Optimizing extracellular vesicles' isolation from chronic lymphocytic leukemia patient plasma and cell line supernatant.

In chronic lymphocytic leukemia (CLL) and very likely all cancer types, extracellular vesicles (EVs) are a common mechanism by which intercellular messages are communicated between normal, diseased, and transformed cells. Studies of EVs in CLL and other cancers have great variability and often lack reproducibility. For CLL patient plasma and cell lines, we sought to characterize current approaches used in isolating EV products and understand whether cell culture-conditioned media or complex biological fluids confound results. Utilizing nanoparticle tracking analysis, protein quantification, and electron microscopy, we show that ultracentrifugation with an OptiPrep cushion can effectively minimize contaminants from starting materials including plasma and conditioned media of CLL cell lines grown in EV-depleted complete RPMI media but not grown in the serum-free media AIM V commonly used in CLL experimental work. Moreover, we confirm the benefit of including 25 mM trehalose in PBS during EV isolation steps to reduce EV aggregation, to preserve function for downstream applications and characterization. Furthermore, we report the highest particles/µg EVs were obtained from our CLL cell lines utilizing the CELLine bioreactor flask. Finally, we optimized a proliferation assay that offers a functional evaluation of our EVs with minimal sample requirements.

Unified platform for genetic and serological detection of COVID-19 with single-molecule technology.

The COVID-19 pandemic raises the need for diverse diagnostic approaches to rapidly detect different stages of viral infection. The flexible and quantitative nature of single-molecule imaging technology renders it optimal for development of new diagnostic tools. Here we present a proof-of-concept for a single-molecule based, enzyme-free assay for detection of SARS-CoV-2. The unified platform we developed allows direct detection of the viral genetic material from patients' samples, as well as their immune response consisting of IgG and IgM antibodies. Thus, it establishes a platform for diagnostics of COVID-19, which could also be adjusted to diagnose additional pathogens.

Temporal analysis of T-cell receptor-imposed forces via quantitative single molecule FRET measurements.

Mechanical forces acting on ligand-engaged T-cell receptors (TCRs) have previously been implicated in T-cell antigen recognition, yet their magnitude, spread, and temporal behavior are still poorly defined. We here report a FRET-based sensor equipped either with a TCR-reactive single chain antibody fragment or peptide-loaded MHC, the physiological TCR-ligand. The sensor was tethered to planar glass-supported lipid bilayers (SLBs) and informed most directly on the magnitude and kinetics of TCR-imposed forces at the single molecule level. When confronting T-cells with gel-phase SLBs we observed both prior and upon T-cell activation a single, well-resolvable force-peak of approximately 5 pN and force loading rates on the TCR of 1.5 pN per second. When facing fluid-phase SLBs instead, T-cells still exerted tensile forces yet of threefold reduced magnitude and only prior to but not upon activation.

Nanomechanics of G-quadruplexes within the promoter of the KIT oncogene.

G-quadruplexes (G4s) are tetrahelical DNA structures stabilized by four guanines paired via Hoogsteen hydrogen bonds into quartets. While their presence within eukaryotic DNA is known to play a key role in regulatory processes, their functional mechanisms are still under investigation. In the present work, we analysed the nanomechanical properties of three G4s present within the promoter of the KIT proto-oncogene from a single-molecule point of view through the use of magnetic tweezers (MTs). The study of DNA extension fluctuations under negative supercoiling allowed us to identify a characteristic fingerprint of G4 folding. We further analysed the energetic contribution of G4 to the double-strand denaturation process in the presence of negative supercoiling, and we observed a reduction in the energy required for strands separation.

Measuring Interactions Between Tau and Aggregation Inducers with Single-Molecule Förster Resonance Energy Transfer.

Tau is an intrinsically disordered protein implicated in the pathogenesis of Alzheimer's disease and other neurodegenerative disorders. Here we describe the application of single-molecule Förster resonance energy transfer (smFRET) for the characterization of the interactions between tau and polyphosphate, an intracellular polymer that accelerates tau aggregation. We describe the design of tau constructs, purification and fluorescent labeling of tau, and details of acquisition and analysis of smFRET data. The protocols provided here outline an approach that may be applied to the study of other intrinsically disordered proteins and their binding partners.

Single-Molecule Arrays for Ultrasensitive Detection of Blood-Based Biomarkers for Immunotherapy.

Single-molecule array (Simoa) technology enables ultrasensitive protein detection that is suited to the development of peripheral blood-based assays for assessing immuno-oncology responses. We adapted a panel of Simoa assays to measure systemic cytokine levels from plasma and characterized physiologic variation in healthy individuals and preanalytic variation arising from processing and handling of patient samples. Insights from these preclinical studies led us to a well-defined set of Simoa assay conditions, a specimen processing protocol, and a data processing approach that we describe here. Simoa enables accurate quantitation of soluble immune signaling molecules in an unprecedented femtomolar range, opening up the potential for liquid biopsy-type approaches in immuno-oncology. We are using the method described here to distinguish PD-1 inhibitor nonresponders as early as after one dose after therapy and envision applications in characterizing PD-1 inhibitor resistance and detection of immune-related adverse effects.

Transient Hybridization Directed Nanoflare for Single-Molecule miRNA Imaging.

Accurate quantifications of cellular miRNAs are important not only for accelerating them becoming reliable diagnostics biomarkers but also for deeply understanding their influence on central signaling pathways. Although single-molecule miRNA imaging permits quantifying biomolecules at the single-molecule level, it is limited by the sensitivity and specificity of hybridization-based probes. We report a miRNA single-molecule imaging method by using conjugated polymer nanoparticle (CPN) labeled short DNA probe termed as a nanoflare. The transient hybridization of the nanoflares and target miRNAs yields a featured single-molecule kinetics signal rendering high single-molecule sensitivity and specificity. miRNA can be detected with a remarkable detection limit of 1 fM without using any amplification steps. The discrimination capability of homologous miRNAs was also demonstrated. Taking advantage of the featured single-molecule signal of the nanoflare, we can directly count single miR-21 molecules in single cells by using highly inclined and laminated optical sheet (HILO) microscopy. The statistics of the counting reveals miR-21's cell-to-cell fluctuation and differential expression of tumor cells and normal cells.

Single-molecule localization microscopy and tracking with red-shifted states of conventional BODIPY conjugates in living cells.

Single-molecule localization microscopy (SMLM) is a rapidly evolving technique to resolve subcellular structures and single-molecule dynamics at the nanoscale. Here, we employ conventional BODIPY conjugates for live-cell SMLM via their previously reported red-shifted ground-state dimers (DII), which transiently form through bi-molecular encounters and emit bright single-molecule fluorescence. We employ the versatility of DII-state SMLM to resolve the nanoscopic spatial regulation and dynamics of single fatty acid analogs (FAas) and lipid droplets (LDs) in living yeast and mammalian cells with two colors. In fed cells, FAas localize to the endoplasmic reticulum and LDs of ~125 nm diameter. Upon fasting, however, FAas form dense, non-LD clusters of ~100 nm diameter at the plasma membrane and transition from free diffusion to confined immobilization. Our reported SMLM capability of conventional BODIPY conjugates is further demonstrated by imaging lysosomes in mammalian cells and enables simple and versatile live-cell imaging of sub-cellular structures at the nanoscale.

Temporal dependence of shifts in mu opioid receptor mobility at the cell surface after agonist binding observed by single-particle tracking.

Agonist binding to the mu opioid receptor (MOR) results in conformational changes that allow recruitment of G-proteins, activation of downstream effectors and eventual desensitization and internalization, all of which could affect receptor mobility. The present study employed single particle tracking (SPT) of quantum dot labeled FLAG-tagged MORs to examine shifts in MOR mobility after agonist binding. FLAG-MORs on the plasma membrane were in both mobile and immobile states under basal conditions. Activation of FLAG-MORs with DAMGO caused an acute increase in the fraction of mobile MORs, and free portions of mobile tracks were partially dependent on interactions with G-proteins. In contrast, 10-minute exposure to DAMGO or morphine increased the fraction of immobile FLAG-MORs. While the decrease in mobility with prolonged DAMGO exposure corresponded to an increase in colocalization with clathrin, the increase in colocalization was present in both mobile and immobile FLAG-MORs. Thus, no single mobility state of the receptor accounted for colocalization with clathrin. These findings demonstrate that SPT can be used to track agonist-dependent changes in MOR mobility over time, but that the mobility states observed likely arise from a diverse set of interactions and will be most informative when examined in concert with particular downstream effectors.

Single-molecule detection of biomarker and localized cellular photothermal therapy using an optical microfiber with nanointerface.

For early-stage diagnostics, there is a strong demand for sensors that can rapidly detect biomarkers at ultralow concentration or even at the single-molecule level. Compared with other types of sensors, optical microfibers are more convenient for use as point-of-care devices in early-stage diagnostics. However, the relatively low sensitivity strongly hinders their use. To this end, an optical microfiber is functionalized with a plasmonic nanointerface consisting of black phosphorus-supported Au nanohybrids. The microfiber is able to detect epidermal growth factor receptor (ErbB2) at concentrations ranging from 10 zM to 100 nM, with a detection limit of 6.72 zM, enabling detection at the single-molecule level. The nanointerface-sensitized microfiber is capable of differentiating cancer cells from normal cells and treating cancer cells through cellular photothermal therapy. This work opens up a possible approach for the integration of cellular diagnosis and treatment.

Determining the Impact of Metabolic Nutrients on Autophagy.

Tumorigenesis relies on the ability of cancer cells to obtain necessary nutrients and fulfill increased energy demands associated with rapid proliferation. However, as a result of increased metabolite consumption and poor vascularization, most cancer cells must survive in a nutrient poor and high cellular stress microenvironment. Cancer cells undergo metabolic reprogramming to evade cell death and ensure proliferation; in particular, cancer cells utilize the catabolic process of autophagy. Autophagy creates an intracellular pool of metabolites by sequestering cytosolic macromolecules in double-membrane vesicles targeted for lysosomal degradation. During times of environmental stress and nutrient starvation, autophagy is upregulated through the dynamic interactions between two nutrient sensing proteins, AMP activated protein kinase (AMPK) and mechanistic target of rapamycin (mTOR), in cooperation with Unc-51 like autophagy activating kinase 1 (ULK1). In this way, a lack of metabolic nutrients plays a critical role in inducing autophagy, while the products of autophagy also serve as readily available fuel for the cell. In this chapter, we describe methods to visualize and quantify autophagy using a fluorescent sensor of autophagic membranes. Thus, the impact of specific nutrients on autophagy can be measured using live-cell fluorescent microscopy.

Quantifying Intramolecular Protein Conformational Dynamics Under Lipid Interaction Using smFRET and FCCS.

FÓ§rster-type resonance energy transfer (FRET) with fluorescence cross-correlation spectroscopy (FCCS) is a powerful combination for observing intramolecular conformational dynamics on the micro- to millisecond timescale. Owing to its sensitivity to various physical parameters, FRET-FCCS has also been used to detect the reagent effects on proteins dynamics. However, FRET-FCCS alone cannot acquire the exact measurements of rate constants. Moreover, this technique is highly model dependent and can be unreliable when determining too many parameters at once. On the contrary, single-molecular FRET (smFRET) can measure the conformational states and their populations directly, although it is extremely challenging for probing fast dynamics under 1 ms. In this chapter, we describe how to realize sub-millisecond conformational dynamics measurements of a SNARE protein Ykt6 under lipid environments by smFRET and FRET-FCCS. This protocol includes sample preparation, microscope designs, data acquisition, and analysis methodology.

Defining Dynamics of Membrane-Bound Pyrophosphatases by Experimental and Computational Single-Molecule FRET.

Membrane-bound pyrophosphatases couple the hydrolysis of inorganic pyrophosphate to the pumping of ions (sodium or protons) across a membrane in order to generate an electrochemical gradient. This class of membrane protein is widely conserved across plants, fungi, archaea, and bacteria, but absent in multicellular animals, making them a viable target for drug design against protozoan parasites such as Plasmodium falciparum. An excellent understanding of many of the catalytic states throughout the enzymatic cycle has already been afforded by crystallography. However, the dynamics and kinetics of the catalytic cycle between these static snapshots remain to be elucidated. Here, we employ single-molecule Förster resonance energy transfer (FRET) measurements to determine the dynamic range and frequency of conformations available to the enzyme in a lipid bilayer during the catalytic cycle. First, we explore issues related to the introduction of fluorescent dyes by cysteine mutagenesis; we discuss the importance of residue selection for dye attachment, and the balance between mutating areas of the protein that will provide useful dynamics while not altering highly conserved residues that could disrupt protein function. To complement and guide the experiments, we used all-atom molecular dynamics simulations and computational methods to estimate FRET efficiency distributions for dye pairs at different sites in different protein conformational states. We present preliminary single-molecule FRET data that points to insights about the binding modes of different membrane-bound pyrophosphatase substrates and inhibitors.

Direct Quantitative Monitoring of Homology-Directed DNA Repair of Damaged Telomeres.

Homology-directed DNA repair (HDR) is an evolutionary conserved mechanism that is required for genome integrity and organismal fitness across species. While a myriad of different factors and mechanisms are able to execute HDR, all forms necessitate common steps of DNA damage recognition, homology search and capture, and assembly of a DNA polymerase complex to conduct templated DNA synthesis. The central question of what determines HDR mechanism utilization in mammalian cells has been limited by an inability to directly monitor the DNA damage response and products of repair as they arise from a defined genomic lesion. In this chapter, we describe several methodologies to delineate major steps of HDR during alternative lengthening of telomeres in human cells. This includes procedures to visualize interchromosomal telomere homology searches in real time and quantitatively detect HDR synthesis of nascent telomeres emanating from synchronous activation of telomere DNA double-strand breaks. We highlight the critical details of these methods and their applicability to monitoring HDR at telomeres in a broad variety of mammalian cell types.

TIRF-Based Single-Molecule Detection of the RecA Presynaptic Filament Dynamics.

RecA is a key protein in homologous DNA repair process. On a single-stranded (ss) DNA, which appears as an intermediate structure at a double-strand break site, RecA forms a kilobase-long presynaptic filament that mediates homology search and strand exchange reaction. RecA requires adenosine triphosphate as a cofactor that confers dynamic features to the filament such as nucleation, end-dependent growth and disassembly, scaffold shift along the ssDNA, and conformational change. Due to the complexity of the dynamics, detailed molecular mechanisms of functioning presynaptic filament have been characterized only recently after the advent of single-molecule techniques that allowed real-time observation of each kinetic process. In this chapter, single-molecule fluorescence resonance energy transfer assays, which revealed detailed molecular pictures of the presynaptic filament dynamics, will be discussed.

Probing Dynamic Assembly and Disassembly of Rad51 Tuned by Srs2 Using smFRET.

The integrity of DNA is critical for sustaining the life of any living organism, as DNA is a reservoir of its genetic information. However, DNA is continuously damaged by either normal metabolic pathways or environmental insults such as ultraviolet exposure or chemicals. Double-stranded DNA break is one of the most common types of DNA damage that requires activation of homologous recombination (HR) pathway mediated by Rad51 in eukaryotes (Paques & Haber, 1999; Symington, 2002). Rad51 protein forms a helical nucleoprotein filament on resected DNA to initiate homology search but also can interact with other single-stranded DNA (ssDNA)-binding proteins including Srs2. Srs2, a well-known antirecombinase in HR, is an ATP-dependent 3'-5' DNA helicase in the budding yeast Saccharomyces cerevisiae as well as an ssDNA translocase. It disrupts Rad51 filaments, preventing HR (Krejci et al., 2003; Le Breton et al., 2008; Veaute et al., 2003). In the following text, we provide detailed experimental platforms employed to investigate the activity of Rad51 and Srs2 using single-molecule Forster resonance energy transfer and protein-induced fluorescence enhancement. First, we demonstrate how to detect Rad51 filament formation to address the binding site size binding kinetic of the Rad51, as well as the directionality of the filament formation. Next, we explain how to visualize ATP-dependent translocation and unwinding activities of Srs2 on DNA. Lastly, we demonstrate the filament forming activity by Rad51 which is counteracted by the filament removal activity of Srs2.

The Recombination Mediator BRCA2: Architectural Plasticity of Recombination Intermediates Revealed by Single-Molecule Imaging (SFM/TIRF).

Cellular functions are defined by dynamic assembly, rearrangement, and disassembly of biomolecules to achieve control and specificity. As an example, effective DNA repair is brought about by the concerted action of several DNA processing proteins. Both changes in the structure of individual proteins and in the arrangement of multiple proteins together (referred to here as architecture) are inherent to biological function. These dynamic changes are exemplified in the breast cancer susceptibility protein 2 (BRCA2). BRCA2 is a DNA repair protein that undergoes changes in its own structure and affects changes in molecular architecture with partners during homologous recombination (HR) repair of DNA double strand breaks. These challenging features of BRCA2 protein, its size and predicted stretches of intrinsically disordered regions, have made it difficult to determine the structural consequences and mechanistic importance of interactions between full-length BRCA2 with RAD51 and other HR proteins. In this chapter, we describe scanning force microscopy (SFM)-based approaches to study DNA-protein complexes involved in HR, the architectural plasticity of full-length BRCA2, and the dynamic reorganization of these molecular components associated with essential steps of HR.

Single-Molecule Dynamics and Localization of DNA Repair Proteins in Cells.

Direct observation of individual protein molecules in their native environment, at nanometer resolution, in a living cell, in motion is not only fascinating but also uniquely informative. Several recent major technological advances in genomic engineering, protein and synthetic fluorophore development, and light microscopy have dramatically increased the accessibility of this approach. This chapter describes the procedures for modifying endogenous genomic loci to producing fluorescently tagged proteins, their high-resolution visualization, and analysis of their dynamics in mammalian cells, using DNA repair proteins BRCA2 and RAD51 as an example.

Diagnostic Potential of Imaging Flow Cytometry.

Imaging flow cytometry (IFC) captures multichannel images of hundreds of thousands of single cells within minutes. IFC is seeing a paradigm shift from low- to high-information-content analysis, driven partly by deep learning algorithms. We predict a wealth of applications with potential translation into clinical practice.

3D single-molecule super-resolution microscopy with a tilted light sheet.

Tilted light sheet microscopy with 3D point spread functions (TILT3D) combines a novel, tilted light sheet illumination strategy with long axial range point spread functions (PSFs) for low-background, 3D super-localization of single molecules as well as 3D super-resolution imaging in thick cells. Because the axial positions of the single emitters are encoded in the shape of each single-molecule image rather than in the position or thickness of the light sheet, the light sheet need not be extremely thin. TILT3D is built upon a standard inverted microscope and has minimal custom parts. The result is simple and flexible 3D super-resolution imaging with tens of nm localization precision throughout thick mammalian cells. We validate TILT3D for 3D super-resolution imaging in mammalian cells by imaging mitochondria and the full nuclear lamina using the double-helix PSF for single-molecule detection and the recently developed tetrapod PSFs for fiducial bead tracking and live axial drift correction.