Abstract
Zinc and copper are involved in neuronal differentiation and synaptic plasticity but the molecular mechanisms behind these processes are still elusive due in part to the difficulty of imaging trace metals together with proteins at the synaptic level. We correlate stimulated-emission-depletion microscopy of proteins and synchrotron X-ray fluorescence imaging of trace metals, both performed with 40 nm spatial resolution, on primary rat hippocampal neurons. We reveal the co-localization at the nanoscale of zinc and tubulin in dendrites with a molecular ratio of about one zinc atom per tubulin-αβ dimer. We observe the co-segregation of copper and F-actin within the nano-architecture of dendritic protrusions. In addition, zinc chelation causes a decrease in the expression of cytoskeleton proteins in dendrites and spines. Overall, these results indicate new functions for zinc and copper in the modulation of the cytoskeleton morphology in dendrites, a mechanism associated to neuronal plasticity and memory formation.
Highlights
The neurobiology of copper and zinc is a matter of intense investigation since they have been recently associated to neuronal signaling and differentiation processes (Chang, 2015; Vergnano et al, 2014; Barr et al, 2017; D’Ambrosi et al, 2015; Xiao et al, 2018; Hatori et al, 2016)
We designed a specific protocol consisting in live-cell stimulated emission depletion (STED) microscopy on silicon nitride (SN) substrates followed by cryogenic processing of the cells before nano-Synchrotron X-Ray Fluorescence (SXRF) imaging as described in
STXM enabled to visualize the dendritic morphology showing the exact superposition of dendrites after cryofixation and freeze drying with silicone rhodamine (SiR)-tubulin fluorescence previously observed by STED on living cells
Summary
The neurobiology of copper and zinc is a matter of intense investigation since they have been recently associated to neuronal signaling and differentiation processes (Chang, 2015; Vergnano et al, 2014; Barr et al, 2017; D’Ambrosi et al, 2015; Xiao et al, 2018; Hatori et al, 2016). Understanding the functions of copper and zinc in the cytoskeleton structure would require to correlate metal localization with respect to relevant cytoskeleton proteins at the sub-dendritic level To this end, we have developed an original method for correlative imaging of metals and proteins at 40 nm spatial resolution described in this paper. We have previously reported a correlative microscopy approach consisting in labeling organelles or proteins with specific fluorophores for live-cell imaging prior to SXRF imaging (Roudeau et al, 2014; Carmona et al, 2019) This correlative approach is limited by the spatial resolution of optical fluorescence microscopy, above 200 nm, which is larger than the spatial resolution achieved today with nano-SXRF and insufficient to resolve synaptic substructures. We explored the effect of zinc deficiency induced by TPEN, an intracellular zinc chelator, on the expression of cytoskeleton proteins in dendrites and dendritic spines
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