OCOCAMKS · Optical control of CaMKII signaling

Optical control of CaMKII signaling

How are memories being formed and stored in the brain? On a cellular level, this is achieved through straightening of connections (synapses) between nerve cells, in a process called long term potentiation (LTP). During LTP, repetitive signals coming from the pre-synaptic neuron to the post-synaptic neuron evoke short calcium (Ca2+) bursts which result in long-term changes of synaptic strength. Ca2+ signals are translated to protein modifications, which enable signaling even long after the initial Ca2+ levels return to baseline. The most notable effector of Ca2+ signaling in postsynaptic neurons is Ca2+ -Calmodulin dependent Protein Kinase II (CaMKII), which comprises 1-2% of total brain protein. There are 4 isoforms of CaMKII (a,b,g,d) in the brain, which all share the same overall structure and mode of activation. After Ca2+ enters the post-synaptic neuron, it binds to a small effector protein called calmodulin (CaM). Next, Ca2+-bound CaM binds to CaMKII and initiates autophosphorylation and activation of CaMKII. A unique feature of CaMKII is its dodecameric assembly. Monomers (or subunits) within a dodecamer are held together by hub (association) domains of CaMKII, and form the so-called CaMKII holoenzyme. It has been postulated that, once activated, CaMKII remains and spreads activity during LTP by allowing exchange of subunits between activated und unactivated holoenzymes, and subsequent autophosphorylation of inactive subunits, thereby bypassing the need for Ca2+. This feature is referred to as autonomous activity of CaMKII. CaMKII deletion from neurons completely abolishes LTP, making it indispensable for this process. The overall objectives of this project focus on better understanding of several aspects of CaMKII signaling: How is CaMKII remaining active after the initial Ca2+ signal is gone? Does CaM have a role in CaMKII activity other than initiation of activation? What is the timing of CaMKII activity during LTP? To date, many clues into the biology of CaMKII were found, but the molecular details underlying the mechanisms of CaMKII activity are still lacking. The aim of this project has been to implement genetic code expansion (GCE) and optogenetic tools to interfere CaMKII activity with unprecedented precision, and with spatial and temporal resolution. GCE relies on replacement of naturally-occurring residues with unnatural amino acids (UAA) at different positions within a protein of interest, thereby allowing optical manipulation of proteins, without disturbing their overall assembly. I am using p-benzoyl-l-phenylalanine (BzF, also known as Bpa), which, upon exposure to UV light, undergoes irreversible cross-linking with neighboring residues. Placing BzF at carefully chosen sites in CaMKII allows us to control different aspects of CaMKII biology with light. Using this approach we found that CaMKII can spread its activity even when its ability to exchange subunits is diminished. Therefore, the exchange of subunits between activated and unactivated holoenzymes is not necessary for the spread of kinase activity, but might play a role in substrate phosphorylation.

Data: CORDIS, © European Union