OCRL regulates PIP₂ homeostasis in diverse endosomal compartments.

<p>(A) Endogenously tagged dOCRL (magenta) co-localizes strongly with Clc-GFP (green), single channel confocal slices shown below in gray. (B) Pearson correlations between endogenously tagged dOCRL and trafficking compartments in live primary hemocytes. dOCRL localizes only moderately with other endosomal compartments. (C-D) <i>docrl</i>^Δ3 hemocytes exhibit increased PH-PLC-Cherry marker of PIP₂ (magenta) in live primary hemocytes in all membrane compartments examined (green). (E) The phosphatase activity of dOCRL is necessary but not sufficient to restrict hemocyte number. Scale bars in A,C are 10 μm. Data are presented as mean +/- SEM. Individual values in B,D represent single cells. <b>Associated with</b> <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007052#pgen.1007052.s004" target="_blank">S4 Fig</a>.</p

Loss of <i>docrl</i> causes increased actin assembly in hemocytes.

<p>(A) <i>docrl</i>^Δ3 hemocytes exhibit greater F-actin assembly (phalloidin, green) and a spikier morphology than control cells. (B-C) Quantification of F-actin intensity (B) and ‘spikiness’ (C). Both phenotypes are rescued by hemocyte-autonomous re-expression of dOCRL-GFP. (D) Infection of larvae with <i>M. luteus</i> promotes assembly of F-actin in hemocytes. (E) Sparse re-expression in hemocytes with HH-LT-GAL4 reduces actin assembly in GFP+ rescue hemocytes, but not GFP- hemocytes that do not express the rescue construct. (F) Expression of phosphatase alone moderately rescues actin accumulation (phalloidin staining, gray), though actin accumulation at ruffles (arrowhead) and intracellular puncta (arrows) are still observed. Data are presented as mean +/- SEM. Sample N in panels B-F represent individual cells pooled equally from three independent samples of 2–4 larvae. Scale bars are 10 μm.</p

dOCRL is required in hemocytes to restrict hemocyte abundance.

<p>(A-B) Absolute quantification of hemocytes in hemolymph extracted from wandering third instar larvae. (A) <i>docrl</i> mutants exhibit increased circulating hemocytes. (B) Re-expression of dOCRL in hemocytes is sufficient to suppress hemocyte number. (C) 2D projection of confocal images showing hemocyte distribution in larvae (left). Red arrows indicate salivary glands (a common site of expression for GAL4 drivers); white arrows indicate hematopoietic pockets. Scale bar is 200 μm. Quantification of circulating (released upon opening of the larval cuticle) and sessile (retained in the larval carcass) hemocytes in control and <i>docrl</i>^Δ3 larvae (right). Control genotype is w; CD8-GFP/+; he-GAL4/+. (D) <i>docrl</i> mutant larvae exhibit melanotic masses, which are rescued by <i>docrl</i> gene duplication Dup(1;3)DC402. (E) Frequency of visible melanotic masses. (F) Epifluorescence and reflected light images of a <i>docrl</i>^Δ3 larva showing GFP-labeled hemocytes clustered around a melanotic mass (arrow). Scale bar is 500 μm. (right) 2D projection of confocal image of hemocytes surrounding a melanotic mass. Scale bar is 50 μm. <b>Associated with</b> <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007052#pgen.1007052.s001" target="_blank">S1</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007052#pgen.1007052.s002" target="_blank">S2</a> Figs.</p

Aberrant endosomal compartment function in <i>docrl</i> mutants.

<p>(A) <i>docrl</i> mutant hemocytes exhibit defective scavenger receptor mediated endocytosis of mBSA (magenta). (B) <i>docrl</i> mutant hemocytes are competent for phagocytosis. Images show 2D maximum intensity projection of confocal stacks, showing myr-mRFP-expressing hemocytes incubated with Alexa-488 labeled <i>E</i>. <i>coli</i>. Lower panels show ZX projections taken at the plane marked by arrows in the respective projections. Z sections show internalized <i>E</i>. <i>coli</i>. (Middle) Quantification of mean number of <i>E</i>. <i>coli</i> particles. (Left) Fraction of particles completely engulfed by hemocytes. Data are presented as mean +/- SEM. (C) The structure and abundance of Lysotracker-positive (acidified) compartments is altered in <i>docrl</i> mutants, and rescued cell-autonomously by re-expression of <i>docrl</i>. Image shows 2D maximum intensity projection of confocal stacks. (D) <i>docrl</i> mutants exhibit defective accumulation of autophagic compartments, marked by GFP-mCherry-Atg8a (GC::Atg8A). Quantification shows Pearson’s Correlation Coefficient per cell +/- s.e.m. for GFP and mCherry (left) and mean fluorescence +/- s.e.m. for each fluorophore/cell (right). All panels are shown with the same brightness and contrast. Individual values in graphs represent single cells. Scale bars are 10 μm. <b>Associated with</b> <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007052#pgen.1007052.s006" target="_blank">S6 Fig</a>.</p

Aberrant endosomal compartment structure in <i>docrl</i> mutants.

<p>(A-C) Loss of <i>docrl</i> disrupts the structure of multiple endosomal compartments, including fragmentation and perinuclear accumulation of Clc (A) and Rab5 (B), and expansion of Rab7 (C). Images show single confocal slices at the plasma membrane and an internal Z plane. Scale bar is 10 μm. (D) Quantification of endosomal compartment levels in <i>docrl</i>^Δ3 mutants. Rab7 levels are increased ~3 fold, while Clc and Rab11 levels are slightly increased. N in panel D represent individual cells equally pooled from three independent sets of larvae. (E) Hemocytes from injured wild-type larvae exhibit defective Rab7-positive endosome structure. Scale bar is 5 μm. All panels are representative single confocal slices from live primary hemocytes. <b>Associated with</b> <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007052#pgen.1007052.s005" target="_blank">S5 Fig</a>.</p

Endosomal sorting defects underlie hemocyte activation in <i>docrl</i> mutants.

<p>(A) Relative hemocyte number in larvae with hemocyte-specific manipulation of the indicated Rab GTPases. Inhibiting the internalization step of endocytosis has no effect on hemocyte number, while manipulation of endosomal sorting recapitulates the <i>docrl</i> mutant phenotype. Statistical significance noted reflects pairwise tests with wild type control. (B) The phosphatase activity of dOCRL is sufficient to rescue scavenger receptor endocytosis of mBSA. (C) Manipulation of endosomal sorting via Rab11 and Rab7 rescue <i>docrl-</i>induced hemocyte overabundance. Data sets were each normalized and statistical significance noted relative to <i>docrl</i> mutant controls taken at the same time. Data are presented as mean +/- SEM. (D) Expression of Rab7^DN, but not Rab11^CA, rescues <i>docrl</i>-induced actin (left) and Lysotracker (right) accumulation. Individual values in A,C are independent samples pooled from 2–4 larvae. Multiple independent experiments are shown together for clarity, all statistical comparisons reflect independent controls. Data points in B and D represent single cells pooled equivalently from three independent larval collections. Statistical comparisons shown in B and D are to the relevant control group (green). <b>Associated with</b> <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007052#pgen.1007052.s007" target="_blank">S7 Fig</a>.</p