Journal: The Journal of Cell Biology

Article Title: TLNRD1 is a CCM complex component and regulates endothelial barrier integrity

doi: 10.1083/jcb.202310030

Figure Lengend Snippet: TLNRD1 is expressed in endothelial cells in vivo and regulates the vascular system. (A and B) TLNRD1 expression in mouse brain (A) and mouse heart (B). This single-cell RNA-Seq data is from the Tabula Muris dataset . For the brain, endothelial cells were defined as Cdh5+, Pecam1+, Slco1c1+, and Ocln+; in the heart, endothelial cells were defined as Cdh5+ and Pecam1+ . (C) Mouse brain slices were stained for TLNRD1, PECAM, and DAPI and imaged using a spinning disk confocal microscope. A single Z-plane is displayed. The yellow square highlights a magnified region of interest (ROI). Scale bars: (main) 50 µm and (inset) 10 µm. (D and E) kdrl:mCherry-CAAX zebrafish embryos were injected with recombinant Cas9 alone or together with sgRNA targeting TLNRD1 or slc45a2. The embryos were then imaged using a fluorescence microscope. (D) Representative images are displayed. The yellow and red squares highlight ROIs, which are magnified. The mesencephalic (MsV), mid-cerebral (MCeV), and caudal (CV) vein plexus are highlighted. Scale bars: (main) 500 µm and (inset) 100 µm. (E) The thickness of the mesencephalic, mid-cerebral, and caudal vein plexus measured from microscopy images are plotted as dot plots (non-injected, n = 17; Cas9, n = 13; slc45a2, n = 14; TLNRD1, n = 16). The gray bar highlights the data distribution, while the black line indicates the mean. The P values were determined using a randomization test.

Article Snippet: The recombinant expression vectors of GST-CCM2(FL), HisSumo-CCM2(413-438), TLNRD1(4H)-2T, and TLNRD1(4H)-2E will be deposited in Addgene ( https://www.addgene.org/Ben_Goult/ ).

Techniques: In Vivo, Expressing, RNA Sequencing, Staining, Microscopy, Injection, Recombinant, Fluorescence

Journal: The Journal of Cell Biology

Article Title: TLNRD1 is a CCM complex component and regulates endothelial barrier integrity

doi: 10.1083/jcb.202310030

Figure Lengend Snippet: The TLNRD1–CCM2 interaction involves the TLNRD1 4-helix bundle and a C-terminal helix in CCM2. (A and B) U2OS cells expressing TLNRD1-GFP and mito-mScarlet (CTRL), mito-PDCD10-mScarlet, or mito-CCM2-mScarlet were imaged using a spinning disk confocal microscope. (A) Representative single Z-planes are displayed. Dashed yellow lines highlight the cell outlines. The yellow squares highlight magnified ROIs. Scale bars: (main) 25 µm and (inset) 5 µm. (B) 3D colocalization analysis was performed using the JACoP Fiji plugin (three biological repeats, n > 31 image stacks per condition). The results are shown as Tukey boxplots. The whiskers (shown here as vertical lines) extend to data points no further from the box than 1.5× the interquartile range. The P values were determined using a randomization test. NS indicates no statistical difference between the mean values of the highlighted condition and the control. (C) HUVECs expressing TLNRD1-GFP and CCM2-mCherry were stained for F-actin and DAPI and imaged using an Airyscan confocal microscope. A single Z-plane is displayed. The yellow squares highlight a magnified ROI. Scale bars: (main) 25 µm and (inset) 5 µm. (D) GFP-pulldown in HEK293T cells expressing GFP-TLNRD1, GFP-TLNRD1 4H . GFP-TLNRD1 5H or GFP alone. CCM2 recruitment to the bait proteins was assessed by western blotting (representative of three biological repeats). (E) CCM2 schematic showing the boundaries of the phosphotyrosine binding (PTB) domain, the harmonin homology domain (HHD), and the C-terminal helix (CTH). (F) A GST-pulldown assay was used where Glutathione agarose-bound GST-CCM2 fragments (beads: B) were incubated with recombinant TLNRD (input: I). After multiple washes, proteins bound to the beads (pellet: P) were eluted. Red boxes highlight areas of interest in the gel. (G) A fluorescence polarization assay was used to determine the K d of the interaction between TLNRD1, TLNRD1 4H , or TLN1 R7R8 with SUMO-CCM2 CTH . K d values (nM) are shown in parentheses. ND, not determined. Source data are available for this figure: .

Article Snippet: The recombinant expression vectors of GST-CCM2(FL), HisSumo-CCM2(413-438), TLNRD1(4H)-2T, and TLNRD1(4H)-2E will be deposited in Addgene ( https://www.addgene.org/Ben_Goult/ ).

Techniques: Expressing, Microscopy, Control, Staining, Western Blot, Binding Assay, GST Pulldown Assay, Incubation, Recombinant, Fluorescence

Journal: The Journal of Cell Biology

Article Title: TLNRD1 is a CCM complex component and regulates endothelial barrier integrity

doi: 10.1083/jcb.202310030

Figure Lengend Snippet: TLNRD1 interacts with CCM2. (A) U2OS cells expressing mito-PDCD10-mScarlet, mito-CCM2-mScarlet, or mito-TLNRD1-mScarlet were imaged using a spinning disk confocal microscope. (B) U2OS cells treated with siRNA targeting KRIT1 or siRNA control. KRIT1 levels were then analyzed using Western blots. A representative western blot is displayed. (C) U2OS cells treated with siRNA targeting KRIT1 or siRNA control and expressing TLNRD1-GFP and mito-CCM2-mScarlet were imaged using a spinning disk confocal microscope. 3D colocalization analyses were performed using the JACoP Fiji plugin, and results are displayed as Tukey boxplots (three biological repeats, n > 21 image stacks per condition). (D) Glutathione agarose-bound GST-CCM2 (beads: B) was incubated with recombinant TLNRD1, TLNRD1 4H , or TLN1 R7R8 (input: I). After multiple washes, proteins bound to the beads (pellet: P) were visualized. The various fractions were then analyzed using SDS-PAGE followed by Coomassie staining. A representative gel of three independent repeats is displayed. Red boxes highlight areas of interest in the gel. (E) U2OS cells expressing various GFP-tagged CCM2 constructs and mito-TLNRD1-mScarlet or mito-mScarlet (CTRL) were imaged using a spinning disk confocal microscope. Representative single Z-planes are displayed. The yellow squares highlight magnified ROIs. Scale bars: (main) 25 µm and (inset) 5 µm. (F) 3D colocalization analyses were performed using the JACoP Fiji plugin, and results are displayed as Tukey boxplots (three biological repeats, n > 38 image stacks per condition). The whiskers (shown here as vertical lines) extend to data points no further from the box than 1.5× the interquartile range. For all panels, the P values were determined using a randomization test. NS indicates no statistical difference between the mean values of the highlighted condition and the control. Source data are available for this figure: .

Article Snippet: The recombinant expression vectors of GST-CCM2(FL), HisSumo-CCM2(413-438), TLNRD1(4H)-2T, and TLNRD1(4H)-2E will be deposited in Addgene ( https://www.addgene.org/Ben_Goult/ ).

Techniques: Expressing, Microscopy, Control, Western Blot, Incubation, Recombinant, SDS Page, Staining, Construct