Glunde Kristine, Guggino Sandra E, Ichikawa Yoshitaka, Bhujwalla Zaver M
Johns Hopkins University School of Medicine, 208C Traylor Building, 720 Rutland Avenue, Baltimore, MD 21205, USA.
Mol Imaging. 2003 Jan;2(1):24-36. doi: 10.1162/15353500200302142.
Cancer cells invade by secreting degradative enzymes which, under normal conditions, are sequestered in lysosomal vesicles. The ability to noninvasively label lysosomes and track lysosomal trafficking would be extremely useful to understand the mechanisms by which degradative enzymes are secreted in the presence of pathophysiological environments, such as hypoxia and acidic extracellular pH, which are frequently encountered in solid tumors. In this study, a novel method of introducing a fluorescent label into lysosomes of human mammary epithelial cells (HMECs) was evaluated. Highly glycosylated lysosomal membrane proteins were labeled with a newly synthesized compound, 5-dimethylamino-naphthalene-1-sulfonic acid 5-amino-3,4,6-trihydroxy-tetrahydro-pyran-2-ylmethyl ester (6-O-dansyl-GlcNH2). The ability to optically image lysosomes using this new probe was validated by determining the colocalization of the fluorescence from the dansyl group with immunofluorescent staining of two well-established lysosomal marker proteins, LAMP-1 and LAMP-2. The location of the dansyl group in lysosomes was also verified by using an anti-dansyl antibody in Western blots of lysosomes isolated using isopycnic density gradient centrifugation. This novel method of labeling lysosomes biosynthetically was used to image lysosomes in living HMECs perfused in a microscopy-compatible cell perfusion system.
癌细胞通过分泌降解酶进行侵袭，在正常情况下，这些酶被隔离在溶酶体囊泡中。对溶酶体进行非侵入性标记并追踪溶酶体运输的能力，对于理解在病理生理环境（如实体瘤中常见的缺氧和细胞外酸性pH值）下，降解酶是如何分泌的机制极为有用。在本研究中，评估了一种将荧光标记引入人乳腺上皮细胞（HMECs）溶酶体的新方法。用新合成的化合物5-二甲基氨基萘-1-磺酸5-氨基-3,4,6-三羟基-四氢吡喃-2-基甲酯（6-O-丹磺酰基-GlcNH2）标记高度糖基化的溶酶体膜蛋白。通过确定丹磺酰基团的荧光与两种成熟的溶酶体标记蛋白LAMP-1和LAMP-2的免疫荧光染色的共定位，验证了使用这种新探针光学成像溶酶体的能力。在使用等密度梯度离心分离的溶酶体的蛋白质免疫印迹中，通过使用抗丹磺酰抗体，也验证了丹磺酰基团在溶酶体中的位置。这种通过生物合成标记溶酶体的新方法，被用于对在显微镜兼容的细胞灌注系统中灌注的活HMECs中的溶酶体进行成像。
