Monitoring the autophagy-endolysosomal system using monomeric Keima-fused MAP1LC3B.

Hideki Hayashi,Yumi Iida,Ayumi Sasaki,Ting Wang,Shinji Hadano,Nahoko Fukunishi,Chisa Okada,Shunji Amano,Kazuhiro Yoshida,Masatoshi Ito,Ayaka Nakamura,Sanae Ogiwara,Masayuki Tanaka,Masato Ohtsuka,Asako Otomo,Vladimir Trajkovic

doi:10.1371/journal.pone.0234180

Abstract

The autophagy-endolysosomal pathway is an evolutionally conserved degradation system that is tightly linked to a wide variety of physiological processes. Dysfunction of this system is associated with many pathological conditions such as cancer, inflammation and neurodegenerative diseases. Therefore, monitoring the cellular autophagy-endolysosomal activity is crucial for studies on the pathogenesis as well as therapeutics of such disorders. To this end, we here sought to create a novel means exploiting Keima, an acid-stable fluorescent protein possessing pH-dependent fluorescence excitation spectra, for precisely monitoring the autophagy-endolysosomal system. First, we generated three lines of transgenic (tg) mouse expressing monomeric Keima-fused MAP1LC3B (mKeima-LC3B). Then, these tg mice were subjected to starvation by food-restriction, and also challenged to neurodegeneration by genetically crossing with a mouse model of amyotrophic lateral sclerosis; i.e., SOD1H46R transgenic mouse. Unexpectedly, despite that a lipidated-form of endogenous LC3 (LC3-II) was significantly increased, those of mKeima-LC3B (mKeima-LC3B-II) were not changed under both stressed conditions. It was also noted that mKeima-LC3B-positive aggregates were progressively accumulated in the spinal cord of SOD1H46R;mKeima-LC3B double-tg mice, suggestive of acid-resistance and aggregate-prone natures of long-term overexpressed mKeima-LC3B in vivo. Next, we characterized mouse embryonic fibroblasts (MEFs) derived from mKeima-LC3B-tg mice. In contrast with in vivo, levels of mKeima-LC3B-I were decreased under starved conditions. Furthermore, when starved MEFs were treated with chloroquine (CQ), the abundance of mKeima-LC3B-II was significantly increased. Remarkably, when cultured medium was repeatedly changed between DMEM (nutrient-rich) and EBSS (starvation), acidic/neutral signal ratios of mKeima-LC3B-positive compartments were rapidly and reversibly shifted, which were suppressed by the CQ treatment, indicating that intraluminal pH of mKeima-LC3B-positive vesicles was changeable upon nutritional conditions of culture media. Taken together, although mKeima-LC3B-tg mice may not be an appropriate tool to monitor the autophagy-endolysosomal system in vivo, mKeima-LC3B must be one of the most sensitive reporter molecules for monitoring this system under in vitro cultured conditions.

Highlights

The autophagy-endolysosomal pathway is an evolutionally conserved degradation system that is tightly linked to a wide variety of physiological processes [1, 2]
light chain 3 (LC3) tagged with a green fluorescent protein, GFP-fused human-MAP1LC3B cDNA (GFP-LC3) [9], has widely been utilized in in vitro culture experiments and in vivo animal studies
GFP-LC3 fusion protein was ubiquitously and highly expressed in GFP-LC3-tg, mKeimaLC3B transgenic (mKeima)-length human MAP1LC3B cDNA (LC3B) was rather preferentially expressed in the central nervous system (CNS), hearts and muscles, but not in other peripheral tissues of mKeima-LC3B-tg mice, with highest in KLC3_44 line (Fig 1A)

Summary

Introduction

The autophagy-endolysosomal pathway is an evolutionally conserved degradation system that is tightly linked to a wide variety of physiological processes [1, 2]. Three different forms of the autophagic pathways; i.e., macroautophagy, microautophagy, and chaperon-mediated autophagy are currently documented. Macroautophagy (hereafter referred to as “autophagy”) together with the endolysosomal pathway plays a crucial role in the removal and degradation of cytoplasmic long-lived as well as misfolded proteins and of damaged or superfluous organelles through a sequential step comprising the autophagosome formation, maturation (fused with endosomes and/or lysosomes) and degradation within autolysosomes/lysosomes [1]. Microtubule-associated protein 1 light chain 3 (LC3), whose lipidated form (LC3-II) is highly enriched onto autophagosomal membrane during entire stages of autophagy; from the formation to maturation of autophagosomes, has reliably been used for such purposes [7, 8]. We and others have previously reported that GFP-LC3 can be used to monitor the autophagic status as well as disease progression in a GFP-LC3-expressing animal model of amyotrophic lateral sclerosis (ALS); i.e., human mutant SOD1-expressing transgenic mice [10, 11]

Methods

Results

Conclusion