Browsing School, Graduate by Subject "PALM"
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Actin Dynamics at nanometer domains within individual dendritic spinesExcitatory 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.
Multiple spatial and kinetic subpopulations of CaMKII in spines and dendrites as resolved by single-molecule tracking PALMCalcium/calmodulin-dependent protein kinase II (CaMKII) is essential for synaptic plasticity underlying memory formation. The existing hypothesis is that with stimuli that induce LTP, activated CaMKII translocates to the PSD and phosphorylates synaptic proteins and thus increases synaptic strength. However, many known CaMKII substrates that regulate synaptic transmission lie away from the PSD, and it is unlikely that PSD-bound CaMKII can phosphorylate such targets. Therefore, I propose an alternative hypothesis that CaMKII interacts with binding partners both at and away from the PSD upon NMDAR stimulation. It has been difficult to study protein-protein interaction in living cells. I considered one way to do this would be to measure CaMKII mobility as an indication of its interaction with the substrate. Confocal imaging does not achieve the resolution necessary to distinguish subpopulations within the small volumes of a spine. Here, I used photo-activated localization microscopy (PALM) to track single molecules of CaMKIIα in live neurons, mapping their spatial distribution and kinetic heterogeneity at high resolution. I found that CaMKIIα exhibits at least three kinetic subpopulations, even within individual spines. Latrunculin application, which depolymerizes actin filaments, or co-expression of CaMKIIβ containing its actin-binding domain, strongly modulated CaMKII diffusion, indicating that a major subpopulation is regulated by the actin cytoskeleton. CaMKII in spines was typically more slowly mobile than in dendrites, consistent with the presence of a higher density of binding partners or obstacles. Importantly, NMDA receptor stimulation that triggered CaMKII activation prompted the immobilization and presumed binding of CaMKII in spines not only at PSDs but also at other points up to several hundred nanometers away, suggesting that activated kinase does not target only the PSD. Consistent with this, single endogenous activated CaMKII molecules detected via STORM immunocytochemistry were concentrated in spines both at the PSD and at points distant from the synapse. Together, these results indicate that CaMKII mobility within spines is determined by association with multiple interacting proteins that could potentially activate different downstream signals, even outside the PSD, The results lend greater support to the alternative hypothesis proposed above, and suggest diverse mechanisms by which CaMKII may regulate synaptic transmission.