Abstract
Glycosylphosphatidylinositol (GPI) is a post-translational modification resulting in the attachment of modified proteins to the outer leaflet of the plasma membrane. Tissue culture experiments have shown GPI-anchored proteins (GPI-APs) to be targeted to the apical membrane of epithelial cells. However, the in vivo importance of this targeting has not been investigated since null mutations in GPI biosynthesis enzymes in mice result in very early embryonic lethality. Missense mutations in the human GPI biosynthesis enzyme pigv are associated with a multiple congenital malformation syndrome with a high frequency of Hirschsprung disease and renal anomalies. However, it is currently unknown how these phenotypes are linked to PIGV function. Here, we identify a temperature-sensitive hypomorphic allele of PIGV in Caenorhabditis elegans, pigv-1(qm34), enabling us to study the role of GPI-APs in development. At the restrictive temperature we found a 75% reduction in GPI-APs at the surface of embryonic cells. Consequently, ~80% of pigv-1(qm34) embryos arrested development during the elongation phase of morphogenesis, exhibiting internal cysts and/or surface ruptures. Closer examination of the defects revealed them all to be the result of breaches in epithelial tissues: cysts formed in the intestine and excretory canal, and ruptures occurred through epidermal cells, suggesting weakening of the epithelial membrane or membrane-cortex connection. Knockdown of piga-1, another GPI biosynthesis enzymes resulted in similar phenotypes. Importantly, fortifying the link between the apical membrane and actin cortex by overexpression of the ezrin/radixin/moesin ortholog ERM-1, significantly rescued cyst formation and ruptures in the pigv-1(qm34) mutant. In conclusion, we discovered GPI-APs play a critical role in maintaining the integrity of the epithelial tissues, allowing them to withstand the pressure and stresses of morphogenesis. Our findings may help to explain some of the phenotypes observed in human syndromes associated with pigv mutations.
Highlights
Proteins can attach to the plasma membrane by intrinsic transmembrane domains or by posttranslational modifications with lipid moieties
The GPI-anchor biosynthesis pathway is highly conserved from yeast to humans and null mutations in any of the key enzymes are lethal at early developmental stages
ethyl methanesulfonate (EMS) mutagenesis results in hypomorphic alleles that are temperature-sensitive. We investigated this possibility by growing pigv-1(qm34) worms at 15, 20 and 25°C and measuring their viability at each temperature
Summary
Proteins can attach to the plasma membrane by intrinsic transmembrane domains or by posttranslational modifications with lipid moieties. One such lipid modification, tethering proteins to the outer leaflet of the plasma membrane (PM), is a glycosylphospatidylinositol (GPI) anchor, whose synthesis and attachment to proteins in the endoplasmic reticulum (ER) is a multi-step process involving >30 enzymes [1]. The basic structure of a GPI anchor consists of a phosphoethanolamine linker, a glycan core and a phospholipid tail. GPI-APs are mostly localized to the apical membrane of polarized cells and are enriched in domains known as lipid rafts [5,6]. GPI-APs are present in non-polarized tissues with equal distribution across the membrane [8]
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