Sequestration of CaMKII in dendritic spines in silico

Abstract

Calcium calmodulin dependent kinase II (CaMKII) is sequestered in dendritic spines within seconds upon synaptic stimulation. The program Smoldyn was used to develop scenarios of single molecule CaMKII diffusion and binding in virtual dendritic spines. We first validated simulation of diffusion as a function of spine morphology. Additional cellular structures were then incorporated to simulate binding of CaMKII to the post-synaptic density (PSD); binding to cytoskeleton; or their self-aggregation. The distributions of GFP tagged native and mutant constructs in dissociated hippocampal neurons were measured to guide quantitative analysis. Intra-spine viscosity was estimated from fluorescence recovery after photo-bleach (FRAP) of red fluorescent protein. Intra-spine mobility of the GFP-CaMKIIα constructs was measured, with hundred-millisecond or better time resolution, from FRAP of distal spine tips in conjunction with fluorescence loss (FLIP) from proximal regions. Different FRAP \ FLIP profiles were predicted from our Scenarios and provided a means to differentiate binding to the PSDs from self-aggregation. The predictions were validated by experiments. Simulated fits of the Scenarios provided estimates of binding and rate constants. We utilized these values to assess the role of self-aggregation during the initial response of native CaMKII holoenzymes to stimulation. The computations revealed that self-aggregation could provide a concentration-dependent switch to amplify CaMKII sequestration and regulate its activity depending on its occupancy of the actin cytoskeleton.

Spatiotemporal maps of CaMKII in dendritic spines

“…GFP fusions of the native and mutant CaMKIIβ forms were expressed in a monkey kidney epithelial cell line (Cos-7) to validate phenotypes as assessed by interactions with the cell cortex ). The association with the cell cortex of the native GFP-CaMKIIβ construct we used was shown previously (Khan et al 2011). GFP-CaMKIIβΔ was uniformly distributed in the cell consistent with a weaker interaction with the actin cortex ( Fig.…”

“…Glutamate does not significantly increase GFP-CaMKIIα times, while the rapid GFP*-CaMKIIβ phase (1) seen in glutamate treated spines is comparable to them decay profiles in order to elucidate the exchange of CaMKII between dendritic shafts and spines. The virtual spine used in our earlier work (Khan et al 2011) was modified (Methods) for 3D simulation of GFP-CaMKIIβ interactions with the neuronal actin cytoskeleton not studied by (Khan et al 2011). The cortical actin along the dendritic shaft was modeled as a layer lining the interior surface of the shaft.…”

“…1d. New features that build on the initial virtual spine (Khan et al 2011) are: 1 -Shaft boundaries reflect PaGFP, adsorb GFP* species; 2 -Molecules convert between forms with different diffusion coefficients upon crossing the spine neck/head boundary; 3 -Molecules partition between the dendrite walls and cytoplasm with coefficient that reflects their affinity to cortical actin; 4 -Spine actin cytoskeletal meshwork is modeled as a 3D lattice of filaments (see section 4 for details). Right Panel: Photo-activation in spine tip.…”

“…This discrepancy could be because the simulations used a higher cytoplasmic viscosity and/or narrower spine neck than the actual values in our experimental samples. Viscosity estimates, including our own (Khan et al 2011) have associated measurement error. In addition, there could be systematic as well as random error introduced, for example, by the assumption of a homogenous dodecameric CaMKIIα species.…”

“…In earlier work, we introduced a novel half-spine FRAP protocol to characterize the intra-spine mobility of native and mutant GFP-CaMKIIα constructs prior and post glutamate stimulation. We developed a framework within the single molecule dynamics particle simulator Smoldyn for interpretation of the half-spine FRAP data, leading to a model for a possible role for CaMKII self-aggregation during stimulus overload (Khan et al 2011) In the present work we characterized binding compartments for the two CaMKII isoforms in dendritic spines with greater spatiotemporal precision than hitherto achieved. We employed a photoactivation protocol that allowed us to distinguish bound and soluble CaMKII fraction within dendritic spines.…”