Akimzhanov Askar M, Boehning Darren
Department of Neuroscience and Cell Biology, University of Texas Medical Branch.
J Vis Exp. 2011 Apr 1(50):2579. doi: 10.3791/2579.
Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, differentiation, and apoptosis(1). During apoptosis, calcium accumulation in mitochondria promotes the release of pro-apoptotic factors from the mitochondria into the cytosol(2). It is therefore of interest to directly measure mitochondrial calcium in living cells in situ during apoptosis. High-resolution fluorescent imaging of cells loaded with dual-excitation ratiometric and non-ratiometric synthetic calcium indicator dyes has been proven to be a reliable and versatile tool to study various aspects of intracellular calcium signaling. Measuring cytosolic calcium fluxes using these techniques is relatively straightforward. However, measuring intramitochondrial calcium levels in intact cells using synthetic calcium indicators such as rhod-2 and rhod-FF is more challenging. Synthetic indicators targeted to mitochondria have blunted responses to repetitive increases in mitochondrial calcium, and disrupt mitochondrial morphology(3). Additionally, synthetic indicators tend to leak out of mitochondria over several hours which makes them unsuitable for long-term experiments. Thus, genetically encoded calcium indicators based upon green fluorescent protein (GFP)(4) or aequorin(5) targeted to mitochondria have greatly facilitated measurement of mitochondrial calcium dynamics. Here, we describe a simple method for real-time measurement of mitochondrial calcium fluxes in response to different stimuli. The method is based on fluorescence microscopy of 'ratiometric-pericam' which is selectively targeted to mitochondria. Ratiometric pericam is a calcium indicator based on a fusion of circularly permuted yellow fluorescent protein and calmodulin(4). Binding of calcium to ratiometric pericam causes a shift of its excitation peak from 415 nm to 494 nm, while the emission spectrum, which peaks around 515 nm, remains unchanged. Ratiometric pericam binds a single calcium ion with a dissociation constant in vitro of ~1.7 μM(4). These properties of ratiometric pericam allow the quantification of rapid and long-term changes in mitochondrial calcium concentration. Furthermore, we describe adaptation of this methodology to a standard wide-field calcium imaging microscope with commonly available filter sets. Using two distinct agonists, the purinergic agonist ATP and apoptosis-inducing drug staurosporine, we demonstrate that this method is appropriate for monitoring changes in mitochondrial calcium concentration with a temporal resolution of seconds to hours. Furthermore, we also demonstrate that ratiometric pericam is also useful for measuring mitochondrial fission/fragmentation during apoptosis. Thus, ratiometric pericam is particularly well suited for continuous long-term measurement of mitochondrial calcium dynamics during apoptosis.
细胞内钙浓度随各种刺激发生的动态变化调节着许多细胞过程，如增殖、分化和凋亡(1)。在凋亡过程中，线粒体中的钙积累会促使促凋亡因子从线粒体释放到细胞质中(2)。因此，在凋亡过程中原位直接测量活细胞中的线粒体钙具有重要意义。已证明，用双激发比率型和非比率型合成钙指示剂染料加载细胞的高分辨率荧光成像，是研究细胞内钙信号传导各个方面的可靠且通用的工具。使用这些技术测量细胞质钙通量相对简单。然而，使用如罗丹明 - 2和罗丹明 - FF等合成钙指示剂测量完整细胞内的线粒体钙水平则更具挑战性。靶向线粒体的合成指示剂对线粒体钙的重复增加反应迟钝，并会破坏线粒体形态(3)。此外，合成指示剂在数小时内往往会从线粒体中泄漏出来，这使得它们不适合长期实验。因此，基于靶向线粒体的绿色荧光蛋白(GFP)(4)或水母发光蛋白(5)的基因编码钙指示剂极大地促进了线粒体钙动力学的测量。在此，我们描述一种实时测量线粒体对不同刺激的钙通量的简单方法。该方法基于对选择性靶向线粒体的“比率型钙敏荧光蛋白”进行荧光显微镜观察。比率型钙敏荧光蛋白是一种基于环状排列的黄色荧光蛋白与钙调蛋白融合的钙指示剂(4)。钙与比率型钙敏荧光蛋白结合会导致其激发峰从415 nm 移至494 nm，而发射光谱在515 nm左右达到峰值，保持不变。比率型钙敏荧光蛋白在体外以约1.7 μM的解离常数结合单个钙离子(4)。比率型钙敏荧光蛋白的这些特性使得能够定量线粒体钙浓度的快速和长期变化。此外，我们描述了将该方法应用于配备常用滤光片组的标准宽视野钙成像显微镜。使用两种不同的激动剂，嘌呤能激动剂ATP和凋亡诱导药物星形孢菌素，我们证明该方法适用于以秒至小时的时间分辨率监测线粒体钙浓度的变化。此外，我们还证明比率型钙敏荧光蛋白也可用于测量凋亡过程中的线粒体分裂/碎片化。因此，比率型钙敏荧光蛋白特别适合在凋亡过程中连续长期测量线粒体钙动力学。
