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Optical control of CaMKII signaling

Periodic Reporting for period 1 - OCOCAMKS (Optical control of CaMKII signaling)

Reporting period: 2018-05-01 to 2020-04-30

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.
We are able to produce 10+ CaMKII BzF mutants in E.Coli and purify them. These mutants mostly behave like wt CaMKII, with respect to the overall dodecameric assembly and activity, but unlike the wt protein, some mutants are susceptible to UV light.
Mutating carefully chosen positions to BzF in the CaMKII hub domain (one at the time) enables us to use UV light to trap higher oligomeric species of CaMKII, and thereby abolish any subunit exchange process and test if it is necessary for the spread of kinase activity. Radioactive activity assays with our oligomeric mutants suggest that the subunit exchange most likely is not necessary for the spread of kinase activity between activated and unactivated CaMKII holoenzymes. Spreading of activity happens regardless of subunit restriction. However, activity assays indicate that restriction of CaMKII to higher oligomeric species plays a role in substrate phosphorylation.
We have also produced a BzF mutant of CaMKII which can be crosslinked to CaM, with 100% efficiency, using UV light. We will use this protein to investigate the influence of CaM binding to the overall holoenzyme assembly, as it has been proposed that CaM binding can initiate the subunit exchange. This CaMKII, covalently crosslinked to CaM, is an excellent candidate for cryo-EM studies, where we can directly observe the influence of CaM binding on CaMKII holoenzyme assembly.
Finally, we are able to make CaMKII BzF mutants in mammalian HEK cells, allowing us to test activity in the cytosol of eukaryotic cells. The final goal of the project is to test observations from in vitro studies under close to native conditions in neurons, using CaMKII BzF mutants and UV light. We continue to develop our methods to produce CaMKII BzF mutants in neurons, which are limited due to several factors. These challenges include optimal introduction of the necessary components of the BzF rescue system to primary neurons. This work is ongoing and will be continued even after the two-year period of the MSCA action.

Dissemination:

June 2018 – “Postdoc Day 2018”; Berlin, Germany (poster)
September 2018 – conference “Membranes and Modules”; Berlin, Germany (poster)
December 2018 - SFB/TRR186 International meeting “Molecular Switches in Spatio-temporal control of Cellular Signal Transmission”; Heidelberg, Germany (poster)
April 2019 - SFB/TRR186 PhD students and postdocs symposium; Heidelberg, Germany (talk)
June 2019 – Berlin Postdoc Day, Berlin, Germany (poster)
July 2019 – FENS regional meeting, Belgrade, Serbia (poster)
At the end of 2019 I took maternity leave for 1 year.
European Research Night - a short video explaining my research for Serbian Research Night in November 2020 (https://nocistrazivaca.rs/programi2020/ococamks/419).
I will participate in 88th Harden conference of Biochemical Society “Beyond catalysis: Kinases and Pseudokinases” in May 2022.
When we looked at the presence of CaMKII from two different batches of purification (one with BzF residue in hub domains, the other one wt) in the same holoenzymes after UV treatment, we could not detect the presence of wt CaMKII in BzF crosslinked complexes, even after long incubations. This indicates that proteins from two different sources (belonging to different holoenzymes) do not directly interact, as one would expect if they would be located within the same holoenzyme due to subunit exchange. This mechanistic insight into the mechanism of CaMKII activity strongly suggests that subunit exchange is not required for normal CaMKII actions in the brain.
We focused our research on CaMKIIa, which is the predominant isoform in the brain. The findings of our study and the methods developed can be used for investigation of other CaMKII isoforms. For example CaMKIIg is predominantly expressed in the heart, where it plays an important role in Ca2+ - driven signaling, which is often disbalanced in cardiac disease. Light manipulation of CaMKIIg in cardiomyocites could prove as a useful tool for studying molecular mechanisms of CaMKII involvement in cardiac pathologies. Finally, this work might encourage other studies with optically controlled kinases via the use of the UAA system which enables studying of kinases under almost native structures, in which mutation effects can be controlled by light.
Revised model of the role of subunit exchange during activation of CaMKII