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Single‐molecule localization microscopy of septin bundles in mammalian cells
Author(s) -
Vissa Adriano,
Giuliani Maximiliano,
Froese Carol D.,
Kim Moshe S.,
Soroor Forooz,
Kim Peter K.,
Trimble William S.,
Yip Christopher M.
Publication year - 2019
Publication title -
cytoskeleton
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.95
H-Index - 86
eISSN - 1949-3592
pISSN - 1949-3584
DOI - 10.1002/cm.21481
Subject(s) - septin , biology , cytoskeleton , microbiology and biotechnology , actin , cytokinesis , fluorescence microscope , actin cytoskeleton , cell division , biophysics , cell cortex , cell , biochemistry , optics , fluorescence , physics
Septins are a conserved family of GTPases that associate with numerous components of the cytoskeleton and the inner leaflet of the plasma membrane. These proteins are involved in many biological processes, including cell division and membrane trafficking, and serving as a scaffolding component of the cytoskeleton used to recruit other proteins and form diffusion barriers to maintain the composition of membrane domains. In order to carry out their cellular functions, septins undergo interactions via their NC or G interfaces to form heteromeric rod‐like structures that can polymerize into filaments and associate laterally into bundles. While electron microscopy studies of affinity‐tagged and purified S accharomyces cerevisiae septin complexes have provided evidence for this periodic organization and in‐registry lateral bundling in vitro, the in‐vivo arrangement of stress fiber‐associated septin bundles in mammalian cells remains poorly characterized. We report here on a direct stochastic optical reconstruction microscopy and photoactivated localization microscopy study of the 2D spatial distribution of septins in mammalian cells. From simulated and experimental results, we show the effects of labeling method, labeling efficiency, and fluorescent emitter photophysics on image reconstruction and interpretation. Our experimental results are consistent with septin organization by polymerization of hetero‐octamers and an approximate 30–35 nm periodicity between subsequent units of SEPT2–SEPT2 or SEPT9–SEPT9.