Ferris A L, Brown J C, Park R D, Storrie B
Department of Biochemistry and Nutrition, Virginia Polytechnic Institute and State University, Blacksburg 24061.
J Cell Biol. 1987 Dec;105(6 Pt 1):2703-12. doi: 10.1083/jcb.105.6.2703.
We have used cell fusion to address the question of whether macromolecules are rapidly exchanged between lysosomes. Donor cell lysosomes were labeled by the long-term internalization of the fluid-phase pinocytic markers, invertase (sucrase), Lucifer Yellow, FITC-conjugated dextran, or Texas red-conjugated dextran. Recipient cells contained lysosomes swollen by long-term internalization of dilute sucrose or marked by an overnight FITC-dextran uptake. Cells were incubated for 1 or 2 h in marker-free media before cell fusion to clear any marker from an endosomal compartment. Recipient cells were infected with vesicular stomatitis virus as a fusogen. Donor and recipient cells were co-cultured for 1 or 2 h and then fused by a brief exposure to pH 5. In all cases, extensive exchange of content between donor and recipient cell lysosomes was observed at 37 degrees C. Incubation of cell syncytia at 17 degrees C blocked lysosome/lysosome exchange, although a "priming" process(es) appeared to occur at 17 degrees C. The kinetics of lysosome/lysosome exchange in fusions between cells containing invertase-positive lysosomes and sucrose-positive lysosomes indicated that lysosome/lysosome exchange was as rapid, if not more rapid, than endosome/lysosome exchange. These experiments suggest that in vivo the lysosome is a rapidly intermixing organellar compartment.
我们利用细胞融合来研究溶酶体之间大分子是否会快速交换。通过长期内化液相胞饮标记物(转化酶(蔗糖酶)、荧光素黄、异硫氰酸荧光素偶联葡聚糖或德克萨斯红偶联葡聚糖)对供体细胞溶酶体进行标记。受体细胞含有因长期内化稀蔗糖而肿胀的溶酶体,或通过过夜摄取异硫氰酸荧光素葡聚糖进行标记。在细胞融合前,将细胞在无标记培养基中孵育1或2小时,以清除内体区室中的任何标记物。用泡状口炎病毒作为融合剂感染受体细胞。将供体细胞和受体细胞共培养1或2小时,然后通过短暂暴露于pH 5使其融合。在所有情况下,在37℃观察到供体细胞和受体细胞溶酶体之间有广泛的内容物交换。将细胞融合体在17℃孵育可阻断溶酶体/溶酶体交换,尽管在17℃似乎发生了一个“引发”过程。含有转化酶阳性溶酶体和蔗糖阳性溶酶体的细胞之间融合时溶酶体/溶酶体交换的动力学表明,溶酶体/溶酶体交换即使不比内体/溶酶体交换更快,也同样迅速。这些实验表明,在体内溶酶体是一个快速混合的细胞器区室。