Endocytic phenotypes in mutant primary hemocytes from <i>Drosophila</i>.

<p>(A–D) dsRNA treated S2R+ cells phenocopy corresponding allelic mutants in primary hemocyte cultures in a secondary assay. Scatter plots (A, B) show normalized fold change in fluorescence intensity of dextran that was pulsed (A) or chased (B) in S2R+ cells treated with different dsRNAs (y axis) or in hemocytes (x axis) from the corresponding mutant flies. In all cases, representative values were normalized to those from negative controls (CS hemocytes or zeo dsRNA treated S2R+ cells) and are plotted as mean± SEM. (n>30 for hemocyte assays, n>200 for S2R+ assays in all cases). For the chase assay in (B), we utilized <i>dor⁴</i> and <i>car¹</i> mutant hemocytes as positive controls (shown in light blue; Sriram et al., 2003). (C) Representative micrographs of hemocyte cultures from flies carrying hypomorphic alleles of <i>vps35</i>, <i>epac</i>, <i>α-cop</i> and <i>CG1418</i> assayed as in (B). (D) Summary of the experiment in (A–B) displaying statistically significant (Student's T-test, p<0.05) changes in uptake/retention of mutant hemocytes or gene-depleted S2R+ cells as colour coded maps. Scale bar in (C) = 5 µm.</p

Role of lysosomal genes.

<p>(A) Network map depicting known and predicted interactions (green lines: genetic; blue lines: physical; brown lines: predicted based on conserved data) between the ‘Granule group’ set of eye colour mutants (pink) and selected hits (gray). In this network, genes encoding Carnation (<i>car</i>; the fly homolog of VPS33), Deep orange (<i>dor</i>), Carmine (<i>cm</i>) and Rab7 were identified with roles in CG endocytosis in this study (denoted by black asterisks), while White (<i>w</i>) depletion affected at least one Tf pathway feature (white asterisk). (B) Localization of Carnation on early fluid endosomes. <i>Drosophila</i> S2R+ cells were pulsed with TMR-Dextran for two minutes and fixed and labeled with antibodies to Carnation (αCar). Micrographs show a representative cell imaged in two channels and a pseudo colour merge image (labeled TMRdex and αCar), in red, green and merge respectively). Carnation (green) is seen enriched on peripheral, small, early fluid endosomes (red). Three examples of such endosomes (white arrows in merge panel) are shown in the magnified inset. (C) Fluorescent micrographs depict the levels of fluid uptake in representative S2R+ cells treated with dsRNA against <i>car</i> (first lower panel) or <i>syx1A</i> (last lower panel) or in hemocytes from <i>car¹</i> mutant flies (middle lower panel), with their respective controls (upper panels). Bar graph represents mean and SD of normalized fluorescent integrated intensity per cell from 2–3 experiments, with 100–150 cells per treatment (S2R+ cells) or 40 cells per genotype (hemocytes). (D) Representative fluorescent micrographs depict fluid uptake measured in hemocytes as in (C), in flies that were: homozygous for a mutant allele of <i>car</i> (<i>car¹</i>); a hetero-allelic combination of <i>car¹</i>/+;<i>syx1</i>/+;or wild type (CS). Also tested were flies heterozygous for <i>syx1</i>/+ and <i>car¹</i>/+. Bar graph represents mean and SD of normalized fluorescent integrated intensity per hemocyte from 2–3 experiments with 40 cells per genotype. (E) Representative micrographs show human AGS cells treated with control siRNA or siRNA to hSYX1A and hVPS33A/B and pulsed with FITC-Dextran for 5 min. Right panel - Bar graphs show population averaged mean fluorescence intensity uptake per cell (representative experiment with n>50 cells per replicate, 2 replicates). Scale bar in (B–E) main panel = 5 µm, inset = 1 µm. Double asterisks denote significance <i>p</i> values lower than 0.01 with the Student's T-Test.</p

Quantitative profiling of two endocytic routes at single cell resolution.

<p>(A) Experimental workflow outline for cell seeding, transfection and multiplex endocytic assays to obtain multifeature data across 7131 gene depletions. The entire procedure was performed on a cell array (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100554#pone.0100554.s001" target="_blank">Figure S1A</a>; details in SOM) and the positions of negative and positive (dsRNA against <i>sec23</i>, <i>arf1</i>, <i>shi</i>) controls are highlighted in their respective colours. (B) Table grouping the 27 quantitative features into categories. The top half of the table contains direct measurements of intensity, while the lower half contains geometric parameters of the cell, endosomes and nucleus. Various measurements are made from each fluorescent channel, including those utilizing different pixel radii for local background subtraction while detecting endosomes. (C) Representative brightfield (bf) and fluorescent micrographs of a field of view of individual cells (zoomed in insets) labeled with four different fluorescent probes: Hoechst; FITC-Dextran (Fdex); Alexa568-Tf (Tf); Alexa647-αOkt9 (Okt9); (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100554#pone.0100554.s018" target="_blank">Methods S1</a> for details). The psuedocolour merge image is a composite of the Fdex (green), TfR (red) and Okt9 (blue) channels. Scale bar = 15 µm; inset = 3×. (D) Grayscale heatmap representing the fraction of four control genes (<i>arf1</i> (<i>arf79f</i>); <i>shi</i>; <i>sec23</i>; <i>chc</i>) picked up as hits (above a Z-score threshold of 3) across all 27 features in the entire dataset. Higher values on the grayscale bar denote higher pick-up rates. The features with higher pick-up rates correspond to the known endocytic roles of these genes.</p

Primary hits validated in a secondary classification assay.

<p>(A–B) Schema (A) and positional patterning (B) on cell arrays of secondary endocytic classification assays carried out for all CG features (upper schema) or a subset of CD features (lower schema).All the test genes were surrounded with local positive controls, and negative controls (see legend in (B)). With this patterning, each gene was tested in triplicate, with three local positive controls and six local negative controls. (C) Heatmap representing raw mean fluorescence intensities (in the pulse channel) across a test cell array used to validate the CG secondary endocytic assay described in (A). Only the means of control wells are shown in the top panel and the inter-control variation in means is representative of a typical experiment. For comparison, the lower panel depicts the mean fluorescence intensities of test genes. (D) The green bars show the fraction of genes predicted as hits for each feature in the primary screen that were also picked up as hits for that feature in the secondary. The gray bars show the fraction of genes not predicted as a hit for each feature in the primary screen that were nevertheless picked up as hits for that feature in the secondary. With a single exception (Tnum) we find that the green bars exceed the gray (p-value 5×10⁻⁶ for 22 fair coin flips) demonstrating the selectivity and reproducibility of our primary assay. (E) Psuedocoloured fluorescence micrographs of representative control and <i>drab5</i>- and <i>dvps4</i>- dsRNA treated populations of cells that were subjected to the CG pulse-chase assay from (<b>A</b>). Both Drab5 and Dvps4 depleted cells were affected in the chase (with Fdex, green) portion of the assay, while the pulse portion (with Rdex, red) was unaffected (see quantitation in bar graphs on the right, normalized to control). Scale bar = 10 µm.</p