Superresolution microscopy reveals distinct localisation of full length IRSp53 and its I-BAR domain protein within filopodia

Scientific Reports
Thankiah SudhaharanSohail Ahmed

Abstract

Superresolution microscopy offers the advantage of imaging biological structures within cells at the nano-scale. Here we apply two superresolution microscopy techniques, specifically 3D structured illumination microscopy (3D-SIM) and direct stochastic optical reconstruction microscopy (dSTORM), a type of single molecule localisation microscopy, to localise IRSp53 protein and its I-BAR domain in relation to F-actin within filopodia. IRSp53 generates dynamic (extending and retracting) filopodia 300 nm wide with a distinct gap between IRSp53 and F-actin. By contrast, protrusions induced by the I-BAR domain alone are non-dynamic measuring between 100-200 nm in width and exhibit a comparatively closer localisation of the I-BAR domain with the F-actin. The data suggest that IRSp53 membrane localisation is spatially segregated to the lateral edges of filopodia, in contrast to the I-BAR domain is uniformly distributed throughout the membranes of protrusions. Modeling of fluorescence recovery after photobleaching (FRAP) data suggests that a greater proportion of I-BAR domain is associated with membranes when compared to full length IRSp53. The significance of this new data relates to the role filopodia play in cell migration and its imp...Continue Reading

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Citations

Jun 25, 2020·Molecular Biology of the Cell·Karen W Cheng, R Dyche Mullins
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Jun 8, 2021·The Journal of Cell Biology·Anne PipathsoukOrion D Weiner

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Datasets Mentioned

BETA
BC015619

Methods Mentioned

BETA
superresolution microscopy
GTPases
confocal microscopy
chip

Software Mentioned

FIJI
MATLAB
QuickPALM
SIM
MetaMorph
3D
ThunderSTORM
Bitplane Imaris
OMX
dSTORM

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