murine magi2  (Addgene inc)

Journal: Human molecular genetics

Article Title: Phosphorylation of the Usher syndrome 1G protein SANS controls Magi2-mediated endocytosis.

doi: 10.1093/hmg/ddu104

Figure Lengend Snippet: Figure 1. Validation of the direct interaction between SANS and Magi2. (A) Domain structure schemes: SANS is composed of ankyrin repeats, CENT, SAM and a C-terminalPBM(asterisk).Magi2ischaracterizedbyGuK(guanylatekinase),PDZ1-6domainsandtwoWW(tryptophan)repeats.P:predictedphosphorylationsites (SANSS422,MagiS1152).(B)Y2H:linesin(A)indicatebaitandidentifiedprey.(C–E)GST-pulldowns:(C)FLAG-taggedSANSfull-lengthwith(3×FLAG-SANS) and without PBM (3×FLAG-SANSDPBM) were assayed with immobilized GST-PDZ6 or GST alone. Western blots revealed recovery of SANS irrespective of the PBM. (D) SANS’ N-terminus (3×FLAG-Nterm), central domain (3×FLAG-CENT), and C-terminus with/without PBM (3×FLAG-SAM-PBM/3×FLAG-SAM) were assayed. Recovery was restricted to SANS C-terminus irrespective of PBM. (E) Recovery of FLAG-tagged SANS full-length assayed with GST-PDZ4 and 5 of Magi2 failed. (F, G) Trapw assays: (F) 3×FLAG-SANS was assayed with immobilized mRFP-PDZ6. SANS was co-precipitated with Magi2-PDZ6. (G) Magi2-HA wasassayedwithimmobilizedGFP-SANS.Magi2wasco-precipitatedwith SANS.(H)Membranetargetingassay:IMCD3cellsweresingly(a;b)or co-transfected(c) with eCFP-tagged SANS N-terminally fused to a membrane-anchoring signal (MyrPalm-eCFP-SANS) or/and Magi2 C-terminally HA-tagged (Magi2-HA). (a) IMCD3 cells singly transfected with MyrPalm-eCFP-SANS (green) showed SANS enriched at the plasma membrane. (b) Cells single-transfected with Magi2-HA (red) showed a perinuclear, cytoplasmic staining. (c) In co-transfected cells, Magi2 co-localized with SANS (yellow). Nuclei were stained with DAPI. Scale bars: 10 and 2.5 mm. TCL: total cell lysate.

Article Snippet: Human SANS (amino acid 2–461) and murine Magi2 (amino acid 2–1112) were subcloned in the MyrPalm-eCFP vector (plasmid 14867, Addgene, Cambridge, MA, USA) (64) containing an N-terminal membrane-anchoring peptide and eCFP.

Techniques: Biomarker Discovery, Western Blot, Transfection, Membrane, Clinical Proteomics, Staining

Journal: Human molecular genetics

Article Title: Phosphorylation of the Usher syndrome 1G protein SANS controls Magi2-mediated endocytosis.

doi: 10.1093/hmg/ddu104

Figure Lengend Snippet: Figure 2. Phosphorylation dependency of SANS-Magi2 interaction in IMCD3 cells. (A) Cells were co-transfected with MyrPalm-eCFP-SANS and Magi2-HA and incubatedeitherwithDMSOascontrolorwithDRB.(A,a)ImmunocytochemistryofDMSO-treatedIMCD3cellsco-transfectedwithMyrPalm-eCFP-SANS(green) and Magi2-HA (red) revealed the recruitment of Magi2 towards the cell membrane. In the high magnification, the co-localization of Magi2-HA with MyrPalm-eCFP-SANS at the membrane was demonstrated (yellow). (B, b) In co-transfected cells incubated with DRB, no altered localization of Magi2-HA could be detected. High-magnification image showed no co-localization of Magi2-HA with MyrPalm-eCFP-SANS at the cell membrane. Scale bars: 10 and 1 mm.

Article Snippet: Human SANS (amino acid 2–461) and murine Magi2 (amino acid 2–1112) were subcloned in the MyrPalm-eCFP vector (plasmid 14867, Addgene, Cambridge, MA, USA) (64) containing an N-terminal membrane-anchoring peptide and eCFP.

Techniques: Phospho-proteomics, Transfection, Membrane, Incubation

Journal: Human molecular genetics

Article Title: Phosphorylation of the Usher syndrome 1G protein SANS controls Magi2-mediated endocytosis.

doi: 10.1093/hmg/ddu104

Figure Lengend Snippet: Figure 3. Phosphorylation dependency of SANS-Magi2 interaction in cells transfected with phospho-/dephospho-constructs. (A) IMCD3 cells were co-transfected with a phospho-mimicking (serine mutated to glutamic acid) SANS construct (MyrPalm-eCFP-SANS S422E, green) and wild-type Magi2-HA (red). (a) High mag- nification revealed recruitment of Magi2 to the plasma membrane (yellow). (B) Cells were co-transfected with the dephospho (serine mutated to alanine)-SANS con- struct MyrPalm-eCFP-SANS S422A (green) and wild-type Magi2-HA (red). (b) High magnification demonstrated that Magi2 was no longer recruited to the membrane. (C) Cells were co-transfected with wild-type SANS (MyrPalm-eCFP-SANS, green) and a dephospho-Magi2 construct (Magi2-HA S1152A, red, serine mutated to alanine). (c) High magnification demonstrates that Magi2 was still recruited towards SANS at the membrane (yellow). Scale bars: 10 and 1 mm.

Article Snippet: Human SANS (amino acid 2–461) and murine Magi2 (amino acid 2–1112) were subcloned in the MyrPalm-eCFP vector (plasmid 14867, Addgene, Cambridge, MA, USA) (64) containing an N-terminal membrane-anchoring peptide and eCFP.

Techniques: Phospho-proteomics, Transfection, Construct, Clinical Proteomics, Membrane

Journal: Human molecular genetics

Article Title: Phosphorylation of the Usher syndrome 1G protein SANS controls Magi2-mediated endocytosis.

doi: 10.1093/hmg/ddu104

Figure Lengend Snippet: Figure 5. Association of Magi2 with transferrin-positive vesicles in IMCD3 cells. (A) Uptake of Alexa647 labeled transferrin (Tf647, red) by IMCD3 cells was fluor- escence microscopically monitored during a time course of 5, 15 and 30 min, counterstained with anti-Magi2 (green). Magi2 was associated with Tf647 vesicles after 5 min of endocytoticuptakeand accumulateswith Tf647within30 min in the perinuclearcytoplasm. (B) In cells treatedwiththe GTPase inhibitordynasore uptakeof Tf647 was inhibited. Tf647 remained at the plasma membrane and Magi2 was localized in the cytoplasm. Scale bars: 10 and 1 mm.

Article Snippet: Human SANS (amino acid 2–461) and murine Magi2 (amino acid 2–1112) were subcloned in the MyrPalm-eCFP vector (plasmid 14867, Addgene, Cambridge, MA, USA) (64) containing an N-terminal membrane-anchoring peptide and eCFP.

Techniques: Labeling, Clinical Proteomics, Membrane

Journal: Human molecular genetics

Article Title: Phosphorylation of the Usher syndrome 1G protein SANS controls Magi2-mediated endocytosis.

doi: 10.1093/hmg/ddu104

Figure Lengend Snippet: Figure 6. Endocytosis of transferrin is facilitated by Magi2, but decreased by SANS and phosphorylation. (A) Tf647 uptake (red) was monitored in IMCD3 cells transfected with scrambled shRNA control (green, upper panel) or shRNA against Magi2 (green, lower panel). Knock down of Magi2 diminished endocytosis of Tf647. Quantification of the mean fluorescence intensity of Tf647, normalized to mock-transfected cells revealed significant ≏57% decrease of endocytosis (SD+20.23%, n ¼ 5, P-value 0.001) in cells transfected with shRNA-Magi2 compared with cells transfected with scrambled shRNA (101%, SD+23.35%, P-value 0.032). (B) Tf647 uptake was monitored in cells transfected with scrambled shRNA control or shRNA against SANS. Knock down of SANS increased endo- cytosis of Tf647. Cytochemistry of Tf647 uptake in shRNA-transfected IMCD3 cells shows SANS-dependent endocytosis. Quantification of the mean fluorescence intensity of Tf647, normalized to mock-transfected cells revealed a significant ≏62% increase of endocytosis (SD+20.11%, n ¼ 4, P-value 0.016) in cells trans- fected with shRNA-SANS compared with mock-transfected cells and a slight increase of endocytosis of ≏30% compared with cells transfected with empty shRNA vector (SD+3.26%, P-value 0.051). (C) Tf647 uptake was monitored in IMCD3 cells treated with kinase inhibitor DRB revealing phosphorylation-dependent de- crease of endocytosis. Magi2 staining(green) illustratedthe association with Tf647. Quantification of the meanfluorescence intensity of Tf647, normalized to control revealed significant 1.63-fold increase of endocytosis (SD+0.1310, n ¼ 3, P-value 0.012) in cells treated with DRB compared with cells treated with DMSO (SD+ 0.171). Scale bar: 10 mm.

Article Snippet: Human SANS (amino acid 2–461) and murine Magi2 (amino acid 2–1112) were subcloned in the MyrPalm-eCFP vector (plasmid 14867, Addgene, Cambridge, MA, USA) (64) containing an N-terminal membrane-anchoring peptide and eCFP.

Techniques: Phospho-proteomics, Transfection, shRNA, Control, Knockdown, Plasmid Preparation, Staining

Journal: Human molecular genetics

Article Title: Phosphorylation of the Usher syndrome 1G protein SANS controls Magi2-mediated endocytosis.

doi: 10.1093/hmg/ddu104

Figure Lengend Snippet: Figure8. Ciliogenesisis influencedbyMagi2 anddependsonendocytosis.(A)Immunocytochemicalstainingof ciliabyantibodiesagainstacetylatedtubulin(ac.tub, red) of starved IMCD3 cells transfected with empty scrambled shRNA (control, green, upper panel) or shRNA against Magi2 (green, lower panel). Knock down of Magi2 diminished number of cells with a primary cilium. (B) Analysis of the ciliary shape of starved IMCD3 cells transfected with shRNA (b) or treated with the dynamin-inhibitor dynasore (d) revealed that the few emerging primary cilia in shRNA Magi2-transfected as well as in dynasore-treated cells are shortened (0.73 mm, SD+0.04/0.76 mm, SD+0.07) compared with controls (1.28 mm, SD+0.24/1.73 mm, SD+0.40). (C) Quantification of the total number of primary cilia in three independent experiments revealed the highly significant decrease of ciliogenesis down to 18% (SD+7.09%, n ¼ 3, P-value 0.000001) in cells transfected with shRNA Magi2, normalized to control transfected cells with scrambled shRNA (100%, SD+1.47%). (D) Quantification of the total number of primary cilia in three independent experiments revealed the highly significant decrease of ciliogenesis down to 32% (SD+3.9%, n ¼ 3, P-value 0.0003) in cells treated with dynasore compared with DMSO-treated controls (100%, SD+7.27%). Scale bars: 5 mm.

Article Snippet: Human SANS (amino acid 2–461) and murine Magi2 (amino acid 2–1112) were subcloned in the MyrPalm-eCFP vector (plasmid 14867, Addgene, Cambridge, MA, USA) (64) containing an N-terminal membrane-anchoring peptide and eCFP.

Techniques: Transfection, shRNA, Control, Knockdown

Journal: Human molecular genetics

Article Title: Phosphorylation of the Usher syndrome 1G protein SANS controls Magi2-mediated endocytosis.

doi: 10.1093/hmg/ddu104

Figure Lengend Snippet: Figure 9. In situ localization of Magi2 and SANS-Magi2 in photoreceptor cells of murine retina. (A) Schematic representation of a rod photoreceptor cell. Ax, axoneme; CC, connecting cilium (arrow head); IS, inner segment; N, nucleus; OS, outer segment; S, synapses; 2nd, secondary neurons. (B) Indirect immunofluor- escence demonstrated Magi2 (red) localization in the IS, at the outer limiting membrane (OLM, arrow) and in the synapses of outer plexiform layer (OPL) of mouse retina. (C) Double labeling of Magi2 and compartmental marker proteins. Merged image with centrin (Cen2, green) revealed overlapping staining in the region of the CC. (D) Double labeling of Magi2 (red) and SANS (green) in the apical sub-compartment of photoreceptor cells. Magi2 and SANS were localized in the IS predom- inantlyatthebaseoftheCC(arrowhead).(E)Triplelabelingof SANS,Magi2andcentrin-2(Cen2)revealedthelocalization ofSANSandMagi2at thebaseoftheCC. (F)PLAdemonstratedSANS-Magi2complexesattheciliarybaseinsitu;(G)schematicillustrationofSANS-Magi2complexlocalizationintheciliaryregionofarod photoreceptor cell (yellow, immunofluorescence signal; red, PLA signal). BB, basal body; Ce, adjacent centriole. Scale bars: (C) 10 mm; (D) 5 mm; (F and G) 1 mm.

Article Snippet: Human SANS (amino acid 2–461) and murine Magi2 (amino acid 2–1112) were subcloned in the MyrPalm-eCFP vector (plasmid 14867, Addgene, Cambridge, MA, USA) (64) containing an N-terminal membrane-anchoring peptide and eCFP.

Techniques: In Situ, Membrane, Labeling, Marker, Staining

Journal: Human molecular genetics

Article Title: Phosphorylation of the Usher syndrome 1G protein SANS controls Magi2-mediated endocytosis.

doi: 10.1093/hmg/ddu104

Figure Lengend Snippet: Figure 10. Subcellular localization of Magi2 and TfR in mouse sections through parts of mouse rod photoreceptor cells. (A) Magi2 was detected at the outer segment (OS)base, inthe connecting cilium (CC),the adjacentcentriole(Ce) andthe innersegment(IS). (B) Electronmicrograph demonstrating the association of Magi2with vesicles in the periciliary compartment of the apical IS (white arrows). (b) Higher magnification of Magi2-labelling associated with vesicles. (C) Triple labeling of Magi2, TfR and centrin-2 (Cen2) showed localization of Magi2 and TfR at the base of the CC. (D) Immunoelectron microscopy analyses of TfR localization in a longitudinal section through the periciliary region of a mouse photoreceptor cell demonstrated TfR decoration at membranes of the apical inner segment (arrows) and the membrane (white arrowheads) of the ciliary pocket indicated by black arrowheads. (E) Immunoelectron microscopy of the cross section through the CC showed accumulation of TfR labeling in the ciliary pocket. Scale bars: (A, B, D, E) 0.25 mm; (C) 1 mm.

Article Snippet: Human SANS (amino acid 2–461) and murine Magi2 (amino acid 2–1112) were subcloned in the MyrPalm-eCFP vector (plasmid 14867, Addgene, Cambridge, MA, USA) (64) containing an N-terminal membrane-anchoring peptide and eCFP.

Techniques: Labeling, Immuno-Electron Microscopy, Membrane

Journal: Human molecular genetics

Article Title: Phosphorylation of the Usher syndrome 1G protein SANS controls Magi2-mediated endocytosis.

doi: 10.1093/hmg/ddu104

Figure Lengend Snippet: Figure 11. Model for the SANS-Magi2 complex function in the periciliary region of photoreceptor cells. (A) Magi2 molecules mediate endocytosis from the ciliary pocket.SANSis associatedwithvesicularcargotransportthroughthe cytoplasmof theinner segment (IS)towardsthe ciliarybase. (B) In the periciliaryregion, SANS is phosphorylated by protein kinase CK2 increasing its affinity to Magi2. (C) Phosphorylated SANS recruits Magi2 from the endocytosis module of the ciliary pocket and facilitates cargo vesicle targeting to the periciliary membrane and thereby exocytosis. OS, outer segment.

Article Snippet: Human SANS (amino acid 2–461) and murine Magi2 (amino acid 2–1112) were subcloned in the MyrPalm-eCFP vector (plasmid 14867, Addgene, Cambridge, MA, USA) (64) containing an N-terminal membrane-anchoring peptide and eCFP.

Techniques: Membrane