Automated high-throughput high-content autophagy and mitophagy analysis platform

Jonathan Arias-Fuenzalida,Jonas Walter,Paul M A Antony,Javier Jarazo,Rejko Krueger,Julia I Forster,Gemma Gomez-Giro,Jens C Schwamborn

doi:10.1038/s41598-019-45917-2

Abstract

Autophagic processes play a central role in cellular homeostasis. In pathological conditions, the flow of autophagy can be affected at multiple and distinct steps of the pathway. Current analyses tools do not deliver the required detail for dissecting pathway intermediates. The development of new tools to analyze autophagic processes qualitatively and quantitatively in a more straightforward manner is required. Defining all autophagy pathway intermediates in a high-throughput manner is technologically challenging and has not been addressed yet. Here, we overcome those requirements and limitations by the developed of stable autophagy and mitophagy reporter-iPSC and the establishment of a novel high-throughput phenotyping platform utilizing automated high-content image analysis to assess autophagy and mitophagy pathway intermediates.

Highlights

Autophagy and mitophagy play central roles in normal development and disease[1,2]
The mitochondrial sensor ATP5C1-Rosella allows the quantification of the rate of mitophagy events (Fig. 1B,D,F) as accounted by acidic DsRedpospHluorinneg vesicles derived from degraded mitochondria
We identified and categorized the subcellular structures observed during autophagy (Fig. 1G)

Summary

Introduction

Autophagy and mitophagy play central roles in normal development and disease[1,2]. Increasing interest and research in the field point out the need of pathway reconstitution tools and reliable quantification methods of autophagy and mitophagy[3,4]. Our approach relies on the stable expression of the Rosella reporter, which is integrated into the AAVS1 locus[13] using TALE nucleases guided targeting It represents an additional step in the technical improvement of the Rosella system and enables a high level of detail in the analysis. Automated tools allow applying classification algorithms to specimens in an unbiased manner, and provide high statistical power Those tools provide an insight to the flow of these pathways that would otherwise be missed in population-based analyses. We present such automated tools to assess autophagy and mitophagy intermediates

Methods

Results

Conclusion