Media 1: Numerical far field simulations with the fast Fourier transformation and Fourier space interpolation

Originally published in Optics Express on 09 February 2015 (oe-23-3-3341

Loh induces Prc matrix formation <i>in vivo</i>.

<p>(<b>A</b>) Prc (green) is absent from the ECM of larval muscles or the fat body in wild type third instar larvae. (<b>B</b>) Schematic representation of ectopically expressed LohA wild type protein or the secretion defective variant LohA^ΔSP. (<b>C</b>) Expression of LohA (red) in adipocytes or myocytes results in the formation of a dense Prc matrix (green) along both cell types in third instar larvae, while secretion of LohA is essential to mediate Prc recruitment. Inset shows accumulation of LohA at muscle attachment sites. (<b>D</b>) Loh and Prc partially co-localize in an artificial matrix around adipocytes. Loh distributes as weak fibers accumulating in a spotted fashion along cellular contacts (arrowheads). (<b>E</b>) Single optical slice of adipocytes. Loh co-localizes to Prc (arrowhead) at the root of Prc fibers (double arrowhead). (<b>F</b>) Loh co-immunoprecipitates with Prc and vice versa from total adult protein extracts demonstrating a biochemical interaction of both proteins.</p

The Conserved ADAMTS-like Protein Lonely heart Mediates Matrix Formation and Cardiac Tissue Integrity

<div><p>Here we report on the identification and functional characterization of the ADAMTS-like homolog <i>lonely heart</i> (<i>loh</i>) in <i>Drosophila melanogaster</i>. Loh displays all hallmarks of ADAMTSL proteins including several thrombospondin type 1 repeats (TSR1), and acts in concert with the collagen Pericardin (Prc). Loss of either <i>loh</i> or <i>prc</i> causes progressive cardiac damage peaking in the abolishment of heart function. We show that both proteins are integral components of the cardiac ECM mediating cellular adhesion between the cardiac tube and the pericardial cells. Loss of ECM integrity leads to an altered myo-fibrillar organization in cardiac cells massively influencing heart beat pattern. We show evidence that Loh acts as a secreted receptor for Prc and works as a crucial determinant to allow the formation of a cell and tissue specific ECM, while it does not influence the accumulation of other matrix proteins like Nidogen or Perlecan. Our findings demonstrate that the function of ADAMTS-like proteins is conserved throughout evolution and reveal a previously unknown interaction of these proteins with collagens.</p></div

Embryonic expression and localization of Loh and Prc.

<p>(<b>A–F</b>) Whole mount <i>in situ</i> hybridization revealing the expression patterns of <i>loh</i> (A–C) and <i>prc</i> (D–F) during embryogenesis. Chordotonal organs (double arrowhead in C), the ring gland (arrowhead in E, F) and oenocytes (double arrowhead in F) are indicated. (<b>G, H</b>) Double labeling of <i>loh</i> transcripts and Tinman (G) or <i>odd</i>-LacZ (H) in stage 17 embryos. (<b>I, J</b>) Co-staining of either Prc (I) or Loh (J) with Vkg::GFP in stage 17 embryos demonstrates localization within the cardiac ECM at the basal side of cardiomyocytes. (<b>K, L</b>) Co-staining of Loh and Prc shows co-localization of both proteins.</p

Localization of Prc depends on Loh but not vice versa.

<p>(<b>A–I</b>) Localization of Perlecan and Prc in stage 17 control embryos (A–C), compared to homozygous <i>loh^MB05750</i> (D–F) or homozygous <i>prc^MB03017</i> mutants (G–I). Prc but not Perlecan becomes mis-localized by the absence of Loh. (<b>J–O</b>) Localization of Loh in stage 17 control embryos (J–L) compared to homozygous <i>prc^MB03017</i> mutants (M–O). The localization of Loh to the ECM is not affected by the absence of Prc. Of note, the anti-Loh antiserum needs heat fixation leading to a different appearance of Prc in the stained control animals compared to chemical fixation as shown in A. (<b>P–R</b>) The luminal ECM of cardiomyocytes at embryonic stage 17 is not altered in either homozygous <i>loh^MB05750</i> or <i>prc^MB03017</i> mutants. The arrowheads indicate the thickness of the ECM. (<b>S</b>) Quantification of luminal basement membrane (BM) thickness in animals of the indicated genotypes. Mutants do not show significant alterations in ECM thickness. Error bars shown are standard deviation (s.d.) (<b>T–V</b>) TEM section of the adhesion area between cardiomyocytes (CC) and pericardial cells (PC). Lack of either <i>loh</i> or <i>prc</i> cause gaps between the cells. Scale bars are 250 nm.</p

Loss of pericardial cell adhesion causes loss of heart function.

<p>(<b>A</b>) Scheme depicting the basics of dye angiography in pharate adults. The main body parts, the dorsal vessel (DV) and the injection area are indicated. (<b>B</b>) Head of a wild type animal (corresponding to dashed box in scheme A) showing the accumulation of the tracer at four consecutive time points after injection. (<b>C</b>) Mean pixel intensities measured at four consecutive time points showing cardiac output in wild type (<i>white¹¹¹⁸</i>) and homozygous <i>prc^MB03017</i> and <i>loh^MB05750</i> pharate adult animals. Error bars are s.e.m. The region used for measurement is indicated in the lowest panel in B.</p

Molecular characterization of <i>loh</i> and <i>prc</i>.

<p>(<b>A</b>) Developmental Northern blots showing <i>loh</i> and <i>prc</i> expression in total RNA samples of 0–24 h old embryos (E), first, second or third instar larvae (L1–L3), mid-stage pupae (P) or adults (A) using gene specific riboprobes (indicated in B and C). (<b>B, C</b>) Schematic representation of <i>loh</i> (B) and <i>prc</i> (C) gene loci and transcripts. The schemes indicate the position of transposons, location of hairpins (IR) used for knock down and riboprobes used for Northern analysis (NB) and <i>in situ</i> hybridization (ISH). (<b>D</b>) Immunoblot of total protein extracts obtained from stage 17 control, homozygous Df(2L)Exel7048 or homozygous <i>loh^MB05750</i> embryos probed with antibodies against Loh or βTub. Loh is undetectable in homozygous deficiency or mutant extracts. (<b>E</b>) Immunoblot of total protein extracts obtained from control or homozygous <i>prc^MB03017</i> 0–24 h old embryos (E), third instar larvae (L3) mid-stage pupae (P) or adults (A) probed with antibodies against Prc or βTub. Prc is undetectable in extracts of homozygous mutants.</p

Prc becomes secreted by the larval fat body and recruited to the heart.

<p>(<b>A, B</b>) Embryonic (stage 17) and larval (L1) expression pattern of the <i>prc</i>>GFP reporter. (<b>C, D</b>) Prc protein becomes trapped in adipocytes of <i>prc</i>><i>sar1</i>-IR knock down first instar larvae, proving that the protein becomes expressed by the fat body. Scale bar is 10 µm. (<b>E</b>) Immunoblots of whole larval extracts probed against Prc. While <i>hand</i>C-Gal4 driven knock down does not alter the total amount of Prc the protein is nearly undetectable in <i>prc</i>-Gal4 driven knock down animals. Total protein was stained with amido black 10B. (<b>F</b>) Percentage of third instar larvae showing pericardial cell detachment induced by <i>prc</i> knock down either driven by <i>hand</i>C-Gal4 or <i>prc</i>-Gal4. <i>n</i> indicates the total number of tested animals. (<b>G</b>) Scheme of Prc matrix formation. Secreted Prc from different sources (heart cells in the embryo, adipocytes in the larva) becomes incorporated into the cardiac ECM dependent on Loh.</p

<i>loh</i> and <i>prc</i> are essential for cellular adhesion of cardiac cells.

<p>(<b>A–I</b>) Progressive loss of pericardial cell adhesion (cells marked by <i>hand</i>C-GFP) in <i>prc^MB03017</i> (D–F) and <i>loh^MB05750</i>/Df(2L)Exel7048 (G–I) mutants indicated by pericardial cell detachment (arrow heads). (<b>J, K</b>) Connection between the larval alary muscle (AM) and the cardiomyocytes (CC) is mainly facilitated by a reticular Prc matrix, covering the pericardial cells (PC). (<b>L–N</b>) Loss of cell adhesion includes the detachment of alary muscles (AM) from the heart in <i>loh^MB05750</i>/Df(2L)Exel7048 (M) or <i>prc^MB03017</i>/Df(3L)vin6 (N) third instar larvae. (<b>O–Q</b>) F-actin arrangement in control (O) <i>loh^MB05750</i>/Df(2L)Exel7048 (P) and <i>prc^MB03017</i>/Df(3L)<i>vin6</i> (Q) mutants third instar larvae. The orientation of the actin fibers is altered by loss of pericardial cell adhesion.</p

Comparison of phase unwrap with different algorithms for various noise levels.

<p><b>A,B,C:</b> Wrapped phase distributions for different noise levels. <b>D,E,F:</b> Tile-based unwrap with a tessellation into 20 × 20 tiles. Tile unwrapping was performed with the model-based least squares unwrapper (MLSQU) using <i>P</i> = 2, <i>N</i>_<i>ρ</i> = 40. Merging was performed with the tile-based <i>τ</i>SRNCP merger. <b>G,H,I:</b> Tile-based unwrap with a tessellation into 40 × 40 tiles. Tile unwrapping was done with Strand’s unwrapper (<i>N</i>_<i>r</i><i>ho</i> = 20) and a unidirectional merger. This corresponds to Strand’s original algorithm. <b>J,K,L:</b> Unwrapped phase map using the pixel-based SRNCP algorithm. For a full resolution graphic see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143186#pone.0143186.s001" target="_blank">S1 Fig</a>. Algorithms proposed in this paper are in black boxes.</p