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.
