PLD2KO mice suffer from late-onset anosmia.

<p>Independent tests indicate that PLD2 KO mice have olfaction defect and that these defects are age-dependent and begin after 13 weeks of age. <b>A</b>: Buried food test; mice were timed for the retrieval of a small piece of chocolate hidden under bedding. In 72% of trials WT mice find the bait in less than 600 seconds, in contrast in only 34.8% of trials are PLD2KO mice able to find it; p<0.005. <b>B and B’</b>: Immunostaining of olfactory bulbs cryosections against c-fos (green) and counterstained with Hoechst (blue) identifies activated mitral cells in the mitral cell layer (arrow). Scale bar = 50μm. There is no difference in the number of active, c-fos positive, mitral cells between WT and PLD2KO male mice (blue and red, respectively) that have been challenged with vanilla or chocolate 30 minutes prior to sampling. <b>C</b>: Habituation-deshabituation; mice were used for three days to a cotton bud smelling of orange and on the fourth day were presented with a cotton bud smelling of vanilla. While PLD2KO mice (blue line) do not differentiate the smell, WT mice (orange line) show a regain interest in the new odour; p<0.001. <b>D</b>: The difference in habituation deshabituation response to olfactory cues is age dependent as there is no difference between WT and PLD2KO in mice that are 11 weeks of age, unlike the 14 week-old cohort presented in (C). <b>E</b>: When different objects that have the same smell are presented to the mice, WT and PLD2KO react similarly. <b>F</b>: PLD2KO mice are as good as WT mice at learning and sustaining the accelerating rotarod test.</p

Lipidomic analysis indicates lipid imbalance in PLD2KO mouse brain.

<p><b>A-F</b> Lipidomic analysis of six regions of the brain indicate that PLD2KO mice have significant changes in their pools of PA. Percentage of PA species are calculated compared to the total sum of PA species identified. A) 32: species, B) 34: species, C) 36: species, D) 38: species, E) Polyunsaturated species, F) Saturated- and monounsaturated species. <i>CRB</i>, Cerebellum; <i>CTX</i>, Cortex; <i>OLF</i>, Olfactory bulbs; <i>TH</i>, Thalamus-Hypothalamus and Striatum; <i>HP</i>, Hippocampus; <i>Rest</i>, Midbrain-Hindbrain and Medulla. <b>G</b> Comparison of difference between percentage of species in WT and PLD2KO indicate that the overall proportion of 32:, 34: and 36: species have significantly changed. A-F significance is calculated by Univariate Analysis of Variance test over two different experiments. G significance is calculated as paired t-test comparing the profile of all brain regions. When p<0.05, *; p<0.01, **; p<0.005, ***</p

The cerebellar architecture is affected in PLD2KO.

<p>A: Normally, the Purkinje cells (Pjc) form a monolayer sandwiched between the granular layer (Gr) and the molecular layer (Mol). In PLD2KO, ectopic Purkinje cells are also found either in the arbor vitae (arb, arrowhead) or clustered on the surface of the molecular layer (arrow). Scale bar = 100μm. B: PLD2KO mice have significantly more ectopic Purkinje cells than WT mice (p<0.005)</p

Screenshot of the LipidHome “Browser” view.

<p>The LipidHome structural hierarchy can be navigated in the far left tree panel. Clicking on a lipid record produces two vertically stacked panels in the right hand panel. The top panel shows general information about the selected record including an image. The bottom panel displays a table of the selected records’ children lipids, i.e. selecting the “<i>Sub Class</i>” “Diacylglycerophosphocholines” provides a list of its “<i>Species</i>”. These lists are exportable to a number of file formats.</p

The structural hierarchy of lipid records.

<p><b>a)</b><i>The structural hierarchy of lipid records in the Lipid Maps Structural Database (LMSD)</i>. The Lipid Maps classification system organises all lipids into “<i>Categories</i>”, “<i>Main Classes</i>” and “<i>Sub Classes</i>”. The lipid records are stored at the “<i>Geometric Isomer</i>” level where the total number of carbons, total number of double bonds, the position of double bonds and the stereochemistry of double bonds are defined for each fatty acid. The transparent lipid identification hierarchy levels are not supported by the LMSD. <b>b)</b><i>The structural hierarchy of lipid records in the LipidHome database</i>. Similar to the LIPID MAPS classification system, lipids are organised into “<i>Categories</i>”, “<i>Main Classes</i>” and “<i>Sub Classes</i>”. Lipid records are stored at four levels. Each level relates to a typical type of identification from a high throughput mass spectrometry experiment. From structurally undefined “<i>Species</i>” typically identified from a single precursor ion mass, to structurally resolved “<i>Isomer</i>” level identifications.</p

A diagram of in silico construction of theoretical diradyl lipid “Sub Species”.

<p>Steps: 1. All viable potential fatty acids are generated from a set of starting parameters; 2. They are combined all against all; 3. The head groups with alpha-carbons and linkages are generated; 4. The head groups are crossed with the fatty acid pairs to produce all viable lipid structures within the predefined chemical space.</p

Chemotaxis of cells from different melanoma stages.

<p>(A) Chemotaxis of a panel of six cell lines from different melanoma stages (RGP, green; VGP, purple; metastatic, red) up a 0%–10% FBS gradient was measured as above (<i>n</i>≥45 cells per cell line). (B) Chemotactic index of cells from different stages. Data from (A) were collated by melanoma stage. Chemotaxis improves as the stage of melanoma progresses, although even the earliest RGP cells show clear chemotaxis. (C) Speeds of cells from different stages. Data from (A) were collated by melanoma stage. Metastatic lines are conspicuously faster (<i>p</i>-values from unpaired <i>t</i>-tests), although again the speed of RGP and VGP cells is still relatively high for non-haematopoietic cells.</p

1,2- and 1,3-DAG rescue the fragmented NE phenotype.

<p>(A) Confocal images of live HeLa cells 1 min after addition of small unilamellar vesicles (SUVs) containing BODIPY-PtdCho and unsaturated 1,2 DAG (80∶20 mole% respectively). Incorporation of SUVs (green) into interphase and metaphase (white arrows) cells are shown. (B) LBR localisation during mitosis in rapalogue-treated, LBR and DGKε-expressing HeLa cells after addition at metaphase of SUVs containing PtdCho and unsaturated 1,2 DAG (80∶20 mole%). NE reformation (yellow arrows) was rescued. (C) Ultrastructure of the NE (yellow arrow) of the same cell at cytokinesis imaged using CLEM (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051150#pone.0051150.s004" target="_blank">Fig. S4</a>). LBR localisation in green, DGKε in red. Serial sections are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051150#pone.0051150.s013" target="_blank">Movie S8</a>. (D) Comparison of 3D models reconstructed from serial images of DAG-depleted (left panel) and DAG-rescued (right panel) cells shows the NE reformed in the presence of 1,2 DAG. (E–G) Similar results as in (A–C) respectively were obtained with SUVs of the non-C1 domain-binding DAG isomer 1,3 DAG. (H) CLEM images of a rapalogue-treated, dark LBR and DGKε-expressing HeLa cell fixed at cytokinesis, after addition of (60∶40 mole %) SUVs with BODIPY-PtdCho and unsaturated 1,3 DAG. Incorporation of the SUVs into cell membranes in green, DGKε in red. EM images show 1,3 DAG completely rescued NE reformation. Images representative of n = 11 experiments. Scale bars: confocal 10 μm, CLEM as indicated on the images.</p

Diacylglycerol localises to the NE, Golgi and ER in mammalian cells.

<p>HeLa (A) and COS-7 (B) cells were transfected with EGFP-PKCεC1aC1b (DAG probe-green), fixed and imaged by confocal microscopy. DAG was localised at the NE (yellow arrows), ER (green arrow) and Golgi (white arrows). (C–D) Calreticulin (ER marker) was detected by indirect immunofluorescence (red). Apart from a minor detection of the Golgi, the signal at the ER and NE (insets) was absent in cells transfected with the DAG non-binding mutant (C1b W264G) (D). HeLa (E-H) and COS-7 (I) cells were followed through mitosis by live confocal microscopy. EGFP-PKCεC1aC1b in HeLa (F) and COS-7 (I) presented similar distributions as DiOC₆ (E), GFP-POM121 (G), and ER tracker (H). ER tubules (green arrows) and NE reformation (yellow arrows) were observed. To label chromatin, cells were incubated with Hoechst 333432 or transfected with mCherry-H2B. Scale bars: 10 μm.</p

LPA responses are essential for serum chemotaxis in 2-D and 3-D assays.

<p>(A) LPA receptor antagonist Ki16425 blocks chemotaxis to serum. Chemotaxis of WM239A cells was compared with and without 10 µM Ki16425. Inhibitor-treated cells showed no chemotaxis despite essentially normal random migration. (B) Quantitative analysis of Ki16425 activity. Data from three experiments, including the one in (A). The chemotactic index of inhibitor-treated cells is essentially zero. (C) Melanoma cell lines from all stages chemotaxing up a 10% serum gradient with and without Ki16425. Colours represent melanoma stage. In RGP and VGP cells, chemotaxis is totally blocked, while in metastatic lines it is substantially inhibited. Bars show SEM. (D–E) 3-D organotypic assays. The cell lines WM98-1 and WM1158 are shown ±Ki16425. LPA receptor antagonist greatly inhibits invasion. In (D), invasion index is calculated as the percentage of total cells on the organotypic matrix that invaded beyond ∼30 µm as a ratio of cells on top of the matrix (<i>n</i>>1,000 cells per condition). (E) shows haematoxylin and eosin-stained vertical sections through gels, showing downward invasion of melanoma cells.</p