Background

Cytosolic Ca²⁺ is an important factor for regulating various cellular events such as gene expression, neurotransmitter release at synaptic terminal sites, muscle contraction, and hormone secretion (Clapham, 2007). Excessive amounts of Ca²⁺ in the cytoplasm provoke excitotoxic necrosis, often seen in neurodegenerative diseases (Yamashima, 2004; Wojda et al., 2008), heart failures (Nakayama et al., 2007), and cancer (Danese et al., 2017). Recently, we have found that a transient increase in Ca²⁺ level in the cytoplasm of neurons undergoing necrosis is necessary for the exposure of phosphatidylserine (PS), which acts as an “eat me” signal to attract engulfing cells on the surfaces of necrotic cells in the nematode Caenorhabditis elegans (Furuta et al., 2021). In previous studies, in Caenorhabditis elegans larvae, Ca²⁺ dynamics was monitored in the cell bodies of mechanosensory neurons (touch neurons) with Cameleon, a genetically encoded calcium indicator (GECI) (Suzuki et al., 2003). Different generations of GCaMP, another series of GECIs, were also reported to monitor Ca²⁺ in the neuronal processes of different sensory neurons in the Caenorhabditis elegans larvae (Tian et al., 2009; Ghosh-Roy et al., 2010; Akerboom et al., 2012). These Ca²⁺ recording experiments were performed in Caenorhabditis elegans larvae, and recording time ranged from milliseconds to seconds. However, recording of Ca²⁺ signal in Caenorhabditis elegans for longer periods of time or during embryonic development has not been reported previously.

We study the necrosis and clearance of Caenorhabditis elegans touch neurons during embryogenesis. In Caenorhabditis elegans, dominant mutations in the DEG/ENaC sodium channel subunit MEC-4 induce the six touch neurons to undergo excitotoxic necrosis (Figure 1A) (Chalfie and Sulston, 1981; Driscoll and Chalfie, 1991). These necrotic neurons expose PS and are subsequently engulfed and digested by neighboring hypodermal cells (Li et al., 2015). Previously, we developed secreted fluorescence PS reporters by tagging MFG-E8, a secreted PS-binding protein, with fluorescent proteins such as mCherry, and expressing each reporter under the control of the dyn-1 promoter (P_dyn-1) (Li et al., 2015). The secreted MFG-E8::mCherry protein associates with PS exposed on cell surfaces and, in this manner, labels the surfaces of necrotic and apoptotic cells (Figure 1B). We established a time-lapse recording protocol that allowed us to monitor, for hours, the dynamics of PS externalization on the surface of necrotic cells in Caenorhabditis elegans embryo (Li et al., 2015).

Figure 1. Caenorhabditis elegans six touch neurons and PS exposed on the surface of a necrotic cell. (A) Diagram of a hermaphrodite indicating the positions and names of the six touch neurons. (B) A necrotic cell caused by mec-4(e1611) mutation in Differential Interference Contrast (DIC) image (left) and exposed PS visualized by MFG-E8::mCherry reporter (right). The scale bar is 15 µm.

To investigate the effect of cytoplasmic Ca²⁺ in triggering PS exposure on the surfaces of necrotic neurons, we constructed an in vivo Ca²⁺ reporter CGaMP5 that was expressed under the control of promoter P_mec-7, a promoter that is specifically active in touch neurons (Furuta et al., 2021). We generated a strain that co-expressed this Ca²⁺ reporter and the PS reporter MFG-E8::mCherry, and established a time-lapse recording protocol that allowed us to simultaneously monitor the level of Ca²⁺ in the cytoplasm of two touch neurons (PLML and PLMR) that undergo necrosis and the level of PS on the outer surfaces of these neurons during embryogenesis. This protocol, together with genetic and pharmacological approaches, enabled us to discover that a transient increase in the level of cytoplasmic Ca²⁺ triggers the exposure of PS on the surfaces of touch neurons during necrosis and that the endoplasmic reticulin (ER) contributes to the PS exposure by releasing Ca²⁺ to the cytoplasm (Furuta et al., 2021).

Monitoring cytoplasmic Ca²⁺ and cell surface PS in necrotic cells simultaneously is challenging because long-time imaging of embryos tends to cause photo-damage that impairs proper embryonic development. To overcome this problem, we operated our protocol using the DeltaVision Elite Deconvolution Imaging System from GE Healthcare, Inc., and optimized our experimental settings to minimize photo-damage. Our protocol uses the DeltaVision Elite Deconvolution Imaging System or an equivalent imaging system, the Caenorhabditis elegans strain carrying the Ca²⁺ and PS reporters, and requires the routine laboratory setup for propagating Caenorhabditis elegans. This protocol can also be adapted to monitor cytoplasmic Ca²⁺ or PS exposure individually. Our protocol is thus applicable to other Ca²⁺- and/or PS exposure-related cellular and developmental events. Furthermore, if the P_mec-7 promoter is replaced by other promoters, this protocol can be used to monitor cytosolic Ca²⁺ in cell types other than the touch neurons.