of its effects in all the single particle tracking analysis steps is still lacking. Here we present tools to quantify its impact on the estimation of diffusivity and of its distribution using one of the most popular tracking software for biological applications on simulated data and movies. We found important shifts and different widths for diffusivity distributions, depending on the interplay of temporal sampling conditions with various parameters, such as simulated diffusivity, density of spots, signal-to-noise ratio, lengths of trajectories, and kind of boundaries in the simulation. We examined conditions starting from the ones of experiments on the fluorescently labelled receptor p75NTR, a relatively fast-diffusing membrane receptor (diffusivity around 0.5–1 µm2/s), visualized by TIRF microscopy on the basal membrane of living cells. From the analysis of the simulations, we identified the best conditions in cases similar to these ones; considering also the experiments, we could confirm a range of values of temporal resolution suitable for obtaining reliable diffusivity results. The procedure we present can be exploited in different single particle/molecule tracking applications to find an optimal temporal resolution.

Molecular dissection of PI3Kβ synergistic activation by receptor tyrosine kinases, GβGγ, and Rho-family GTPases.

Phosphoinositide 3-kinase (PI3K) beta (PI3Kβ) is functionally unique in the ability to integrate signals derived from receptor tyrosine kinases (RTKs), G-protein coupled receptors, and Rho-family GTPases. The mechanism by which PI3Kβ prioritizes interactions with various membrane-tethered signaling inputs, however, remains unclear. Previous experiments did not determine whether interactions with membrane-tethered proteins primarily control PI3Kβ localization versus directly modulate lipid kinase activity. To address this gap in our knowledge, we established an assay to directly visualize how three distinct protein interactions regulate PI3Kβ when presented to the kinase in a biologically relevant configuration on supported lipid bilayers. Using single molecule Total Internal Reflection Fluorescence (TIRF) Microscopy, we determined the mechanism controlling PI3Kβ membrane localization, prioritization of signaling inputs, and lipid kinase activation. We find that auto-inhibited PI3Kβ prioritizes interactions with RTK-derived tyrosine phosphorylated (pY) peptides before engaging either GβGγ or Rac1(GTP). Although pY peptides strongly localize PI3Kβ to membranes, stimulation of lipid kinase activity is modest. In the presence of either pY/GβGγ or pY/Rac1(GTP), PI3Kβ activity is dramatically enhanced beyond what can be explained by simply increasing membrane localization. Instead, PI3Kβ is synergistically activated by pY/GβGγ and pY/Rac1 (GTP) through a mechanism consistent with allosteric regulation.

Molecular dissection of PI3Kβ synergistic activation by receptor tyrosine kinases, GβGγ, and Rho-family GTPases

Phosphoinositide 3-kinase (PI3K) beta (PI3Kβ) is functionally unique in the ability to integrate signals derived from receptor tyrosine kinases (RTKs), G-protein coupled receptors, and Rho-family GTPases. The mechanism by which PI3Kβ prioritizes interactions with various membrane-tethered signaling inputs, however, remains unclear. Previous experiments did not determine whether interactions with membrane-tethered proteins primarily control PI3Kβ localization versus directly modulate lipid kinase activity. To address this gap in our knowledge, we established an assay to directly visualize how three distinct protein interactions regulate PI3Kβ when presented to the kinase in a biologically relevant configuration on supported lipid bilayers. Using single molecule Total Internal Reflection Fluorescence (TIRF) Microscopy, we determined the mechanism controlling PI3Kβ membrane localization, prioritization of signaling inputs, and lipid kinase activation. We find that auto-inhibited PI3Kβ prioritizes interactions with RTK-derived tyrosine phosphorylated (pY) peptides before engaging either GβGγ or Rac1(GTP). Although pY peptides strongly localize PI3Kβ to membranes, stimulation of lipid kinase activity is modest. In the presence of either pY/GβGγ or pY/Rac1(GTP), PI3Kβ activity is dramatically enhanced beyond what can be explained by simply increasing membrane localization. Instead, PI3Kβ is synergistically activated by pY/GβGγ and pY/Rac1 (GTP) through a mechanism consistent with allosteric regulation.

Prediction of the essential intermolecular contacts for side-binding of VASP on F-actin.

Vasodilator-stimulated phosphoprotein (VASP) family proteins play a crucial role in mediating the actin network architecture in the cytoskeleton. The Ena/VASP homology 2 (EVH2) domain in each of the four identical arms of the tetrameric VASP consists of a loading poly-Pro region, a G-actin-binding domain (GAB), and an F-actin-binding domain (FAB). Together, the poly-Pro, GAB, and FAB domains allow VASP to bind to sides of actin filaments in a bundle, and recruit profilin-G-actin to processively elongate the filaments. The atomic resolution structure of the ternary complex, consisting of the loading poly-Pro region and GAB domain of VASP with profilin-actin, has been solved over a decade ago; however, a detailed structure of the FAB-F-actin complex has not been resolved to date. Experimental insights, based on homology of the FAB domain with the C region of WASP, have been used to hypothesize that the FAB domain binds to the cleft between subdomains 1 and 3 of F-actin. Here, in order to develop our understanding of the VASP-actin complex, we first augment known structural information about the GAB domain binding to actin with the missing FAB domain-actin structure, which we predict using homology modeling and docking simulations. In earlier work, we used mutagenesis and kinetic modeling to study the role of domain-level binding-unbinding kinetics of Ena/VASP on actin filaments in a bundle, specifically on the side of actin filaments. We further look at the nature of the side-binding of the FAB domain of VASP at the atomistic level using our predicted structure, and tabulate effective mutation sites on the FAB domain that would disrupt the VASP-actin complex. We test the binding affinity of Ena with mutated FAB domain using total internal reflection fluorescence microscopy experiments. The binding affinity of VASP is affected significantly for the mutant, providing additional support for our predicted structure.

Effect of Particle Heterogeneity in Catalytic Copper-Containing Single-Chain Polymeric Nanoparticles Revealed by Single-Particle Kinetics.

Single-chain polymeric nanoparticles (SCPNs) have been extensively explored as a synthetic alternative to enzymes for catalytic applications. However, the inherent structural heterogeneity of SCPNs, arising from the dispersity of the polymer backbone and stochastic incorporation of different monomers as well as catalytic moieties, is expected to lead to variations in catalytic activity between individual particles. To understand the effect of structural heterogeneities on the catalytic performance of SCPNs, techniques are required that permit researchers to directly monitor SCPN activity at the single-polymer level. In this study, we introduce the use of single-molecule fluorescence microscopy to study the kinetics of Cu(I)-containing SCPNs towards depropargylation reactions. We developed Cu(I)-containing SCPNs that exhibit fast kinetics towards depropargylation and Cu-catalyzed azide-alkyne click reactions, making them suitable for single-particle kinetic studies. SCPNs were then immobilized on the surface of glass coverslips and the catalytic reactions were monitored at a single-particle level using total internal reflection fluorescence (TIRF) microscopy. Our studies revealed the interparticle turnover dispersity for Cu(I)-catalyzed depropargylations. In the future, our approach can be extended to different polymer designs which can give insights into the intrinsic heterogeneity of SCPN catalysis and can further aid in the rational development of SCPN-based catalysts.

Probing actin-activated ATP turnover kinetics of human cardiac myosin II by single molecule fluorescence.

Mechanistic insights into myosin II energy transduction in striated muscle in health and disease would benefit fromfunctional studies of a wide range of point-mutants. This approach is, however, hampered by the slow turnaround of myosin II expression that usually relies on adenoviruses for gene transfer. A recently developed virus-free method is more time effective but would yield too small amounts of myosin for standard biochemical analyses. However, if the fluorescent adenosine triphosphate (ATP) and single molecule (sm) total internal reflection fluorescence microscopy previously used to analyze basal ATP turnover by myosin alone, can be expanded to actin-activated ATP turnover, it would appreciably reduce the required amount of myosin. To that end, we here describe zero-length cross-linking of human cardiac myosin II motor fragments (sub-fragment 1 long [S1L]) to surface-immobilized actin filaments in a configuration with maintained actin-activated ATP turnover. After optimizing the analysis of sm fluorescence events, we show that the amount of myosin produced from C2C12 cells in one 60 mm cell culture plate is sufficient to obtain both the basal myosin ATP turnover rate and the maximum actin-activated rate constant (kcat). Our analysis of many single binding events of fluorescent ATP to many S1L motor fragments revealed processes reflecting basal and actin-activated ATPase, but also a third exponential process consistent with non-specific ATP-binding outside the active site.

Simultaneous TIRF imaging of subplasmalemmal Ca2+ dynamics and granule fusions in insulin-secreting INS-1 cells reveals coexistent synchronized and asynchronous release

The basal and glucose-induced insulin secretion from pancreatic beta cells is a tightly regulated process that is triggered in a Ca2+-dependent fashion and further positively modulated by substances that raise intracellular levels of adenosine 3′,5′-cyclic monophosphate (cAMP) or by certain antidiabetic drugs. In a previous study, we have temporally resolved the subplasmalemmal [Ca2+]i dynamics in beta cells that are characterized by trains of sharply delimited spikes, reaching peak values up to 5 µM. Applying total internal reflection fluorescence (TIRF) microscopy and synaptopHluorin to visualize fusion events of individual granules, we found that several fusion events can coincide within 50 to 150 ms. To test whether subplasmalemmal [Ca2+]i microdomains around single or clustered Ca2+ channels may cause a synchronized release of insulin-containing vesicles, we applied simultaneous dual-color TIRF microscopy and monitored Ca2+ fluctuations and exocytotic events in INS-1 cells at high frame rates. The results indicate that fusions can be triggered by subplasmalemmal Ca2+ spiking. This, however, does account for a minority of fusion events. About 90 %-95 % of fusion events either happen between Ca2+ spikes or incidentally overlap with subplasmalemmal Ca2+ spikes. We conclude that only a fraction of exocytotic events in glucose-induced and tolbutamide- or forskolin-enhanced insulin release from INS-1 cells is tightly coupled to Ca2+ microdomains around voltage-gated Ca2+ channels.

Mitotic spindle positioning protein (MISP) preferentially binds to aged F-actin

Actin bundling proteins crosslink filaments into polarized structures that shape and support membrane protrusions including filopodia, microvilli, and stereocilia. In the case of epithelial microvilli, mitotic spindle positioning protein (MISP) is an actin bundler that localizes specifically to the basal rootlets, where the pointed ends of core bundle filaments converge. Previous studies established that MISP is prevented from binding more distal segments of the core bundle by competition with other actin-binding proteins. Yet whether MISP holds a preference for binding directly to rootlet actin remains an open question. By immunostaining native intestinal tissue sections, we found that microvillar rootlets are decorated with the severing protein, cofilin, suggesting high levels of ADP-actin in these structures. Using total internal reflection fluorescence microscopy assays, we also found that purified MISP exhibits a binding preference for ADP- versus ADP-Pi-actin-containing filaments. Consistent with this, assays with actively growing actin filaments revealed that MISP binds at or near their pointed ends. Moreover, although substrate attached MISP assembles filament bundles in parallel and antiparallel configurations, in solution MISP assembles parallel bundles consisting of multiple filaments exhibiting uniform polarity. These discoveries highlight nucleotide state sensing as a mechanism for sorting actin bundlers along filaments and driving their accumulation near filament ends. Such localized binding might drive parallel bundle formation and/or locally modulate bundle mechanical properties in microvilli and related protrusions.

A colour-encoded nanometric ruler for axial super-resolution microscopies

Recent progress has boosted the resolving power of optical microscopies to spatial dimensions well below the diffraction limit. Yet, axial super-resolution and axial single-molecule localisation typically require more complicated implementations than their lateral counterparts. Also, comparison between techniques remains a major challenge due to the absence of suitable calibration tools that would permit a metrology along the microscope's optical axis. Likewise, such test samples would be useful as well for quantifying nanometric drift, or axial fluorophore mobility, or again for quantifying the sub-wavelength light confinement in near-field microscopies. In the present work, we propose a colour-encoded nanoscopic axial ruler. We provide a multi-layered single-excitation, dual-emission test slide, which translates the axial-distance problem into a spectral measurement. Our test slide combines, on a standard microscope coverslip substrate, two flat, thin, uniform and brightly emitting fluorophore layers, separated by a nanometric transparent spacer layer having a refractive index close to a biological cell. The ensemble is sealed in an index-matched protective polymer. As a proof-of-principle, we demonstrate the light confinement resulting from evanescent-wave excitation in total internal reflection fluorescence (TIRF) microscopy. Our test sample permits, even for the non-expert user, a facile axial metrology at the sub-100-nm scale, a critical requirement for axial super-resolution, as well as near-surface imaging, spectroscopy and sensing. (201 words).

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.