Correlating STED and synchrotron XRF nano-imaging unveils cosegregation of metals and cytoskeleton proteins in dendrites
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.
Data availability
Synchrotron datasets (SXRF and PCI images) are available from the ESRF data portal in open mode with the following DOI numbers: doi:10.15151/ESRF-ES-162248067 (https://doi.esrf.fr/10.15151/ESRF-ES-162248067) and doi:10.15151/ESRF-ES-101127303 (https://doi.esrf.fr/10.15151/ESRF-ES-101127303). Figure 1-source data 1. Data are available at https://doi.esrf.fr/10.15151/ESRF-ES-162248067 datasets M20_zone67_nfp3_015nm and M20_zone67_fine01. Table 1-source data 1. Table1 Source data 1.xlsx. Figure 2-source data 1. Data are available at https://doi.esrf.fr/10.15151/ESRF-ES-101127303 datasets TA15_neu64_fine2 and TA15_neu64_fine5. Figure 3-source data 1. Data are available at https://doi.esrf.fr/10.15151/ESRF-ES-162248067 datasets M8_neur43_sted44_nfp_015nm and M8_neu43_fine03. Figure 4-source data 1. Data are available at https://doi.esrf.fr/10.15151/ESRF-ES-101127303 dataset TA15_neu71_fine01. Figure 4-source data 2. Data for Pearson's correlation coefficients are included in Figure 4 source data 2.zip Figure 5-source data 1. Data are available at https://doi.esrf.fr/10.15151/ESRF-ES-101127303 datasets TA15- neu 26 fine 01 and TA15_neu23_fine02. Figure 6-source data 1. Data for F-actin are available in file Figure 6 source data 1.xlxs. Figure 6-source data 2. Data for β-tubulin are available in file Figure 6 source data 2.xlxs. Figure 2-source data 2. Synchrotron XRF data for Figure 2-figure supplement 1 are available at https://doi.esrf.fr/10.15151/ESRF-ES-101127303 datasets TA15_neu64_fine4 and TA15_neu64_fine3. Figure 2-source data 3. Data for Pearson's correlation coefficients of Figure 2-figure supplement 1 panel h are provided in Figure 2 source data 3.zip Figure 2-source data 4. Data for Pearson's correlation coefficients of of Figure 2-figure supplement 1 panel o are provided in Figure 2 source data 4.zip Figure3-source data 2. Synchrotron XRF data for Figure 3-figure supplement 1 are available at https://doi.esrf.fr/10.15151/ESRF-ES-101127303 dataset SiTA1_neu7_fine01. Figure 4-figure source data 2. Synchrotron XRF and PCI data for Figure 4-figure supplement 1 are available at https://doi.esrf.fr/10.15151/ESRF-ES-162248067 datasets M20_zone67_fine01, M20_zone67_fine02, and M20_zone67_fine06. Figure 5-source data 2. Synchrotron XRF data for Figure 5-figure supplement 1 are available at https://doi.esrf.fr/10.15151/ESRF-ES-162248067 datasets M20_zone67_nfp3_015nm and M20_zone67_fine01. Figure 6-source data 3. F-actin data for Figure 6-figure supplement 1 are available in file Figure 6 source data 3.xlxs. Figure 6-source data 4. Tubulin data for Figure 6-figure supplement 1 are available in file Figure6 source data 4.xlxs. Supplementary File 1. Raw data provided in Source Data 1, file Source data 1.xlsx.
Article and author information
Author details
Funding
Centre National de la Recherche Scientifique
- Richard Ortega
H2020 European Research Council
- Daniel Choquet
IDEX Bordeaux
- Richard Ortega
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2020, Domart et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Metrics
-
- 2,515
- views
-
- 289
- downloads
-
- 23
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
Download links
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Further reading
-
- Neuroscience
At many vertebrate synapses, presynaptic functions are tuned by expression of different Cav2 channels. Most invertebrate genomes contain only one Cav2 gene. The Drosophila Cav2 homolog, cacophony (cac), induces synaptic vesicle release at presynaptic active zones (AZs). We hypothesize that Drosophila cac functional diversity is enhanced by two mutually exclusive exon pairs that are not conserved in vertebrates, one in the voltage sensor and one in the loop binding Caβ and Gβγ subunits. We find that alternative splicing in the voltage sensor affects channel activation voltage. Only the isoform with the higher activation voltage localizes to AZs at the glutamatergic Drosophila larval neuromuscular junction and is imperative for normal synapse function. By contrast, alternative splicing at the other alternative exon pair tunes multiple aspects of presynaptic function. While expression of one exon yields normal transmission, expression of the other reduces channel number in the AZ and thus release probability. This also abolishes presynaptic homeostatic plasticity. Moreover, reduced channel number affects short-term plasticity, which is rescued by increasing the external calcium concentration to match release probability to control. In sum, in Drosophila alternative splicing provides a mechanism to regulate different aspects of presynaptic functions with only one Cav2 gene.
-
- Neuroscience
- Structural Biology and Molecular Biophysics
We present near-atomic-resolution cryoEM structures of the mammalian voltage-gated potassium channel Kv1.2 in open, C-type inactivated, toxin-blocked and sodium-bound states at 3.2 Å, 2.5 Å, 3.2 Å, and 2.9 Å. These structures, all obtained at nominally zero membrane potential in detergent micelles, reveal distinct ion-occupancy patterns in the selectivity filter. The first two structures are very similar to those reported in the related Shaker channel and the much-studied Kv1.2–2.1 chimeric channel. On the other hand, two new structures show unexpected patterns of ion occupancy. First, the toxin α-Dendrotoxin, like Charybdotoxin, is seen to attach to the negatively-charged channel outer mouth, and a lysine residue penetrates into the selectivity filter, with the terminal amine coordinated by carbonyls, partially disrupting the outermost ion-binding site. In the remainder of the filter two densities of bound ions are observed, rather than three as observed with other toxin-blocked Kv channels. Second, a structure of Kv1.2 in Na+ solution does not show collapse or destabilization of the selectivity filter, but instead shows an intact selectivity filter with ion density in each binding site. We also attempted to image the C-type inactivated Kv1.2 W366F channel in Na+ solution, but the protein conformation was seen to be highly variable and only a low-resolution structure could be obtained. These findings present new insights into the stability of the selectivity filter and the mechanism of toxin block of this intensively studied, voltage-gated potassium channel.