Name: dissecting mechanoenzymatic properties of processive myosins with ultrafast force-clamp spectroscopy
Uploaded: 2021-07-01T13:30:00
Description: Watch this Scientific Journal Video about Dissecting Mechanoenzymatic Properties of Processive Myosins with Ultrafast Force-Clamp Spectroscopy at JoVE.com

This work was supported by the European Union&#8217;s Horizon 2020 research and innovation programme under Grant Agreement No. 871124 Laserlab-Europe, by the Italian Ministry of University and Research (FIRB &#8220;Futuro in Ricerca&#8221; 2013 Grant No. RBFR13V4M2), and by Ente Cassa di Risparmio di Firenze. A.V. Kashchuk was supported by Human Frontier Science Program Cross-Disciplinary Fellowship LT008/2020-C.

1. Optical setup
NOTE: The experimental setup is composed of double optical tweezers with nanometer pointing stability and &#60; 1% laser intensity fluctuations. Under these conditions, stability of the dumbbell at the nanometer level is guaranteed under typical trap stiffness (0.1 pN/nm) and tension (1 pN - few tens of pN). Figure 2 shows a detailed scheme of the optical setup.
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Although single molecule techniques, such as the three-bead assay, are technically challenging and low throughput, UFFCS improves the detection of molecular interactions thanks to the high signal-to-noise ratio of the data. UFFCS allows the study of the load-dependence of motor proteins, with the main advantages of applying the force very rapidly upon binding of the motor to the filament to probe early and very rapid events in force production and weak binding states under controlled force; maintaining the force constant...

An erratum was issued for:&#160;<a href="https://www.jove.com/t/62388">Dissecting Mechanoenzymatic Properties of Processive Myosins with Ultra Force-Clamp Spectroscopy</a>. The title was updated.
The title was updated from:
Dissecting Mechanoenzymatic Properties of Processive Myosins with Ultra Force-Clamp Spectroscopy
to:
Dissecting Mechanoenzymatic Properties of Processive Myosins with Ultrafast Force-Clamp Spectroscopy

An erratum was issued for:&#160;<a href="https://www.jove.com/t/62388">Dissecting Mechanoenzymatic Properties of Processive Myosins with Ultra Force-Clamp Spectroscopy</a>. The title was updated.

Erratum: Dissecting Mechanoenzymatic Properties of Processive Myosins with Ultra Force-Clamp Spectroscopy

In the last decades optical tweezers have been a valuable tool to elucidate the mechanochemistry of protein interactions at the single molecule level, due to the striking possibility of concurrent manipulation and measurement of conformational changes and enzymatic kinetics 1,2. In particular, the capability to apply and measure forces in the range of those exerted by molecular motors in the cell, together with the capacity to measure sub-nanometer conformational changes, made optical tweezers a unique single-molecule tool for unraveling the chemomechanical properties of motor proteins and their mechanical regulation.
Ultrafast force-clamp spectroscopy (UFFCS) is a single-molecule force-spectroscopy technique based on optical tweezers, developed to study the fast kinetics of molecular motors under load in a three-bead geometry (Figure 1a)3,4. UFFCS reduces the time lag for force application to the motor protein to the physical limit of optical tweezers, i.e., the mechanical relaxation time of the system, thus allowing the application of the force rapidly after the beginning of a myosin run (few tens of microseconds)3. This capability has been exploited to investigate the early mechanical events in fast skeletal 3 and cardiac5 muscle myosin to reveal the load dependence of the powerstroke, the weak- and strong-binding states, as well as the order of biochemical (Pi) and mechanical (powerstroke) events.
The three-bead geometry is usually employed to study non-processive motors, a single bead geometry with a force-clamp has been commonly used to investigate processive non-conventional myosins such as myosin Va6. However, there are several reasons to prefer a three-bead UFFCS assay also for processive myosins. First, the rapid application of load right after actin-myosin binding allows the measurement of the early events in force development as in non-processive motors. In addition, in the case of processive motors it also allows an accurate measurement of the motor&#39;s run lengths and run durations under constant force all through their progression (Figure 1b). Moreover, because of the high rate of the force feedback, the system can maintain the force constant during fast changes in position, such as the myosin working stroke, thereby guaranteeing a constant load during motor stepping. The high-temporal resolution of the system allows the detection of sub-ms interactions, opening the possibility of investigating weak binding of myosin to actin. Finally, the assay geometry guarantees that the force is applied along the actin filament, with negligible transverse and vertical components of the force. This point is of particular relevance since the vertical force component has been shown to influence significantly the load-dependence of motor&#39;s kinetics7,8. By using this technique, we could apply a range of assistive and resistive loads to processive myosin-5B and directly measure the load dependence of its processivity for a wide range of forces4.
As shown in Figure 1a, in this system a single actin filament is suspended between two polystyrene beads trapped in the focus of double optical tweezers (the &#34;dumbbell&#34;). An imbalanced net force F= F1-F2 is imposed on the filament, through a fast feedback system, which makes the filament move at constant velocity in one direction until it reaches a user-defined inversion point where the net force is reversed in the opposite direction. When the motor protein is not interacting with the filament, the dumbbell is free to move back and forth in a triangular wave shape (Figure 1b, bottom panel) spanning the pedestal bead on which a single motor protein is attached. Once the interaction is established the force carried by the dumbbell is very rapidly transferred to the motor protein and the motor starts displacing the filament by stepping under the force intensity and direction that was applied by the feedback system at the time of the interaction, until myosin detaches from actin. Being the displacement produced by the stepping of the motor dependent on the polarity of the trapped actin filament, according to the direction of the applied force the load can be either assistive, i.e., pushing in the same direction of the motor displacement (push in Figure 1b upper panel), or resistive, i.e., pulling in the opposite direction with respect to the motor displacement (pull in Figure 1b upper panel) making it possible to study the chemomechanical regulation of the motor processivity by both the intensity and the directionality of the applied load.
In the next sections all the steps to measure actin-myosin-5B interactions under different loads with an ultrafast force-clamp spectroscopy setup are fully described, including 1) the setting up of the optical setup, optical traps alignment and calibration procedures, 2) the preparations of all the components and their assembly in the sample chamber, 3) the measurement procedure, 4) representative data and data analysis to extract important physical parameters, such as the run length, the step size and the velocity of the motor protein.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>&#160;Aliphatic Amine Latex Beads</td>
			<td>ThermoFisher</td>
			<td>A37362</td>
			<td>1.0-&#956;m diameter, 2% (w/v)</td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>Sigma</td>
			<td>32201</td>
			<td></td>
		</tr>
		<tr>
			<td>Actin polymerization buffer</td>
			<td>Cytoskeleton</td>
			<td>BSA02</td>
			<td>10X</td>
		</tr>
		<tr>
			<td>AODs (acousto-optic deflectors)</td>
			<td>AA Opto Electronic</td>
			<td>DTS-XY 250</td>
			<td>Laser beam deflectors</td>
		</tr>
		<tr>
			<td>ATP</td>
			<td>Sigma</td>
			<td>A7699</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotinylated-BSA</td>
			<td>ThermoFisher</td>
			<td>29130</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma</td>
			<td>B4287</td>
			<td></td>
		</tr>
		<tr>
			<td>Calmodulin from porcine brain (CaM)</td>
			<td>Merck Millipore</td>
			<td>208783</td>
			<td></td>
		</tr>
		<tr>
			<td>Catalase from bovine liver</td>
			<td>Sigma</td>
			<td>C40</td>
			<td></td>
		</tr>
		<tr>
			<td>Condenser</td>
			<td>Olympus</td>
			<td>OlympusU-AAC, Aplanat, Achromat</td>
			<td>NA 1.4, oil immersion</td>
		</tr>
		<tr>
			<td>Creatine phosphate disodium salt tetrahydrate</td>
			<td>Sigma</td>
			<td>27920</td>
			<td></td>
		</tr>
		<tr>
			<td>Creatine Phosphokinase from rabbit muscle</td>
			<td>Sigma</td>
			<td>C3755</td>
			<td></td>
		</tr>
		<tr>
			<td>DDs</td>
			<td>AA Opto Electronic</td>
			<td>AA.DDS.XX</td>
			<td>Two-channel digital synthesizer</td>
		</tr>
		<tr>
			<td>DL-Dithiothreitol (DTT)/td&#62;</td>
			<td>Sigma</td>
			<td>43819</td>
			<td></td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Sigma</td>
			<td>E4378</td>
			<td></td>
		</tr>
		<tr>
			<td>G-actin protein</td>
			<td>Cytoskeleton</td>
			<td>AKL99</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma</td>
			<td>G7528</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose Oxidase from Aspergillus niger</td>
			<td>Sigma</td>
			<td>&#160;G7141</td>
			<td></td>
		</tr>
		<tr>
			<td>HaloTag succinimidyl ester O2 ligand</td>
			<td>Promega</td>
			<td>P1691</td>
			<td></td>
		</tr>
		<tr>
			<td>High vacuum silicone grease heavy</td>
			<td>Merck Millipore</td>
			<td>107921</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma</td>
			<td>P9541</td>
			<td></td>
		</tr>
		<tr>
			<td>KH2PO4/K2HPO4</td>
			<td>Sigma</td>
			<td>P5379/ P8281</td>
			<td></td>
		</tr>
		<tr>
			<td>Labview</td>
			<td>National Instruments</td>
			<td>version 8.1</td>
			<td>Data acquisition</td>
		</tr>
		<tr>
			<td>Labview FPGA module</td>
			<td>National Instruments</td>
			<td>version 8.1</td>
			<td>Fast Force-Clamp</td>
		</tr>
		<tr>
			<td>Matlab</td>
			<td>MathWorks</td>
			<td>2016</td>
			<td>Data analysis</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Fluka</td>
			<td>63020</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope Objective</td>
			<td>Nikon</td>
			<td>Plan-Apo 60X</td>
			<td>NA 1.2, WD 0.2 mm, water imm.</td>
		</tr>
		<tr>
			<td>MOPS</td>
			<td>Sigma</td>
			<td>M1254</td>
			<td></td>
		</tr>
		<tr>
			<td>Nitrocellulose</td>
			<td>Sigma</td>
			<td>N8267</td>
			<td>0.45 pore size</td>
		</tr>
		<tr>
			<td>Pentyl acetate solution</td>
			<td>Sigma</td>
			<td>46022</td>
			<td></td>
		</tr>
		<tr>
			<td>Pure Ethanol</td>
			<td>&#160;Sigma</td>
			<td>2860</td>
			<td></td>
		</tr>
		<tr>
			<td>QPDs</td>
			<td>UDT</td>
			<td>DLS-20</td>
			<td>D Position Detecto</td>
		</tr>
		<tr>
			<td>Rhodamine BSA</td>
			<td>Molecular Probes</td>
			<td>A23016</td>
			<td></td>
		</tr>
		<tr>
			<td>Rhodamine Phalloidin</td>
			<td>&#160;Sigma</td>
			<td>P1951</td>
			<td></td>
		</tr>
		<tr>
			<td>Silica beads</td>
			<td>Bangslabs</td>
			<td>SS04N</td>
			<td>1.21 mm, 10% solids</td>
		</tr>
		<tr>
			<td>Sodium azide</td>
			<td>&#160;Sigma</td>
			<td>S2002</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptavidin protein</td>
			<td>&#160;Sigma</td>
			<td>189730</td>
			<td></td>
		</tr>
	</tbody>
</table>

dissecting mechanoenzymatic properties of processive myosins with ultrafast force-clamp spectroscopy

Ultrafast force-clamp spectroscopy (UFFCS) is a single molecule technique based on laser tweezers that allows the investigation of the chemomechanics of both conventional and unconventional myosins under load with unprecedented time resolution. In particular, the possibility to probe myosin motors under constant force right after the actin-myosin bond formation, together with the high rate of the force feedback (200 kHz), has shown UFFCS to be a valuable tool to study the load dependence of fast dynamics such as the myosin working stroke. Moreover, UFFCS enables the study of how processive and non-processive myosin-actin interactions are influenced by the intensity and direction of the applied force.
By following this protocol, it will be possible to perform ultrafast force-clamp experiments on processive myosin-5 motors and on a variety of unconventional myosins. By some adjustments, the protocol could also be easily extended to the study of other classes of processive motors such as kinesins and dyneins. The protocol includes all the necessary steps, from the setup of the experimental apparatus to sample preparation, calibration procedures, data acquisition and analysis.

Representative data consist in position records over time as shown in Figure 4. In the position record two kinds of displacement are visible. Firstly, when the myosin motor is not interacting with the actin filament the trapped beads are moving at constant velocity against the viscous drag force of the solution showing a linear displacement oscillating within the oscillation range set by the operator in a triangular wave3 (not visible in <strong class...

Watch this Scientific Journal Video about Dissecting Mechanoenzymatic Properties of Processive Myosins with Ultrafast Force-Clamp Spectroscopy at JoVE.com

Dissecting Mechanoenzymatic Properties of Processive Myosins with Ultrafast Force-Clamp Spectroscopy

Presented here is a comprehensive protocol to perform ultrafast force-clamp experiments on processive myosin-5 motors, which could be easily extended to the study of other classes of processive motors. The protocol details all the necessary steps, from the setup of the experimental apparatus to sample preparation, data acquisition and analysis.

Ultrafast force-clamp spectroscopy (UFFCS) is a single molecule technique based on laser tweezers that allows the investigation of the chemomechanics of ...

Ultrafast force-clamp spectroscopy is a single molecule technique based on laser tweezers that makes it possible to investigate the chemomechanics of myosins motors under load with the highest time resolution. This technique allows probing very rapid events in force production by maintaining the force constant during every phase of the motor interaction with full control on force directionality. By making appropriate adjustments, this protocol could be easily applied to other kinds of processive motors, including kinesins and dyneins, to uncover the regulation by force.After mounting one set of acousto-optic deflectors onto a linear translator, take an iris and adjust its aperture to fit the objective back aperture size. Replace the objective with the iris centered on the threaded objective housing. Move the translator toward the laser beam until the portion of the beam blocked by the piezo crystal is visible after the iris, and turn the translator slightly backward until the beam completely fills the iris aperture.To prepare silica beads in pentyl acetate, first dilute 20 microliters of 1.2 micron 10%solid silica beads in 1-1.5 milliliters of acetone with vortexing and 30 seconds of sonication. After sonicating, collect the beads by centrifugation and resuspend the bead pellet in 1 milliliter of fresh acetone for a second wash. After the second acetone wash, wash the beads three times in 1 milliliter of fresh pentyl acetate per wash under the same centrifugation conditions, and resuspend the pellet in 100 microliters of 1%of nitrocellulose and 900 microliters pentyl acetate.Next, use a pure ethanol-soaked piece of lab tissue to carefully wipe a 24 x 24 millimeter glass coverslip before using clean tweezers to rinse the glass directly in the ethanol and allowing the coverslip to dry under gentle nitrogen flow. Vortex and sonicate the prepared silica bead stock for 30 seconds and use an ethanol-cleaned 24 x 60 millimeter coverslip to smear 2 microliters of silica bead solution onto the surface of the smaller coverslip. While the beads are drying, clean a 26 x 76 millimeter slide as demonstrated, and place two 60-100 micron-thick lines of 3 millimeter double-sided taped onto the long edges of the slide.Using clean tweezers, place the bead-coated coverslip onto the tape with the beads facing the inside of the slide chamber, and fill the chamber with 15 millimolar phosphate buffer and floating polystyrene bead solution. Then, seal the chamber with silicon grease. To calibrate the pixel-to-nanometer ratio of the brightfield camera, acquire a first image of a silica bead on the coverslip surface approximately 5 microns from the center of the field of view.Move the stage to displace the bead 10 microns toward the center of the field of view and take a second image. Calculate the camera calibration constant by measuring the pixel distance between two positions of the bead. To calibrate the trap position, trap a single floating particle in one trap and move the acousto-optic deflectors in 0.2 megahertz steps, acquiring an image of the particle and the corresponding frequency of the deflectors for each step.Then, repeat the calibration for the second trap in the same manner. To calibrate the trap power and stiffness, trap a single particle in each trap and displace both traps using the acousto-optic deflectors in 0.2 megahertz steps, recording a Brownian motion of the particle in both traps with Quadrant Photodiode Detectors and the corresponding frequency of the acousto-optic deflectors. Obtain calibration constants by fitting a Lorentzian function to a power spectrum of the recorded Brownian motion.To assemble a sample for measurement, add 1 milligram per milliliter of biotinylated BSA to a new chamber with silica beads on the coverslip surface. After 5 minutes, wash the chamber with AB buffer and add 1 milligram per milliliter of streptavidin to the chamber. After 5 minutes, wash the chamber as demonstrated, and add 3 nanomolar biotinylated Myosin-5B heavy meromyosin in M5B buffer supplemented with 2 micromolar Calmodulin.After 5 minutes, wash the chamber three times with 1 milligram per milliliter of biotinylated BSA supplemented with 2 micromolar Calmodulin in AB buffer and wait 3 minutes after the final wash. Flow the reaction mixture and seal the chamber with silicon grease. To measure the sample, place the chamber onto the microscope stage and move the objective until the floating alpha-actinin beads are in focus.Switch on one trap and trap one bead. Once the first trap is occupied, move the translator to position the trapped bead close to the coverslip surface to avoid trapping multiple beads, and trap a bead in the second trap. Displace the sample through the long-range translators to locate actin filaments of at least 5 microns in length within the solution.When a filament has been spotted, move the sample to let a trapped bead approach one end of the filament. Once the filament has been attached, adjust the bead's distance to the approximate filament length and move the stage to create a flow in the unbound bead direction. The filament will become stretched by the flow and eventually bind to the second bead.The resulting complex is called a dumbbell"To establish actin-myosin contact, gently separate the two traps and set the filament to pretension levels up to about 3 piconewtons. To probe the rigidity of the dumbbell, make one trap oscillate in a triangular wave by changing the frequency of one acousto-optic deflector and verifying the consequent transmission of the motion to the trailing bead through its position signal. Move the stage to position the dumbbell in the proximity of a pedestal silica bead and adjust the height of the dumbbell so that the actin filament gets in contact with the silica bead surface.As observed in this representative position record, when the myosin motor is not interacting with the actin filament, the trapped beads move at a constant velocity against the viscous drag force of the solution. Once the myosin motor begins interacting with the filament, the force carried by the moving filament is very rapidly transferred to the protein, and the system velocity drops to zero, with the stepping events occurring under constant force till the end of the run. The force is switched from the positive to the negative direction by the feedback system, which switches the force direction when the bead reaches the edge of the oscillation range set by the user.When the myosin binds and displaces the filament toward the positive direction, it pushes the bead toward the upper edge of the oscillation range. If this happens under assistive force, the run of the myosin will be interrupted by the force direction inversion at the oscillation edge, limiting the length of the run to the amplitude of the dumbbell oscillation. Precise alignment of the optical system is necessary to achieve the optimal spatial and temporal resolution, while an accurate calibration is necessary to precisely determine the values of the applied forces.This technique has been adapted to study the sliding and targeted search of transcription factors in DNA as well as dynamic interactions between mechanotransduction proteins inducting over microtubule filaments.

dissecting-mechanoenzymatic-properties-processive-myosins-with

Research

JoVE Journal

Biology

Dissecting and Recording from The C. Elegans Neuromuscular Junction

Neurotransmission is the process by which neurons transfer information via chemical signals to their post-synaptic targets, on a rapid time scale. This complex process requires the coordinated activity of many pre- and post-synaptic proteins to ensure appropriate synaptic connectivity, conduction of electrical signals, targeting and priming of secretory vesicles, calcium sensing, vesicle fusion, localization and function of postsynaptic receptors and finally, recycling mechanisms. As neuroscientists it is our goal to elucidate which proteins function in each of these steps and understand their mechanisms of action. Electrophysiological recordings from synapses provide a quantifiable read out of the underlying electrical events that occur during synaptic transmission. By combining this technique with the powerful array of molecular and genetic tools available to manipulate synaptic proteins in C. elegans, we can analyze the resulting functional changes in synaptic transmission. 
The C. elegans NMJs formed between motor neurons and body wall muscles control locomotion, therefore, mutants with uncoordinated locomotory phenotypes (known as unc s) often perturb synaptic transmission at these synapses 1. Since unc mutants are maintained on a rich supply of a bacterial food source, they remain viable as long as they retain some pharyngeal pumping ability to ingest food. This, together with the fact that C. elegans exist as hermaphrodites, allows them to pass on mutant progeny without the need for elaborate mating behaviors. These attributes, coupled with our recent ability to record from the worms NMJs 2,3,7 make this an excellent model organism in which to address precisely how unc mutants impact neurotransmission. 
The dissection method involves immobilizing adult worms using a cyanoacrylic glue in order to make an incision in the worm cuticle exposing the NMJs. Since C. elegans adults are only 1 mm in length the dissection is performed with the use of a dissecting microscope and requires excellent hand-eye coordination. NMJ recordings are made by whole-cell voltage clamping individual body wall muscle cells and neurotransmitter release can be evoked using a variety of stimulation protocols including electrical stimulation, light-activated channel-rhodopsin-mediated depolarization 4 and hyperosmotic saline, all of which will be briefly described.

Application of electrophysiology to accessible synapses provides a quantifiable measure of synaptic activity, useful in analyzing synaptic mutants. This article describes a dissection method used to expose the neuromuscular junctions (NMJ) of Caenorhabditis elegans (C. elegans) and briefly discusses some of the uses to which this preparation can be applied.

Neurotransmission is the process by which neurons transfer information via chemical signals to their post-synaptic targets, on a rapid time scale.   This ...

Dissecting the Non-human Primate Brain in Stereotaxic Space

The use of non-human primates provides an excellent translational model for our understanding of developmental and aging processes in humans1-6. In addition, the use of non-human primates has recently afforded the opportunity to naturally model complex psychiatric disorders such as alcohol abuse7. Here we describe a technique for blocking the brain in the coronal plane of the vervet monkey (Chlorocebus aethiops sabeus) in the intact skull in stereotaxic space. The method described here provides a standard plane of section between blocks and subjects and minimizes partial sections between blocks. Sectioning a block of tissue in the coronal plane also facilitates the delineation of an area of interest. This method provides manageable sized blocks since a single hemisphere of the vervet monkey yields more than 1200 sections when slicing at 50&mu;m. Furthermore by blocking the brain into 1cm blocks, it facilitates penetration of sucrose for cyroprotection and allows the block to be sliced on a standard cryostat.

The non-human primate is an important translational species for our understanding of the normal processing of the brain. The anatomical organization of the primate brain can provide important insights into normal and pathological conditions in humans.

The use of non-human primates provides an excellent translational model for our understanding of developmental and aging processes in humans1-6. In ...

The Gateway to the Brain: Dissecting the Primate Eye

The visual system in humans is considered the gateway to the world and plays a principal role in the plethora of sensory, perceptual and cognitive processes. It is therefore not surprising that quality of vision is tied to quality of life . Despite widespread clinical and basic research surrounding the causes of visual disorders, many forms of visual impairments, such as retinitis pigmentosa and macular degeneration, lack effective treatments. Non-human primates have the closest general features of eye development to that of humans. Not only do they have a similar vascular anatomy, but amongst other mammals, primates have the unique characteristic of having a region in the temporal retina specialized for high visual acuity, the fovea1. Here we describe a general technique for dissecting the primate retina to provide tissue for retinal histology, immunohistochemistry, laser capture microdissection, as well as light and electron microscopy. With the extended use of the non-human primate as a translational model, our hope is that improved understanding of the retina will provide insights into effective approaches towards attenuating or reversing the negative impact of visual disorders on the quality of life of affected individuals.

The non-human primate is an important translational species for our understanding of development and aging. The anatomical organization of the primate retina may provide important insights into normal and pathological conditions in humans.

The visual system in humans is considered the  gateway  to the world and plays a principal role in the plethora of sensory, perceptual and cognitive ...

Exploring Cognitive Functions in Babies, Children &amp; Adults with Near Infrared Spectroscopy

An explosion of functional Near Infrared Spectroscopy (fNIRS) studies investigating cortical activation in relation to higher cognitive processes, such as language1,2,3,4,5,6,7,8,9,10, memory11, and attention12 is underway worldwide involving adults, children and infants 3,4,13,14,15,16,17,18,19 with typical and atypical cognition20,21,22. The contemporary challenge of using fNIRS for cognitive neuroscience is to achieve systematic analyses of data such that they are universally interpretable23,24,25,26, and thus may advance important scientific questions about the functional organization and neural systems underlying human higher cognition.
Existing neuroimaging technologies have either less robust temporal or spatial resolution. Event Related Potentials and Magneto Encephalography (ERP and MEG) have excellent temporal resolution, whereas Positron Emission Tomography and functional Magnetic Resonance Imaging (PET and fMRI) have better spatial resolution. Using non-ionizing wavelengths of light in the near-infrared range (700-1000 nm), where oxy-hemoglobin is preferentially absorbed by 680 nm and deoxy-hemoglobin is preferentially absorbed by 830 nm (e.g., indeed, the very wavelengths hardwired into the fNIRS Hitachi ETG-400 system illustrated here), fNIRS is well suited for studies of higher cognition because it has both good temporal resolution (~5s) without the use of radiation and good spatial resolution (~4 cm depth), and does not require participants to be in an enclosed structure27,28. Participants cortical activity can be assessed while comfortably seated in an ordinary chair (adults, children) or even seated in mom s lap (infants). Notably, NIRS is uniquely portable (the size of a desktop computer), virtually silent, and can tolerate a participants subtle movement. This is particularly outstanding for the neural study of human language, which necessarily has as one of its key components the movement of the mouth in speech production or the hands in sign language. 
The way in which the hemodynamic response is localized is by an array of laser emitters and detectors. Emitters emit a known intensity of non-ionizing light while detectors detect the amount reflected back from the cortical surface. The closer together the optodes, the greater the spatial resolution, whereas the further apart the optodes, the greater depth of penetration. For the fNIRS Hitachi ETG-4000 system optimal penetration / resolution the optode array is set to 2cm. 
Our goal is to demonstrate our method of acquiring and analyzing fNIRS data to help standardize the field and enable different fNIRS labs worldwide to have a common background.

Here we describe a data collection and data analysis method for functional Near Infrared Spectroscopy (fNIRS), a novel non-invasive brain imaging system used in cognitive neuroscience, particularly in studying child brain development. This method provides a universal standard of data acquisition and analysis vital to data interpretation and scientific discovery.

An explosion of functional Near Infrared Spectroscopy (fNIRS) studies investigating cortical activation in relation to higher cognitive processes, such as ...

Assessing Signaling Properties of Ectodermal Epithelia During Craniofacial Development

The accessibility of avian embryos has helped experimental embryologists understand the fates of cells during development and the role of tissue interactions that regulate patterning and morphogenesis of vertebrates (e.g., 1, 2, 3, 4). Here, we illustrate a method that exploits this accessibility to test the signaling and patterning properties of ectodermal tissues during facial development. In these experiments, we create quail-chick 5 or mouse-chick 6 chimeras by transplanting the surface cephalic ectoderm that covers the upper jaw from quail or mouse onto either the same region or an ectopic region of chick embryos. The use of quail as donor tissue for transplantation into chicks was developed to take advantage of a nucleolar marker present in quail but not chick cells, thus allowing investigators to distinguish host and donor tissues 7. Similarly, a repetitive element is present in the mouse genome and is expressed ubiquitously, which allows us to distinguish host and donor tissues in mouse-chick chimeras 8. The use of mouse ectoderm as donor tissue will greatly extend our understanding of these tissue interactions, because this will allow us to test the signaling properties of ectoderm derived from various mutant embryos.

This article describes a tissue transplantation technique that was designed to test the signaling and patterning properties of surface cephalic ectoderm during craniofacial development.

The accessibility of avian embryos has helped experimental embryologists understand the fates of cells during development and the role of tissue ...

Procedures for Rat in situ Skeletal Muscle Contractile Properties

There are many circumstances where it is desirable to obtain the contractile response of skeletal muscle under physiological circumstances: normal circulation, intact whole muscle, at body temperature. This includes the study of contractile responses like posttetanic potentiation, staircase and fatigue. Furthermore, the consequences of disease, disuse, injury, training and drug treatment can be of interest. This video demonstrates appropriate procedures to set up and use this valuable muscle preparation.
To set up this preparation, the animal must be anesthetized, and the medial gastrocnemius muscle is surgically isolated, with the origin intact. Care must be taken to maintain the blood and nerve supplies. A long section of the sciatic nerve is cleared of connective tissue, and severed proximally. All branches of the distal stump that do not innervate the medial gastrocnemius muscle are severed. The distal nerve stump is inserted into a cuff lined with stainless steel stimulating wires. The calcaneus is severed, leaving a small piece of bone still attached to the Achilles tendon. Sonometric crystals and/or electrodes for electromyography can be inserted. Immobilization by metal probes in the femur and tibia prevents movement of the muscle origin. The Achilles tendon is attached to the force transducer and the loosened skin is pulled up at the sides to form a container that is filled with warmed paraffin oil. The oil distributes heat evenly and minimizes evaporative heat loss. A heat lamp is directed on the muscle, and the muscle and rat are allowed to warm up to 37&deg;C. While it is warming, maximal voltage and optimal length can be determined. These are important initial conditions for any experiment on intact whole muscle. The experiment may include determination of standard contractile properties, like the force-frequency relationship, force-length relationship, and force-velocity relationship.
With care in surgical isolation, immobilization of the origin of the muscle and alignment of the muscle-tendon unit with the force transducer, and proper data analysis, high quality measurements can be obtained with this muscle preparation.

Procedures for Rat in situ Skeletal Muscle Contractile Properties

This video demonstrates the surgical preparation and procedures needed to study the contractile responses of the rat medial gastrocnemius muscle preparation in situ. This preparation allows measurement of skeletal muscle contractile properties under physiological conditions. The animal is anesthetized and the muscle is separated from surrounding tissue at its distal end. The Achilles tendon is attached to a force transducer, allowing measurement of the muscle&#x2019;s contractile response at 37 degrees C with an intact circulation.

There are many circumstances where it is desirable to obtain the contractile response of skeletal muscle under physiological circumstances: normal ...

Combining Single-molecule Manipulation and Imaging for the Study of Protein-DNA Interactions

The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method offers the opportunity of investigating interactions occurring in solution (thus avoiding problems due to closeby surfaces as in other single molecule methods), controlling the DNA extension and tracking interaction dynamics as a function of both mechanical parameters and DNA sequence. The methods for establishing successful optical trapping and nanometer localization of single molecules are illustrated. We illustrate the experimental conditions allowing the study of interaction of lactose repressor (lacI), labeled with Atto532, with a DNA molecule containing specific target sequences (operators) for LacI binding. The method allows the observation of specific interactions at the operators, as well as one-dimensional diffusion of the protein during the process of target search. The method is broadly applicable to the study of protein-DNA interactions but also to molecular motors, where control of the tension applied to the partner track polymer (for example actin or microtubules) is desirable.

Here we describe the instrumentation and methods for detecting single fluorescently-labeled protein molecules interacting with a single DNA molecule suspended between two optically trapped microspheres.

The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method ...

Photostimulation by Femtosecond Laser Activates Extracellular-signal-regulated Kinase (ERK) Signaling or Mitochondrial Events in Target Cells

Direct control of cellular defined molecular events is important to life science. Recently, studies have demonstrated that femtosecond laser stimulation can simultaneously activate multiple cellular molecular signaling pathways. In this protocol, we show that through coupling femtosecond laser into a confocal microscope, cells can be stimulated precisely by the tightly-focused laser. Some molecular processes that can be simultaneously observed are subsequently activated. We present detailed protocols of the photostimulation to activate extracellular signal regulated kinase (ERK) signaling pathway in Hela cells. Mitochondrial flashes of reactive oxygen species (ROS) and other mitochondrial events can be also stimulated if focusing the femtosecond laser pulse on a certain mitochondrial tubular structure. This protocol includes pretreating cells before photostimulation, delivering the photostimulation by a femtosecond laser flash onto the target, and observing/identifying molecular changes afterwards. This protocol represents an all-optical tool for related biological researches.

Photostimulation by Femtosecond Laser Activates Extracellular-signal-regulated Kinase ERK Signaling or Mitochondrial Events in Target Cells

Tightly-focused femtosecond laser can deliver precise stimulation to cells by being coupled into a confocal microscopy enabling the real-time observation and photostimulation. The photostimulation can activate cell molecular events including ERK signaling pathway and mitochondrial flashes of reactive oxygen species.

Direct control of cellular defined molecular events is important to life science. Recently, studies have demonstrated that femtosecond laser stimulation ...

Quantification of Cellular Densities and Antigenic Properties using Magnetic Levitation

The described method was developed based on the principles of magnetic levitation, which separates cells and particles based on their density and magnetic properties. Density is a cell type identifying property, directly related to its metabolic rate, differentiation, and activation status. Magnetic levitation allows a one-step approach to successfully separate, image and characterize circulating blood cells, and to detect anemia, sickle cell disease, and circulating tumor cells based on density and magnetic properties. This approach is also amenable to detecting soluble antigens present in a solution by using sets of low- and high-density beads coated with capture and detection antibodies, respectively. If the antigen is present in solution, it will bridge the two sets&#160;of beads, generating a new bead-bead complex, which will levitate in between the rows of antibody-coated beads. Increased concentration of the target antigen in solution will generate a larger number of bead-bead complexes when compared to lower concentrations of antigen, thus allowing for quantitative measurements of the target antigen. Magnetic levitation is advantageous to other methods due to its decreased sample preparation time and lack of dependance on classical readout methods. The image generated is easily captured and analyzed using a standard microscope or mobile device, such as a smartphone or a tablet.

This paper describes a magnetic levitation-based method that can specifically detect the presence of antigens, either soluble or membrane-bound, by quantifying changes in the levitation height of capture beads with fixed densities.

The described method was developed based on the principles of magnetic levitation, which separates cells and particles based on their density and magnetic ...

Detection of Protein Aggregation using Fluorescence Correlation Spectroscopy

Protein aggregation is a hallmark of neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), and so on. To detect and analyze soluble or diffuse protein oligomers or aggregates, fluorescence correlation spectroscopy (FCS), which can detect the diffusion speed and brightness of a single particle with a single molecule sensitivity, has been used. However, the proper procedure and know-how for protein aggregation detection have not been widely shared. Here, we show a standard procedure of FCS measurement for diffusion properties of aggregation-prone proteins in cell lysate and live cells: ALS-associated 25 kDa carboxyl-terminal fragment of TAR DNA/RNA-binding protein 43 kDa (TDP25) and superoxide dismutase 1 (SOD1). The representative results show that a part of aggregates of green fluorescent protein (GFP)-tagged TDP25 was slightly included in the soluble fraction of murine neuroblastoma Neuro2a cell lysate. Moreover, GFP-tagged SOD1 carrying ALS-associated mutation shows a slower diffusion in live cells. Accordingly, we here introduce the procedure to detect the protein aggregation via its diffusion property using FCS.

We here introduce a procedure to measure protein oligomers and aggregation in cell lysate and live cells using fluorescence correlation spectroscopy.

Protein aggregation is a hallmark of neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's ...