Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, 30332, USA.
Integr Biol (Camb). 2010 Oct;2(10):536-44. doi: 10.1039/c0ib00035c. Epub 2010 Sep 20.
The intracellular vesicle-mediated degradation of extracellular cargo is an essential cellular function. Using two-color single particle tracking fluorescence microscopy, we have probed the intracellular degradation of low-density lipoprotein (LDL) in living cells. To detect degradation, individual LDL particles were heavily labeled with multiple fluorophores resulting in a quenched fluorescent signal. The degradation of the LDL particle then resulted in an increase in fluorescence. Endocytic vesicles were fluorescently labeled with variants of GFP. We imaged the transient colocalization of LDL with endocytic vesicles while simultaneously measuring the intensity of the LDL particle as an indicator of degradation. These studies demonstrate that late endosomes are active sites of degradation for LDL. Measurement of the time from colocalization with lysosome-associated membrane protein 1 (LAMP1) vesicles to degradation suggests that LAMP1-vesicles initiate the degradative event. Observing degradation as it occurs in living cells makes it possible to describe the complete endocytic pathway of LDL from internalization to degradation. More generally, this research provides a model for the intracellular degradation of extracellular cargo and a method for its study in living cells.
细胞内囊泡介导的细胞外货物降解是一种基本的细胞功能。我们使用双色单颗粒跟踪荧光显微镜技术，研究了活细胞中低密度脂蛋白（LDL）的细胞内降解。为了检测降解，我们使用多个荧光团对单个 LDL 颗粒进行了重度标记，从而导致荧光信号猝灭。LDL 颗粒的降解随后导致荧光增加。内吞小泡用 GFP 的变体进行荧光标记。我们在同时测量 LDL 颗粒的强度作为降解指标的情况下，对 LDL 与内吞小泡的瞬时共定位进行成像。这些研究表明，晚期内体是 LDL 降解的活性部位。从与溶酶体相关膜蛋白 1（LAMP1）囊泡的共定位到降解的时间测量表明，LAMP1 囊泡启动了降解事件。在活细胞中观察降解使得描述 LDL 从内化到降解的完整内吞途径成为可能。更一般地说，这项研究为细胞外货物的细胞内降解提供了模型，并为在活细胞中研究它提供了一种方法。
