4 March 2014 Using dSTORM to probe the molecular architecture of filopodia
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Abstract
IRSp53 is a Cdc42 effector and a member of the Inverse-Bin-Amphiphysins-Rvs (I-BAR) domain family which can induce negative membrane curvature. IRSp53 generates filopodia by coupling membrane protrusion (I-BAR domain) with actin dynamics through its SH3 domain binding partners. Dynamin 1 (Dyn1), a large GTPase associated with endocytosis, is a novel interacting partner of IRSp53 that localises to filopodia. Using rapid time-lapse TIRF microscopy we have shown that Dyn1 localized to a subcellular region just behind Mena at the leading edge, or in filopodial tip complexes when co-expressed with IRSp53. Dyn1-GFP was strongly localized in the filopodial shaft during the early phase of elongation, after which it moved rearward, suggestive of a role in early filopodia assembly. Mena and Eps8, accumulate at the tip complex in sequence and are involved in filopodial extension and retraction, respectively (Chou at al, 2014 submitted). Here we describe the use of dSTORM to investigate the molecular architecture of filopodia and in particular the size of the F-actin bundle in these structures. The data suggest that direct Stochastic Optical Reconstruction Microscopy (dSTORM) in combination with other techniques will allow the molecular architecture of
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Sohail Ahmed, Sohail Ahmed, Amy Chou, Amy Chou, K. P. Sem, K. P. Sem, Sudaharan Thankiah, Sudaharan Thankiah, Graham Wright, Graham Wright, John Lim, John Lim, Srivats Hariharan, Srivats Hariharan, } "Using dSTORM to probe the molecular architecture of filopodia", Proc. SPIE 8950, Single Molecule Spectroscopy and Superresolution Imaging VII, 89500Y (4 March 2014); doi: 10.1117/12.2058123; https://doi.org/10.1117/12.2058123
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