Calcium signalling in Drosophila photoreceptors measured with GCaMP6f.
Calcium signalling in Drosophila photoreceptors measured with GCaMP6f.
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DOI：
10.1016/j.ceca.2017.02.006
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发表时间：
2017-07
期刊：
影响因子：
4
通讯作者：
Hardie RC
中科院分区：
文献类型：
--
作者：
Asteriti S;Liu CH;Hardie RC
果蝇使用视蛋白启动子在光感受器中表达 GCaMP6f。完整的果蝇以及解离细胞的体内 Ca2+ 定量成像。 Ca2+ 自由浴中的 Ca2+ 升高在 Na+/Ca2+ 交换突变体中被消除。由于 Na+ 流入后 Na+/Ca2+ 交换重新平衡，Ca2+ 自由升高。内部储存的 Ca2+ 没有明显的光诱导释放。果蝇光转导由磷脂酶 C 介导，导致 30000 个微绒毛中的阳离子通道（TRP 和 TRPL）激活，形成光吸收弹状体。这些通道在光的作用下介导大量的 Ca2+ 流入，但 Ca2+ 是否从内部储存中释放仍然存在争议。我们生成了在其光感受器中表达 GCaMP6f 的果蝇，并测量了来自分离细胞的 Ca2+ 信号，以及通过对完整果蝇中的横纹肌进行成像来测量体内的 Ca2+ 信号。为了响应短暂的闪光，GCaMP6f 信号的延迟为 10–25 ms，在 ∼1200 个有效吸收光子时达到 50% Fmax，并在 10000–30000 个光子时达到饱和 (ΔF/F0 ∼ 10–20)。在无 Ca2+ 浴中，仍然可以检测到较小的 (ΔF/F0 ~4)、长潜伏期 (~200 ms) 光诱导的 Ca2+ 升高。这些在 InsP3 受体突变体中不受影响，但当 Na+ 也从浴中省略时，或在缺乏光敏通道的 trpl;trp 突变体中，这些几乎被消除。 Na+/Ca2+交换器突变体中Ca2+自由升高也被消除，但在过表达交换器的果蝇中大大加速。这些结果表明，Ca2+ 自由升高严格依赖于 Na+ 流入和交换器活性，表明它们反映了大量 Na+ 流入后跨质或细胞内膜的 Na+/Ca2+ 交换的重新平衡。任何没有交换活性的微小 Ca2+ 自由上升相当于 <10 nM (ΔF/F0 ∼0.1)，并且不太可能在光转导中发挥任何作用。
Fruitflies generated expressing GCaMP6f in photoreceptors using the opsin promoter. Quantitative in vivo Ca2+ imaging in completely intact flies as well as dissociated cells. Ca2+ rise in Ca2+ free bath abolished in Na+/Ca2+ exchanger mutants. Ca2+ free rise due to re-equilibration of Na+/Ca2+ exchange following Na+ influx. No significant light-induced release of Ca2+ from internal stores. Drosophila phototransduction is mediated by phospholipase C leading to activation of cation channels (TRP and TRPL) in the 30000 microvilli forming the light-absorbing rhabdomere. The channels mediate massive Ca2+ influx in response to light, but whether Ca2+ is released from internal stores remains controversial. We generated flies expressing GCaMP6f in their photoreceptors and measured Ca2+ signals from dissociated cells, as well as in vivo by imaging rhabdomeres in intact flies. In response to brief flashes, GCaMP6f signals had latencies of 10–25 ms, reached 50% Fmax with ∼1200 effectively absorbed photons and saturated (ΔF/F0 ∼ 10–20) with 10000–30000 photons. In Ca2+ free bath, smaller (ΔF/F0 ∼4), long latency (∼200 ms) light-induced Ca2+ rises were still detectable. These were unaffected in InsP3 receptor mutants, but virtually eliminated when Na+ was also omitted from the bath, or in trpl;trp mutants lacking light-sensitive channels. Ca2+ free rises were also eliminated in Na+/Ca2+ exchanger mutants, but greatly accelerated in flies over-expressing the exchanger. These results show that Ca2+ free rises are strictly dependent on Na+ influx and activity of the exchanger, suggesting they reflect re-equilibration of Na+/Ca2+ exchange across plasma or intracellular membranes following massive Na+ influx. Any tiny Ca2+ free rise remaining without exchanger activity was equivalent to <10 nM (ΔF/F0 ∼0.1), and unlikely to play any role in phototransduction.