Cell polarity in fission yeast: a matter of confining, positioning, and switching growth zones

The two key processes in growth polarisation are the generation of a confined region and the correct positioning of that region. Fission yeast has greatly contributed to the study of cell polarisation, particularly in the aspect of growth site positioning, which involves the interphase microtubule cytoskeleton. Here we review the mechanisms of growth polarity in vegetatively growing fission yeast cells. These seemingly simple cells show astonishingly complex growth polarity behaviour, including polarity switching and integrating multiple levels of control by the cell cycle machinery. We aim to extract and highlight the underlying concepts and discuss these in context of current understanding; showing how relevant proteins are networked to integrate the various machineries.     

Cre recombinase resources for conditional mouse mutagenesis

Large scale international activities for systematic conditional mouse mutagenesis, exploiting advances in the sophisticated manipulation of the mouse genome, has established the mouse as the premier organism for developing models of human disease and drug action. Conditional mutagenesis is critical for the elucidation of the gene functions that exert pleiotropic effects in a variety of cell types and tissues throughout the life of the animal. The majority of new mouse mutants are therefore designed as conditional, activated only in a specific tissue (spatial control) and/or life stage (temporal control) through biogenic Cre/loxP technologies. The full power of conditional mutant mice can therefore only be exploited with the availability of well characterized mouse lines expressing Cre-recombinase in tissue, organ and cell type-specific patterns, to allow the creation of somatic mutations in defined genes. This chapter provides an update on the current state of Cre driver mouse lines worldwide, and reviews the available public databases and portals that capture critical details of Cre driver lines such as the efficiency of recombination, cell tissue specificity, or genetic background effects. The continuously changing landscape of these mouse resources reflects the rapid progression of research and development in conditional and inducible mouse mutagenesis.     

Effects of vitamin E and beta-carotene on sperm competitiveness

Sperm are particularly prone to oxidative damage because they generate reactive oxygen species (ROS), have a high polyunsaturated fat content and a reduced capacity to repair DNA damage. The dietary compounds vitamin E and beta-carotene are argued to have antioxidant properties that help to counter the damaging effects of excess ROS. Here in, we tested the post-copulatory consequences for male crickets (Teleogryllus oceanicus) of dietary intake of these two candidate antioxidants. During competitive fertilisation trials, vitamin E, but not beta-carotene, singularly enhanced sperm competitiveness. However, the diet combining a high vitamin E dose and beta-carotene produced males with the most competitive ejaculates, possibly due to the known ability of beta-carotene to recycle vitamin E. Our results provide support for the idea that these two common dietary compounds have interactive antioxidant properties in vivo, by affecting the outcomes of male reproductive success under competitive conditions.     

Knockdown of NPY expression in the dorsomedial hypothalamus promotes development of brown adipocytes and prevents diet-induced obesity

Hypothalamic neuropeptide Y (NPY) has been implicated in control of energy balance, but the physiological importance of NPY in the dorsomedial hypothalamus (DMH) remains unclear. Here we report that knockdown of NPY expression in the DMH by adeno-associated virus-mediated RNAi reduced fat depots in rats fed regular chow and ameliorated high-fat diet-induced hyperphagia and obesity. DMH NPY knockdown resulted in development of brown adipocytes in inguinal white adipose tissue through the sympathetic nervous system. This knockdown increased uncoupling protein 1 expression in both inguinal fat and interscapular brown adipose tissue (BAT). Consistent with the activation of BAT, DMH NPY knockdown increased energy expenditure and enhanced the thermogenic response to a cold environment. This knockdown also increased locomotor activity, improved glucose homeostasis, and enhanced insulin sensitivity. Together, these results demonstrate critical roles of DMH NPY in body weight regulation through affecting food intake, body adiposity, thermogenesis, energy expenditure, and physical activity.     

An integrated disease/pharmacokinetic/pharmacodynamic model suggests improved interleukin-21 regimens validated prospectively for mouse solid cancers

Interleukin (IL)-21 is an attractive antitumor agent with potent immunomodulatory functions. Yet thus far, the cytokine has yielded only partial responses in solid cancer patients, and conditions for beneficial IL-21 immunotherapy remain elusive. The current work aims to identify clinically-relevant IL-21 regimens with enhanced efficacy, based on mathematical modeling of long-term antitumor responses. For this purpose, pharmacokinetic (PK) and pharmacodynamic (PD) data were acquired from a preclinical study applying systemic IL-21 therapy in murine solid cancers. We developed an integrated disease/PK/PD model for the IL-21 anticancer response, and calibrated it using selected "training" data. The accuracy of the model was verified retrospectively under diverse IL-21 treatment settings, by comparing its predictions to independent "validation" data in melanoma and renal cell carcinoma-challenged mice (R(2)>0.90). Simulations of the verified model surfaced important therapeutic insights: (1) Fractionating the standard daily regimen (50 μg/dose) into a twice daily schedule (25 μg/dose) is advantageous, yielding a significantly lower tumor mass (45% decrease); (2) A low-dose (12 μg/day) regimen exerts a response similar to that obtained under the 50 μg/day treatment, suggestive of an equally efficacious dose with potentially reduced toxicity. Subsequent experiments in melanoma-bearing mice corroborated both of these predictions with high precision (R(2)>0.89), thus validating the model also prospectively in vivo. Thus, the confirmed PK/PD model rationalizes IL-21 therapy, and pinpoints improved clinically-feasible treatment schedules. Our analysis demonstrates the value of employing mathematical modeling and in silico-guided design of solid tumor immunotherapy in the clinic.     

A negative feedback loop that limits the ectopic activation of a cell type-specific sporulation sigma factor of Bacillus subtilis

Two highly similar RNA polymerase sigma subunits, σ(F) and σ(G), govern the early and late phases of forespore-specific gene expression during spore differentiation in Bacillus subtilis. σ(F) drives synthesis of σ(G) but the latter only becomes active once engulfment of the forespore by the mother cell is completed, its levels rising quickly due to a positive feedback loop. The mechanisms that prevent premature or ectopic activation of σ(G) while discriminating between σ(F) and σ(G) in the forespore are not fully comprehended. Here, we report that the substitution of an asparagine by a glutamic acid at position 45 of σ(G) (N45E) strongly reduced binding by a previously characterized anti-sigma factor, CsfB (also known as Gin), in vitro, and increased the activity of σ(G) in vivo. The N45E mutation caused the appearance of a sub-population of pre-divisional cells with strong activity of σ(G). CsfB is normally produced in the forespore, under σ(F) control, but sigGN45E mutant cells also expressed csfB and did so in a σ(G)-dependent manner, autonomously from σ(F). Thus, a negative feedback loop involving CsfB counteracts the positive feedback loop resulting from ectopic σ(G) activity. N45 is invariant in the homologous position of σ(G) orthologues, whereas its functional equivalent in σ(F) proteins, E39, is highly conserved. While CsfB does not bind to wild-type σ(F), a E39N substitution in σ(F) resulted in efficient binding of CsfB to σ(F). Moreover, under certain conditions, the E39N alteration strongly restrains the activity of σ(F) in vivo, in a csfB-dependent manner, and the efficiency of sporulation. Therefore, a single amino residue, N45/E39, is sufficient for the ability of CsfB to discriminate between the two forespore-specific sigma factors in B. subtilis.     

A modified host-cell reactivation assay to quantify DNA repair capacity in cryopreserved peripheral lymphocytes

The host-cell reactivation assay (HCRA) is a functional assay that allows the identification of the genes responsible for DNA repair-deficient syndromes, such as Xeroderma pigmentosum, by cross-complementation experiments. It has also been used in molecular epidemiology studies to correlate the low nucleotide excision repair pathway function in peripheral blood lymphocytes with an increased risk of bladder, head and neck, skin and lung cancers. Herein, we present the technical validation of a newly modified HCRA, where nucleofection is used for the transfection of the pmaxGFP plasmid into cryopreserved peripheral blood lymphocytes (PBLs) or lymphoblastoid cell lines. In each sample, 20-24h after transfection, the relative DNA repair capacity (DRC) was quantified by flow cytometry, comparing the transfection efficiency of nucleoporated cells with undamaged plasmid to those transfected with UV-light damaged plasmid in the seven cell lines that were characterized by different DNA repair phenotypes. Dead cells were excluded from the analysis. We observed a high reproducibility of the relative DRC, transfection efficiency and cell viability. The inter-experimental normalization of the flow cytometry resulted in an increased data accuracy and reproducibility. The amount of cells required for each transfection reaction was reduced fourfold, without affecting the final relative DRC. Furthermore, our HCRA demonstrated strong discrimination power in the UV-light dose-response, both in lymphoblastoid cell lines and cryopreserved PBLs. We also observed a strong correlation of the relative DRC data, when samples were measured against two independent batches of both damaged and undamaged plasmid DNA. The relative DRC variable shows a normal distribution when analyzed in the cryopreserved PBLs from a cohort of 35 lung cancer patients and a 5.59-fold variation in the relative DRC is identified among our patients. The mitotic dynamic was discarded as a confounding factor for the relative DRC measurement in this cohort of patients. The results indicate that our method is highly sensitive, reliable and reproducible, and thus, it suitable for population-based studies to quantify in vitro DNA-repair deficiencies.     

Mouse strains for the ubiquitous or conditional overexpression of the Flii gene

The gelsolin related actin binding protein, Flii, is able to regulate wound healing; mice with decreased Flii expression show improved wound healing whereas mice with elevated Flii expression exhibit impaired wound healing. In both mice and humans Flii expression increases with age and amelioration of FLII activity represents a possible therapeutic strategy for improved wound healing in humans. Despite analysis of Flii function in a variety of organisms we know little of the molecular mechanisms underlying Flii action. Two new murine alleles of Flii have been produced to drive constitutive or tissue-specific expression of Flii. Each strain is able to rescue the embryonic lethality associated with a Flii null allele and to impair wound healing. These strains provide valuable resources for ongoing investigation of Flii function in a variety of biological processes.     

SLIT/ROBO1 signaling suppresses mammary branching morphogenesis by limiting basal cell number

In the field of breast biology, there is a growing appreciation for the "gatekeeping function" of basal cells during development and disease processes yet mechanisms regulating the generation of these cells are poorly understood. Here, we report that the proliferation of basal cells is controlled by SLIT/ROBO1 signaling and that production of these cells regulates outgrowth of mammary branches. We identify the negative regulator TGF-β1 upstream of Robo1 and show that it induces Robo1 expression specifically in the basal layer, functioning together with SLIT2 to restrict branch formation. Loss of SLIT/ROBO1 signaling in this layer alone results in precocious branching due to a surplus of basal cells. SLIT2 limits basal cell proliferation by inhibiting canonical WNT signaling, increasing the cytoplasmic and membrane pools of β-catenin at the expense of its nuclear pool. Together, our studies provide mechanistic insight into how specification of basal cell number influences branching morphogenesis.