Telomerase Interaction Partners–Insight from Plants

Telomerase, an essential enzyme that maintains chromosome ends, is important for genome integrity and organism development. Various hypotheses have been proposed in human, ciliate and yeast systems to explain the coordination of telomerase holoenzyme assembly and the timing of telomerase performance at telomeres during DNA replication or repair. However, a general model is still unclear, especially pathways connecting telomerase with proposed non-telomeric functions. To strengthen our understanding of telomerase function during its intracellular life, we report on interactions of several groups of proteins with the Arabidopsis telomerase protein subunit (AtTERT) and/or a component of telomerase holoenzyme, POT1a protein. Among these are the nucleosome assembly proteins (NAP) and the minichromosome maintenance (MCM) system, which reveal new insights into the telomerase interaction network with links to telomere chromatin assembly and replication. A targeted investigation of 176 candidate proteins demonstrated numerous interactions with nucleolar, transport and ribosomal proteins, as well as molecular chaperones, shedding light on interactions during telomerase biogenesis. We further identified protein domains responsible for binding and analyzed the subcellular localization of these interactions. Moreover, additional interaction networks of NAP proteins and the DOMINO1 protein were identified. Our data support an image of functional telomerase contacts with multiprotein complexes including chromatin remodeling and cell differentiation pathways.

Flavivirus replication kinetics in early-term placental cell lines with different differentiation pathways

Background The uncontrollable spread of Zika virus (ZIKV) in the Americas during 2015–2017, and its causal link to microcephaly in newborns and Guillain-Barré syndrome in adults, led the World Health Organisation to declare it a global public health emergency. One of the most notable features of ZIKV pathogenesis was the ability of the virus to pass the placental barrier to infect the growing foetus. This pathogenic trait had not been observed previously for medically important flaviviruses, including dengue and yellow fever viruses. Methods In this study we evaluated the replication kinetics of ZIKV and the related encephalitic flavivirus West Nile strain Kunjin virus (WNVKUN) in early-term placental cell lines. Results We have observed that WNVKUN in fact replicates with a greater rate and to higher titres that ZIKV in these cell lines. Conclusions These results would indicate the potential for all flaviviruses to replicate in placental tissue but it is the ability to cross the placenta itself that is the restrictive factor in the clinical progression and presentation of congenital Zika syndrome.

Mechanics-driven nuclear localization of YAP can be reversed by N-cadherin ligation in mesenchymal stem cells

Mesenchymal stem cells adopt differentiation pathways based upon cumulative effects of mechanosensing. A cell’s mechanical microenvironment changes substantially over the course of development, beginning from the early stages in which cells are typically surrounded by other cells and continuing through later stages in which cells are typically surrounded by extracellular matrix. How cells erase the memory of some of these mechanical microenvironments while locking in memory of others is unknown. Here, we develop a material and culture system for modifying and measuring the degree to which cells retain cumulative effects of mechanosensing. Using this system, we discover that effects of the RGD adhesive motif of fibronectin (representative of extracellular matrix), known to impart what is often termed “mechanical memory” in mesenchymal stem cells via nuclear YAP localization, are erased by the HAVDI adhesive motif of the N-cadherin (representative of cell-cell contacts). These effects can be explained by a motor clutch model that relates cellular traction force, nuclear deformation, and resulting nuclear YAP re-localization. Results demonstrate that controlled storage and removal of proteins associated with mechanical memory in mesenchymal stem cells is possible through defined and programmable material systems.

Profiling of mature stage human breastmilk cells identifies host-defense lactocyte sub-populations

Breastmilk is chock-full of nutrients, immunological factors, and cells that aid infant development. Maternal cells are the least studied breastmilk component, and their unique properties are difficult to identify using traditional techniques. Here, we characterized the cells in mature stage breastmilk from healthy donors at the protein, gene, and transcriptome levels. Holistic analysis of flow cytometry, qPCR, and single cell RNA sequencing data identified the predominant cell population as epithelial with smaller populations of macrophages and T cells. Two percent of epithelial cells expressed four stem cell markers: SOX2, TRA-1-60, NANOG, and SSEA4. Furthermore, milk contained six distinct epithelial lactocyte sub-populations, including three previously unidentified sub-populations programmed towards host-defense and intestinal development. Pseudotime analysis delineated the differentiation pathways of epithelial progenitors. Together, these data define healthy human maternal breastmilk cells and provide a basis for their application in maternal and infant medicine.

Single-cell transcriptomic analysis reveals disparate effector differentiation pathways in human Treg compartment

Human FOXP3+ regulatory T (Treg) cells are central to immune tolerance. However, their heterogeneity and differentiation remain incompletely understood. Here we use single-cell RNA and T cell receptor sequencing to resolve Treg cells from healthy individuals and patients with or without acute graft-versus-host disease (aGVHD) who undergo stem cell transplantation. These analyses, combined with functional assays, separate Treg cells into naïve, activated, and effector stages, and resolve the HLA-DRhi, LIMS1hi, highly suppressive FOXP3hi, and highly proliferative MKI67hi effector subsets. Trajectory analysis assembles Treg subsets into two differentiation paths (I/II) with distinctive phenotypic and functional programs, ending with the FOXP3hi and MKI67hi subsets, respectively. Transcription factors FOXP3 and SUB1 contribute to some Path I and Path II phenotypes, respectively. These FOXP3hi and MKI67hi subsets and two differentiation pathways are conserved in transplanted patients, despite having functional and migratory impairments under aGVHD. These findings expand the understanding of Treg cell heterogeneity and differentiation and provide a single-cell atlas for the dissection of Treg complexity in health and disease.

Single-cell RNA-seq analysis reveals penaeid shrimp hemocyte subpopulations and cell differentiation process

Crustacean aquaculture is expected to be a major source of fishery commodities in the near future. Hemocytes are key players of the immune system in shrimps; however, their classification, maturation, and differentiation are still under debate. To date, only discrete and inconsistent information on the classification of shrimp hemocytes has been reported, showing that the morphological characteristics are not sufficient to resolve their actual roles. Our present study using single-cell RNA sequencing, revealed six types of hemocytes of Marsupenaeus japonicus based on their transcriptional profiles. We identified markers of each subpopulation and predicted the differentiation pathways involved in their maturation. We also predicted cell growth factors that might play crucial roles in hemocyte differentiation. Different immune roles among these subpopulations were suggested from the analysis of differentially expressed immune-related genes. These results provide a unified classification of shrimp hemocytes, which improves the understanding of its immune system.

NF-κB and TNF Affect the Astrocytic Differentiation from Neural Stem Cells

The NF-κB signaling pathway is crucial during development and inflammatory processes. We have previously shown that NF-κB activation induces dedifferentiation of astrocytes into neural progenitor cells (NPCs). Here, we provide evidence that the NF-κB pathway plays also a fundamental role during the differentiation of NPCs into astrocytes. First, we show that the NF-κB pathway is essential to initiate astrocytic differentiation as its early inhibition induces NPC apoptosis and impedes their differentiation. Second, we demonstrate that persistent NF-κB activation affects NPC-derived astrocyte differentiation. Tumor necrosis factor (TNF)-treated NPCs show NF-κB activation, maintain their multipotential and proliferation properties, display persistent expression of immature markers and inhibit astrocyte markers. Third, we analyze the effect of NF-κB activation on the main known astrocytic differentiation pathways, such as NOTCH and JAK-STAT. Our findings suggest that the NF-κB pathway plays a dual fundamental role during NPC differentiation into astrocytes: it promotes astrocyte specification, but its persistent activation impedes their differentiation.

Early germline differentiation in bivalves: TDRD7 as a candidate investigational unit for Ruditapes philippinarum germ granule assembly

The germline is a key feature of sexual animals and the ways in which it separates from the soma differ widely across Metazoa. However, at least at some point during germline differentiation, some cytoplasmic supramolecular structures (collectively called germ plasm-related structures) are present and involved in its specification and/or differentiation. The factors involved in the assembly of these granular structures are various and non-ubiquitous among animals, even if some functional patterns and the presence of certain domains appear to be shared among some. For instance, the LOTUS domain is shared by Oskar, the Holometabola germ plasm master regulator, and some Tudor-family proteins assessed as being involved in the proper assembly of germ granules of different animals. Here, we looked for the presence of LOTUS-containing proteins in the transcriptome of Ruditapes philippinarum (Bivalvia). Such species is of particular interest because it displays annual renewal of gonads, sided by the renewal of germline differentiation pathways. Moreover, previous works have identified in its early germ cells cytoplasmic granules containing germline determinants. We selected the orthologue of TDRD7 as a candidate involved in the early steps of germline differentiation through bioinformatic predictions and immunohistological patterning (immunohistochemistry and immunofluorescence). We observed the expression of the protein in putative precursors of germline cells, upstream to the germline marker Vasa. This, added to the fact that orthologues of this protein are involved in the assembly of germ granules in mouse, zebrafish, and fly, makes it a worthy study unit for investigations on the formation of such structures in bivalves.

A bird-like genome from a frog: Mechanisms of genome size reduction in the ornate burrowing frog, Platyplectrum ornatum

The diversity of genome sizes across the tree of life is of key interest in evolutionary biology. Various correlates of variation in genome size, such as accumulation of transposable elements (TEs) or rate of DNA gain and loss, are well known, but the underlying molecular mechanisms driving or constraining genome size are poorly understood. Here, we study one of the smallest genomes among frogs characterized thus far, that of the ornate burrowing frog (Platyplectrum ornatum) from Australia, and compare it to other published frog and vertebrate genomes to examine the forces driving reduction in genome size. At ∼1.06 gigabases (Gb), the P. ornatum genome is like that of birds, revealing four major mechanisms underlying TE dynamics: reduced abundance of all major classes of TEs; increased net deletion bias in TEs; drastic reduction in intron lengths; and expansion via gene duplication of the repertoire of TE-suppressing Piwi genes, accompanied by increased expression of Piwi-interacting RNA (piRNA)-based TE-silencing pathway genes in germline cells. Transcriptomes from multiple tissues in both sexes corroborate these results and provide insight into sex-differentiation pathways in Platyplectrum. Genome skimming of two closely related frog species (Lechriodus fletcheri and Limnodynastes fletcheri) confirms a reduction in TEs as a major driver of genome reduction in Platyplectrum and supports a macroevolutionary scenario of small genome size in frogs driven by convergence in life history, especially rapid tadpole development and tadpole diet. The P. ornatum genome offers a model for future comparative studies on mechanisms of genome size reduction in amphibians and vertebrates generally.