Biology Reference
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if multiple components are located at distances
300 nm. As such, NSOM
is capable of bridging the gap between 10 and 300 nm, providing valuable
information at these important spatial scales.
NSOM in dry conditions has also been used to spatially relate topographical
features to two different lipid species.
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Both GM1 and GM3 were seen to
cluster in 40-360 nm domains that distributed randomly on the plasma
membrane of epithelial cells. However, upon closer examination, it appeared
that the GM3 clusters were localized on the peaks of microvillus-like
structures.
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In contrast, the majority of the GM1 lipid clusters were found
in the valleys or slopes of these topographical protrusions.
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These results
highlight the importance of correlating topography and optical information
uniquely afforded by NSOM. Along these lines, it is worthy to mention that
several groups have also implemented AFM in combination with confocal
microscopy to correlate topography with luorescence information, albeit
at lower optical resolution (diffraction-limited). On the other hand, a
combination of AFM and confocal FCS can also provide complementary
information on the dynamics of different nano-environments on membranes
and correlate it with topographic information as afforded by AFM.
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More recently, our group has applied dual-colour NSOM in physiological
conditions together with detailed statistical analysis to follow the spatial
nano-scale organization of the integrin receptor LFA-1 and its association
with membrane “rafts” along different stages of integrin activation.
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Rafts
have been implicated in regulation of integrin-mediated cell adhesion,
although the underlying mechanism has remained elusive. We used single-
molecule NSOM with localization accuracy of ~3 nm, to capture the spatio-
functional relationship between the integrin LFA-1 and raft components (GPI-
APs) on immune cells. Our experiments showed that in resting cells, LFA-1
organizes in small nanoclusters of about 80 nm, while GPI-APs organized
largely as monomers. Interestingly, a 20% subpopulation of GPI-APs formed
small oligomers on the cell surface and concentrated in regions smaller than
250 nm, suggesting a hierarchical pre-arrangement of GPI-APs on resting
monocytes. Dual-colour NSOM demonstrated that integrin nanoclusters
are spatially different but reside proximal to GPI-AP nanodomains, forming
resulted in an interconversion from monomers to nanodomains of GPI-APs
and the generation of nascent adhesion sites where integrin and GPI-APs
colocalized at the nanoscale. Cholesterol depletion signiicantly affected the
reciprocal distribution pattern of LFA-1 and GPI-APs in the resting state, and
LFA-1 adhesion to its ligand. As such, our data demonstrated the existence
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