Methods for the detection of intracellular calcium in filamentous fungi

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.