Endolysosomes Are the Principal Intracellular Sites of Acid Hydrolase Activity.

The endocytic delivery of macromolecules from the mammalian cell surface for degradation by lysosomal acid hydrolases requires traffic through early endosomes to late endosomes followed by transient (kissing) or complete fusions between late endosomes and lysosomes. Transient or complete fusion results in the formation of endolysosomes, which are hybrid organelles from which lysosomes are re-formed. We have used synthetic membrane-permeable cathepsin substrates, which liberate fluorescent reporters upon proteolytic cleavage, as well as acid phosphatase cytochemistry to identify which endocytic compartments are acid hydrolase active. We found that endolysosomes are the principal organelles in which acid hydrolase substrates are cleaved. Endolysosomes also accumulated acidotropic probes and could be distinguished from terminal storage lysosomes, which were acid hydrolase inactive and did not accumulate acidotropic probes. Using live-cell microscopy, we have demonstrated that fusion events, which form endolysosomes, precede the onset of acid hydrolase activity. By means of sucrose and invertase uptake experiments, we have also shown that acid-hydrolase-active endolysosomes and acid-hydrolase-inactive, terminal storage lysosomes exist in dynamic equilibrium. We conclude that the terminal endocytic compartment is composed of acid-hydrolase-active, acidic endolysosomes and acid hydrolase-inactive, non-acidic, terminal storage lysosomes, which are linked and function in a lysosome regeneration cycle.This work was supported by MRC research grant MR/M010007/1. The CIMR is supported by Wellcome Trust Strategic Award 100140. The Cellomics ArrayScan™ VTi High Content Screening Microscope, Zeiss LSM710 confocal microscope and FEI Tecnai G2 Spirit BioTWIN transmission EM were purchased with Wellcome Trust grants 079919 and 093026. LJD is supported by a BBSRC industrial CASE studentship with GSK Research and Development Ltd. We thank Sally Gray for preparing and sequencing pLXIN constructs and Matthew Gratian for help with light microscopy and analytical software.This is the final version of the article. It first appeared from Elsevier via https://doi.org/ 10.1016/j.cub.2016.06.04

Clathrin-mediated endocytosis in AP-2–depleted cells

We have used RNA interference to knock down the AP-2 μ2 subunit and clathrin heavy chain to undetectable levels in HeLaM cells. Clathrin-coated pits associated with the plasma membrane were still present in the AP-2–depleted cells, but they were 12-fold less abundant than in control cells. No clathrin-coated pits or vesicles could be detected in the clathrin-depleted cells, and post-Golgi membrane compartments were swollen. Receptor-mediated endocytosis of transferrin was severely inhibited in both clathrin- and AP-2–depleted cells. Endocytosis of EGF, and of an LDL receptor chimera, were also inhibited in the clathrin-depleted cells; however, both were internalized as efficiently in the AP-2–depleted cells as in control cells. These results indicate that AP-2 is not essential for clathrin-coated vesicle formation at the plasma membrane, but that it is one of several endocytic adaptors required for the uptake of certain cargo proteins including the transferrin receptor. Uptake of the EGF and LDL receptors may be facilitated by alternative adaptors

Comparing plasma and faecal measures of steroid hormones in Adelie penguins Pygoscelis adeliae

Physiological measurements of both stress and sex hormones are often used to estimate the consequences of natural or human-induced change in ecological studies of various animals. Different methods of hormone measurement exist, potentially explaining variation in results across studies; methods should be cross-validated to ensure that they correlate. We directly compared faecal and plasma hormone measurements for the first time in a wild free-living species, the Adelie penguin (Pygoscelis adeliae). Blood and faecal samples were simultaneously collected from individual penguins for comparison and assayed for testosterone and corticosterone (or their metabolites). Sex differences and variability within each measure, and correlation of values across measures were compared. For both hormones, plasma samples showed greater variation than faecal samples. Males had higher mean corticosterone concentrations than females, but the difference was only statistically significant in faecal samples. Plasma testosterone, but not faecal testosterone, was significantly higher in males than females. Correlation between sample types was poor overall, and weaker in females than in males, perhaps because measures from plasma represent hormones that are both free and bound to globulins, whereas measures from faeces represent only the free portion. Faecal samples also represent a cumulative measure of hormones over time, as opposed to a plasma ‘snapshot’ concentration. Our data indicate that faecal sampling appears more suitable for assessing baseline hormone concentrations, whilst plasma sampling may best define immediate responses to environmental events. Consequently, future studies should ensure that they select the most appropriate matrix and method of hormone measurement to answer their research questions

A non-canonical ESCRT pathway, including histidine domain phosphotyrosine phosphatase (HD-PTP), is used for down-regulation of virally ubiquitinated MHC class I.

The Kaposi's sarcoma-associated herpes virus (KSHV) K3 viral gene product effectively down-regulates cell surface MHC class I. K3 is an E3 ubiquitin ligase that promotes Lys(63)-linked polyubiquitination of MHC class I, providing the signal for clathrin-mediated endocytosis. Endocytosis is followed by sorting into the intralumenal vesicles (ILVs) of multivesicular bodies (MVBs) and eventual delivery to lysosomes. The sorting of MHC class I into MVBs requires many individual proteins of the four endosomal sorting complexes required for transport (ESCRTs). In HeLa cells expressing the KSHV K3 ubiquitin ligase, the effect of RNAi-mediated depletion of individual proteins of the ESCRT-0 and ESCRT-I complexes and three ESCRT-III proteins showed that these are required to down-regulate MHC class I. However, depletion of proteins of the ESCRT-II complex or of the ESCRT-III protein, VPS20 (vacuolar protein sorting 20)/CHMP6 (charged MVB protein 6), failed to prevent the loss of MHC class I from the cell surface. Depletion of histidine domain phosphotyrosine phosphatase (HD-PTP) resulted in an increase in the cell surface concentration of MHC class I in HeLa cells expressing the KSHV K3 ubiquitin ligase. Rescue experiments with wild-type (WT) and mutant HD-PTP supported the conclusion that HD-PTP acts as an alternative to ESCRT-II and VPS20/CHMP6 as a link between the ESCRT-I and those ESCRT-III protein(s) necessary for ILV formation. Thus, the down-regulation of cell surface MHC class I, polyubiquitinated by the KSHV K3 ubiquitin ligase, does not employ the canonical ESCRT pathway, but instead utilizes an alternative pathway in which HD-PTP replaces ESCRT-II and VPS20/CHMP6.This work was supported by an MRC research grant to J.P.L. (G0900113). M.D.J.P. and J.L.E. were MRC research students and S.P. a Wellcome Trust research student. K.B. was a British Heart Foundation Intermediate Fellow and P.J.L. is a Wellcome Trust Principal Fellow. The CIMR is supported by a Wellcome Trust Strategic Award 100140 and an electron microscope was purchased with Wellcome Trust grant 093026.This is the final version of the article. It first appeared from Portland Press via http://dx.doi.org/10.1042/BJ2015033

The multifactorial nature of beak and skull shape evolution in parrots and cockatoos (Psittaciformes)

Background The Psittaciformes (parrots and cockatoos) are characterised by their large beaks, and are renowned for their ability to produce high bite forces. These birds also possess a suite of modifications to their cranial architecture interpreted to be adaptations for feeding on mechanically resistant foods, yet the relationship between cranial morphology and diet has never been explicitly tested. Here, we provide a three-dimensional geometric morphometric analysis of the developmental and biomechanical factors that may be influencing the evolution of psittaciformes’ distinctive cranial morphologies. Results Contrary to our own predictions, we find that dietary preferences for more- or less- mechanically resistant foods have very little influence on beak and skull shape, and that diet predicts only 2.4% of the shape variation in psittaciform beaks and skulls. Conversely, evolutionary allometry and integration together predict almost half the observed shape variation, with phylogeny remaining an important factor in shape identity throughout our analyses, particularly in separating cockatoos (Cacatuoidea) from the true parrots (Psittacoidea). Conclusions Our results are similar to recent findings about the evolutionary trajectories of skull and beak shape in other avian families. We therefore propose that allometry and integration are important factors causing canalization of the avian head, and while diet clearly has an influence on beak shape between families, this may not be as important at driving evolvability within families as is commonly assumed

The Primacy of Public Health Considerations in Defining Poor Quality Medicines

Paul Newton and colleagues argue that public health, and not intellectual property or trade issues, should be the prime consideration in defining and combating counterfeit medicines, and that the World Health Organization (WHO) should take a more prominent role

CALM regulates clathrin-coated vesicle size and maturation by directly sensing and driving membrane curvature.

The size of endocytic clathrin-coated vesicles (CCVs) is remarkably uniform, suggesting that it is optimized to achieve the appropriate levels of cargo and lipid internalization. The three most abundant proteins in mammalian endocytic CCVs are clathrin and the two cargo-selecting, clathrin adaptors, CALM and AP2. Here we demonstrate that depletion of CALM causes a substantial increase in the ratio of "open" clathrin-coated pits (CCPs) to "necked"/"closed" CCVs and a doubling of CCP/CCV diameter, whereas AP2 depletion has opposite effects. Depletion of either adaptor, however, significantly inhibits endocytosis of transferrin and epidermal growth factor. The phenotypic effects of CALM depletion can be rescued by re-expression of wild-type CALM, but not with CALM that lacks a functional N-terminal, membrane-inserting, curvature-sensing/driving amphipathic helix, the existence and properties of which are demonstrated. CALM is thus a major factor in controlling CCV size and maturation and hence in determining the rates of endocytic cargo uptake.S.E.M. and D.J.O. are funded by a Wellcome Trust Fellowship (to D.J.O. no. 090909/Z). N.A.B. is funded by MRC grant MR/M010007/1, and S.H. is funded by a grant from the German Science Foundation (SFB 635, TP A3). D.S. and S.M. acknowledge financial support from the Lundbeck Foundation and the Danish Councils for Independent and Strategic Research. C.J.M. and F.P. were funded by the Fondation pour la Recherche Medicale.This is the final published version. It first appeared at http://www.cell.com/developmental-cell/fulltext/S1534-5807%2815%2900144-6