Deficiencies of the Lipid-Signaling Enzymes Phospholipase D1 and D2 Alter Cytoskeletal Organization, Macrophage Phagocytosis, and Cytokine-Stimulated Neutrophil Recruitment

Cell migration and phagocytosis ensue from extracellular-initiated signaling cascades that orchestrate dynamic reorganization of the actin cytoskeleton. The reorganization is mediated by effector proteins recruited to the site of activity by locally-generated lipid second messengers. Phosphatidic acid (PA), a membrane phospholipid generated by multiple enzyme families including Phospholipase D (PLD), has been proposed to function in this role. Here, we show that macrophages prepared from mice lacking either of the classical PLD isoforms PLD1 or PLD2, or wild-type macrophages whose PLD activity has been pharmacologically inhibited, display isoform-specific actin cytoskeleton abnormalities that likely underlie decreases observed in phagocytic capacity. Unexpectedly, PA continued to be detected on the phagosome in the absence of either isoform and even when all PLD activity was eliminated. However, a disorganized phagocytic cup was observed as visualized by imaging PA, F-actin, Rac1, an organizer of the F-actin network, and DOCK2, a Rac1 activator, suggesting that PLD-mediated PA production during phagocytosis is specifically critical for the integrity of the process. The abnormal F-actin reorganization additionally impacted neutrophil migration and extravasation from the vasculature into interstitial tissues. Although both PLD1 and PLD2 were important in these processes, we also observed isoform-specific functions. PLD1-driven processes in particular were observed to be critical in transmigration of macrophages exiting the vasculature during immune responses such as those seen in acute pancreatitis or irritant-induced skin vascularization

Lepidopteran wing scales contain abundant cross-linked film-forming histidine-rich cuticular proteins

Scales are symbolic characteristic of Lepidoptera; however, nothing is known about the contribution of cuticular proteins (CPs) to the complex patterning of lepidopteran scales. This is because scales are resistant to solubilization, thus hindering molecular studies. Here we succeeded in dissolving developing wing scales from Bombyx mori, allowing analysis of their protein composition. We identified a distinctive class of histidine rich (His-rich) CPs (6%–45%) from developing lepidopteran scales by LC-MS/MS. Functional studies using RNAi revealed CPs with different histidine content play distinct and critical roles in constructing the microstructure of the scale surface. Moreover, we successfully synthesized films in vitro by crosslinking a 45% His-rich CP (BmorCPR152) with laccase2 using N-acetyl- dopamine or N-β-alanyl-dopamine as the substrate. This molecular study of scales provides fundamental information about how such a fine microstructure is constructed and insights into the potential application of CPs as new biomaterials

Functional Evolution of Duplicated Odorant-Binding Protein Genes, Obp57d and Obp57e, in Drosophila

Odorant-binding proteins (OBPs) are extracellular proteins found in insect chemosensilla, where they participate in the sensing of odors, tastes, and pheromones. Although a large number of OBP genes have been identified in insect genomes, their molecular functions and biological roles have been clarified in limited cases. Two OBP genes, Obp57d and Obp57e, were involved in the evolution of host-plant preference in Drosophila sechellia. Comparative analyses of the Obp57d/e genomic sequences from 27 closely related species suggested that the two genes arose by tandem gene duplication and functionally diverged from each other. In this study, the functional evolution of Obp57d and Obp57e was examined by in vitro binding assays using recombinant proteins synthesized in a bacterial system. Compared to the ancestral Dpse\OBP57de, Dmel\OBP57d was more specialized to tridecanoic acid while Dmel\OBP57e was generalized regarding their binding affinity, suggesting that the two OBP genes underwent subfunctionalization and neofunctionalization. A behavioral analysis using knockout flies supported that the biological role is different between OBP57d and OBP57e in vivo. Site-directed mutagenesis of the evolutionarily conserved amino acids revealed that these residues play an important role in protein folding. These findings provide a clue to understanding how the repertoire of OBP genes is maintained in a genome under natural selection

Loss of either isoform of PLD decreases phagocytosis.

<p>A) 40x brightfield images of BMDM cultured with human IgG-coated beads for 5 min and then fixed following washes to remove unbound beads. B) Graph of phagocytic index for WT, <i>Pld1</i>^−/−, and <i>Pld2</i>^−/− BMDM as well as WT BMDM treated with FIPI (750 nM, pre-treated for 1 hr). Number of beads/cell was calculated as total number of beads/field divided by total number of cells/field. Index was defined as % of beads/cell phagocytosed by untreated WT BMDM. Extracellular (bound but not phagocytosed) beads were visualized by Cy5-anti human IgG immunostaining and excluded from the quantitation. Three fields were quantitated for each condition/day and the experiment was performed at least 5 times with BMDM prepared from different mice. C) Confocal images of WT, <i>Pld1</i>^−/−, and <i>Pld2</i>^−/− BMDM that were cultured with GFP-expressing <i>Yersinia pseudotuberculosis</i> strain IP32777c at an MOI of 20 for 20 min, washed, fixed, permeabilized, and stained with rhodamine phalloidin (red). Images are ones typical of at least 3 experiments. Bar, 25 µm D) Graph of IP32777c uptake. Bacteria/cell was calculated as above for beads/cell. p-values: *  =  p<0.05, **  =  p<0.005, ***  =  p<0.0001.</p

F-actin organization in resting and activated macrophages.

<p>A) Confocal images of BMDM transfected via nucleofection with the Spo20-GFP PA sensor (green), serum starved for 2 hrs, and then plated on fibronectin-coated coverslips for 30 min, fixed, and stained for F-actin using rhodamine phalloidin (red). Images are representative best of at least 3 separate experiments from which at least 3 macrophages were visualized. Arrow, podosome; arrowhead, cortical actin; *, region magnified in inset. B) Western blot analysis of BMDM macrophages treated as above. The experiment was repeated at least 3 times using BMDM prepared from different mice. C) Confocal images of intraperitoneal (IP) macrophages from WT, <i>Pld1</i>^−/−, and <i>Pld2</i>^−/− mice serum-starved for 2 hrs and plated on fibronectin-coated coverslips. Macrophages were stained with rhodamine phalloidin to visualize F-actin. Podosomes are circled and arrows indicate cortical actin. Images are representatives of at least 3 experiments. D) Graph summarizing average cell diameter of IP macrophages on fibronectin. Data collected from at least 3 fields of vision from 3 independent mice. E) Graph representing the diameters of podosomes from IP macrophages plated on fibronectin. Data collected from at least 3 fields of vision from 3 separate mice. Bar, 7.5 µm. *  =  p<0.05, **  =  p<0.005, ***  =  p<0.0001.</p

Rac1 recruitment and localization is abnormal in the absence of PLD1 or PLD2.

<p>A) Confocal microscopy of WT, <i>Pld1</i>^−/−, and <i>Pld2</i>^−/− BMDM stimulated with human IgG-coated beads for 5 min followed by immunostaining for Rac1 (green) and staining for F-actin (rhodamine phalloidin, red). *, shown magnified in inset. B) Graph of distinct (example: top panel, left) and indistinct (examples: top panel, right) Rac1 cups. Type of cup (distinct versus indistinct) was normalized to the number of beads per field. Quantitation was performed using Zeiss intensity maps as well as MATLAB parameters as described in Materials and Methods. At least 3 fields and 20 cups were scored for each condition from 5 independent experiments. C) Confocal microscopy using an activation state-sensitive anti-Rac1 monoclonal antibody to visualize activated Rac1 in WT, <i>Pld1</i>^−/−, and <i>Pld2</i>^−/− BMDM stimulated with human IgG-coated beads for 5 min. D) Western blot of PBD-pull down assay to assess GTP-bound Rac1 in WT, <i>Pld1</i>^−/−, and <i>Pld2</i>^−/− BMDM stimulated with human IgG-coated beads for 5 min; total Rac1 in the lysates is shown as a loading control. The western was repeated at least 3 times with similar results. Bar, 7.5 µm. *  =  p<0.05, **  =  p<0.005, ***  =  p<0.0001.</p

<i>Pld1</i>^−/− and <i>Pld2</i>^−/− neutrophils exhibit impaired migration but only <i>Pld1</i>^−/− neutrophils have impaired tissue extravasation.

<p>A) Quantitation of chemoattractant-stimulated <i>in vitro</i> migration through 4 µm pore transwell filters of neutrophils isolated from the blood of WT, <i>Pld1</i>^−/− and <i>Pld2</i>^−/− mice. WT macrophages stimulated with LPS (1 µg/mL for 2 hrs) were used to generate conditioned media that was used as the chemoattractant. Blood neutrophils were allowed to migrate towards the conditioned media for 30 min. The experiment was performed at least 3 times using independently prepared BMDM. B) Quantitation of circulating neutrophil recruitment to the peritoneum. LPS (1 mg/kg) was injected into the peritoneum 4 hrs prior to sacrifice. The peritoneum was lavaged using HBSS to collect the recruited neutrophils; n = 3 for each type. C) IHC of pancreata stained with Ly6B.2 to detect neutrophils (circled) following induction of pancreatitis. Arrows indicate blood vessels and fibrotic border. D) Quantitation of neutrophil recruitment to the pancreas following acute pancreatitis. WT, n = 9; <i>Pld1</i>^−/−, n = 5; <i>Pld2</i>^−/−, n = 7. E) Quantitation of Evan's blue dye leakage into the ear following irritation with mineral oil. Results are the average of 3 independent experiments. *  =  p<0.05, **  =  p<0.005.</p