Surface Plasmon resonance of calumenin with different divalent ions.

<p>SPR analysis of Ca²⁺-binding to calumenin. Sensorgrams obtained at 8 different ion concentrations (a) in quadruplicate were processed with the Biacore analysis software. The curve (b) provided a K_d,Ca value of 201 ± 13 μM (n = 32) for the interaction between Ca²⁺ and immobilised calumenin.</p

Ca²⁺-induced shift in size exclusion chromatography.

<p>Comparison between the SEC-MALS profiles of calumenin in the presence (blue and black) or in the absence (red) of Ca²⁺. The dashed lines crossing the peaks represent the MALS calculated molecular weight (right vertical axis). Calculations for the hydrodynamic radius (R_H) and molecular weight are provided in table for the two peaks.</p

Annotated sequence of calumenin.

<p>Primary structure of calumenin with predicted secondary structure elements (alpha-helices indicated in green below amino acid sequence). Six canonical EF-hand domains are identified (orange boxes) located between an N-terminal ER signal peptide (aa. 1–19) and a C-terminal ER retention sequence (HDEF). An “orphan loop” (aa. 46–58), lacking the flanking alpha-helices found in EF-domains is identified as an additional Ca²⁺-binding motif.</p

Calumenin 3D structure prediction and fitting with experimental data.

<p>Overlap of the calumenin model obtained from iTasser (coloured ribbons) with the envelope calculated from the SAXS data (white volume). Three orthogonal projections are shown confirming a good concordance between the trilobal model of calumenin and the experimental data.</p

SDS-PAGE overview of calumenin purification.

<p>His-tagged calumenin was first isolated from the lysate using a HisTrap affinity column (a). The eluted protein was further purified on a SEC column (b). The resulting tagged protein was submitted to SUMO-protease digestion (c), and the tagged sample (- SUMO-prot.) was treated until the formation of a 30 kDa band in the protease-treated sample (+ SUMO-prot.). Reverse affinity was used to eliminate any uncleaved protein and the tag (not shown). Finally, the now untagged calumenin was purified on a second SEC column (d). M = Benchmark molecular weight markers, L = loaded sample, FT = unbound flow-through fraction, eluted fractions are indicated with cardinal numbers. For the column, these numbers correspond to the retention volume (in ml). Fractions indicated with an asterisk were kept for the next stages of purification.</p

Small-angle X-ray scattering of calumenin.

<p>SAXS experimental I(q) curves for apo- (red) and Ca²⁺-bound calumenin (blue) (a). The slope of the Guinier plot, i.e. the ln(q) vs q² function at low q provides the radius of gyration R_g of the molecule (b). The P(r) function representing the distribution of the length of interatomic vectors (c) and Kratky plot(d) were used respectively to calculate the shape of the molecular envelope and to estimate the flexibility of calumenin.</p

Phenotyping of <i>Pkd1l1</i>^{<i>tm1/tm1</i>} Mutants.

<p>(A) Schematic diagram of PKD1L1 and PKD2 showing protein domains and the nature of the <i>Pkd1l1</i>^<i>rks</i> and <i>Pkd2</i>^<i>lrm4</i> point mutations. The double headed red arrow denotes the site of interaction between PKD1L1 and PKD2. PKD—Polycystic Kidney Disease; REJ—Receptor for Egg Jelly; GPS—G-protein Coupled Receptor Proteolytic Site; PLAT—Polycystin-1, Liopoxygenase, Alpha-Toxin. (B) <i>Pkd1l1</i>^{<i>tm1/tm1</i>} and sibling control showing reversed and normal situs, respectively. White arrows indicate stomach position. (C) Heart-stomach discordance (H-S Disc.) in <i>Pkd1l1</i>^{<i>tm1/tm1</i>}, <i>Dnah11</i>^<i>iv/iv</i> and <i>Pkd1l1</i>^{<i>rks/rks</i>} mutants scored at E13.5. Normally, the heart apex and stomach are positioned to the left. H-S Disc. is defined as the heart apex and stomach being on opposite sides. ns—not significant; *—p<0.05; **—p<0.001, Fisher’s Exact Test applied. (D-F) Lung situs assessed at E13.5 for embryos of the indicated genotypes with the ratio of lung lobes between left and right sides given. The percentage and total numbers of embryos showing each phenotype are indicated in <i>(F)</i>. (G-P) Expression patterns of <i>Nodal</i>, <i>Pitx2</i>, and <i>Lefty1/2</i> in embryos at E8.5 of the indicated genotypes, with the percentage number of embryos exhibiting each phenotype and the total number given. Embryos exhibiting bilateral marker expression are further categorized by whether they show equal or biased expression between the left and right sides. The inset in <i>(M)</i> shows a <i>Pkd1l1</i>^{<i>tm1/tm1</i>} embryo with bilateral <i>Pitx2</i> expression but with a right-sided bias. Arrowheads in <i>(N)</i> and <i>(O)</i> indicate midline <i>Lefty1</i> expression. <i>t</i> is shorthand for <i>Pkd1l1</i>^<i>tm1</i>. (Q-R) Sonic hedgehog (<i>Shh</i>) expression in the node (n) and notochord (nc) at E8.5.</p

The Genetic Relationship between <i>Pkd1l1</i>, <i>Pkd2</i>, and Cilia.

<p>(A-F) <i>Cerl2</i> (<i>A-C</i>) and <i>Nodal</i> (<i>D-F</i>) expression at the node of <i>Pkd1l1</i>^{<i>tm1/tm1</i>} and control embryos. Quantitation of <i>in situ</i> signal reveals expression of both genes to be more symmetrical in mutant embryos (<i>C</i>, <i>F</i>). *—p<0.05, unpaired <i>t</i>-test applied. Error bars represent 95% confidence intervals. (G-H) Lung situs <i>(G)</i> (assessed at E13.5) and <i>Pitx2</i> expression <i>(H)</i> (assessed at E8.5) for embryos of the indicated genotypes, with the percentage of embryos exhibiting each phenotype and the total number given.</p

The Relationship Between Nodal Flow and <i>Pkd1l1/Pkd2</i> Function.

<p>(A-C) Nodal flow in embryos of indicated genotypes was examined at the 1–3 somite stages by means of PIV analysis. Flow was normal in <i>Pkd1l1</i>^{<i>tm1/tm1</i>} mutants and wild-type controls but was absent in <i>Dnah11</i>^<i>iv/iv</i> mutants. Black arrowheads denote the direction and speed of flow at that position while the false coloring indicates the direction and magnitude of the flow. Red indicates leftward and blue rightward fluid movements. (D) Lung situs (assessed at E13.5) and <i>Pitx2</i> expression (assessed at E8.5) for embryos of the indicated genotypes, with the percentage of embryos exhibiting each phenotype and the total number given. (E) <i>Pitx2</i> expression for <i>Pkd1l1</i>^{<i>tm1/tm1</i>}, <i>Dnah11</i>^<i>iv/iv</i> and control embryos for each of the 1–7 somite stages. The onset of <i>Pitx2</i> expression is delayed in <i>Dnah11</i>^<i>iv/iv</i> mutants but not in <i>Pkd1l1</i>^{<i>tm1/tm1</i>} embryos.</p

Cilia and PKD2 Localization and Function.

<p>(A-E) PKD2 localization in nodal cilia of embryos of the indicated genotype. Staining was divided into categories and quantitation is given in (<i>A</i>). In <i>(A)</i>, all genotypes are statistically significantly different from each other (p<0.001) except for <i>Pkd2</i>^{<i>+/lrm4</i>} and <i>Pkd1l1</i>^<i>+/tm1</i> which are statistically not significantly different.</p