B Calvo, P Torres-Vidal, A Delrio-Lorenzo, C Rodriguez, F J Aulestia, J Rojo-Ruiz, B M McVeigh, V Moiseenkova-Bell, D I Yule, J Garcia-Sancho, S Patel, M T Alonso
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引用次数: 0
摘要
内含溶酶体被认为是酸性 Ca 2+ 储存库,但对其中腔内 Ca 2+ 的直接测量却很有限。在这里,我们报告了对 Ca 2+ 敏感的发光蛋白 aequorin 在高酸性 pH 下不能与其辅助因子重组,但当针对内-溶酶体系统时,探针的很大一部分在微酸性区室中起作用。我们利用该探针(ELGA)报告了该区室中 Ca 2+ 的动态变化。我们发现 Ca 2+ 的吸收是 ATP 依赖性的,并且对内质网 Ca 2+ 泵的阻断剂敏感。我们发现,通常以内质网为目标的钙2+动员信使IP 3能在野生型细胞中唤起强烈的管腔反应,但在IP 3受体敲除细胞中却不能。这些反应与激活内溶酶体离子通道 TRPML1 所引起的反应相当。形成 IP 3 的激动剂也能调动完整细胞中的储存。超分辨率显微镜分析证实,在活细胞和固定细胞中,内溶酶体系统中都存在 IP 3 受体。我们的数据揭示了内溶酶体系统中与生理相关的、对 IP 3 敏感的 Ca 2+ 储存。
Direct measurements of luminal Ca 2+ with endo-lysosomal GFP-aequorin reveal functional IP 3 receptors.
Endo-lysosomes are considered acidic Ca 2+ stores but direct measurements of luminal Ca 2+ within them are limited. Here we report that the Ca 2+ -sensitive luminescent protein aequorin does not reconstitute with its cofactor at highly acidic pH but that a significant fraction of the probe is functional within a mildly acidic compartment when targeted to the endo-lysosomal system. We leveraged this probe (ELGA) to report Ca 2+ dynamics in this compartment. We show that Ca 2+ uptake is ATP-dependent and sensitive to blockers of endoplasmic reticulum Ca 2+ pumps. We find that the Ca 2+ mobilizing messenger IP 3 which typically targets the endoplasmic reticulum evokes robust luminal responses in wild type cells, but not in IP 3 receptor knock-out cells. Responses were comparable to those evoked by activation of the endo-lysosomal ion channel TRPML1. Stimulation with IP 3 -forming agonists also mobilized the store in intact cells. Super-resolution microscopy analysis confirmed the presence of IP 3 receptors within the endo-lysosomal system, both in live and fixed cells. Our data reveal a physiologically-relevant, IP 3 -sensitive store of Ca 2+ within the endo-lysosomal system.