TY  - GEN
T1  - N-terminally endogenous mKate2-tagged HIM-4 in Caenorhabditis elegans
AU  - Ihara, Shinji
DO  - 10.17912/micropub.biology.000386
UR  - http://beta.micropublication.org/journals/biology/micropub-biology-000386/
AB  - The gene him-4 in Caenorhabditis elegans encodes hemicentin, which is evolutionally conserved (Vogel & Hedgecock, 2001). Hemicentins are characterized by multiple domains, including a conserved von Willebrand A domain, a long chain of immunoglobulin modules, a series of EGF-like modules, and a carboxyl-terminal fibulin-type module(Whittaker & Hynes, 2002). Despite being an extracellular matrix protein, mutant alleles of hemicentin exhibit both pleiotropic defects during development and tissue fragility (Hodgkin et al., 1979; Vogel & Hedgecock, 2001). HMCN1, a human ortholog of him-4, is involved in age-related macular degeneration 1(Thompson et al., 2007). Previous studies in C. elegans have shown that HIM-4 is secreted from the skeletal muscle and gonads and is recruited to specific extracellular sites using a transgenic strain (Vogel & Hedgecock, 2001). A recent study reported the construction of a C-terminally endogenous mNeonGreen-tagged him-4 using CRISPR/Cas9 genome editing (Keeley et al., 2020). Using the bright far-red fluorescence protein mKate2, I generated a homozygous mkate2::HIM-4 strain, where mKate2 is inserted after the 109th glycine residue of the him-4 genomic locus via CRISPR/Cas9 genome editing. As expected, localization of mkate2::HIM-4 to the pharyngeal basement membrane was observed at the L1 stage (Figure 1B). In contrast to the uniform localization on the basement membrane of type IV collagen or nidogen, which are major components of the basement membrane, that of mkate2::HIM-4 was polarized at the anterior part of the pharyngeal basement membrane (Kang & Kramer, 2013; Matsuo et al., 2019)(Figure 1B). A unique polarized localization pattern was also observed in the pharyngeal basement membrane at stage L4 (Figure 1D). These observations indicate that polarized HIM-4 localization in the anterior part of the pharyngeal BM is maintained throughout the L1–L4 stages, although it remains unclear whether polarized localization is associated with pharynx function. The localization was also observed at the rachis during gonadal development (Figure 1F) at the L4 stage; this observation was consistent with that of previous reports (Vogel & Hedgecock, 2001). As the repellent behavior of C. elegans is observed after exposure to blue light (488 nm) that excites GFP but not after exposure to light (588 nm) that excites red fluorescent protein(Ward et al., 2008), live imaging may be useful for visualizing mkate2::HIM-4 localization in vivo.
PY  - 2021
JO  - microPublication Biology
ER  -