The apical compartment: trafficking pathways,regulators and scaffolding proteins

Defects in the trafficking of apical membrane proteins in polarized epithelial cells are often associated with diseases, including cystic fibrosis, Liddle's syndrome, nephrogenic diabetes insipidus and Dubin-Johnson syndrome. In recent years, we have learned much about the specialized apical trafficking pathways in polarized cells. Many laboratories have identified signals that direct proteins within these pathways and have defined protein interactions that mediate specific steps in the sorting and stabilization of these proteins. In addition, many cytosolic proteins, including lipid kinases, GTPases, ATPases and scaffolding/adaptor proteins that lack enzymatic activity, regulate the trafficking of proteins through these pathways. Recent advances in the field include the role of small GTPases, unconventional myosins and lipid kinases in apical endocytosis and transcytosis, and the identification of PDZ proteins that regulate apical membrane trafficking of receptors, transporters and ion channels.     

Detection of QTL in Rainbow Trout Affecting Survival When Challenged with Flavobacterium psychrophilum

Bacterial cold water disease (BCWD) causes significant economic loss in salmonid aquaculture. We previously detected genetic variation in survival following challenge with Flavobacterium psychrophilum (Fp), the causative agent of BCWD in rainbow trout (Oncorhynchus mykiss). A family-based selection program to improve resistance was initiated in 2005 at the USDA National Center for Cool and Cold Water Aquaculture. Select crosses were made in 2007 and 2009 to evaluate family-based disease survival using Fp injection challenges. From each putative F₂/BC₁ family generated in 2009, 200–260 fish were challenged in 4–7 replicates per family. Whole genome QTL scans of three F₂/BC₁ families were conducted with about 270 informative microsatellite loci per family spaced at an average interval size of 6 cM throughout the rainbow trout genome. Markers on chromosomes containing QTL were further evaluated in three additional F₂/BC₁ families. The additional F₂/BC₁ families were sire or dam half-sibs (HS) of the initially genome scanned families. Overall, we identified nine major QTL on seven chromosomes that were significant or highly significant with moderate to large effects of at least 13 % of the total phenotypic variance. The largest effect QTL for BCWD resistance explaining up to 40 % of the phenotypic variance was detected on chromosome OMY8 in family 2009070 and in the combined dam HS family 2009069–070. The nine major QTL identified in this study are candidates for fine mapping to identify new markers that are tightly linked to disease resistance loci for using in marker assisted selection strategies.     

Taste and Physiological Responses to Glucosinolates: Seed Predator versus Seed Disperser

In contrast to most other plant tissues, fleshy fruits are meant to be eaten in order to facilitate seed dispersal. Although fleshy fruits attract consumers, they may also contain toxic secondary metabolites. However, studies that link the effect of fruit toxins with seed dispersal and predation are scarce. Glucosinolates (GLSs) are a family of bitter-tasting compounds. The fleshy fruit pulp of Ochradenus baccatus was previously found to harbor high concentrations of GLSs, whereas the myrosinase enzyme, which breaks down GLSs to produce foul tasting chemicals, was found only in the seeds. Here we show the differential behavioral and physiological responses of three rodent species to high dose (80%) Ochradenus’ fruits diets. Acomys russatus, a predator of Ochradenus’ seeds, was the least sensitive to the taste of the fruit and the only rodent to exhibit taste-related physiological adaptations to deal with the fruits’ toxins. In contrast, Acomys cahirinus, an Ochradenus seed disperser, was more sensitive to a diet containing the hydrolyzed products of the GLSs. A third rodent (Mus musculus) was deterred from Ochradenus fruits consumption by the GLSs and their hydrolyzed products. We were able to alter M. musculus avoidance of whole fruit consumption by soaking Ochradenus fruits in a water solution containing 1% adenosine monophosphate, which blocks the bitter taste receptor in mice. The observed differential responses of these three rodent species may be due to evolutionary pressures that have enhanced or reduced their sensitivity to the taste of GLSs.     

The App-Runx1 region is critical for birth defects and electrocardiographic dysfunctions observed in a Down syndrome mouse model

Down syndrome (DS) leads to complex phenotypes and is the main genetic cause of birth defects and heart diseases. The Ts65Dn DS mouse model is trisomic for the distal part of mouse chromosome 16 and displays similar features with post-natal lethality and cardiovascular defects. In order to better understand these defects, we defined electrocardiogram (ECG) with a precordial set-up, and we found conduction defects and modifications in wave shape, amplitudes, and durations in Ts65Dn mice. By using a genetic approach consisting of crossing Ts65Dn mice with Ms5Yah mice monosomic for the App-Runx1 genetic interval, we showed that the Ts65Dn viability and ECG were improved by this reduction of gene copy number. Whole-genome expression studies confirmed gene dosage effect in Ts65Dn, Ms5Yah, and Ts65Dn/Ms5Yah hearts and showed an overall perturbation of pathways connected to post-natal lethality (Coq7, Dyrk1a, F5, Gabpa, Hmgn1, Pde10a, Morc3, Slc5a3, and Vwf) and heart function (Tfb1m, Adam19, Slc8a1/Ncx1, and Rcan1). In addition cardiac connexins (Cx40, Cx43) and sodium channel sub-units (Scn5a, Scn1b, Scn10a) were found down-regulated in Ts65Dn atria with additional down-regulation of Cx40 in Ts65Dn ventricles and were likely contributing to conduction defects. All these data pinpoint new cardiac phenotypes in the Ts65Dn, mimicking aspects of human DS features and pathways altered in the mouse model. In addition they highlight the role of the App-Runx1 interval, including Sod1 and Tiam1, in the induction of post-natal lethality and of the cardiac conduction defects in Ts65Dn. These results might lead to new therapeutic strategies to improve the care of DS people.     

Temperature trends and recent decline in body size of the stone marten Martes foina in Denmark

We studied a sample of 131 skulls of the stone marten Martes foina that were collected in Denmark between 1858 and 1999. Data were available for 37 years, but collection effort was not uniform throughout the study period and annual sample size varied between 1 and 27. We used principal component analysis (PCA) to combine the information of four skull measurements into a single variable (PC1). PC1 was then corrected for factors that significantly affected it (sex and longitude), and residual PC1 was used for further analysis in which we calculated trends in PC1 values during the study period. During the study period there was an increase in mean annual temperature in Denmark, but this increase was not continuous, as there was slight decrease in temperature between 1947 and 1965.We found that skull size (and by implication body size) of the stone marten in Denmark had two periods of decrease and these two periods coincide with the periods of increase in mean annual temperature. These results may indicate that body size of the stone marten is sensitive to the change in ambient temperature, either due to a change in food availability that was caused by the increase in temperature, or decreased its size in accordance with Bergmann's rule.     

State-dependent electrostatic interactions of S4 arginines with E1 in S2 during Kv7.1 activation

The voltage-sensing domain of voltage-gated channels is comprised of four transmembrane helices (S1–S4), with conserved positively charged residues in S4 moving across the membrane in response to changes in transmembrane voltage. Although it has been shown that positive charges in S4 interact with negative countercharges in S2 and S3 to facilitate protein maturation, how these electrostatic interactions participate in channel gating remains unclear. We studied a mutation in Kv7.1 (also known as KCNQ1 or KvLQT1) channels associated with long QT syndrome (E1K in S2) and found that reversal of the charge at E1 eliminates macroscopic current without inhibiting protein trafficking to the membrane. Pairing E1R with individual charge reversal mutations of arginines in S4 (R1–R4) can restore current, demonstrating that R1–R4 interact with E1. After mutating E1 to cysteine, we probed E1C with charged methanethiosulfonate (MTS) reagents. MTS reagents could not modify E1C in the absence of KCNE1. With KCNE1, (2-sulfonatoethyl) MTS (MTSES)⁻ could modify E1C, but [2-(trimethylammonium)ethyl] MTS (MTSET)⁺ could not, confirming the presence of a positively charged environment around E1C that allows approach by MTSES⁻ but repels MTSET⁺. We could change the local electrostatic environment of E1C by making charge reversal and/or neutralization mutations of R1 and R4, such that MTSET⁺ modified these constructs depending on activation states of the voltage sensor. Our results confirm the interaction between E1 and the fourth arginine in S4 (R4) predicted from open-state crystal structures of Kv channels and reveal an E1–R1 interaction in the resting state. Thus, E1 engages in electrostatic interactions with arginines in S4 sequentially during the gating movement of S4. These electrostatic interactions contribute energetically to voltage-dependent gating and are important in setting the limits for S4 movement.     

Dynamics of β-Catenin Interactions with APC Protein Regulate Epithelial Tubulogenesis

Epithelial tubulogenesis involves complex cell rearrangements that require control of both cell adhesion and migration, but the molecular mechanisms regulating these processes during tubule development are not well understood. Interactions of the cytoplasmic protein, β-catenin, with several molecular partners have been shown to be important for cell signaling and cell–cell adhesion. To examine if β-catenin has a role in tubulogenesis, we tested the effect of expressing NH₂-terminal deleted β-catenins in an MDCK epithelial cell model for tubulogenesis. After one day of treatment, hepatocyte growth factor/scatter factor (HGF/ SF)-stimulated MDCK cysts initiated tubulogenesis by forming many long cell extensions. Expression of NH₂-terminal deleted β-catenins inhibited formation of these cell extensions. Both ΔN90 β-catenin, which binds to α-catenin, and ΔN131 β-catenin, which does not bind to α-catenin, inhibited formation of cell extensions and tubule development, indicating that a function of β-catenin distinct from its role in cadherin-mediated cell–cell adhesion is important for tubulogenesis. In cell extensions from parental cysts, adenomatous polyposis coli (APC) protein was localized in linear arrays and in punctate clusters at the tips of extensions. Inhibition of cell extension formation correlated with the colocalization and accumulation of NH₂-terminal deleted β-catenin in APC protein clusters and the absence of linear arrays of APC protein. Continued HGF/ SF treatment of parental cell MDCK cysts resulted in cell proliferation and reorganization of cell extensions into multicellular tubules. Similar HGF/SF treatment of cysts derived from cells expressing NH₂-terminal deleted β-catenins resulted in cells that proliferated but formed cell aggregates (polyps) within the cyst rather than tubules. Our results demonstrate an unexpected role for β-catenin in cell migration and indicate that dynamic β-catenin–APC protein interactions are critical for regulating cell migration during epithelial tubulogenesis.     

Qualitative and Quantitative Analysis of DNA Fragmentation Using Digital Imaging

Apoptosis is an important and common pathway of cellular death. Differentiation from cellular necrosis and quantitation of apoptosis within the milieu of necrosis are analytical challenges. We describe the use of the RIT120 digital imaging software package for quantitative and qualitative analysis of apoptotic DNA ladders induced by a variety of agents, such as serum, tumor necrosis factor-α, transforming growth factor-β1, and nitric oxide. Autoradiographs of DNA ladders are densitometrically scanned to yield a set of curves with peaks corresponding to specific DNA fragments, thereby allowing quantitative subtraction of concurrent DNA degradation from necrotic death. Integration of the areas specifically under the peaks yields a quantitative measure of apoptosis. We provide a useful, rapid, and objective means to quantitate apoptosis, using relatively inexpensive hardware and software.     

The role of pre-symbiotic auxin signaling in ectendomycorrhiza formation between the desert truffle <Emphasis Type="Italic">Terfezia boudieri</Emphasis> and <Emphasis Type="Italic">Helianthemum sessiliflorum</Emphasis>

The ectendomycorrhizal fungus Terfezia boudieri is known to secrete auxin. While some of the effects of fungal auxin on the plant root system have been described, a comprehensive understanding is still lacking. A dual culture system to study pre mycorrhizal signal exchange revealed previously unrecognized root–fungus interaction mediated by the fungal auxin. The secreted fungal auxin induced negative taproot gravitropism, attenuated taproot growth rate, and inhibited initial host development. Auxin also induced expression of Arabidopsis carriers AUX1 and PIN1, both of which are involved in the gravitropic response. Exogenous application of auxin led to a root phenotype, which fully mimicked that induced by ectomycorrhizal fungi. Co-cultivation of Arabidopsis auxin receptor mutants tir1-1, tir1-1 afb2-3, tir1-1 afb1-3 afb2-3, and tir1-1 afb2-3 afb3-4 with Terfezia confirmed that auxin induces the observed root phenotype. The finding that auxin both induces taproot deviation from the gravity axis and coordinates growth rate is new. We propose a model in which the fungal auxin induces horizontal root development, as well as the coordination of growth rates between partners, along with the known auxin effect on lateral root induction that increases the availability of accessible sites for colonization at the soil plane of fungal spore abundance. Thus, the newly observed responses described here of the root to Terfezia contribute to a successful encounter between symbionts.