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

Blom H, Rönnlund D, Scott L, Spicarova Z, Widengren J, Bondar A, Aperia A, Brismar H - BMC Neurosci (2011)

Bottom Line: Despite this, there is as yet little known about the isoform specific distribution in neurons.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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Applied Physics, Royal Institute of Technology, Stockholm, Sweden.

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.

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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
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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

Mentions: The use of STED microscopy made it possible to visualize the spine localization of NKA in unprecedented detail (n = 20 spines were analyzed). Figure 3 shows spineneck widths having FWHM-values of 82 +/-7 nm. Neuronal α3 NKA subunit was fluorescently immunolabeled with Alexa-594, a dye well suited for STED microscopy [14]. The STED images also revealed discernible pools of NKA complexes both in heads and necks of spines and also within the connecting dendritic structures. Notably, there are areas in the necks where there appears to be empty patches (cf. Figure 3A-E). Parts of the spine-necks are thus filled, but just below the head and/or above the shaft there are zones without α3 NKA subunits. Similar topological variations are also seen in the heads. Spine-heads viewed from the side, tilted, as well as from above (cf. Figure 3A-E), reveals a modulated distribution of α3 NKA complexes. The spatial size of these individual pools have FWHM-values of 58 +/- 11 nm, which physically limits the number of sodium pumps (size 65 × 75 × 150 Å) in a head-complex pool to contain not more than 20-30 subunits, assuming a mean density of 4-5 PSD-95 molecules per 1000 nm2 and only one single sodium pump per scaffolding protein [15]. Steric hindrance by the relatively large antibody complexes may influence the estimate of NKA per nanocluster. It may on one hand give an overestimate if the antibodies physically increase the size of the imaged fluorescence cluster. On the other hand if quantification is based on the fluorescence intensity it may give an underestimate.


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

Blom H, Rönnlund D, Scott L, Spicarova Z, Widengren J, Bondar A, Aperia A, Brismar H - BMC Neurosci (2011)

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
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3040715&req=5

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
Mentions: The use of STED microscopy made it possible to visualize the spine localization of NKA in unprecedented detail (n = 20 spines were analyzed). Figure 3 shows spineneck widths having FWHM-values of 82 +/-7 nm. Neuronal α3 NKA subunit was fluorescently immunolabeled with Alexa-594, a dye well suited for STED microscopy [14]. The STED images also revealed discernible pools of NKA complexes both in heads and necks of spines and also within the connecting dendritic structures. Notably, there are areas in the necks where there appears to be empty patches (cf. Figure 3A-E). Parts of the spine-necks are thus filled, but just below the head and/or above the shaft there are zones without α3 NKA subunits. Similar topological variations are also seen in the heads. Spine-heads viewed from the side, tilted, as well as from above (cf. Figure 3A-E), reveals a modulated distribution of α3 NKA complexes. The spatial size of these individual pools have FWHM-values of 58 +/- 11 nm, which physically limits the number of sodium pumps (size 65 × 75 × 150 Å) in a head-complex pool to contain not more than 20-30 subunits, assuming a mean density of 4-5 PSD-95 molecules per 1000 nm2 and only one single sodium pump per scaffolding protein [15]. Steric hindrance by the relatively large antibody complexes may influence the estimate of NKA per nanocluster. It may on one hand give an overestimate if the antibodies physically increase the size of the imaged fluorescence cluster. On the other hand if quantification is based on the fluorescence intensity it may give an underestimate.

Bottom Line: Despite this, there is as yet little known about the isoform specific distribution in neurons.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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Applied Physics, Royal Institute of Technology, Stockholm, Sweden.

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.

Show MeSH