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

Faraz Ahmad; Ping Liu

doi:10.3791/62268

JoVE Journal > Neuroscience

Neurowissenschaften

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

Published: June 03, 2021

doi:

10.3791/62268

Faraz Ahmad¹, Ping Liu¹

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

Summary

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.

Abstract

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⁺.

Introduction

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.

Protocol

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 families.

1. Preparation of buffers and reagents

Prepare the following buffers for the homogenization of brain tissue and the preparation of enriched fraction of synaptic terminals:
Homogenization buffer: 5 mM HEPES, pH 7.4 supplemented with 0.32 M sucrose
Resuspension buffer: 5 mM Tris, pH 7.4 supplemented with 0.32 M sucrose
Washing buffer: 5 mM Tris, pH 8.1
1.2 M sucrose
1.0 M sucrose
0.85 M sucrose
Add homemade or commercial mix of protease and phosphatase inhibitors.
NOTE: We have used EDTA-free version of Complete Protease Inhibitor mix (1 tablet per 10 mL buffer) and Phosphatase inhibitor cocktail IV (1:100; volume: volume).
Prepare the following buffers and reagents for the fixation, permeabilization and binding of phalloidin:
Krebs buffer: 118.5 mM NaCl, 4.7 mM KCl, 1.2 mM MgCl₂, 2 mM CaCl₂, 0.1 mM KH₂PO₄, 5 mM NaHCO₃, 10 mM glucose, 20 mM HEPES, pH 7.4
1 M KCl
25% glutaraldehyde (stock solution)
Krebs buffer containing 0.1 % Triton X-100 and 1 mg/mL NaBH₄
400x Alexa Fluor 647 Phalloidin in DMSO
Krebs buffer containing 0.32 M sucrose
50 μM latrunculin A in DMSO (stock solution)
1 M KCl (stock solution)
CAUTION: Phalloidin is toxic (LD₅₀ = 2 mg/kg) and must be handled with care. Inhalation of glutaraldehyde is toxic and it should be handled in a fume hood.
Store the buffers at 4 °C and phalloidin and latrunculin A at -20 °C for long-term storage.
NOTE: Long-term storage of buffers is not recommended. The protocol can be paused here.

2. Brain tissue homogenization

Homogenize the cryopreserved or freshly dissected rat brain tissue in 10 volumes of homogenization buffer in a Potter-Elvehjem glass tube and pestle on ice.
NOTE: Optimal homogenization is achieved by 15-20 strokes of the pestle by hand for brain tissues. Successful homogenization can be confirmed by smooth flow of the suspension through the glass tube. The protocol can be paused here.
Determine the protein concentration in the homogenate using a Bradford assay³¹.
NOTE: Alternate homemade or commercial assays for protein estimation can also be used.
Dilute homogenate samples in Krebs buffer at a concentration of 2-3 mg protein/mL in a volume of 50 µL.
NOTE: In some of our experiments, we incubate homogenates with 2 μM latrunculin A or DMSO (controls) at 37 °C for 1 h. For this, homogenates are resuspended in 48 µL of Krebs buffer, and 2 µL of 50 μM latrunculin A or 2 µL of DMSO is added. Further for immunoblotting, a small amount of sample (10 μg) is collected after the incubation.
Fix the homogenate samples (section 5).

3. Preparation of isolated nerve terminals

Preparation of synaptosomes
NOTE: Alternative protocols³²^,³³ for synaptosomes can also be used.
1. Centrifuge brain homogenate at 1,200 x g for 10 min at 4 °C.
2. Discard the pellet, which is the crude nuclear fraction.
3. Further centrifuge the supernatant (S1) obtained in step 3.1.1 at 12,000 x g for 15 min at 4 °C.
4. Remove the supernatant (S2), which is the soluble cytosolic fraction.
5. Resuspend the pellet (P2) obtained in step 3.1.3, which is the crude synaptosomal fraction in resuspension buffer.
  NOTE: The volume of resuspension buffer depends on the amount of starting tissue and the amount of pellet obtained. For example, when starting with 150-300 mg of brain tissues, the pellet obtained can be resuspended in 200 µL of resuspension buffer.
6. Load the resuspended crude synaptosomes onto a discontinuous sucrose gradient made of equal volumes of 0.85-1.0-1.2 M sucrose.
  NOTE: We typically use 1 mL each of the sucrose solution (for 150-300 mg tissue). This can be changed according for larger tissue amounts. Discontinuous gradients can be made using a 25G syringe pressed against the internal wall of the ultracentrifuge tube and gentle layering of the sucrose layers.
7. Centrifuge at 85,000 x g for 2 h at 4 °C.
  NOTE: Because of the high-speed of centrifugation, an ultracentrifuge capable of creating a vacuum to reduce heating is required.
8. Collect the synaptosomal fraction at the interface between 1.0 and 1.2 M sucrose using a 200 µL pipet tip.
9. Wash the synaptosomal fraction with ice-cold washing buffer by centrifugation at 18,000 x g for 10 minutes at 4 °C.
  NOTE: For washing, the synaptosomal fraction obtained at the interface of 1.0 and 1.2 M sucrose is collected in a fresh 1.5 mL tube, and an equal volume of washing buffer is added, ensuring the removal of high sucrose from the medium.
10. Wash the synaptosomal pellet again with ice-cold homogenization buffer at 12,000 g for 10 minutes at 4 °C.
11. Resuspend the synaptosomes in homogenization buffer on ice.
  NOTE: The protocol can be briefly paused here.
12. Determine the protein concentration using a Bradford assay.
13. Resuspend the synaptosomes in Krebs buffer at a concentration of 2-3 mg protein/mL in a volume of 50 µL (47.5 µL if synaptosomes are to be depolarized by KCl; see section 4).
  NOTE: For resuspension, the synaptosomal fraction is first spun at 12,000 x g for 5 min at 4 °C and the supernatant (buffer) is removed. The synaptosomal pellet is then resuspended in Krebs buffer by gentle pipetting using a 200 µL pipet tip.
14. Proceed with depolarization (section 4).
Preparation of synaptoneurosomes
NOTE: Alternative protocols³⁴^,³⁵ for synaptoneurosomes can also be used.
1. Pass the brain homogenate through a pre-wetted net filter of 100 μm pore size in a filter holder using a 1 mL syringe.
  NOTE: Pre-wetting of all filters is important to avoid loss of sample and should be done using homogenization buffer. For this, homogenization buffer is passed through the filters in the filter holder using a 1 mL syringe until the buffer flow out.
2. Collect the filtrate (F1) in a pre-chilled 1.5 mL tube on ice.
3. Repeat the process (steps 3.2.1 and 3.2.2) for F1 fraction to obtain the second filtrate (F2).
4. Pass F2 filtrate through a net filter of 5 μm pore size.
5. Collect the filtrate (F3) in a pre-chilled 1.5 mL tube on ice.
6. Centrifuge filtrate F3 at 1,500 x g for 10 min at 4 °C.
7. Resuspend the pellet (synaptoneurosomes) in Krebs buffer on ice at a concentration of 2-3 mg protein/mL in a volume of 50 µL (47.5 µL if synaptosomes are to be depolarized by KCl; see section 4).
  NOTE: The protocol can be briefly paused here.
8. Estimate the protein concentration using a Bradford assay.
9. Proceed with depolarization (section 4).

4. KCl-mediated depolarization of isolated synaptic terminals

Equilibrate synaptosomes/synaptoneurosomes at 37 °C for 5-10 min.
Stimulate synaptosomes/synaptoneurosomes by adding KCl to increase extracellular K⁺ to 50 mM for 30 s at 37 °C and add equal volume of Krebs buffer to the respective unstimulated control set.
NOTE: For example, add 2.5 µL of 1 M KCl to synaptosomes resuspended in 47.5 µL of Krebs buffer; and add 2.5 µL of Krebs buffer to the respective unstimulated control synaptosome. For experiments wherein a large number of samples are involved, proceed with no more than 2 samples at a time so that the depolarization time does not exceed 30 s.
Terminate stimulation by adding glutaraldehyde (section 5).

5. Fixation and phalloidin staining of samples

Add glutaraldehyde to homogenate/synaptosomal/synaptoneurosomal samples to a final concentration of 2.5% for 2-3 min at room temperature.
NOTE: We added 6 µL of 25% glutaraldehyde solution so that the final concentration of glutaraldehyde in the 50 µL samples (homogenates/synaptosomes/synaptoneurosomes) was ca. 2.5%. Fixation is critical and should be fast and hence immediately after adding glutaraldehyde, the sample should be vigorously vortexed.
Sediment the samples at 20,000 x g for 5 min.
Remove the supernatant.
NOTE: Discard the supernatant in a fume hood as glutaraldehyde is toxic.
Permeabilize the pellet by resuspension in 100 µL of Krebs buffer containing 0.1% Triton X-100 and 1 mg/mL NaHB₄ for 2-3 min at room temperature.
Sediment the samples at 20,000 x g for 5 min.
Remove the permeabilization buffer.
Wash the pellet with 200 µL of Krebs buffer by centrifugation at 20,000 x g for 5 min.
Resuspend and stain the pellet with 1x Alexa Fluor 647 Phalloidin (corresponding to 500 μU) in 100 µL of Krebs buffer for 10 minutes in dark at room temperature.
NOTE: Other varieties of fluorescent phalloidin analogs are commercially available and can be replaced for the assay. Concentration of phalloidin and total sample volume of incubation may have to be modified according to the amount and type of tissue/sample being tested and optimal conditions should be standardized accordingly.
Centrifuge the stained samples at 20,000 x g for 5 min.
Remove the unbound phalloidin (supernatant).
Wash the sample with 200 µL of Krebs buffer by centrifugation at 20,000 x g for 5 min.
Resuspend in 200 µL of Krebs buffer containing 0.32 M sucrose.
NOTE: The protocol can be briefly paused here.

6. Fluorometric analysis and light scattering

Dispense Alexa Fluor 647 Phalloidin-stained samples in a black 96-well plate.
Measure the fluorescence intensity at an excitation wavelength of 645 nm and an emission wavelength of 670 nm in a plate reader at room temperature.
Transfer the samples from the black 96-well plate to the transparent 96-well plate using 200 µL pipet tips.
Measure the light scattering at 540 nm to correct for any losses that might have occurred during the previous steps of fixation, permeabilization and staining.
NOTE: Variations in biological material retained in the stained samples might be more prominent for smaller amounts of starting material (see Discussion).
Include a set of Alexa Fluor 647 Phalloidin in Krebs buffer at different concentrations (0.05x, 0.1x, 0.25x, 0.35x, 0.75x, 0.5x and 1x corresponding to 25, 50, 125, 175, 250, 375 and 500 μU) for each batch of the assay as a standard curve.
NOTE: This is an optional step and does not affect the results of the assay particularly when F-actin levels are being expressed in a relative manner (Section 7). The protocol can be paused here.

7. Data analysis

The amount of F-actin in the samples is directly proportional to the fluorescence intensity of bound phalloidin. Express in absolute terms of units of phalloidin bound calculated from the linear curve of the tagged phalloidin standard.
NOTE: As an example, see Figures 2A-B, 3A, 4A-B and Supplementary Figure 2.
Express F-actin levels as a fraction of the control samples.
NOTE: As an example, see Figures 5A-B.

Representative Results

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) were processed. The assay was found to be linear in the range of 50-200 μg of protein as assessed by amounts of labeled phalloidin retained. Light scattering at 540 nm was used to confirm the different amounts of samples (Figure 2C and Figure 3C).

Latrunculin, an actin depolymerizing agent reduces binding of labelled phalloidin
Latrunculin A is known to depolymerize actin filaments and reduce the levels of F-actin³⁶^,³⁷^,³⁸^,³⁹. Homogenates from either rodent or human brain tissues were incubated with 2 μM latrunculin A for 1 hour at 37 °C to depolymerize actin filaments. Respective untreated control sets were incubated with DMSO for the same duration of 1 hour at 37 °C. The assay robustly measured the loss of F-actin levels from 95.7 ± 6.6 (mean ± SEM) in control samples to 72.0 ± 3.2 (mean ± SEM) μU of bound phalloidin in latrunculin A-treated samples in rodent brain homogenates (Figure 4A). A similar decrease (from 83.7 ± 3.9 to 66.9 ± 4.2 μU) in retention of labeled phalloidin was also observed when homogenates from human brain tissues were subjected to latrunculin A treatment (Figure 4B). Noteworthy, total actin levels, as assessed by immunoblotting, did not alter upon treatment with latrunculin A in both rat (Supplementary Figure 1A-B) and human (Supplementary Figure 1C-D) brain homogenates.

Depolarization of isolated synaptic terminalsstimulates actin polymerization and filament formation
Ex vivo depolarization of isolated synaptic terminals has been shown to result in rapid stimulation of actin polymerization⁶^,³⁰^,⁴⁰, and this phenomenon was used as a further confirmation of the assay reported herein. Biochemical fractions enriched in synaptic terminals were prepared in two different manners; a gradient-based ultracentrifugation method to obtain "synaptosomes"⁴¹^,⁴²^,⁴³^,⁴⁴ and a sequential filtration-based protocol to obtain "synaptoneurosomes"⁴³^,⁴⁵^,⁴⁶. Because the yield for the latter is higher, we used it for human post-mortem brain tissues wherein the tissue amounts are often limiting. On the other hand, we preferred to use synaptosomes with a higher degree of enrichment of synaptic fragments⁴¹ for our rat brain tissue experiments.

Depolarization of synaptosomes or synaptoneurosomes and stimulation of actin polymerization were achieved by a short (30 second) burst of increase in extracellular K⁺ to 50 mM. KCl exposure resulted in increased phalloidin binding by almost 40% in rodent brain synaptosomes compared to the respective mock-stimulated controls (Figure 5A; see also Supplementary Figure 2A). A smaller (around 20%) but consistent increase was also observed in human synaptoneurosomes treated with KCl (Figure 5B; see also Supplementary Figure 2A). These experiments validate the robustness of our assay in determining alterations in F-actin levels in brain tissue samples, including isolated synaptic terminals.

Figure 1. Standard curve for fluorescence of Alexa Fluor 647 Phalloidin. Linearity of fluorescence emission of different amounts (25-500 μU) of labeled phalloidin was confirmed by fluorescence spectroscopy at an excitation of 645 nm and emission of 670 nm (R² = 0.9942). Data are represented as mean ± SEM (n=3). Please click here to view a larger version of this figure.

Figure 2. Linearity of phalloidin binding to whole-cell homogenates from rat brain tissue. (A) Binding of phalloidin to different amounts of homogenates (50-300 μg protein) was assessed. (B) Binding was linear in the range of 50-200 μg protein (R² = 0.9602). (C) Scattering at 540 nm was used to confirm different amounts of the samples (R² = 0.8319). Data are represented as mean ± SEM (n=4). Please click here to view a larger version of this figure.

Figure 3. Phalloidin binding to whole-cell homogenates from post-mortem human brain tissue. (A) Phalloidin retention in a range of amounts of homogenates (50-300 μg protein) was evaluated. (B) Phalloidin binding was found to be linear in the range of 50-200 μg protein (R² = 0.8832). (C) Scattering at 540 nm confirmed the varying amounts of the samples (R² = 0.9730). Data are represented as mean ± SEM (n=4). Please click here to view a larger version of this figure.

Figure 4. Effects of latrunculin A on F-actin levels. Treatment of brain homogenates with actin depolymerizing agent Latrunculin A (2 μM, 1 h at 37°C) resulted in significant decrease in the amounts of actin filaments compared to the respective mock-treated control samples as assessed by retention of labeled phalloidin both in rodent (p = 0.0034; paired two-tailed Student's t-test) (A) and post-mortem human tissues (p = 0.0011; paired two-tailed Student's t-test) (B). Data are represented as mean ± SEM (n=6 pairs). Please click here to view a larger version of this figure.

Figure 5. Effects of KCl-mediated depolarization on F-actin amounts in isolated synaptic terminals. (A) Incubation of synaptosomes from rat brain with 50 mM KCl for 30 s at 37°C stimulated actin polymerization which consequently resulted in an increase in phalloidin binding (p = 0.0014; paired two-tailed Student's t-test). (B) A smaller increase was also observed in synaptoneurosomal fraction from post-mortem human brain tissues (p = 0.014; paired two-tailed Student's t-test). Data are represented as mean ± SEM (n=6 pairs). Please click here to view a larger version of this figure.

Supplementary Figure 1. Effects of latrunculin A on total actin levels. Brain homogenates were incubated with latrunculin A (2 μM, 1 h at 37°C) or equal volume of DMSO (1 h at 37°C). 10 μg protein per sample (latrunculin A treated and DMSO mock-treated controls) were collected prior to fixation. Total actin levels were assessed by immunoblotting. Representative blots are shown for rat (A) and human (C) brain homogenates. Latrunculin A did not alter the total actin levels in both rat (B; p = 0.40; paired two-tailed Student's t-test) and human (D; p = 0.42; paired two-tailed Student's t-test) brain homogenates. Data are represented as mean ± SEM (n=3 pairs). Please click here to download this File.

Supplementary Figure 2. Effects of KCl-mediated depolarization on F-actin levels in rat synaptosomes and human synaptoneurosomes. (A) Incubation of synaptosomes from rat brain with 50 mM KCl (30 s at 37 °C) stimulated actin polymerization and a consequent increase in phalloidin binding (p = 0.0019; paired two-tailed Student's t-test). (B) Increase in phalloidin retention was also observed in human synaptoneurosomal fraction depolarized by KCl compared to the respective unstimulated controls (p = 0.015; paired two-tailed Student's t-test). Data are represented as mean ± SEM (n=6 pairs). Please click here to download this File.

Discussion

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 most widely used instruments to detect actin filaments particularly for subsequent fluorescence microscopy-based analyses. Importantly, phalloidin has been shown to stain even loose, irregular meshwork of short actin filaments⁵¹, indicating that phalloidin binding is not dependent on the filament length. Our protocol, on the other hand, relies on fluorescence spectroscopy to analyze actin dynamics in ex vivo biological samples, for example brain tissues from rodents and humans.

The major advantage of the protocol is that it considerably reduces the time taken with respect to the existing protocols that first require a high-speed centrifugation-based biochemical isolation of F-actin (separation from G-actin based on insolubility of actin filaments in certain detergents such as Triton X-100) and subsequent analysis of immunoreactive levels using Western blotting⁶^,²⁸^,²⁹. The time-efficiency of our assay is also an advantage with regards to phalloidin-based immunocytochemistry techniques⁴⁰^,⁵², although there might be some other benefits associated with the latter. Another advantage is that it can be applied to cryopreserved post-mortem human brain tissues, procurement of which is always associated with some post-mortem delay. Further, with respect to the original protocol³⁰ from which this methodology has been modified, we have considerably reduced the requirement for the tissue amount from 1 mL cuvette to a single well of a 96-well plate. Moreover, because of the inclusion of a phalloidin standard curve in each set of experiment, our protocol can quantitate absolute levels of actin filaments in units of phalloidin bound (Figures 2A-B, 3A, 4A-B; see also Supplementary Figure 2), as well as the levels relative to control samples (Figure 5A-B).

It should be noted that the application of phalloidin to assess F-actin levels however is restricted to fixed cells and samples, both for our assay protocol as well as microscopy-based protocols. This is because phalloidin is essentially a toxic bicyclic heptapeptide that binds specifically at the interface between F-actin subunits with high affinity and stabilizes these actin filaments, rendering them incapable to depolymerize and in fact increasing the net conversion of G-actin into F-actin⁴⁰^,⁵³^,⁵⁴. Hence, phalloidin stabilizes actin filaments in vivo and in vitro, and can result in significant changes in the equilibrium status of actin per se⁴⁷^,⁵⁵. As such evaluation of actin polymerization status mediated by fluorescent phalloidin is based upon arrested filament structures. Moreover, because of its low permeability through the lipid bilayer, phalloidin-based methodologies rely on permeabilization of the cells or biological samples. Infeasibility of a time course live-cell assay is hence a major limitation of the protocol as with immunocytochemistry-based methods employing phalloidin.

Infeasibility of phalloidin-based methods to evaluate dynamic changes in actin filaments in live unfixed cells begs the question of whether there are alternative procedures to do so. Indeed, advances have been made in this regard with exogenous expression of fluorescent-actin analogs prior to analysis using protocols such as video micrography³⁶^,⁵⁶, fluorescence recovering after photobleaching (FRAP)³⁸ or fluorescence resonance energy transfer (FRET)³⁹. Heterologous expression of fluorescently tagged peptides and proteins that bind actin filaments are also employed to study actin dynamics in live cells; however there are disadvantages and limitations associated with them as well as with the heterologous expression of fluorescently tagged actin⁴⁷^,⁴⁹^,⁵⁷. For example, G-actin that comprises 50-70% of total actin in most cells is soluble and freely diffusible in the cytosol, resulting in a higher background causing challenges in differentiating signals from actin filaments specifically⁵⁷.

A critical factor in the assay is that as shown in Figure 2 and Figure 3; it is not linear throughout the range of protein amounts tested. Hence, an optimal amount of protein suitable for the assay should be determined first or the amount of phalloidin analog should be adjusted such that it is no longer limiting (at higher amounts of proteins). Another critical aspect of the protocol is that the multiple centrifugation-based steps for removal of fixative, permeabilization agent and unbound phalloidin can lead to varying loss of proteins (and F-actin bound phalloidin) from the samples, particularly when lower amounts of samples are used. Hence it is important to normalize the amount of sample retained by monitoring light scattering at 540 nm. Lastly, since actin is in a dynamic state of interconversion between its F- and G-forms, fixing should be fast. A minor related critical aspect of the assay is that we could not evaluate its efficiency in assessing pharmacological actin polymerization. As opposed to pharmacological actin depolymerization by latrunculin A, jasplakinolide (a reliable and widely used actin polymerizing agent) has overlapping binding sites with phalloidin and competitively inhibits its binding to actin filaments⁵⁸^,⁵⁹. Nevertheless, employment of KCl-stimulated synaptic terminals as an ex vivo model for increased actin polymerization indicates that our assay can also detect increases in F-actin levels.

In conclusion, we describe a robust time-efficient and high-throughput assay for analysis of actin filaments (F-actin) and its alternations in physiological and pathophysiological states suitable for a 96-well plate format. In combination with other existing methods for evaluation of F-actin in fixed and unfixed samples, the protocol will prove to be an essential tool in actin-related studies in the neuroscience field, as well as other areas of biological science research.

Offenlegungen

The authors have nothing to disclose.

Acknowledgements

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.

Materials

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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