Quantitative Multicolour Nanoscopy for the Investigation of the Postsynaptic Actin CytoskeletonDoctoral thesis
Date of Examination:2023-04-19
Date of issue:2023-08-16
Advisor:Prof. Dr. Stefan Hell
Referee:Prof. Dr. Stefan Hell
Referee:Prof. Dr. Thomas Dresbach
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EnglishCognitive processes like the formation of memory and learning originate in the correct functioning of synapses. Dendritic spines are highly plastic, specialised actin-rich structures harbouring the postsynaptic site in excitatory glutamatergic synapses. Mechanisms crucial for synaptic physiology, like spine plasticity and neurotransmitter receptor mobilisation depend on synaptic activity and are subjected to actin cytoskeleton dynamics. However, a detailed outline of how the postsynaptic actin architecture changes at the nanoscale in response to distinct synaptic activity states is vastly lacking. The implementation of fluorescence nanoscopy in synaptic research has allowed exploring previously inaccessible subcellular organizations of various synaptic components. Nevertheless, accessing the fine actin organization in the spine has remained a challenge due to the dense actin network formed at the micron-sized postsynaptic site. In this study, we establish a robust method based on high-throughput multicolour STED nanoscopy to quantitatively evaluate the composition and organisation of the postsynaptic actin architecture based on four actin regulating proteins: the capping protein CapZ, the branching complex Arp2/3 and two proteins related to the membrane-associated periodic skeleton, β-II-spectrin and α-adducin. In basal activity conditions, we report that the proteins, present in vast majority of the spine population, distribute in a polarised manner, with the MPS components preferentially locating in the spine neck, while CapZ and Arp2/3 populate mainly the spine head positioning within few hundred of nanometers of distance to the PSD. By means of dual-colour 3D MINFLUX nanoscopy, for the first time implemented at synaptic sites, we were able to confirm this observation and demonstrate the presence of some CapZ and Arp2/3 molecules within tens of nanometres of distance to the PSD. These findings suggested a close relationship between the PSD and the actin architectural components in mature spines, which was further corroborated by a robust positive correlation between a marker of postsynaptic strength and the presence of actin regulating proteins. To prove the connection between synaptic activity and actin structure, we characterised the effects of synaptic activity on the actin proxies under short- and long-term activity inhibiting or stimulating conditions. We could demonstrate that individual activity states induce specific architectural rearrangements on the postsynaptic actin framework. The evoked structural changes depended on the exposure time and character of the activity modulation, and on the spine morphology. Importantly, we noted that acute activity modulation affected the levels of the actin components, while chronic activity modulation modified rather the correlation between their abundance and the postsynaptic strength. Prompted by the insights of our quantitative four-colour STED approach and by the urge of amplifying the number of simultaneously imaged synaptic targets to better comprehend their complex interplay, we implemented the use of newly developed photoactivatable dyes for multiplexed nanoscopy purposes achieving six-colour STED imaging in fixed neuronal samples. Altogether, this work evidences the potential of a joint use of two nanoscopy techniques, STED and MINFLUX, in a quantitative way. The highest benefit can be achieved by combining the high-throughput capability of STED and the unprecedented molecular resolution attainable with MINFLUX. Most importantly, this work puts forward the importance of multicolour nanoscopy in quantitative synaptic studies, exposing the relationship between the postsynaptic actin architectural state and synaptic activity. The detailed portrayal of the postsynaptic actin scaffold in dependence of activity will aid in the modelling of an average synapse and its response to different synaptic input, contributing to a better comprehension of processes like synaptic plasticity.
Keywords: super-resolution; nanoscopy; fluorescence microscopy; actin; MINFLUX; STED; cytoskeleton; spines; actin binding proteins; multiplexing; multicolor; postsynapse; synapse; neuronal activity; synaptic plasticity; quantitiative; Arp2/3; actin capping; spectrin; adducin; Cap; neuron; PSD