Abstract
Cortical actin plays a key role in cell movement and division, but has also been implicated in the organisation of cell surface receptors such as G protein-coupled receptors. The actin mesh proximal to the inner membrane forms small fenced regions, or ‘corrals’, in which receptors can be constrained. Quantification of the actin mesh at the nanoscale has largely been attempted in single molecule datasets and electron micrographs. This work describes the development and validation of workflows for analysis of super resolved fixed cortical actin images obtained by Super Resolved Radial Fluctuations (SRRF), Structured Illumination Microscopy (3D-SIM) and Expansion Microscopy (ExM). SRRF analysis was used to show a significant increase in corral area when treating cells with the actin disrupting agent cytochalasin D (increase of 0.31 µm2 ± 0.04 SEM), and ExM analysis allowed for the quantitation of actin filament densities. Thus, this work allows complex actin networks to be quantified from super-resolved images and is amenable to both fixed and live cell imaging.
Highlights
Cortical actin plays a key role in cell movement and division, but has been implicated in the organisation of cell surface receptors such as G protein-coupled receptors
Fourier Ring Correlation (FRC) measurements show a significant increase in resolution over standard TIRF images, as previously reported[17,18]
In this study we have developed and tested the application of two workflows for quantifying cortical actin organisation in super-resolved microscopy images
Summary
Cortical actin plays a key role in cell movement and division, but has been implicated in the organisation of cell surface receptors such as G protein-coupled receptors. Super resolution microscopy studies have indicated that cortical actin can lie between < 10 nm and a maximum of 20 nm from the m embrane[2] This close association between actin and the plasma membrane are in agreement with the picket fence model proposed by Fujiwara et al.[3]. Single molecule localisation microscopy (SMLM) techniques can resolve structures down to ~ 20 nm, almost rivalling EM, and capable of resolving individual actin filaments in the dense mesh of the cortical actin[11]. By focussing on the empty space the actin structure creates in SRRF and SIM images, as opposed to the filaments themselves, the work described here sets up analysis workflows that can be used to assess the cortical actin mesh in fixed cell super resolved images, as well as quantifying the response of the actin network to disruption
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