Name: visualizing diffusional dynamics of gold nanorods on cell membrane using single nanoparticle darkfield microscopy
Uploaded: 2021-03-05T15:00:00
Description: Watch this Scientific Journal Video about Visualizing Diffusional Dynamics of Gold Nanorods on Cell Membrane using Single Nanoparticle Darkfield Microscopy at JoVE.com

This work was supported by the National Natural Science Foundation of China with grant numbers of 21425519, 91853105 and 21621003.

1. Cell culture
<ol>
	<li>Prepare complete medium for U87 MG cells by adding fetal bovine serum (final concentration 10%) and penicillin-streptomycin (final concentration 1%) to the minimum essential medium (MEM). Use plastic cell culture dish for cells subculture.</li>
	<li>Passage cells 2 to 3 times a week.
	<ol>
		<li>Remove the culture medium and rinse the cell layer with Dulbecco&#39;s phosphate-buffered saline (D-PBS) 2~3 times when confluent (80%~90%).</li>
		<li>Add 1.0 to 2.0 mL of Trypsin-EDTA solution to the...

<ol>
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The presented protocol is used to study the dynamics of AuNRs on cell membrane. The protocol consists of four parts, including microscopic imaging, data extraction, dynamic parameters calculation and data analysis methods, and each part is flexible and universal. Therefore, there are many possible future applications, for instance, studying movement of NP-linked membrane molecules on membrane, endocytosis dynamics of NP-labeled receptors, dynamic analysis of intracellular NPs and vesicle-coated NPs transportation along m...

The dynamics of nanoparticles (NPs) on the membrane is closely associated with the cellular uptake process, which is essential for the understanding of cell functions, viral or bacterial infections and the development of artificial nanomedical delivery systems1,2. Single particle tracking (SPT) technique is a robust tool for characterizing the heterogeneous behaviors of NPs3,4. In general, cell membrane is fluidic, which means that the components such as proteins and lipids can move laterally in the plasma membrane plane5,6,7. The spatiotemporal complexity of membrane organization and structure may lead to spatiotemporal heterogeneity of the interaction between NPs and membrane. Hence, direct visualization of the movement of NPs on the membrane requires both high spatial and temporal resolution.
Single particle tracking microscopy that monitors the localization of individual particles in living cells with a spatial resolution of tens of nanometers and a time resolution of milliseconds has been well developed to study the NPs or membrane molecules dynamics8,9. Fluorescence-based microscopic imaging techniques have become valuable tools for observing NPs/molecules in living cell environment9,10,11,12. For example, total internal reflection fluorescence microscopy, which images thin layers (~100 nm) of the sample at the substrate/solution interface with a high spatiotemporal resolution has been widely used in studies of membrane molecules dynamics13,14. However, the inherent disadvantages of single fluorophores, such as low intensity and rapid irreversible photobleaching reduce the accuracy and duration of tracking13. Therefore, non-fluorescent plasmonic NPs, which replace the fluorescent probes, have attracted more and more attention in long-term imaging studies due to their unique optical characteristics15. Based on the scattering signals of plasmonic NP probes, several kinds of optical microscopic imaging technologies have been used to study the mechanism of biological processes, such as dark-field microscopy (DFM)16, interferometric scattering (iSCAT) microscopy17 and differential interference contrast microscopy (DICM)18. In addition, the motion and rotation dynamic of AuNRs can be obtained using DFM and DICM18,19,20,21,22. Typically, in an SPT experiment, the motion of the object is recorded by the optical microscope, and then analyzed by SPT analysis methods3. The time-resolved trajectories and orientational angles generated by individual NPs are normally stochastic and heterogeneous, so it is necessary to present abundant dynamic information with various analysis methods.
Here, we provide an integrated protocol that monitors the dynamics of AuNRs on cell membrane using DFM, extracts the location and orientation of AuNRs with ImageJ and MATLAB and characterizes the diffusion of AuNRs with SPT analysis methods. As a demonstration, we show here how to use the SPT protocol to visualize dynamics of unmodified AuNRs (CTAB-AuNRs, synthesized by cetyltrimethylammonium ammonium bromide molecule as protective agent) on U87 MG cell membrane. It has been demonstrated that CTAB-AuNRs can adsorb proteins in biological environment, move on cell membrane and then enter cells2,20,22. U87 MG cell is the most common and most malignant tumor of the central nervous system, and its membrane receptors are abnormally expressed. The membrane receptors can interact with proteins on AuNRs, which influence the dynamics of AuNRs. Our protocol is generally applicable to other SPT experiments in the field of biology.

<table>
	<tbody>
		<tr>
			<td>CTAB coated gold nanorods(CTAB-AuNRs)</td>
			<td>Nanoseedz</td>
			<td>NR-40-650</td>
			<td>85 nm * 40 nm</td>
		</tr>
		<tr>
			<td>Color CMOS camera</td>
			<td>Olympus</td>
			<td>DP74</td>
			<td>Japan</td>
		</tr>
		<tr>
			<td>Coverslips</td>
			<td>Citoglas</td>
			<td>z10212222C</td>
			<td>22*22 mm</td>
		</tr>
		<tr>
			<td>Dark-field microscopy</td>
			<td>Nikon</td>
			<td>80i</td>
			<td>upright microscope</td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Gibco</td>
			<td>10099141</td>
			<td></td>
		</tr>
		<tr>
			<td>Fiji</td>
			<td>National Institutes of Health</td>
			<td>2.0.0-rc-69/1.52 p</td>
			<td>a distribution of ImageJ</td>
		</tr>
		<tr>
			<td>Grooved glass slide</td>
			<td>Sail brand</td>
			<td>7103</td>
			<td>Single concave</td>
		</tr>
		<tr>
			<td>Image J</td>
			<td>National Institutes of Health</td>
			<td>1.52 j</td>
			<td></td>
		</tr>
		<tr>
			<td>MATLAB</td>
			<td>MathWorks</td>
			<td>R2019b</td>
			<td></td>
		</tr>
		<tr>
			<td>MATLAB Code</td>
			<td></td>
			<td></td>
			<td>https://github.com/fenggeqd/JOVE-2020</td>
		</tr>
		<tr>
			<td>Minimum essential medium (MEM)</td>
			<td>Gibco</td>
			<td>10-010-CVR</td>
			<td>with phenol red</td>
		</tr>
		<tr>
			<td>Minimum essential medium (MEM)</td>
			<td>Gibco</td>
			<td>51200038</td>
			<td>no phenol red</td>
		</tr>
		<tr>
			<td>Origin</td>
			<td>OriginLab</td>
			<td>Origin Pro 2018C</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic cell culture dishes</td>
			<td>Falcon</td>
			<td>353002</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic cell culture dishes</td>
			<td>Falcon</td>
			<td>353001</td>
			<td>35*10 mm</td>
		</tr>
		<tr>
			<td>U87 MG cell</td>
			<td>American Type Culture Collection</td>
			<td>ATCC HTB-14</td>
			<td>a human primary glioblastoma cell line</td>
		</tr>
	</tbody>
</table>

visualizing diffusional dynamics of gold nanorods on cell membrane using single nanoparticle darkfield microscopy

Analyzing the diffusional dynamics of nanoparticles on cell membrane plays a significant role in better understanding the cellular uptake process and provides a theoretical basis for the rational design of nano-medicine delivery. Single particle tracking (SPT) analysis could probe the position and orientation of individual nanoparticles on cell membrane, and reveal their translational and rotational states. Here, we show how to use traditional dark-field microscopy to monitor the dynamics of gold nanorods (AuNRs) on live cell membrane. We also show how to extract the location and orientation of AuNRs using ImageJ and MATLAB, and how to characterize the diffusive states of AuNRs. Statistical analysis of hundreds of particles show that single AuNRs perform Brownian motion on the surface of U87 MG cell membrane. However, individual long trajectory analysis shows that AuNRs have two distinctly different types of motion states on the membrane, namely long-range transport and limited-area confinement. Our SPT methods can be potentially used to study the surface or intracellular particle diffusion in different biological cells and can become a powerful tool for investigations of complex cellular mechanisms.

In the protocol, the unmodified 40 x 85 nm CTAB-AuNRs were used. As shown in Figure 2B, its longitudinal plasmonic maximum at is ~650 nm (red region) and transverse resonance is at 520 nm (green region). Previous literatures have revealed that the optical properties (such as LSPR intensity) of plasmonic AuNRs will change significantly with their diameter20,22. In Figure 2C, the scattering intensity from ...

Watch this Scientific Journal Video about Visualizing Diffusional Dynamics of Gold Nanorods on Cell Membrane using Single Nanoparticle Darkfield Microscopy at JoVE.com

Visualizing Diffusional Dynamics of Gold Nanorods on Cell Membrane using Single Nanoparticle Darkfield Microscopy

Here, we show the use of traditional dark-field microscopy to monitor the dynamics of gold nanorods (AuNRs) on cell membrane. The location and orientation of single AuNRs are detected using ImageJ and MATLAB, and the diffusive states of AuNRs are characterized by single particle tracking analysis.

Analyzing the diffusional dynamics of nanoparticles on cell membrane plays a significant role in better understanding the cellular uptake process and ...

Our protocol uses single particle checking analysis to monitor the dynamics the location and the orientation and characterizes the diffusion of golden nanorods on cell membranes. Using this method, both the translational and the rotational dynamics of golden nanorods can be obtained and the dynamic can be comprehensively analyzed and the presented. This protocol has the potential to be used for the study of other types of complex biological systems.Begin by burning an ethanol dipped glass coverslip on the flame and placing the coverslip in a 35 by 10 millimeter cell culture dish containing two milliliters of cell culture medium without phenol red. Add 50 microliters of the cell suspension of interest onto the coverslip and gently shake the dish back and forth and left and right to evenly distribute the cells. Place the dish into the cell culture incubator for approximately 12 hours until the cells reach 20 to 40%confluency before adding 20 microliters of CTAB coated gold nanorods to the dish.After gentle shaking to disperse the nanorods evenly across the dish, place the dish in a humidified atmosphere for five minutes. At the end of the incubation, slowly transfer 100 microliters of supernatant from the dish into the groove of a glass slide and carefully place the coverslip cell side down onto the slide groove, then seal the edge of the coverslip with nail polish and let the nail polish dry before placing the slide onto the darkfield microscope stage. For single particle tracking by darkfield microscopy, place a drop of oil onto the oil immerse darkfield condenser and turn the knob until the condenser contacts the glass slide.Place a drop of oil onto the top of the cover glass and turn the focusing knob until the 60X oil immersion objective touches the oil. Turn on the light source and slightly turn the focusing knob to focus the imaging plane. Then click the camera icon in the microscope software to record a sample scattering light image time series using the color CMOS camera and save the image in a TIFF format.To extract a single long-term trajectory, open the image in ImageJ and click image, type, and 8-bit to convert the image from RGB mode to 8-bit mode. To adjust the contrast, click image, adjust, brightness contrast and set the parameters. Select a target particle and use Control X to cut off the time series background.Click plugins, particle tracker classic, and particle tracker to open the particle detection and particle linking window and set the radius to six, the cutoff to zero, and the percentile to 0.01%Set the link range to 10 and the displacement to 10 and click OK to open the particle tracker results window to see the results. Click visualize all trajectories to inspect the generated trajectories. If the software generated trajectory and the moving trajectory of the gold nanorods are matched, click save full report to save the results.If the software generated trajectory does not match the moving trajectory of the gold nanorods, click relink particles to relink the detected particles with different link range and percentile parameters. To find the center pixel coordinate of the gold nanorod in each frame according to the XY coordinate, use the xycoordination. m function.To delimit a three by three pixels matrix, extract the nine scattering intensity values of the red or green channels and calculate an average value, use the rgextraction. m function. Then use the polarangle.m function to calculate the polar angles using the dual channel differential method. To calculate dynamic parameters using the formulas in the table, run the two analysis scripts. For visual analysis of the trajectory, set the X coordinate as X, the Y coordinate as Y, and the time as Y, then click plot, scatter, and color map, set the graph parameters, and add the color bar.To generate mean square displacement time interval figures, set the time interval as X and the mean square displacement as Y.Click plot, scatter, analysis, fitting, nonlinear curve fitting and open dialogue and set the graph parameters. For multi-particle statistical analysis, set the dynamic parameters of interest as Y and click plot and histogram. Double-click the histogram to set the division size or the number of divisions and click apply.Then add a column and set the graph parameters. For a time series analysis, set the time as X and the time series parameters as Y.Click plot, multi-pane, and stack. In the popup window, select line and OK.Then set the parameters for the graph.The longitudinal plasmonic maximum of 40 by 85 nanometers CTAB coated gold nanorods is approximately 650 nanometers and the transverse resonance is 520 nanometers. The scattering intensity of the CTAB coated gold nanorods on U87 cell membranes demonstrates a typical Gaussian distribution with a narrow width consistent with that of CTAB coated gold nanorods on glass indicating that CTAB coated gold nanorods tracked in this experiment are well monodispersed. As illustrated, more than 500 CTAB coated gold nanorod trajectories can be tracked by darkfield microscopy and can be divided into long range diffusion trajectories and confined diffusion trajectories.The radius of gyration of all 500 trajectories in this representative analysis showed a small value distribution with a mean radius of gyration of 0.5 micrometers and the max displacement was more distributed at small values. As demonstrated in this ensemble time average mean square displacement analysis, the CTAB coated gold nanorods normally diffuse with an alpha of approximately one. The density distribution of the diffusion coefficient and the alpha obtained from all of the trajectories, however, reveals that the dynamic of the gold nanorods exhibits a heterogeneous distribution with superdiffusion, Brownian, and subdiffusion motions.In addition, single particle statistical analysis and time series parameter analysis can be used to characterize two representative long-term confined and moving trajectories. It is necessary to summarize and extract a single novel interpretation of data from on the single particle tracking analysis readout as there are typically some difficult tradeoffs to make.

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Biochemistry

A Fluorescence Fluctuation Spectroscopy Assay of Protein-Protein Interactions at Cell-Cell Contacts

A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring cells. Of interest, only few assays are capable of specifically probing such interactions directly in living cells. Here, we present an assay to measure the binding of proteins expressed at the surfaces of neighboring cells, at cell-cell contacts. This assay consists of two steps: mixing of cells expressing the proteins of interest fused to different fluorescent proteins, followed by fluorescence fluctuation spectroscopy measurements at cell-cell contacts using a confocal laser scanning microscope. We demonstrate the feasibility of this assay in a biologically relevant context by measuring the interactions of the amyloid precursor-like protein 1 (APLP1) across cell-cell junctions. We provide detailed protocols on the data acquisition using fluorescence-based techniques (scanning fluorescence cross-correlation spectroscopy, cross-correlation number and brightness analysis) and the required instrument calibrations. Further, we discuss critical steps in the data analysis and how to identify and correct external, spurious signal variations, such as those due to photobleaching or cell movement.
In general, the presented assay is applicable to any homo- or heterotypic protein-protein interaction at cell-cell contacts, between cells of the same or different types and can be implemented on a commercial confocal laser scanning microscope. An important requirement is the stability of the system, which needs to be sufficient to probe diffusive dynamics of the proteins of interest over several minutes.

This protocol describes a fluorescence fluctuation spectroscopy-based approach to investigate interactions among proteins mediating cell-cell interactions, i.e. proteins localized in cell junctions, directly in living cells. We provide detailed guidelines on instrument calibration, data acquisition and analysis, including corrections to possible artefact sources.

A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring ...

Probing The Structure And Dynamics Of Nucleosomes Using Atomic Force Microscopy Imaging

Chromatin, which is a long chain of nucleosome subunits, is a dynamic system that allows for such critical processes as DNA replication and transcription to take place in eukaryotic cells. The dynamics of nucleosomes provides access to the DNA by replication and transcription machineries, and critically contributes to the molecular mechanisms underlying chromatin functions. Single-molecule studies such as atomic force microscopy (AFM) imaging have contributed significantly to our current understanding of the role of nucleosome structure and dynamics. The current protocol describes the steps enabling high-resolution AFM imaging techniques to study the structural and dynamic properties of nucleosomes. The protocol is illustrated by AFM data obtained for the centromere nucleosomes in which H3 histone is replaced with its counterpart centromere protein A (CENP-A). The protocol starts with the assembly of mono-nucleosomes using a continuous dilution method. The preparation of the mica substrate functionalized with aminopropyl silatrane (APS-mica) that is used for the nucleosome imaging is critical for the AFM visualization of nucleosomes described and the procedure to prepare the substrate is provided. Nucleosomes deposited on the APS-mica surface are first imaged using static AFM, which captures a snapshot of the nucleosome population. From analyses of these images, such parameters as the size of DNA wrapped around the nucleosomes can be measured and this process is also detailed. The time-lapse AFM imaging procedure in the liquid is described for the high-speed time-lapse AFM that can capture several frames of nucleosome dynamics per second. Finally, the analysis of nucleosome dynamics enabling the quantitative characterization of the dynamic processes is described and illustrated.

Here, we present a protocol to characterize nucleosome particles at the single-molecule level using static and time-lapse atomic force microscopy (AFM) imaging techniques. The surface functionalization method described allows for the capture of the structure and dynamics of nucleosomes in high-resolution at the nanoscale.

Chromatin, which is a long chain of nucleosome subunits, is a dynamic system that allows for such critical processes as DNA replication and transcription ...

Single Liposome Measurements for the Study of Proton-Pumping Membrane Enzymes Using Electrochemistry and Fluorescent Microscopy

Proton-pumping enzymes of electron transfer chains couple redox reactions to proton translocation across the membrane, creating a proton-motive force used for ATP production. The amphiphilic nature of membrane proteins requires particular attention to their handling, and reconstitution into the natural lipid environment is indispensable when studying membrane transport processes like proton translocation. Here, we detail a method that has been used for the investigation of the proton-pumping mechanism of membrane redox enzymes, taking cytochrome bo3 from Escherichia coli as an example. A combination of electrochemistry and fluorescence microscopy is used to control the redox state of the quinone pool and monitor pH changes in the lumen. Due to the spatial resolution of fluorescent microscopy, hundreds of liposomes can be measured simultaneously while the enzyme content can be scaled down to a single enzyme or transporter per liposome. The respective single enzyme analysis can reveal patterns in the enzyme functional dynamics that might be otherwise hidden by the behavior of the whole population. We include a description of a script for automated image analysis.

Here, we present a protocol to study the molecular mechanism of proton translocation across lipid membranes of single liposomes, using cytochrome bo3 as an example. Combining electrochemistry and fluorescence microscopy, pH changes in the lumen of single vesicles, containing single or multiple enzyme, can be detected and analyzed individually.

Proton-pumping enzymes of electron transfer chains couple redox reactions to proton translocation across the membrane, creating a proton-motive force used ...

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 ...

Evaluation of Protein&#8211;Protein Interactions using an On-Membrane Digestion Technique

Numerous intracellular proteins physically interact in accordance with their intracellular and extracellular circumstances. Indeed, cellular functions largely depend on intracellular protein&#8211;protein interactions. Therefore, research regarding these interactions is indispensable to facilitating the understanding of physiologic processes. Co-precipitation of associated proteins, followed by mass spectrometry (MS) analysis, enables the identification of novel protein interactions. In this study, we have provided details of the novel technique of immunoprecipitation-liquid chromatography (LC)-MS/MS analysis combined with on-membrane digestion for the analysis of protein&#8211;protein interactions. This technique is suitable for crude immunoprecipitants and can improve the throughput of proteomic analyses. Tagged recombinant proteins were precipitated using specific antibodies; next, immunoprecipitants blotted onto polyvinylidene difluoride membrane pieces were subjected to reductive alkylation. Following trypsinization, the digested protein residues were analyzed using LC-MS/MS. Using this technique, we were able to identify several candidate associated proteins. Thus, this method is convenient and useful for the characterization of novel protein&#8211;protein interactions.

Evaluation of Protein&amp;#8211;Protein Interactions using an On-Membrane Digestion Technique

Here, we present a protocol for an on-membrane digestion technique for the preparation of samples for mass spectrometry. This technique facilitates the convenient analysis of protein&#8211;protein interactions.

Numerous intracellular proteins physically interact in accordance with their intracellular and extracellular circumstances. Indeed, cellular functions ...

Visualizing Surface T-Cell Receptor Dynamics Four-Dimensionally Using Lattice Light-Sheet Microscopy

The signaling and function of a cell are dictated by the dynamic structures and interactions of its surface receptors. To truly understand the structure-function relationship of these receptors in situ, we need to visualize and track them on the live cell surface with enough spatiotemporal resolution. Here we show how to use recently developed Lattice Light-Sheet Microscopy (LLSM) to image T-cell receptors (TCRs) four-dimensionally (4D, space and time) at the live cell membrane. T cells are one of the main effector cells of the adaptive immune system, and here we used T cells as an example to show that the signaling and function of these cells are driven by the dynamics and interactions of the TCRs. LLSM allows for 4D imaging with unprecedented spatiotemporal resolution. This microscopy technique therefore can be generally applied to a wide array of surface or intracellular molecules of different cells in biology.

The goal of this protocol is to show how to use Lattice Light-Sheet Microscopy to four-dimensionally visualize surface receptor dynamics in live cells. Here T cell receptors on CD4+ primary T cells are shown.

The signaling and function of a cell are dictated by the dynamic structures and interactions of its surface receptors. To truly understand the ...

Production of Dynein and Kinesin Motor Ensembles on DNA Origami Nanostructures for Single Molecule Observation

Cytoskeletal motors are responsible for a wide variety of functions in eukaryotic cells, including mitosis, cargo transport, cellular motility, and others. Many of these functions require motors to operate in ensembles. Despite a wealth of knowledge about the mechanisms of individual cytoskeletal motors, comparatively less is known about the mechanisms and emergent behaviors of motor ensembles, examples of which include changes to ensemble processivity and velocity with changing motor number, location, and configuration. Structural DNA nanotechnology, and the specific technique of DNA origami, enables the molecular construction of well-defined architectures of motor ensembles. The shape of cargo structures as well as the type, number and placement of motors on the structure can all be controlled. Here, we provide detailed protocols for producing these ensembles and observing them using total internal reflection fluorescence microscopy. Although these techniques have been specifically applied for cytoskeletal motors, the methods are generalizable to other proteins that assemble in complexes to accomplish their tasks. Overall, the DNA origami method for creating well-defined ensembles of motor proteins provides a powerful tool for dissecting the mechanisms that lead to emergent motile behavior.

The goal of this protocol is to form ensembles of molecular motors on DNA origami nanostructures and observe the ensemble motility using total internal reflection fluorescence microscopy.

Cytoskeletal motors are responsible for a wide variety of functions in eukaryotic cells, including mitosis, cargo transport, cellular motility, and ...

A Model Membrane Platform for Reconstituting Mitochondrial Membrane Dynamics

Mitochondrial dynamics is essential for the organelle&#8217;s diverse functions and cellular responses. The crowded, spatially complex, mitochondrial membrane is a challenging environment to distinguish regulatory factors. Experimental control of protein and lipid components can help answer specific questions of regulation. Yet, quantitative manipulation of these factors is challenging in cellular assays. To investigate the molecular mechanism of mitochondria inner-membrane fusion, we introduced an in vitro reconstitution platform that mimics the lipid environment of the mitochondrial inner-membrane. Here we describe detailed steps for preparing lipid bilayers and reconstituting mitochondrial membrane proteins. The platform allowed analysis of intermediates in mitochondrial inner-membrane fusion, and the kinetics for individual transitions, in a quantitative manner. This protocol describes the fabrication of bilayers with asymmetric lipid composition and describes general considerations for reconstituting transmembrane proteins into a cushioned bilayer. The method may be applied to study other membrane systems.

Mitochondrial fusion is an important homeostatic reaction underlying mitochondrial dynamics. Described here is an in vitro reconstitution system to study mitochondrial inner-membrane fusion that can resolve membrane tethering, docking, hemifusion, and pore opening. The versatility of this approach in exploring cell membrane systems is discussed.

Mitochondrial dynamics is essential for the organelle&#8217;s diverse functions and cellular responses. The crowded, spatially complex, mitochondrial ...

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;

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 ...

Simultaneous Visualization of the Dynamics of Crosslinked and Single Microtubules In Vitro by TIRF Microscopy

Microtubules are polymers of &#945;&#946;-tubulin heterodimers that organize into distinct structures in cells. Microtubule-based architectures and networks often contain subsets of microtubule arrays that differ in their dynamic properties. For example, in dividing cells, stable bundles of crosslinked microtubules coexist in close proximity to dynamic non-crosslinked microtubules. TIRF-microscopy-based in vitro reconstitution studies enable the simultaneous visualization of the dynamics of these different microtubule arrays. In this assay, an imaging chamber is assembled with surface-immobilized microtubules, which are either present as single filaments or organized into crosslinked&#160;bundles. Introduction of tubulin, nucleotides, and protein regulators allows direct visualization of associated proteins and of dynamic properties of single and crosslinked microtubules. Furthermore, changes that occur as dynamic single microtubules organize into bundles can be monitored in real-time. The method described here allows for a systematic evaluation of the activity and localization of individual proteins, as well as synergistic effects of protein regulators on two different microtubule subsets under identical experimental conditions, thereby providing mechanistic insights that are inaccessible by other methods.

Simultaneous Visualization of the Dynamics of Crosslinked and Single Microtubules In Vitro by TIRF Microscopy

Here, a TIRF microscopy-based in vitro reconstitution assay is presented to simultaneously quantify and compare the dynamics of two microtubule populations. A method is described to simultaneously view the collective activity of multiple microtubule-associated proteins on crosslinked microtubule bundles and single microtubules.

Microtubules are polymers of &#945;&#946;-tubulin heterodimers that organize into distinct structures in cells. Microtubule-based architectures and ...