Spatial distribution of Na+-K+-ATPase in dendritic spines dissected by nanoscale superresolution STED microscopy

Abstract

Background: The Na+,K+-ATPase plays an important role for ion homeostasis in virtually all mammalian cells, including neurons. Despite this, there is as yet little known about the isoform specific distribution in neurons.

Results: With help of superresolving stimulated emission depletion microscopy the spatial distribution of Na+,K+-ATPase in dendritic spines of cultured striatum neurons have been dissected. The found compartmentalized distribution provides a strong evidence for the confinement of neuronal Na+,K+-ATPase (α3 isoform) in the postsynaptic region of the spine.

Conclusions: A compartmentalized distribution may have implications for the generation of local sodium gradients within the spine and for the structural and functional interaction between the sodium pump and other synaptic proteins. Superresolution microscopy has thus opened up a new perspective to elucidate the nature of the physiological function, regulation and signaling role of Na+,K+-ATPase from its topological distribution in dendritic spines.

Figure 1

Co-immunoprecipitation and GST pull down assay of PSD-95 and α3 NKA. Co-immunoprecipitation (CoIP) shows interaction between PSD-95 and α3 NKA. GST pull down assays shows interaction of PSD-95 with the α3 NKA N-terminus and a specific PDZ3 interaction.

Figure 2

Principle of STED microscopy. The red depletion beam is phase-modulated to form a focal doughnut shown in the top right panel. Superimposition of this STED focus onto the diffraction limited excitation focus, shown in the adjacent panel, sharpens the effective fluorescence spot, which allows nanoscale imaging. The lower panels show well discernable fluorescent beads (diameter 40 nm) and a line profile across one of them with FWHM-value below 50 nm. Scale bar: 200 nm

Figure 3

STED microscopy dissecting the localization of Na⁺,K⁺-ATPase in cultured striatal neurons. The 5 × 3 mosaic shows a comparision of the confocal (left) and STED images (middle) of the Alexa-594 immunolabelled α3 NKA in dendritic spines. Postprocessing of the raw STED data by a Richardson-Lucy deconvolution further enhances the details as shown in the right row of images. Scale bar: 500 nm