Fast-Response Calmodulin-Based Fluorescent Indicators Reveal Rapid Intracellular Calcium Dynamics.
Fast-Response Calmodulin-Based Fluorescent Indicators Reveal Rapid Intracellular Calcium Dynamics.
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DOI：
10.1038/srep15978
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发表时间：
2015-11-03
影响因子：
4.6
通讯作者：
Török K
中科院分区：
文献类型：
--
作者：
Helassa N;Zhang XH;Conte I;Scaringi J;Esposito E;Bradley J;Carter T;Ogden D;Morad M;Török K
细胞内Ca 2+动态的时间模式的忠实报告需要工作范围的指标，以匹配的信号。目前遗传编码的钙调素为基础的荧光指示剂可能会扭曲快Ca 2+信号的表观饱和和整合，由于其限制荧光上升和衰减动力学。一系列的探针被设计成具有一系列的Ca 2+亲和力，并通过减弱Ca 2 +-钙调素-肽相互作用来加速动力学。在37 °C下，GCaMP 3衍生的探针GCaMP 3fast比GCaMP 3快40倍，Ca 2+衰减和上升时间t1/2分别为3.3 ms和0.9 ms，使其成为迄今为止最快的。GCaMP 3fast揭示了与GCaMP 6 f相比，新生心肌细胞中Ca 2+动力学显著更快的离散瞬变。GCaMP 3fast在940 nm处的Ca 2+双光子荧光截面增加了5倍，适用于深部组织研究。该绿色荧光蛋白作为一个记者提供了重要的新见解的动力学机制的目标识别钙调蛋白。我们的策略，以匹配探针的信号，通过调整的亲和力，因此Ca 2+动力学的指示剂是适用于新兴的新一代钙调素为基础的探针。
Faithful reporting of temporal patterns of intracellular Ca2+ dynamics requires the working range of indicators to match the signals. Current genetically encoded calmodulin-based fluorescent indicators are likely to distort fast Ca2+ signals by apparent saturation and integration due to their limiting fluorescence rise and decay kinetics. A series of probes was engineered with a range of Ca2+ affinities and accelerated kinetics by weakening the Ca2+-calmodulin-peptide interactions. At 37 °C, the GCaMP3-derived probe termed GCaMP3fast is 40-fold faster than GCaMP3 with Ca2+ decay and rise times, t1/2, of 3.3 ms and 0.9 ms, respectively, making it the fastest to-date. GCaMP3fast revealed discreet transients with significantly faster Ca2+ dynamics in neonatal cardiac myocytes than GCaMP6f. With 5-fold increased two-photon fluorescence cross-section for Ca2+ at 940 nm, GCaMP3fast is suitable for deep tissue studies. The green fluorescent protein serves as a reporter providing important novel insights into the kinetic mechanism of target recognition by calmodulin. Our strategy to match the probe to the signal by tuning the affinity and hence the Ca2+ kinetics of the indicator is applicable to the emerging new generations of calmodulin-based probes.