Geminin Is Required for Zygotic Gene Expression at the Xenopus Mid-Blastula Transition

In many organisms early development is under control of the maternal genome and zygotic gene expression is delayed until the mid-blastula transition (MBT). As zygotic transcription initiates, cell cycle checkpoints become activated and the tempo of cell division slows. The mechanisms that activate zygotic transcription at the MBT are incompletely understood, but they are of interest because they may resemble mechanisms that cause stem cells to stop dividing and terminally differentiate. The unstable regulatory protein Geminin is thought to coordinate cell division with cell differentiation. Geminin is a bi-functional protein. It prevents a second round of DNA replication during S and G2 phase by binding and inhibiting the essential replication factor Cdt1. Geminin also binds and inhibits a number of transcription factors and chromatin remodeling proteins and is thought to keep dividing cells in an undifferentiated state. We previously found that the cells of Geminin-deficient Xenopus embryos arrest in G2 phase just after the MBT then disintegrate at the onset of gastrulation. Here we report that they also fail to express most zygotic genes. The gene expression defect is cell-autonomous and is reproduced by over-expressing Cdt1 or by incubating the embryos in hydroxyurea. Geminin deficient and hydroxyurea-treated blastomeres accumulate DNA damage in the form of double stranded breaks. Bypassing the Chk1 pathway overcomes the cell cycle arrest caused by Geminin depletion but does not restore zygotic gene expression. In fact, bypassing the Chk1 pathway by itself induces double stranded breaks and abolishes zygotic transcription. We did not find evidence that Geminin has a replication-independent effect on transcription. We conclude that Geminin is required to maintain genome integrity during the rapid cleavage divisions, and that DNA damage disrupts zygotic gene transcription at the MBT, probably through activation of DNA damage checkpoint pathways

Integrin α5β1 Function Is Regulated by XGIPC/kermit2 Mediated Endocytosis during Xenopus laevis Gastrulation

During Xenopus gastrulation α5β1 integrin function is modulated in a temporally and spatially restricted manner, however, the regulatory mechanisms behind this regulation remain uncharacterized. Here we report that XGIPC/kermit2 binds to the cytoplasmic domain of the α5 subunit and regulates the activity of α5β1 integrin. The interaction of kermit2 with α5β1 is essential for fibronectin (FN) matrix assembly during the early stages of gastrulation. We further demonstrate that kermit2 regulates α5β1 integrin endocytosis downstream of activin signaling. Inhibition of kermit2 function impairs cell migration but not adhesion to FN substrates indicating that integrin recycling is essential for mesoderm cell migration. Furthermore, we find that the α5β1 integrin is colocalized with kermit2 and Rab 21 in embryonic and XTC cells. These data support a model where region specific mesoderm induction acts through kermit2 to regulate the temporally and spatially restricted changes in adhesive properties of the α5β1 integrin through receptor endocytosis

When What's Left Is Right: Visuomotor Transformations in an Aged Population

Background: There has been little consensus as to whether age-related visuomotor adaptation effects are readily observable. Some studies have found slower adaptation, and/or reduced overall levels. In contrast, other methodologically similar studies have found no such evidence of aging effects on visuomotor adaptation. A crucial early step in successful adaptation is the ability to perform the necessary transformation to complete the task at hand. The present study describes the use of a viewing window paradigm to examine the effects of aging in a visuomotor transformation task. Methods: Two groups of participants, a young adult control group (age range 18–33 years old, mean age = 22) and an older adult group (age range 62–74, mean age = 68) completed a viewing window task that was controlled by the user via a computer touchscreen. Four visuomotor ‘‘flip’ ’ conditions were created by varying the relationship between the participant’s movement, and the resultant on-screen movement of the viewing window: 1) No flip 2) X-Axis and Y-axis body movements resulted in the opposite direction of movement of the viewing window. In each of the 3) Flip-X and 4) Flip-Y conditions, the solitary X- or Y-axes were reversed. Response times and movement of the window were recorded. Conclusions: Older participants demonstrated impairments in performing a required visuomotor transformation, as evidenced by more complex scanning patterns and longer scanning times when compared to younger control participants. These results provide additional evidence that the mechanisms involved in visuomotor transformation are negatively affected by age

Akt1-associated actomyosin remodelling is required for nuclear lamina dispersal and nuclear shrinkage in epidermal terminal differentiation

Keratinocyte cornification and epidermal barrier formation are tightly controlled processes, which require complete degradation of intracellular organelles, including removal of keratinocyte nuclei. Keratinocyte nuclear destruction requires Akt1-dependent phosphorylation and degradation of the nuclear lamina protein, Lamin A/C, essential for nuclear integrity. However, the molecular mechanisms that result in complete nuclear removal and their regulation are not well defined. Post-confluent cultures of rat epidermal keratinocytes (REKs) undergo spontaneous and complete differentiation, allowing visualisation and perturbation of the differentiation process in vitro. We demonstrate that there is dispersal of phosphorylated Lamin A/C to structures throughout the cytoplasm in differentiating keratinocytes. We show that the dispersal of phosphorylated Lamin A/C is Akt1-dependent and these structures are specific for the removal of Lamin A/C from the nuclear lamina; nuclear contents and Lamin B were not present in these structures. Immunoprecipitation identified a group of functionally related Akt1 target proteins involved in Lamin A/C dispersal, including actin, which forms cytoskeletal microfilaments, Arp3, required for actin filament nucleation, and Myh9, a component of myosin IIa, a molecular motor that can translocate along actin filaments. Disruption of actin filament polymerisation, nucleation or myosin IIa activity prevented formation and dispersal of cytoplasmic Lamin A/C structures. Live imaging of keratinocytes expressing fluorescently tagged nuclear proteins showed a nuclear volume reduction step taking less than 40 min precedes final nuclear destruction. Preventing Akt1-dependent Lamin A/C phosphorylation and disrupting cytoskeletal Akt1-associated proteins prevented nuclear volume reduction. We propose keratinocyte nuclear destruction and differentiation requires myosin II activity and the actin cytoskeleton for two intermediate processes: Lamin A/C dispersal and rapid nuclear volume reduction

Assembly and dynamics of the bacteriophage T4 homologous recombination machinery

Homologous recombination (HR), a process involving the physical exchange of strands between homologous or nearly homologous DNA molecules, is critical for maintaining the genetic diversity and genome stability of species. Bacteriophage T4 is one of the classic systems for studies of homologous recombination. T4 uses HR for high-frequency genetic exchanges, for homology-directed DNA repair (HDR) processes including DNA double-strand break repair, and for the initiation of DNA replication (RDR). T4 recombination proteins are expressed at high levels during T4 infection in E. coli, and share strong sequence, structural, and/or functional conservation with their counterparts in cellular organisms. Biochemical studies of T4 recombination have provided key insights on DNA strand exchange mechanisms, on the structure and function of recombination proteins, and on the coordination of recombination and DNA synthesis activities during RDR and HDR. Recent years have seen the development of detailed biochemical models for the assembly and dynamics of presynaptic filaments in the T4 recombination system, for the atomic structure of T4 UvsX recombinase, and for the roles of DNA helicases in T4 recombination. The goal of this chapter is to review these recent advances and their implications for HR and HDR mechanisms in all organisms

Msb2 Shedding Protects Candida albicans against Antimicrobial Peptides

Msb2 is a sensor protein in the plasma membrane of fungi. In the human fungal pathogen C. albicans Msb2 signals via the Cek1 MAP kinase pathway to maintain cell wall integrity and allow filamentous growth. Msb2 doubly epitope-tagged in its large extracellular and small cytoplasmic domain was efficiently cleaved during liquid and surface growth and the extracellular domain was almost quantitatively released into the growth medium. Msb2 cleavage was independent of proteases Sap9, Sap10 and Kex2. Secreted Msb2 was highly O-glycosylated by protein mannosyltransferases including Pmt1 resulting in an apparent molecular mass of >400 kDa. Deletion analyses revealed that the transmembrane region is required for Msb2 function, while the large N-terminal and the small cytoplasmic region function to downregulate Msb2 signaling or, respectively, allow its induction by tunicamycin. Purified extracellular Msb2 domain protected fungal and bacterial cells effectively from antimicrobial peptides (AMPs) histatin-5 and LL-37. AMP inactivation was not due to degradation but depended on the quantity and length of the Msb2 glycofragment. C. albicans msb2 mutants were supersensitive to LL-37 but not histatin-5, suggesting that secreted rather than cell-associated Msb2 determines AMP protection. Thus, in addition to its sensor function Msb2 has a second activity because shedding of its glycofragment generates AMP quorum resistance

A quantitative analysis of complexity of human pathogen-specific CD4 T cell responses in healthy M. tuberculosis infected South Africans

Author Summary: Human pathogen-specific immune responses are tremendously complex and the techniques to study them ever expanding. There is an urgent need for a quantitative analysis and better understanding of pathogen-specific immune responses. Mycobacterium tuberculosis (Mtb) is one of the leading causes of mortality due to an infectious agent worldwide. Here, we were able to quantify the Mtb-specific response in healthy individuals with Mtb infection from South Africa. The response is highly diverse and 66 epitopes are required to capture 80% of the total reactivity. Our study also show that the majority of the identified epitopes are restricted by multiple HLA alleles. Thus, technical advances are required to capture and characterize the complete pathogen-specific response. This study demonstrates further that the approach combining identified epitopes into "megapools" allows capturing a large fraction of the total reactivity. This suggests that this technique is generally applicable to the characterization of immunity to other complex pathogens. Together, our data provide for the first time a quantitative analysis of the complex pathogen-specific T cell response and provide a new understanding of human infections in a natural infection setting

Aggravation of Chronic Stress Effects on Hippocampal Neurogenesis and Spatial Memory in LPA1 Receptor Knockout Mice

The lysophosphatidic acid LPA₁ receptor regulates plasticity and neurogenesis in the adult hippocampus. Here, we studied whether absence of the LPA₁ receptor modulated the detrimental effects of chronic stress on hippocampal neurogenesis and spatial memory.Male LPA₁-null (NULL) and wild-type (WT) mice were assigned to control or chronic stress conditions (21 days of restraint, 3 h/day). Immunohistochemistry for bromodeoxyuridine and endogenous markers was performed to examine hippocampal cell proliferation, survival, number and maturation of young neurons, hippocampal structure and apoptosis in the hippocampus. Corticosterone levels were measured in another a separate cohort of mice. Finally, the hole-board test assessed spatial reference and working memory. Under control conditions, NULL mice showed reduced cell proliferation, a defective population of young neurons, reduced hippocampal volume and moderate spatial memory deficits. However, the primary result is that chronic stress impaired hippocampal neurogenesis in NULLs more severely than in WT mice in terms of cell proliferation; apoptosis; the number and maturation of young neurons; and both the volume and neuronal density in the granular zone. Only stressed NULLs presented hypocortisolemia. Moreover, a dramatic deficit in spatial reference memory consolidation was observed in chronically stressed NULL mice, which was in contrast to the minor effect observed in stressed WT mice.These results reveal that the absence of the LPA₁ receptor aggravates the chronic stress-induced impairment to hippocampal neurogenesis and its dependent functions. Thus, modulation of the LPA₁ receptor pathway may be of interest with respect to the treatment of stress-induced hippocampal pathology

Multi-messenger observations of a binary neutron star merger

On 2017 August 17 a binary neutron star coalescence candidate (later designated GW170817) with merger time 12:41:04 UTC was observed through gravitational waves by the Advanced LIGO and Advanced Virgo detectors. The Fermi Gamma-ray Burst Monitor independently detected a gamma-ray burst (GRB 170817A) with a time delay of ~1.7 s with respect to the merger time. From the gravitational-wave signal, the source was initially localized to a sky region of 31 deg2 at a luminosity distance of 40+8-8 Mpc and with component masses consistent with neutron stars. The component masses were later measured to be in the range 0.86 to 2.26 Mo. An extensive observing campaign was launched across the electromagnetic spectrum leading to the discovery of a bright optical transient (SSS17a, now with the IAU identification of AT 2017gfo) in NGC 4993 (at ~40 Mpc) less than 11 hours after the merger by the One- Meter, Two Hemisphere (1M2H) team using the 1 m Swope Telescope. The optical transient was independently detected by multiple teams within an hour. Subsequent observations targeted the object and its environment. Early ultraviolet observations revealed a blue transient that faded within 48 hours. Optical and infrared observations showed a redward evolution over ~10 days. Following early non-detections, X-ray and radio emission were discovered at the transient’s position ~9 and ~16 days, respectively, after the merger. Both the X-ray and radio emission likely arise from a physical process that is distinct from the one that generates the UV/optical/near-infrared emission. No ultra-high-energy gamma-rays and no neutrino candidates consistent with the source were found in follow-up searches. These observations support the hypothesis that GW170817 was produced by the merger of two neutron stars in NGC4993 followed by a short gamma-ray burst (GRB 170817A) and a kilonova/macronova powered by the radioactive decay of r-process nuclei synthesized in the ejecta