A new Caenorhabditis elegans model to study copper toxicity in Wilson disease.

Wilson disease (WD) is caused by mutations in the ATP7B gene that encodes a copper (Cu) transporting ATPase whose trafficking from the Golgi to endo-lysosomal compartments drives sequestration of excess Cu and its further excretion from hepatocytes into the bile. Loss of ATP7B function leads to toxic Cu overload in the liver and subsequently in the brain, causing fatal hepatic and neurological abnormalities. The limitations of existing WD therapies call for the development of new therapeutic approaches, which require an amenable animal model system for screening and validation of drugs and molecular targets. To achieve this objective, we generated a mutant Caenorhabditis elegans strain with a substitution of a conserved histidine (H828Q) in the ATP7B ortholog cua-1 corresponding to the most common ATP7B variant (H1069Q) that causes WD. cua-1 mutant animals exhibited very poor resistance to Cu compared to the wild-type strain. This manifested in a strong delay in larval development, a shorter lifespan, impaired motility, oxidative stress pathway activation, and mitochondrial damage. In addition, morphological analysis revealed several neuronal abnormalities in cua-1 mutant animals exposed to Cu. Further investigation suggested that mutant CUA-1 is retained and degraded in the endoplasmic reticulum, similarly to human ATP7B-H1069Q. As a consequence, the mutant protein does not allow animals to counteract Cu toxicity. Notably, pharmacological correctors of ATP7B-H1069Q reduced Cu toxicity in cua-1 mutants indicating that similar pathogenic molecular pathways might be activated by the H/Q substitution and, therefore, targeted for rescue of ATP7B/CUA-1 function. Taken together, our findings suggest that the newly generated cua-1 mutant strain represents an excellent model for Cu toxicity studies in WD.

Enteric neurons increase maternal food intake during reproduction.

Reproduction induces increased food intake across females of many animal species1-4, providing a physiologically relevant paradigm for the exploration of appetite regulation. Here, by examining the diversity of enteric neurons in Drosophila melanogaster, we identify a key role for gut-innervating neurons with sex- and reproductive state-specific activity in sustaining the increased food intake of mothers during reproduction. Steroid and enteroendocrine hormones functionally remodel these neurons, which leads to the release of their neuropeptide onto the muscles of the crop-a stomach-like organ-after mating. Neuropeptide release changes the dynamics of crop enlargement, resulting in increased food intake, and preventing the post-mating remodelling of enteric neurons reduces both reproductive hyperphagia and reproductive fitness. The plasticity of enteric neurons is therefore key to reproductive success. Our findings provide a mechanism to attain the positive energy balance that sustains gestation, dysregulation of which could contribute to infertility or weight gain.

Systemic muscle wasting and coordinated tumour response drive tumourigenesis.

Cancer cells demand excess nutrients to support their proliferation, but how tumours exploit extracellular amino acids during systemic metabolic perturbations remain incompletely understood. Here, we use a Drosophila model of high-sugar diet (HSD)-enhanced tumourigenesis to uncover a systemic host-tumour metabolic circuit that supports tumour growth. We demonstrate coordinate induction of systemic muscle wasting with tumour-autonomous Yorkie-mediated SLC36-family amino acid transporter expression as a proline-scavenging programme to drive tumourigenesis. We identify Indole-3-propionic acid as an optimal amino acid derivative to rationally target the proline-dependency of tumour growth. Insights from this whole-animal Drosophila model provide a powerful approach towards the identification and therapeutic exploitation of the amino acid vulnerabilities of tumourigenesis in the context of a perturbed systemic metabolic network.

Hyaluronic acid matrices show matrix stiffness in 2D and 3D dictates cytoskeletal order and myosin-II phosphorylation within stem cells.

Physical features of microenvironments such as matrix elasticity E can clearly influence cell morphology and cell phenotype, but many differences between model matrices raise questions as to whether a standard biological scale for E exists, especially in 3D as well as in 2D. An E-series of two distinct types of hydrogels are ligand-functionalized here with non-fibrous collagen and used to elucidate wide-ranging cell and cytoskeletal responses to E in both 2D and 3D matrix geometries. Cross-linked hyaluronic acid (HA) based matrices as well as standard polyacrylamide (PA) hydrogels show that, within hours of initial plating, the adhesion, asymmetric shape, and cytoskeletal order within mesenchymal stem cells generally depend on E nonmonotonically over a broad range of physiologically relevant E. In particular, with overlays of a second matrix the stiffer of the upper or lower matrix dominates key cell responses to 3D: the cell invariably takes an elongated shape that couples to E in driving cytoplasmic stress fiber assembly. In contrast, embedding cells in homogeneous HA matrices constrains cells to spherically symmetric shapes in which E drives the assembly of a predominantly cortical cytoskeleton. Non-muscle myosin II generates the forces required for key cell responses and is a target of a phospho-Tyrosine signaling pathway that likely regulates contractile assemblies and also depends nonmonotonically on E. The results can be understood in part from a theory for stress fiber polarization that couples to matrix elasticity as well as cell shape and accurately predicts cytoskeletal order in 2D and 3D, regardless of polymer system.

How deeply cells feel: methods for thin gels.

Tissue cells lack the ability to see or hear but have evolved mechanisms to feel into their surroundings and sense a collective stiffness. A cell can even sense the effective stiffness of rigid objects that are not in direct cellular contact - like the proverbial princess who feels a pea placed beneath soft mattresses. How deeply a cell feels into a matrix can be measured by assessing cell responses on a controlled series of thin and elastic gels that are affixed to a rigid substrate. Gel elasticity E is readily varied with polymer concentrations of now-standard polyacrylamide hydrogels, but to eliminate wrinkling and detachment of thin gels from an underlying glass coverslip, vinyl groups are bonded to the glass before polymerization. Gel thickness is nominally specified using micron-scale beads that act as spacers, but gels swell after polymerization as measured by z-section, confocal microscopy of fluorescent gels. Atomic force microscopy (AFM) is used to measure E at gel surfaces, employing stresses and strains that are typically generated by cells and yielding values for E that span a broad range of tissue microenvironments. To illustrate cell sensitivities to a series of thin-to-thick gels, the adhesive spreading of mesenchymal stem cells was measured on gel mimics of a very soft tissue (eg. brain, E ~ 1 kPa). Initial results show that cells increasingly respond to the rigidity of an underlying 'hidden' surface starting at about 10-20 microm gel thickness with a characteristic tactile length of less than about 5 microm.

Cross-correlated TIRF/AFM reveals asymmetric distribution of force-generating heads along self-assembled, "synthetic" myosin filaments.

Myosin-II's rod-like tail drives filament assembly with a head arrangement that is often considered to be a symmetric bipole that generates equal and opposite contractile forces on actin. Self-assembled myosin filaments are shown here to be asymmetric in physiological buffer based on cross-correlated images from both atomic force microscopy and total internal reflection fluorescence. Quantitative cross-correlation of these orthogonal methods produces structural information unavailable to either method alone in showing that fluorescence intensity along the filament length is proportional to height. This implies that myosin heads form a shell around the filament axis, consistent with F-actin binding. A motor density of approximately 50-100 heads/micrometer is further estimated but with an average of 32% more motors on one half of any given filament compared to the other, regardless of length. A purely entropic pyramidal lattice model is developed and mapped onto the Dyck paths problem that qualitatively captures this lack of length dependence and the distribution of filament asymmetries. Such strongly asymmetric bipoles are likely to produce an unbalanced contractile force in cells and in actin-myosin gels and thereby contribute to motility as well as cytoskeletal tension.