Biology subjects

Di Minin, G.

Publications and source records attributed to Di Minin, G..

4 recordsLinked to original sources

bioRxiv2023

The scramblases VMP1 and TMEM41b are required for primitive endoderm specification by targeting WNT signaling

The ER resident proteins VMP1 and TMEM41b share a conserved DedA domain, which confers lipid scramblase activity. Loss of either gene results in embryonic lethality in mice and defects in autophagy and lipid droplet metabolism. We set out to investigate their role in pluripotency and specification. For this purpose, we generated Vmp1 and Tmem41b mutations in mouse embryonic stem cells (ESCs). We observed that ESCs carrying mutations in Vmp1 and Tmem41b show robust self-renewal and an unperturbed pluripotent expression profile but accumulate LC3-positive autophagosomes and lipid droplets consistent with defects in autophagy and lipid metabolism. ESCs carrying combined mutations in Vmp1 and Tmem41b can differentiate into a wide range of embryonic cell types. However, differentiation into primitive endoderm-like cells in culture is impaired, and the establishment of extra- embryonic endoderm stem (XEN) cells is delayed. Mechanistically, we show the deregulation of genes that are associated with WNT signaling. This is further confirmed by cell surface proteome profiling, which identified a significant reduction of the WNT receptor FZD2 at the plasma membrane in Vmp1 and Tmem41b double mutant ESCs. Importantly, we show that transgenic expression of Fzd2 rescues XEN differentiation. Our findings identify the role of the lipid scramblases VMP1 and TMEM41b in WNT signaling during extra-embryonic endoderm development and characterize their distinct and overlapping functions.

developmental biology↗

bioRxiv2020

Polyploidy of semi-cloned embryos generated from parthenogenetic haploid embryonic stem cells

In mammals, the fusion of two gametes, an oocyte and a spermatozoon, during fertilization forms a totipotent zygote. There has been no reported case of natural parthenogenesis, in which embryos develop from unfertilized oocytes. The genome and epigenetic information of haploid gametes are crucial for the proper development of embryos. Haploid embryonic stem cells (haESCs) are unique stem cells established from uniparental blastocysts and possess only one set of chromosomes. Previous studies have shown that sperm or oocyte genome can be replaced by haESCs with or without manipulation of genomic imprinting for generation of mice. Recently, these remarkable semi-cloning methods have been applied for screening of key factors of mouse embryonic development. While haESCs have been applied as substitute of gametic genome, the fundamental mechanism how haESCs contribute to the genome of totipotent embryos is unclear. Here, we show the generation of fertile semi-cloned mice by injection of parthenogenetic haESCs (phaESCs) into oocytes after deletions of two differentially methylated regions (DMRs), the IG-DMR and H19-DMR. For characterizing the genome of semi-cloned embryos further we establish ESC lines from semi-cloned blastocysts. We report that polyploid karyotypes are observed frequently in semi-cloned ESCs (scESCs). Our results confirm that mitotically arrested phaESCs provide high efficiency for semi-cloning when the IG-DMR and H19-DMR are deleted. In addition, we highlight the occurrence of polyploidy that needs to be considered for further improvement for development of semi-cloned embryos derived by haESC injection.

developmental biology↗

bioRxiv2020

Tmed2 regulates Smoothened trafficking and Hedgehog signalling

Hedgehog (HH) signalling plays a key role in embryonic pattering and stem cell differentiation. Compounds that selectively bind Smoothened (SMO) can induce cell death in mouse embryonic stem cells (ESCs). Here we perform a genetic screen in haploid ESCs and discover that SMO inhibits a cell death pathway that resembles dissociation induced death of human ESCs and Anoikis. In mouse ESCs, SMO acts through a G-protein coupled mechanism that is independent of GLI activation. Our screen also identifies the Golgi proteins Tmed2 and Tmed10. We show that TMED2 binds SMO and controls its abundance at the plasma membrane. In neural differentiation and neural tube pattering Tmed2 acts as a repressor of HH signalling strength. We demonstrate that the interaction between SMO and TMED2 is regulated by HH signalling suggesting SMO release form the ER-Golgi is critical for controlling G-protein and GLI mediated functions of mammalian HH signalling.

cell biology↗

bioRxiv2018

Structural basis of sterol recognition by human hedgehog receptor PTCH1

Hedgehog signaling is central in embryonic development and tissue regeneration. Disruption of the pathway is linked to genetic diseases and cancer. Binding of the secreted ligand, Sonic hedgehog (ShhN) to its receptor Patched (PTCH1) activates the signaling pathway. Here, we describe a 3.4 [A] cryo-EM structure of the human PTCH1 bound to ShhNC24II, a modified hedgehog ligand mimicking its palmitoylated form. The membrane-embedded part of PTCH1 is surrounded by ten sterol molecules at the inner and outer lipid bilayer portion of the protein. The annular sterols interact at multiple sites with both the sterol sensing domain, SSD, and the SSD-like domain, SSDL, which are located on opposite sides of PTCH1. The structure reveals a possible route for sterol translocation across the lipid bilayer by PTCH1 and homologous transporters.

biochemistry↗