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Expansion microscopy (ExM) is a novel super-resolution microscopy method that enables nano-scale biophysical studies by increasing the distance among biomolecules. Such effect is achieved through the physical magnification of the specimen obtained by its embedding inside a swellable polymer network, eventually expanded in water. Several improvements to the original ExM technique were made to optimize the imaging of proteins and the isotropicity of their expansion. However, few works have been done to investigate what happens to the DNA-chromatin architecture once it’s subjected to the mechanical strain associated with the expansion process. To understand whether this technique could be suitable for investigating the crowded nuclear environment, we aim to study the induced changes in the DNA conformation by the application of different expansion microscopy protocols to fixed mammalian cells and single mononucleosomes. Optical imaging will be performed using a combination of confocal, two-photon excitation and super-resolution microscopy techniques, and the performances of different fluorophores for DNA staining will also be compared. Finally, we aim to develop a reproducible protocol for the precise quantification of the expansion factor of the specimens at the microscale.

Mutations in PRoline Rich Transmembrane protein 2 (PRRT2) cause pleiotropic syndromes including benign infantile epilepsy, paroxysmal kinesigenic dyskinesia, episodic ataxia, that share the paroxysmal character of the clinical manifestations. PRRT2 is a neuronal protein that plays multiple roles in the regulation of neuronal development, excitability, and neurotransmitter release. To better understand the physiopathology of these clinical phenotypes, we investigated PRRT2 interactome in mouse brain by a pulldown-based proteomic approach and identified α1 and α3 Na+/K+ ATPase (NKA) pumps as major PRRT2-binding proteins. We confirmed PRRT2 and NKA interaction by biochemical approaches and showed their colocalization at neuronal plasma membrane. The acute or constitutive inactivation of PRRT2 had a functional impact on NKA. While PRRT2-deficiency did not modify NKA expression and surface exposure, it caused an increased clustering of α3-NKA on the plasma membrane. Electrophysiological recordings showed that PRRT2-deficiency in primary neurons impaired NKA function during neuronal stimulation without affecting pump activity under resting conditions. Both phenotypes were fully normalized by re-expression of PRRT2 in PRRT2-deficient neurons. In addition, the NKA-dependent afterhyperpolarization that follows high-frequency firing was also reduced in PRRT2-silenced neurons. Taken together, these results demonstrate that PRRT2 is a physiological modulator of NKA function and suggest that an impaired NKA activity contributes to the hyperexcitability phenotype caused by PRRT2 deficiency.