Components of the Engulfment Machinery Have Distinct Roles in Corpse Processing.

Billions of cells die in our bodies on a daily basis and are engulfed by phagocytes. Engulfment, or phagocytosis, can be broken down into five basic steps: attraction of the phagocyte, recognition of the dying cell, internalization, phagosome maturation, and acidification. In this study, we focus on the last two steps, which can collectively be considered corpse processing, in which the engulfed material is degraded. We use the Drosophila ovarian follicle cells as a model for engulfment of apoptotic cells by epithelial cells. We show that engulfed material is processed using the canonical corpse processing pathway involving the small GTPases Rab5 and Rab7. The phagocytic receptor Draper is present on the phagocytic cup and on nascent, phosphatidylinositol 3-phosphate (PI(3)P)- and Rab7-positive phagosomes, whereas integrins are maintained on the cell surface during engulfment. Due to the difference in subcellular localization, we investigated the role of Draper, integrins, and downstream signaling components in corpse processing. We found that some proteins were required for internalization only, while others had defects in corpse processing as well. This suggests that several of the core engulfment proteins are required for distinct steps of engulfment. We also performed double mutant analysis and found that combined loss of draper and αPS3 still resulted in a small number of engulfed vesicles. Therefore, we investigated another known engulfment receptor, Crq. We found that loss of all three receptors did not inhibit engulfment any further, suggesting that Crq does not play a role in engulfment by the follicle cells. A more complete understanding of how the engulfment and corpse processing machinery interact may enable better understanding and treatment of diseases associated with defects in engulfment by epithelial cells

Host factor Rab11a is critical for efficient assembly of influenza A virus genomic segments

It is well documented that influenza A viruses selectively package 8 distinct viral ribonucleoprotein complexes (vRNPs) into each virion; however, the role of host factors in genome assembly is not completely understood. To evaluate the significance of cellular factors in genome assembly, we generated a reporter virus carrying a tetracysteine tag in the NP gene (NP-Tc virus) and assessed the dynamics of vRNP localization with cellular components by fluorescence microscopy. At early time points, vRNP complexes were preferentially exported to the MTOC; subsequently, vRNPs associated on vesicles positive for cellular factor Rab11a and formed distinct vRNP bundles that trafficked to the plasma membrane on microtubule networks. In Rab11a deficient cells, however, vRNP bundles were smaller in the cytoplasm with less co-localization between different vRNP segments. Furthermore, Rab11a deficiency increased the production of non-infectious particles with higher RNA copy number to PFU ratios, indicative of defects in specific genome assembly. These results indicate that Rab11a+ vesicles serve as hubs for the congregation of vRNP complexes and enable specific genome assembly through vRNP:vRNP interactions, revealing the importance of Rab11a as a critical host factor for influenza A virus genome assembly.</p

Host factor Rab11a is critical for efficient assembly of influenza A virus genomic segments.

It is well documented that influenza A viruses selectively package 8 distinct viral ribonucleoprotein complexes (vRNPs) into each virion; however, the role of host factors in genome assembly is not completely understood. To evaluate the significance of cellular factors in genome assembly, we generated a reporter virus carrying a tetracysteine tag in the NP gene (NP-Tc virus) and assessed the dynamics of vRNP localization with cellular components by fluorescence microscopy. At early time points, vRNP complexes were preferentially exported to the MTOC; subsequently, vRNPs associated on vesicles positive for cellular factor Rab11a and formed distinct vRNP bundles that trafficked to the plasma membrane on microtubule networks. In Rab11a deficient cells, however, vRNP bundles were smaller in the cytoplasm with less co-localization between different vRNP segments. Furthermore, Rab11a deficiency increased the production of non-infectious particles with higher RNA copy number to PFU ratios, indicative of defects in specific genome assembly. These results indicate that Rab11a+ vesicles serve as hubs for the congregation of vRNP complexes and enable specific genome assembly through vRNP:vRNP interactions, revealing the importance of Rab11a as a critical host factor for influenza A virus genome assembly

Croquemort becomes enriched in the follicle cells during engulfment and has defects in follicle cell health.

<p>(A-C) Egg chambers expressing <i>UAS-GFP</i> driven by <i>Crq-GAL4</i> (<i>Crq-GAL4/CyO; UAS-mCD8-GFP/TM2</i>) stained with DAPI (cyan). (A-A’) Healthy egg chambers show little to no Crq expression in the germline or follicle cells. (B-B’) Phase 3 egg chambers show an increase in Crq expression in the follicle cells. (C-C’) Phase 5 egg chambers show considerably more Crq expression in the remaining follicle cells. (D-E) Egg chambers from starved flies stained with DAPI (cyan). (D) A wild-type egg chamber (<i>w</i>^<i>1118</i>) has a germline surrounded by a monolayer of healthy follicle cells. (E) <i>crq</i> null egg chambers (<i>crq</i>^<i>KO</i>) show undead germline with no remaining healthy follicle cells. (F) Quantification of the number of “undead” egg chambers per 100 ovarioles in the indicated genotypes. Scale bar is 50μm.</p

Candidate dsRNA Screen Results.

<p>Candidate dsRNA Screen Results.</p

Maturing phagosomes are marked with FYVE and Draper, but not integrins.

<p>Dying mid-stage egg chambers expressing FYVE (red) and Rab7GFP (green) labeled with DAPI (cyan) and antibodies (blue) against Draper (A) or αPS3 (B). (A-A”‘) A mid-stage dying egg chamber (<i>UAS-GAL4/FYVE-mCherry; GR1-GAL4</i>, <i>UAS-Rab7GFP/+</i>) stained with α-Draper shows normal engulfment, Rab7 association, and Draper enrichment. (A’-A”‘) Single channel zooms show that FYVE-mCherry co-localizes with some Rab7GFP-positive vesicles (white arrowhead) and clusters near the apical surface of the follicle cells. Several of the FYVE- and Rab7-positive vesicles are also positive for Draper (white arrowhead). Some FYVE- and Rab7-negative vesicles were positive for Draper (green arrowhead). (B-B”‘) A mid-stage dying egg chamber (<i>UAS-GAL4/FYVE-mCherry; GR1-GAL4</i>, <i>UAS-Rab7GFP/+</i>) stained with anti-αPS3 shows normal engulfment, Rab7 association, and αPS3 enrichment. (B’-B”‘) Single channel zooms show that αPS3 is present only on the apical surface and not within the cell. A Rab7-positive vesicle with low FYVE (green arrowhead) and a Rab7- and FYVE-positive vesicle (white arrowhead) are shown, both negative for αPS3. Egg chamber scale bar is 50μm. Zoom scale bar is 10μm.</p

Model of engulfment throughout the initial phases of engulfment.

<p>A diagram depicting our model for the molecular changes within an engulfing epithelial follicle cell. (A) In phase 1 egg chambers, Draper is already present on the apical surface of the follicle cells and initiates corpse processing and activation of Rac1 by activating Ced-12. Ced-12 is required for efficient corpse processing. Integrins and Draper are also being trafficked to the apical surface and Dor activity is required for inhibiting excessive uptake. (B) In phase 2 egg chambers, integrins are potentially required for adhesion to the dying germline. (C) In phase 3 egg chambers, both integrins and Draper are fully active and working in concert to promote engulfment. The JNK pathway is active and serves as an amplification signal, increasing engulfment genes such as Draper.</p

The active Dcp-1 antibody marks engulfed vesicles that are processed in an endocytic fashion.

<p>Mid-stage egg chambers stained with DAPI (cyan) and antibodies against cleaved Dcp-1 (magenta). (A-C’) Egg chambers expressing Rab5GFP (<i>UAS-GAL4/+; GR1-GAL4</i>, <i>UAS-Rab5GFP/UAS-luciferase</i>^<i>dsRNA</i>) show normal engulfment and Rab5 association. (A-A’) In healthy egg chambers, Dcp-1 is not detectable and small Rab5GFP-positive vesicles are enriched at the apical region of the follicle cells (Rab5 only in zoom in A’). (B-C’) In dying phase 3 egg chambers, the germline is Dcp-1-positive. Many of the engulfed vesicles are also Dcp-1-positive (zoom in B’). (C, zoom in C’) Some vesicles in a phase 3 egg chambers become Rab5-positive (white arrowhead). The association does not make a complete circle around the vesicle, but rather several puncta associate with the vesicle. Several Rab5-negative vesicles are also still present (orange arrowhead). * indicates autofluorescence of dorsal appendage. (D-F’) Egg chambers expressing Rab7GFP (<i>UAS-GAL4/+; GR1-GAL4</i>, <i>UAS-Rab7GFP/UAS-luciferase</i>^<i>dsRNA</i>) show normal engulfment and Rab7 association. (D-D’) In healthy egg chambers, Dcp-1 is not detectable and small Rab7GFP-positive vesicles are enriched at the apical region of the follicle cells (Rab7 only in zoom in D’). (E-F’) In phase 3 egg chambers, the germline is Dcp-1-positive. Many of the engulfed vesicles are also Dcp-1-positive. (E, zoom in E’) Some vesicles in phase 3 egg chambers become Rab7-positive (F, zoom in F’, white arrowhead). The association is seen as a bright circle completely surrounding a Dcp-1-positive vesicle (E’). Several Rab7-negative vesicles are also present (orange arrowhead). (G-I’) Egg chambers stained with LysoTracker (genotype: <i>UAS-GAL4/+; GR1-GAL4</i>, <i>UAS-Rab5GFP/UAS-luciferase</i>^<i>dsRNA</i>) (G-G’) In healthy egg chambers, LysoTracker is not detectable within the germline or surrounding follicle cells (LysoTracker only in zoom in G’). (H-I’) In phase 3 egg chambers, the germline is not LysoTracker-positive but many of the engulfed vesicles are LysoTracker-positive (H, I, zooms in H’, I’, vesicle indicated by white arrowhead). Egg chamber scale bar is 50μm. Zoom scale bar is 10μm.</p

Loss of the canonical corpse processing genes, <i>shi</i> and <i>dor</i>, result in opposite defects.

<p>(A-D) Dying egg chambers from the indicated genotypes stained with DAPI (cyan) and antibodies against cleaved Dcp-1 or Dlg (magenta). (A-A”) Loss of <i>shi</i> (<i>UAS-GAL4/tub-GAL80</i>^<i>ts</i><i>; GR1-GAL4</i>, <i>UAS-Rab7GFP/UAS-shi</i>^<i>dsRNA</i>) results in reduced vesicle uptake, and the few vesicles that are engulfed are not Rab7-positive. (B-B”) Loss of <i>shi</i> (<i>UAS-GAL4/tub-GAL80</i>^<i>ts</i><i>; GR1-GAL4</i>, <i>UAS-Rab7GFP/UAS-shi</i>^<i>dsRNA</i>) results in little to no LysoTracker-positive vesicles. (C-C”) Loss of <i>dor</i> (<i>UAS-GAL4/+; GR1-GAL4</i>, <i>UAS-Rab7GFP/UAS-dor</i>^<i>dsRNA</i>) results in elevated numbers of vesicles, and many are Rab7-positive. (D-D”) Loss of <i>dor</i> (<i>UAS-GAL4/+; GR1-GAL4</i>, <i>UAS-Rab7GFP/UAS-dor</i>^<i>dsRNA</i>) results in elevated numbers of LysoTracker-positive vesicles. (E) Average number of Dcp-1-positive vesicles engulfed per central slice. (F) Ratio of Rab7-positive to Dcp-1-positive vesicles in phase 2 egg chambers and (G) ratio of LysoTracker-positive vesicles to Dcp-1-positive vesicles in phase 3 egg chambers. Egg chamber scale bar is 50μm. Zoom scale bar is 10μm. At least three egg chambers were quantified for each genotype, phase, and quantification method in E-G, except those noted here. For E, 47 egg chambers were quantified for the control (<i>UAS-GAL4/+; GR1-GAL4</i>, <i>UAS-Rab5GFP/UAS-luciferase</i>^<i>dsRNA</i> and <i>UAS-GAL4/+; GR1-GAL4</i>, <i>UAS-Rab7GFP/UAS-luciferase</i>^<i>dsRNA</i>); 33 for <i>shi</i>^<i>dsRNA</i>; 15 for <i>Rab5</i>^<i>dsRNA</i>; 41 for <i>Rab7</i>^<i>dsRNA</i>; and 48 for <i>dor</i>^<i>dsRNA</i>. For F, 8 egg chambers were quantified for control (<i>UAS-GAL4/+; GR1-GAL4</i>, <i>UAS-Rab7GFP/UAS-luciferase</i>^<i>dsRNA</i>); 2 for <i>shi</i>^<i>dsRNA</i>; 2 for <i>Rab5</i>^<i>dsRNA</i>; and 6 for <i>dor</i>^<i>dsRNA</i>. For G, 7 egg chambers were quantified for control (the mixture from E); 8 for <i>shi</i>^<i>dsRNA</i>; 1 for <i>Rab5</i>^<i>dsRNA</i>; 18 for <i>Rab7</i>^<i>dsRNA</i>; and 8 for <i>dor</i>^<i>dsRNA</i>. Phase 2 for <i>shi</i>^<i>dsRNA</i> and <i>Rab5</i>^<i>dsRNA</i> and phase 4 for <i>Rab5</i>^<i>dsRNA</i> have less than 3 egg chambers quantified. Phase 3 for <i>Rab5</i>^<i>dsRNA</i>, LT only, has less than 3 egg chambers quantified. The data for control were first shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158217#pone.0158217.g003" target="_blank">Fig 3</a>. Two-tailed <i>t</i>-tests were performed: ***–<i>P</i><0.005, **–<i>P</i><0.01, *–<i>P</i><0.05.</p

Croquemort is not required for engulfment by the follicle cells.

<p>(A-J) Mid-stage healthy and dying egg chambers from the indicated genotypes stained with DAPI (cyan) and antibodies against cleaved Dcp-1 (yellow) and Discs large (red). The Discs large channel was brightened in C-D and G-H to better visualize follicle cell enlargement. (A-B) Control (<i>w</i>^<i>1118</i>) egg chambers show normal follicle cell enlargement and engulfment. (C-D) Loss of <i>crq</i> alone (<i>crq</i>^<i>KO</i>) shows no defects in follicle cell enlargement or engulfment. (E-F) Loss of <i>crq</i> and <i>draper</i> (<i>crq</i>^<i>KO</i>; <i>draper</i>^<i>Δ5</i>) results in strong engulfment defects, similar to loss of <i>draper</i> alone. (G-H) Loss of <i>crq</i> and <i>αPS3</i> (<i>crq</i>^<i>KO</i><i>; GR1-GAL4/UAS-αPS3</i>^<i>dsRNA</i>) results in strong engulfment defects, but not stronger than loss of <i>αPS3</i> alone. (I-J) Loss of <i>crq</i>, <i>draper</i>, and <i>αPS3</i> (<i>crq</i>^<i>KO</i><i>; draper</i>^<i>Δ5</i> <i>UAS-αPS3</i>^<i>dsRNA</i><i>/draper</i>^<i>Δ5</i>, <i>GR1-GAL4</i>) results in strong engulfment defects, but not stronger than loss of <i>draper</i> and <i>αPS3</i>. (K) Average number of Dcp-1-positive vesicles engulfed per central slice for each phase of death. Scale bar is 50μm. All data are mean ± s.e.m. At least three egg chambers were quantified for each genotype and phase in K-M, except those noted here. For K, 19 egg chambers were quantified for <i>w</i>^<i>1118</i>; 32 for <i>crq</i>^<i>KO</i>; 44 for <i>αPS3</i>^<i>dsRNA</i>; 46 for <i>draper</i>^<i>Δ5</i>; 85 for <i>draper</i>^<i>Δ5</i> <i>αPS3</i>^<i>dsRNA</i>; 20 for <i>crq</i>^<i>KO</i><i>; draper</i>^<i>Δ5</i>; 7 for <i>crq</i>^<i>KO</i> <i>αPS3</i>^<i>dsRNA</i>; 86 for <i>crq</i>^<i>KO</i><i>; draper</i>^<i>Δ5</i> <i>αPS3</i>^<i>dsRNA</i>. Phase 2 for <i>crq</i>^<i>KO</i><i>; draper</i>^<i>Δ5</i> and <i>crq</i>^<i>KO</i><i>; αPS3</i>^<i>dsRNA</i> and phase 3 of <i>crq</i>^<i>KO</i><i>; αPS3</i>^<i>dsRNA</i> have less than 3 egg chambers quantified. The data for <i>αPS3</i>^<i>dsRNA</i>, <i>draper</i>^<i>Δ5</i>, and <i>draper</i>^<i>Δ5</i> <i>αPS3</i>^<i>dsRNA</i> were first shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158217#pone.0158217.g003" target="_blank">Fig 3</a>. Two-tailed <i>t</i>-tests were performed: ***–<i>P</i><0.005, **–<i>P</i><0.01, *–<i>P</i><0.05.</p