PEDIGREE ERROR DUE TO EXTRA‐PAIR REPRODUCTION SUBSTANTIALLY BIASES ESTIMATES OF INBREEDING DEPRESSION

Understanding the evolutionary dynamics of inbreeding and inbreeding depression requires unbiased estimation of inbreeding depression across diverse mating systems. However, studies estimating inbreeding depression often measure inbreeding with error, for example, based on pedigree data derived from observed parental behavior that ignore paternity error stemming from multiple mating. Such paternity error causes error in estimated coefficients of inbreeding (f) and reproductive success and could bias estimates of inbreeding depression. We used complete “apparent” pedigree data compiled from observed parental behavior and analogous “actual” pedigree data comprising genetic parentage to quantify effects of paternity error stemming from extra‐pair reproduction on estimates of f, reproductive success, and inbreeding depression in free‐living song sparrows (Melospiza melodia). Paternity error caused widespread error in estimates of f and male reproductive success, causing inbreeding depression in male and female annual and lifetime reproductive success and juvenile male survival to be substantially underestimated. Conversely, inbreeding depression in adult male survival tended to be overestimated when paternity error was ignored. Pedigree error stemming from extra‐pair reproduction therefore caused substantial and divergent bias in estimates of inbreeding depression that could bias tests of evolutionary theories regarding inbreeding and inbreeding depression and their links to variation in mating system.     

Integrin-linked kinase controls microtubule dynamics required for plasma membrane targeting of caveolae

Caveolae are specialized compartments of the plasma membrane that are involved in signaling, endocytosis, and cholesterol transport. Their formation requires the transport of caveolin-1 to the plasma membrane, but the molecular mechanisms regulating the transport are largely unknown. Here, we?identify a critical role for adhesion-mediated signaling through β1 integrins and integrin-linked kinase (ILK) in caveolae formation. Mice lacking β1 integrins or ILK in keratinocytes have dramatically reduced numbers of plasma membrane caveolae in?vivo, which is due to impaired transport of caveolin-1-containing vesicles along microtubules (MT) to the plasma membrane. Mechanistically, ILK promotes the recruitment of the F-actin binding protein IQGAP1 to the cell cortex, which, in turn, cooperates with its?effector mDia1 to locally stabilize MTs and to allow?stable insertion of caveolae into the plasma membrane. Our results assign an important role to the integrin/ILK complex for caveolar trafficking to the cell surface.     

Neuron Specific Rab4 Effector GRASP-1 Coordinates Membrane Specialization and Maturation of Recycling Endosomes

The endosomal pathway in neuronal dendrites is essential for membrane receptor trafficking and proper synaptic function and plasticity. However, the molecular mechanisms that organize specific endocytic trafficking routes are poorly understood. Here, we identify GRIP-associated protein-1 (GRASP-1) as a neuron-specific effector of Rab4 and key component of the molecular machinery that coordinates recycling endosome maturation in dendrites. We show that GRASP-1 is necessary for AMPA receptor recycling, maintenance of spine morphology, and synaptic plasticity. At the molecular level, GRASP-1 segregates Rab4 from EEA1/Neep21/Rab5-positive early endosomal membranes and coordinates the coupling to Rab11-labelled recycling endosomes by interacting with the endosomal SNARE syntaxin 13. We propose that GRASP-1 connects early and late recycling endosomal compartments by forming a molecular bridge between Rab-specific membrane domains and the endosomal SNARE machinery. The data uncover a new mechanism to achieve specificity and directionality in neuronal membrane receptor trafficking.     

RNF8 ubiquitylates histones at DNA double-strand breaks and promotes assembly of repair proteins

Accumulation of repair proteins on damaged chromosomes is required to restore genomic integrity. However, the mechanisms of protein retention at the most destructive chromosomal lesions, the DNA double-strand breaks (DSBs), are poorly understood. We show that RNF8, a RING-finger ubiquitin ligase, rapidly assembles at DSBs via interaction of its FHA domain with the phosphorylated adaptor protein MDC1. This is accompanied by an increase in DSB-associated ubiquitylations and followed by accumulation of 53BP1 and BRCA1 repair proteins. Knockdown of RNF8 or disruption of its FHA or RING domains impaired DSB-associated ubiquitylation and inhibited retention of 53BP1 and BRCA1 at the DSB sites. In addition, we show that RNF8 can ubiquitylate histone H2A and H2AX, and that its depletion sensitizes cells to ionizing radiation. These data suggest that MDC1-mediated and RNF8-executed histone ubiquitylation protects genome integrity by licensing the DSB-flanking chromatin to concentrate repair factors near the DNA lesions.     

Apoplastic barriers to radial oxygen loss and solute penetration: a chemical and functional comparison of the exodermis of two wetland species, Phragmites australis and Glyceria maxima

Few studies have examined exodermal development in relation to the formation of barriers to both radial oxygen loss (ROL) and solute penetration along growing roots. Here, we report on the structural development, chemical composition and functional properties of the exodermis in two diverse wetland grasses, Glyceria maxima and Phragmites australis. Anatomical features, development, the biochemical composition of exodermal suberin and the penetration of apoplastic tracers and oxygen were examined. Striking interspecific differences in exodermal structure, suberin composition and quantity per unit surface area, and developmental changes along the roots were recorded. Towards the root base, ROL and periodic acid (H(5)IO(6)) penetration were virtually stopped in P. australis; in G. maxima, a tight ROL barrier restricted but did not stop H(5)IO(6) penetration and the exodermis failed to stain with lipidic dyes. Cultivation in stagnant deep hypoxia conditions or oxygenated circulating solution affected the longitudinal pattern of ROL profiles in G. maxima but statistically significant changes in exodermal suberin composition or content were not detected. Interspecific differences in barrier performance were found to be related to hypodermal structure and probably to qualitative as well as quantitative variations in suberin composition and distribution within exodermal cell walls. Implications for root system function are discussed, and it is emphasized that sufficient spatial resolution to identify the effects of developmental changes along roots is crucial for realistic evaluation of exodermal barrier properties.     

Composition of the cuticle of developing sweet cherry fruit

The composition of wax and cutin from developing sweet cherry (Prunus avium) fruit was studied by GC-MS between 22 and 85 days after full bloom (DAFB). In this and our previous study, fruit mass and surface area increased in a sigmoidal pattern with time, but mass of the cuticular membrane (CM) per unit fruit surface area decreased. On a whole fruit basis, mass of CM increased up to 36 DAFB and remained constant thereafter. At maturity, triterpenes, alkanes and alcohols accounted for 75.6%, 19.1% and 1.2% of total wax, respectively. The most abundant constituents were the triterpenes ursolic (60.0%) and oleanolic acid (7.5%), the alkanes nonacosane (13.0%) and heptacosane (3.0%), and the secondary alcohol nonacosan-10-ol (1.1%). In developing fruit triterpenes per unit area decreased, but alkanes and alcohols remained essentially constant. The cutin fraction of mature fruit consisted of mostly C16 (69.5%) and, to a lower extent, C18 monomers (19.4%) comprising alkanoic, omega-hydroxyacids, alpha,omega-dicarboxylic and midchain hydroxylated acids. The most abundant constituents were 9(10),16-dihydroxy-hexadecanoic acid (53.6%) and 9,10,18-trihydroxy-octadecanoic acid (7.8%). Amounts of C16 and C18 monomers per unit area decreased in developing fruit, but remained approximately constant on a whole fruit basis. Within both classes of monomers, opposing changes occurred. Amounts of hexadecandioic, 16-hydroxy-hexadecanoic, 9(10)-hydroxy-hexadecane-1,16-dioic and 9,10-epoxy-octadecane-1,18-dioic acids increased, but 9,10,18-trihydroxy-octadecanoic and 9,10,18-trihydroxy-octadecenoic acids decreased. There were no qualitative and minor quantitative differences in wax and cutin composition between cultivars at maturity. Our data indicate that deposition of some constituents of wax and cutin ceased during early fruit development.     

Mechanisms of programmed cell death during oogenesis in <Emphasis Type="Italic">Drosophila virilis</Emphasis>

We describe the features of programmed cell death occurring in the egg chambers of Drosophila virilis during mid-oogenesis and late oogenesis. During mid-oogenesis, the spontaneously degenerating egg chambers exhibit typical characteristics of apoptotic cell death. As revealed by propidium iodide, rhodamine-conjugated phalloidin staining, and the TUNEL assay, respectively, the nurse cells contain condensed chromatin, altered actin cytoskeleton, and fragmented DNA. In vitro caspase activity assays and immunostaining procedures demonstrate that the atretic egg chambers possess high levels of caspase activity. Features of autophagic cell death are also observed during D. virilis mid-oogenesis, as shown by monodansylcadaverine staining, together with an ultrastructural examination by transmission electron microscopy. During the late stages of oogenesis in D. virilis, once again, the two mechanisms, viz., nurse cell cluster apoptosis and autophagy, operate together, manifesting features of cell death similar to those detailed above. Moreover, an altered form of cytochrome c seems to be released from the mitochondria in the nurse cells proximal to the oocyte. We propose that apoptosis and autophagy function synergistically during oogenesis in D. virilis in order to achieve a more efficient elimination of the degenerated nurse cells and abnormal egg chambers. The present study was co-financed within Op. Education by the European Social Fund and by National Resources via a grant (HRAKLEITOS 70/3/7164) to Professor L.H. Margaritis.