Roberts R L, Barbieri M A, Ullrich J, Stahl P D
Department of Cell Biology and Physiology, Washington University, School of Medicine, St. Louis, Missouri 63110, USA.
J Leukoc Biol. 2000 Nov;68(5):627-32.
Fluid-phase endocytosis is stimulated by H-ras-linked growth factor receptors and this stimulation requires activation of rab5. We utilized a GFP-rab5a:wt fusion protein to monitor GFP-rab5a:wt activation in living fibroblasts and in J774 macrophages. Control CHO cells that expressed GFP-rab5a:wt were cultured in serum-free conditions and showed GFP-rab5a:wt localized to endosomal vesicles with a mean diameter of 0.3 +/- 0.1 microm. Endosome fusion, membrane ruffling, and pinosome formation were rarely detected in these cells. Coexpression of H-ras:G12V, a constitutively active H-ras mutant that activates rab5a, in cells resulted in marked enlargement of labeled endosomes (mean diameter 0.7 +/- 0.2 microm) and large numbers of giant GFP-rab5a:wt-positive endosomes were present. Time-lapse recordings showed abundant fusion among giant labeled endosomes, and membrane ruffling and pinosome formation were commonly observed. Alterations in GFP-rab5a:wt endosome structure and activity in cells expressing H-ras:G12V were linked to rab5a activation because these changes were identical to those found in cells expressing GFP-rab5a:Q79L, a constitutively activated rab5a mutant. Furthermore, cells co-expressing H-ras:G12V and GFP-rab5a:S34N, an inactive rab5a mutant, exhibited no evidence of H-ras:G12V-induced endosome enlargement. To observe changes in endosome structure and activity that directly followed activation of GFP-rab5a:wt, we performed time-lapse recordings of cells cultured overnight in serum-free media after addition of EGF. EGF caused a rapid increase in endosome fusion and in membrane ruffling activity. Membrane ruffling was often associated with GFP-rab5a:wt-positive vesicle (pinosome) formation at the base of membrane ruffles. Endosome and pinosome fusion were common in EGF-stimulated cells. Phagocytosis is also regulated by GFP-rab5a:wt. J774 macrophages that expressed GFP-rab5a:wt showed transiently activation and recruitment of GFP-rab5a:wt to newly formed phagosomes that contained rhodamine-labeled Escherichia coli. These studies show that GFP-rab5a:wt activation results in dynamic alterations in the structure and activity of the early endosomal and early phagosomal elements.
液相内吞作用由与H-ras相关的生长因子受体刺激，这种刺激需要rab5的激活。我们利用GFP-rab5a:wt融合蛋白来监测活的成纤维细胞和J774巨噬细胞中GFP-rab5a:wt的激活情况。表达GFP-rab5a:wt的对照CHO细胞在无血清条件下培养，显示GFP-rab5a:wt定位于平均直径为0.3±0.1微米的内体小泡。在这些细胞中很少检测到内体融合、膜皱襞和吞噬体形成。在细胞中共表达H-ras:G12V（一种激活rab5a的组成型活性H-ras突变体）导致标记的内体显著增大（平均直径0.7±0.2微米），并且存在大量巨大的GFP-rab5a:wt阳性内体。延时记录显示巨大的标记内体之间有大量融合，并且普遍观察到膜皱襞和吞噬体形成。在表达H-ras:G12V的细胞中GFP-rab5a:wt内体结构和活性的改变与rab5a激活有关，因为这些变化与在表达GFP-rab5a:Q79L（一种组成型激活的rab5a突变体）的细胞中发现的变化相同。此外，共表达H-ras:G12V和GFP-rab5a:S34N（一种无活性的rab5a突变体）的细胞没有显示出H-ras:G12V诱导的内体增大的证据。为了观察直接跟随GFP-rab5a:wt激活后的内体结构和活性变化，我们在添加表皮生长因子（EGF）后对在无血清培养基中过夜培养的细胞进行了延时记录。EGF导致内体融合和膜皱襞活性迅速增加。膜皱襞通常与膜皱襞基部的GFP-rab5a:wt阳性小泡（吞噬体）形成相关。在EGF刺激的细胞中内体和吞噬体融合很常见。吞噬作用也受GFP-rab5a:wt调节。表达GFP-rab5a:wt的J774巨噬细胞显示GFP-rab5a:wt短暂激活并募集到含有罗丹明标记的大肠杆菌的新形成的吞噬体中。这些研究表明GFP-rab5a:wt激活导致早期内体和早期吞噬体成分的结构和活性发生动态改变。
