Characterization of Novel Proteins Modulating the Nanoscale Organization of the Membrane-Associated Periodic Skeleton in Neurons
Dissertation
Datum der mündl. Prüfung:2023-09-29
Erschienen:2024-09-19
Betreuer:Prof. Dr. Stefan Hell
Gutachter:Prof. Dr. Stefan Hell
Gutachter:Prof. Dr. Fred Wouters
Gutachter:PD Dr. Hauke Werner
Gutachter:Dr. Katrin Willig
Gutachter:Dr. Christian Vogl
Gutachter:Prof. Dr. Henning Urlaub
Dateien
Name:VM_PhD-Thesis.pdf
Size:18.8Mb
Format:PDF
Zusammenfassung
Englisch
The Membrane-associated Periodic Skeleton (MPS) is a lattice located underneath the neuronal plasma membrane, characterized by a distinctive ~190 nm periodic architecture imposed by the nanoscale arrangement of its main components actin, spectrin, and adducin. To date, over 400 proteins have been found to interact with the MPS. However, despite the enormous progresses that have been made in the last decade, the underlying mechanisms of MPS regulation are only starting to emerge. Inspired by preliminary biochemical data inferring an interaction between a neuronal spectrin isoform and Palm1, this thesis identifies two proteins of the Paralemmin family, Palm1 and Palm2, as novel components of the MPS, elucidating their key roles over the course of neuronal development. Palm1 it is anchored to the inner side of the plasma membrane with a reported role in membrane expansion, filopodia formation, and spine maturation. By leveraging cutting-edge nanoscopy techniques (STED and MINFLUX), this study proposes a dual role for Palm1 in neurons. The first role is related to neuritogenesis, cellular growth and neurite expansion. Indeed, Palm1 overexpression increased neuronal arborization, whereas depletion of Palm1 delayed neuritogenesis, primarily suggesting a function in the regulation of actin dynamics. These processes are mainly mediated by a Palm1 isoform lacking exon 8 (Palm1ΔEx8), whose mRNA levels decay during neuron maturation. The second role is related to the regulation of the MPS architecture, which is mainly mediated by the full length Palm1 isoform. Palm1 is recruited into the already formed MPS to populate the actin rings and flank adducin, likely interacting with the N-terminus of ßII spectrin. Examination of overexpression, knock-out, and rescue experiments revealed that Palm1 levels alone regulate the periodic architecture of the MPS without altering the local concentrations of its components, showing that the nanoscale organization of the MPS is dictated by the amount of Palm1. Herein, this thesis describes for the first time a novel mechanism to modulate the MPS. Lastly, Palm1 knock-out neurons exhibited altered electrophysiological properties, with lower excitatory post synaptic currents and lower rheobase, probably as a result of the disturbance of both roles of Palm1. Since the MPS is present in virtually all neuronal cells and neuron types and Palm1 is maintained through vertebrates, these mechanisms might be conserved across brain regions and organisms, which would have significant implications for the understanding of the MPS regulation in the human brain. Palm1 is ubiquitously expressed in hippocampal neurons but its levels are reduced in the axon initial segment (AIS). This evidence raised the possibility that another member of the Paralemmin family might be present in neurons. Indeed, we identified Palm2 as a novel component of the AIS. Palm2 is present along the premature axon and is restricted into the AIS from DIV 5. However, it is not essential for axon specification and AIS assembly. Although Palm2 does not show a periodic organization at endogenous levels, it becomes periodic upon overexpression in all neuronal compartments, assuming a localization at the MPS comparable to Palm1. Interestingly, overexpression of Palm2 negatively impacted the AIS-specific MPS component ßIV spectrin both in terms of expression levels and periodic organization, suggesting a function in the modulation of the AIS structure. This hypothesis is supported by the evidence that neurons lacking Palm2 require higher currents to initiate an action potential. Altogether, this thesis provides an exhaustive characterization of both Palm1 and Palm2 in hippocampal neurons, identifying two novel proteins interacting with the MPS. Importantly, a novel approach to regulate this lattice is elucidated, opening up new opportunities for future studies on axon degeneration and neurological diseases, in which the MPS is involved.
Keywords: Paralemmins; membrane-associated periodic skeleton; spectrins; primary hippocampal neurons; cytoskeleton; axon; nanoscopy