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The ancestral gamete fusion protein, HAP2/GCS1, plays an essential role in fertilization in a broad range of taxa. To identify factors that may regulate HAP2/GCS1 activity, we screened mutants of the ciliate Tetrahymena thermophila for behaviors that mimic Δhap2/gcs1 knockout phenotypes in this species. Using this approach, we identified two new genes, GFU1 and GFU2 , whose products are necessary for membrane pore formation following mating type recognition and adherence. GFU2 is predicted to be a single-pass transmembrane protein, while GFU1, though lacking obvious transmembrane domains, has the potential to interact directly with membrane phospholipids in the cytoplasm. Like Tetrahymena HAP2/GCS1, expression of GFU1 is required in both cells of a mating pair for efficient fusion to occur. To explain these bilateral requirements, we propose a model that invokes cooperativity between the fusion machinery on apposed membranes of mating cells and accounts for successful fertilization in Tetrahymena's multiple mating type system., Competing Interests: The authors declare no competing interests., (© 2024 The Authors.)

The ancestral gamete fusion protein, HAP2, catalyzes sperm-egg fusion in a broad range of taxa dating to the last eukaryotic common ancestor. Remarkably, HAP2 orthologs are structurally related to the class II fusogens of modern-day viruses, and recent studies make clear that these proteins utilize similar mechanisms to achieve membrane merger. To identify factors that may regulate HAP2 activity, we screened mutants of the ciliate Tetrahymena thermophila for behaviors that mimic Δhap2 knockout phenotypes in this species. Using this approach, we identified two new genes, GFU1 and GFU2 , whose products are necessary for the formation of membrane pores during fertilization and show that the product of a third gene, namely ZFR1 , may be involved in pore maintenance and/or expansion. Finally, we propose a model that explains cooperativity between the fusion machinery on apposed membranes of mating cells and accounts for successful fertilization in T. thermophila's multiple mating type system., Competing Interests: Competing Interests The authors declare no competing conflicts of interest.

Most, if not all the cellular requirements for fertilization and sexual reproduction arose early in evolution and are retained in extant lineages of single-celled organisms including a number of important model organism species. In recent years, work in two such species, the green alga, Chlamydomonas reinhardtii , and the free-living ciliate, Tetrahymena thermophila , have lent important new insights into the role of HAP2/GCS1 as a catalyst for gamete fusion in organisms ranging from protists to flowering plants and insects. Here we summarize the current state of knowledge around how mating types from these algal and ciliate systems recognize, adhere and fuse to one another, current gaps in our understanding of HAP2-mediated gamete fusion, and opportunities for applying what we know in practical terms, especially for the control of protozoan parasites., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Pinello and Clark.)

Union of two gametes to form a zygote is a defining event in the life of sexual eukaryotes, yet the mechanisms that underlie cell-cell fusion during fertilization remain poorly characterized. Here, in studies of fertilization in the green alga, Chlamydomonas, we report identification of a membrane protein on minus gametes, Minus Adhesion Receptor 1 (MAR1), that is essential for the membrane attachment with plus gametes that immediately precedes lipid bilayer merger. We show that MAR1 forms a receptor pair with previously identified receptor FUS1 on plus gametes, whose ectodomain architecture we find is identical to a sperm adhesion protein conserved throughout plant lineages. Strikingly, before fusion, MAR1 is biochemically and functionally associated with the ancient, evolutionarily conserved eukaryotic Class II fusion protein HAP2 on minus gametes. Thus, the integral membrane protein MAR1 provides a molecular link between membrane adhesion and bilayer merger during fertilization in Chlamydomonas., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Recognition and fusion between gametes during fertilization is an ancient process. Protein HAP2, recognized as the primordial eukaryotic gamete fusogen, is a structural homolog of viral class II fusion proteins. The mechanisms that regulate HAP2 function, and whether virus-fusion-like conformational changes are involved, however, have not been investigated. We report here that fusion between plus and minus gametes of the green alga Chlamydomonas indeed requires an obligate conformational rearrangement of HAP2 on minus gametes from a labile, prefusion form into the stable homotrimers observed in structural studies. Activation of HAP2 to undergo its fusogenic conformational change occurs only upon species-specific adhesion between the two gamete membranes. Following a molecular mechanism akin to fusion of enveloped viruses, the membrane insertion capacity of the fusion loop is required to couple formation of trimers to gamete fusion. Thus, species-specific membrane attachment is the gateway to fusion-driving HAP2 rearrangement into stable trimers., (© 2021. The Author(s).)

The conserved transmembrane protein, HAP2/GCS1, has been linked to fertility in a wide range of taxa and is hypothesized to be an ancient gamete fusogen. Using template-based structural homology modeling, we now show that the ectodomain of HAP2 orthologs from Tetrahymena thermophila and other species adopt a protein fold remarkably similar to the dengue virus E glycoprotein and related class II viral fusogens. To test the functional significance of this predicted structure, we developed a flow-cytometry-based assay that measures cytosolic exchange across the conjugation junction to rapidly probe the effects of HAP2 mutations in the Tetrahymena system. Using this assay, alterations to a region in and around a predicted "fusion loop" in T. thermophila HAP2 were found to abrogate membrane pore formation in mating cells. Consistent with this, a synthetic peptide corresponding to the HAP2 fusion loop was found to interact directly with model membranes in a variety of biophysical assays. These results raise interesting questions regarding the evolutionary relationships of class II membrane fusogens and harken back to a long-held argument that eukaryotic sex arose as the byproduct of selection for the horizontal transfer of a "selfish" genetic element from cell to cell via membrane fusion., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Neonates often generate incomplete immunity against intracellular pathogens, although the mechanism of this defect is poorly understood. An important question is whether the impaired development of memory CD8+ T cells in neonates is due to an immature priming environment or lymphocyte-intrinsic defects. In this article, we show that neonatal and adult CD8+ T cells adopted different fates when responding to equal amounts of stimulation in the same host. Whereas adult CD8+ T cells differentiated into a heterogeneous pool of effector and memory cells, neonatal CD8+ T cells preferentially gave rise to short-lived effector cells and exhibited a distinct gene expression profile. Surprisingly, impaired neonatal memory formation was not due to a lack of responsiveness, but instead because neonatal CD8+ T cells expanded more rapidly than adult cells and quickly became terminally differentiated. Collectively, these findings demonstrate that neonatal CD8+ T cells exhibit an imbalance in effector and memory CD8+ T cell differentiation, which impairs the formation of memory CD8+ T cells in early life., (Copyright © 2014 by The American Association of Immunologists, Inc.)