用于对酵母中快速细胞反应进行成像的4D实时荧光显微镜技术方案

Protocol for 4D live fluorescence microscopy to image rapid cellular responses in yeast.

作者信息

Alvarado-Cartagena Yaneris M, Huppenbauer Christopher, Segev Nava

机构信息

Department of Biochemistry and Molecular Genetics, College of Medicine, University of Illinois Chicago, Chicago, IL, USA.

Advanced Imaging and Research Applications W. Nuhsbaum, Inc., McHenry, IL, USA.

出版信息

STAR Protoc. 2025 Aug 13;6(3):104026. doi: 10.1016/j.xpro.2025.104026.

DOI:10.1016/j.xpro.2025.104026

PMID:40815568

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC12365329/

Abstract

Micro-autophagy is thought to occur through cargo engulfment by the lysosomal membrane based on electron microscopy of fixed cells. We present a protocol that follows micro-ER (endoplasmic reticulum)-phagy in live yeast cells that can be used for following other rapid cellular processes. We describe steps for inducing the accumulation of misfolded proteins and their selective shuttling for degradation during nutritional stress. We then detail procedures for live-cell time-lapse confocal microscopy and 3D reconstruction showing individual events of cargo engulfment and internalization into the lysosome that occur within seconds. For complete details on the use and execution of this protocol, please refer to Gyurkovska et al..

摘要

基于固定细胞的电子显微镜观察，微自噬被认为是通过溶酶体膜对货物的吞噬作用而发生的。我们提供了一种在活酵母细胞中追踪微内质网自噬的方案，该方案可用于追踪其他快速的细胞过程。我们描述了在营养应激期间诱导错误折叠蛋白积累及其选择性转运以进行降解的步骤。然后，我们详细介绍了活细胞延时共聚焦显微镜和三维重建的程序，展示了在几秒钟内发生的货物被吞噬并内化到溶酶体中的个别事件。有关该方案的使用和执行的完整详细信息，请参考久尔科夫斯卡等人的研究。