Forty Years of Clathrin-coated Vesicles.

The purification of coated vesicles and the discovery of clathrin by Barbara Pearse in 1975 was a landmark in cell biology. Over the past 40 years, work from many labs has uncovered the molecular details of clathrin and its associated proteins, including how they assemble into a coated vesicle and how they select cargo. Unexpected connections have been found with signalling, development, neuronal transmission, infection, immunity and genetic disorders. But there are still a number of unanswered questions, including how clathrin-mediated trafficking is regulated and how the machinery evolved.This is the author accepted manuscript. The final version is available from Wiley via http://dx.doi.org/10.1111/tra.1233

Rag GTPases and phosphatidylinositol 3-phosphate mediate recruitment of the AP-5/SPG11/SPG15 complex.

Adaptor protein complex 5 (AP-5) and its partners, SPG11 and SPG15, are recruited onto late endosomes and lysosomes. Here we show that recruitment of AP-5/SPG11/SPG15 is enhanced in starved cells and occurs by coincidence detection, requiring both phosphatidylinositol 3-phosphate (PI3P) and Rag GTPases. PI3P binding is via the SPG15 FYVE domain, which, on its own, localizes to early endosomes. GDP-locked RagC promotes recruitment of AP-5/SPG11/SPG15, while GTP-locked RagA prevents its recruitment. Our results uncover an interplay between AP-5/SPG11/SPG15 and the mTORC1 pathway and help to explain the phenotype of AP-5/SPG11/SPG15 deficiency in patients, including the defect in autophagic lysosome reformation

Assembly and function of AP-3 complexes in cells expressing mutant subunits

The mouse mutants mocha and pearl are deficient in the AP-3 δ and β3A subunits, respectively. We have used cells from these mice to investigate both the assembly of AP-3 complexes and AP-3 function. In mocha cells, the β3 and μ3 subunits coassemble into a heterodimer, whereas the σ3 subunit remains monomeric. In pearl cells, the δ and σ3 subunits coassemble into a heterodimer, whereas μ3 gets destroyed. The yeast two hybrid system was used to confirm these interactions, and also to demonstrate that the A (ubiquitous) and B (neuronal-specific) isoforms of β3 and μ3 can interact with each other. Pearl cell lines were generated that express β3A, β3B, a β3Aβ2 chimera, two β3A deletion mutants, and a β3A point mutant lacking a functional clathrin binding site. All six constructs assembled into complexes and were recruited onto membranes. However, only β3A, β3B, and the point mutant gave full functional rescue, as assayed by LAMP-1 sorting. The β3Aβ2 chimera and the β3A short deletion mutant gave partial functional rescue, whereas the β3A truncation mutant gave no functional rescue. These results indicate that the hinge and/or ear domains of β3 are important for function, but the clathrin binding site is not needed

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

The WDR11 complex facilitates the tethering of AP-1-derived vesicles.

Vesicluar transport of proteins from endosomes to the trans-Golgi network (TGN) is an essential cellular pathway, but much of its machinery is still unknown. A screen for genes involved in endosome-to-TGN trafficking produced two hits, the adaptor protein-1 (AP-1 complex), which facilitates vesicle budding, and WDR11. Here we demonstrate that WDR11 forms a stable complex with two other proteins, which localises to the TGN region and does not appear to be associated with AP-1, suggesting it may act downstream from budding. In a vesicle tethering assay, capture of vesicles by golgin-245 was substantially reduced in WDR11-knockout cells. Moreover, structured illumination microscopy and relocation assays indicate that the WDR11 complex is initially recruited onto vesicles rather than the TGN, where it may in turn recruit the golgin binding partner TBC1D23. We propose that the complex acts together with TBC1D23 to facilitate the golgin-mediated capture of vesicles that were generated using AP-1

The H-alpha and Infrared Star Formation Rates for the Nearby Field Galaxy Survey

We investigate the H-alpha and infrared star formation rate (SFR) diagnostics for galaxies in the Nearby Field Galaxy Survey (NFGS). For the 81 galaxies in our sample, we derive H-alpha fluxes (included here) from integrated spectra. There is a strong correlation between the ratio of far-infrared to optical luminosities L(FIR)/L(H-alpha) and the extinction E(B-V) measured with the Balmer decrement. Before reddening correction, the SFR(IR) and SFR(H-alpha) are related to each other by a power-law. Correction of the SFR(H-alpha) for extinction using the Balmer decrement and a classical reddening curve both reduces the scatter in the SFR(IR)-SFR(H-alpha) correlation and results in a much closer agreement (within ~10%) between the two SFR indicators. This SFR relationship spans 4 orders of magnitude and holds for all Hubble types with IRAS detections in the NFGS. A constant ratio between the SFR(IR) and SFR(H-alpha) for all Hubble types, including early types (S0-Sab), suggests that the IR emission in all of these objects results from a young stellar population.Comment: 23 pages, 5 figures, 1 table. Accepted for publication in the Astronomical Journal. V2: Important changes: IRAS fluxes updated. Only moderate and good quality IRAS FIR fluxes are now used, resulting in slight changes to the equations and figures. The IR and H-alpha SFRs now agree to within ~10%, rather than ~30% as quoted previousl

How do early-life adverse childhood experiences mediate the relationship between childhood socioeconomic conditions and adolescent health outcomes in the UK?

Background Both adverse childhood experiences (ACEs) and adverse childhood socioeconomic conditions (SECs) in early life are associated with poor outcomes across the life course. However, the complex interrelationships between childhood SECs and ACEs are unclear, as are the consequences for health outcomes beyond childhood. We therefore assessed the extent to which early-life ACEs mediate the relationship between SECs and socioemotional behavioural problems, cognitive disability and overweight/obesity in adolescence. Methods We used longitudinal data from the UK Millennium Cohort Study (MSC). Outcomes assessed at age 14 were socioemotional behavioural problems, cognitive disability and overweight/obesity. SECs at birth were measured by maternal education. Potentially mediating ACEs measured up to 5 years were verbal and physical maltreatment, parental drug use, domestic violence, parental divorce, maternal mental illness and high frequency of parental alcohol use. We used counterfactual mediation analysis to assess the extent to which ACEs mediate the association between SECs at birth and behavioural, cognitive and physical outcomes at age 14, estimating total (TE), natural direct and indirect effects, and mediated proportions. Results Children with disadvantaged SECs were more likely to have socioemotional behavioural problems (relative risk (RR) 3.85, 95% CI 2.48 to 5.97), cognitive disability (RR 3.87, 95% CI 2.33 to 6.43) and overweight/obesity (RR 1.61, 95% CI 1.32 to 1.95), compared to those with more advantaged SECs. Overall, 18% of the TE of SECs on socioemotional behavioural problems was mediated through all ACEs investigated. For cognitive disability and overweight/obese, the proportions mediated were 13% and 19%, respectively. Conclusion ACEs measured up to age 5 years in the MCS explained about one-sixth of inequalities in adolescents behavioural, cognitive and physical outcomes