Intra- and intermolecular domain interactions of the C-terminal GTPase effector domain of the multimeric dynamin-like GTPase Drp1

Mammalian Drp1 is a dynamin-like GTPase required for mitochondrial fission. Although it exists primarily as a cytosolic homo-tetramer in vivo, it can also self-assemble into higher order structures on the mitochondrial outer membrane, where it is required for proper mitochondrial division. Functional studies and sequence comparisons have revealed four different structural domains in Drp1, comprising N-terminal GTP-binding, middle, insert B, and C-terminal GTPase effector (GED) domains. Here we describe an intramolecular interaction within Drp1 between the GED and the N-terminal GTP-binding and middle domains. A point mutation (K679A) within the C-terminal GED domain inhibits this intramolecular association, without affecting the formation of Drp1 tetramers or the intermolecular associations among isolated C-terminal domains. Mutant Drp1 K679A exhibits impaired GTPase activity, and when overexpressed in mammalian cells it decreases mitochondrial division. Sedimentation experiments indicate that the K679A mutation either increases Drp1 complex formation or, more likely, decreases complex disassembly as compared with wild-type Drp1. Taken together, these data suggest that the C-terminal GED domain is important for stimulation of GTPase activity, formation and stability of higher order complexes, and efficient mitochondrial division.     

Potency of a tau fibrillization inhibitor is influenced by its aggregation state

Tau fibrillization is a potential therapeutic target for Alzheimer's and other neurodegenerative diseases. Several small-molecule inhibitors of tau aggregation have been developed for this purpose. One of them, 3,3'-bis(beta-hydroxyethyl)-9-ethyl-5,5'-dimethoxythiacarbocyanine iodide (N744), is a cationic thiacarbocyanine dye that inhibits recombinant tau filament formation when present at submicromolar concentrations. To prepare dosing regimens for testing N744 activity in biological models, its full concentration-effect relationship in the range 0.01-60muM was examined in vitro by electron microscopy and laser light scattering methods. Results revealed that N744 concentration dependence was biphasic, with fibrillization inhibitory activity appearing at submicromolar concentration, but with relief of inhibition and increases in fibrillization apparent above 10muM. Therefore, fibrillization was inhibited 50% only over a narrow concentration range, which was further reduced by filament stabilizing modifications such as tau pseudophosphorylation. N744 inhibitory activity also was paralleled by changes in its aggregation state, with dimer predominating at inhibitory concentrations and large dye aggregates appearing at high concentrations. Ligand dimerization was promoted by the presence of tau protein, which lowered the equilibrium dissociation constant for dimerization more than an order of magnitude relative to controls. The results suggest that ligand aggregation may play an important role in both inhibitory and disinhibitory phases of the concentration-effect curve, and may lead to complex dose-response relationships in model systems.     

Charles Manning Child (1869-1954): the past, present, and future of metabolic signaling

Charles Manning Child's work focused on metabolic gradients and their influence on organismal development. Early in the 20th century, his work had considerable currency, but by the second half of the century he had become little more than a historical footnote. Yet today Child's ideas are once again topical. While there were issues of cause and effect that Child and his students were never able to address adequately, in hindsight the extent of his eclipse hardly seems warranted. In fact, the demise of Child's theories may have resulted from larger changes in the nature of biology in the early 20th century. Child frequently studied planarians, hydroids, and other animals that are capable of asexual, agametic reproduction, and his theories most clearly apply to such organisms. In contrast, Thomas Hunt Morgan, initially one of Child's competitors in studies of regeneration, later developed the field of transmission genetics based on fruit flies, which can only reproduce via gametes. Child's theories and model systems were largely casualties of the success of Morgan's mechanistic paradigm. Nevertheless, in modern biology metabolic gradients, recast in terms of redox signaling, have become central to understanding both normal and pathological development.     

Appalachian spring: variations on ancient gastro-entero-pancreatic themes in New World mammals

Studies of guinea pig genomic and/or cDNA clones encoding the gastro-entero-pancreatic (GEP) hormones--insulin, glucagon and pancreatic polypeptide--as well as portions of the insulin receptor, are described. Multiple clustered substitutions (localized rapid mutation acceptance) altering the biological properties of both insulin and glucagon have been revealed, but this does not appear to be the case with either pancreatic polypeptide or those regions of guinea pig insulin receptor cDNAs that have been examined thus far. These findings suggest that novel selective pressures operative in the New World environment, in which these animals evolved in isolation from Old World mammalian species, have led to altered solutions to problems related to the regulation of growth and carbohydrate metabolism.     

Experimental heterochrony in hydractiniid hydroids: Why mechanisms matter

As with most clonal organisms, hydractiniid hydroids display a range of morphological variation from sheet-like to runner-like forms. Life history differences correlate with these morphological traits, exhibiting patterns commonly found in studies of heterochrony. Experimental studies of two hydractiniid species show that both morphological and life history heterochronies correlate with patterns of gastrovascular circulation. Similar experimental perturbations of energy metabolism, however, have opposite heterochronic effects on the two species. Treatment with 2,4-dinitrophenol, an uncoupler of oxidative phosphorylation, produced diminished peripheral circulation and sheet-like peramorphic morphologies in the runner-like species, Podocoryne carnea. In contrast, similar manipulations of the sheet-like species, Hydractinia symbiolongicarpus, produced variable peripheral circulation and more runner-like, juvenilized morphologies. These results diverge at the level of morphological and life history pattern, but are consistent when viewed in terms of the physiology of the gastrovascular system. Interpretations of patterns of heterochrony must focus on the physiological and developmental mechanisms which mediate the expression of heterochronic traits.     

Structure and signaling in polyps of a colonial hydroid

Abstract. After feeding, polyps of colonial hydroids contract regularly, dispersing food throughout the colony via the gastrovascular fluid. Such contractions may trigger signaling pathways that allow colonies to grow in an adaptive manner, i.e., to initiate development of more polyps in food‐rich areas and to suppress polyp development in food‐poor areas. In this context, we investigated the structure and potential signaling of the junction between polyps and stolons in colonies of the hydroid Podocoryna carnea. Using transmission electron microscopy, we found that the density of mitochondrion‐rich epitheliomuscular cells was low in polyp and stolon tissues except at or near the polyp‐stolon junction, where many of these mitochondrion‐rich cells occur in ectodermal tissue. In vivo fluorescence microscopy suggests that these mitochondria are a principal source of the metabolic signals of the colony. Both native fluorescence of NAD(P)H and fluorescence from peroxides (visualized with H₂DCFDA) co‐localize to this region of the polyp. Rhodamine 123 fluorescence suggests that both these metabolic signals emanate from mitochondria. To test whether such metabolic signals may be involved in colony pattern formation, inbred lines of P. carnea were used. Colonies of a runner‐like inbred line grow with widely spaced polyps and long stolonal connections, much like wild‐type colonies in a food‐poor environment. Colonies of a sheet‐like inbred line grow with closely spaced polyps and short stolonal connections, similar to wild‐type colonies in a food‐rich environment. Polyp‐stolon junctions in runner‐like and sheet‐like colonies were imaged for the fluorescence of H₂DCFDA. Densitometric analysis of this signal indicates that the mitochondria in epitheliomuscular cells of runner‐like polyps emit greater amounts of peroxides. Because peroxides and other reactive oxygen species are frequently intermediaries in metabolic signaling pathways, we suspect that such signaling may indeed occur at polyp‐stolon junctions, affecting colony pattern formation in these inbred lines and possibly in hydroid colonies in general.     

Quantitative measures of gastrovascular flow in octocorals and hydroids: toward a comparative biology of transport systems in cnidarians

Using microscopy, the gastrovascular systems of four hydroids (Eirene viridula, Cordylophora lacustris, Hydractinia symbiolongicarpus, and Podocoryna carnea) and two distantly related alcyonacean octocorals (Acrossota amboinensis and Sarcothelia sp.) were examined and compared within a phylogenetic framework. Despite a range of stolon widths (means 53–160 μm), the hydroid species exhibited similar patterns of gastrovascular flow: sequentially bidirectional flow in the stolons, driven by myoepithelial contractions emanating from the center of the colony. Unlike the hydroids, the gastrovascular system of A. amboinensis (mean stolon widths for 5 colonies, 0.57–1.21 mm) exhibited simultaneously bidirectional flow with incomplete, medial baffles (width 4–20 μm) separating the flow. Baffles visualized with transmission electron microscopy consisted of endoderm, mesoglea, and occasionally another layer of tissue. Mean flow rates of the gastrovascular fluid for seven stolons ranged from 125 to 275 μm s^?1, with maximum rates of 225–700 μm s^?1. In Sarcothelia sp., stolons were of comparable width (means for 13 colonies 0.55–1.4 mm) to those of A. amboinensis. These stolons, however, were divided by several partitions (width 8–25 μm), both complete and incomplete, which were spaced every 100.5±5.1 μm (mean±SE; range 27.1–283.7 μm) and appeared structurally similar to baffles. In lanes defined by these partitions, ciliary motion was visible in image sequences, and flow was unidirectional. Within a single stolon, flow moved in different directions in different lanes and changed direction by moving from lane to lane via occasional spaces between the partitions. Mean flow rates for 30 stolons ranged from 75 to 475 μm s^?1, with maximum rates of 85–775 μm s^?1. For both octocorals, flow rates of the gastrovascular fluid were not correlated with the width of the stolon lumen. While octocoral gastrovascular systems probably exhibit differences based on phylogenetic affinities, in all species studied thus far, gastrovascular flow is entirely driven by cilia, in contrast to the hydroid taxa.     

The novel Smad protein Expansion regulates the receptor tyrosine kinase pathway to control Drosophila tracheal tube size

Tubes with distinct shapes and sizes are critical for the proper function of many tubular organs. Here we describe a unique phenotype caused by the loss of a novel, evolutionarily-conserved, Drosophila Smad-like protein, Expansion. In expansion mutants, unicellular and intracellular tracheal branches develop bubble-like cysts with enlarged apical membranes. Cysts in unicellular tubes are enlargements of the apical lumen, whereas cysts in intracellular tubes are cytoplasmic vacuole-like compartments. The cyst phenotype in expansion mutants is similar to, but weaker than, that observed in double mutants of Drosophila type III receptor tyrosine phosphatases (RPTPs), Ptp4E and Ptp10D. Ptp4E and Ptp10D negatively regulate the receptor tyrosine kinase (RTK) pathways, especially epithelial growth factor receptor (EGFR) and fibroblast growth factor receptor/breathless (FGFR, Btl) signaling to maintain the proper size of unicellular and intracellular tubes. We show Exp genetically interacts with RTK signaling, the downstream targets of RPTPs. Cyst size and number in expansion mutants is enhanced by increased RTK signaling and suppressed by reduced RTK signaling. Genetic interaction studies strongly suggest that Exp negatively regulates RTK (EGFR, Btl) signaling to ensure proper tube sizes. Smad proteins generally function as intermediate components of the transforming growth factor-β (TGF-β, DPP) signaling pathway. However, no obvious genetic interaction between expansion and TGF-β (DPP) signaling was observed. Therefore, Expansion does not function as a typical Smad protein. The expansion phenotype demonstrates a novel role for Smad-like proteins in epithelial tube formation.