Name: generation and assembly of virus-specific nucleocapsids of the respiratory syncytial virus
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The research programs in the Liang laboratory at Emory are supported by the US National Institute of General Medical Sciences (NIGMS), National Institutes of Health (NIH) under award number R01GM130950, and the Research Start-Up Fund at Emory University School of Medicine. The author acknowledges the members of the Liang laboratory for helpful support and critical discussion.

1. Molecular cloning
NOTE: Ligase Independent Cloning (LIC) was used to make an RSV bi-cistronic coexpression construct plasmid. LIC is a method developed in the early 1990s, which uses the 3&#8217;-5&#8217; Exo activity of the T4 DNA&#160; polymerase to create overhangs with complementarity between the vector and the DNA insert21,22. The constructs were made using the 2BT-10 vector DNA, which consists of a 10x His tag a...

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The known nucleocapsid-like particle (NCLP) structures of the nonsegmented negative-sense (NNS) RNA viruses show that the assembled NCLPs are the complex N with host cellular RNAs when overexpressed in bacterial or eukaryotic expression systems15,16,17,18,19. Previous studies have attempted to get the RNA free N with a variety of methods, such as the RNase A d...

Nonsegmented negative-sense (NNS) RNA viruses include many significant human pathogens, such as rabies, Ebola, and respiratory syncytial virus (RSV)1,2. RSV is the leading cause of respiratory illness such as bronchiolitis and pneumonia in young children and older adults worldwide3. Currently, no effective vaccines or antiviral therapies are available to prevent or treat RSV4. As part of the life cycle, the RSV genome serves as the template for replication by the RSV RNA dependent RNA polymerase to produce an antigenome, which in turn acts as the template to generate a progeny genome. Both genome and antigenome RNAs are entirely encapsidated by the nucleoprotein (N) to form the nucleocapsids (NCs)3. Because the NCs serve as the templates for both replication and transcription by the RSV polymerase, proper NC assembly is crucial for the polymerase to gain access to the templates for RNA synthesis5. Interestingly, based on the structural analyses of the NNS viral polymerases, it is hypothesized that several N proteins transiently dissociate from the NCs to allow the access of the polymerase and rebind to RNA after the RNA synthesis6,7,8,9,10,11,12. &#160;
Currently, the RSV RNA polymerization assay has been established using purified RSV polymerase on short naked RNA templates13,14. However, the activities of the RSV polymerase do not reach optimal, as observed in the non-processive and abortive products generated by the RSV polymerase when using naked RNA templates. The lack of NC with virus-specific RNA is a primary barrier for the further mechanistic understanding of the RSV RNA synthesis. Therefore, using an authentic RNA template becomes a critical need to advance the fundamental knowledge of RSV RNA synthesis. The known structures of the nucleocapsid-like particles (NCLPs) from RSV and other NNS RNA viruses reveal that the RNAs in the NCLPs are either random cellular RNAs or average viral genomic RNAs15,16,17,18,19. Together, the main hurdle is that N binds non-specifically to cellular RNAs to form NCLPs when N is overexpressed in the host cells.
To overcome this hurdle, we established a protocol to obtain RNA-free (N0) first and assemble N0 with authentic viral genomic RNA into NCLPs20. The principle of this protocol is to obtain a large quantity of recombinant RNA-free N (N0) by co-expressing N with a chaperone, the N-terminal domain of RSV phosphoprotein (PNTD). The purified N0P could be stimulated and assembled into NCLPs by adding RSV-specific RNA oligos, and during the assembly process, the chaperone PNTD is displaced upon the addition of RNA oligos.
Here, we detail a protocol for the generation and assembly of RSV RNA-specific NCs. In this protocol, we describe the molecular cloning, protein preparation, in vitro assembly, and validation of the complex assembly. We highlight the cloning strategy to generate bi-cistronic constructs for protein coexpression for molecular cloning. For protein preparation, we describe the procedures of cell culture, protein extraction, and the purification of the protein complex. Then we discuss the method for in vitro assembly of the RSV RNA-specific NCs. Finally, we use size exclusion chromatography (SEC) and negative stain electron microscopy (EM) to characterize and visualize the assembled NCLPs.

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	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Agarose</td>
			<td style="width:302px;">SIgma</td>
			<td style="width:301px;">A9539-500G</td>
			<td style="width:319px;">making construct using LIC method</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Amicon Ultra-15 Centrifugal Filter Unit</td>
			<td>Millipore</td>
			<td>UFC901024</td>
			<td>concentrate the protein sample</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Ampicillin sodium</td>
			<td style="width:302px;">GOLD BIOTECHNOLOGY</td>
			<td style="width:301px;">5118.111317A</td>
			<td>antibiotic for cell culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">AseI</td>
			<td style="width:302px;">NEB</td>
			<td style="width:301px;">R0526S</td>
			<td>making construct using LIC method</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cobalt (High Density) Agarose Beads</td>
			<td style="width:302px;">Gold Bio</td>
			<td>H-310-500</td>
			<td>For purification of His-tag protein</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Corning LSE Digital Dry Bath Heater</td>
			<td style="width:302px;">CORNING</td>
			<td style="width:301px;">6885-DB</td>
			<td>Heate the sample</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">dCTP</td>
			<td style="width:302px;">Invitrogen</td>
			<td style="width:301px;">10217016</td>
			<td>making construct using LIC method</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">dGTP</td>
			<td style="width:302px;">Invitrogen</td>
			<td style="width:301px;">10218014</td>
			<td>making construct using LIC method</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Glycerol</td>
			<td style="width:302px;">Sigma</td>
			<td style="width:301px;">G5516-4L</td>
			<td>making solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">HEPES</td>
			<td style="width:302px;">Sigma</td>
			<td style="width:301px;">H3375-100G</td>
			<td>making solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">HiTrap Q HP</td>
			<td style="width:302px;">Sigma</td>
			<td style="width:301px;">GE29-0513-25</td>
			<td>Protein purification</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Imidazole</td>
			<td style="width:302px;">Sigma</td>
			<td style="width:301px;">I5513-100G</td>
			<td>making solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">IPTG (Isopropyl-beta-D-thiogalactopyranoside)</td>
			<td style="width:302px;">GOLD BIOTECHNOLOGY</td>
			<td style="width:301px;">1116.071717A</td>
			<td>induce the expression of protein</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Microcentrifuge Tubes</td>
			<td style="width:302px;">VWR</td>
			<td style="width:301px;">47730-598</td>
			<td>for PCR</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Misonix Sonicator XL2020 Ultrasonic Liquid Processor</td>
			<td style="width:302px;">SpectraLab</td>
			<td style="width:301px;">MSX-XL-2020</td>
			<td>sonicator for lysing cell</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Negative stain grids</td>
			<td style="width:302px;">Electron Microscopy Sciences</td>
			<td style="width:301px;">CF400-Cu-TH</td>
			<td>For making negative stain grids</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">New Brunswick Innova 44/44R</td>
			<td style="width:302px;">eppendorf</td>
			<td style="width:301px;">M1282-0000</td>
			<td>Shaker for culturing the cell</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Nonidet P 40 Substitute</td>
			<td style="width:302px;">Sigma</td>
			<td style="width:301px;">74385-1L</td>
			<td>making solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">OneTaq DNA Polymerase</td>
			<td style="width:302px;">NEB</td>
			<td style="width:301px;">M0480L</td>
			<td>PCR</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">QIAquick Gel Extraction Kit</td>
			<td style="width:302px;">QIAGEN</td>
			<td style="width:301px;">28706</td>
			<td>Purify DNA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">SSPI-HF</td>
			<td style="width:302px;">NEB</td>
			<td style="width:301px;">R3132S</td>
			<td>making construct using LIC method</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Superose 6 Increase 10/300 GL</td>
			<td style="width:302px;">Sigma</td>
			<td>GE29-0915-96</td>
			<td>Protein purification</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">T4 DNA polymerase</td>
			<td style="width:302px;">Sigma</td>
			<td>70099-3</td>
			<td>making construct using LIC method</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Thermo Scientific Sorvall RC 6 Plus Centrifuge</td>
			<td style="width:302px;">Fisher Scientific</td>
			<td style="width:301px;">36-101-0816</td>
			<td>Centrifuge, highest speed 20,000 rpm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Trizma hydrochloride</td>
			<td style="width:302px;">Sigma</td>
			<td style="width:301px;">T3253-250G</td>
			<td>making solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Uranyl Formate</td>
			<td style="width:302px;">Electron Microscopy Sciences</td>
			<td style="width:301px;">22451</td>
			<td>making negative stain solution</td>
		</tr>
	</tbody>
</table>

generation and assembly of virus-specific nucleocapsids of the respiratory syncytial virus

The use of an authentic RNA template is critical to advance the fundamental knowledge of viral RNA synthesis that can guide both mechanistic discovery and assay development in virology. The RNA template of nonsegmented negative-sense (NNS) RNA viruses, such as the respiratory syncytial virus (RSV), is not an RNA molecule alone but rather a nucleoprotein (N) encapsidated ribonucleoprotein complex. Despite the importance of the authentic RNA template, the generation and assembly of such a ribonucleoprotein complex remain sophisticated and require in-depth elucidation. The main challenge is that the overexpressed RSV N binds non-specifically to cellular RNAs to form random nucleocapsid-like particles (NCLPs). Here, we established a protocol to obtain RNA-free N (N0) first by co-expressing N with a chaperone phosphoprotein (P), then assembling N0 with RNA oligos with the RSV-specific RNA sequence to obtain virus-specific nucleocapsids (NCs). This protocol shows how to overcome the difficulty in the preparation of this traditionally challenging viral ribonucleoprotein complex.

Purification of RNA-free N0P protein 
With this protocol, a large-scale soluble heterodimeric RSV N0P complex can be obtained. The full length of N and N terminal part of P proteins were co-expressed with 10X His-Tag on the N protein in E. coli. N0P was purified using a cobalt column, ion exchange, and size exclusion chromatography. N0P contains both the full-length N and N terminal P but did not contain cellular RNA based on the UV absorbance A<s...

Watch this Scientific Journal Video about Generation and Assembly of Virus-Specific Nucleocapsids of the Respiratory Syncytial Virus at JoVE.com

Generation and Assembly of Virus-Specific Nucleocapsids of the Respiratory Syncytial Virus

For in-depth mechanistic analysis of the respiratory syncytial virus (RSV) RNA synthesis, we report a protocol of utilizing the chaperone phosphoprotein (P) for coexpression of the RNA-free nucleoprotein (N0) for subsequent in vitro assembly of the virus-specific nucleocapsids (NCs).

The use of an authentic RNA template is critical to advance the fundamental knowledge of viral RNA synthesis that can guide both mechanistic discovery and ...

This method utilize the various way to tackle the recombinant viral protein expression challenges using one viral protein P'as a chaperone for another viral protein N'to obtain RNA-free N proteins. The RNA-free RSV N protein is obtained before assembling the virus specific RNA into nucleocapsid in vitro. The method can also be used with other non-segmented negative sense RNA viruses.The ligation independent cloning method is a quick technique for acquiring co-expression constructs. Why or electives stain electron microscope is a quick method for checking large assemble in raw cell. We want to give you two tips.First, get high yield and solid results for each step before moving to the next step. Second, set up proper programs for chromatography and practice picking up grids with forceps. For bi-cistronic construction of the N and P co-expression, grow four one liter cultures of E coli BL21DE3 strain cells in LB medium at 37 degrees Celsius.When the optical density at 600 nanometers reaches 0.6, lower the temperature to 16 degrees Celsius. After one hour, induce protein expression by treatment with 0.5 millimolar IPTG overnight. The next morning, collect the cells by centrifugation and resuspend the pellets in 200 milliliters of lysis buffer.lyse the cells by sonication for 15 minutes, with three seconds on, three seconds off pulses. And collect the lysates by centrifugation. Load the supernatant into a 2.5 by 10 centimeter cobalt gravity column, with approximately 10 milliliters of beads.Pre-equilibrated with 5 to 10 column volumes of lysis buffer. And wash the column with five column volumes of buffer B and five volumes of buffer C.Use two volumes of buffer D to elute the protein from the beads. And equilibrate the Q column with five one milliliter volumes of QA buffer.Use a peristaltic pump to load the diluted sample onto the column. Melt the loaded Q column onto the HPLC machine, along with fresh QA and QB buffers. Run the pump wash to successfully wash the machine with one to two volumes of QB and QA buffers.After the last wash, set the flow rate to one milliliter per minute, and set UV1 to 280 nanometers and UV2 to 260 nanometers, using a 96 deep well plate to collect the fractions. Then use a stepwise gradient application of three to four volumes of each concentration of elution agent to elute the proteins, starting at a 0%QB concentration and increasing the percentage by 5%each time. The N-0 P protein complex will elute at the 15%QB buffer concentration.Isolate the protein by gel filtration, in a one by 30 centimeters small scale purification column, equilibrated with buffer E.Then analyze the protein containing fractions by SDS page. For virus specific nucleocapsid assembly, mix and incubate the purified N-0 P complex with RNA oligo at a 1:1.5 molecular ratio at room temperature for one hour. Pre-equilibrate a small scale purification gel filtration column with buffer E.And remove any precipitation by centrifugation.Load the supernatant, to the size exclusion chromatography column. And compare the size exclusion chromatography images of the protein to RNA assembly, and protein alone control samples, combining the nucleic acid purity ratios, to identify which peaks are the assembled N RNA, N-0 P and free RNA. To assemble an N RNA complex, collect all of the N RNA peak fractions, perform an RNA extraction, and run a urea page gel to double-check the length of the specific RNA.To prepare a negative stain grid for negative stain electron microscopy, Use a heat plate to heat 5 mL of double distilled water to boiling and add 37.5 milligrams of uranyl formate to the water to obtain a 0.75%uranyl formate staining solution. Transfer the solution to an aluminum foil covered beaker. And add four microliters of 10 molar sodium hydroxide.After 15 minutes of stirring protected from light, filter the solution through a 0.22 micron filter into a test tube. To make the continuous carbon coated electron microscopy grids hydrophilic, place the grids in a chamber connected to a power supply and apply negatively charged ions to the grids. Cut and fold a two by two inch parafilm strip.Add two 40 microliter drops of distilled water to one end of the strip. And two 40 microliter drops of 0.75%uranyl formate staining solution to the other end. Add three microliters of protein sample to each grid.After one minute, block the grids against blotting paper and dip the grids two times in the distilled water droplets. And once in the first staining solution droplet. Then immerse the grid in the second staining solution droplet for 30 seconds, before blotting the grids against blotting paper to remove any excess stain solution and allowing the grids to air-dry before imaging.Using this protocol, a large-scale soluble hetero dimeric respiratory syncytial virus N0 P complex can be obtained. In E coli, the full length of N and N-terminal portions of the P proteins are co-expressed with a 10 X His tag on the N protein. Based on the UV absorbance nucleic acid purity ratio, N0 P contained both the full length N and N-terminal P, but did not contain cellular RNA.The purified N0 P could then be stimulated and assembled into nucleocapsid like particles on electron microscopy grids via incubation with specific RNA oligos as demonstrated. When purifying the protein avoid air bubbles during the column purification and dilute the sample with QA buffer to adjust the pH and salt concentration before loading the sample onto the column. Take care to hold the electron microscopy grids at the outer edge to avoid contaminating the grids.This will help us to determine the non-positive RSV genome packing questions, whether it is left handed or right handed helical structure. Following those procedures and authentic an RNA template for the RSV polymerase activity assay can be performed. And this RSV viral specific nucleocapsid can be used for the high resolution cryo EM analysis.

generation-assembly-virus-specific-nucleocapsids-respiratory

Research

JoVE Journal

Biochemistry

Examining Proteasome Assembly with Recombinant Archaeal Proteasomes and Nondenaturing PAGE:  The Case for a Combined Approach

Proteasomes are found in all domains of life. They provide the major route of intracellular protein degradation in eukaryotes, though their assembly is not completely understood. All proteasomes contain a structurally conserved core particle (CP), or 20S proteasome, containing two heptameric&#160;&#946; subunit rings sandwiched between two heptameric&#160;&#945; subunit rings. Archaeal 20S proteasomes are compositionally simpler compared to their eukaryotic counterparts, yet they both share a common assembly mechanism. Consequently, archaeal 20S proteasomes continue to be important models for eukaryotic proteasome assembly. Specifically, recombinant expression of archaeal 20S proteasomes coupled with nondenaturing polyacrylamide gel electrophoresis (PAGE) has yielded many important insights into proteasome biogenesis. Here, we discuss a means to improve upon the usual strategy of coexpression of archaeal proteasome &#945; and &#946; subunits prior to nondenaturing PAGE. We demonstrate that although rapid and efficient, a coexpression approach alone can miss key assembly intermediates. In the case of the proteasome, coexpression may not allow detection of the half-proteasome, an intermediate containing one complete &#945;-ring and one complete &#946;-ring. However, this intermediate is readily detected via lysate mixing. We suggest that combining coexpression with lysate mixing yields an approach that is more thorough in analyzing assembly, yet remains labor nonintensive. This approach may be useful for the study of other recombinant multiprotein complexes.

This protocol uses both subunit coexpression and postlysis subunit mixing for a more thorough examination of recombinant proteasome assembly.

Proteasomes are found in all domains of life. They provide the major route of intracellular protein degradation in eukaryotes, though their assembly is ...

Isolation and Respiratory Measurements of Mitochondria from Arabidopsis thaliana

Mitochondria are essential organelles involved in numerous metabolic pathways in plants, most notably the production of adenosine triphosphate (ATP) from the oxidation of reduced compounds such as nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FADH2). The complete annotation of the Arabidopsis thaliana genome has established it as the most widely used plant model system, and thus the need to purify mitochondria from a variety of organs (leaf, root, or flower) is necessary to fully utilize the tools that are now available for Arabidopsis to study mitochondrial biology. Mitochondria are isolated by homogenization of the tissue using a variety of approaches, followed by a series of differential centrifugation steps producing a crude mitochondrial pellet that is further purified using continuous colloidal density gradient centrifugation. The colloidal density material is subsequently removed by multiple centrifugation steps. Starting from 100 g of fresh leaf tissue, 2 - 3 mg of mitochondria can be routinely obtained. Respiratory experiments on these mitochondria display typical rates of 100 - 250 nmol O2 min-1 mg total mitochondrial protein-1 (NADH-dependent rate) with the ability to use various substrates and inhibitors to determine which substrates are being oxidized and the capacity of the alternative and cytochrome terminal oxidases. This protocol describes an isolation method of mitochondria from Arabidopsis thaliana leaves using continuous colloidal density gradients and an efficient respiratory measurements of purified plant mitochondria.

Isolation and Respiratory Measurements of Mitochondria from Arabidopsis thaliana

As mitochondria are only a small percentage of the plant cell, they need to be purified for a range of studies. Mitochondria can be isolated from a variety of plant organs by homogenization, followed by differential and density gradient centrifugation to obtain a highly purified mitochondrial fraction.

Mitochondria are essential organelles involved in numerous metabolic pathways in plants, most notably the production of adenosine triphosphate (ATP) from ...

Manipulating Living Cells to Construct Stable 3D Cellular Assembly Without Artificial Scaffold

Regenerative medicine and tissue engineering offer several advantages for the treatment of intractable diseases, and several studies have demonstrated the importance of 3-dimensional (3D) cellular assemblies in these fields. Artificial scaffolds have often been used to construct 3D cellular assemblies. However, the scaffolds used to construct cellular assemblies are sometimes toxic and may change the properties of the cells. Thus, it would be beneficial to establish a non-toxic method for facilitating cell-cell contact. In this paper, we introduce a novel method for constructing stable cellular assemblies by using optical tweezers with dextran. One of the advantages of this method is that it establishes stable cell-to-cell contact within a few minutes. This new method allows the construction of 3D cellular assemblies in a natural hydrophilic polymer and is expected to be useful for constructing next-generation 3D single-cell assemblies in the fields of regenerative medicine and tissue engineering.

We demonstrate a novel method for constructing a single-cell-based 3-dimensional (3D) assembly without an artificial scaffold.

Regenerative medicine and tissue engineering offer several advantages for the treatment of intractable diseases, and several studies have demonstrated the ...

Hybrid Clear/Blue Native Electrophoresis for the Separation and Analysis of Mitochondrial Respiratory Chain Supercomplexes

Complexes of the oxidative phosphorylation machinery form supramolecular protein arrangements named supercomplexes (SCs), which are believed to confer structural and functional advantages to mitochondria. SCs have been identified in many species, from yeast to mammal, and an increasing number of studies report disruption of their organization in genetic and acquired human diseases. As a result, an increasing number of laboratories are interested in analyzing SCs, which can be methodologically challenging. This article presents an optimized protocol that combines the advantages of Blue- and Clear-Native PAGE methods to resolve and analyze SCs in a time-effective manner. With this hybrid CN/BN-PAGE method, mitochondrial SCs extracted with optimal amounts of the mild detergent digitonin are exposed briefly to the anionic dye Coomassie Blue (CB) at the beginning of the electrophoresis, without exposure to other detergents. This short exposure to CB allows to separate and resolve SCs as effectively as with traditional BN-PAGE methods, while avoiding the negative impact of high CB levels on in-gel activity assays, and labile protein-protein interactions within SCs. With this protocol it is thus possible to combine precise and rapid in gel activity measurements with analytical techniques involving 2D electrophoresis, immuno-detection, and/or proteomics for advanced analysis of SCs.

Here we present a protocol to extract, resolve and identify mitochondrial supercomplexes which minimizes exposure to detergents and Coomassie Blue. This protocol offers an optimal balance between resolution, and preservation of enzyme activities, while minimizing the risk of losing labile protein-protein interactions.

Complexes of the oxidative phosphorylation machinery form supramolecular protein arrangements named supercomplexes (SCs), which are believed to confer ...

Real-Time, Semi-Automated Fluorescent Measurement of the Airway Surface Liquid pH of Primary Human Airway Epithelial Cells

In recent years, the importance of mucosal surface pH in the airways has been highlighted by its ability to regulate airway surface liquid (ASL) hydration, mucus viscosity and activity of antimicrobial peptides, key parameters involved in innate defense of the lungs. This is of primary relevance in the field of chronic respiratory diseases such as cystic fibrosis (CF) where these parameters are dysregulated. While different groups have studied ASL pH both in vivo and in vitro, their methods report a relatively wide range of ASL pH values and even contradictory findings regarding any pH differences between non-CF and CF cells. Furthermore, their protocols do not always provide enough details in order to ensure reproducibility, most are low throughput and require expensive equipment or specialized knowledge to implement, making them difficult to establish in most labs. Here we describe a semi-automated fluorescent plate reader assay that enables the real-time measurement of ASL pH under thin film conditions that more closely resemble the in vivo situation. This technique allows for stable measurements for many hours from multiple airway cultures simultaneously and, importantly, dynamic changes in ASL pH in response to agonists and inhibitors can be monitored. To achieve this, the ASL of fully differentiated primary human airway epithelial cells (hAECs) are stained overnight with a pH-sensitive dye in order to allow for the reabsorption of the excess fluid to ensure thin film conditions. After fluorescence is monitored in the presence or absence of agonists, pH calibration is performed in situ to correct for volume and dye concentration. The method described provides the required controls to make stable and reproducible ASL pH measurements, which ultimately could be used as a drug discovery platform for personalized medicine, as well as adapted to other epithelial tissues and experimental conditions, such as inflammatory and/or host-pathogen models.

We present a protocol to make dynamic measurements of the airway surface liquid pH under thin film conditions using a plate-reader.

In recent years, the importance of mucosal surface pH in the airways has been highlighted by its ability to regulate airway surface liquid (ASL) ...

Deciphering Molecular Mechanism of Histone Assembly by DNA Curtain Technique

Chromatin is a higher-order structure that packages eukaryotic DNA. Chromatin undergoes dynamic alterations according to the cell cycle phase and in response to environmental stimuli. These changes are essential for genomic integrity, epigenetic regulation, and DNA metabolic reactions such as replication, transcription, and repair. Chromatin assembly is crucial for chromatin dynamics and is catalyzed by histone chaperones. Despite extensive studies, the mechanisms by which histone chaperones enable chromatin assembly remains elusive. Moreover, the global features of nucleosomes organized by histone chaperones are poorly understood. To address these problems, this work describes a unique single-molecule imaging technique named DNA curtain, which facilitates the investigation of the molecular details of nucleosome assembly by histone chaperones. DNA curtain is a hybrid technique that combines lipid fluidity, microfluidics, and total internal reflection fluorescence microscopy (TIRFM) to provide a universal platform for real-time imaging of diverse protein-DNA interactions.Using DNA curtain, the histone chaperone function of Abo1, the Schizosaccharomyces pombe bromodomain-containing AAA+ ATPase, is investigated, and the molecular mechanism underlying histone assembly of Abo1 is revealed. DNA curtain provides a unique approach for studying chromatin dynamics.

DNA curtain, a high-throughput single-molecule imaging technique, provides a platform for real-time visualization of diverse protein-DNA interactions. The present protocol utilizes the DNA curtain technique to investigate the biological role and molecular mechanism of Abo1, a Schizosaccharomyces pombe bromodomain-containing AAA+ ATPase.

Chromatin is a higher-order structure that packages eukaryotic DNA. Chromatin undergoes dynamic alterations according to the cell cycle phase and in ...

Advancing High-Resolution Imaging of Virus Assemblies in Liquid and Ice

Interest in liquid-electron microscopy (liquid-EM) has skyrocketed in recent years as scientists can now observe real-time processes at the nanoscale. It is extremely desirable to pair high-resolution cryo-EM information with dynamic observations as many events occur at rapid timescales - in the millisecond range or faster. Improved knowledge of flexible structures can also assist in the design of novel reagents to combat emerging pathogens, such as SARS-CoV-2. More importantly, viewing biological materials in a fluid environment provides a unique glimpse of their performance in the human body. Presented here are newly developed methods to investigate the nanoscale properties of virus assemblies in liquid and vitreous ice. To accomplish this goal, well-defined samples were used as model systems. Side-by-side comparisons of sample preparation methods and representative structural information are presented. Sub-nanometer features are shown for structures resolved in the range of ~3.5-&#197;-10 &#197;. Other recent results that support this complementary framework include dynamic insights of vaccine candidates and antibody-based therapies imaged in liquid. Overall, these correlative applications advance our ability to visualize molecular dynamics, providing a unique context for their use in human health and disease.

Here protocols are described to prepare virus assemblies suitable for liquid-EM and cryo-EM analysis at the nanoscale using transmission electron microscopy.

Interest in liquid-electron microscopy (liquid-EM) has skyrocketed in recent years as scientists can now observe real-time processes at the nanoscale. It ...

Using Microfluidics and Fluorescence Microscopy to Study the Assembly Dynamics of Single Actin Filaments and Bundles

In order to decipher the complex molecular mechanisms that regulate the assembly and disassembly of actin filaments, it is a great asset to monitor individual reactions live in well-controlled conditions. To do so, live single-filament experiments have emerged over the past 20 years, mostly using total internal reflection fluorescence (TIRF) microscopy, and have provided a trove of key results. In 2011, in order to further expand the possibilities of these experiments and to avoid recurring problematic artifacts, we introduced simple microfluidics in these assays. This study details our basic protocol, where individual actin filaments are anchored by one end to the passivated coverslip surface, align with the flow, and can be successively exposed to different protein solutions. We also present the protocols for specific applications and explain how controlled mechanical forces can be applied, thanks to the viscous drag of the flowing solution. We highlight the technical caveats of these experiments and briefly present possible developments based on this technique. These protocols and explanations, along with today&#39;s availability of easy-to-use microfluidics equipment, should allow non-specialists to implement this assay in their labs.

We present protocols for simple actin filament microfluidic assays, in combination with fluorescence microscopy, that allow one to accurately monitor individual actin filaments in real-time while sequentially exposing them to different protein solutions.

In order to decipher the complex molecular mechanisms that regulate the assembly and disassembly of actin filaments, it is a great asset to monitor ...

Self-Assembly of Microtubule Tactoids

The cytoskeleton is responsible for major internal organization and re-organization within the cell, all without a manager to direct the changes. This is especially the case during mitosis or meiosis, where the microtubules form the spindle during cell division. The spindle is the machinery used to segregate genetic material during cell division. Toward creating self-organized spindles in vitro, we recently developed a technique to reconstitute microtubules into spindle-like assemblies with a minimal set of microtubule-associated proteins and crowding agents. Specifically, MAP65 was used, which is an antiparallel microtubule crosslinker from plants, a homolog of Ase1 from yeast and PRC1 from mammalian organisms. This crosslinker self-organizes microtubules into long, thin, spindle-like microtubule self-organized assemblies. These assemblies are also similar to liquid crystal tactoids, and microtubules could be used as mesoscale mesogens. Here, protocols are presented for creating these microtubule tactoids, as well as for characterizing the shape of the assemblies using fluorescence microscopy and the mobility of the constituents using fluorescence recovery after photobleaching.

This article presents a protocol for the formation of microtubule assemblies in the shape of tactoids using MAP65, a plant-based microtubule crosslinker, and PEG as a crowding agent.

The cytoskeleton is responsible for major internal organization and re-organization within the cell, all without a manager to direct the changes. This is ...

Reconstitution of Septin Assembly at Membranes to Study Biophysical Properties and Functions

Most cells can sense and change their shape to carry out fundamental cell processes. In many eukaryotes, the septin cytoskeleton is an integral component in coordinating shape changes like cytokinesis, polarized growth, and migration. Septins are filament-forming proteins that assemble to form diverse higher-order structures and, in many cases, are found in different areas of the plasma membrane, most notably in regions of micron-scale positive curvature. Monitoring the process of septin assembly in vivo is hindered by the limitations of light microscopy in cells, as well as the complexity of interactions with both membranes and cytoskeletal elements, making it difficult to quantify septin dynamics in living systems. Fortunately, there has been substantial progress in the past decade in reconstituting the septin cytoskeleton in a cell-free system to dissect the mechanisms controlling septin assembly at high spatial and temporal resolutions. The core steps of septin assembly include septin heterooligomer association and dissociation with the membrane, polymerization into filaments, and the formation of higher-order structures through interactions between filaments. Here, we present three methods to observe septin assembly in different contexts: planar bilayers, spherical supports, and rod supports. These methods can be used to determine the biophysical parameters of septins at different stages of assembly: as single octamers binding the membrane, as filaments, and as assemblies of filaments. We use these parameters paired with measurements of curvature sampling and preferential adsorption to understand how curvature sensing operates at a variety of length and time scales.

Cell-free reconstitution has been a key tool to understand the cytoskeleton assembly, and work in the last decade has established approaches to study septin dynamics in minimal systems. Presented here are three complementary methods to observe septin assembly in different membrane contexts: planar bilayers, spherical supports, and rod supports.

Most cells can sense and change their shape to carry out fundamental cell processes. In many eukaryotes, the septin cytoskeleton is an integral component ...