SNAP-23 regulates phagosome formation and maturation in macrophages.
SNAP-23 regulates phagosome formation and maturation in macrophages.
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DOI：
10.1091/mbc.e12-01-0069
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发表时间：
2012-12
影响因子：
3.3
通讯作者：
Hatsuzawa K
中科院分区：
文献类型：
--
作者：
Sakurai C;Hashimoto H;Nakanishi H;Arai S;Wada Y;Sun-Wada GH;Wada I;Hatsuzawa K
使用过表达或减少SNAP-23的巨噬细胞，该研究表明SNAP-23与吞噬体形成和成熟有关，推测是通过介导基于SNARE的膜运输。事实上，在过表达溶酶体SNARE VAMP 7的细胞的吞噬体膜上观察到基于FRET信号的SNAP-23结构的构象变化。突触体相关蛋白23 kDa（SNAP-23），质膜定位的可溶性N-乙基马来酰亚胺敏感因子附着蛋白受体（SNARE），已涉及巨噬细胞的吞噬作用。为了阐明其在该过程中的确切作用，建立了过表达单体Venus标记的SNAP-23的巨噬细胞系。这些细胞表现出增强的Fc受体介导的吞噬作用。对吞噬作用的每个过程的详细分析显示，吞噬体内活性氧的产生显著增加。此外，增强的积累溶嗜性染料，以及增强淬灭的pH敏感的荧光团进行了观察。对分离的吞噬体的分析表明SNAP-23在NADPH氧化酶复合物和空泡型H+-ATP酶向吞噬体的功能性募集中起关键作用。来自过表达实验的数据通过SNAP-23敲低得到证实，这证明了吞噬体成熟的显著延迟和摄取活性的降低。最后，为了分析吞噬体SNAP-23是否引起蛋白质的结构变化，构建了分子内Förster共振能量转移（FRET）探针，其中TagGFP 2-TagRFP内的距离在其两个SNARE基序的N-末端接近时改变。FRET效率吞噬体显着增强，只有当VAMP 7，溶酶体陷阱，共表达。总之，我们的研究结果强烈表明SNAP-23参与巨噬细胞吞噬体的形成和成熟，可能是通过介导基于SNARE的膜交通。
Using macrophages overexpressing or reducing SNAP-23, this study shows that SNAP-23 is implicated in phagosome formation and maturation, presumably by mediating SNARE-based membrane traffic. Indeed, a conformational change in SNAP-23 structure based on FRET signal is observed on the phagosome membrane of cells overexpressing the lysosomal SNARE VAMP7. Synaptosomal associated protein of 23 kDa (SNAP-23), a plasma membrane–localized soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE), has been implicated in phagocytosis by macrophages. For elucidation of its precise role in this process, a macrophage line overexpressing monomeric Venus–tagged SNAP-23 was established. These cells showed enhanced Fc receptor–mediated phagocytosis. Detailed analyses of each process of phagocytosis revealed a marked increase in the production of reactive oxygen species within phagosomes. Also, enhanced accumulation of a lysotropic dye, as well as augmented quenching of a pH-sensitive fluorophore were observed. Analyses of isolated phagosomes indicated the critical role of SNAP-23 in the functional recruitment of the NADPH oxidase complex and vacuolar-type H+-ATPase to phagosomes. The data from the overexpression experiments were confirmed by SNAP-23 knockdown, which demonstrated a significant delay in phagosome maturation and a reduction in uptake activity. Finally, for analyzing whether phagosomal SNAP-23 entails a structural change in the protein, an intramolecular Förster resonance energy transfer (FRET) probe was constructed, in which the distance within a TagGFP2-TagRFP was altered upon close approximation of the N-termini of its two SNARE motifs. FRET efficiency on phagosomes was markedly enhanced only when VAMP7, a lysosomal SNARE, was coexpressed. Taken together, our results strongly suggest the involvement of SNAP-23 in both phagosome formation and maturation in macrophages, presumably by mediating SNARE-based membrane traffic.