Actin Dynamics at nanometer domains within individual dendritic spines

Abstract

Excitatory synapses within the brain occur on dendritic spines. Within the spine the branching, filamentous network of the actin cytoskeleton anchors receptors in the postsynaptic density and is critical to the regulation of synaptic transmission. The mechanisms whereby actin regulates synaptic function are vague and may consist of direct interaction with receptors in the synaptic or extrasynaptic membrane, propulsive forces at the base or sides of the postsynaptic density (PSD), enhancement of local recycling, or selective filtering of membrane protein mobility through the spine neck. Furthermore, it is unclear where polymerization takes place within spines, and whether regulation of the network occurs en bloc as it has been measured in the past or if specific subregions of the network may be regulated in response to local demands. I propose a model in which actin networks within individual dendritic spines have multiple points of control at distributed locations, allowing regulation of parts of the network within specific spine subdomains. The submicron dimensions of spines have limited examination of actin dynamics within spines and prevented live-cell discrimination of perisynaptic actin filaments. To overcome this I used Photoactivated Localization Microscopy (PALM) to localize with a high degree of accuracy and track single polymerized actin molecules within dendritic spines. I use Monte Carlo simulations to test the effect of diffusion on localization accuracy, demonstrating that motion blur from rapidly moving molecules negatively impacts localization accuracy. In addition, when imaging freely diffusing molecules, the distance molecules travel during the course of excitation affects their localized position with respect to the edge of a bounded region, again necessitating very short excitation pulses to accurately measure morphology. On the other hand, slowly moving molecules, such as polymerized actin are mostly spared from these effects. Thus, this motion blur provides a mechanism to specifically track polymerized actin molecules by using long exposures in which freely diffusing molecules are blurred. Using PALM I show that the velocity of single actin molecules along filaments, an index of filament polymerization rate, was highly heterogeneous within individual spines. Most strikingly, molecular velocity was elevated in discrete, well-separated foci occurring not principally at the spine tip, but in subdomains throughout the spine, including the neck. Whereas actin velocity on filaments at the synapse was substantially elevated, those at the endocytic zone showed no enhanced polymerization activity. I conclude that actin subserves spatially diverse, independently regulated processes throughout spines. Perisynaptic actin forms a uniquely dynamic structure well suited for direct, active regulation of the synapse.