The small-eye trait induced by expression of the <i>UAS-endoA^A66W</i> mutant transgene.

<p>A, eye of a control fly carrying the <i>elav-GAL4</i> driver but no <i>endoA</i> transgene. B, <i>endoA</i> null fly rescued to the pharate adult stage by <i>UAS-endoA^A66W</i> expression, driven by <i>elav-GAL4</i> (<i>elav-GAL4/w</i>; <i>UAS-endoA^{A66W 4.1}/+</i>; <i>endoA^Δ4/endoA^Δ4</i>). Note that the eye size is reduced and that the lower eye tip is pointy rather than rounded. C, The small-eye trait also appears when <i>UAS-endoA^A66W</i> expression occurs on a wild-type <i>endoA</i> background (<i>elav-GAL4/w</i>; <i>UAS-endoA^{A66W 4.1}/+</i>; <i>endoA⁺/endoA⁺</i>). D–G, Scanning electron micrographs of <i>elav-GAL4</i> (D, F) and <i>elav-GAL4/w</i>; <i>UAS-endoA^{A66W 4.1}/+</i>; <i>endoA⁺/endoA⁺</i> (E, G) eyes. In G, some examples of ommatidia that lack bristles are indicated by <i>asterisks</i>, and aberrant dual bristles by <i>arrowheads</i>. Pitting is indicated by an <i>arrow</i>. Scale bars: C, 100 µm (applies to A–C); E, 50 µm (D, E); G, 20 µm (F, G).</p

Ability of transgenic <i>endoA</i> constructs to rescue the development of <i>endoA</i> null mutants to adulthood.

<p>Shown is the proportion of eclosed adult rescuants (genotype <i>elav-GAL4/Y or w</i>; <i>UAS-endoA</i>*<i>/+; endoA^Δ4/endoA^Δ4</i>) relative to the total number of adult progeny resulting from the rescue cross. The <i>UAS-endoA</i>* transgene carried the mutations indicated on the abscissa and in some cases also encoded a hemagglutinin epitope tag (indicated by the suffix “−HA”). Also shown is the proportion of rescuants in which the <i>endoA</i> transgene encoded either wild type EndoA (“wt”), or HA-tagged wild type EndoA (“wt-HA”). Each <i>bar</i> represents one transgenic integration line, specified <i>below the abscissa</i>. The total number of adult progeny resulting from the rescue cross is indicated for each line (<i>numbers above the bars</i>). The lower and upper 95% confidence intervals are given. ^*<i>P</i><0.01. N.s., not significant. ^†Besides <i>UAS-endoA^{A66W 4.1}</i>, the rescue efficiency of two other <i>UAS-endoA^A66W</i> transgenes was evaluated (<i>UAS-endoA^{A66W 41.3}</i> and <i>UAS-endoA^89.1</i>). They both caused lethality of all the progeny from the rescue cross, as detailed in the text.</p

Neurotransmission at the neuromuscular junction in larval mutant rescuants.

<p>Intracellular recordings were made from the somatic muscles of <i>Elav-GAL4/Y or w</i>; <i>UAS-endoA</i>*<i>/+</i>; <i>endoA^Δ4/endoA^Δ4</i> third instar larvae, where <i>UAS-endoA</i>* represents a mutated <i>endoA</i> transgene, or one encoding wild type EndoA (“wt”), as indicated in A–F. The suffix “−HA” signifies the presence of an additional HA tag. Raw recordings are not shown. A, Ability to sustain neurotransmitter release during a tetanus (10 min at 10 Hz) and immediately following tetanic stimulation (10 min at 0.2 Hz). The amplitude of the excitatory junctional potential (EJP), relative to the amplitude prior to the tetanus (0.2 Hz, not shown), is plotted. Error bars are omitted in A for clarity; the variability can be judged from B–D. B, The EJP amplitude (mean and 95% confidence interval) at the end of the 10 Hz tetanus, just before switching back to stimulation at 0.2 Hz (<i>arrow</i> in A). C, The maximal EJP amplitude (mean and 95% confidence interval) observed in the 10 min post-tetanic recovery period. D, The proportion of cases, in which the EJP took less than two minutes to recover from the end-tetanic level (arrow in A) to at least 75% of the maximal post-tetanic EJP amplitude, after switching from 10 Hz to 0.2 Hz stimulation. The bars indicate 95% confidence intervals. E and F, Frequency and amplitude of miniature excitatory junctional potentials (mEJPs). For each genotype and line, <i>n</i> is shown above the bars. N.s., not significant.</p

Expression capability of <i>UAS-endoA</i>* transgenes.

<p>A, Western blots of extracts from late-stage embryos, probed simultaneously with anti-EndoA and anti-Elav primary antibodies. Shown are genotypes without transgenes (<i>w¹¹¹⁸</i> and <i>elav-GAL4</i>), <i>EndoA^Δ4</i> null mutants, and null mutants carrying the indicated <i>endoA</i> transgenes driven by <i>elav-GAL4</i>. Note that some genotypes appear more than once. The EndoA immunosignal (wild type or mutant) generally runs as a doublet with the lower band matching the predicted size of EndoA (41.4 kDa). The signals from Elav and an unidentified protein (<i>asterisk</i>) both serve as loading controls. The <i>UAS-endoA^{EndoA(Arf)-HA}</i> product runs distinctly lower than other products, due to the deletion of the entire BAR appendage. B, Extracts from null mutants carrying the indicated HA-tagged <i>endoA</i> transgenes driven by <i>elav-GAL4</i>, probed simultaneously with anti-HA and anti-Elav. C, Extracts from fly heads of EndoA nulls, rescued to adulthood with mutant EndoA transgenes (“successful transgenes”) and probed with anti-EndoA and anti-Elav. Numbers above lanes in all panels are for reference only.</p

Supplementary figure 1

Supplementary figure 1. Association between the levels of circulating inflammatory proteome and diabetes complications in a cohort matched for diabetes duration, HbA1c levels, and insulin need</p

Supplementary table 1-6

Supplementary tables 1-6. Details on the statistical analyses performed on the relation between diabetes duration, insulin need, HbA1c levels, and diabetes complications with circulating immune cells (Supp table 1 & 2), PBMCs ex vivo stimulation (Supp table 3 & 4), and circulating inflammatory proteome (Supp table 5 & 6)</p

Life span and locomotor activity of adult rescuants.

<p>A, Kaplan-Meier survival curves showing the post-eclosion life span of the rescuants. The <i>UAS-endoA</i>* transgene either carried the mutations indicated in the Figure, or wild type <i>endoA</i> (“wt”). B, survival curves for the <i>EndoA(Arf)-HA</i> rescuants (<i>closed squares</i>) and control rescuants carrying an HA-tagged wild type <i>endoA</i> transgene (“wt-HA”, <i>open squares</i>). C, The median survival time (MST) of individual transgenic integration lines. On average, 32 flies per line were included in the survival analysis (range 12–52). D, The locomotor activity period (LAP; mean ± SEM). On average, 27 flies per line were included in the locomotion analysis (range 12–37). For statistical analysis, the rescue of EndoA(Arf)-HA was compared with the rescue provided by the HA-tagged <i>endoA⁺</i> transgene (“wt-HA”). Otherwise, the rescuants associated with the untagged <i>endoA⁺</i> transgene were used as controls (“wt”). ^*<i>P</i><0.01, ^**<i>P</i><10⁻⁹. N.s., not significant.</p

Targeted mutations in dEndoA-BAR and their relationship to the structure of hEndoA1-BAR.

<p>A, Schematic representation of the mutations introduced in the rescue constructs encoding dEndoA-BAR. B, Mutations homologous to the mutations in dEndoA-BAR (A), mapped onto the tertiary structure of hEndoA1-BAR monomer <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009492#pone.0009492-Masuda1" target="_blank">[PDB code 1X03A, 8]</a>. The central helix-loop appendage (<i>red</i>) and the residues constituting the hydrophobic ridge (<i>yellow</i>) are indicated. The residues mutated to change the BAR domain curvature are also indicated (<i>pink</i>), as are the three electropositive lysine residues that were mutated to electronegative glutamic acid residues (<i>light green</i>). The <i>inset</i> at the lower right shows the BAR dimer, with the two monomers colored <i>gray</i> and <i>blue</i>. C, Primary structure alignment of hEndoA1-BAR (accession BAE44459.1; <i>top</i>) and dEndoA-BAR (accession CAD24682.1; <i>bottom</i>). The alpha-helical secondary structure is indicated by <i>squiggles</i>, based on the hEndoA1-BAR structure. The residues associated with the hydrophobic ridge are also indicated (<i>closed triangles</i>).</p

PICK1 Deficiency Impairs Secretory Vesicle Biogenesis and Leads to Growth Retardation and Decreased Glucose Tolerance

<div><p>Secretory vesicles in endocrine cells store hormones such as growth hormone (GH) and insulin before their release into the bloodstream. The molecular mechanisms governing budding of immature secretory vesicles from the trans-Golgi network (TGN) and their subsequent maturation remain unclear. Here, we identify the lipid binding BAR (Bin/amphiphysin/Rvs) domain protein PICK1 (protein interacting with C kinase 1) as a key component early in the biogenesis of secretory vesicles in GH-producing cells. Both PICK1-deficient <i>Drosophila</i> and mice displayed somatic growth retardation. Growth retardation was rescued in flies by reintroducing PICK1 in neurosecretory cells producing somatotropic peptides. PICK1-deficient mice were characterized by decreased body weight and length, increased fat accumulation, impaired GH secretion, and decreased storage of GH in the pituitary. Decreased GH storage was supported by electron microscopy showing prominent reduction in secretory vesicle number. Evidence was also obtained for impaired insulin secretion associated with decreased glucose tolerance. PICK1 localized in cells to immature secretory vesicles, and the PICK1 BAR domain was shown by live imaging to associate with vesicles budding from the TGN and to possess membrane-sculpting properties in vitro. In mouse pituitary, PICK1 co-localized with the BAR domain protein ICA69, and PICK1 deficiency abolished ICA69 protein expression. In the <i>Drosophila</i> brain, PICK1 and ICA69 co-immunoprecipitated and showed mutually dependent expression. Finally, both in a <i>Drosophila</i> model of type 2 diabetes and in high-fat-diet-induced obese mice, we observed up-regulation of PICK1 mRNA expression. Our findings suggest that PICK1, together with ICA69, is critical during budding of immature secretory vesicles from the TGN and thus for vesicular storage of GH and possibly other hormones. The data link two BAR domain proteins to membrane remodeling processes in the secretory pathway of peptidergic endocrine cells and support an important role of PICK1/ICA69 in maintenance of metabolic homeostasis.</p></div

Rescue of the phenotype of PICK1-deficient mice by GH administration.

<p>(A) Lean body mass was evaluated by MRI scanning before GH administration and subsequently once every week both in PICK1-deficient mice (black squares) and in saline-treated WT littermates (white circles). Before treatment the difference between the two groups was highly significant (**<i>p</i> = 0.0027), after a week the difference decreased but was still significant (*<i>p</i> = 0.050), and after 2 and 3 wk no significant difference were observed. (B) IGF-1 mRNA level in the liver as determined by RT-PCR was significantly lower in PICK1-deficient mice compared to untreated mice (*<i>p</i> = 0.0045), however this difference was not observed after GH-treatment for 3 wk. (C) OGTT and ITT on GH-treated PICK1-deficient mice (black circles) and the saline-treated WT littermate controls (white circles). Significant difference was observed in the OGTT, whereas no difference was observed in ITT. Data are expressed as mean ± SE and analyzed by two-way ANOVA in (A) and Student's <i>t</i> test in (B–D) (<i>n</i> = 4–8 in all the experiments). All experiments have been reproduced in another cohort of mice (<i>n</i> = 3–4), where the age of the mice was 4 wk higher.</p