This material is based upon work supported by the National Science Foundation under Grant No. 0842736.

Introduction to the Novel GFS Configuration
 The GFS consists of several essential components: A regular light microscope, an equatorial mount, a camera, and a computer [Figure 1]. The sealed flow-cell chamber which holds the sample is also indispensible according to the GFS design. The Light microscope is mounted onto the equatorial mount so the scope can be rotated into different orientations in space. This ability allows the static vector of gravity to be exploited so that the samples can be dynamically oriented in relation to the vector so the force of gravity can impart piconewton-range force loads to the samples. The camera replaces the light microscope's ocular lens so it can record changes in the orientation of the sample. This raw data is digitized and manipulated by the computer to interpret the data into actual force and distance measurements. The sealed flow-chamber is designed to allow all degrees of freedom in space without sample loss. In the chamber resides the sample molecule which is tethered near one terminus to an "anchored" bead that is glued to the surface of the chamber. The opposite terminus is tethered to a "mobile" bead that is free from the surface of the chamber. It is this mobile bead, free in the assay buffer that can be acted upon by gravitational force thereby stretching the tethered molecule at such low force loads [Figure 2]. The sample simply looks like a pair of microspheres under the microscope, although it does take some experience to discern good usable pairs coupled by their attachment to a molecule from pairs where both beads are sitting on the surface of the flow-chamber. One modification to the system is the addition of a floating platform which holds the GFS and is suspended by springs. In this configuration, once the sample has been rotated into a position where gravitational force can act on the sample, the platform and all its components can be dropped against the spring constant. Near free-fall, the force acting on the mobile bead is close to zero and at the springs' maximum extension, the gravitational force is multiplied by as much as two times. In this way, a graded force/distance response can be graphed to measure the behavior of a single molecule at different force loads.
1. Microsphere Preparation
<ol>
<li>Submerge about 10 mg of glass or silica beads in 0.04% 3-Aminopropyltriethoxysilane (cut with acetone) for two minutes.</li>
<li>Rinse with two changes of double distilled water and centrifuge to pellet at 2000 X G for 5 minutes. Discard supernatant.</li>
<li>Add 5 mL of coupling buffer (0.01 M pyridine cut with double distilled water and adjust pH to 6.0), and shake this mixture vigorously. Centrifuge as described above. Repeat this step three times.</li>
<li>To wet cake of beads, add 2 mL of 5% gluteraldehyde solution (cut gluteraldehyde with coupling buffer). Shake vigorously.</li>
<li>Under a hood, rotate bead/gluteraldehyde mixture for 3 hours at room temperature.</li>
<li>Centrifuge as above and aspirate the supernatant.</li>
<li>Wash the beads in 5 mL of coupling buffer by shaking them vigorously and centrifuge and aspirate the supernatant. Repeat this three more times.</li>
<li>Add about 15 &mu;L of the desired antibody to beads and shake vigorously. The beads should be rotated for 16-24 hours.</li>
<li>Under the hood, add 5 mL of 1 M glycine quenching solution (cut glycine with double distilled water and adjust pH to 7.0). Shake this mixture vigorously and rotate for 30 minutes.</li>
<li>Centrifuge and aspirate the supernatant.</li>
<li>Add 5 mL of wash buffer (0.01 M Tris, pH 7.0; 0.1% sodium azide; 0.1% BSA; 0.15 M NaCl; and 0.001 M EDTA). Shake this vigorously, centrifuge and aspirate the supernatant. Repeat this step three additional times.</li>
<li>Change buffer to low-salt buffer (0.1 M KCl; 0.02 M imidazole; 5 mM MgCl2; adjust to pH 7.0). Repeat three times.</li>
</ol>
2. Sample Attachment to Microspheres
<ol>
<li>Take a small amount of prepared beads (about 2 &mu;L from each cake of beads however, if there is a large discrepancy in diameter between batches, it is advantageous to use around an 8:1 ratio of large to small beads) and add them to a microcentrifuge tube with assay buffer. Reduce the concentration of your protein to around 5 &mu;M, using the assay buffer. Prepare at least a total volume of 400 &mu;L including the buffer, the protein, and the beads.</li>
<li>Rotate this mixture at around 1 RPM for 3 hours (if the protein is agitated too much, it will aggregate and become useless).</li>
</ol>
3. Slide Chamber Preparation
<ol>
<li>Coat a thick microscope slide with 0.01% nitrocellulose (in amyl acetate). Let this slide dry for about 10 minutes.</li>
<li>With a sharp glass cutter, cut cover slide so as to make a chamber. This requires four strips of glass.</li>
<li>Use the factory edge of the glass and rake it across a smear of vacuum grease on both sides of the edge.</li>
<li>Press grease coated strips onto dried nitrocellulose covered slide to create a box on the surface of the slide.</li>
<li>Pipette about 2 &mu;L of bead/protein mixture by tapping pipetter onto surface of glass inside the box.</li>
<li>Add about 20-400 &mu;L of low salt buffer depending on how large the chamber is.</li>
<li>Press a cover slip on top of the box which is already coated with vacuum grease to finish the sealed and buffered chamber.</li>
<li>Let slide sit on a level place so the beads have ample time to anchor themselves to the nitrocellulose.</li>
</ol>
4. GFS Data Acquisition
<ol>
<li>Mount slide onto GFS stage</li>
<li>Monitor what the GFS camera is recording and search for legitimate "bead pairs," in which the small microsphere is attached near the equator of the large microsphere.</li>
<li>When a potential bead pair is found, go through the depth of focus to determine if the "mobile" bead is not resting on the surface of the slide.</li>
<li>Once a suitable pair is identified, note angle mobile bead is away from dmax (dmax = the maximum distance between the centroids of the coupled microspheres). Move the scope into position to acquire video.</li>
<li>The angle of travel of the GFS should be sufficient to record dmin, dmax, and dmin which might call for 25-90 degrees depending on the length of the molecule.</li>
<li>RECORD as the pair travels from dmin to dmax and back to dmin.</li>
<li>It is a good idea to also shoot a movie of the background so it can be subtracted later for analysis.</li>
<li>If performing a GFS drop, move the scope back to dmax and record the drop at at least 60 frames per second. The critical part is the first oscillation, but long record times can also be used for dynamic study.</li>
</ol>
5. GFS Data Analysis
<ol>
<li>Transform raw video into digitally "thresholded" image and run macro in imageJ to determine the centroid position of each bead in every frame of video. This is also for the drop video.</li>
<li>Dump the X,Y and area data from ImageJ into Excel and plot the points.</li>
<li>If a proper bead pair was acquired by video, a noticeable hump in the graph is indicative of the beads being at their closest (dmin) and at the apex of the graph is the position of dmax.</li>
<li>Using this data, the radius of each bead should be determined precisely in ImageJ and all this information is put into the nested equation: 
d = [(g sin &alpha;)2 + (g cos &alpha; + dmax - g)2]0.5 
g = [dmin2 + rb 2 - (ra + rb)2]0.5 = rb sin &beta; 
c = dmax - ra - g 
(d = distance between centroids; g = force of gravity; &alpha; = the angle in degrees parallel to the objective lens; rb = radius of mobile bead; ra = radius of anchored bead; &beta; = angle of attachment off the axis of the equator of the anchored bead.</li>
<li>Using the fitted radius of the mobile bead which also yields its volume, and given the density of the bead, the force the mobile bead imparts on the molecule can be calculated in piconewtons after the buoyancy of the solvent is subtracted. This method measures the force on the tethered molecule in piconewtons and calculates the absolute molecule length between antibody attachments in nanometers. F = V (d-b) a (F = force, V = volume, d = density of the glass microsphere, a = acceleration due to gravity, b = the density of the displaced water). </li>
</ol>
6. Representative Results:
If the bead prep is done correctly, there will be minimum bead aggregation although there may still be an occasional bead clump. When viewed through the scope, there should be a reasonable distribution of beads whether paired or not in the chamber.
It is important to minimize vibrations as much as possible; to do this either an air table, special shock absorbing feet for a tripod that holds an EQ mount, or on the system that utilizes springs can be used for vibration isolation.
Another useful tip concerning the sealed flow chamber is to let it stand for about five minutes on a level table after it has been fully constructed. This allows any unattached larger beads to float down through the buffer and rest in the layer of nitrocellulose. If the slide were instead mounted directly upon completion, the investigator would continually have to deal with beads literally flying through the field of view; and if this happens during video acquisition it can corrupt the experiment. If this is done properly, bead flight is significantly minimized and cleaner video results.
When a potential bead pair is identified by the GFS operator, it is useful to put it through a preliminary rotation to monitor the behavior of the pair. Rarely, the large bead is not firmly affixed to the slide. If this happens, there is no use in utilizing the pair because it is critical that the larger anchored bead stay in a fixed position through the duration of the acquisition. If the pair is stable and does not exhibit "anchored bead roll," then it is suitable for experimentation.
A plot of bead separation distances versus change in angle relative to gravity can be used to evaluate the acquired data. A good representative result would show little separation on a plateau and as the mobile bead frees itself from the anchored bead because of the influence of gravity, the graph will show more separation. This continues until a nice peak is reached which is called dmax and the curve starts downward again returning to the baseline dmin [Figure 1]. In ideal conditions, this signature is symmetrical. A non-representative result would show a graph with incoherent patterns showing no distinct dmin-dmax-dmin pattern; or it would show separations that are orders of magnitude too high for a single molecule indicating maybe there was a piece of dust between the beads, or maybe that the mobile bead was simply not attached at all. The process of finding the pair, shooting it, processing it, and analyzing it has many stop gaps where improper bead pairs are culled out. So, if you get to the end of the entire process and you have a molecule length that is consistent with previous results, one can be very confident that the original bead pair is representative and can be included in the results. On a good day, about half of the bead pairs shot can be taken through to become a legitimate data point. For instance, the length of the coiled coil of myosin between MF20 and MF30 antibodies, is known based on EM data and AFM, data to approach 100 nm 2,7,8,9. If the result is several times this length, the sample has aggregated. The results presented here are from standard GFS rotation experiments and demonstrate a distance of 96 nm &plusmn; 5 nm, [Figure 2] which agrees closely with the measurements of the MF30 (which binds at the N-terminus of myosin subfragment-2) and MF20 (which binds in the light meromyosin) antibody separation distance on myosin deduced from the literature values [Figure 3].
<img src="/files/ftp_upload/2624/2624fig1.jpg" alt="Figure 1" /> Figure 1. GFS configuration. Major parts of GFS are labeled.
<img src="/files/ftp_upload/2624/2624fig2.jpg" alt="Figure 2" /> Figure 2. Schematic of GFS principle. Left side shows centroid of mobile bead at a minimum distance from centroid of anchored bead. As GFS is rotated, mobile bead aligns with vector of gravity which is also running parallel with the axis of the tethered molecule. In this position, the distance between the centroids of the mobile and anchored beads is at a maximum. Top right shows a representative slice from a GFS movie. Bottom right is an image of the GFS undergoing rotation.
<img src="/files/ftp_upload/2624/2624fig3.jpg" alt="Figure 3" /> Figure 3. Representative result showing dmin on the left of the graph; dmax at a relative separation of 17.78 microns; and a return to dmin around the baseline of about 17.75 microns of relative separation between the beads.
<img src="/files/ftp_upload/2624/2624fig4.jpg" alt="Figure 4" /> Figure 4. Representative result of GFS rotation experiment showing distance between MF20 and MF30 antibodies average 96 nm. This represents the approximate length of S2.
<img src="/files/ftp_upload/2624/2624fig5.jpg" alt="Figure 5" /> Figure 5. Myosin II dimer used to show possible attachments for use with the GFS including antibody and/or actin attachments. Different attachment possibilities allows for different GFS strategies to measure different regions, or to apply force perpendicular or parallel to the rod domain of the myosin dimer.

<ol>
	<li>Schwaiger, I., Sattler, C., Hostetter, D. R., Rief, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+myosin+coiled-coil+is+a+truly+elastic+protein+structure.">The myosin coiled-coil is a truly elastic protein structure.</a> Nat. Mater. 1, 232-235 (2002).</li><li>Root, D. D., Yadavalli, V. M., Forbes, J. G., Wang, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coiled-coil+nanomechanics+and+uncoiling+and+unfolding+of+the+superhelix+and+alpha-helices+of+myosin.">Coiled-coil nanomechanics and uncoiling and unfolding of the superhelix and alpha-helices of myosin.</a> Biophysical Journal. 90, 2852-2866 (2006).</li><li>Nishizaka, T., Miyata, H., Yoshikawa, H., Ishiwata, S., Kinosita, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unbinding+force+of+a+single+motor+molecule+of+muscle+measured+using+optical+tweezers.">Unbinding force of a single motor molecule of muscle measured using optical tweezers.</a> Nature. 377, 251-254 (1995).</li><li>Gawalapu, R. K., Root, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+labeling+and+computational+analysis+of+the+strut+of+myosin's+50+kDa+cleft.">Fluorescence labeling and computational analysis of the strut of myosin's 50 kDa cleft.</a> Arch. Biochem. Biophys. 456, 102-111 (2006).</li><li>Kellermayer, M. S. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+and+manipulating+individual+protein.">Visualizing and manipulating individual protein.</a> Molecules Physiol. Meas. 26, R119-R153 (2005).</li><li>Shimizu, T., Dennis, J. E., Masaki, T., Fischman, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axial+arrangement+of+the+myosin+rod+in+vertebrate+thick+filaments:+immunoelectron+microscopy+with+a+monoclonal+antibody+to+light+meromyosin.">Axial arrangement of the myosin rod in vertebrate thick filaments: immunoelectron microscopy with a monoclonal antibody to light meromyosin.</a> J. Cell Biol. 101, 1115-1123 (1985).</li><li>Godfrey, J. E., Harrington, W. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-association+in+the+myosin+system+at+high+ionic+strength.+I.+Sensitivity+of+the+interaction+to+pH+and+ionic+environment.">Self-association in the myosin system at high ionic strength. I. Sensitivity of the interaction to pH and ionic environment.</a> Biochemistry. 9, 886-893 (1970).</li><li>Root, D. D., Stewart, S., Xu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+docking+of+myosin+and+actin+observed+with+resonance+energy+transfer.">Dynamic docking of myosin and actin observed with resonance energy transfer.</a> Biochemistry. 41, 1786-1794 (2002).</li><li>Xu, J., Root, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conformational+Selection+during+Weak+Binding+at+the+Actin+and+Myosin+Interface.">Conformational Selection during Weak Binding at the Actin and Myosin Interface.</a> Biophys. J. 79, 1498-1510 (2000).</li><li>Sattin, B. D., Pelling, A. E., Goh, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+base+pair+resolution+by+single+molecule+force+spectroscopy.">DNA base pair resolution by single molecule force spectroscopy.</a> Nucleic Acids Res. 32, 4876-4883 (2004).</li></ol>

No conflicts of interest declared.

When converting a movie to a digitally thresholded representation, it is crucial for the thresholded image to maintain the same area in each frame of video. Because the beads in a bead pair move independently of one another, any drift in the thresholded areas can also cause the relative distances between the centroids of the beads to drift and introduce significant error. Controlling the threshold area reduced the error five-fold in the distance measurements from 26 nm down to 5 nm. It is also crucial to obtain an acc...

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		<th>Comment</th>
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<td>3-Aminopropyltriethoxysilane</td>
<td>&nbsp;</td>
<td>Poly Sciences</td>
<td>919-30-2</td>
<td>&nbsp;</td>
<tr>
<td>Acetone</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>A18P-4</td>
<td>&nbsp;</td>
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<td>Pyridine</td>
<td>&nbsp;</td>
<td>Sigma Aldrich</td>
<td>110-86-1</td>
<td>&nbsp;</td>
<tr>
<td>Glutaraldehyde</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>G7776</td>
<td>&nbsp;</td>
<tr>
<td>Glycine</td>
<td>&nbsp;</td>
<td>Research Organics</td>
<td>BP381-1</td>
<td>&nbsp;</td>
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<td>Tris</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>9682T</td>
<td>&nbsp;</td>
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<td>Sodium azide</td>
<td>&nbsp;</td>
<td>Amresco</td>
<td>71289</td>
<td>&nbsp;</td>
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<td>BSA</td>
<td>&nbsp;</td>
<td>Sigma Aldrich</td>
<td>AMR-0332-100G</td>
<td>&nbsp;</td>
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<td>NaCl</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S7653</td>
<td>&nbsp;</td>
<tr>
<td>EDTA</td>
<td>&nbsp;</td>
<td>MSI</td>
<td>E9884</td>
<td>&nbsp;</td>
<tr>
<td>Nitrocellulose</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>60443</td>
<td>&nbsp;</td>
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<td>N-N Dimethyl Formamide</td>
<td>&nbsp;</td>
<td>Extracted from Large New </td>
<td>D4254</td>
<td>&nbsp;</td>
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<td>Rabbit skeletal myosin II</td>
<td>&nbsp;</td>
<td>Zealand White Rabbits (7-8)</td>
<td>NA</td>
<td>&nbsp;</td>
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<td>MF30 antibody (9-10)</td>
<td>&nbsp;</td>
<td>Developmental Studies </td>
<td>MF30</td>
<td>&nbsp;</td>
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<td>MF20 antibody (6)</td>
<td>&nbsp;</td>
<td>Hybridoma Bank</td>
<td>MF20</td>
<td>&nbsp;</td>
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<td>Lab microscope</td>
<td>&nbsp;</td>
<td>Boreal</td>
<td>WW57905M00</td>
<td>&nbsp;</td>
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<td>Equatorial mount</td>
<td>&nbsp;</td>
<td>Celestron</td>
<td>CG-5</td>
<td>&nbsp;</td>
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<td>Digital video cam</td>
<td>&nbsp;</td>
<td>Sony</td>
<td>XCDV60</td>
<td>&nbsp;</td>
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<td>Caliper release</td>
<td>&nbsp;</td>
<td>Cabelas</td>
<td>IA-415482</td>
<td>&nbsp;</td>
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<td>Compression spring</td>
<td>&nbsp;</td>
<td>Jones Spring Co.</td>
<td>723</td>
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<td>Extension spring</td>
<td>&nbsp;</td>
<td>Jones Spring Co. </td>
<td>770</td>
<td>&nbsp;</td>
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<td>ImageJ</td>
<td>&nbsp;</td>
<td>NIH</td>
<td>NA</td>
<td>&nbsp;</td>
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<td>Fire-i drivers &amp; application </td>
<td>&nbsp;</td>
<td>Unibrain</td>
<td>3.80</td>
<td>&nbsp;</td>
<tr>
<td>Excel</td>
<td>&nbsp;</td>
<td>Microsoft</td>
<td>NA</td>
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demonstrating the uses of the novel gravitational force spectrometer to stretch and measure fibrous proteins

The study of macromolecular structure has become critical to the elucidation of molecular mechanisms and function. There are several limited, but important bioinstruments capable of testing the force dependence of structural features in proteins. Scale has been a limiting parameter on how accurately researchers can peer into the nanomechanical world of molecules, such as nucleic acids, enzymes, and motor proteins that perform life-sustaining work. Atomic force microscopy (AFM) is well tuned to determine native structures of fibrous proteins with a distance resolution on par with electron microscopy. However, in AFM force studies, the forces are typically much higher than a single molecule might experience 1, 2. Optical traps (OT) are very good at determining the relative distance between the trapped beads and they can impart very small forces 3. However, they do not yield accurate absolute lengths of the molecules under study. Molecular simulations provide supportive information to such experiments, but are limited in the ability to handle the same large molecular sizes, long time frames, and convince some researchers in the absence of other supporting evidence2, 4.
The gravitational force spectrometer (GFS) fills a critical niche in the arsenal of an investigator by providing a unique combination of abilities. This instrument is capable of generating forces typically with 98% or better accuracy from the femtonewton range to the nanonewton range. The distance measurements currently are capable of resolving the absolute molecular length down to five nanometers, and relative bead pair separation distances with a precision similar to an optical trap. Also, the GFS can determine stretching or uncoiling where the force is near equilibrium, or provide a graded force to juxtapose against any measured structural changes. It is even possible to determine how many amino acid residues are involved in uncoiling events under physiological force loads 2. Unlike in other methods where there is extensive force calibration that must precede any assay, the GFS requires no such force calibration 5. By complementing the strengths of other methods, the GFS will bridge gaps in understanding the nanomechanics of vital proteins and other macromolecules.

Watch this Scientific Journal Video about Demonstrating the Uses of the Novel Gravitational Force Spectrometer to Stretch and Measure Fibrous Proteins at JoVE.com

Demonstrating the Uses of the Novel Gravitational Force Spectrometer to Stretch and Measure Fibrous Proteins

This is a step-by step guide showing the purpose, operation, and representative results from the novel gravitational force spectrometer.

The study of macromolecular structure has become critical to the elucidation of molecular mechanisms and function. There are several limited, but ...

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10.3K vues.  University of North Texas. Ceci est une étape-par étape indiquant l'objet, l'exploitation et des résultats représentatifs du spectromètre force gravitationnelle roman.

Vidéo: Demonstrating the Uses of the Novel Gravitational Force Spectrometer to Stretch and Measure Fibrous Proteins

Axon Stretch Growth: The Mechanotransduction of Neuronal Growth

During pre-synaptic embryonic development, neuronal processes traverse short distances to reach their targets via growth cone. Over time, neuronal somata are separated from their axon terminals due to skeletal growth of the enlarging organism (Weiss 1941; Gray, Hukkanen et al. 1992). This mechanotransduction induces a secondary mode of neuronal growth capable of accommodating continual elongation of the axon (Bray 1984; Heidemann and Buxbaum 1994; Heidemann, Lamoureux et al. 1995; Pfister, Iwata et al. 2004).
Axon Stretch Growth (ASG) is conceivably a central factor in the maturation of short embryonic processes into the long nerves and white matter tracts characteristic of the adult nervous system. To study ASG in vitro, we engineered bioreactors to apply tension to the short axonal processes of neuronal cultures (Loverde, Ozoka et al. 2011). Here, we detail the methods we use to prepare bioreactors and conduct ASG. First, within each stretching lane of the bioreactor, neurons are plated upon a micro-manipulated towing substrate. Next, neurons regenerate their axonal processes, via growth cone extension, onto a stationary substrate. Finally, stretch growth is performed by towing the plated cell bodies away from the axon terminals adhered to the stationary substrate; recapitulating skeletal growth after growth cone extension.
Previous work has shown that ASG of embryonic rat dorsal root ganglia neurons are capable of unprecedented growth rates up to 10mm/day, reaching lengths of up to 10cm; while concurrently resulting in increased axonal diameters (Smith, Wolf et al. 2001; Pfister, Iwata et al. 2004; Pfister, Bonislawski et al. 2006; Pfister, Iwata et al. 2006; Smith 2009). This is in dramatic contrast to regenerative growth cone extension (in absence of mechanical stimuli) where growth rates average 1mm/day with successful regeneration limited to lengths of less than 3cm (Fu and Gordon 1997; Pfister, Gordon et al. 2011). Accordingly, further study of ASG may help to reveal dysregulated growth mechanisms that limit regeneration in the absence of mechanical stimuli.

A unique tissue engineering method was developed to elongate numerous nerve fibers in culture by recapitulating axon stretch growth; a form of nervous system growth whereby nerves elongate in conjunction with growth of the enlarging body.

During pre-synaptic embryonic development, neuronal processes traverse short distances to reach their targets via growth cone. Over time, neuronal somata ...

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

Quantifying the Mechanical Properties of the Endothelial Glycocalyx with Atomic Force Microscopy

Our understanding of the interaction of leukocytes and the vessel wall during leukocyte capture is limited by an incomplete understanding of the mechanical properties of the endothelial surface layer. It is known that adhesion molecules on leukocytes are distributed non-uniformly relative to surface topography 3, that topography limits adhesive bond formation with other surfaces 9, and that physiological contact forces (&asymp; 5.0 &minus; 10.0 pN per microvillus) can compress the microvilli to as little as a third of their resting length, increasing the accessibility of molecules to the opposing surface 3, 7. We consider the endothelium as a two-layered structure, the relatively rigid cell body, plus the glycocalyx, a soft protective sugar coating on the luminal surface 6. It has been shown that the glycocalyx can act as a barrier to reduce adhesion of leukocytes to the endothelial surface 4. In this report we begin to address the deformability of endothelial surfaces to understand how the endothelial mechanical stiffness might affect bond formation. Endothelial cells grown in static culture do not express a robust glycocalyx, but cells grown under physiological flow conditions begin to approximate the glycocalyx observed in vivo 2. The modulus of the endothelial cell body has been measured using atomic force microscopy (AFM) to be approximately 5 to 20 kPa 5. The thickness and structure of the glycocalyx have been studied using electron microscopy 8, and the modulus of the glycocalyx has been approximated using indirect methods, but to our knowledge, there have been no published reports of a direct measurement of the glycocalyx modulus in living cells. In this study, we present indentation experiments made with a novel AFM probe on cells that have been cultured in conditions to maximize their glycocalyx expression to make direct measurements of the modulus and thickness of the endothelial glycocalyx.

The mechanical characteristics of endothelial glycocalyx were measured by indentation using micron sized spheres on AFM cantilevers. Endothelial cells were cultured in a custom chamber under physiological flow conditions to induce glycocalyx expression. Data were analyzed using a thin film model to determine the glycocalyx thickness and modulus.

Our  understanding of the interaction of leukocytes and the vessel wall during  leukocyte capture is limited by an incomplete understanding of the ...

In situ Compressive Loading and Correlative Noninvasive Imaging of the Bone-periodontal Ligament-tooth Fibrous Joint

This study demonstrates a novel biomechanics testing protocol. The advantage of this protocol includes the use of an in situ loading device coupled to a high resolution X-ray microscope, thus enabling visualization of internal structural elements under simulated physiological loads and wet conditions. Experimental specimens will include intact bone-periodontal ligament (PDL)-tooth fibrous joints. Results will illustrate three important features of the protocol as they can be applied to organ level biomechanics: 1) reactionary force vs. displacement: tooth displacement within the alveolar socket and its reactionary response to loading, 2) three-dimensional (3D) spatial configuration and morphometrics: geometric relationship of the tooth with the alveolar socket, and 3) changes in readouts 1 and 2 due to a change in loading axis, i.e. from concentric to eccentric loads. Efficacy of the proposed protocol will be evaluated by coupling mechanical testing readouts to 3D morphometrics and overall biomechanics of the joint. In addition, this technique will emphasize on the need to equilibrate experimental conditions, specifically reactionary loads prior to acquiring tomograms of fibrous joints. It should be noted that the proposed protocol is limited to testing specimens under ex vivo conditions, and that use of contrast agents to visualize soft tissue mechanical response could lead to erroneous conclusions about tissue and organ-level biomechanics.

In situ Compressive Loading and Correlative Noninvasive Imaging of the Bone-periodontal Ligament-tooth Fibrous Joint

In this study, the use of an in situ loading device coupled with micro-X-ray computed tomography for fibrous joint biomechanics will be discussed. Experimental readouts identifiable with an overall change in joint biomechanics will include: 1) reactionary force vs. displacement, i.e. tooth displacement within the alveolar socket and its reactionary response to loading, 2) three-dimensional (3D) spatial configuration and morphometrics, i.e.&#160;geometric relationship of the tooth with the alveolar socket, and 3) changes in readouts 1 and 2 due to a change in loading axis, i.e. concentric or eccentric loads.

This study demonstrates a novel biomechanics testing protocol. The advantage of this protocol includes the use of an in situ loading device coupled to a ...

A Novel Method for Localizing Reporter Fluorescent Beads Near the Cell Culture Surface for Traction Force Microscopy

PA gels have long been used as a platform to study cell traction forces due to ease of fabrication and the ability to tune their elastic properties. When the substrate is coated with an extracellular matrix protein, cells adhere to the gel and apply forces, causing the gel to deform. The deformation depends on the cell traction and the elastic properties of the gel. If the deformation field of the surface is known, surface traction can be calculated using elasticity theory. Gel deformation is commonly measured by embedding fluorescent marker beads uniformly into the gel. The probes displace as the gel deforms. The probes near the surface of the gel are tracked. The displacements reported by these probes are considered as surface displacements. Their depths from the surface are ignored. This assumption introduces error in traction force evaluations. For precise measurement of cell forces, it is critical for the location of the beads to be known. We have developed a technique that utilizes simple chemistry to confine fluorescent marker beads, 0.1 and 1 &#181;m in diameter, in PA gels, within 1.6 &#956;m of the surface. We coat a coverslip with poly-D-lysine (PDL) and fluorescent beads. PA gel solution is then sandwiched between the coverslip and an adherent surface. The fluorescent beads transfer to the gel solution during curing. After polymerization, the PA gel contains fluorescent beads on a plane close to the gel surface.

Traditional techniques for fabricating polyacrylamide (PA) gels containing fluorescent probes involve sandwiching a gel between an adherent surface and a glass slide. Here, we show that coating this slide with poly-D-lysine (PDL) and fluorescent probes localizes the probes to within 1.6 &#181;m from the gel surface.

PA gels have long been used as a platform to study cell traction forces due to ease of fabrication and the ability to tune their elastic properties. When ...

An In Vitro Enzymatic Assay to Measure Transcription Inhibition by Gallium(III) and H3 5,10,15-tris(pentafluorophenyl)corroles

Chemotherapy often involves broad-spectrum cytotoxic agents with many side effects and limited targeting. Corroles are a class of tetrapyrrolic macrocycles that exhibit differential cytostatic and cytotoxic properties in specific cell lines, depending on the identities of the chelated metal and functional groups. The unique behavior of functionalized corroles towards specific cell lines introduces the possibility of targeted chemotherapy.
Many anticancer drugs are evaluated by their ability to inhibit RNA transcription. Here we present a step-by-step protocol for RNA transcription in the presence of known and potential inhibitors. The evaluation of the RNA products of the transcription reaction by gel electrophoresis and UV-Vis spectroscopy provides information on inhibitive properties of potential anticancer drug candidates and, with modifications to the assay, more about their mechanism of action.
Little is known about the molecular mechanism of action of corrole cytotoxicity. In this experiment, we consider two corrole compounds: gallium(III) 5,10,15-(tris)pentafluorophenylcorrole (Ga(tpfc)) and freebase analogue 5,10,15-(tris)pentafluorophenylcorrole (tpfc). An RNA transcription assay was used to examine the inhibitive properties of the corroles. Five transcription reactions were prepared: DNA treated with Actinomycin D, triptolide, Ga(tpfc), tpfc at a [complex]:[template DNA base] ratio of 0.01, respectively, and an untreated control.
The transcription reactions were analyzed after 4 hr using agarose gel electrophoresis and UV-Vis spectroscopy. There is clear inhibition by Ga(tpfc), Actinomycin D, and triptolide.
This RNA transcription assay can be modified to provide more mechanistic detail by varying the concentrations of the anticancer complex, DNA, or polymerase enzyme, or by incubating the DNA or polymerase with the complexes prior to RNA transcription; these modifications would differentiate between an inhibition mechanism involving the DNA or the enzyme. Adding the complex after RNA transcription can be used to test whether the complexes degrade or hydrolyze the RNA. This assay can also be used to study additional anticancer candidates.

An In Vitro Enzymatic Assay to Measure Transcription Inhibition by GalliumIII and H3 5,10,15-trispentafluorophenylcorroles

Gallium(III) 5,10,15-(tris)pentafluorophenylcorrole and its freebase analogue exhibit low micromolar cell cytotoxicity. This manuscript describes an RNA transcription reaction, imaging RNA with an ethidium bromide-stained gel, and quantifying RNA with UV-Vis spectroscopy, in order to assess transcription inhibition by corroles and demonstrates a straightforward method of evaluating anticancer candidate properties.

Chemotherapy often involves broad-spectrum cytotoxic agents with many side effects and limited targeting. Corroles are a class of tetrapyrrolic ...

Live Cell Imaging during Mechanical Stretch

There is currently a significant interest in understanding how cells and tissues respond to mechanical stimuli, but current approaches are limited in their capability for measuring responses in real time in live cells or viable tissue. A protocol was developed with the use of a cell actuator to distend live cells grown on or tissues attached to an elastic substrate while imaging with confocal and atomic force microscopy (AFM). Preliminary studies show that tonic stretching of human bronchial epithelial cells caused a significant increase in the production of mitochondrial superoxide. Moreover, using this protocol, alveolar epithelial cells were stretched and imaged, which showed direct damage to the epithelial cells by overdistention simulating one form of lung injury in vitro. A protocol to conduct AFM nano-indentation on stretched cells is also provided.

A novel imaging protocol was developed using a custom motor-driven mechanical actuator to allow the measurement of real time responses to mechanical strain in live cells. Relevant to mechanobiology, the system can apply strains up to 20% while allowing near real-time imaging with confocal or atomic force microscopy.

There is currently a significant interest in understanding how cells and tissues respond to mechanical stimuli, but current approaches are limited in ...

Fluorescence Recovery after Merging a Droplet to Measure the Two-dimensional Diffusion of a Phospholipid Monolayer

We introduce a new method to measure the lateral diffusivity of a surfactant monolayer at the fluid-fluid interface, called fluorescence recovery after merging (FRAM). FRAM adopts the same principles as the fluorescence recovery after photobleaching (FRAP) technique, especially for measuring fluorescence recovery after bleaching a specific area, but FRAM uses a drop coalescence instead of photobleaching dye molecules to induce a chemical potential gradient of dye molecules. Our technique has several advantages over FRAP: it only requires a fluorescence microscope rather than a confocal microscope equipped with high power lasers; it is essentially free from the selection of fluorescence dyes; and it has far more freedom to define the measured diffusion area. Furthermore, FRAM potentially provides a route for studying the mixing or inter-diffusion of two different surfactants, when the monolayers at a surface of droplet and at a flat air/water interface are prepared with different species, independently.

We present a new technique to measure the lateral diffusion of a surface active species at the fluid-fluid interface by merging a droplet monolayer onto a flat monolayer.

We introduce a new method to measure the lateral diffusivity of a surfactant monolayer at the fluid-fluid interface, called fluorescence recovery after ...

High Speed Sub-GHz Spectrometer for Brillouin Scattering Analysis

The goal of this protocol is to build a parallel high-extinction and high-resolution optical Brillouin spectrometer. Brillouin spectroscopy is a non-contact measurement method that can be used to obtain direct readouts of viscoelastic material properties. It has been a useful tool in material characterization, structural monitoring and environmental sensing. In the past, Brillouin spectroscopy has usually employed scanning Fabry-Perot etalons to perform spectral analysis. This process requires high illumination power and long acquisition times, making the technique unsuitable for biomedical applications. A recently introduced novel spectrometer overcomes this challenge by employing two VIPAs in a cross-axis configuration. This innovation enables sub-Gigahertz (GHz) resolution spectral analysis with sub-second acquisition time and illumination power within the safety limits of biological tissue. The multiple new applications facilitated by this improvement are currently being explored in biological research and clinical application.

Here we present a protocol to build a rapid Brillouin spectrometer. Cascading virtually imaged phase array (VIPA) etalons achieve a measurement speed more than 1,000 times faster than traditional scanning Fabry-Perot spectrometers. This improvement provides the means for Brillouin analysis of tissue and biomaterials at low power levels in vivo.

The goal of this protocol is to build a parallel high-extinction and high-resolution optical Brillouin spectrometer. Brillouin spectroscopy is a ...

A Protocol for Using F&#246;rster Resonance Energy Transfer (FRET)-force Biosensors to Measure Mechanical Forces across the Nuclear LINC Complex

The LINC complex has been hypothesized to be the critical structure that mediates the transfer of mechanical forces from the cytoskeleton to the nucleus. Nesprin-2G is a key component of the LINC complex that connects the actin cytoskeleton to membrane proteins (SUN domain proteins) in the perinuclear space. These membrane proteins connect to lamins inside the nucleus. Recently, a F&#246;rster Resonance Energy Transfer (FRET)-force probe was cloned into mini-Nesprin-2G (Nesprin-TS (tension sensor)) and used to measure tension across Nesprin-2G in live NIH3T3 fibroblasts. This paper describes the process of using Nesprin-TS to measure LINC complex forces in NIH3T3 fibroblasts. To extract FRET information from Nesprin-TS, an outline of how to spectrally unmix raw spectral images into acceptor and donor fluorescent channels is also presented. Using open-source software (ImageJ), images are pre-processed and transformed into ratiometric images. Finally, FRET data of Nesprin-TS is presented, along with strategies for how to compare data across different experimental groups.

A Protocol for Using F&amp;#246;rster Resonance Energy Transfer (FRET)-force Biosensors to Measure Mechanical Forces across the Nuclear LINC Complex

A number of FRET-based force biosensors have recently been developed, enabling the protein-specific resolution of intracellular force. In this protocol, we demonstrate how one of these sensors, designed for the linker of the nucleoskeleton-cytoskeleton (LINC) complex protein Nesprin-2G can be used to measure actomyosin forces on the nuclear LINC complex.

The LINC complex has been hypothesized to be the critical structure that mediates the transfer of mechanical forces from the cytoskeleton to the nucleus. ...