Nickerson Daniel P, Merz Alexey J
Department of Biochemistry, University of Washington, Seattle, WA, 98195-7350, USA.
Department of Physiology and Biophysics, University of Washington, Seattle, WA, 98195-7350, USA.
Traffic. 2015 Dec;16(12):1318-29. doi: 10.1111/tra.12331. Epub 2015 Nov 16.
Endosomes are transportation nodes, mediating selective transport of soluble and transmembrane cargos to and from the Golgi apparatus, plasma membrane and lysosomes. As endosomes mature to become multivesicular bodies (MVBs), Endosomal Sorting Complexes Required for Transport (ESCRTs) selectively incorporate transmembrane cargos into vesicles that bud into the endosome lumen. Luminal vesicles and their cargoes are targeted for destruction when MVBs fuse with lysosomes. Common assays of endosomal luminal targeting, including fluorescence microscopy and monitoring of proteolytic cargo maturation, possess significant limitations. We present a quantitative assay system called LUCID (LUCiferase reporter of Intraluminal Deposition) that monitors exposure of chimeric luciferase-cargo reporters to cytosol. Luciferase-chimera signal increases when sorting to the endosome lumen is disrupted, and silencing of signal from the chimera depends upon luminal delivery of the reporter rather than proteolytic degradation. The system presents several advantages, including rapidity, microscale operation and a high degree of reproducibility that enables detection of subtle phenotypic differences. Luciferase reporters provide linear signal over an extremely broad dynamic range, allowing analysis of reporter traffic even at anemic levels of expression. Furthermore, LUCID reports transport kinetics when applied to inducible trafficking reporters.
内涵体是运输节点，介导可溶性和跨膜货物在高尔基体、质膜和溶酶体之间的选择性运输。当内涵体成熟为多囊泡体（MVBs）时，运输所需的内体分选复合物（ESCRTs）将跨膜货物选择性地整合到向内体腔内出芽的囊泡中。当MVBs与溶酶体融合时，腔内囊泡及其货物会被定向降解。常见的内涵体腔内靶向检测方法，包括荧光显微镜和蛋白水解货物成熟监测，都有显著局限性。我们提出了一种名为LUCID（腔内沉积的荧光素酶报告基因）的定量检测系统，该系统可监测嵌合荧光素酶-货物报告基因暴露于细胞质中的情况。当向内体腔的分选被破坏时，荧光素酶-嵌合体信号会增加，并且嵌合体信号的沉默取决于报告基因的腔内递送而非蛋白水解降解。该系统具有几个优点，包括快速、微尺度操作和高度可重复性，能够检测细微的表型差异。荧光素酶报告基因在极宽的动态范围内提供线性信号，即使在低表达水平下也能分析报告基因的运输情况。此外，当应用于诱导性运输报告基因时，LUCID可报告运输动力学。
