Automated quantification of protein periodic nanostructures in fluorescence nanoscopy images: abundance and regularity of neuronal spectrin membrane-associated skeleton

Abstract

Fluorescence nanoscopy imaging permits the observation of periodic supramolecular protein structures in their natural environment, as well as the unveiling of previously unknown protein periodic structures. Deciphering the biological functions of such protein nanostructures requires systematic and quantitative analysis of large number of images under different experimental conditions and specific stimuli. Here we present a method and an open source software for the automated quantification of protein periodic structures in super-resolved images. Its performance is demonstrated by analyzing the abundance and regularity of the spectrin membrane-associated periodic skeleton (MPS) in hippocampal neurons of 2 to 40 days in vitro, imaged by STED and STORM nanoscopy. The automated analysis reveals that both the abundance and the regularity of the MPS increase over time and reach maximum plateau values after 14 DIV. A detailed analysis of the distributions of correlation coefficients provides indication of dynamical assembly and disassembly of the MPS.

Quantitative expansion microscopy for the characterization of the spectrin periodic skeleton of axons using fluorescence microscopy

“…4a). The regularity of the MPS along axons was determined using Gollum 22 , an open-source image-analysis software recently developed by our group for quantitatively assessing the regularity of periodic structures. The algorithm calculates Pearson's correlation of axonal segments against a predefined modeled MPS (Fig.…”

“…Images were obtained at a single plane, and with the pinhole open to ~1 Airy unit (more details on STED acquisitions using this instrument can be found in the supplier´s website). 2) A custom-built nanoscope at the Center for Bionanoscience Research (CIBION, CONICET) for which a detailed description was published before 22 . Briefly, samples were excited with a linearly polarized pulsed (200 ps) laser at 640 nm (PicoQuant LDH-P-C-640B) operating at 40 MHz repetition and depleted with a linearly polarized pulsed (1 ns) laser at 775 nm (Onefive Katana HP).…”

“…MpS evaluation: Assessment of organization and abundance. Regions of interest were divided in 1 μm × 1 μm sub-regions and analyzed using the Gollum software 22 , which is accessible at https://github.com/ cibion-conicet/Gollum. The correlation value obtained corresponds to a Pearson's correlation between the subregion and a modeled MPS period and is indicative of how preserved the periodic organization of the MPS is in that 1 μm section.…”

Clinical-pathological study on β-APP, IL-1β, GFAP, NFL, Spectrin II, 8OHdG, TUNEL, miR-21, miR-16, miR-92 expressions to verify DAI-diagnosis, grade and prognosis

“…Spectrin plays important roles in nervous cells, it exhibited periodic structures alternating with those of actin and adducin, and the distance between adjacent actin-adducin rings was comparable to the length of a spectrin tetramer 56 , 57 .…”

The functional architecture of axonal actin

“…Actin and spectrin thus form a membrane-associated periodic scaffold (MPS) with actin rings connected by head-to-head tetramers of spectrin that bind actin on each side via the aminoterminus of the ß-spectrin subunits (Rasband, 2013;Xu et al, 2013). This periodic scaffold has been observed in living neurons (D'Este et al, 2015;Zhong et al, 2014) and detected in various neuronal types and organisms, including worm, fly, rodent and human (Barabas et al, 2017;D'Este et al, 2016;He et al, 2016). Interestingly, a mono-dimensional periodic scaffold is also detected along a minority of dendritic segments, along dendritic spine necks (Bär et al, 2016;D'Este et al, 2015;He et al, 2016), and within fine processes of oligodendrocytes (D'Este et al, 2016).…”