Signaling through tyrosine kinase receptors (TKRs) is thought to be modulated by receptor-mediated endocytosis and degradation of the receptor in the lysosome. However, factors that regulate endosomal sorting of TKRs are largely unknown. Here, we demonstrate that Hrs (Hepatocyte growth factor-regulated tyrosine kinase substrate) is one such factor. Electron microscopy studies of hrs mutant larvae reveal an impairment in endosome membrane invagination and formation of multivesicular bodies (MVBs). hrs mutant animals fail to degrade active epidermal growth factor (EGF) and Torso TKRs, leading to enhanced signaling and altered embryonic patterning. These data suggest that Hrs and MVB formation function to downregulate TKR signaling.

We have identified mutations in Drosophila endophilin to study its function in vivo. Endophilin is required presynaptically at the neuromuscular junction, and absence of Endophilin dramatically impairs endocytosis in vivo. Mutant larvae that lack Endophilin fail to take up FM1-43 dye in synaptic boutons, indicating an inability to retrieve synaptic membrane. This defect is accompanied by an expansion of the presynaptic membrane, and a depletion of vesicles from the bouton lumen. Interestingly, mutant larvae are still able to sustain release at 15%-20% of the normal rate during high-frequency stimulation. We propose that kiss-and-run maintains neurotransmission at active zones of the larval NMJ in endophilin animals.

Antimicrobial peptides (AMPs) are a potential source of new molecules to counter the increase in antimicrobial resistant infections but a better understanding of their properties is required for effective translation as therapeutics. Details of the mechanism of their interaction with the bacterial plasma membrane are desired since damage or penetration of this structure is considered essential for AMP activity. Relatively modest modifications to AMP primary sequence can induce substantial changes in potency and/or spectrum of activity but, hitherto, have not been predicted to substantially alter the mechanism of interaction with the bacterial plasma membrane. Here we use a combination of molecular dynamics simulations, circular dichroism, liquid-and solid-state NMR and patch clamp to investigate the extent to which temporin B and its analogues can be distinguished both in vitro and in silico on the basis of their interactions with model membranes. Enhancing the hydrophobicity of the N-terminus and cationicity of the C-terminus in temporin B improves its membrane activity and potency against both Gram-negative and Grampositive bacteria. In contrast, enhancing the cationicity of the N-terminus abrogates its ability to trigger channel conductance and renders it ineffective against Staphylococcus aureus while nevertheless enhancing its potency against Escherichia coli. Our findings suggest even closely related AMPs may target the same bacterium with fundamentally differing mechanisms of action.

1H‐NMR and circular dichroism studies have been carried out on osteocalcin, a 49‐residue, calcium‐binding protein, the sequence of which contains a disulphide bridge, a proline‐rich segment and three γ‐carboxyglutamic acid (Gla) residues. These latter residues have been proposed to lie on one face of an α helix and interact with the mineral phase, leading to incorporation of the protein into the bone matrix. Circular dichroism shows an increase in the α‐helical structure on Ca2+ binding to bovine osteocalcin. This induced structure is lost on heating the protein, giving a spectrum close to that of the Ca2+‐free protein. 1H‐NMR studies of rabbit osteocalcin gave a set of resonance assignments and NOEs which could be interpreted in terms of distance constraints. These did not allow a single conformation to be defined for the protein in solution but reflect rather a flexible structure which may be essential for the function of the protein. The calculated structures contain a hydrophobic core (comprising Leu2, Leu32, Val36 and Tyr42, seen to be slowly flipping in the Ca2+‐bound form) and have the γ‐carboxyglutamic acid side chains exposed on one face of the molecule.