OrgaMapper: a robust and easy-to-use workflow for analyzing organelle positioning

Area of Science:

• Cell Biology
• Microscopy
• Image Analysis

Background:

• Eukaryotic cells utilize organelles for specific functions, with their precise positioning being crucial for cellular processes.
• Organelle positioning is vital for function, as seen with lysosomes and the nucleus, and alterations are linked to disease.
• Understanding the molecular mechanisms of organelle localization is limited, partly due to challenges in accurately measuring positioning phenotypes.

Purpose of the Study:

• To develop and present a robust, quantitative, and user-friendly image analysis workflow for assessing organelle positioning.
• To provide tools that facilitate the identification of molecular components regulating organelle localization.

Main Methods:

• Developed a Fiji plugin and an R Shiny App for automated image analysis.
• Workflow includes segmentation of cells, nuclei, and organelles; measurement of cell size and organelle-nucleus distance; intensity measurements; and radial profile analysis.
• Validated using simulated data and immunofluorescence images of perturbed lysosome positioning.

Main Results:

• The workflow accurately and robustly measures organelle positioning phenotypes.
• It is resilient to common confounding factors like changes in cell/organelle size and background intensity.
• Demonstrated utility in analyzing known factors affecting lysosome positioning.

Conclusions:

• OrgaMapper is a versatile, robust, and easy-to-use automated workflow for microscopy-based research.
• It enables quantitative mapping of intracellular space and discovery of novel organelle positioning regulators.