Scheffler Julia M, Schiefermeier Natalia, Huber Lukas A
Biocenter, Division of Cell Biology, Innsbruck Medical University, Innsbruck, Austria.
Biocenter, Division of Cell Biology, Innsbruck Medical University, Innsbruck, Austria.
Methods Enzymol. 2014;535:93-102. doi: 10.1016/B978-0-12-397925-4.00006-7.
Intracellular membrane trafficking is a highly dynamic process to sort proteins into either the recycling or degradation pathway. The late endosome is a major component of this endosomal biogenesis toward degradation by the lysosome. The endocytotic system is spread throughout the cytoplasm, and vesicle motility is achieved by multiple proteins including Rabs, motor proteins, and cytostructural elements. The subcellular localization of the late endosome is distributed from the accumulation in the perinuclear region toward the cell periphery. Using immunofluorescence methods combined with live-cell microscopy, we want to show that the preservation of the peripheral late endosomal compartment can be successfully achieved by two different techniques. On one hand, we compare two different widely used permeabilization methods: Triton X-100 and saponin. Comparing live-cell microscopic pictures of the same cell with immunofluorescences after fixation and permeabilization revealed improved results by the use of saponin. On the other hand, we present here a protocol of mild fixation to preserve peripheral structures like focal adhesion in combination with endosomes and actin filaments.
细胞内膜运输是一个高度动态的过程，用于将蛋白质分选到再循环或降解途径中。晚期内体是这种内体生物发生向溶酶体降解转变的主要组成部分。内吞系统遍布整个细胞质，囊泡运动由包括Rabs、运动蛋白和细胞结构元件在内的多种蛋白质实现。晚期内体的亚细胞定位从核周区域的积累分布到细胞周边。使用免疫荧光方法结合活细胞显微镜，我们想要表明通过两种不同的技术可以成功实现外周晚期内体区室的保存。一方面，我们比较两种广泛使用的通透化方法：Triton X-100和皂素。比较同一细胞在固定和通透化后的免疫荧光活细胞显微镜图像，结果显示使用皂素能得到更好的结果。另一方面，我们在此提出一种温和固定方案，以保存外周结构，如与内体和肌动蛋白丝结合的粘着斑。
