Cellular Calcium Levels Influenced by NCA-2 Impact Circadian Period Determination in Neurospora.
Cellular Calcium Levels Influenced by NCA-2 Impact Circadian Period Determination in Neurospora.
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DOI：
10.1128/mbio.01493-21
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发表时间：
2021-06-29
期刊：
影响因子：
6.4
通讯作者：
Dunlap JC
中科院分区：
文献类型：
--
作者：
Wang B;Zhou X;Gerber SA;Loros JJ;Dunlap JC
细胞内钙信号转导与动物和植物的各种昼夜节律过程的控制有关，但其在微生物生物钟中的作用在很大程度上仍然是神秘的。为了研究胞内Ca 2+在脉孢菌时钟中的作用，我们筛选了敲除钙转运蛋白基因的突变体，并确定了一个编码钙输出蛋白的基因，nca-2，唯一具有显着的周期效应。NCA-2的丢失导致细胞溶质钙水平的增加，这导致核心时钟成分FRQ和WC-1的过度磷酸化，以及短周期，如通过核心振荡器和外显时钟测量的。遗传分析表明，在某些frq磷酸化位点和Ca 2 +-钙调蛋白依赖性激酶2（camk-2）的突变是上位nca-2在控制的步伐振荡器。这些数据与细胞内Ca 2+升高导致CAMK-2活性增加的模型一致，导致FRQ磷酸化增强，昼夜节律反馈环加速闭合，昼夜节律周期长度缩短。在机制水平上，一些CAMK在Δnca-2突变体中经历更多的自磷酸化，这与Δnca-2突变体中影响CAMK酶活性的高钙水平一致。NCA-2与多种蛋白质相互作用，包括CSP-6，一种已知为昼夜节律输出所需的蛋白质。最重要的是，nca-2的表达在转录和翻译水平上都受到昼夜节律钟的控制，这与缺乏NCA-2的菌株中观察到的周期效应相结合，将钙信号传导牢固地置于更大的昼夜节律系统中，在该系统中，它既作为核心时钟的输入又作为核心时钟的输出。
Intracellular calcium signaling has been implicated in the control of a variety of circadian processes in animals and plants, but its role in microbial clocks has remained largely cryptic. To examine the role of intracellular Ca2+ in the Neurospora clock, we screened mutants with knockouts of calcium transporter genes and identified a gene encoding a calcium exporter, nca-2, uniquely as having significant period effects. The loss of NCA-2 results in an increase in the cytosolic calcium level, and this leads to hyper-phosphorylation of core clock components, FRQ and WC-1, and a short period, as measured by both the core oscillator and the overt clock. Genetic analyses showed that mutations in certain frq phospho-sites and in Ca2+-calmodulin-dependent kinase 2 (camk-2) are epistatic to nca-2 in controlling the pace of the oscillator. These data are consistent with a model in which elevated intracellular Ca2+ leads to the increased activity of CAMK-2, leading to enhanced FRQ phosphorylation, accelerated closure of the circadian feedback loop, and a shortened circadian period length. At a mechanistic level, some CAMKs undergo more auto-phosphorylations in the Δnca-2 mutant, consistent with high calcium levels in the Δnca-2 mutant influencing the enzymatic activities of CAMKs. NCA-2 interacts with multiple proteins, including CSP-6, a protein known to be required for circadian output. Most importantly, the expression of nca-2 is circadian clock-controlled at both the transcriptional and translational levels, and this in combination with the period effects seen in strains lacking NCA-2 firmly places calcium signaling within the larger circadian system, where it acts as both an input to and an output from the core clock.