Biology subjects

Kennedy, M. B.

Publications and source records attributed to Kennedy, M. B..

5 recordsLinked to original sources

bioRxiv2025

SynGAP forms biocondensates at sub-micromolar concentrations and recruits PSD95 and receptor oligomers, functioning as a key initiator of PSD formation

A key issue in neuronal circuit regulation is how synapse formation is initiated. Synapse formation could start when one or more synaptic scaffold proteins that can initiate synapse formation reach certain threshold concentrations in the dendritic shaft, which might lead to their oligomerization or even liquid-liquid phase separation (LLPS). By combining in vitro reconstitution of purified proteins with live-cell single-molecule and confocal imaging, we demonstrated that SynGAP alone forms assemblies of nanoscale clusters containing several to several tens of molecules at 10-nM order concentrations and micron-scale LLPS hydrogel-like condensates at submicromolar concentrations. The trimers of SynGAP's intrinsically disordered region (IDR) induced by its coiled-coil domain are responsible for SynGAP condensation. CaMKII-mediated phosphorylation moderately suppresses SynGAP condensation, and also increases condensate liquidity. While PSD95 fails to form assemblies under these conditions, it is recruited to SynGAP condensates by specifically binding to the PDZ-binding motif of SynGAP. SynGAP[PSD95] condensates selectively immobilize postsynaptic transmembrane proteins, Neuroligin1 and AMPAR-TARP2 complexes, in a manner dependent on their oligomerization state, indicating cooperative recruitment dynamics among SynGAP, PSD95, and transmembrane components, which might mimic initial PSD assembly. These findings suggest that SynGAP may act as a primary nucleator of postsynaptic density assembly, challenging the PSD95-centered models.

neuroscience↗

bioRxiv2024

A spatial model of autophosphorylation of CaMKII in a glutamatergic spine suggests a network-driven kinetic mechanism for bistable changes in synaptic strength.

Long-term potentiation (LTP) is a biochemical process that underlies learning in excitatory glutamatergic synapses in the Central Nervous System (CNS). The critical early driver of LTP is autophosphorylation of the abundant postsynaptic enzyme, Ca2+/calmodulin-dependent protein kinase II (CaMKII). Autophosphorylation is initiated by Ca2+ flowing through NMDA receptors activated by strong synaptic activity. Its lifetime is ultimately determined by the balance of the rates of autophosphorylation and of dephosphorylation by protein phosphatase 1 (PP1). Here we have modeled the autophosphorylation and dephosphorylation of CaMKII during synaptic activity in a spine synapse using MCell4, an open source computer program for creating particle-based stochastic, and spatially realistic models of cellular microchemistry. The model integrates four earlier detailed models of separate aspects of regulation of spine Ca2+ and CaMKII activity, each of which incorporate experimentally measured biochemical parameters and have been validated against experimental data. We validate the composite model by showing that it accurately predicts previous experimental measurements of effects of NMDA receptor activation, including high sensitivity of induction of LTP to phosphatase activity in vivo, and persistence of autophosphorylation for a period of minutes after the end of synaptic stimulation. We then use the model to probe aspects of the mechanism of regulation of autophosphorylation of CaMKII that are difficult to measure in vivo. We examine the effects of "CaM-trapping," a process in which the affinity for Ca2+/CaM increases several hundred-fold after autophosphorylation. We find that CaM-trapping does not increase the proportion of autophosphorylated subunits in holoenzymes after a complex stimulus, as previously hypothesized. Instead, CaM-trapping may dramatically prolong the lifetime of autophosphorylated CaMKII through steric hindrance of dephosphorylation by protein phosphatase 1. The results provide motivation for experimental measurement of the extent of suppression of dephosphorylation of CaMKII by bound Ca2+/CaM. The composite MCell4 model of biochemical effects of complex stimuli in synaptic spines is a powerful new tool for realistic, detailed dissection of mechanisms of synaptic plasticity.

neuroscience↗

bioRxiv2019

Interactions between calmodulin and neurogranin govern the dynamics of CaMKII as a leaky integrator

Calmodulin-dependent kinase II (CaMKII) has long been known to play an important role in learning and memory as well as long term potentiation (LTP). More recently it has been suggested that it might be involved in the time averaging of synaptic signals, which can then lead to the high precision of information stored at a single synapse. However, the role of the scaffolding molecule, neurogranin (Ng), in governing the dynamics of CaMKII is not yet fully understood. In this work, we adopt a rule-based modeling approach through the Monte Carlo method to study the effect of Ca2+ signals on the dynamics of CaMKII phosphorylation in the postsynaptic density (PSD). Calcium surges are observed in synaptic spines during an EPSP and back-propagating action potential due to the opening of NMDA receptors and voltage dependent calcium channels. We study the differences between the dynamics of phosphorylation of CaMKII monomers and dodecameric holoenzymes. The scaffolding molecule Ng, when present in significant concentration, limits the availability of free calmodulin (CaM), the protein which activates CaMKII in the presence of calcium. We show that it plays an important modulatory role in CaMKII phosphorylation following a surge of high calcium concentration. We find a non-intuitive dependence of this effect on CaM concentration that results from the different affinities of CaM for CaMKII depending on the number of calcium ions bound to the former. It has been shown previously that in the absence of phosphatase CaMKII monomers integrate over Ca2+ signals of certain frequencies through autophosphorylation (Pepke et al, Plos Comp. Bio., 2010). We also study the effect of multiple calcium spikes on CaMKII holoenzyme autophosphorylation, and show that in the presence of phosphatase CaMKII behaves as a leaky integrator of calcium signals, a result that has been recently observed in vivo. Our models predict that the parameters of this leaky integrator are finely tuned through the interactions of Ng, CaM, CaMKII, and PP1. This is a possible mechanism to precisely control the sensitivity of synapses to calcium signals.

neuroscience↗

bioRxiv2019

A sex difference in the composition of the rodent postsynaptic density

SynGAP is a postsynaptic density (PSD) protein that binds to PDZ domains of the scaffold protein PSD-95. We previously reported that heterozygous deletion of synGAP in mice is correlated with increased steady-state levels of other key PSD proteins that bind PSD-95, although the level of PSD-95 remains constant (Walkup et al., 2016). For example, the ratio to PSD-95 of Transmembrane AMPA-Receptor-associated Proteins (TARPs), which mediate binding of AMPA-type glutamate receptors to PSD-95, was increased in young synGAP+/- mice. Here we show that a highly significant increase in TARP in the PSDs of young synGAP+/- rodents is present only in females and not in males. The data reveal a sex difference in the adaptation of the PSD scaffold to synGAP heterozygosity.

neuroscience↗

bioRxiv2019

A multi-state model of the CaMKII dodecamer suggests a role for calmodulin in maintenance of autophosphorylation

Ca2+/calmodulin-dependent protein kinase II (CaMKII) accounts for up to 2 percent of all brain protein and is essential to memory function. CaMKII activity is known to regulate dynamic shifts in the size and signaling strength of neuronal connections, a process known as synaptic plasticity. Increasingly, computational models are used to explore synaptic plasticity and the mechanisms regulating CaMKII activity. Conventional modeling approaches may exclude biophysical detail due to the impractical number of state combinations that arise when explicitly monitoring the conformational changes, ligand binding, and phosphorylation events that occur on each of the CaMKII holoenzymes twelve subunits. To manage the combinatorial explosion without necessitating bias or loss in biological accuracy, we use a specialized syntax in the software MCell to create a rule-based model of the twelve-subunit CaMKII holoenzyme. Here we validate the rule-based model against previous measures of CaMKII activity and investigate molecular mechanisms of CaMKII regulation. Specifically, we explore how Ca2+/CaM-binding may both stabilize CaMKII subunit activation and regulate maintenance of CaMKII autophosphorylation. Noting that Ca2+/CaM and protein phosphatases bind CaMKII at nearby or overlapping sites, we compare model scenarios in which Ca2+/CaM and protein phosphatase do or do not structurally exclude each others binding to CaMKII. Our results suggest a functional mechanism for the so-called \"CaM trapping\" phenomenon, such that Ca2+/CaM structurally excludes phosphatase binding and thereby prolongs CaMKII autophosphorylation. We conclude that structural protection of autophosphorylated CaMKII by Ca2+/CaM may be an important mechanism for regulation of synaptic plasticity.\n\nAuthor summaryIn the hippocampus, the dynamic fluctuation in size and strength of neuronal connections is thought to underlie learning and memory processes. These fluctuations, called synaptic plasticity, are in-part regulated by the protein calcium/calmodulin-dependent kinase II (CaMKII). During synaptic plasticity, CaMKII becomes activated in the presence of calcium ions (Ca2+) and calmodulin (CaM), allowing it to interact enzymatically with downstream binding partners. Interestingly, activated CaMKII can phosphorylate itself, resulting in state changes that allow CaMKII to be functionally active independent of Ca2+/CaM. Phosphorylation of CaMKII at Thr-286/287 has been shown to be a critical component of learning and memory. To explore the molecular mechanisms that regulate the activity of CaMKII holoenzymes, we use a rule-based approach that reduces computational complexity normally associated with representing the wide variety of functional states that a CaMKII holoenzyme can adopt. Using this approach we observe regulatory mechanisms that might be obscured by reductive approaches. Our results newly suggest that CaMKII phosphorylation at Thr-286/287 is stabilized by a mechanism in which CaM structurally excludes phosphatase binding at that site.

neuroscience↗