Nanoscale elucidation of Na,K-ATPase isoforms in dendritic spines

Abstract

Background: The dimensions of neuronal synapses suggest that optical super-resolution imaging methods are necessary for thorough investigation of protein distributions and interactions. Nanoscopic evaluation of neuronal samples has presented practical hurdles, but advancing methods are making synaptic protein topology and quantification measurements feasible. This work explores the application of Photoactivated Localization Microscopy (PALM) pointillistic super-resolution imaging for investigation of the membrane bound sodium pump, the Na,K-ATPase, in matured neurons. Results: Two isoforms of the sodium pump (ATP1a1 and ATP1a3) were studied in cultured neurons using the PALM-compatible fluorescent proteins PAGFP and mEos. Nanoscopic imaging reveals a compartmentalized distribution of sodium pumps in dendritic spines. Several nanoclusters of pumps are typically found in the spine head and fewer in the spine neck. The density of sodium pumps was estimated from a quantification of detected single molecules at 450-650 pump copies/μm2 in the spine heads. Conclusions: We have utilized PALM for dissection of nanoscale localization in mature cultured neurons and demonstrated similar topology and quantification estimates with PAGFP and mEos. PALM topology assessments of the sodium pump appeared similar to previous STED studies, though quantification estimates varied, implying that labeling strategies, sample analysis and choice of nanoscopic imaging method can be critical factors for correct molecular quantification. © 2013 Liebmann et al.