Genome-wide CRISPR screen reveals CLPTM1L as a lipid scramblase required for efficient glycosylphosphatidylinositol biosynthesis.

SignificanceScramblases translocate lipids across the lipid bilayer without consumption of ATP, thereby regulating lipid distributions in cellular membranes. Cytosol-to-lumen translocation across the endoplasmic reticulum (ER) membrane is a common process among lipid glycoconjugates involved in posttranslational protein modifications in eukaryotes. These translocations are thought to be mediated by specific ER-resident scramblases, but the identity of these proteins and the underlying molecular mechanisms have been elusive. Here, we show that CLPTM1L, an integral membrane protein with eight putative transmembrane domains, is the major lipid scramblase involved in efficient glycosylphosphatidylinositol biosynthesis in the ER membrane. Our results validate the long-standing hypothesis that lipid scramblases ensure the efficient translocations of lipid glycoconjugates across the ER membrane for protein glycosylation pathways.

Rescue of Glycosylphosphatidylinositol-Anchored Protein Biosynthesis Using Synthetic Glycosylphosphatidylinositol Oligosaccharides.

The attachment of proteins to the cell membrane using a glycosylphosphatidylinositol (GPI) anchor is a ubiquitous process in eukaryotic cells. Deficiencies in the biosynthesis of GPIs and the concomitant production of GPI-anchored proteins lead to a series of rare and complicated disorders associated with inherited GPI deficiencies (IGDs) in humans. Currently, there is no treatment for patients suffering from IGDs. Here, we report the design, synthesis, and use of GPI fragments to rescue the biosynthesis of GPI-anchored proteins (GPI-APs) caused by mutation in genes involved in the assembly of GPI-glycolipids in cells. We demonstrated that the synthetic fragments GlcNAc-PI (1), Man-GlcN-PI (5), and GlcN-PI with two (3) and three lipid chains (4) rescue the deletion of the GPI biosynthesis in cells devoid of the PIGA, PIGL, and PIGW genes in vitro. The compounds allowed for concentration-dependent recovery of GPI biosynthesis and were highly active on the cytoplasmic face of the endoplasmic reticulum membrane. These synthetic molecules are leads for the development of treatments for IGDs and tools to study GPI-AP biosynthesis.

Perturbation of DNA repair gene expression due to interspecies hybridization.

The effect of interspecies hybridization on gene regulation was examined using real-time polymerase chain reaction (RT-PCR) to measure the expression of five base-excision repair genes in brain, eye, gill, liver, and tailfin tissues from Xiphophorus parental species and F(1) hybrids. Relative mRNA levels of uracil N-glycosylase (Ung), Apurinic/apyrimidinic endonuclease (Ape1), polymerase-beta (Polb), flap endonuclease (Fen1), and DNA ligase (Lig1) were measured in three parental Xiphophorus species (X. maculatus Jp 163 B, X. helleri Sarabia, and X. andersi andC) and in two interspecies F(1) hybrids, the Sp-helleri hybrid (X. maculatus Jp 163 BxX. helleri Sarabia) and the Sp-andersi hybrid (X. maculatus Jp 163 BxX. andersi) to identify genes that undergo changes in expression levels upon interspecies hybridization. Significant differences in gene expression were observed between parental animals and their respective F(1) hybrids in both interspecies crosses. Generally, marked increases in DNA repair gene mRNA levels were observed across all tissues in F(1) hybrid animals from the Sp-helleri cross compared to either X. maculatus or X. helleri parents. In contrast, the Sp-andersi F(1) hybrid animals generally exhibited decreased base-excision repair gene expression, although this trend was more specific to individual tissues than observed for Sp-helleri hybrids.

Production of F1 interspecies hybrid offspring with cryopreserved sperm from a live-bearing fish, the swordtail Xiphophorus helleri.

Despite study of sperm cryopreservation in more than 200 fish species, production of broods from cryopreserved sperm in live-bearing fish has not been demonstrated. This has not been due to a lack of effort, but instead is a result of the unique morphology, biology, and biochemistry of reproduction in viviparous fishes. For example, sperm of Xiphophorus helleri have a cylindrical nucleus, can swim for days after being activated, have glycolytic capabilities, and can reside in the female reproduction tract for months before fertilization. These traits are not found in fishes with external fertilization. The long-standing research use of the genus Xiphophorus has led to development of over 60 pedigreed lines among the 26 species maintained around the world. These species and lines serve as contemporary models in medical research, although they must be maintained as live populations. Previous attempts at establishing sperm cryopreservation protocols for Xiphophorus have not produced live young. To address this we have been studying the parameters surrounding cryobiology of Xiphophorus sperm and applying this information to an improved understanding of internal fertilization and reproduction. Here we report the first successful fertilization and offspring production by cryopreserved sperm in any live-bearing fish. This claim is supported by our use of artificial insemination between two species that yield distinct hybrid offspring to verify paternity via cryopreserved sperm. We provide a practical approach for preservation of valuable genetic resources from live-bearing fish species, a group that is rapidly being lost due to destruction of native habitats.