A genetically-encoded fluorescence-based reporter to spatiotemporally investigate mannose-6-phosphate pathway.

IF 3.1 3区 生物学 Q3 CELL BIOLOGY
Molecular Biology of the Cell Pub Date : 2024-08-01 Epub Date: 2024-06-18 DOI:10.1091/mbc.E23-09-0344
Mallika Bhat, Akshaya Nambiar, Lakshmi Edakkandiyil, Irine Maria Abraham, Ritoprova Sen, Mamta Negi, Ravi Manjithaya
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引用次数: 0

Abstract

Maintenance of a pool of active lysosomes with acidic pH and degradative hydrolases is crucial for cell health. Abnormalities in lysosomal function are closely linked to diseases, such as lysosomal storage disorders, neurodegeneration, intracellular infections, and cancer among others. Emerging body of research suggests the malfunction of lysosomal hydrolase trafficking pathway to be a common denominator of several disease pathologies. However, available conventional tools to assess lysosomal hydrolase trafficking are insufficient and fail to provide a comprehensive picture about the trafficking flux and location of lysosomal hydrolases. To address some of the shortcomings, we designed a genetically-encoded fluorescent reporter containing a lysosomal hydrolase tandemly tagged with pH sensitive and insensitive fluorescent proteins, which can spatiotemporally trace the trafficking of lysosomal hydrolases. As a proof of principle, we demonstrate that the reporter can detect perturbations in hydrolase trafficking, that are induced by pharmacological manipulations and pathophysiological conditions like intracellular protein aggregates. This reporter can effectively serve as a probe for mapping the mechanistic intricacies of hydrolase trafficking pathway in health and disease and is a utilitarian tool to identify genetic and pharmacological modulators of this pathway, with potential therapeutic implications.

基于基因编码的荧光报告器,用于对 6-磷酸甘露糖途径进行时空研究。
维持具有酸性 pH 值和降解水解酶的活性溶酶体池对细胞健康至关重要。溶酶体功能异常与疾病密切相关,如溶酶体贮积症(LSD)、神经变性、细胞内感染和癌症等。新近的研究表明,溶酶体水解酶贩运途径的功能失常是多种疾病病理的共同点。然而,用于评估溶酶体水解酶贩运的现有传统工具并不充分,无法全面反映溶酶体水解酶的贩运通量和位置。为了弥补上述不足,我们设计了一种基因编码的荧光报告物,它含有一种溶酶体水解酶,并串联了 pH 敏感和不敏感的荧光蛋白。作为原理验证,我们证明了该报告物可以检测水解酶迁移的扰动,这种扰动是由药理操作和病理生理条件(如细胞内蛋白质聚集)诱发的。这种报告器可以有效地作为一种探针,用于绘制健康和疾病中水解酶贩运途径的复杂机理图,也是一种实用的工具,可用于鉴定该途径的遗传和药理调节剂,具有潜在的治疗意义。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
Molecular Biology of the Cell
Molecular Biology of the Cell 生物-细胞生物学
CiteScore
6.00
自引率
6.10%
发文量
402
审稿时长
2 months
期刊介绍: MBoC publishes research articles that present conceptual advances of broad interest and significance within all areas of cell, molecular, and developmental biology. We welcome manuscripts that describe advances with applications across topics including but not limited to: cell growth and division; nuclear and cytoskeletal processes; membrane trafficking and autophagy; organelle biology; quantitative cell biology; physical cell biology and mechanobiology; cell signaling; stem cell biology and development; cancer biology; cellular immunology and microbial pathogenesis; cellular neurobiology; prokaryotic cell biology; and cell biology of disease.
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