Journal: eLife

Article Title: GIPC proteins negatively modulate Plexind1 signaling during vascular development

doi: 10.7554/eLife.30454

Figure Lengend Snippet: ( A ) Confocal lateral images of the trunk vasculature (green) of 32 hpf embryos (region dorsal to the yolk extension). Anterior, left; dorsal, up. Scale bars (white horizontal lines), 100 μm. Genotypes indicated on top of each image in yellow font. Angiogenesis deficits are indicated as follows: white asterisks (DLAV gaps), magenta asterisks (truncated Se), white greater-than sign (thin Se). Maternal-zygotic (MZ) removal of gipc activity is denoted by the designation ‘MZ’ in superscript. In the WT image (top left), the vessels are designated with the white font as follows: DLAV (Dorsal Longitudinal Anastomotic Vessel), Se (Segmental Vessel), DA (Dorsal Aorta), and PCV (Posterior Cardinal Vein). ( B ) Bar graph. Percentage of Se in 32 hpf embryos of the indicated genotypes belonging to each of the following four phenotypic classes. Truncated: severe (includes missing Se), medium (yellow), and weak (gray). Non-truncated: complete (black). Significance values were calculated using a two-sided Fisher Exact test and significant differences (p<0.0033) assigned using a Bonferroni-type adjustment for 15 pairwise genotype comparisons (0.05/15 = 0.0033). Brackets and asterisks indicate pairs of genotypes with significantly different distributions of these four phenotypic classes. Quantifications. We scored Se angiogenesis in embryos of the following six genotypes: WT (138 Se, 12 embryos; an average of 11.5 Se/embryo), gipc1 skt1 (130 Se, 11 embryos; an average of 11.8 Se/embryo) , gipc1 skt1(MZ) (380 Se, 33 embryos; an average of 11.5 Se/embryo) , gipc2 skt3/skt4 (130 Se, 11 embryos; an average of 11.8 Se/embryo) , gipc1 skt1 ; gipc2 skt3/skt4 (152 Se, 13 embryos; an average of 11.6 Se/embryo), and gipc1 skt1(MZ) ; gipc2 skt3/skt4 (220 Se, 19 embryos; an average of 11.5 Se/embryo). For additional data, graphs, and statistical comparisons related to this figure, see , and . Please note that given the use of different scales for scoring angiogenesis deficits, it is unfeasible to compare the quantifications in and directly.

Article Snippet: Antibody , Rabbit anti-GIPC1 , Proteintech Group , Cat. #14822–1-AP. RRID: AB_2263269 , WB (1:3,000). This antibody detects GIPC1, GIPC2, and GIPC3 (our data).

Techniques: Activity Assay

Journal: eLife

Article Title: GIPC proteins negatively modulate Plexind1 signaling during vascular development

doi: 10.7554/eLife.30454

Figure Lengend Snippet: ( A–D ) Confocal lateral images of the trunk vasculature (green) of 32 hpf embryos (region dorsal to the yolk extension). Anterior, left; dorsal, up. Scale bars (white horizontal lines), 100 μm. Morpholino injection (un-injected or injected with plxnd1 morpholino) indicated on top, genotypes (WT or gipc1 skt1(MZ) ; gipc2 skt4(MZ) ) indicated on the left. The un-injected WT picture ( A ) shows the names of the major vessels in white font: DLAV (Dorsal Longitudinal Anastomotic Vessel), Se (Segmental Vessel), DA (Dorsal Aorta), and PCV (Posterior Cardinal Vein). Vascular defects highlighted as follows: truncated or missing Se (magenta asterisk), thin Se (white greater/less-than signs), DLAV gaps (white asterisk). Quantifications. The following number of embryos were analyzed: WT (four embryos), WT injected with plxnd1 morpholino (four embryos; 4/4 showed a vascular phenotype similar to that of plxnd1 fov01b nulls), gipc1 skt1(MZ) ; gipc2 skt4(MZ) (12 embryos; 7/12 showed angiogenesis deficits), and gipc1 skt1(MZ) ; gipc2 skt4(MZ) injected with plxnd1 morpholino (11 embryos; 11/11 showed a vascular phenotype similar to that of plxnd1 fov01b nulls).

Article Snippet: Antibody , Rabbit anti-GIPC1 , Proteintech Group , Cat. #14822–1-AP. RRID: AB_2263269 , WB (1:3,000). This antibody detects GIPC1, GIPC2, and GIPC3 (our data).

Techniques: Injection

Journal: eLife

Article Title: GIPC proteins negatively modulate Plexind1 signaling during vascular development

doi: 10.7554/eLife.30454

Figure Lengend Snippet: ( A–F ). Representative fluorescent images of HUVEC morphology in cell collapse experiments under the following conditions. No ligand ( A–C ; top) or 45 min stimulation with 10 nM of SEMA3E ( D–F ; bottom). In each picture, the square area marked by yellow dotted sides is shown at twice the magnification at the bottom left corner and delimited by white sides. shRNA treatments as follows. Non-targeting, control ( A, D ), GIPC ( GIPC1, GIPC2, and GIPC3 ; B, E ), and PLXND1 ( C, F ). Scale bars (white horizontal lines), 100 μm. ( A–C ) Without ligand stimulation cells are uncollapsed regardless of the knockdown condition. ( D, E ) Cell collapse under ligand stimulation. Cells treated with non-targeting, control shRNA collapse ( D ). GIPC knockdown cells hypercollapse ( E ). SEMA3E-induced collapse is PLXND1-dependent. PLXND1 knockdown abrogates the morphological response ( F ). Cell collapse data collected from three independent experiments. This figure is related to .

Article Snippet: Antibody , Rabbit anti-GIPC1 , Proteintech Group , Cat. #14822–1-AP. RRID: AB_2263269 , WB (1:3,000). This antibody detects GIPC1, GIPC2, and GIPC3 (our data).

Techniques: shRNA, Control, Knockdown

Journal: eLife

Article Title: GIPC proteins negatively modulate Plexind1 signaling during vascular development

doi: 10.7554/eLife.30454

Figure Lengend Snippet: Western blots for GIPC1, GIPC2, PLXND1, and GAPDH (loading control) from TCLs of cells infected with the indicated shRNA lentiviral particles. Note the effective decrease of GIPC1-2 and PLXND1 levels. GIPC3 expression was absent under all the experimental conditions assayed. Hence, for brevity, the corresponding Western blots are not shown. This figure is related to .

Article Snippet: Antibody , Rabbit anti-GIPC1 , Proteintech Group , Cat. #14822–1-AP. RRID: AB_2263269 , WB (1:3,000). This antibody detects GIPC1, GIPC2, and GIPC3 (our data).

Techniques: Western Blot, Control, Infection, shRNA, Expressing

Journal: eLife

Article Title: GIPC proteins negatively modulate Plexind1 signaling during vascular development

doi: 10.7554/eLife.30454

Figure Lengend Snippet:

Article Snippet: Antibody , Rabbit anti-GIPC1 , Proteintech Group , Cat. #14822–1-AP. RRID: AB_2263269 , WB (1:3,000). This antibody detects GIPC1, GIPC2, and GIPC3 (our data).

Techniques: Transgenic Assay, Plasmid Preparation, Mutagenesis, Derivative Assay, Selection, Stable Transfection, Knock-Out, Recombinant, Control, shRNA, Sequencing, Construct, Synthesized, Positive Control, Sterility, Concentration Assay

Journal: eLife

Article Title: GIPC proteins negatively modulate Plexind1 signaling during vascular development

doi: 10.7554/eLife.30454

Figure Lengend Snippet:

Article Snippet: Antibody , Rabbit anti-GIPC1 , Proteintech Group , Cat. #14822–1-AP. RRID: AB_2263269 , WB (1:3,000). This antibody detects GIPC1, GIPC2, and GIPC3 (our data).

Techniques: Sequencing

Journal: eLife

Article Title: Structure analyses reveal a regulated oligomerization mechanism of the PlexinD1/GIPC/myosin VI complex

doi: 10.7554/eLife.27322

Figure Lengend Snippet: ( A ) Domain structure of mouse myosin VI. The HCBD is drawn larger than its proportion. ( B ) Structure of the GIPC1-GH2/myosin VI-HCBD complex. The five GH2/HCBD units in the asymmetric unit are shown. The N- and C-termini of one HCBD are labeled. Interfaces I and II between the GIPC1-GH2 and myosin VI-HCBD domains are displayed in detail in the two expanded views, respectively. ( C ) Overall structure of the GIPC2-GH2/myosin VI-HCBD complex. The five GH2/HCBD units in the asymmetric unit are shown. ( D ) Superimposition of one GH2/HCBD unit to the apo-GIPC1 structure. The superimposition is based on the GH2 domain. For clarity, only one GH2 and PDZ from the apo-GIPC1 structure are shown. It is evident that the PDZ domain in the apo-GIPC1 structure clashes with the myosin VI-HCBD bound to interface I of the GH2 domain. DOI: http://dx.doi.org/10.7554/eLife.27322.012

Article Snippet: The coding regions for mouse PlexinD1 cyto with (residues 1297–1925) and without the JM-segment (residues 1339–1925), mouse GIPC1 GH1-PDZ-GH2 version 1 (residues 52–333), GIPC1 GH1-PDZ-GH2 version 2 (residues 52–327), GIPC1 linker-GH2 (residues 217–333), GIPC1 GH2 (residues 258–333), mouse GIPC2 GH1-PDZ-GH2 (residues 40–314), GIPC2 GH2 (residues 238–314) and mouse GIPC3 GH1-PDZ-GH2 (residues 20–307) were sub-cloned into a modified pET-28a vector (Novagen).

Techniques: Labeling

Journal: eLife

Article Title: Structure analyses reveal a regulated oligomerization mechanism of the PlexinD1/GIPC/myosin VI complex

doi: 10.7554/eLife.27322

Figure Lengend Snippet: ( A ) The five unique copies of the HCBD in the asymmetric unit of the crystal of the GIPC1-GH2/myosin VI-HCBD complex are superimposed. This operation led to well alignment of all the GH2 domains bound to these HCBD molecules, highlighting the fact that all the HCBD molecules in the crystal interact with the GIPC1-GH2 domain in the same modes through either interface I or II. ( B ) The same superimposition as in ( A ) was applied to the GIPC2-GH2/myosin VI-HCBD structure, showing that both interfaces I and II are highly similar to those in the GIPC1-GH2/myosin VI-HCBD structure. DOI: http://dx.doi.org/10.7554/eLife.27322.015

Article Snippet: The coding regions for mouse PlexinD1 cyto with (residues 1297–1925) and without the JM-segment (residues 1339–1925), mouse GIPC1 GH1-PDZ-GH2 version 1 (residues 52–333), GIPC1 GH1-PDZ-GH2 version 2 (residues 52–327), GIPC1 linker-GH2 (residues 217–333), GIPC1 GH2 (residues 258–333), mouse GIPC2 GH1-PDZ-GH2 (residues 40–314), GIPC2 GH2 (residues 238–314) and mouse GIPC3 GH1-PDZ-GH2 (residues 20–307) were sub-cloned into a modified pET-28a vector (Novagen).

Techniques:

Journal: eLife

Article Title: Structure analyses reveal a regulated oligomerization mechanism of the PlexinD1/GIPC/myosin VI complex

doi: 10.7554/eLife.27322

Figure Lengend Snippet: ( A ) Pull-down of GIPC1, GIPC2 and GIPC3 by GST-HCBD in the absence or presence of PlexinD1 cyto . ( B ) Effects of mutations in PlexinD1 and GIPC1 on the interactions among PlexinD1 cyto , GIPC1 and myosin VI-HCBD. PlxD1, PlexinD1 cyto . DOI: http://dx.doi.org/10.7554/eLife.27322.017

Article Snippet: The coding regions for mouse PlexinD1 cyto with (residues 1297–1925) and without the JM-segment (residues 1339–1925), mouse GIPC1 GH1-PDZ-GH2 version 1 (residues 52–333), GIPC1 GH1-PDZ-GH2 version 2 (residues 52–327), GIPC1 linker-GH2 (residues 217–333), GIPC1 GH2 (residues 258–333), mouse GIPC2 GH1-PDZ-GH2 (residues 40–314), GIPC2 GH2 (residues 238–314) and mouse GIPC3 GH1-PDZ-GH2 (residues 20–307) were sub-cloned into a modified pET-28a vector (Novagen).

Techniques:

Journal: eLife

Article Title: Structure analyses reveal a regulated oligomerization mechanism of the PlexinD1/GIPC/myosin VI complex

doi: 10.7554/eLife.27322

Figure Lengend Snippet: Structural mapping of disease-associated mutations in GIPCs. Listed mutations are based on ( Ammar-Khodja et al., 2015 ; Katoh, 2013 ). HNSCC, head and neck squamous cell carcinoma. Mutations are mapped to GIPC1 based on the sequence alignment of GIPC1, 2 and 3 as shown in Figure 1—figure supplement 2 . DOI: http://dx.doi.org/10.7554/eLife.27322.025

Article Snippet: The coding regions for mouse PlexinD1 cyto with (residues 1297–1925) and without the JM-segment (residues 1339–1925), mouse GIPC1 GH1-PDZ-GH2 version 1 (residues 52–333), GIPC1 GH1-PDZ-GH2 version 2 (residues 52–327), GIPC1 linker-GH2 (residues 217–333), GIPC1 GH2 (residues 258–333), mouse GIPC2 GH1-PDZ-GH2 (residues 40–314), GIPC2 GH2 (residues 238–314) and mouse GIPC3 GH1-PDZ-GH2 (residues 20–307) were sub-cloned into a modified pET-28a vector (Novagen).

Techniques: Sequencing, Mutagenesis, Binding Assay

Journal: eLife

Article Title: Structure analyses reveal a regulated oligomerization mechanism of the PlexinD1/GIPC/myosin VI complex

doi: 10.7554/eLife.27322

Figure Lengend Snippet: The secondary structure and domain boundary assignments are based on the apo-GIPC1 structure. Residue numbering is based on mouse GIPC1. The GH1, PDZ, PDZ-GH2 linker and GH2 domains are denoted by background color of yellow, orange, light green and magenta, respectively. The black circles and stars highlight key residues in binding interfaces I and II for myosin VI-HCBD that were tested by mutational analyses. h, human; m, mouse. DOI: http://dx.doi.org/10.7554/eLife.27322.004

Article Snippet: The coding regions for mouse PlexinD1 cyto with (residues 1297–1925) and without the JM-segment (residues 1339–1925), mouse GIPC1 GH1-PDZ-GH2 version 1 (residues 52–333), GIPC1 GH1-PDZ-GH2 version 2 (residues 52–327), GIPC1 linker-GH2 (residues 217–333), GIPC1 GH2 (residues 258–333), mouse GIPC2 GH1-PDZ-GH2 (residues 40–314), GIPC2 GH2 (residues 238–314) and mouse GIPC3 GH1-PDZ-GH2 (residues 20–307) were sub-cloned into a modified pET-28a vector (Novagen).

Techniques: Binding Assay

Journal: eLife

Article Title: Structure analyses reveal a regulated oligomerization mechanism of the PlexinD1/GIPC/myosin VI complex

doi: 10.7554/eLife.27322

Figure Lengend Snippet: ( A ) Interactions of the linker with the GH1 and GH2 domains. ( B ) Interaction between the GH2 and PDZ domains. The loop between helices α1 and α2 in the GH2 domain that partially blocks the PBM binding site is labeled ‘loop(α1-α2)’ and highlighted in cyan. DOI: http://dx.doi.org/10.7554/eLife.27322.006

Article Snippet: The coding regions for mouse PlexinD1 cyto with (residues 1297–1925) and without the JM-segment (residues 1339–1925), mouse GIPC1 GH1-PDZ-GH2 version 1 (residues 52–333), GIPC1 GH1-PDZ-GH2 version 2 (residues 52–327), GIPC1 linker-GH2 (residues 217–333), GIPC1 GH2 (residues 258–333), mouse GIPC2 GH1-PDZ-GH2 (residues 40–314), GIPC2 GH2 (residues 238–314) and mouse GIPC3 GH1-PDZ-GH2 (residues 20–307) were sub-cloned into a modified pET-28a vector (Novagen).

Techniques: Binding Assay, Labeling

Journal: eLife

Article Title: Structure analyses reveal a regulated oligomerization mechanism of the PlexinD1/GIPC/myosin VI complex

doi: 10.7554/eLife.27322

Figure Lengend Snippet: ( A ) Detailed views of the interfaces. The top right panel highlights the packing interactions between the PlexinD1-PBM and GIPC1. The I1918/Y1919 motif in the PMB is accommodated by a hydrophobic pocket between the GH1 and PDZ domains in GIPC1. The lower right panel shows detailed interactions between the PlexinD1-PBM and GIPC1. The lower left panel shows the additional interface between the PlexinD1-GAP domain and the GIPC1-PDZ domain. ( B ) Sequence alignment of the C-terminal region of PlexinD1 from different species and other plexin family members. Residue numbers are based on mouse PlexinD1. ( C ) Superimposition of the structures of the PlexinD1 cyto /GIPC1 complex and apo-GIPC1 based on the PDZ domain. It is evident that the GIPC1-GH2 domain in the apo-GIPC1 structure clashes with PlexinD1 bound to GIPC1. Plx, Plexin. DOI: http://dx.doi.org/10.7554/eLife.27322.011

Article Snippet: The coding regions for mouse PlexinD1 cyto with (residues 1297–1925) and without the JM-segment (residues 1339–1925), mouse GIPC1 GH1-PDZ-GH2 version 1 (residues 52–333), GIPC1 GH1-PDZ-GH2 version 2 (residues 52–327), GIPC1 linker-GH2 (residues 217–333), GIPC1 GH2 (residues 258–333), mouse GIPC2 GH1-PDZ-GH2 (residues 40–314), GIPC2 GH2 (residues 238–314) and mouse GIPC3 GH1-PDZ-GH2 (residues 20–307) were sub-cloned into a modified pET-28a vector (Novagen).

Techniques: Sequencing

Journal: eLife

Article Title: Structure analyses reveal a regulated oligomerization mechanism of the PlexinD1/GIPC/myosin VI complex

doi: 10.7554/eLife.27322

Figure Lengend Snippet: ( A ) Pull-down of GIPC1, GIPC2 and GIPC3 by GST-HCBD in the absence or presence of PlexinD1 cyto . ( B ) Effects of mutations in PlexinD1 and GIPC1 on the interactions among PlexinD1 cyto , GIPC1 and myosin VI-HCBD. PlxD1, PlexinD1 cyto . DOI: http://dx.doi.org/10.7554/eLife.27322.017

Article Snippet: The coding regions for mouse PlexinD1 cyto with (residues 1297–1925) and without the JM-segment (residues 1339–1925), mouse GIPC1 GH1-PDZ-GH2 version 1 (residues 52–333), GIPC1 GH1-PDZ-GH2 version 2 (residues 52–327), GIPC1 linker-GH2 (residues 217–333), GIPC1 GH2 (residues 258–333), mouse GIPC2 GH1-PDZ-GH2 (residues 40–314), GIPC2 GH2 (residues 238–314) and mouse GIPC3 GH1-PDZ-GH2 (residues 20–307) were sub-cloned into a modified pET-28a vector (Novagen).

Techniques:

Journal: eLife

Article Title: Structure analyses reveal a regulated oligomerization mechanism of the PlexinD1/GIPC/myosin VI complex

doi: 10.7554/eLife.27322

Figure Lengend Snippet: Structural mapping of disease-associated mutations in GIPCs. Listed mutations are based on ( Ammar-Khodja et al., 2015 ; Katoh, 2013 ). HNSCC, head and neck squamous cell carcinoma. Mutations are mapped to GIPC1 based on the sequence alignment of GIPC1, 2 and 3 as shown in Figure 1—figure supplement 2 . DOI: http://dx.doi.org/10.7554/eLife.27322.025

Article Snippet: The coding regions for mouse PlexinD1 cyto with (residues 1297–1925) and without the JM-segment (residues 1339–1925), mouse GIPC1 GH1-PDZ-GH2 version 1 (residues 52–333), GIPC1 GH1-PDZ-GH2 version 2 (residues 52–327), GIPC1 linker-GH2 (residues 217–333), GIPC1 GH2 (residues 258–333), mouse GIPC2 GH1-PDZ-GH2 (residues 40–314), GIPC2 GH2 (residues 238–314) and mouse GIPC3 GH1-PDZ-GH2 (residues 20–307) were sub-cloned into a modified pET-28a vector (Novagen).

Techniques: Sequencing, Mutagenesis, Binding Assay

Journal: eLife

Article Title: Structure analyses reveal a regulated oligomerization mechanism of the PlexinD1/GIPC/myosin VI complex

doi: 10.7554/eLife.27322

Figure Lengend Snippet: ( A ) Domain structure of mouse GIPC1 and cartoon representation of the overall architecture of the domain-swapped dimer. The color scheme for one subunit in the dimer of the cartoon representation is the same as in the domain structure. The other subunit is colored gray. ( B ) Two orthogonal views of the GIPC1 structure. The dotted lines indicate the disordered portion of the linker between the PDZ and GH2 domains. The color scheme is the same as in ( A ). ( C ) Expanded views of the individual domains. DOI: http://dx.doi.org/10.7554/eLife.27322.002

Article Snippet: The coding regions for mouse PlexinD1 cyto with (residues 1297–1925) and without the JM-segment (residues 1339–1925), mouse GIPC1 GH1-PDZ-GH2 version 1 (residues 52–333), GIPC1 GH1-PDZ-GH2 version 2 (residues 52–327), GIPC1 linker-GH2 (residues 217–333), GIPC1 GH2 (residues 258–333), mouse GIPC2 GH1-PDZ-GH2 (residues 40–314), GIPC2 GH2 (residues 238–314) and mouse GIPC3 GH1-PDZ-GH2 (residues 20–307) were sub-cloned into a modified pET-28a vector (Novagen).

Techniques:

Journal: eLife

Article Title: Structure analyses reveal a regulated oligomerization mechanism of the PlexinD1/GIPC/myosin VI complex

doi: 10.7554/eLife.27322

Figure Lengend Snippet: The sigma-A weighted 2F o -F c map for helix α1 and loop(α1- α2) in the GIPC1-GH2 domain is shown, contoured at 1.0 sigma. DOI: http://dx.doi.org/10.7554/eLife.27322.003

Article Snippet: The coding regions for mouse PlexinD1 cyto with (residues 1297–1925) and without the JM-segment (residues 1339–1925), mouse GIPC1 GH1-PDZ-GH2 version 1 (residues 52–333), GIPC1 GH1-PDZ-GH2 version 2 (residues 52–327), GIPC1 linker-GH2 (residues 217–333), GIPC1 GH2 (residues 258–333), mouse GIPC2 GH1-PDZ-GH2 (residues 40–314), GIPC2 GH2 (residues 238–314) and mouse GIPC3 GH1-PDZ-GH2 (residues 20–307) were sub-cloned into a modified pET-28a vector (Novagen).

Techniques:

Journal: eLife

Article Title: Structure analyses reveal a regulated oligomerization mechanism of the PlexinD1/GIPC/myosin VI complex

doi: 10.7554/eLife.27322

Figure Lengend Snippet: Data collection and refinement statistics. DOI: http://dx.doi.org/10.7554/eLife.27322.005

Article Snippet: The coding regions for mouse PlexinD1 cyto with (residues 1297–1925) and without the JM-segment (residues 1339–1925), mouse GIPC1 GH1-PDZ-GH2 version 1 (residues 52–333), GIPC1 GH1-PDZ-GH2 version 2 (residues 52–327), GIPC1 linker-GH2 (residues 217–333), GIPC1 GH2 (residues 258–333), mouse GIPC2 GH1-PDZ-GH2 (residues 40–314), GIPC2 GH2 (residues 238–314) and mouse GIPC3 GH1-PDZ-GH2 (residues 20–307) were sub-cloned into a modified pET-28a vector (Novagen).

Techniques:

Journal: eLife

Article Title: Structure analyses reveal a regulated oligomerization mechanism of the PlexinD1/GIPC/myosin VI complex

doi: 10.7554/eLife.27322

Figure Lengend Snippet: The secondary structure and domain boundary assignments are based on the apo-GIPC1 structure. Residue numbering is based on mouse GIPC1. The GH1, PDZ, PDZ-GH2 linker and GH2 domains are denoted by background color of yellow, orange, light green and magenta, respectively. The black circles and stars highlight key residues in binding interfaces I and II for myosin VI-HCBD that were tested by mutational analyses. h, human; m, mouse. DOI: http://dx.doi.org/10.7554/eLife.27322.004

Article Snippet: The coding regions for mouse PlexinD1 cyto with (residues 1297–1925) and without the JM-segment (residues 1339–1925), mouse GIPC1 GH1-PDZ-GH2 version 1 (residues 52–333), GIPC1 GH1-PDZ-GH2 version 2 (residues 52–327), GIPC1 linker-GH2 (residues 217–333), GIPC1 GH2 (residues 258–333), mouse GIPC2 GH1-PDZ-GH2 (residues 40–314), GIPC2 GH2 (residues 238–314) and mouse GIPC3 GH1-PDZ-GH2 (residues 20–307) were sub-cloned into a modified pET-28a vector (Novagen).

Techniques: Binding Assay

Journal: eLife

Article Title: Structure analyses reveal a regulated oligomerization mechanism of the PlexinD1/GIPC/myosin VI complex

doi: 10.7554/eLife.27322

Figure Lengend Snippet: ( A ) Domain structure of mouse PlexinD1. The extracellular and transmembrane regions in PlexinD1 are omitted. The crystallization construct includes the regions denoted by the solid boxes. The single-letter amino acid sequence of the PBM is shown. ( B ) Comparison of the structures of apo-PlexinD1 cyto and PlexinD1 cyto from the PlexinD1 cyto /GIPC1 complex. ( C ) Two orthogonal views of the PlexinD1 cyto /GIPC1 complex structure. The dotted lines in green indicate the connection to the membrane by the transmembrane and juxtamembrane (JM) regions of PlexinD1. The linker-GH2 domains in GIPC1, invisible in the structures, are drawn as cartoons. Plx, Plexin. DOI: http://dx.doi.org/10.7554/eLife.27322.007

Article Snippet: The coding regions for mouse PlexinD1 cyto with (residues 1297–1925) and without the JM-segment (residues 1339–1925), mouse GIPC1 GH1-PDZ-GH2 version 1 (residues 52–333), GIPC1 GH1-PDZ-GH2 version 2 (residues 52–327), GIPC1 linker-GH2 (residues 217–333), GIPC1 GH2 (residues 258–333), mouse GIPC2 GH1-PDZ-GH2 (residues 40–314), GIPC2 GH2 (residues 238–314) and mouse GIPC3 GH1-PDZ-GH2 (residues 20–307) were sub-cloned into a modified pET-28a vector (Novagen).

Techniques: Crystallization Assay, Construct, Sequencing

Journal: eLife

Article Title: Structure analyses reveal a regulated oligomerization mechanism of the PlexinD1/GIPC/myosin VI complex

doi: 10.7554/eLife.27322

Figure Lengend Snippet: Many crystals were collected, washed with the crystallization buffer three times and analyzed by SDS-PAGE. PlexinD1 cyto (~66 kDa) and GIPC1 (~36 kDa) ran at their respective expected molecular weight, demonstrating that both proteins were intact in crystals. DOI: http://dx.doi.org/10.7554/eLife.27322.009

Article Snippet: The coding regions for mouse PlexinD1 cyto with (residues 1297–1925) and without the JM-segment (residues 1339–1925), mouse GIPC1 GH1-PDZ-GH2 version 1 (residues 52–333), GIPC1 GH1-PDZ-GH2 version 2 (residues 52–327), GIPC1 linker-GH2 (residues 217–333), GIPC1 GH2 (residues 258–333), mouse GIPC2 GH1-PDZ-GH2 (residues 40–314), GIPC2 GH2 (residues 238–314) and mouse GIPC3 GH1-PDZ-GH2 (residues 20–307) were sub-cloned into a modified pET-28a vector (Novagen).

Techniques: Crystallization Assay, SDS Page, Molecular Weight

Journal: eLife

Article Title: Structure analyses reveal a regulated oligomerization mechanism of the PlexinD1/GIPC/myosin VI complex

doi: 10.7554/eLife.27322

Figure Lengend Snippet: The two structures are superimposed on the PDZ domain on the left. The red arrows indicate the conformational difference at the other end of the dimer. The PDZ-GH2 linker and GH2 domain in the apo-GIPC1 structure are omitted for clarity. DOI: http://dx.doi.org/10.7554/eLife.27322.010

Article Snippet: The coding regions for mouse PlexinD1 cyto with (residues 1297–1925) and without the JM-segment (residues 1339–1925), mouse GIPC1 GH1-PDZ-GH2 version 1 (residues 52–333), GIPC1 GH1-PDZ-GH2 version 2 (residues 52–327), GIPC1 linker-GH2 (residues 217–333), GIPC1 GH2 (residues 258–333), mouse GIPC2 GH1-PDZ-GH2 (residues 40–314), GIPC2 GH2 (residues 238–314) and mouse GIPC3 GH1-PDZ-GH2 (residues 20–307) were sub-cloned into a modified pET-28a vector (Novagen).

Techniques:

Journal: eLife

Article Title: Structure analyses reveal a regulated oligomerization mechanism of the PlexinD1/GIPC/myosin VI complex

doi: 10.7554/eLife.27322

Figure Lengend Snippet: ( A ) Detailed views of the interfaces. The top right panel highlights the packing interactions between the PlexinD1-PBM and GIPC1. The I1918/Y1919 motif in the PMB is accommodated by a hydrophobic pocket between the GH1 and PDZ domains in GIPC1. The lower right panel shows detailed interactions between the PlexinD1-PBM and GIPC1. The lower left panel shows the additional interface between the PlexinD1-GAP domain and the GIPC1-PDZ domain. ( B ) Sequence alignment of the C-terminal region of PlexinD1 from different species and other plexin family members. Residue numbers are based on mouse PlexinD1. ( C ) Superimposition of the structures of the PlexinD1 cyto /GIPC1 complex and apo-GIPC1 based on the PDZ domain. It is evident that the GIPC1-GH2 domain in the apo-GIPC1 structure clashes with PlexinD1 bound to GIPC1. Plx, Plexin. DOI: http://dx.doi.org/10.7554/eLife.27322.011

Article Snippet: The coding regions for mouse PlexinD1 cyto with (residues 1297–1925) and without the JM-segment (residues 1339–1925), mouse GIPC1 GH1-PDZ-GH2 version 1 (residues 52–333), GIPC1 GH1-PDZ-GH2 version 2 (residues 52–327), GIPC1 linker-GH2 (residues 217–333), GIPC1 GH2 (residues 258–333), mouse GIPC2 GH1-PDZ-GH2 (residues 40–314), GIPC2 GH2 (residues 238–314) and mouse GIPC3 GH1-PDZ-GH2 (residues 20–307) were sub-cloned into a modified pET-28a vector (Novagen).

Techniques: Sequencing

Journal: eLife

Article Title: Structure analyses reveal a regulated oligomerization mechanism of the PlexinD1/GIPC/myosin VI complex

doi: 10.7554/eLife.27322

Figure Lengend Snippet: ( A ) Domain structure of mouse myosin VI. The HCBD is drawn larger than its proportion. ( B ) Structure of the GIPC1-GH2/myosin VI-HCBD complex. The five GH2/HCBD units in the asymmetric unit are shown. The N- and C-termini of one HCBD are labeled. Interfaces I and II between the GIPC1-GH2 and myosin VI-HCBD domains are displayed in detail in the two expanded views, respectively. ( C ) Overall structure of the GIPC2-GH2/myosin VI-HCBD complex. The five GH2/HCBD units in the asymmetric unit are shown. ( D ) Superimposition of one GH2/HCBD unit to the apo-GIPC1 structure. The superimposition is based on the GH2 domain. For clarity, only one GH2 and PDZ from the apo-GIPC1 structure are shown. It is evident that the PDZ domain in the apo-GIPC1 structure clashes with the myosin VI-HCBD bound to interface I of the GH2 domain. DOI: http://dx.doi.org/10.7554/eLife.27322.012

Article Snippet: The coding regions for mouse PlexinD1 cyto with (residues 1297–1925) and without the JM-segment (residues 1339–1925), mouse GIPC1 GH1-PDZ-GH2 version 1 (residues 52–333), GIPC1 GH1-PDZ-GH2 version 2 (residues 52–327), GIPC1 linker-GH2 (residues 217–333), GIPC1 GH2 (residues 258–333), mouse GIPC2 GH1-PDZ-GH2 (residues 40–314), GIPC2 GH2 (residues 238–314) and mouse GIPC3 GH1-PDZ-GH2 (residues 20–307) were sub-cloned into a modified pET-28a vector (Novagen).

Techniques: Labeling

Journal: eLife

Article Title: Structure analyses reveal a regulated oligomerization mechanism of the PlexinD1/GIPC/myosin VI complex

doi: 10.7554/eLife.27322

Figure Lengend Snippet: ( A ) The five unique copies of the HCBD in the asymmetric unit of the crystal of the GIPC1-GH2/myosin VI-HCBD complex are superimposed. This operation led to well alignment of all the GH2 domains bound to these HCBD molecules, highlighting the fact that all the HCBD molecules in the crystal interact with the GIPC1-GH2 domain in the same modes through either interface I or II. ( B ) The same superimposition as in ( A ) was applied to the GIPC2-GH2/myosin VI-HCBD structure, showing that both interfaces I and II are highly similar to those in the GIPC1-GH2/myosin VI-HCBD structure. DOI: http://dx.doi.org/10.7554/eLife.27322.015

Article Snippet: The coding regions for mouse PlexinD1 cyto with (residues 1297–1925) and without the JM-segment (residues 1339–1925), mouse GIPC1 GH1-PDZ-GH2 version 1 (residues 52–333), GIPC1 GH1-PDZ-GH2 version 2 (residues 52–327), GIPC1 linker-GH2 (residues 217–333), GIPC1 GH2 (residues 258–333), mouse GIPC2 GH1-PDZ-GH2 (residues 40–314), GIPC2 GH2 (residues 238–314) and mouse GIPC3 GH1-PDZ-GH2 (residues 20–307) were sub-cloned into a modified pET-28a vector (Novagen).

Techniques:

Journal: eLife

Article Title: Structure analyses reveal a regulated oligomerization mechanism of the PlexinD1/GIPC/myosin VI complex

doi: 10.7554/eLife.27322

Figure Lengend Snippet: The structure of the longer version of myosin VI (PDB ID: 2N12) is superimposed to the HCBD in the GIPC1-GH2/myosin VI-HCBD complex. The two molecules of the GH2 domain, interacting with the HCBD through interfaces I and II, respectively, are shown. The insert helix (red) clashes severely with the GH2 domain bound at interface II, and with that bound at interface I to a lesser extent. DOI: http://dx.doi.org/10.7554/eLife.27322.016

Article Snippet: The coding regions for mouse PlexinD1 cyto with (residues 1297–1925) and without the JM-segment (residues 1339–1925), mouse GIPC1 GH1-PDZ-GH2 version 1 (residues 52–333), GIPC1 GH1-PDZ-GH2 version 2 (residues 52–327), GIPC1 linker-GH2 (residues 217–333), GIPC1 GH2 (residues 258–333), mouse GIPC2 GH1-PDZ-GH2 (residues 40–314), GIPC2 GH2 (residues 238–314) and mouse GIPC3 GH1-PDZ-GH2 (residues 20–307) were sub-cloned into a modified pET-28a vector (Novagen).

Techniques:

Journal: eLife

Article Title: Structure analyses reveal a regulated oligomerization mechanism of the PlexinD1/GIPC/myosin VI complex

doi: 10.7554/eLife.27322

Figure Lengend Snippet: ( A ) Pull-down of GIPC1, GIPC2 and GIPC3 by GST-HCBD in the absence or presence of PlexinD1 cyto . ( B ) Effects of mutations in PlexinD1 and GIPC1 on the interactions among PlexinD1 cyto , GIPC1 and myosin VI-HCBD. PlxD1, PlexinD1 cyto . DOI: http://dx.doi.org/10.7554/eLife.27322.017

Article Snippet: The coding regions for mouse PlexinD1 cyto with (residues 1297–1925) and without the JM-segment (residues 1339–1925), mouse GIPC1 GH1-PDZ-GH2 version 1 (residues 52–333), GIPC1 GH1-PDZ-GH2 version 2 (residues 52–327), GIPC1 linker-GH2 (residues 217–333), GIPC1 GH2 (residues 258–333), mouse GIPC2 GH1-PDZ-GH2 (residues 40–314), GIPC2 GH2 (residues 238–314) and mouse GIPC3 GH1-PDZ-GH2 (residues 20–307) were sub-cloned into a modified pET-28a vector (Novagen).

Techniques:

Journal: eLife

Article Title: Structure analyses reveal a regulated oligomerization mechanism of the PlexinD1/GIPC/myosin VI complex

doi: 10.7554/eLife.27322

Figure Lengend Snippet: ( A ) The interaction of the GIPC1-GH2 and myosin VI-HCBD studied by AUC. Shown are c ( s ) distributions, colored as indicated in the inset. The concentrations of the complex given represent those of the equimolar mixtures of the GH2 and HCBD. All distributions have been normalized by the respective areas under the curves. ( B ) Multi-signal sedimentation velocity (MSSV) analysis of the GH2/HCBD mixture at 30 μM. The areas under the c k ( s ) distributions represent the concentrations of the respective mixture components. The molar ratio of GH2:HCBD sedimenting faster than 1 S is 1:1. ( C ) MSSV analysis of the GH2/HCBD mixture at 150 μM. The molar ratio of GH2:HCBD in the region of 1.5–6 S is 1.1:1. ( D ) AUC analysis of the linker-GH2/HCBD titration. Data are presented in the same fashion as in ( A ). ( E ) Comparison of linker-GH2 sedimentation at low and high concentrations. ( F ) Comparison of HCBD sedimentation at low and high concentrations. Note that the experiment of the HCBD at 20 μM was conducted under low-salt conditions (150 mM NaCl), while the 150 μM sample contained 500 mM NaCl. DOI: http://dx.doi.org/10.7554/eLife.27322.019

Article Snippet: The coding regions for mouse PlexinD1 cyto with (residues 1297–1925) and without the JM-segment (residues 1339–1925), mouse GIPC1 GH1-PDZ-GH2 version 1 (residues 52–333), GIPC1 GH1-PDZ-GH2 version 2 (residues 52–327), GIPC1 linker-GH2 (residues 217–333), GIPC1 GH2 (residues 258–333), mouse GIPC2 GH1-PDZ-GH2 (residues 40–314), GIPC2 GH2 (residues 238–314) and mouse GIPC3 GH1-PDZ-GH2 (residues 20–307) were sub-cloned into a modified pET-28a vector (Novagen).

Techniques: Sedimentation, Titration

Journal: eLife

Article Title: Structure analyses reveal a regulated oligomerization mechanism of the PlexinD1/GIPC/myosin VI complex

doi: 10.7554/eLife.27322

Figure Lengend Snippet: ( A ) Representative fluorescence images of cells. Arrow heads highlight large puncta in which Sema3E/PlexinD1 (red), GIPC1 (purple) and myosin VI (green) co-localize. Nuclei were stained with DAPI (blue). Scale bar, 5 µm. Images shown are representative from three independent samples of each group. ( B ) Quantification of puncta in each group of cells. Puncta larger than 1 µm 2 and containing Sema3E/PlexinD1, GIPC1 and myosin VI are counted for 20 cells from each group. Each dot in the plot represents one cell. Mean and standard deviations are shown as the blue and magenta bars, respectively. p-Values are determined by two-tailed Student’s t-test. DOI: http://dx.doi.org/10.7554/eLife.27322.021

Article Snippet: The coding regions for mouse PlexinD1 cyto with (residues 1297–1925) and without the JM-segment (residues 1339–1925), mouse GIPC1 GH1-PDZ-GH2 version 1 (residues 52–333), GIPC1 GH1-PDZ-GH2 version 2 (residues 52–327), GIPC1 linker-GH2 (residues 217–333), GIPC1 GH2 (residues 258–333), mouse GIPC2 GH1-PDZ-GH2 (residues 40–314), GIPC2 GH2 (residues 238–314) and mouse GIPC3 GH1-PDZ-GH2 (residues 20–307) were sub-cloned into a modified pET-28a vector (Novagen).

Techniques: Fluorescence, Staining, Two Tailed Test

Journal: eLife

Article Title: Structure analyses reveal a regulated oligomerization mechanism of the PlexinD1/GIPC/myosin VI complex

doi: 10.7554/eLife.27322

Figure Lengend Snippet: Structural mapping of disease-associated mutations in GIPCs. Listed mutations are based on ( Ammar-Khodja et al., 2015 ; Katoh, 2013 ). HNSCC, head and neck squamous cell carcinoma. Mutations are mapped to GIPC1 based on the sequence alignment of GIPC1, 2 and 3 as shown in Figure 1—figure supplement 2 . DOI: http://dx.doi.org/10.7554/eLife.27322.025

Article Snippet: The coding regions for mouse PlexinD1 cyto with (residues 1297–1925) and without the JM-segment (residues 1339–1925), mouse GIPC1 GH1-PDZ-GH2 version 1 (residues 52–333), GIPC1 GH1-PDZ-GH2 version 2 (residues 52–327), GIPC1 linker-GH2 (residues 217–333), GIPC1 GH2 (residues 258–333), mouse GIPC2 GH1-PDZ-GH2 (residues 40–314), GIPC2 GH2 (residues 238–314) and mouse GIPC3 GH1-PDZ-GH2 (residues 20–307) were sub-cloned into a modified pET-28a vector (Novagen).

Techniques: Sequencing, Mutagenesis, Binding Assay

Journal: iScience

Article Title: Spatial-temporal regulation of the prostanoid receptor EP2 co-ordinates PGE2-mediated cAMP signaling in decidualizing human endometrium

doi: 10.1016/j.isci.2024.111170

Figure Lengend Snippet:

Article Snippet: GIPC: sense 5′-GTCCAGACAGCAGCCAGAAT-3′ GIPC: antisense 5′-GAGGCCCTGTGATGTGAAGT-3′ , Merck , KSPQ12012G.

Techniques: Recombinant, Bioassay, Sequencing, Expressing, RNA Sequencing Assay, Cell Culture, Software, esiRNA, Negative Control