Jahraus A, Tjelle T E, Berg T, Habermann A, Storrie B, Ullrich O, Griffiths G
Cell Biology Programme, European Molecular Biology Laboratory, Postfach 10.2209, D-69012 Heidelberg, Germany.
J Biol Chem. 1998 Nov 13;273(46):30379-90. doi: 10.1074/jbc.273.46.30379.
We describe novel biochemical and electron microscopy assays to investigate in vitro fusion of latex bead phagosomes with three different endocytic organelle fractions from J774 macrophages. After formation, early phagosomes fuse avidly with early and late endosomes and for a longer period of time with lysosomes, but they subsequently become fusion-incompetent. The fusion of early, but not late, phagosomes with all three endocytic fractions could be significantly stimulated by Rab5. In contrast to other cell types investigated, this Rab is uniquely enriched on both early and late endosomes in J774 macrophages. Moreover, exogenous Rab5 stimulates homotypic fusion between both sets of organelles. This was shown by a quantitative electron microscopy fusion assay that can directly assay fusion between any combination of morphologically defined organelles. By the same approach, we discovered an unexpected Rab5-stimulatable fusion between early and late endosomes in J774, but not in BHK cells. Thus, in J774 cells both Rab5 and the endocytic pathway seem to have evolved additional functions not yet seen in nonphagocytic cells.
我们描述了新颖的生化和电子显微镜检测方法，以研究乳胶珠吞噬体与来自J774巨噬细胞的三种不同内吞细胞器组分在体外的融合情况。形成后，早期吞噬体与早期和晚期内体大量融合，并与溶酶体融合更长时间，但随后它们变得无法融合。Rab5可显著刺激早期而非晚期吞噬体与所有三种内吞组分的融合。与其他研究的细胞类型不同，这种Rab在J774巨噬细胞的早期和晚期内体中均独特富集。此外，外源性Rab5刺激两组细胞器之间的同型融合。这通过定量电子显微镜融合检测得以证明，该检测可直接检测形态学定义的细胞器的任何组合之间的融合。通过相同方法，我们发现在J774细胞中早期和晚期内体之间存在意外的Rab5刺激的融合，但在BHK细胞中不存在。因此，在J774细胞中，Rab5和内吞途径似乎都进化出了非吞噬细胞中尚未见到的额外功能。
