Single‐molecule localization microscopy of septin bundles in mammalian cells

Abstract

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 Saccharomyces 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.

Human septins in cells organize as octamer-based filaments mediating actin-membrane anchoring

“…Septins at the budding yeast neck organize in arrays made of single and paired filaments (Bertin et al, 2012; Ong et al, 2014); bundles of more than two filaments were not observed. However, single-molecule localization microscopy of SF-associated septins in U2OS cells, i.e., in the same system we use in our study, reported that septin bundles may comprise as few as 25 to as many as 150 filaments (Vissa et al, 2019). However, we note that this study assumed septin GTP-binding domains associating laterally without any spacing, without considering the ∼5-20 nm spacing occupied by coiled-coil pairing used for bundling (Leonardo et al, 2021), so the number of filaments could be substantially overestimated.…”

Human septins organize as octamer-based filaments and mediate actin-membrane anchoring in cells

“…Our findings suggest that actin-associated septin filaments in mammalian cells organize as paired septin filaments, with thicker septin fibers consisting of two to three double septin filaments. One single-molecule localization microscopy study of SF-associated septins has reported septin bundles with 25–150 filaments ( Vissa et al, 2019 ), but this study assumed septin GTP-binding domains associating laterally without considering the presence of coiled-coils and the spacing occupied by coiled-coil pairing ( Leonardo et al, 2021 ), so the number of filaments could be overestimated. We thus hypothesize that human septins in cells, like budding yeast septins, organize in a rather narrow range of assembly geometries.…”

A homozygous genome-edited Sept2-EGFP fibroblast cell line

“…Taken together, we conclude that the cell line presented here will be a useful tool for septin biology and due to the homogenous, endogenous expression level will simplify the quantitative analysis of phenotypes observed in microscopy experiments. Especially in connection with single molecule localization based superresolution microscopy methods, this cell line will contribute to the advancement of our understanding of septin organization in cells (Kaplan et al, 2015; Ries et al, 2012; Vissa et al, 2018). More generally, it will simplify quantitative microscopy and microscopy-based screening and help with biochemical and proteomic approaches based on protein purification via the GFP tag (Trinkle-Mulcahy et al, 2008).…”