Sannerud Ragna, Marie Michaël, Hansen Bodil Berger, Saraste Jaakko
Department of Biomedicine, University of Bergen, Bergen, Norway.
Methods Mol Biol. 2008;457:253-65. doi: 10.1007/978-1-59745-261-8_19.
A prerequisite for understanding the cellular functions of an unknown protein is the establishment of its subcellular localization. As increasing numbers of novel proteins of the biosynthetic pathway are currently being identified, accessible new methods are required to facilitate their localization. Differentiating rat pheochromocytoma (PC12) cells reorganize their biosynthetic membrane compartments as they develop neurite-like processes. The authors recently showed that polarization of these cells involves the expansion of the intermediate compartment (IC) between the rough endoplasmic reticulum (RER) and the Golgi apparatus. Tubules emerging from the vacuolar parts of the IC move to the developing neurites accumulating in their growth cones, whereas the vacuoles, like RER and Golgi, remain in the cell body. Thus, polarized PC12 cells enhance the resolution for immunofluorescence microscopic mapping of protein localization in the early biosynthetic pathway. The authors also describe here a rapid cell fractionation protocol employing velocity sedimentation in iodixanol gradients that allows one-step separation of the pre-Golgi vacuoles, tubules, and RER.
了解未知蛋白质的细胞功能的一个前提条件是确定其亚细胞定位。随着目前越来越多的生物合成途径新蛋白质被鉴定出来，需要有可用的新方法来促进它们的定位。分化的大鼠嗜铬细胞瘤（PC12）细胞在形成神经突样突起时会重新组织其生物合成膜区室。作者最近表明，这些细胞的极化涉及粗面内质网（RER）和高尔基体之间中间区室（IC）的扩张。从IC的液泡部分伸出的小管会移动到正在发育的神经突，并聚集在其生长锥中，而液泡，就像RER和高尔基体一样，留在细胞体中。因此，极化的PC12细胞提高了早期生物合成途径中蛋白质定位的免疫荧光显微镜映射分辨率。作者在此还描述了一种快速细胞分级分离方案，该方案采用碘克沙醇梯度中的速度沉降，可实现高尔基体前液泡、小管和RER的一步分离。
