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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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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
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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

Mentions: The localization of α3 NKA subunits in dendritic spines was then studied in cultured neurons, derived from E18.5 day rat striatum (three separate cultures from three embryos of different litters were used). Due to the small dimensions of spines and the diffraction limit of light, it would not be possible to resolve the distribution of a protein within a single spine with classical fluorescence microscopy techniques. To overcome this inherent problem of limited resolution, we used the diffraction unlimited STED microscopy technique [12]. This superresolution technique shrinks a fluorescently activated spot by depleting the fluorescent state in a doughnut-shaped STED area, superimposed onto the excitation (cf. Figure 2). With sufficiently intense powers, the imaged fluorescent spot in a STED microscope can basically be scaled down to molecular sizes. By scanning the subdiffraction spot across the sample, in the same manner as in conventional confocal microscopy, fluorescence images with nanoscale resolution can thus be generated.


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)

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

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

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
Mentions: The localization of α3 NKA subunits in dendritic spines was then studied in cultured neurons, derived from E18.5 day rat striatum (three separate cultures from three embryos of different litters were used). Due to the small dimensions of spines and the diffraction limit of light, it would not be possible to resolve the distribution of a protein within a single spine with classical fluorescence microscopy techniques. To overcome this inherent problem of limited resolution, we used the diffraction unlimited STED microscopy technique [12]. This superresolution technique shrinks a fluorescently activated spot by depleting the fluorescent state in a doughnut-shaped STED area, superimposed onto the excitation (cf. Figure 2). With sufficiently intense powers, the imaged fluorescent spot in a STED microscope can basically be scaled down to molecular sizes. By scanning the subdiffraction spot across the sample, in the same manner as in conventional confocal microscopy, fluorescence images with nanoscale resolution can thus be generated.

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
Related in: MedlinePlus