Methods to measure actin treadmilling rate in dendritic spines

Dendritic spines are small protrusions in neuronal dendrites where the postsynaptic components of most excitatory synapses reside in the brain. The actin cytoskeleton is the structural element underlying changes in dendritic spine morphology and synapse strength. The proper morphology of spines and proper regulation of the actin cytoskeleton have been shown to be important in memory and learning; defects in regulation lead to various memory disorders. Thus, understanding actin cytoskeleton regulation in dendritic spines is of central importance to studies of synaptic and neuronal function. The dynamics of filamentous actin in spines can be studied with fluorescence redistribution assays. In fluorescence recovery after photobleaching (FRAP) experiments, the overexpressed green fluorescent protein (GFP)-actin fluorescence is rapidly photobleached by the application of a high-power laser beam to the area of one spine. The bleached fusion proteins incorporated into actin filaments continue treadmilling through the actin filaments and ultimately depolymerize and diffuse out of the spine. Simultaneously, unbleached GFP-actin fusion proteins diffuse into the spine and are incorporated into the filaments. The rate of actin filament treadmilling can be quantified by following the fluorescence recovery. In a photoactivation assay, the fluorescence intensity of photoactivatable-GFP-actin can be rapidly increased by a short laser pulse. The treadmilling rate of these activated actin monomers can be quantified by following the fluorescence decay. Here, we present our FRAP and photoactivation protocols to measure actin treadmilling rate in dendritic spines of living neurons.