Quantitative analysis of subcellular distributions with an open-source, object-based tool

Abstract

The subcellular localization of objects, such as organelles, proteins, or other molecules, instructs cellular form and function. Understanding the underlying spatial relationships between objects through colocalization analysis of microscopy images is a fundamental approach used to inform biological mechanisms. We generated an automated and customizable computational tool, the SubcellularDistribution pipeline, to facilitate object-based image analysis from 3D fluorescence microcopy images. To test the utility of the SubcellularDistribution pipeline, we examined the subcellular distribution of mRNA relative to centrosomes within syncytial Drosophila embryos. Centrosomes are microtubule-organizing centers, and RNA enrichments at centrosomes are of emerging importance. Our open-source and freely available software detected RNA distributions comparably to commercially available image analysis software. The SubcellularDistribution pipeline is designed to guide the user through the complete process of preparing image analysis data for publication, from image segmentation and data processing to visualization.

c entrocortin RNA localization to centrosomes is regulated by FMRP and facilitates error-free mitosis

“…We selected 10 and 4 µm as the upper boundary for the pseudocell radius for NC 10 and NC 13, respectively, based on measuring the centrosome-to-centrosome distances from a set of representative images. A detailed protocol for RNA analysis was recently described ( Ryder and Lerit, 2020 ).…”

Orb-dependent polyadenylation contributes to PLP expression and centrosome scaffold assembly

“…To quantitatively compare the distributions of plp versus gapdh mRNAs, we measured the percent of mRNA residing within 1 μm from the centrosome surface using a custom Python package (Ryder et al, 2020; Ryder and Lerit, 2020). From this analysis, we observed significantly more plp mRNA localized to centrosomes relative to gapdh mRNA (2∼3-fold more plp mRNA than gapdh mRNA at interphase versus 1.5∼2-fold more at metaphase centrosomes; Figure 1C, Table S1 ).…”

“…Stellaris plp and gapdh smFISH probes conjugated to Quasar 570 dye (LGC Biosearch Technologies; Middlesex, UK) were designed against the coding region for each gene using the Stellaris RNA FISH probe designer (Ryder et al, 2020; Ryder and Lerit, 2020). smFISH probes were dissolved in nuclease-free water at 25 μM and stored at -20°C before use.…”

“…smFISH experiments were performed as previously described (Ryder et al, 2020; Ryder and Lerit, 2020). All the following steps were performed with RNase-free solutions.…”

“…smFISH signals were detected and single molecule normalization was performed as recently described (Ryder et al, 2020; Ryder and Lerit, 2020). Briefly, single-channel .tif raw images were segmented in three dimensions using Python scripts adapted from the Allen Institute for Cell Science Cell Segmenter (Chen et al, 2018).…”