Name: a time-efficient fluorescence spectroscopy-based assay for evaluating actin polymerization status in rodent and human brain tissues
Uploaded: 2021-06-03T14:30:00
Description: Watch this Scientific Journal Video about A Time-Efficient Fluorescence Spectroscopy-Based Assay for Evaluating Actin Polymerization Status in Rodent and Human Brain Tissues at JoVE.com

This work was supported by the Neurological Foundation of New Zealand (1835-PG), the New Zealand Health Research Council (#16-597) and the Department of Anatomy, University of Otago, New Zealand. We are indebted to the Neurological Tissue Bank of HCB-IDIBAPS BioBank (Spain) for human brain tissues. We thank Jiaxian Zhang for her help in recording and editing of the video.

All experimental procedures were carried out in accordance with the regulations of the University of Otago Committee on Ethics in the Care and Use of Laboratory Animals (Ethics Protocol No. AUP95/18 and AUP80/17) and New Zealand legislature. Human brain tissues were obtained from the Neurological Tissue Bank of Hospital Cl&#237;nic-IDIBAPS BioBank in Barcelona, Spain. All tissue collection protocols were approved by the Ethics Committee of Hospital Cl&#237;nic, Barcelona, and informed consent was obtained from the famili...

The assay described here, essentially adapted from a previous study30 with modifications, employs a phallotoxin, phalloidin tagged with a fluorescent label. Fluorescent phalloidin analogs are considered to be the gold standard for staining actin filaments in fixed tissues47,48,49. In fact, they are the oldest tools to specifically identify actin filaments50 and still remain the mos...

Cytoskeletal protein actin is involved in multiple cellular functions, including structural support, cellular transport, cell motility and division. Actin occurs in equilibrium in two forms: monomeric globular actin (G-actin) and polymerized filamentous actin (F-actin). Rapid changes in the polymerization status of actin (interconversion between its G- and F- forms) result in rapid filament assembly and disassembly and underlie its regulatory roles in cellular physiology. Actin forms the major component of the neuronal cytoskeletal structure and influences a wide range of neuronal functions1,2. Of note, the actin cytoskeleton forms an integral part of the structural platform of the synaptic terminals. As such, it is a major determinant of synaptic morphogenesis and physiology and plays a fundamental role in control of the size, number and morphology of synapses3,4,5. In particular, dynamic actin polymerization-depolymerization is a key determinant of the synaptic remodelling associated with synaptic plasticity underlying the memory and learning processes. Indeed, both presynaptic (such as neurotransmitter release6,7,8,9,10) and postsynaptic functions (plasticity related dynamic remodeling11,12,13,14) critically rely on dynamic changes in the polymerization status of the actin cytoskeleton.
Under physiological conditions, F-actin levels are dynamically and tightly regulated through a multimodal pathway involving posttranslational modification4,15,16 as well as actin-binding proteins (ABPs)4,17. ABPs can influence actin dynamics at multiple levels (such as initiating or inhibiting polymerization, inducing filament branching, severing of filaments to smaller pieces, promoting depolymerization, and protecting against depolymerization), and are in-turn under a stringent modulatory control sensitive to various extra- and intracellular signals18,19,20. Such regulatory checks at multiple levels dictate a strict regulation of actin dynamics at the synaptic cytoskeleton, fine-tuning pre- and postsynaptic aspects of neuronal physiology both at the basal and activity-induced states.
Given the important roles of actin in neuronal physiology, it is not surprising that several studies have provided evidence for alterations in actin dynamics as critical pathogenic events linked to a wide range of neurological disorders including neurodegeneration, psychological diseases as well as neurodevelopmental ailments3,21,22,23,24,25,26,27. In spite of the wealth of research data pointing to key roles of actin in neuronal physiology and pathophysiology, however, significant gaps still remain in the understanding of actin dynamics, particularly at the synaptic cytoskeleton. More research studies are needed to have a better comprehension of neuronal actin and its alterations under pathological conditions. One major area of focus in this context is the assessment of actin polymerization status. There are Western blotting-based commercial kits (G-Actin/F-Actin in vivo assay biochemical kit; Cytoskeleton SKU BK03728,29) and home-made assays for the assessment of F-actin levels6. However, because these require biochemical isolation of F-actin and G-actin and because their subsequent quantification is based upon immunoblotting protocols, they can be time consuming. We herein report a fluorescence spectroscopy-based assay adapted from a previous study30 with modifications that can be used to evaluate both basal levels of F-actin, as well as dynamic changes in its assembly-disassembly. Notably, we have efficiently modified the original protocol that requires samples suitable for a 1 mL cuvette to the current 96-well plate format. The modified protocol has therefore significantly reduced the tissue/sample amount required for the assay. Further, we provide evidence that the protocol is suitable for not only brain tissue homogenates, but also subcellular fractions such as isolated synaptic terminals (synaptosomes and synaptoneurosomes). Lastly, the assay can be employed for freshly dissected rodent brain tissues and long-term stored post-mortem human brain samples. Of note, while the assay is presented in a neuronal context, it can be suitably extended to other cell-types and physiological processes associated with them.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>3.5 mL, open-top thickwall polycarbonate tube</td>
			<td>Beckman Coulter</td>
			<td>349622</td>
			<td>For gradient centrifugation (synaptosome prep)</td>
		</tr>
		<tr>
			<td>Alexa Fluor 647 Phalloidin</td>
			<td>Thermo Fisher Scientific</td>
			<td>A22287</td>
			<td>F-actin specific ligand</td>
		</tr>
		<tr>
			<td>Antibody against &#160;b-actin</td>
			<td>Santa Cruz Biotechnology</td>
			<td>Sc-47778</td>
			<td>For evaluation of total actin levels by immunoblotting</td>
		</tr>
		<tr>
			<td>Antibody against GAPDH</td>
			<td>Abcam</td>
			<td>Ab181602</td>
			<td>For evaluation of GAPDH levels by immunoblotting</td>
		</tr>
		<tr>
			<td>Bio-Rad Protein Assay Dye Reagent Concentrate</td>
			<td>Bio-Rad</td>
			<td>5000006</td>
			<td>Bradford based protein estimation</td>
		</tr>
		<tr>
			<td>Calcium chloride dihydrate (CaCl2&#183;2H2O)</td>
			<td>Sigma-Aldrich</td>
			<td>C3306</td>
			<td>Krebs buffer component</td>
		</tr>
		<tr>
			<td>cOmplete, Mini, EDTA-free Protease Inhibitor Cocktail</td>
			<td>Sigma-Aldrich</td>
			<td>4693159001</td>
			<td>For inhibition of endogenous protease activity during sample preparation</td>
		</tr>
		<tr>
			<td>Corning 96-well Clear Flat Bottom Polystyrene</td>
			<td>Corning</td>
			<td>3596</td>
			<td>For light-scattering measurements</td>
		</tr>
		<tr>
			<td>D-(+)-Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G8270</td>
			<td>Krebs buffer component</td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide</td>
			<td>Sigma-Aldrich</td>
			<td>D5879</td>
			<td>Solvent for phalloidin and latrunculin A</td>
		</tr>
		<tr>
			<td>Fluorescent flatbed scanner (Odyssey Infrared Scanner)</td>
			<td>Li-Cor Biosciences</td>
			<td></td>
			<td>For detection of immunoreactive signals on immunoblots</td>
		</tr>
		<tr>
			<td>Glutaraldehyde solution (25% in water) Grade II</td>
			<td>Sigma-Aldrich</td>
			<td>G6257</td>
			<td>Fixative</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H3375</td>
			<td>Buffer ingredient for sample preparation and Krebs buffer component</td>
		</tr>
		<tr>
			<td>Latrunculin A</td>
			<td>Sigma-Aldrich</td>
			<td>L5163</td>
			<td>Depolymerizer of actin filaments</td>
		</tr>
		<tr>
			<td>Magnesium chloride hexahydrate (MgCl2&#183;6H2O)</td>
			<td>Sigma-Aldrich</td>
			<td>M2670</td>
			<td>Krebs buffer component</td>
		</tr>
		<tr>
			<td>Microplates</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mitex membrane filter 5 mm</td>
			<td>Millipore</td>
			<td>LSWP01300</td>
			<td>Preparation of synaptoneurosomes</td>
		</tr>
		<tr>
			<td>Nunc F96 MicroWell Black Plate</td>
			<td>Thermo Fisher Scientific</td>
			<td>237105</td>
			<td>For fluorometric measurements</td>
		</tr>
		<tr>
			<td>Nylon net filter 100 mm</td>
			<td>Millipore</td>
			<td>NY1H02500</td>
			<td>Preparation of synaptoneurosomes</td>
		</tr>
		<tr>
			<td>Phosphatase Inhibitor Cocktail IV</td>
			<td>Abcam</td>
			<td>ab201115</td>
			<td>For inhibition of endogenous phosphatase activity during sample preparation</td>
		</tr>
		<tr>
			<td>Potassium chloride (KCl)</td>
			<td>Sigma-Aldrich</td>
			<td>P9541</td>
			<td>Krebs buffer component and for depolarization of synaptic terminals</td>
		</tr>
		<tr>
			<td>Potassium phosphate monobasic ((KH2PO4)</td>
			<td>Sigma-Aldrich</td>
			<td>P9791</td>
			<td>Krebs buffer component</td>
		</tr>
		<tr>
			<td>Sodium borohydride (NaBH4)</td>
			<td>Sigma-Aldrich</td>
			<td>71320</td>
			<td>Component of Permeabilization buffer</td>
		</tr>
		<tr>
			<td>Sodium chloride (NaCl)</td>
			<td>LabServ (Thermo Fisher Scientific)</td>
			<td>BSPSL944</td>
			<td>Krebs buffer component</td>
		</tr>
		<tr>
			<td>Sodium hydrogen carbonate (NaHCO3)</td>
			<td>LabServ (Thermo Fisher Scientific)</td>
			<td>BSPSL900</td>
			<td>Krebs buffer component</td>
		</tr>
		<tr>
			<td>SpectraMax i3x</td>
			<td>Molecular Devices</td>
			<td></td>
			<td>For fluorometric measurements</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Fisher Chemical</td>
			<td>S/8600/60</td>
			<td>Buffer ingredient for sample preparation</td>
		</tr>
		<tr>
			<td>Swimnex Filter Holder</td>
			<td>Millipore</td>
			<td>Sx0001300</td>
			<td>Preparation of synaptoneurosomes</td>
		</tr>
		<tr>
			<td>Tissue grinder 5 mL Potter-Elvehjem</td>
			<td>Duran Wheaton Kimble</td>
			<td>358034</td>
			<td>For tissue homogenization</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>X100</td>
			<td>Component of Permeabilization buffer</td>
		</tr>
		<tr>
			<td>Trizma base</td>
			<td>Sigma-Aldrich</td>
			<td>T6066</td>
			<td>Buffer ingredient for sample preparation</td>
		</tr>
	</tbody>
</table>

a time-efficient fluorescence spectroscopy-based assay for evaluating actin polymerization status in rodent and human brain tissues

Actin, the major component of cytoskeleton, plays a critical role in the maintenance of neuronal structure and function. Under physiological states, actin occurs in equilibrium in its two forms: monomeric globular (G-actin) and polymerized filamentous (F- actin). At the synaptic terminals, actin cytoskeleton forms the basis for critical pre- and post-synaptic functions. Moreover, dynamic changes in the actin polymerization status (interconversion between globular and filamentous forms of actin) are closely linked to plasticity-related alterations in synaptic structure and function. We report here a modified fluorescence-based methodology to assess polymerization status of actin in ex vivo conditions. The assay employs fluorescently labelled phalloidin, a phallotoxin that specifically binds to actin filaments (F-actin), providing a direct measure of polymerized filamentous actin. As a proof of principle, we provide evidence for the suitability of the assay both in rodent and post-mortem human brain tissue homogenates. Using latrunculin A (a drug that depolymerizes actin filaments), we confirm the utility of the assay in monitoring alterations in F-actin levels. Further, we extend the assay to biochemical fractions of isolated synaptic terminals wherein we confirm increased actin polymerization upon stimulation by depolarization with high extracellular K+.

Linearity of the assay for evaluation of F-actin levels 
First, a standard curve for the linear increase in fluorescence of Alexa Fluor 647 Phalloidin was ascertained and was repeated for each set of experiments (Figure 1). To investigate the linear range of the assay, different amounts of brain homogenates from rodents (Figures 2A and 2B) and post-mortem human subjects (Figure 3A and 3B) w...

Watch this Scientific Journal Video about A Time-Efficient Fluorescence Spectroscopy-Based Assay for Evaluating Actin Polymerization Status in Rodent and Human Brain Tissues at JoVE.com

A Time-Efficient Fluorescence Spectroscopy-Based Assay for Evaluating Actin Polymerization Status in Rodent and Human Brain Tissues

We report a simple, time-efficient and high-throughput fluorescence spectroscopy-based assay for the quantification of actin filaments in ex vivo biological samples from brain tissues of rodents and human subjects.

Actin, the major component of cytoskeleton, plays a critical role in the maintenance of neuronal structure and function. Under physiological states, actin ...

Actin is the major component of cytoskeleton and critically regulates several aspects of cellular morphology and physiology, including in neurons. Actin occurs in equilibrium between its two forms, monomeric globular G-actin or polymeric filamentous F-actin. Polymerization status of actin is stringently regulated at multiple levels both by actin-binding proteins, as well as its own post-translational regulation.At the interneuronal synaptic contacts, dynamic alterations in F-actin levels are a key regulatory event, controlling signaling and plasticity at both pre-and post synaptic terminals. Not surprisingly, actin dysfunction is linked to several neuropathological states. Recent advances have led to a wealth of knowledge of actin in neuronal physiology and pathophysiology.However much is still unknown and needs to be focused on. In this regard, fluorescent analogs of phalloidin have proven to be a major tool in actin research. This phallotoxin specifically binds to filamentous actin, and hence can be used as a direct measure of the quantities of F-actin, and its alteration in pathophysiological states.This study presents a fast and efficient fluorometric assay for evaluation of actin polymerization status in ex-vivo biological samples, such as wholesale homogenates and biochemically isolated synaptic terminals from brain tissues. Of note, the assay can be applied to other cell and tissue types and their associated physiological phenomenon. For the recipes of the buffers used in this study, please refer to the detailed text.Brain tissue samples from either rats or human subjects were homogenized in a Potter-Elvehjem glass tube and pestle on ice in 10 volumes of homogenization buffer. For synaptosome preparation, the homogenate was spun first at a slow speed and then at a higher speed to obtain crude synaptosomal mitochondrial fraction. Enriched synaptosomes from this crude fraction was obtained following a discontinuous sucrose gradient.For the synaptoneurosomal preparation, homogenates were sequentially passed through two 100 microns and one 5 micron filter. Protein estimation was performed using Bradford reagent and samples were diluted in Krebs buffer at a concentration of two to three milligrams per mL protein in a final volume of 50 microliters. Stimulation of isolated synaptic terminals, either synaptosomes or synaptoneurosomes, were carried out by a brief 30 second increase in extracellular potassium at 37 degrees.For this, one molar potassium chloride was added to the samples to a final concentration of 15 millimolar. Homogenates are unstimulated and depolarized synaptic fragments were fixed with 2.5%glutaraldehyde, by the addition of the required volume of 25%glutaraldehyde stock solution. The samples were incubated for two, three minutes at room temperature.Fixative was removed by centrifugation at 20, 000 G for five minutes. The samples were then proceeded for permeabilization by resuspension in Krebs buffer containing 0.1%Triton X-100 and 1 mg per mL sodium borohydride for two to three minutes at room temperature. Permeabilization buffer was removed again by centrifugation and the pellet was superficially washed with Krebs buffer and resuspended in Krebs buffer containing 1X Alexa Fluor 647 phalloidin, corresponding to 500 micro units of phalloidin.Binding was allowed to proceed for 10 minutes at room temperature in dark. Excess unbound phalloidin was removed from the samples by centrifugation at 20, 000 G for five minutes, and the pellets were superficially washed with Krebs buffer, and resuspended again in Krebs buffer containing 0.32 molar sucrose to maintain the buoyancy of synaptosomes or synaptoneurosomes. Fluorescent phalloidin bound samples were dispensed in a 96 well black plate for fluorometric reading in a plate reader.The excitation and emission wavelengths were set at 645 nanometer and 670 nanometer respectively. For each set of experiments we included different amounts of Alexa Fluor 647 phalloidin in Krebs buffer containing 0.32 molar sucrose. To account for any losses of samples during the washing steps, the samples were transferred from the black to the transparent 96 well plate.Capturing of the samples was then examined at 440 nanometers. Retention of fluorescent phalloidin is directly proportional to the amount of actin filaments or F-actin levels in the samples. Linearity of phalloidin binding was observed in the range of 50 to 200 microgram proteins.As a proof of principle, we also tested efficacy of our assay in models for F-actin depolymerization using pharmacological disruption of actin filaments by Latrunculin A, as well as stimulation of F-actin formation by KCl mediated stimulation of isolated synaptic terminals. Fluorescence emission from bound phalloidin can be expressed as either units of bound phalloidin generated from the standard linear curve and are relative to the control samples. For Figure 4, F-actin levels are expressed as micro units of bound phalloidin.On the other hand, for Figure 5 F-actin levels in the depolarized synaptic fragments are expressed relative to the control undepolarized samples. We describe a robust, time-efficient and high throughput assay for analysis of actin filaments, or F-actin, and its alterations in physiological and pathophysiological states suitable for a 96 well plate format. The assay is much faster than existing alternative protocols and can serve as an essential tool in actin-related studies, either alone or in combination with existing immunohistochemistry and Western blotting based assays.

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