TMEM16F phospholipid scramblase mediates trophoblast fusion and placental development

Abstract

<jats:title>Abstract</jats:title><jats:p>Cell-cell fusion or syncytialization is fundamental to the reproduction, development and homeostasis of multicellular organisms. In addition to various cell-type specific fusogenic proteins, cell surface externalization of phosphatidylserine (PS), a universal eat-me signal in apoptotic cells, has been observed in different cell-fusion events. Nevertheless, molecular underpinnings of PS externalization and cellular mechanisms of PS-facilitated cell-cell fusion are unclear. Here we report that TMEM16F, a Ca<jats:sup>2+</jats:sup>-activated phospholipid scramblase (CaPLSase), plays an indispensable role in placental trophoblast fusion by translocating PS to the cell surface independent of apoptosis. Consistent with its essential role in trophoblast fusion, the placentas from TMEM16F-deficient mice exhibit deficiency in syncytialization, placental developmental defects and perinatal lethality. Our findings thus identify a cell-cell fusion mechanism by which TMEM16F CaPLSase-dependent externalization of PS serves as a critical cell fusion signal to facilitate trophoblast syncytialization and placental development.</jats:p>

Functional coupling between TRPV4 channel and TMEM16F modulates human trophoblast fusion

“…The BeWo cell line was a gift from Dr. Sallie Permar at Duke University and was authenticated by the Duke University DNA Analysis Facility. The TMEM16F KO BeWo cell line was generated by sgRNAs targeting exon 2 as described previously (12). The Human placenta tissue and primary cultured human trophoblast cells.…”

“…For each treatment group, six random fields of view were acquired using Zeiss 780 inverted confocal microscope. Cell fusion is quantified by calculating the fusion index (FI) as described previously (12). FI is calculated as:…”

“…Immunostaining. The mCherry-TMEM16F expressing BeWo TMEM16F knockout (KO) cells were generated by transduction of mCherry-TMEM16F containing lentiviral particles as described previously (12). Flag-tagged R. norvegicus TRPV4 plasmids (Addgene, # 45751) were transiently transfected to the HEK293T cells by using X-tremeGENE360 transfection reagent (Millipore-Sigma).…”

“…Different from the CaCCs that can be readily activated by both Ca 2+ entry through Ca 2+ channels and Ca 2+ release from internal stores (9,(19)(20)(21)(22)(23)(24), TMEM16F activation usually requires more robust and sustained intracellular Ca 2+ elevation (3,4,8,9,(25)(26)(27)(28)(29). To induce higher intracellular Ca 2+ levels, Ca 2+ ionophores have been widely used to artificially activate TMEM16F and study its biophysical properties or its functional expression in native cells (3,5,8,12).…”

Gating and Regulatory Mechanisms of TMEM16 Ion Channels and Scramblases

“…As passive phospholipid transporters, TMEM16 CaPLSases can efficiently translocate phospholipids at high speed (4.5 × 10 4 phospholipids per second for TMEM16F; Watanabe et al, 2018). Therefore, activation of TMEM16 CaPLSases leads to rapid collapse of membrane phospholipid asymmetry, which can trigger a plethora of cellular responses and physiological functions, such as blood coagulation (Suzuki et al, 2010;Yang et al, 2012), microparticle release (Fujii et al, 2015), membrane repair (Wu et al, 2020), sheddase activation (Sommer et al, 2016;Veit et al, 2018;Bleibaum et al, 2019), endosomal sorting (Petkovic et al, 2020), cell-cell fusion (Griffin et al, 2016;Whitlock et al, 2018;Zhang et al, 2020;Braga et al, 2021), and viral infection (Bevers and Williamson, 2016;Zaitseva et al, 2017;Younan et al, 2018). While the list of new biological functions of TMEM16 CaPLSases and CaCCs keeps growing, their importance in human health and disease has become apparent, as malfunctions in TMEM16 proteins have been implicated in human diseases, including asthma, cancer, bleeding disorders, muscular dystrophy, arthritis, epilepsy, dystonia, and ataxia (Duran and Hartzell, 2011;Pedemonte and Galietta, 2014;Oh and Jung, 2016;Crottes and Jan, 2019).…”