Name: single cell analysis of transcriptionally active alleles by single molecule fish
Uploaded: 2020-09-20T14:00:00
Description: Watch this Scientific Journal Video about Single Cell Analysis Of Transcriptionally Active Alleles By Single Molecule FISH at JoVE.com

MAM, FS and MGM are funded by NIEHS (Project 4, P42ES027704, PI, Rusyn). MAM, FS, RMM, MGM and PKS are supported by the CPRIT-funded GCC Center for Advanced Microscopy and Image Informatics (RP170719). Imaging is also supported by the Integrated Microscopy Core at Baylor College of Medicine with funding from NIH (DK56338, and CA125123), CPRIT (RP150578), the Dan L. Duncan Comprehensive Cancer Center, and the John S. Dunn Gulf Coast Consortium for Chemical Genomics.

To ensure optimal results, the wet lab portion of this protocol requires standard RNase-free precautions at all steps. For example, the use of filtered pipette tips, sterile vessels and RNase-free buffers is highly encouraged. Using RNase inhibitors such as vanadyl ribonucleoside complexes is also suggested, especially for low abundance target RNAs.
1. Cell culture and experimental setup
<ol>
	<li>Maintain adherent MCF-7 breast cancer cells (ATCC #HTB-22) in a T75 tissue cul...

<ol>
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The smFISH methodology described is based upon previously-published protocols7,12,14. In this protocol, we explain the critical steps that were optimized from the wet-lab (including seeding density, fixation time, permeabilization, and probe concentration), to imaging and image analysis, providing a full experimental pipeline for laboratories interested in performing single cell analysis of gene transcription.
<p class="jove...

Gene transcription and translation are the two major processes involved in the central dogma of biology, going from the DNA sequence to RNA to protein1. In this study, we focus on the production of messenger RNA (mRNA) during transcription. The nascent RNA molecule includes both non-coding introns and coding exons. Introns are co-transcriptionally spliced out before the mRNA moves into the cytoplasm for translation on the ribosomes2,3.
Traditionally, the measurement of mRNA is performed in bulk populations of cells (hundreds to millions) with classic assays such as RT-qPCR or Northern blotting. While very powerful, these methods are limited as they do not provide insights into cell-to-cell variation (phenotypic heterogeneity), identification of the number of transcriptionally active alleles, and, perhaps more importantly, lack spatial information. More recently, genome wide technologies allowing for single cell RNA sequencing (RNAseq) began to bridge the gap between imaging methods and sequencing4. However, RNAseq suffers from relatively low detection efficiencies and the processing steps result in complete loss of spatial information5. Although single cell RNAseq can provide insights into phenotypic heterogeneity amongst a population of isogenic cells, RNA fluorescence in situ hybridization (RNA-FISH) can facilitate a more complete exploration of target gene expression at the spatial level, and on an allele-by-allele basis6,7.
RNA FISH is a technique that allows for the detection and localization of target RNA molecules in fixed cells. Unlike earlier, laborious methods, current state-of-the-art RNA-FISH utilizes commercially available nucleic acid probes that are complementary to the target RNA sequences, and these probes hybridize to their targets by Watson-Crick base pairing8. The early in situ hybridization techniques involved a similar protocol established by Gall and Pardue in 1969, as a sample was processed with nucleic acid probes that specifically hybridized to a target RNA9. Originally the assay was performed using radioactive or colorimetric detection; however, in the 1980s, the development of fluorescence in situ hybridization (FISH) protocols to DNA FISH and later RNA FISH opened the route towards more sensitive detection, and the potential for multiplexing10,11.
Further developments in RNA FISH have led to the ability to detect and quantify single RNA molecules (smFISH)12,13. Direct detection is a method in which the probes themselves are labeled with fluorophores. A challenge with smFISH is that there is a need for enough hybridizing probes/target to facilitate the detection of fluorescent signals as distinct, diffraction-limited spots. To address this issue, one can use a probe set of short single-stranded DNA oligonucleotides complementary to various regions of the target RNA8,12,14. The binding of multiple probes increases local fluorophore density, making the RNA visible as a distinct, high intensity spot by fluorescence microscopy. This method is advantageous because off-target binding of oligonucleotides in the probe set pool can be distinguishable from the true RNA spot signal by quantitatively analyzing the spot size and intensity to differentiate between true signal and spurious oligonucleotide binding, thus facilitating more accurate analysis by reducing false positives12.
Alternative methods to detect mRNA are the classic BAC clone-based nick translation method and the more recently developed branched DNA system. In the BAC-cloned, nick-translation method, the DNA to be labeled is enzymatically-nicked and a new nucleotide is added to the exposed 3&#8217; end. The activity of DNA polymerase I adds a labeled nucleotide resulting in the desired probe15,16. The branched DNA technique involves oligonucleotide probes that hybridize in pairs to RNA targets and these probes are amplified utilizing multiple signal amplification molecules. This method can significantly improve the signal-to-noise ratio but the amplification can skew accurate quantitation since fluorescent spots can be wildly different in size and intensity17.
As a model system for this protocol, which is fully-employed as part of the NIEHS Superfund Research Program participation to quantify the effects of endocrine disrupting chemicals in Galveston Bay/Houston Ship Channel, we used the estrogen receptor (ER) positive breast cancer cell line MCF-7 treated overnight with an ER agonist, 17&#946;-estradiol (E2)7,18,19. E2 regulates many ER-target genes, including the prototypical ER-target gene, GREB17,20,21,22. The smFISH protocol described here utilizes a set of two spectrally-separated probe sets targeting GREB1; one that hybridizes to exons and the other to introns, allowing for the measurement of both mature and nascent RNA7. We then use epifluorescence microscopy coupled with deconvolution to image smFISH labeled samples, and then apply image analysis routines to quantify the number of active alleles and mature RNAs per cell.&#160;

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	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">17&#946; Estradiol</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">E2257</td>
			<td style="width:167px;">CAS Number 50-28-2</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Ambion SSC (20X) Buffer, Rnase-free</td>
			<td style="width:71px;">ThermoFisher Scientific</td>
			<td style="width:119px;">AM9770</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Corning DMEM with L-Glutamine and 4.5 g/L Glucose</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">MT10017CV</td>
			<td>Manufactured by Invitrogen</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">DAPI</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">D8417</td>
			<td>Without sodium pyruvate</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Dextran Sulfate, 50% Solution Sterile</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">3730-100ML</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Dulbecco&#39;s Phosphate Buffered Saline Ca++/Mg++</td>
			<td style="width:71px;">Corning</td>
			<td style="width:119px;">21030CV</td>
			<td>Manufactured by Calbiochem</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Ethanol, 200 Proof (100%)</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">07-678-005</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Falcon 24-Well Clear Flat Bottom TC-treated Multiwell Cell Culture Plate</td>
			<td style="width:71px;">Corning</td>
			<td align="right" style="width:119px;">353047</td>
			<td>Manufactured by Decon Laboratories</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Fetal Bovine Serum (FBS), 500 mL</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">50-753-2978</td>
			<td>Manufactured by Gemini Bio Products</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">GE Healthcare DeltaVision LIVE High Resolution Deconvolution Microscope</td>
			<td style="width:71px;">GE Healthcare</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Hyclone Water, Molecular Biology Grade</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">SH3053802</td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Immersion Oil 1.516</td>
			<td style="width:71px;">GE Healthcare Life Sciences</td>
			<td align="right" style="width:119px;">29162940</td>
			<td>Manufactured by GE Healthcare Bio-Science</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">L-glutamine Solution</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">MT25005Cl</td>
			<td>Manufactured by Corning</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MCF-7 cells</td>
			<td style="width:71px;">ATCC</td>
			<td style="width:119px;">HTB-22</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Molecular Grade Formamide</td>
			<td style="width:71px;">Promega</td>
			<td style="width:119px;">PAH5051</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Olympus 60x/1.42 NA Objective</td>
			<td style="width:71px;">Olympus</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Parafilm</td>
			<td style="width:71px;">Bemis Company, Inc.</td>
			<td style="width:119px;">52858-076</td>
			<td>Distributed by VWR</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Paraformaldehyde, 16% Solution, EM Grade</td>
			<td style="width:71px;">Electron Microscopy Sciences</td>
			<td align="right" style="width:119px;">15710</td>
			<td></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Penicilin-Streptomycin Solution</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">MT3001Cl</td>
			<td>Manufactured by Corning</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Ribonucleoside Vanadyl Complex</td>
			<td style="width:71px;">VWR</td>
			<td style="width:119px;">101229-716</td>
			<td>Manufactured by New England Biosciences</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">RNase Away</td>
			<td style="width:71px;">Ambion</td>
			<td align="right" style="width:119px;">9780</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Sodium pyruvate, 100 mM</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">11-360-070</td>
			<td>Manufactured by Gibco</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Thermo Scientific Glass Coverslips, #1.5</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">12-545-81P</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Triton X-100, 100 mL</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">BP151-100</td>
			<td>Manufactured by Fisher BioReagents</td>
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			<td height="42" style="height:42px;width:216px;">Trypsin-EDTA Solution 0.25%</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">T4049-500ML</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Vectashield Antifade Mounting Medium 10 mL</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">NC9265087</td>
			<td>Manufactured by Vector Laboratories</td>
		</tr>
	</tbody>
</table>

single cell analysis of transcriptionally active alleles by single molecule fish

Gene transcription is an essential process in cell biology, and allows cells to interpret and respond to internal and external cues. Traditional bulk population methods (Northern blot, PCR, and RNAseq) that measure mRNA levels lack the ability to provide information on cell-to-cell variation in responses. Precise single cell and allelic visualization and quantification is possible via single molecule RNA fluorescence in situ hybridization (smFISH). RNA-FISH is performed by hybridizing target RNAs with labeled oligonucleotide probes. These can be imaged in medium/high throughput modalities, and, through image analysis pipelines, provide quantitative data on both mature and nascent RNAs, all at the single cell level. The fixation, permeabilization, hybridization and imaging steps have been optimized in the lab over many years using the model system described herein, which results in successful and robust single cell analysis of smFISH labeling. The main goal with sample preparation and processing is to produce high quality images characterized by a high signal-to-noise ratio to reduce false positives and provide data that are more accurate. Here, we present a protocol describing the pipeline from sample preparation to data analysis in conjunction with suggestions and optimization steps to tailor to specific samples.&#160;

To analyze hormonal responses via smFISH, as an example, we chose the estrogen receptor (ER) model used in high throughput assays to determine the presence of endocrine disrupting chemicals in environmental disasters in the context of participation in the NIEHS Superfund Research Program7. In this experiment, adherent MCF-7 breast cancer cells were treated with the ER agonist 17&#946;-estradiol (E2, 10 nM) or vehicle (DMSO) for 24 hours. Spectrally-separated probe sets against GREB1 intronic (Atto...

Watch this Scientific Journal Video about Single Cell Analysis Of Transcriptionally Active Alleles By Single Molecule FISH at JoVE.com

Single Cell Analysis Of Transcriptionally Active Alleles By Single Molecule FISH

Single molecule RNA fluorescence in situ hybridization (smFISH) is a method to accurately quantify levels and localization of specific RNAs at the single cell level. Here, we report our validated lab protocols for wet-bench processing, imaging and image analysis for single cell quantification of specific RNAs.

Gene transcription is an essential process in cell biology, and allows cells to interpret and respond to internal and external cues. Traditional bulk ...

SM FISH accurately quantifies levels and localization of specific RNase. This method is particularly useful for analyzing cell to cell and allele by allele variation in RNA. Gene transcription is an essential process in cell biology, SM FISH allows for the investigation of RNA content in response to external stimuli at the single cell level and provide spatial information.Following estrogen receptor agonist treatment, wash the cells with cold sterile PBS with calcium and magnesium and fix the cells with 500 microliters of fixation buffer for 30 minutes on ice. At the end of the incubation, wash the cells two times with cold sterile PBS with calcium and magnesium for three minutes before adding 500 microliters of 70%ethanol to each well. Then seal the 24 well plate with a paraffin plastic film and place the plate on a rotator at four degrees Celsius for at least four and up to 16 hours.For RNA FISH hybridization, at the end of the incubation wash the cells one time with 500 microliters of 2X SSC buffer supplemented with 10%formamide for five minutes at room temperature on a rotator. During the incubation, place a piece of parffin plastic film unexposed side up into a glass Petri dish cleaned with an RNA surface decontaminate and place to sterile water-saturated paper towels along the edges of the dish. At the end of the incubation, place evenly spaced 30 microliter droplets of freshly prepared to GREB1 intron and GREB1 exon probe buffer onto the clean side of the paraffin plastic film and use sterile forceps to gently place one cover slip of cells cell side down onto each droplet without bubbles.After sealing the dish with paraffin plastic film, cover the plate with foil and incubate the plate overnight at 37 degrees Celsius on a flat non-rotating surface. The next morning, use forceps to return the cover slips to the 24 well plate cell side up for two 15 minute washes in 500 microliters of fresh 2X SSC buffer supplemented with 10%formamide per wash at 37 degrees Celsius on a heated rotator. After the second wash, counterstain the DNA with one microgram per milliliter of DAPI in 2X SSC buffer for 10 minutes at room temperature on a rotator followed by a five minute wash in 2X SSC buffer at room temperature.Use a paper towel to remove the excess 2X SSC buffer and rinse the samples in nuclease-free water to eliminate excess salts. Then use non-hardening mounting medium to mount the cover slips onto glass slides and seal the cover slips with clear nail polish. For sample imaging, load the slide with the highest expected intensity onto a widefield epifluorescence microscope stage equipped with a 60 or 100 times oil objective in an sCOMS camera and place a droplet of immersion oil onto the objective.For Zstack acquisition, focus on the sample in the DAPI channel and select the top and bottom of the Zstack at the distances at which the DAPI-stained nuclei become out of focus. Use the slide to optimize the amount of illumination from the light source and exposure times for each channel and acquire images from all of the slides under conditions that avoid photo bleaching and or camera pixel saturation. After the acquisition, use 10 cycles of an aggressive restorative algorithm to deconvolve the images and save all of the projection images according to the bit depth of the camera used.For image analysis, first download the Jupiter Notebook from GitHub and install Python Anaconda Distribution version 3.6 or higher. Open the Anaconda prompt and enter the installation command to install SimpleITK for Anaconda. Open the Anaconda Navigator and launch Jupiter Notebook.A web browser will open showing directories and files. Browse through the directory structures to reach the directory containing the downloaded Jupiter Notebook from GitHub. Then open the notebook and press Shift and Enter to run each cell.In this representative experiment, adherent breast cancer cells were treated with an estrogen receptor agonist, or vehicle, for 24 hours. Spectrally separated probe sets against GREB1 intronic and exonic sequences allowed the simultaneous visualization and quantification of nascent and mature mRNA. Importantly, the number of transcriptionally active alleles in each cell that has been shown to be part of the estrogen response time course can be marked.Counting the overlapping intron and exon spots, the E2-treated cells were determined to have a four-fold increased fraction of cells that exhibit two or more active GREB1 alleles compared to vehicle-treated cells. As introns are spliced out of nascent mRNA to produce mature mRNA that consists of only exon sequences, the number of mature mRNA per cell can be counted by quantifying the number of green spots. Plotting these data then allow the shift in distribution of mature GREB1 mRNA cell and the aggregate fold change in the cell population after E2 treatment to be observed.C-FISH allows for investigating multiple RNA species sequentially, MERFISH is a barcoding technique to massively increase multiplexing, and iFISH is used to simultaneously study RNA and proteins.

single-cell-analysis-transcriptionally-active-alleles-single-molecule

Research

JoVE Journal

Biochemistry

Single-molecule Manipulation of G-quadruplexes by Magnetic Tweezers

Non-canonical nucleic acid secondary structure G-quadruplexes (G4) are involved in diverse cellular processes, such as DNA replication, transcription, RNA processing, and telomere elongation. During these processes, various proteins bind and resolve G4 structures to perform their function. As the function of G4 often depends on the stability of its folded structure, it is important to investigate how G4 binding proteins regulate the stability of G4. This work presents a method to manipulate single G4 molecules using magnetic tweezers, which enables studies of the regulation of G4 binding proteins on a single G4 molecule in real time. In general, this method is suitable for a wide scope of applications in studies for proteins/ligands interactions and regulations on various DNA or RNA secondary structures.

A single-molecule magnetic tweezers platform to manipulate G-quadruplexes is reported, which allows for the study of G4 stability and regulation by various proteins.

Non-canonical nucleic acid secondary structure G-quadruplexes (G4) are involved in diverse cellular processes, such as DNA replication, transcription, RNA ...

Covalent Immobilization of Proteins for the Single Molecule Force Spectroscopy

In recent years, atomic force microscopy (AFM) based single molecule force spectroscopy (SMFS) extended our understanding of molecular properties and functions. It gave us the opportunity to explore a multiplicity of biophysical mechanisms, e.g., how bacterial adhesins bind to host surface receptors in more detail. Among other factors, the success of SMFS experiments depends on the functional and native immobilization of the biomolecules of interest on solid surfaces and AFM tips. Here, we describe a straightforward protocol for the covalent coupling of proteins to silicon surfaces using silane-PEG-carboxyls and the well-established N-hydroxysuccinimid/1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimid (EDC/NHS) chemistry in order to explore the interaction of pilus-1 adhesin RrgA from the Gram-positive bacterium Streptococcus pneumoniae (S. pneumoniae) with the extracellular matrix protein fibronectin (Fn). Our results show that the surface functionalization leads to a homogenous distribution of Fn on the glass surface and to an appropriate concentration of RrgA on the AFM cantilever tip, apparent by the target value of up to 20% of interaction events during SMFS measurements and revealed that RrgA binds to Fn with a mean force of 52 pN. The protocol can be adjusted to couple via site specific free thiol groups. This results in a predefined protein or molecule orientation and is suitable for other biophysical applications besides the SMFS.

This protocol describes the covalent immobilization of proteins with a heterobifunctional silane coupling agent to silicon-oxide surfaces designed for the atomic force microscopy based single molecule force spectroscopy which is exemplified by the interaction of RrgA (pilus-1 tip adhesin of S. pneumoniae) with fibronectin.

In recent years, atomic force microscopy (AFM) based single molecule force spectroscopy (SMFS) extended our understanding of molecular properties and ...

Proteome-wide Quantification of Labeling Homogeneity at the Single Molecule Level

Cell proteomes are often characterized using electrophoresis assays, where all species of proteins in the cells are non-specifically labeled with a fluorescent dye and are spotted by a photodetector following their separation. Single molecule fluorescence imaging can provide ultrasensitive protein detection with its ability for visualizing individual fluorescent molecules. However, the application of this powerful imaging method to electrophoresis assays is hampered by the lack of ways to characterize the homogeneity of fluorescent labeling of each protein species across the proteome. Here, we developed a method to evaluate the labeling homogeneity across the proteome based on a single molecule fluorescence imaging assay. In our measurement using a HeLa cell sample, the proportion of proteins labeled with at least one dye, which we termed &#8216;labeling occupancy&#8217; (LO), was determined to range from 50% to 90%, supporting the high potential of the application of single molecule imaging to sensitive and precise proteome analysis.

Here, we present a protocol to assess the labeling homogeneity for each protein species in a complex protein sample at the single molecule level.

Cell proteomes are often characterized using electrophoresis assays, where all species of proteins in the cells are non-specifically labeled with a ...

Conventional BODIPY Conjugates for Live-Cell Super-Resolution Microscopy and Single-Molecule Tracking

Single molecule localization microscopy (SMLM) techniques overcome the optical diffraction limit of conventional fluorescence microscopy and can resolve intracellular structures and the dynamics of biomolecules with ~20 nm precision. A prerequisite for SMLM are fluorophores that transition from a dark to a fluorescent state in order to avoid spatio-temporal overlap of their point spread functions in each of the thousands of data acquisition frames. BODIPYs are well-established dyes with numerous conjugates used in conventional microscopy. The transient formation of red-shifted BODIPY ground-state dimers (DII) results in bright single molecule emission enabling single molecule localization microscopy (SMLM). Here we present a simple but versatile protocol for SMLM with conventional BODIPY conjugates in living yeast and mammalian cells. This procedure can be used to acquire super-resolution images and to track single BODIPY-DII states to extract spatio-temporal information of BODIPY conjugates. We apply this procedure to resolve lipid droplets (LDs), fatty acids, and lysosomes in living yeast and mammalian cells at the nanoscopic length scale. Furthermore, we demonstrate the multi-color imaging capability with BODIPY dyes when used in conjunction with other fluorescent&#160;probes. Our representative results show the differential spatial distribution and mobility of BODIPY-fatty acids and neutral lipids in yeast under fed and fasted conditions. This optimized protocol for SMLM can be used with hundreds of commercially available BODIPY conjugates and is a useful resource to study biological processes at the nanoscale far beyond the applications of this work.&#160;

Conventional BODIPY conjugates can be used for live-cell single-molecule localization microscopy (SMLM) through exploitation of their transiently forming, red-shifted ground state dimers. We present an optimized SMLM protocol to track and resolve subcellular neutral lipids and fatty acids in living mammalian and yeast cells at the nanoscopic length scale.

Single molecule localization microscopy (SMLM) techniques overcome the optical diffraction limit of conventional fluorescence microscopy and can resolve ...

An In Vitro Single-Molecule Imaging Assay for the Analysis of Cap-Dependent Translation Kinetics

Cap-dependent protein synthesis is the predominant translation pathway in eukaryotic cells. While various biochemical and genetic approaches have allowed extensive studies of cap-dependent translation and its regulation, high resolution kinetic characterization of this translation pathway is still lacking. Recently, we developed an in vitro assay to measure cap-dependent translation kinetics with single-molecule resolution. The assay is based on fluorescently labeled antibody binding to nascent epitope-tagged polypeptide. By imaging the binding and dissociation of antibodies to and from nascent peptide&#8211;ribosome&#8211;mRNA complexes, the translation progression on individual mRNAs can be tracked. Here, we present a protocol for establishing this assay, including mRNA and PEGylated slide preparations, real-time imaging of translation, and analysis of single molecule trajectories. This assay enables tracking of individual cap-dependent translation events and resolves key translation kinetics, such as initiation and elongation rates. The assay can be widely applied to distinct translation systems and should broadly benefit in vitro studies of cap-dependent translation kinetics and translational control mechanisms.

An In Vitro Single-Molecule Imaging Assay for the Analysis of Cap-Dependent Translation Kinetics

Tracking individual translation events allows for high-resolution kinetic studies of cap-dependent translation mechanisms. Here we demonstrate an in vitro single-molecule assay based on imaging interactions between fluorescently labeled antibodies and epitope-tagged nascent peptides. This method enables single-molecule characterization of initiation and peptide elongation kinetics during active in vitro cap-dependent translation.

Cap-dependent protein synthesis is the predominant translation pathway in eukaryotic cells. While various biochemical and genetic approaches have allowed ...

Single-Molecule Dwell-Time Analysis of Restriction Endonuclease-Mediated DNA Cleavage

This novel total internal reflection fluorescence microscopy-based assay facilitates the simultaneous measurement of the length of the catalytic cycle for hundreds of individual restriction endonuclease (REase) molecules in one experiment. This assay does not require protein labeling and can be carried out with a single imaging channel. In addition, the results of multiple individual experiments can be pooled to generate well-populated dwell-time distributions. Analysis of the resulting dwell-time distributions can help elucidate the DNA cleavage mechanism by revealing the presence of kinetic steps that cannot be directly observed. Example data collected using this assay with the well-studied REase, EcoRV - a dimeric Type IIP restriction endonuclease that cleaves the palindromic sequence GAT&#8595;ATC (where &#8595; is the cut site) - are in agreement with prior studies. These results suggest that there are at least three steps in the pathway to DNA cleavage that is initiated by introducing magnesium after EcoRV binds DNA in its absence, with an average rate of 0.17 s-1 for each step.

Using quantum-dot-labeled DNA and total internal reflection fluorescence microscopy, we can investigate the reaction mechanism of restriction endonucleases while using unlabeled protein. This single-molecule technique allows for massively multiplexed observation of individual protein-DNA interactions, and data can be pooled to generate well-populated dwell-time distributions.

This novel total internal reflection fluorescence microscopy-based assay facilitates the simultaneous measurement of the length of the catalytic cycle for ...

Single Molecule Fluorescence Energy Transfer Study of Ribosome Protein Synthesis

The ribosome is a large ribonucleoprotein complex that assembles proteins processively along mRNA templates. The diameter of the ribosome is approximately 20 nm to accommodate large tRNA substrates at the A-, P- and E-sites. Consequently, the ribosome dynamics are naturally de-phased quickly. Single molecule method can detect each ribosome separately and distinguish inhomogeneous populations, which is essential to reveal the complicated mechanisms of multi-component systems. We report the details of a smFRET method based on the Nikon Ti2 inverted microscope to probe the ribosome dynamics between the ribosomal protein L27 and tRNAs. The L27 is labeled at its unique Cys 53 position and reconstituted into a ribosome that is engineered to lack L27. The tRNA is labeled at its elbow region. As the tRNA moves to different locations inside the ribosome during the elongation cycle, such as pre- and post- translocation, the FRET efficiencies and dynamics exhibit differences, which have suggested multiple subpopulations. These subpopulations are not detectable by ensemble methods. The TIRF-based smFRET microscope is built on a manual or motorized inverted microscope, with home-built laser illumination. The ribosome samples are purified by ultracentrifugation, loaded into a home-built multi-channel sample cell and then illuminated via an evanescent laser field. The reflection laser spot can be used to achieve feedback control of perfect focus. The fluorescence signals are separated by a motorized filter-turret and collected by two digital CMOS cameras. The intensities are retrieved via the NIS-Elements software.

Single molecule fluorescence energy transfer is a method that tracks the tRNA dynamics during ribosomal protein synthesis. By tracking individual ribosomes, inhomogeneous populations are identified, which shed light on mechanisms. This method can be used to track biological conformational changes in general to reveal dynamic-function relationships in many other complexed biosystems. Single molecule methods can observe non-rate limiting steps and low-populated key intermediates, which are not accessible by conventional ensemble methods due to the average effect.

The ribosome is a large ribonucleoprotein complex that assembles proteins processively along mRNA templates. The diameter of the ribosome is approximately ...

Single-Molecule Imaging of EWS-FLI1 Condensates Assembling on DNA

The fusion genes resulting from chromosomal translocation have been found in many solid tumors or leukemia. EWS-FLI1, which belongs to the FUS/EWS/TAF15 (FET) family of fusion oncoproteins, is one of the most frequently involved fusion genes in Ewing sarcoma. These FET family fusion proteins typically harbor a low-complexity domain (LCD) of FET protein at their N-terminus and a DNA-binding domain (DBD) at their C-terminus. EWS-FLI1 has been confirmed to form biomolecular condensates at its target binding loci due to LCD-LCD and LCD-DBD interactions, and these condensates can recruit RNA polymerase II to enhance gene transcription. However, how these condensates are assembled at their binding sites remains unclear. Recently, a single-molecule biophysics method-DNA Curtains-was applied to visualize these assembling processes of EWS-FLI1 condensates. Here, the detailed experimental protocol and data analysis approaches are discussed for the application of DNA Curtains in studying the biomolecular condensates assembling on target DNA.

Here, we describe the use of the single-molecule imaging method, DNA Curtains, to study the biophysical mechanism of EWS-FLI1 condensates assembling on DNA.

The fusion genes resulting from chromosomal translocation have been found in many solid tumors or leukemia. EWS-FLI1, which belongs to the FUS/EWS/TAF15 ...

Single-Molecule Analysis of Sf9 Purified Superprocessive Kinesin-3 Family Motors

A complex cellular environment poses challenges for single-molecule motility analysis. However, advancement in imaging techniques have improved single-molecule studies and has gained immense popularity in detecting and understanding the dynamic behavior of fluorescent-tagged molecules. Here, we describe a detailed method for&#160;in vitro single-molecule studies of kinesin-3 family motors using Total Internal Reflection Fluorescence (TIRF) microscopy. Kinesin-3 is a large family that plays critical roles in cellular and physiological functions ranging from intracellular cargo transport to cell division to development. We have shown previously that constitutively active dimeric kinesin-3 motors exhibit fast and superprocessive motility with high microtubule affinity at the single-molecule level using cell lysates prepared by expressing motor in mammalian cells. Our lab studies kinesin-3 motors and their regulatory mechanisms using cellular, biochemical and biophysical approaches, and such studies demand purified proteins at a large scale. Expression and purification of these motors using mammalian cells would be expensive and time-consuming, whereas expression in a prokaryotic expression system resulted in significantly aggregated and&#160;inactive protein. To overcome the limitations posed by bacterial purification systems and mammalian cell lysate, we have established a robust Sf9-baculovirus expression system to express and purify these motors. The kinesin-3 motors are C-terminally tagged with 3-tandem fluorescent proteins (3xmCitirine or 3xmCit) that provide enhanced signals and decreased photobleaching. In vitro single-molecule and multi-motor gliding analysis of Sf9 purified proteins demonstrate that kinesin-3 motors are fast and superprocessive akin to our previous studies using mammalian cell lysates. Other applications using these assays include detailed knowledge of oligomer conditions of motors, specific binding partners paralleling biochemical studies, and their kinetic state.

This study details purification of KIF1A(1-393LZ), a member of kinesin-3 family, using Sf9-baculovirus expression system. In vitro single-molecule and multi-motor gliding analysis of these purified motors exhibited robust motility properties comparable to motors from mammalian cell lysate. Thus, Sf9-baculovirus system is amenable to express and purify motor protein of interest.

A complex cellular environment poses challenges for single-molecule motility analysis. However, advancement in imaging techniques have improved ...

Single-Molecule Diffusion and Assembly on Polymer-Crowded Lipid Membranes

Cellular membranes are highly crowded environments for biomolecular reactions and signaling. Yet, most in vitro experiments probing protein interaction with lipids employ naked bilayer membranes. Such systems lack the complexities of crowding by membrane-embedded proteins and glycans and exclude the associated volume effects encountered on cellular membrane surfaces. Also, the negatively charged glass surface onto which the lipid bilayers are formed prevents the free diffusion of transmembrane biomolecules. Here, we present a well-characterized polymer-lipid membrane as a mimic for crowded lipid membranes. This protocol utilizes polyethylene glycol (PEG)-conjugated lipids as a generalized approach for incorporating crowders into the supported lipid bilayer&#160;(SLB). First, a cleaning procedure of the microscopic slides and coverslips for performing single-molecule experiments is presented. Next, methods for characterizing the PEG-SLBs and performing single-molecule experiments of the binding, diffusion, and assembly of biomolecules using single-molecule tracking and photobleaching are discussed. Finally, this protocol demonstrates how to monitor the nanopore assembly of bacterial pore-forming toxin Cytolysin A (ClyA) on crowded lipid membranes with single-molecule photobleaching analysis. MATLAB codes with example datasets are also included to perform some of the common analyses such as particle tracking, extracting diffusive behavior, and subunit counting.

Here, a protocol to perform and analyze the binding, mobility, and assembly of single molecules on artificial crowded lipid membranes using single-molecule total internal reflection fluorescence (smTIRF) microscopy is presented.

Cellular membranes are highly crowded environments for biomolecular reactions and signaling. Yet, most in vitro experiments probing protein interaction ...