DOI: 10.6084/m9.figshare.26984663
title: Mito-kaede photoactivation and chase experiment for mitophagy: optimizing flux measurement via fluid exchange system

description: Modulating autophagy and mitophagy, vital cellular quality control systems, offer therapeutic potential for critical illnesses. However, limited drug screening options hinder progress. We present a novel assay using the photoswitchable fluorescent reporter, mito-Kaede, to quantify mitophagy flux. Mito-Kaede's superior UV-induced photoconversion and brightness post-conversion make it ideal for prolonged mitochondrial dynamics tracking. Its specificity in responding to mitophagy, confirmed by parkin-knockout cells, adds value. When coupled with a custom fluid exchange system, enabling efficient medium changes, precise mitophagy observations become feasible. This mitophagy assay, alongside our methodological insights, can decipher mitophagy's role in pathology and supports drug screening efforts. Our method introduces a novel systematic approach for chronologically tracking the fluorescent decay of a photoactivatable fluorescent protein, mito-Kaede. This is combined with a fluid-exchange method to enable fixed-point observations before and after mitophagy stimulation. Our body has a built-in quality control system called autophagy and mitophagy that helps maintain cellular health. Scientists are exploring ways to use this system as a potential treatment for many diseases. However, finding the right drugs has been challenging due to a lack of effective screening methods. Researchers have now developed a new test using a special fluorescent protein called Kaede. This test has unique advantages. It allows us to track cellular components more effectively. The use of this test, along with a custom fluid exchange system, provides a simple and quick way to study cellular responses in mitophagy. Mitophagy or the autophagic degradation of mitochondria plays critical roles in maintaining the quality of mitochondria and cellular homeostasis. Simple and versatile methodologies for quantifying autophagy and mitophagy flux are limited. Monitoring mitochondria kinetics was confirmed in the new C2C12 cell line expressing mito-Kaede. The photoactivation profile between mito-Kaede and mito-Dendra2 was compared. A photoconversion and chase assay was conducted to monitor mitophagy under time-lapse microscopy after CCCP stimulation to monitor the mitophagy flux response. The effectiveness of real-time fluid exchange was compared with manual fluid exchange by pipetting in serum ± conditions. Mito-Kaede showed more efficient photoconversion than mito-Dendra2. Chronological mitophagy flux quantification was successful, confirming its value as a new mitophagy assay alongside currently available options. Comparing serum ± conditions revealed that a serum-free environment induces more robust mitophagy. This study demonstrates the advantages of the newly developed device for real-time fluid exchange compared with manual fluid exchange by pipetting. Comparing Mito-Kaede with pH-sensitive mitophagy reporters suggests that the newly developed method offers novel advantages over existing reporters. We have established a novel real-time flux measurement assay using mito-Kaede expressed in C2C12 myocytes by photoactivating and monitoring the alterations in the fluorescent intensity under time-lapse microscopy using our fluid exchange system.