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Abstract：
Calcium (Ca2+), a critical secondary messenger, is also known as the molecule of life and death. The cell responds to a minute change in Ca2+ concentration and tightly maintains Ca2+ homeostasis. Therefore, determining the cell Ca2+ level is critical to understand Ca2+ distribution in the cell and various cell processes. Many techniques have been developed to measure Ca2+ in the cell. We review here different methods used to detect and measure Ca2+ in filamentous fungi. Ca2+-sensitive fluorescent chlortetracycline hydrochloride (CTC), Ca2+-selective microelectrode, Ca2+ isotopes, aequorins, and RGECOs are commonly used to measure the Ca2+ level in filamentous fungi. The use of CTC was one of the earliest methods, developed in 1988, to measure the Ca2+ gradient in the filamentous fungus Neurospora crassa. Subsequently, Ca2+-specific microelectrodes were developed later in the 1990s to identify Ca2+ ion flux variations, and to measure Ca2+ concentration. Another method for quantifying Ca2+ is by using radio-labeled Ca2+ as a tracer. The usage of 45Ca to measure Ca2+ in Saccharomyces cerevisiae was reported previously and the same methodology was also used to detect Ca2+ in N. crassa recently. Subsequently, genetically engineered Ca2+ indicators (GECIs) like aequorins and RGECOs have been developed as Ca2+ indicators to detect and visualize Ca2+ inside the cell. In this review, we summarize various methodologies used to detect and measure Ca2+ in filamentous fungi with their advantages and limitations. •Chlortetracycline (CTC) fluorescence assay is used for visualizing Ca2+ level, whereas microelectrodes technique is used to determine Ca2+ flux in the cell.•Radioactive 45Ca is useful for quantification of Ca2+ in the cellular compartments.•Genetically modified calcium indicators (GECIs) are used to study Ca2+ dynamics in the cell.
摘要：
钙(Ca2+),一个关键的二级信使，也被称为生与死的分子。细胞响应于Ca2+浓度的微小变化并紧密维持Ca2+稳态。因此,确定细胞Ca2+水平对于了解Ca2+在细胞中的分布和各种细胞过程至关重要。已经开发了许多技术来测量细胞中的Ca2+。我们在这里回顾了用于检测和测量丝状真菌中Ca2的不同方法。Ca2+敏感荧光盐酸金霉素(CTC),Ca2+-选择性微电极,Ca2+同位素，aequorins,和RGECO通常用于测量丝状真菌中的Ca2水平。使用CTC是最早的方法之一，1988年开发，用于测量丝状真菌Neurosporacrassa中的Ca2梯度。随后，1990年代后期开发了Ca2特异性微电极，以识别Ca2离子通量的变化，并测量Ca2+浓度。定量Ca2+的另一种方法是使用放射性标记的Ca2+作为示踪剂。以前曾报道过使用45Ca测量酿酒酵母中的Ca2，最近也使用相同的方法检测了N.crassa中的Ca2。随后，已经开发了基因工程化的Ca2指示剂（GECIs），例如水母发光蛋白和RGECO，作为Ca2指示剂，以检测和可视化细胞内的Ca2。在这次审查中,我们总结了用于检测和测量丝状真菌中Ca2+的各种方法及其优点和局限性。•金霉素（CTC）荧光测定法用于可视化Ca2+水平，而微电极技术用于确定细胞中的Ca2通量。•放射性45Ca可用于定量细胞区室中的Ca2+。•使用遗传修饰的钙指示剂(GECIs)来研究细胞中的Ca2+动力学。
