We present a DNA library preparation method that has allowed us to reconstruct a high-coverage (30×) genome sequence of a Denisovan, an extinct relative of Neandertals. The quality of this genome allows a direct estimation of Denisovan heterozygosity indicating that genetic diversity in these archaic hominins was extremely low. It also allows tentative dating of the specimen on the basis of “missing evolution” in its genome, detailed measurements of Denisovan and Neandertal admixture into present-day human populations, and the generation of a near-complete catalog of genetic changes that swept to high frequency in modern humans since their divergence from Denisovans.

https://science.sciencemag.org/content/sci/338/6104/222.full.pdf

A high-coverage genome sequence from an archaic Denisovan individual

Membrane fusion can occur between cells, between different intracellular compartments, between intracellular compartments and the plasma membrane and between lipid-bound structures such as viral particles and cellular membranes In order for membranes to fuse they must first be brought together The more highly curved a membrane is, the more fusogenic it becomes We discuss how proteins, including SNAREs, synaptotagmins and viral fusion proteins, might mediate close membrane apposition and induction of membrane curvature to drive diverse fusion processes We also highlight common principles that can be derived from the analysis of the role of these proteins

Mechanisms of membrane fusion: disparate players and common principles

Tumours progress through a cascade of events that enable the formation of metastases. Some of the components that are required for this fatal process are well established. Tetraspanins, however, have only recently received attention as both metastasis suppressors and metastasis promoters. This late appreciation is probably due to their capacity to associate with various molecules, which they recruit into special membrane microdomains, and their abundant presence in tumour-derived small vesicles that aid intercellular communication. It is reasonable to assume that differences in the membrane and vesicular web components that associate with individual tetraspanins account for their differing abilities to promote and suppress metastasis.

Tetraspanins: push and pull in suppressing and promoting metastasis

Fertilization occurs when sperm and egg recognize each other and fuse to form a new, genetically distinct organism. The molecular basis of sperm-egg recognition is unknown, but is likely to require interactions between receptor proteins displayed on their surface. Izumo1 is an essential sperm cell-surface protein, but its receptor on the egg has not been described. Here we identify folate receptor 4 (Folr4) as the receptor for Izumo1 on the mouse egg, and propose to rename it Juno. We show that the Izumo1-Juno interaction is conserved within several mammalian species, including humans. Female mice lacking Juno are infertile and Juno-deficient eggs do not fuse with normal sperm. Rapid shedding of Juno from the oolemma after fertilization suggests a mechanism for the membrane block to polyspermy, ensuring eggs normally fuse with just a single sperm. Our discovery of an essential receptor pair at the nexus of conception provides opportunities for the rational development of new fertility treatments and contraceptives.

/pdf/juno-is-the-egg-izumo-receptor-and-is-essential-for-4yu3eain2q.pdf

Juno is the egg Izumo receptor and is essential for mammalian fertilization

Despite high expression levels at the plasma membrane or in intracellular vesicles, tetraspanins remain among the most mysterious transmembrane molecules 20 years after their discovery. Several genetic studies in mammals and invertebrates have demonstrated key physiological roles for some of these tetraspanins, in particular in the immune response, sperm-egg fusion, photoreceptor function and the normal function of certain epithelia. Other studies have highlighted their ability to modulate cell migration and metastasis formation. Their role in the propagation of infectious agents has drawn recent attention, with evidence for HIV budding in tetraspanin-enriched plasma membrane domains. Infection of hepatocytic cells by two major pathogens, the hepatitis C virus and the malaria parasite, also requires the tetraspanin CD81. The function of tetraspanins is thought to be linked to their ability to associate with one another and a wealth of other integral proteins, thereby building up an interacting network or 'tetraspanin web'. On the basis of the biochemical dissection of the tetraspanin web and recent analysis of the dynamics of some of its constituents, we propose that tetraspanins tightly regulate transient interactions between a variety of molecules and as such favour the efficient assembly of specialized structures upon proper stimulation.

Lateral organization of membrane proteins: tetraspanins spin their web.

Oocyte CD9 is enriched on the microvillar membrane and required for normal microvillar shape and distribution

Izumo, a sperm membrane protein, is essential for gamete fusion in the mouse. It has an Immunoglobulin (Ig) domain and an N-terminal domain for which neither the functions nor homologous sequences are known. In the present work we identified three novel proteins showing an N-terminal domain with significant homology to the N-terminal domain of Izumo. We named this region "Izumo domain," and the novel proteins "Izumo 2," "Izumo 3," and "Izumo 4," retaining "Izumo 1" for the first described member of the family. Izumo 1-3 are transmembrane proteins expressed specifically in the testis, and Izumo 4 is a soluble protein expressed in the testis and in other tissues. Electrophoresis under mildly denaturing conditions, followed by Western blot analysis, showed that Izumo 1, 3, and 4 formed protein complexes on sperm, Izumo 1 forming several larger complexes and Izumo 3 and 4 forming a single larger complex. Studies using different recombinant Izumo constructs suggested the Izumo domain possesses the ability to form dimers, whereas the transmembrane domain or the cytoplasmic domain or both of Izumo 1 are required for the formation of multimers of higher order. Co-immunoprecipitation studies showed the presence of other sperm proteins associated with Izumo 1, suggesting Izumo 1 forms a multiprotein membrane complex. Our results raise the possibility that Izumo 1 might be involved in organizing or stabilizing a multiprotein complex essential for the function of the membrane fusion machinery.

Izumo is part of a multiprotein family whose members form large complexes on mammalian sperm

Pancreatic cancer is a highly aggressive disease with rapid invasion and early encasement of blood vessels. Hence, levels of circulating nucleic acids and tumor-associated mutations in them may have clinical importance. We analyzed the levels of circulating tumor DNA and oncogenic k-ras mutation in plasma of patients with pancreatic cancer and correlated their levels with survival and clinicopathological parameters. Higher levels of plasma DNA (>62 ng/mL) was found to associate significantly with lower overall survival time (p=.002), presence of vascular encasement (p=.030) and metastasis (p=.001). However, k-ras mutation status did not correlate with any of the clinicopathological parameters or survival. We conclude that circulating DNA in plasma can be an important predictor of prognosis in pancreatic cancer.

High Levels of Cell-Free Circulating Nucleic Acids in Pancreatic Cancer are Associated With Vascular Encasement, Metastasis and Poor Survival

Network of cytokines, integrins and hormones in human trophoblast cells.

Several labs have shown that oocytes lacking the tetraspanin membrane protein, CD9, rarely fuse with sperm (reviewed in Primakoff and Myles, 2007). Recently, Miyado et al. (2008) reported striking results, aimed at further defining the mechanism of sperm–oocyte fusion. They found that fusion capability of CD9 null oocytes was rescued when they were inseminated in the presence of wild-type oocytes. Their interpretation of this result is that wild-type oocytes release CD9-containing membrane vesicles, termed ‘‘exosomes,’’ that associate with sperm and this association is required to produce sperm capable of fusion. In particular, exosomes derived from wild-type CD9 oocytes can associate with sperm and make them able to fuse with either wild-type or CD9 null oocytes (Miyado et al., 2008). Our lab has been studying the role of CD9 in mouse sperm–oocyte fusion and has been interested in extending these results. Following the experimental design used by Miyado et al., CD9 null oocytes were inseminated in the presence of wild-type oocytes, and fusion with both types of oocytes was scored. In three separate experiments, we found the expected rates of fusion between sperm and wild -type oocytes (78/108 oocytes fused). In contrast to the results of Miyado et al., we found that only one of 67 CD9 null oocytes fused with a sperm (Table 1). Since we observed robust fusion of sperm with wild-type oocytes in these tests, we believe our assay conditions allow definitive findings (Table 1). Therefore, we draw attention of other investigators to the fact that there are now two distinct datasets concerning the possible rescue of fusion of CD9 null oocytes in the presence of wild-type oocytes. While we can only speculate why different results are found, it is possible that some difference in the experimental conditions used by Miyado et al. and our experiments is responsible. Though such a difference in the precise experimental conditions might be expected to yield small differences in the data, the discrepancy between the datasets is large. In order to explain this large difference between the two sets of results, a new idea or additional experiments in other laboratories may need to be developed.

Surabhi Gupta

Papers

Oocyte CD9 is enriched on the microvillar membrane and required for normal microvillar shape and distribution

Izumo is part of a multiprotein family whose members form large complexes on mammalian sperm

High Levels of Cell-Free Circulating Nucleic Acids in Pancreatic Cancer are Associated With Vascular Encasement, Metastasis and Poor Survival

Network of cytokines, integrins and hormones in human trophoblast cells.

Can the presence of wild‐type oocytes during insemination rescue the fusion defect of CD9 null oocytes?