Comparison of the genomic organization of amphioxus () and mouse () genes

<p><b>Copyright information:</b></p><p>Taken from "Evolutionary genomics of the recently duplicated amphioxus Hairy genes"</p><p>International Journal of Biological Sciences 2006;2(2):66-72.</p><p>Published online 10 Apr 2006</p><p>PMCID:PMC1458425.</p><p>© Ivyspring International Publisher. This is an open access article. Reproduction is permitted for personal and noncommerical use, provided that the article is in whole, unmodified, and properly cited.</p> Dashed lines indicated identical positions in the coding sequence. The diagram is not drawn to scale

Schematic representation of the 5' region of amphioxus , , and genes showing conserved non-coding regions

<p><b>Copyright information:</b></p><p>Taken from "Evolutionary genomics of the recently duplicated amphioxus Hairy genes"</p><p>International Journal of Biological Sciences 2006;2(2):66-72.</p><p>Published online 10 Apr 2006</p><p>PMCID:PMC1458425.</p><p>© Ivyspring International Publisher. This is an open access article. Reproduction is permitted for personal and noncommerical use, provided that the article is in whole, unmodified, and properly cited.</p> These are named Box1 to 4 and shown in different colours. The small green oval represents a core region conserved in all four genes and located inside the conserved Box3 (yellow) present in all but the gene. Putative RBP-Jκ binding sites are shown as grey lines below each gene and are clustered within the conserved boxes

-Methyl-D-aspartic Acid (NMDA) in the nervous system of the amphioxus -1

<p><b>Copyright information:</b></p><p>Taken from "-Methyl-D-aspartic Acid (NMDA) in the nervous system of the amphioxus "</p><p>http://www.biomedcentral.com/1471-2202/8/109</p><p>BMC Neuroscience 2007;8():109-109.</p><p>Published online 20 Dec 2007</p><p>PMCID:PMC2241627.</p><p></p>before purification by OPA treatment. The same sample after purification with OPA, which eliminates all the amino acids (or almost all) except NMDA. Note that it is not possible to see the NMDA in this graphic because it does not react with OPA-mercaptoethanol, that is the reagent used for the determination of free amino acids at HPLC. The same sample as B, but after treatment with D-AspO. In this case, the D-AspO oxidizes NMDA producing the CHNHwhich reacts with OPA-mercaptoethanol to give a well-defined sharp peak at the end of the chromatogram at retention time 11.8–12.0 min

Oxidation reaction of NMDA by D-Aspartate oxidase and production of methylamine (CHNH)

<p><b>Copyright information:</b></p><p>Taken from "-Methyl-D-aspartic Acid (NMDA) in the nervous system of the amphioxus "</p><p>http://www.biomedcentral.com/1471-2202/8/109</p><p>BMC Neuroscience 2007;8():109-109.</p><p>Published online 20 Dec 2007</p><p>PMCID:PMC2241627.</p><p></p

Alex3 degradation by Wnt1 is independent of the proteasome, JNK, CAMKII and Calcineurin pathways.

<p>(<b>A</b>) Proteasome inhibition with 10 µM MG-132 treatment blocks the normal turnover of Alex3 protein but not its Wnt1-induced degradation. (<b>B</b>) Numerous Alex3-overexpressing HEK293AD cells treated with the proteasomal inhibitor MG132 show the most severe mitochondrial aggregating phenotype. (<b>C</b>) Inhibition of JNK with 10 µM SP600125 (downstream effector of the Wnt/PCP pathway), CAMKII with 25 µM KN62 or Calcineurin with 10 µM Cypermetrin (downstream effectors of the Wnt/Ca²⁺ pathway) do not induce Alex3 protein degradation. Scale bar: 10 µm.</p

PKC and CKII phosphorylation protects against Wnt/Frizzled degradation of Alex3.

<p>(<b>A</b>) Inhibition of CKII (with 100 µM casein kinase II inhibitor I), downstream effector of the Wnt signaling pathway, is sufficient to trigger Alex3 degradation. (<b>B</b>) In contrast, PKC activation with 1 µM TPA protects against Wnt1-induced degradation of Alex3 protein. (<b>C</b>,<b>D</b>) Inhibition of PKC (with 1 µM Calphostin C) and treatment with 20 µM BAPTA/AM, an intracellular calcium chelator, also reproduces Wnt1 degradation. (<b>E</b>) Photomicrographs demonstrating that treatment with TPA prevents Alex3 degradation induced by Wnt1 and the reversion to normal mitochondrial phenotypes. (<b>F</b>) Quantification and graphical representation (mean ± standard deviation) of mitochondrial phenotypes in HEK293AD cells in the conditions shown in (<b>E</b>); note that incubation with TPA prevents the rescue of mitochondrial phenotypes induced by Wnt1. Scale bar: 10 µm. The quantification of Alex3 protein levels is shown at the bottom.</p

The N-terminal domain of Alex3 is sufficient to induce mitochondrial aggregation.

<p>(<b>A–D</b>) Overexpression of Alex3-GFP (green) in HEK293T cells induces severe alterations of the mitochondrial network when compared with the expression of control GFP (<b>A</b>). (<b>B</b>) Illustrates an Alex3-transfected cell displaying normal mitochondrial morphology; (<b>C,D</b>) Alex3-overexpressing cells showing mild aggregating phenotypes (<b>C</b>) and severe aggregating mitochondrial phenotypes (<b>D</b>); Alex3 protein was visualized in green, mitochondria in red (MitDsRed), and nuclei were labeled with bisbenzimide (blue). (<b>E</b>) Quantification and graphical representation (mean ± standard deviation) of mitochondrial phenotypes in control (GFP) and Alex3-GFP-overexpressing cells. (<b>F</b>) Top: Scheme of the Alex3-GFP deletion constructs used for transfection. Bottom: Western Blot showing representative truncated Alex3-GFP constructs at the predicted protein sizes. (<b>G–J</b>) Photomicrographs illustrating that expression of the Alex3(1–200)-GFP (<b>G</b>), Alex3(1–106)-GFP (<b>H</b>) and Alex3(1–30)-GFP (<b>I</b>) constructs leads to mitochondrial aggregation; in contrast, deletion of the first N terminal 12 aa (GFP-Alex3ΔNt) targets Alex3 protein to the nucleus (<b>J</b>). Note that the 30 aa N-terminus deletion construct has a truncated outer mitochondrial membrane localization sequence, which may interfere with its mitochondrial targeting, thereby leading to nuclear localization. (<b>K</b>) Quantification and graphical representation (mean ± standard deviation) of mitochondrial phenotypes in HEK293T cells after transfection with several truncated Alex3-GFP constructs; the data show that all the constructs containing the N terminal region cause mitochondrial aggregation. Alex3 protein was visualized in green (GFP), mitochondria in red (MitDsRed) and nuclei in blue (bisbenzimide). Scale bar: 10 µm.</p

The Wnt/Frizzled pathway induces the degradation of Alex3 protein.

<p>(<b>A</b>) WBs showing that Wnt1 co-transfection (left) and incubation with Wnt1-conditioned media (CM, right), but not treatment with Wnt3a (200 ng/ml) (middle), induces the degradation of Alex3 protein. (<b>B</b>) WB showing that co-transfection with Wnt1, Fz2, Wnt5a and Wnt11 lead to different reductions in Alex3 protein levels (left). Recombinant Wnt5a also induces Alex3 degradation in a concentration-dependent manner (right).</p

Schematic representation of Alex3 protein.

<p>Predicted domains are annotated on the basis of databases such as Pfam, Smart or Wolfpsort and bibliographic references. The stars show the position of putative phosphorylation sites in serine or threonine residues by CK2, PKC and PKA kinases.</p

Alex3 degradation is independent of the canonical Wnt/β-catenin pathway.

<p>(<b>A,B</b>) Constitutively active β-catenin (red) neither induces Alex3 protein degradation, as seen in WB (<b>A</b>), nor reverts the aggregated mitochondrial phenotypes induced by Alex3 overexpression (green in <b>B</b>). Nuclei were visualized in blue (bisbenzimide) (<b>B</b>). (<b>C,D</b>) Neither co-transfection with Dvl2 (<b>C</b>) nor the inhibition of GSK3β with 10 mM LiCl or with 10 µM SB212763 (<b>D</b>) induces Alex3 protein degradation. Wnt1 transfection was used as a control for Alex3 degradation. Scale bar: 10 µm.</p