This work was supported by the University of Central Florida start-up funds and the National Heart, Lung, And Blood Institute of the National Institute of Health under award K25HL132098.

1. Making polyacrylamide (PA) gels
<ol>
	<li>Preparation of Petri dish
	<ol>
		<li>Prepare bind silane solution by mixing 200 mL ultrapure water with 80 &#181;L acetic acid and 50 &#181;L of 3-(trimethoxysilyl)propyl methacrylate. Bind silane is a solution used to functionalize the glass bottom Petri dish surface for hydrogel attachment.</li>
		<li>Stir the bind silane solution on a stir plate for at least 1 h.</li>
		<li>Treat the center well of the Petri dish with bind silane solution for 45 min.</li>
		<li>Remove bind silane solution and rinse the Petri dish with ultra-pure water 2x-3x.</li>
		<li>Dry the Petri dish surface and store at room temperature for future use.</li>
	</ol>
	</li>
	<li>Preparation of hydrogel solution
	<ol>
		<li>Mix ultra-pure water, 40% acrylamide, and 2% bis-acrylamide in a 15 mL centrifuge tube according to Table 1.</li>
		<li>Add 80 &#181;L of fluorescent beads to the hydrogel solution.</li>
		<li>Gently shake the tube to mix beads with the gel solution.</li>
		<li>Lightly tighten the tube cap on the centrifuge tube and place in a vacuum chamber.</li>
		<li>Degas the gel solution for at least 45 min in vacuum chamber.</li>
	</ol>
	</li>
	<li>Hydrogel polymerization
	<ol>
		<li>First, add 75 &#181;L of 10% ammonium persulfate (dissolved in ultra-pure water) and then add 8 &#181;L of TEMED (N,N,N&#39;,N&#39;-tetramethylethane-1,2-diamine) to the hydrogel solution.</li>
		<li>Place 24 &#181;L of hydrogel solution at the center of the Petri dish (see Table 2).</li>
		<li>Use an 18 mm coverslip to flatten the hydrogel. This will give a height of ~100 &#181;m.</li>
		<li>Wait at least 30-40 min for gel polymerization.</li>
		<li>Submerge the polymerized hydrogel in ultra-pure water to prevent gel dehydration.</li>
		<li>Cover the Petri dish with aluminum foil to prevent photobleaching of fluorescent beads and store at 4 &#176;C. 
		NOTE: Hydrogels can be stored a period of up to 3 months, but it is suggested that these gels be used no longer than 1 week after fabrication to obtain the best results.</li>
	</ol>
	</li>
</ol>
2. Cell culture
<ol>
	<li>Culture human umbilical vein endothelial cells (HUVECs) in cell culture medium 200 (see Table of Materials) supplemented with 1% penicillin-streptomycin on 0.1% gelatin-coated flasks at 37 &#176;C and 5% CO2.</li>
</ol>
3. Micropattern stencil preparation
<ol>
	<li>Cure a thin layer of polydimethylsiloxane (PDMS) in a 100 mm Petri dish by mixing the silicone base with the silicone curing agent at a ratio of 20:1 (base: curing agent). 
	NOTE: It is possible to use other base to curing agent ratios (ex. 10:1 or 30:1). However, a lower base to curing agent ratio will yield a stiffer pattern, while a higher base to curing agent ratio will produce a softer pattern.</li>
	<li>Prepare a 20:1 (base:curing agent) PDMS solution and mix carefully in a 50 mL centrifuge tube. Invert the tube upside down and shake vigorously multiple times to ensure proper mixing of the PDMS solution, as nonuniform mixing will result in incomplete polymerization.</li>
	<li>Remove bubbles that have been introduced from the above step by centrifuging the PDMS solution for 1 min at 190 x g.</li>
	<li>Pour 5-6 mL of PDMS solution in the center of a 100 mm Petri dish and agitate the dish until the PDMS solution covers the entire Petri dish surface.</li>
	<li>Cure the PDMS solution overnight at 50-60 &#176;C. PDMS can also be cured at room temperature.</li>
	<li>Remove a circular, 16 mm diameter PDMS stencil with a hole puncher.</li>
	<li>Use a biopsy punch to create small holes in the PDMS stencil. This protocol uses a 1.25 mm diameter biopsy punch.</li>
	<li>Sterilize PDMS stencils by first submerging them in 70% ethanol for 2-3 min, aspirating off excess ethanol and then placing under a UV light for 5 min.</li>
</ol>
4. Collagen-I hydrogel coating
<ol>
	<li>Remove the coverslip from the hydrogel and aspirate any excess liquid.</li>
	<li>Place the PDMS stencil on the hydrogel surface. 
	NOTE: Tweezers can be used to apply light pressure to PDMS stencil to ensure a water-tight seal between the PDMS stencil and hydrogel surface. Our PDMS stencils are water-tight and thus prevent water access between the gel top surface and PDMS stencil bottom surface. Also, the hydrogel stiffness does not need to be adjusted with respect to the PDMS stiffness.</li>
	<li>Cover the hydrogel surface with sulfosuccinimidyl-6-(4-azido-2-nitrophenylamino) hexanoate (sulfo-SANPAH) dissolved in a 0.1 M HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer solution at a concentration of 1 : 1000 and place under a UV lamp (power 36 W) for 8 min.</li>
	<li>Aspirate the excess sulfo-SANPAH and HEPES solution and rinse the hydrogel twice with 0.1 M HEPES followed by an additional two rinses with ultra-pure water.</li>
	<li>Aspirate excess ultra-pure water and coat hydrogels with 0.1 mg/mL collagen-I overnight at 4 &#176;C.</li>
	<li>Cover the dishes and protect fluorescent beads from photobleaching. 
	NOTE: PDMS stencils are used to create micropatterned monolayers. Micropatterned monolayers are utilized as they allow for multiple monolayers of the same geometry and dimensions to be observed simultaneously during each experiment. However, should micropatterns not be desired the above steps can be followed with the exception of step 4.2.</li>
</ol>
5. Creating HUVEC monolayers on hydrogels
<ol>
	<li>Use 1x trypsin to detach cells from tissue culture flasks for 3-5 min in the incubator.</li>
	<li>After trypsinization, add cell culture media to the trypsin solution and add to the 15 mL centrifuge tube.</li>
	<li>Centrifuge the cell solution for 3 min at 1710 x g. A small, white pellet of cells should be visible at the bottom of the centrifuge tube.</li>
	<li>Aspirate the supernatant and resuspend cells in media to a concentration of 50 x 104 cells/ mL.</li>
	<li>Remove collagen-I from the hydrogel and rinse 1x with PBS.</li>
	<li>Add 75 x 103 cells to the top of the PDMS stencil and allow cells to attach to the hydrogel surface for at least 1 h in the incubator at 37 &#176;C and 5% CO2.</li>
	<li>Remove the PDMS stencil and add at least 2 mL of media to the Petri dish. Submerge the PDMS stencil in 10x trypsin to remove any attached cells and sterilize by spraying with 70% ethanol and then placing under the UV light for 5 min.</li>
	<li>Place the Petri dish in the incubator and wait at least 36 h or until a confluent monolayer is observed.</li>
</ol>
6. 2,5 dihydroxychalcone treatment for Cx43 disruption
<ol>
	<li>Dissolve 2,5 dihydroxychalcone (chalcone) in dimethylsulfoxide (DMSO) to make a 0.1875 mg/mL stock solution.</li>
	<li>Dilute the stock solution with cell culture media to make a low chalcone concentration (0.2 &#181;g/mL) aliquot and a high chalcone concentration (2 &#181;g/mL) aliquot.</li>
</ol>
7. Data acquisition
<ol>
	<li>Locate cell islands with a microscope.</li>
	<li>Acquire phase contrast and bead images to image cell morphology and hydrogel displacements, respectively. 
	NOTE: This protocol used a 10x objective for data acquisition.</li>
	<li>At the end of the experiment, detach cells with 10x trypsin and acquire an image of the gel surface without cells (reference image).</li>
</ol>
8. Immunostaining
<ol>
	<li>Fix monolayers with 4% formaldehyde and incubate at 37 &#176;C for 15 min.</li>
	<li>Remove 4% formaldehyde and add 0.2% Triton X-100 for 5 min at 37 &#176;C to permeabilize cells.</li>
	<li>Remove 0.2% Triton X-100 and rinse monolayers with PBS 2x-3x.</li>
	<li>Cover monolayer with 2% bovine serum albumin (BSA) solution for 45 min at 37 &#176;C.</li>
	<li>Remove the 2% BSA solution and rinse monolayers with PBS 2x-3x.</li>
	<li>Add primary Cx43 antibody at a concentration of 1:400 to the sample and incubate overnight at 4 &#176;C.</li>
	<li>Remove primary antibody and rinse sample with PBS 2x-3x.</li>
	<li>Add secondary antibody at a concentration of 3:200 and incubate for 2 h at 37 &#176;C. 
	NOTE: Samples should be covered to prevent photobleaching.</li>
	<li>Remove secondary antibody and rinse with PBS 2x-3x.</li>
	<li>Cover sample with mounting medium (Fluromount-G DAPI) and seal with an 18 mm cover slip.</li>
</ol>
9. Implementation of traction force microscopy (TFM) and monolayer stress microscopy (MSM)
<ol>
	<li>Hydrogel deformation calculation 
	NOTE: A step-by-step displacement calculation procedure using MATLAB is given below.
	<ol>
		<li>Open the main traction.m file in MATLAB (for all the MATLAB routines see Supplementary Materials). 
		NOTE: Follow instructions provided in the code to set the MATLAB directory.</li>
		<li>Define the following variables: image format, pixel-to-micron conversion, Young&#39;s modulus, Poisson&#39;s ratio, and microscope objective.</li>
		<li>Locate the bead, trypsin image, and phase image using the OpenFiles subroutine. 
		NOTE: For large data sets, it is best to name files in sequential order. For example, files should be named &#34;filename1.tif&#34;, &#34;filename2.tif&#34;, etc.</li>
		<li>Define a square ROI (region of interest) around the cell monolayer and execute the cell_cropper subroutine to crop the original image.</li>
		<li>Execute the displacement_finder subroutine to compute displacements.</li>
		<li>Execute the Dedrift subroutine to remove any additional non-cellular displacements that may be due to microscope stage drift.</li>
	</ol>
	</li>
	<li>Computation of tractions 
	NOTE: Here, unconstrained tractions are calculated using our custom-written MATLAB routine. Step by step traction computation using the MATLAB routine previously mentioned is given below.
	<ol>
		<li>Define the following variables within the traction.m routine: boundary condition, gel thickness, gel height, and dedrift.</li>
		<li>Execute the traction_finder subroutine to calculate tractions and execute the plot_traction subroutine to plot tractions. All the tractions in x-direction (Tx) and y-direction (Ty) along with their corresponding pixel locations can be found in a traction.dat file that will be generated by the code.</li>
	</ol>
	</li>
	<li>Computation of intercellular stresses 
	NOTE: Step by step instructions for intercellular stress computation using the MATLAB routine previously mentioned are given below.
	<ol>
		<li>Execute the mark_circular_domain subroutine to specify the monolayer boundary. This routine will prompt all cropped phase images sequentially for the user to manually draw a boundary around the monolayer. Keep a note of the nXPts generated in command window and use them later as grid parameters in both X axis and Y axis for FEM analysis (see step 9.3.2).</li>
		<li>Execute Run_StressCode to compute intercellular stresses. This subroutine directly reads parameters from &#34;model.in&#34; file and executes &#34;island.exe&#34; to perform FEM analysis. Before running this routine, make sure all parameters in the model.in file, i.e. grid parameters in X and Y, pixel to micron conversion, Young&#39;s modulus of the gel, Poisson&#39;s ratio, monolayer height, and monolayer pattern (strip or hole), are edited correctly.</li>
		<li>Plot all the FEM results using the plot_FEM_results subroutine. 
		NOTE: All results generated will automatically be stored in the Results folder in the MATLAB directory.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

Our group, as well as others, has been successfully using TFM and MSM to probe the influence of cell-cell junctions in various pathological and physiological cellular processes in vitro7,15,18,27. For example, Hardin et al. presented a very insightful study that suggests intercellular stress transmission guides paracellular gap formation in endothelial cells15. While it ...

The field that refers to the study of the effects of physical forces and of mechanical properties on cellular and tissue physiology and pathology is known as mechanobiology1. A few useful techniques that have been utilized in mechanobiology are monolayer stress microscopy and traction force microscopy. Traction force microscopy allows for the computation of tractions generated at the cell-substrate interface, while monolayer stress microscopy allows for the computation of intercellular stresses generated between adjacent cells within a monolayer2,3,4,5,6. Results yielded from previous methods have suggested that cell-derived mechanical stresses play a crucial role in determining the fate of a host of cellular processes3,4,5. For example, upon exposure to an external mechanical force, a group of cells migrating as a collective can alter their morphology and polarize their shape to align and migrate along the direction of applied force by, in part, generating tractions7,8. Tractions provide a metric that can be used to evaluate cell contractility and are calculated using traction force microscopy (TFM). Traction force microscopy (TFM) begins with the determination of cell-induced substrate deformations followed by the calculation of the traction field using a mathematically rigorous, mechanics-based computational approach. Since the ability to calculate tractions has been around for quite some time, researchers have utilized TFM to reveal the impact tractions have on a host of processes, including cancer9, wound healing10 and assessment of engineered cardiac tissue11.
Implementation of TFM and MSM together can be divided into three essential steps that must be executed in the following order: first, the hydrogel deformations produced by the cells are determined; second, tractions are recovered from hydrogel deformations; and third, a finite element approach is used to compute normal and shear intercellular stresses within the entire monolayer. To compute gel displacements, fluorescence bead images with cells were compared with the reference bead image (without cells) by using a custom-written particle image velocimetry (PIV) routine. The cross-correlation window size and overlap for PIV analysis were chosen to be 32 x 32 pixels and 0.5, respectively. At this time, pixel shifts were converted into microns by multiplying with a pixel-to-micron conversion factor (for our microscope, this conversion factor is 0.65) to obtain in-plane displacements. Errors associated with ignoring out-of-plane displacements are negligible12,13. After computation of gel displacements, there are two types of traction measurements that can be utilized, constrained tractions and unconstrained tractions8,14. Unconstrained tractions provide the traction field for the entire field of view (including regions with and without cells), while constrained tractions provide the traction field only for regions that include cells14. Then, intercellular stresses are calculated using monolayer stress microscopy (MSM), which is an extension of traction force microscopy. Implementation of MSM is based off the assumption that local tractions exerted by a monolayer of cells at the cell-substrate interface must be balanced by mechanical forces transmitted between cells at the cell-cell interface as demanded by Newton&#39;s laws7,12,13. A key assumption here is that the cell monolayer can be treated as a thin elastic sheet because the traction distribution in the monolayer is known and the force balance does not depend on cell material properties. Another key assumption is that the traction forces are balanced by local intercellular stresses within the optical field of view (within the monolayer) and the influence of this force balance is minimal in the distal region (outside of the monolayer)13. Therefore, the boundary conditions defined by intercellular stresses, displacements, or a combination of both at the monolayer boundary are crucial to perform MSM13.Taking into account the above information, we utilize MSM to perform a finite element analysis (FEM) to recover the maximum principal stress (&#963;max) and minimum principal stress (&#963;min) by rotating the stress plane at every point within the monolayer. These principal stresses are subsequently used to compute the 2D average normal intercellular stress [(&#963;max + &#963;min) /2] and 2D maximum shear intercellular stress [(&#963;max - &#963;min) /2] within the entire monolayer12,13. This procedure is described in more detail by Tambe et al.12,13
Monolayer stress microscopy (MSM) allows for the calculation of cell-cell intercellular stresses generated within a monolayer6,7,8,12,13. These intercellular stresses have been suggested to be important for tissue growth and repair, wound healing, and cancer metastasis12,15,16,17. In addition, intercellular stresses have been suggested to also be important in endothelial cell migration and endothelial barrier function17,18. While cell-cell junctions such as tight junctions and adherens junctions have both been suggested to play a critical role in endothelial intercellular stress generation and transmission, the role of gap junctions remains elusive. Gap junctions physically connect adjacent cells and provide a pathway for electrical current and molecules (&#60;1 KDa) to pass between neighboring cells19,20,21. Although endothelial cells express Cx37, Cx40, and Cx43 gap junctions19,22, Cx43 is arguably the most important in terms of disease progression23. Evidence of Cx43&#39;s importance may be found in the fact that genetic deletion of Cx43 in mice results in hypotension24 and has adverse effects on angiogenesis25. In addition, Cx43 has been documented to be important for cell migration and proliferation and in the progression of atherosclerosis18,22,23,24,25.
In this protocol, we used TFM and MSM to investigate whether traction and intercellular stress generation within the confluent, endothelial monolayer would be impacted by the disruption of the endothelial gap junction Cx43. We disrupted Cx43 with 2,5-dihydroxychalcone (chalcone), a molecule documented to inhibit Cx43 expression26. Chalcone was used to disrupt Cx43 instead of siRNA as chalcone has been reported previously by Lee et al. to disrupt Cx43 expression26. In addition, we were particularly interested in chalcone&#39;s influence on the endothelium as it has also been reported to be an anti-inflammatory and anti-platelet compound that can potentially be used for the prevention and treatment of various vascular pathologies26. Chalcone treatments were performed an hour after the experiment onset, chalcone-treated monolayers were imaged for a total of six hours, and image processing was performed with a custom-written MATLAB code to determine tractions and subsequently intercellular stresses. Our results showed an overall decrease in tractions and intercellular stresses, suggesting Cx43 plays a key role in endothelial biomechanics.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>18 mm coverslip</td>
			<td>ThermoFisher</td>
			<td>18CIR-1</td>
			<td>Essential to flatten polyacrylamide gels</td>
		</tr>
		<tr>
			<td>2% bis-acrylamide</td>
			<td>BIO-RAD</td>
			<td>1610143</td>
			<td>Component of polyacrylamide gel</td>
		</tr>
		<tr>
			<td>2&#8242;,5&#8242;-Dihydroxychalcone</td>
			<td>SIGMA</td>
			<td>IDF00046</td>
			<td>To disrupt Cx43 structure</td>
		</tr>
		<tr>
			<td>3-(Trimethoxysilyl)propyl methacrylate</td>
			<td>SIGMA</td>
			<td>2530-85-0</td>
			<td>Stock solution to make bind silane mixture with acetic acid and ultra-pure water</td>
		</tr>
		<tr>
			<td>40% Acrylamide</td>
			<td>BIO-RAD</td>
			<td>1610140</td>
			<td>Component of polyacrylamide gel</td>
		</tr>
		<tr>
			<td>Acetic acid</td>
			<td>Fisher-Sceintific</td>
			<td>64-19-7</td>
			<td>Essential to make bind saline solution</td>
		</tr>
		<tr>
			<td>Alexa Fluro 488 goat anti-mouse IgG;</td>
			<td>ThermoFisher</td>
			<td>Catalog # A-11001</td>
			<td>Secondary antibody</td>
		</tr>
		<tr>
			<td>Ammonium persulfate</td>
			<td>BIO-RAD</td>
			<td>1610700</td>
			<td>Polyacrylamide gel polymerizing agent</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>SIGMA</td>
			<td>9048-46-8</td>
			<td>To make blocking solution</td>
		</tr>
		<tr>
			<td>Bovine Type I Atelo-Collagen Solution, 3 mg/mL, 100 mL</td>
			<td>Advance Biomatrix</td>
			<td>5005-100ML</td>
			<td>Use as a extracellular matrix</td>
		</tr>
		<tr>
			<td>Corning Cell Culture Phosphate Buffered Saline (1x)</td>
			<td>Fisher-Sceintific</td>
			<td>21040CV</td>
			<td>Buffer Saline needed for cell culture</td>
		</tr>
		<tr>
			<td>Dimethyl Sulfoxide, Fisher BioReagents</td>
			<td>Fisher-Sceintific</td>
			<td>67-68-5</td>
			<td>To dissolve chalcone and make stock solution</td>
		</tr>
		<tr>
			<td>Fluoromount-G with DAPI</td>
			<td>ThermoFisher</td>
			<td>00-4959-52</td>
			<td>Mounting medium for immunostaing used to stain for DAPI</td>
		</tr>
		<tr>
			<td>Fluroscent microsphere Carboxylate-modified beads</td>
			<td>ThermoFisher</td>
			<td>F8812</td>
			<td>0.5 micron carboxylate-modified beads (red), 2% solids</td>
		</tr>
		<tr>
			<td>HEPES buffer solution 1 M</td>
			<td>SIGMA</td>
			<td>7365-45-9</td>
			<td>Essential to</td>
		</tr>
		<tr>
			<td>LVES</td>
			<td>ThermoFisher</td>
			<td>A1460801</td>
			<td>Essential HUEVC media 200 supplement</td>
		</tr>
		<tr>
			<td>Medium 200</td>
			<td>ThermoFisher</td>
			<td>M200500</td>
			<td>Essential media for HUVEC cell culture</td>
		</tr>
		<tr>
			<td>Mouse monoclonal Cx43 antibody (CX - 1B1)</td>
			<td>ThermoFisher</td>
			<td>Catalog #13-8300</td>
			<td>Primary antibody for Cx43</td>
		</tr>
		<tr>
			<td>Petri dish (35 mm dia)</td>
			<td>CellVis</td>
			<td>D35-20-1.5H</td>
			<td>35 mm petri dish with a 20 mm center well</td>
		</tr>
		<tr>
			<td>Sulfo-SANPAH Crosslinker 100 mg</td>
			<td>Proteochem</td>
			<td>102568-43-4</td>
			<td>Essential to functionalize polyacrylamide gel surface</td>
		</tr>
		<tr>
			<td>SYLGARD 184 Silicone Elastomer Kit</td>
			<td>DOW corning</td>
			<td>2646340</td>
			<td>Silicon elastomer with curing agent to make PDMS</td>
		</tr>
		<tr>
			<td>TEMED</td>
			<td>BIO-RAD</td>
			<td>1610801</td>
			<td>Polyacrylamide gel polymerizing agent</td>
		</tr>
		<tr>
			<td>Triton-X 100</td>
			<td>SIGMA</td>
			<td>9002-93-1</td>
			<td>To permeabilize cells</td>
		</tr>
		<tr>
			<td>Trypsin -EDTA</td>
			<td>ThermoFisher</td>
			<td>25300054</td>
			<td>Used to detach cells</td>
		</tr>
	</tbody>
</table>

perturbing endothelial biomechanics via connexin 43 structural disruption

Endothelial cells have been established to generate intercellular stresses and tractions, but the role gap junctions play in endothelial intercellular stress and traction generation is currently unknown. Therefore, we present here a mechanics-based protocol to probe the influence of gap junction connexin 43 (Cx43) has on endothelial biomechanics by exposing confluent endothelial monolayers to a known Cx43 inhibitor 2,5-dihydroxychalcone (chalcone) and measuring the impact this inhibitor has on tractions and intercellular stresses. We present representative results, which show a decrease in both tractions and intercellular stresses under a high chalcone dosage (2 &#181;g/mL) when compared to control. This protocol can be applied to not just Cx43, but also other gap junctions as well, assuming the appropriate inhibitor is used. We believe this protocol will be useful in the fields of cardiovascular and mechanobiology research.

Phase contrast images of control, 0.2 &#181;g/mL, and 2 &#181;g/mL chalcone treated monolayers were taken 30 minutes before chalcone treatment (Figure 1A-C) and 2 hours after chalcone treatment (Figure 1D-F). Cell-induced bead displacements (&#181;m) were observed to decrease in both low dose chalcone and high dose chalcone conditions (Figure 2E,F) w...

Watch this Scientific Journal Video about Perturbing Endothelial Biomechanics via Connexin 43 Structural Disruption at JoVE.com

Perturbing Endothelial Biomechanics via Connexin 43 Structural Disruption

Here, we present a mechanics-based protocol to disrupt the gap junction connexin 43 and measure the subsequent impact this has on endothelial biomechanics via observation of tractions and intercellular stresses.

Endothelial cells have been established to generate intercellular stresses and tractions, but the role gap junctions play in endothelial intercellular ...

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Research

JoVE Journal

Biochemistry

5.4K Views.  University of Central Florida. Here, we present a mechanics-based protocol to disrupt the gap junction connexin 43 and measure the subsequent impact this has on endothelial biomechanics via observation of tractions and intercellular stresses.

Video: Perturbing Endothelial Biomechanics via Connexin 43 Structural Disruption

Reconstitution of Nucleosomes with Differentially Isotope-labeled Sister Histones

Asymmetrically modified nucleosomes contain the two copies of a histone (sister histones) decorated with distinct sets of Post-translational Modifications (PTMs). They are newly identified species with unknown means of establishment and functional implications. Current analytical methods are inadequate to detect the copy-specific occurrence of PTMs on the nucleosomal sister histones. This protocol presents a biochemical method for the in vitro reconstitution of nucleosomes containing differentially isotope-labeled sister histones. The generated complex can be also asymmetrically modified, after including a premodified histone pool during refolding of histone subcomplexes. These asymmetric nucleosome preparations can be readily reacted with histone-modifying enzymes to study modification cross-talk mechanisms imposed by the asymmetrically pre-incorporated PTM using nuclear magnetic resonance (NMR) spectroscopy. Particularly, the modification reactions in real-time can be mapped independently on the two sister histones by performing different types of NMR correlation experiments, tailored for the respective isotope type. This methodology provides the means to study crosstalk mechanisms that contribute to the formation and propagation of asymmetric PTM patterns on nucleosomal complexes.

This protocol describes the reconstitution of nucleosomes containing differentially isotope-labeled sister histones. At the same time, asymmetrically post-translationally modified nucleosomes can be generated after using a premodified histone copy. These preparations can be readily used to study modification crosstalk mechanisms, simultaneously on both sister histones, by using high-resolution NMR spectroscopy.

Asymmetrically modified nucleosomes contain the two copies of a histone (sister histones) decorated with distinct sets of Post-translational Modifications ...

LERLIC-MS/MS for In-depth Characterization and Quantification of Glutamine and Asparagine Deamidation in Shotgun Proteomics

Characterization of protein deamidation is imperative to decipher the role(s) and potentialities of this protein posttranslational modification (PTM) in human pathology and other biochemical contexts. In order to perform characterization of protein deamidation, we have recently developed a novel long-length electrostatic repulsion-hydrophilic interaction chromatography-tandem mass spectrometry (LERLIC-MS/MS) method which can separate the glutamine (Gln) and asparagine (Asn) isoform products of deamidation from model compounds to highly complex biological samples. LERLIC-MS/MS is, therefore, the first shotgun proteomics strategy for the separation and quantification of Gln deamidation isoforms. We also demonstrate, as a novelty, that the sample processing protocol outlined here stabilizes the succinimide intermediate allowing its characterization by LERLIC-MS/MS. Application of LERLIC-MS/MS as shown in this video article can help to elucidate the currently unknown molecular arrays of protein deamidation. Additionally, LERLIC-MS/MS provides further understanding of the enzymatic reactions that encompass deamidation in distinct biological backgrounds.

Here we present a step-by-step protocol of the long-length electrostatic repulsion-hydrophilic interaction chromatography-tandem mass spectrometry (LERLIC-MS/MS) method. This is a novel methodology that enables for the first time quantification and characterization of the glutamine and asparagine deamidation isoforms by shotgun proteomics.

Characterization of protein deamidation is imperative to decipher the role(s) and potentialities of this protein posttranslational modification (PTM) in ...

Biochemical and Structural Characterization of the Carbohydrate Transport Substrate-binding-protein SP0092

Development of new antimicrobials and vaccines for Streptococcus pneumoniae (pneumococcus) are necessary to halt the rapid rise in multiple resistant strains. Carbohydrate substrate binding proteins (SBPs) represent viable targets for the development of protein-based vaccines and new antimicrobials because of their extracellular localization and the centrality of carbohydrate import for pneumococcal metabolism, respectively. Described here is a rationalized integrated protocol to carry out a comprehensive characterization of SP0092, which can be extended to other carbohydrate SBPs from the pneumococcus and other bacteria. This procedure can aid the structure-based design of inhibitors for this class of proteins. Presented in the first part of this manuscript are protocols for biochemical analysis by thermal shift assay, multi angle light scattering (MALS), and size exclusion chromatography (SEC), which optimize the stability and homogeneity of the sample directed to crystallization trials and so enhance the probability of success. The second part of this procedure describes the characterization of the SBP crystals using a tunable wavelength anomalous diffraction synchrotron beamline, and data collection protocols for measuring data that can be used to resolve the crystallized protein structure.

A streamlined protocol for performing an extensive biochemical and structural characterization of a carbohydrate substrate binding protein from Streptococcus pneumoniae is presented.

Development of new antimicrobials and vaccines for Streptococcus pneumoniae (pneumococcus) are necessary to halt the rapid rise in multiple resistant ...

Structural Characterization of Mannan Cell Wall Polysaccharides in Plants Using PACE

Plant cell wall polysaccharides are notoriously difficult to analyze, and most methods require expensive equipment, skilled operators, and large amounts of purified material. Here, we describe a simple method for gaining detailed polysaccharide structural information, including resolution of structural isomers. For polysaccharide analysis by gel electrophoresis (PACE), plant cell wall material is hydrolyzed with glycosyl hydrolases specific to the polysaccharide of interest (e.g., mannanases for mannan). Large format polyacrylamide gels are then used to separate the released oligosaccharides, which have been fluorescently labeled. Gels can be visualized with a modified gel imaging system (see Table of Materials). The resulting oligosaccharide fingerprint can either be compared qualitatively or, with replication, quantitatively. Linkage and branching information can be established using additional glycosyl hydrolases (e.g., mannosidases and galactosidases). Whilst this protocol describes a method for analyzing glucomannan structure, it can be applied to any polysaccharide for which characterized glycosyl hydrolases exist. Alternatively, it can be used to characterize novel glycosyl hydrolases using defined polysaccharide substrates.

A protocol for the structural analysis of polysaccharides by gel electrophoresis (PACE), using mannan as an example, is described.

Plant cell wall polysaccharides are notoriously difficult to analyze, and most methods require expensive equipment, skilled operators, and large amounts ...

Calibration-free In Vitro Quantification of Protein Homo-oligomerization Using Commercial Instrumentation and Free, Open Source Brightness Analysis Software

Number and brightness is a calibration-free fluorescence fluctuation spectroscopy (FFS) technique for detecting protein homo-oligomerization. It can be employed using a conventional confocal microscope equipped with digital detectors. A protocol for the use of the technique in vitro is shown by means of a use case where number and brightness can be seen to accurately quantify the oligomeric state of mVenus-labelled FKBP12F36V before and after the addition of the dimerizing drug AP20187. The importance of using the correct microscope acquisition parameters and the correct data preprocessing and analysis methods are discussed. In particular, the importance of the choice of photobleaching correction is stressed. This inexpensive method can be employed to study protein-protein interactions in many biological contexts.

Calibration-free In Vitro Quantification of Protein Homo-oligomerization Using Commercial Instrumentation and Free, Open Source Brightness Analysis Software

This protocol describes a calibration-free approach for quantifying protein homo-oligomerization in vitro based on fluorescence fluctuation spectroscopy using commercial light scanning microscopy. The correct acquisition settings and analysis methods are shown.

Number and brightness is a calibration-free fluorescence fluctuation spectroscopy (FFS) technique for detecting protein homo-oligomerization. It can be ...

Modifying Baculovirus Expression Vectors to Produce Secreted Plant Proteins in Insect Cells

It has been a challenge for scientists to express recombinant secretory eukaryotic proteins for structural and biochemical studies. The baculovirus-mediated insect cell expression system is one of the systems used to express recombinant eukaryotic secretory proteins with some post-translational modifications. The secretory proteins need to be routed through the secretory pathways for protein glycosylation, disulfide bonds formation, and other post-translational modifications. To improve the existing insect cell expression of secretory plant proteins, a baculovirus expression vector is modified by the addition of either a GP67 or a hemolin signal peptide sequence between the promoter and multiple-cloning sites. This newly designed modified vector system successfully produced a high yield of soluble recombinant secreted plant receptor proteins of Arabidopsis thaliana. Two of the expressed plant proteins, the extracellular domains of Arabidopsis TDR and PRK3 plasma membrane receptors, were crystallized for X-ray crystallographic studies. The modified vector system is an improved tool that can potentially be used for the expression of recombinant secretory proteins in the animal kingdom as well.

Here, we present the protocols for utilizing the insect cell and baculovirus protein expression system to produce large quantities of plant secreted proteins for protein crystallization. A baculovirus expression vector has been modified with either GP67 or insect hemolin signal peptide for plant protein secretion expression in insect cells.

It has been a challenge for scientists to express recombinant secretory eukaryotic proteins for structural and biochemical studies. The ...

Structural Studies of Macromolecules in Solution using Small Angle X-Ray Scattering

Protein-protein interactions involving proteins with multiple globular domains present technical challenges for determining how such complexes form and how the domains are oriented/positioned. Here, a protocol with the potential for elucidating which specific domains mediate interactions in multicomponent system through ab initio modeling is described. A method for calculating solution structures of macromolecules and their assemblies is provided that involves integrating data from small angle X-ray scattering (SAXS), chromatography, and atomic resolution structures together in a hybrid approach. A specific example is that of the complex of full-length nidogen-1, which assembles extracellular matrix proteins and forms an extended, curved nanostructure. One of its globular domains attaches to laminin &#947;-1, which structures the basement membrane. This provides a basis for determining accurate structures of flexible multidomain protein complexes and is enabled by synchrotron sources coupled with automation robotics and size exclusion chromatography systems. This combination allows rapid analysis in which multiple oligomeric states are separated just prior to SAXS data collection. The analysis yields information on the radius of gyration, particle dimension, molecular shape and interdomain pairing. The protocol for generating 3D models of complexes by fitting high-resolution structures of the component proteins is also given.

Here, we present how Small Angle X-Ray Scattering (SAXS) can be utilized to obtain information on low-resolution envelopes representing the macromolecular structures. When used in conjunction with high-resolution structural techniques such as X-Ray Crystallography and Nuclear Magnetic Resonance, SAXS can provide detailed insights into multidomain proteins and macromolecular complexes in-solution.

Protein-protein interactions involving proteins with multiple globular domains present technical challenges for determining how such complexes form and ...

Analysis of SEC-SAXS data via EFA deconvolution and Scatter

BioSAXS is a popular technique used in molecular and structural biology to determine the solution structure, particle size and shape, surface-to-volume ratio and conformational changes of macromolecules and macromolecular complexes. A high quality SAXS dataset for structural modeling must be from monodisperse, homogeneous samples and this is often only reached by a combination of inline chromatography and immediate SAXS measurement. Most commonly, size-exclusion chromatography is used to separate samples and exclude contaminants and aggregations from the particle of interest allowing SAXS measurements to be made from a well-resolved chromatographic peak of a single protein species. Still, in some cases, even inline purification is not a guarantee of monodisperse samples, either because multiple components are too close to each other in size or changes in shape induced through binding alter perceived elution time. In these cases, it may be possible to deconvolute the SAXS data of a mixture to obtain the idealized SAXS curves of individual components. Here, we show how this is achieved and the practical analysis of SEC-SAXS data is performed on ideal and difficult samples. Specifically, we show the SEC-SAXS analysis of the vaccinia E9 DNA polymerase exonuclease minus mutant.

SEC-BioSAXS measurements of biological macromolecules are a standard approach for determining solution structure of macromolecules and their complexes. Here, we analyze SEC-BioSAXS data from two types of commonly encountered SEC traces&#8212;chromatograms with fully resolved and partially resolved peaks. We demonstrate the analysis and deconvolution using scatter and BioXTAS RAW.

BioSAXS is a popular technique used in molecular and structural biology to determine the solution structure, particle size and shape, surface-to-volume ...

Fast Grid Preparation for Time-Resolved Cryo-Electron Microscopy

The field of cryo-electron microscopy (cryo-EM) is rapidly developing with new hardware and processing algorithms, producing higher resolution structures and information on more challenging systems. Sample preparation for cryo-EM is undergoing a similar revolution with new approaches being developed to supersede the traditional blotting systems. These include the use of piezo-electric dispensers, pin printing and direct spraying. As a result of these developments, the speed of grid preparation is going from seconds to milliseconds, providing new opportunities, especially in the field of time-resolved cryo-EM where proteins and substrates can be rapidly mixed before plunge freezing, trapping short lived intermediate states. Here we describe, in detail, a standard protocol for making grids on our in-house time-resolved EM device both for standard fast grid preparation and also for time-resolved experiments. The protocol requires a minimum of about 50 &#181;L sample at concentrations of &#8805; 2 mg/mL for the preparation of 4 grids. The delay between sample application and freezing can be as low as 10 ms. One limitation is increased ice thickness at faster speeds and compared to the blotting method. We hope this protocol will aid others in designing their own grid making devices and those interested in designing time-resolved experiments.

Here, we provide a detailed protocol for the use of a rapid grid making device for both fast grid-making and for rapid mixing and freezing to conduct time-resolved experiments.

The field of cryo-electron microscopy (cryo-EM) is rapidly developing with new hardware and processing algorithms, producing higher resolution structures ...

gP2S, an Information Management System for CryoEM Experiments

Cryogenic electron microscopy (cryoEM) has become an integral part of many drug-discovery projects because crystallography of the protein target is not always achievable and cryoEM provides an alternative means to support structure-based ligand design. When dealing with a large number of distinct projects, and within each project a potentially large number of ligand-protein co-structures, accurate record keeping rapidly becomes challenging. Many experimental parameters are tuned for each target, including at the sample preparation, grid preparation, and microscopy stages. Therefore, accurate record keeping can be crucially important to enable long-term reproducibility, and to facilitate efficient teamwork, especially when steps of the cryoEM workflow are performed by different operators. To help deal with this challenge, we developed a web-based information management system for cryoEM, called gP2S. 
The application keeps track of each experiment, from sample to final atomic model, in the context of projects, a list of which is maintained in the application, or externally in a separate system. User-defined controlled vocabularies of consumables, equipment, protocols and software help describe each step of the cryoEM workflow in a structured manner. gP2S is widely configurable and, depending on the team's needs, may exist as a standalone product or be a part of a broader ecosystem of scientific applications, integrating via REST APIs with project management tools, applications tracking the production of proteins or of small molecules ligands, or applications automating data collection and storage. Users can register details of each grid and microscopy session including key experimental metadata and parameter values, and the lineage of each experimental artifact (sample, grid, microscopy session, map, etc.) is recorded. gP2S serves as a cryoEM experimental workflow organizer that enables accurate record keeping for teams, and is available under an open-source license.

gP2S is a web application for the tracking of cryoEM experiments. Its main features are described, as are the steps required to install and configure the application. Once configured, the application allows one to accurately record metadata associated with negative stain and cryoEM experiments.

Cryogenic electron microscopy (cryoEM) has become an integral part of many drug-discovery projects because crystallography of the protein target is not ...