Phagosome-lysosome fusion is a calcium-independent event in macrophages.
Phagosome-lysosome fusion is a calcium-independent event in macrophages.
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DOI：
10.1083/jcb.132.1.49
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发表时间：
1996-01
影响因子：
7.8
通讯作者：
Ernst, JD
中科院分区：
文献类型：
--
作者：
Zimmerli, S;Majeed, M;Gustavsson, M;Stendahl, O;Sanan, DA;Ernst, JD
吞噬体-溶酶体膜融合是细胞内杀死微生物所必需的高度调节的事件。在功能上，它代表了一种形式的极化调节分泌，这是经典依赖于细胞内离子钙（[Ca 2 +]i）的增加。事实上，[Ca 2 +]i的增加对于嗜中性粒细胞中的吞噬体-颗粒（溶酶体）融合和介导克氏锥虫锥鞭毛体侵入宿主细胞的溶酶体融合事件是必需的。由于几种细胞内病原体在不与溶酶体融合的巨噬细胞吞噬体中存活，我们研究了巨噬细胞中吞噬体-溶酶体融合的调节。用12.5 μ M双-（2-氨基-S-甲基苯氧基）乙烷- N，N，N ′，N ′，-四乙酸四乙酰氧基甲酯（MAPT/AM）处理巨噬细胞（M phi），MAPT/AM是一种细胞渗透性钙螯合剂，其将静息细胞质[Ca 2 +] i从80 nM降低至≤ 20 nM，并完全阻断响应于多种刺激的[Ca 2 +]i的增加，即使在存在细胞外钙的情况下。随后，M phi吞噬血清调理的酵母聚糖、葡萄球菌或牛分枝杆菌。通过4 '，6-二脒基-2-苯基吲哚二盐酸盐（DAPI）染色计数微生物，并使用溶酶体相关膜蛋白（LAMP-1）作为膜标记物和罗丹明葡聚糖作为溶酶体的含量标记物对吞噬体-溶酶体融合进行评分。通过电子显微镜证实吞噬体-溶酶体融合验证了荧光显微镜的发现。我们发现在极低的[Ca ~（2+）]i（≤ 20 nM）下，M phi的吞噬体-溶酶体融合正常发生。动力学分析表明，在M phi中，从颗粒结合到最终吞噬体-溶酶体融合的步骤均不受[Ca 2 +]i以限速方式调节。此外，共聚焦显微镜显示钙缓冲与对照巨噬细胞中的吞噬溶酶体膜中的LAMP-1免疫荧光强度没有差异。我们的结论是，无论是膜识别，也不融合事件中的巨噬细胞的吞噬体途径依赖于或调节钙。
Phagosome-lysosome membrane fusion is a highly regulated event that is essential for intracellular killing of microorganisms. Functionally, it represents a form of polarized regulated secretion, which is classically dependent on increases in intracellular ionized calcium ([Ca2+]i). Indeed, increases in [Ca2+]i are essential for phagosome- granule (lysosome) fusion in neutrophils and for lysosomal fusion events that mediate host cell invasion by Trypanosoma cruzi trypomastigotes. Since several intracellular pathogens survive in macrophage phagosomes that do not fuse with lysosomes, we examined the regulation of phagosome-lysosome fusion in macrophages. Macrophages (M phi) were treated with 12.5 microM bis-(2-amino-S-methylphenoxy) ethane- N,N,N',N',-tetraacetic acid tetraacetoxymethyl ester (MAPT/AM), a cell- permeant calcium chelator which reduced resting cytoplasmic [Ca2+]; from 80 nM to < or = 20 nM and completely blocked increases in [Ca2+]i in response to multiple stimuli, even in the presence of extracellular calcium. Subsequently, M phi phagocytosed serum-opsonized zymosan, staphylococci, or Mycobacterium bovis. Microbes were enumerated by 4',6- diamidino-2-phenylindole, dihydrochloride (DAPI) staining, and phagosome-lysosome fusion was scored using both lysosome-associated membrane protein (LAMP-1) as a membrane marker and rhodamine dextran as a content marker for lysosomes. Confirmation of phagosome-lysosome fusion by electron microscopy validated the fluorescence microscopy findings. We found that phagosome-lysosome fusion in M phi occurs noramlly at very low [Ca2+]i (< or = 20 nM). Kinetic analysis showed that in M phi none of the steps leading from particle binding to eventual phagosome-lysosome fusion are regulated by [Ca2+]i in a rate- limiting way. Furthermore, confocal microscopy revealed no difference in the intensity of LAMP-1 immunofluorescence in phagolysosome membranes in calcium-buffered vs. control macrophages. We conclude that neither membrane recognition nor fusion events in the phagosomal pathway in macrophages are dependent on or regulated by calcium.
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DOI：
10.1083/jcb.124.5.677
发表时间：
1994-03
期刊：
The Journal of cell biology
影响因子：
--
作者：
Desjardins M;Huber LA;Parton RG;Griffiths G
通讯作者：
Griffiths G
影响因子：
--
作者：
TSUNODA, Y;STUENKEL, EL;WILLIAMS, JA
通讯作者：
WILLIAMS, JA
影响因子：
56.9
作者：
JOINER, KA;FUHRMAN, SA;MELLMAN, I
通讯作者：
MELLMAN, I
DOI：
10.1084/jem.179.3.1017
发表时间：
1994-03-01
期刊：
The Journal of experimental medicine
影响因子：
--
作者：
Tardieux I;Nathanson MH;Andrews NW
通讯作者：
Andrews NW
影响因子：
19
作者：
BERON, W;ALVAREZDOMINGUEZ, C;STAHL, PD
通讯作者：
STAHL, PD