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Neuroscience

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

Published: June 3rd, 2021

DOI:

10.3791/62268

1Department of Anatomy, School of Biomedical Sciences, University of Otago

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 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+.

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 ac....

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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 Clínic-IDIBAPS BioBank in Barcelona, Spain. All tissue collection protocols were approved by the Ethics Committee of Hospital Clínic, Barcelona, and informed consent was obtained from the famili.......

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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.......

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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.......

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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.

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

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