Hrs sorts ubiquitinated proteins into clathrin-coated microdomains of early endosomes

After endocytosis, some membrane proteins recycle from early endosomes to the plasma membrane whereas others are transported to late endosomes and lysosomes for degradation. Conjugation with the small polypeptide ubiquitin is a signal for lysosomal sorting. Here we show that the hepatocyte growth factor-regulated tyrosine kinase substrate, Hrs, is involved in the endosomal sorting of ubiquitinated membrane proteins. Hrs contains a clathrin-binding domain, and by electron microscopy we show that Hrs localizes to flat clathrin lattices on early endosomes. We demonstrate that Hrs binds directly to ubiquitin by way of a ubiquitin-interacting motif (UIM), and that ubiquitinated proteins localize specifically to Hrs- and clathrin-containing microdomains. Whereas endocytosed transferrin receptors fail to colocalize with Hrs and rapidly recycle to the cell surface, transferrin receptors that are fused to ubiquitin interact with Hrs, localize to Hrs- and clathrin-containing microdomains and are sorted to the degradative pathway. Overexpression of Hrs strongly and specifically inhibits recycling of ubiquitinated transferrin receptors by a mechanism that requires a functional UIM. We conclude that Hrs sorts ubiquitinated membrane proteins into clathrin-coated microdomains of early endosomes, thereby preventing their recycling to the cell surface.     

Inhibitors of COP-mediated transport and cholera toxin action inhibit simian virus 40 infection

Simian virus 40 (SV40) is a nonenveloped virus that has been shown to pass from surface caveolae to the endoplasmic reticulum in an apparently novel infectious entry pathway. We now show that the initial entry step is blocked by brefeldin A and by incubation at 20 degrees C. Subsequent to the entry step, the virus reaches a domain of the rough endoplasmic reticulum by an unknown pathway. This intracellular trafficking pathway is also brefeldin A sensitive. Infection is strongly inhibited by expression of GTP-restricted ADP-ribosylation factor 1 (Arf1) and Sar1 mutants and by microinjection of antibodies to betaCOP. In addition, we demonstrate a potent inhibition of SV40 infection by the dipeptide N-benzoyl-oxycarbonyl-Gly-Phe-amide, which also inhibits late events in cholera toxin action. Our results identify novel inhibitors of SV40 infection and show that SV40 requires COPI- and COPII-dependent transport steps for successful infection.     

Ubiquitination and proteasomal activity is required for transport of the EGF receptor to inner membranes of multivesicular bodies

EGF, but not TGF alpha, efficiently induces degradation of the EGF receptor (EGFR). We show that EGFR was initially polyubiquitinated to the same extent upon incubation with EGF and TGF alpha, whereas the ubiquitination was more sustained by incubation with EGF than with TGF alpha. Consistently, the ubiquitin ligase c-Cbl was recruited to the plasma membrane upon activation of the EGFR with EGF and TGF alpha, but localized to endosomes only upon activation with EGF. EGF remains bound to the EGFR upon endocytosis, whereas TGF alpha dissociates from the EGFR. Therefore, the sustained polyubiquitination is explained by EGF securing the kinase activity of endocytosed EGFR. Overexpression of the dominant negative N-Cbl inhibited ubiquitination of the EGFR and degradation of EGF and EGFR. This demonstrates that EGF-induced ubiquitination of the EGFR as such is important for lysosomal sorting. Both lysosomal and proteasomal inhibitors blocked degradation of EGF and EGFR, and proteasomal inhibitors inhibited translocation of activated EGFR from the outer limiting membrane to inner membranes of multivesicular bodies (MVBs). Therefore, lysosomal sorting of kinase active EGFR is regulated by proteasomal activity. Immuno-EM showed the localization of intact EGFR on internal membranes of MVBs. This demonstrates that the EGFR as such is not the proteasomal target.     

Cbl-dependent ubiquitination is required for progression of EGF receptors into clathrin-coated pits

Ligand binding causes the EGF receptor (EGFR) to become ubiquitinated by Cbl upon association with the adaptor protein Grb2. We have investigated the role of ubiquitin and Grb2 in ligand-induced endocytosis of the EGFR. Incubation of cells with EGF on ice caused translocation of Grb2 and Cbl from the cytosol to the rim of coated pits. Grb2 with point mutations in both SH3 domains inhibited recruitment of the EGFR to clathrin-coated pits, in a Ras-independent manner. On overexpression of the Cbl-binding protein Sprouty, ubiquitination of the EGFR was inhibited, the EGFR was recruited only to the rim of coated pits, and endocytosis of the EGFR was inhibited. Conjugation-defective ubiquitin similarly inhibited recruitment of EGF-EGFR to clathrin-coated pits. Even though this does not prove that cargo must be ubiquitinated, this indicates the importance of interaction of ubiquitinated protein(s) with proteins harboring ubiquitin-interacting domains. We propose that Grb2 mediates transient anchoring of the EGFR to an Eps15-containing molecular complex at the rim of coated pits and that Cbl-induced ubiquitination of the EGFR allows relocation of EGFR from the rim to the center of clathrin-coated pits.     

PCB impairs smoltification and seawater performance in anadromous Arctic charr (Salvelinus alpinus)

The impacts of polychlorinated biphenyl (PCB) exposure on smoltification and subsequent seawater performance were investigated in hatchery-reared, anadromous Arctic charr (Salvelinus alpinus). The fish were subjected to a 2-month summer seawater residence, after which they were orally dosed with 0 (Control, C), 1 (Low Dose, LD) or 100 mg Aroclor 1254 kg(-1) body mass (High Dose, HD) in November. They were then held in fresh water, without being fed (to mimic their natural overwintering in freshwater), until they had smolted in June the next year. The smolts were then transferred to seawater and fed to mimic their summer feeding residence in seawater, followed by a period without food in freshwater from August until maturation in October. Compared with C and LD charr, the HD charr had either a transient or a permanent reduction in plasma growth hormone, insulin-like growth factor-1, and thyroxin and triiodothyronine titers during the period of smoltification. These hormonal alterations in the HD charr corresponded with impaired hyposmoregulatory ability in May and June, as well as reduced growth rate and survival after transference to seawater. Consequently, fewer fish in the HD group matured in October compared to the other two treatments. The HD fish had a liver PCB concentration ranging between 14 and 42 mg kg(-1) wet mass, whereas there were similar, and very low, liver PCB concentrations in LD and C fish throughout the smolting period. Our findings suggest that PCB might compromise mechanisms important for fitness in a fish species living in an extreme environment.     

Performance Development in Adolescent Track and Field Athletes According to Age,Sex and Sport Discipline

Introduction

Sex-specific differences that arise during puberty have a pronounced effect on the training process. However, the consequences this should have for goal-setting, planning and implementation of training for boys and girls of different ages remains poorly understood. The aim of this study was to quantify performance developments in athletic running and jumping disciplines in the age range 11-18 and identify progression differences as a function of age, discipline and sex.

Methods

The 100 all-time best Norwegian male and female 60-m, 800-m, long jump and high jump athletes in each age category from 11 to 18 years were analysed using mixed models with random intercept according to athlete.

Results

Male and female athletes perform almost equally in running and jumping events up to the age of 12. Beyond this age, males outperform females. Relative annual performance development in females gradually decreases throughout the analyzed age period. In males, annual relative performance development accelerates up to the age of 13 (for running events) or 14 (for jumping events) and then gradually declines when approaching 18 years of age. The relative improvement from age 11 to 18 was twice as high in jumping events compared to running events. For all of the analyzed disciplines, overall improvement rates were >50% higher for males than for females. The performance sex difference evolves from < 5% to 10-18% in all the analyzed disciplines from age 11 to 18 yr.

Conclusion

To the authors’ knowledge, this is the first study to present absolute and relative annual performance developments in running and jumping events for competitive athletes from early to late adolescence. These results allow coaches and athletes to set realistic goals and prescribe conditioning programs that take into account sex-specific differences in the rate of performance development at different stages of maturation.     

Notch1 Is Pan-Endothelial at the Onset of Flow and Regulated by Flow

Arteriovenous differentiation is a key event during vascular development and hemodynamic forces play an important role. Arteriovenous gene expression is present before the onset of flow, however it remains plastic and flow can alter arteriovenous identity. Notch signaling is especially important in the genetic determination of arteriovenous identity. Nevertheless, the effect of the onset of circulation on Notch expression and signaling has not been studied. The aim of this study is therefore to investigate the interaction of Notch1 signaling and hemodynamic forces during early vascular development. We find that the onset of Notch1 expression coincides with the onset of flow, and that expression is pan-endothelial at the onset of circulation in mouse embryos and only becomes arterial-specific after remodeling has occurred. When we ablate flow in the early embryo, endothelial cells fail to express Notch1. We show that low and disturbed flow patterns upregulate Notch1 expression in endothelial cells in vitro, but that higher shear stress levels do not (≥10 dynes/cm²). Using siRNA, we knocked down Notch1 to investigate the role of Notch1 in mechanotransduction. When we applied shear stress levels similar to those found in embryonic arteries, we found an upregulation of Klf2, Dll1, Dll4, Jag1, Hey1, Nrp1 and CoupTFII but that only Dll4, Hey1, Nrp1 and EphB4 required Notch1 for flow-induced expression. Our results therefore indicate that Notch1 can modulate mechanotransduction but is not a critical mediator of the process since many genes mechanotransduce normally in the absence of Notch1, including genes involved in arteriovenous differentiation.     

Competition on the Rocks: Community Growth and Tessellation

Crustose lichen communities on rocks exhibit fascinating spatial mosaics resembling political maps of nations or municipalities. Although the establishment and development of biological populations are important themes in ecology, our understanding of the formation of such patterns on the rocks is still in its infancy. Here, we present a novel model of the concurrent growth, establishment and interaction of lichens. We introduce an inverse technique based on Monte Carlo simulations to test our model on field samples of lichen communities. We derive an expression for the time needed for a community to cover a surface and predict the historical spatial dynamics of field samples. Lichens are frequently used for dating the time of exposure of rocks in glacial deposits, lake retreats or rock falls. We suggest our method as a way to improve the dating.