Capabilities and Limitations of Tissue Size Control through Passive Mechanical Forces

Embryogenesis is an extraordinarily robust process, exhibiting the ability to control tissue size and repair patterning defects in the face of environmental and genetic perturbations. The size and shape of a developing tissue is a function of the number and size of its constituent cells as well as their geometric packing. How these cellular properties are coordinated at the tissue level to ensure developmental robustness remains a mystery; understanding this process requires studying multiple concurrent processes that make up morphogenesis, including the spatial patterning of cell fates and apoptosis, as well as cell intercalations. In this work, we develop a computational model that aims to understand aspects of the robust pattern repair mechanisms of the Drosophila embryonic epidermal tissues. Size control in this system has previously been shown to rely on the regulation of apoptosis rather than proliferation; however, to date little work has been done to understand the role of cellular mechanics in this process. We employ a vertex model of an embryonic segment to test hypotheses about the emergence of this size control. Comparing the model to previously published data across wild type and genetic perturbations, we show that passive mechanical forces suffice to explain the observed size control in the posterior (P) compartment of a segment. However, observed asymmetries in cell death frequencies across the segment are demonstrated to require patterning of cellular properties in the model. Finally, we show that distinct forms of mechanical regulation in the model may be distinguished by differences in cell shapes in the P compartment, as quantified through experimentally accessible summary statistics, as well as by the tissue recoil after laser ablation experiments.     

Characterization of U2AF6, a Splicing Factor Related to U2AF35

The essential splicing factor U2AF (U2 auxiliary factor) is a heterodimer composed of 65-kDa (U2AF(65)) and 35-kDa (U2AF(35)) subunits. U2AF(35) has multiple functions in pre-mRNA splicing. First, U2AF(35) has been shown to function by directly interacting with the AG at the 3' splice site. Second, U2AF(35) is thought to play a role in the recruitment of U2AF(65) by serine-arginine-rich (SR) proteins in enhancer-dependent splicing. It has been proposed that the physical interaction between the arginine-serine-rich (RS) domain of U2AF(35) and SR proteins is important for this activity. However, other data suggest that this may not be the case. Here, we report the identification of a mammalian gene that encodes a 26-kDa protein bearing strong sequence similarity to U2AF(35), designated U2AF(26). The N-terminal 187 amino acids of U2AF(35) and U2AF(26) are nearly identical. However, the C-terminal domain of U2AF(26) lacks many characteristics of the U2AF(35) RS domain and, therefore, might be incapable of interacting with SR proteins. We show that U2AF(26) can associate with U2AF(65) and can functionally substitute for U2AF(35) in both constitutive and enhancer-dependent splicing, demonstrating that the RS domain of the small U2AF subunit is not required for splicing enhancer function. Finally, we show that U2AF(26) functions by enhancing the binding of U2AF(65) to weak 3' splice sites. These studies identify U2AF(26) as a mammalian splicing factor and demonstrate that distinct U2AF complexes can participate in pre-mRNA splicing. Based on its sequence and functional similarity to U2AF(35), U2AF(26) may play a role in regulating alternative splicing.     

Dissociation of Striatal GTPase and Dopamine Release Responses to Muscarinic Cholinergic Agonists in F344 Rats: Influence of Age and Dietary Manipulation

Abstract: There is evidence that dietary lipids and age both influence neuronal membrane composition and receptor G protein-linked signal transduction, but very little information is available on the interaction between these two factors. To investigate this, we obtained striata from 2, 12, and 22-month-old male F344 rats who were fed either a high-cholesterol, high-saturated fat or low-fat diet for 1 month. The striata were assayed for muscarinic agonist-stimulated low-K_m GTPase activity using 10^?3M carbachol and 10^?5M oxotremorine and for KCl-evoked dopamine release enhancement by 10^?5M oxotremorine. Membrane cholesterol and phospholipid content and phospholipid class composition were also determined. Mature animals showed significant but divergent changes in GTPase activity and dopamine release for high-cholesterol and low-fat diets: GTPase activity decreased, whereas dopamine release increased in these groups. Alterations in GTPase activity but not in dopamine release were inversely correlated with the cholesterol/phospholipid molar ratio. Old control animals showed reductions in both GTPase activity and oxotremorine-enhanced dopamine release compared with young animals. Whereas none of the experimental diets affected GTPase activity in old animals, the low-fat diet produced a marked decrease in dopamine release. In contrast to mature and old groups, young rats showed no significant change in either GTPase or dopamine release, suggesting a relative “resistance” to such dietary lipid modulation. The observed dissociation in GTPase and dopamine release responses to diet may reflect differing effects of these diets on discrete membrane lipid domains that preferentially influence different signal transduction components. The substantial age-related differences in striatal membrane response to dietary lipid modulation may represent the effects of underlying age differences in membrane lipid metabolism, structure, and/or dynamics. Our findings support the work of other groups that have shown that brain membranes are susceptible to modification by exogenous lipids. They also suggest the need for a more systematic examination of the influence of age on the response to other types of dietary lipid changes.     

Miscanthus × giganteus and Arundo donax shoot and rhizome tolerance of extreme moisture stress

Crops grown for bioenergy production are a mandated component of the United States energy portfolio. Giant miscanthus (Miscanthus × giganteus) is a leading bioenergy crop similar in habit to the invasive plant giant reed (Arundo donax). To characterize the environmental tolerance of giant miscanthus, we compared the soil moisture stress tolerance of giant miscanthus and giant reed under glasshouse conditions. We subjected both species to soil moisture conditions of severe drought (?4.2 MPa), mild drought (?0.5 MPa), field‐capacity (control), and flooded soils. These conditions were applied to two cohorts: one in which soil moisture conditions were imposed on newly planted rhizome fragments, and one in which conditions were imposed on established plants after 8 weeks of growth in field‐capacity soil. After 16 weeks, we harvested all plants, measured above‐ and belowground biomass, and evaluated the reproductive viability of rhizome fragments. The total biomass of each species under flooded conditions was not different from the field‐capacity control groups regardless of cohort. However, drought did affect the two cohorts differently. In the cohort treated after 8 weeks of growth, mild and severe drought conditions resulted in 56% and 66% reductions in biomass, averaged over both species, compared with the controls. In the cohort treated for the entire 16 weeks, mild and severe drought conditions resulted in 92% and 94% reductions in biomass. Rhizome fragments from both species and both cohorts showed 100% viability following flooded and control treatments; drought treatments reduced rhizome viability in both species, with a greater impact on giant miscanthus. Although giant miscanthus does not appear to have the potential to escape and establish in relatively dry upland ecosystems, it does show tolerance to flooded conditions similar to giant reed.     

Microassay for Human and Chick Cell Interferon

The microassay for human and chick interferon described in this paper required much smaller amounts of samples and reagents and considerably less time and effort than the plaque reduction assay while yielding comparable results.     

EDC3 phosphorylation regulates growth and invasion through controlling P‐body formation and dynamics

Regulation of mRNA stability and translation plays a critical role in determining protein abundance within cells. Processing bodies (P‐bodies) are critical regulators of these processes. Here, we report that the Pim1 and 3 protein kinases bind to the P‐body protein enhancer of mRNA decapping 3 (EDC3) and phosphorylate EDC3 on serine (S)161, thereby modifying P‐body assembly. EDC3 phosphorylation is highly elevated in many tumor types, is reduced upon treatment of cells with kinase inhibitors, and blocks the localization of EDC3 to P‐bodies. Prostate cancer cells harboring an EDC3 S161A mutation show markedly decreased growth, migration, and invasion in tissue culture and in xenograft models. Consistent with these phenotypic changes, the expression of integrin β1 and α6 mRNA and protein is reduced in these mutated cells. These results demonstrate that EDC3 phosphorylation regulates multiple cancer‐relevant functions and suggest that modulation of P‐body activity may represent a new paradigm for cancer treatment.     

The gene for human complement component C9 mapped to chromosome 5 by polymerase chain reaction

The gene for human complement component C9 has been mapped to chromosome 5. This was achieved by using a novel application of the polymerase chain reaction to amplify specifically the human C9 gene on a background of rodent DNA in somatic cell hybrids. The assignment to chromosome 5 was confirmed by in situ hybridization to human metaphase chromosomes, giving a regional localization of 5p13.