- Title
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Nuclear SUN1 stabilizes endothelial cell junctions via microtubules to regulate blood vessel formation
- Authors
- Buglak, D.B., Bougaran, P., Kulikauskas, M.R., Liu, Z., Monaghan-Benson, E., Gold, A.L., Marvin, A.P., Burciu, A., Tanke, N.T., Oatley, M., Ricketts, S.N., Kinghorn, K., Johnson, B.N., Shiau, C.E., Rogers, S., Guilluy, C., Bautch, V.L.
- Source
- Full text @ Elife
(A) Schematic of tamoxifen-induced excision of exon 4 of Sun1 in pups from cross of Sun1fl/fl × Sun1fl/+;Cdh5-CreERT2 mice. (B) Representative images of postnatal day (P)7 mouse retinas of indicated genotypes, stained for IB4 (isolectin). Scale bar, 500 µm. Inset shows vascular plexus ahead of vein. Red line shows expansion of vascular front. Scale bar inset, 150 µm. (C) Quantification of vessel network radial expansion in (B). n=186 ROIs from 44 retinas (controls) and 63 ROIs from 16 retinas (Sun1iECKO) from six independent litters. ****, p<0.0001 by Student’s two-tailed unpaired t-test. (D) Quantification of vascular density ahead of either arteries or veins. n=87 ROIs (controls, artery), 38 ROIs (Sun1iECKO, artery), 84 ROIs (controls, vein), and 37 ROIs (Sun1iECKO, vein) from 27 retinas (controls) and 12 retinas (Sun1iECKO) from three independent litters. **, p<0.01 by Student’s two-tailed unpaired t-test. (E) Representative images of IB4 (isolectin) (green, vessels) and VE-cadherin (white, junctions) staining in P7 retinas of indicated genotypes. Scale bar, 50 µm. (F) Quantification of disorganized VE-cadherin as shown in (E). n=160 junctions (10 retinas, controls) and 160 junctions (10 retinas, Sun1iECKO). ****, p<0.0001 by Student’s two-tailed unpaired t-test. For all graphs, boxes represent the upper quartile, lower quartile, and median; whiskers represent the minimum and maximum values.
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(A) Representative images of human umbilical vein endothelial cells (HUVEC) with indicated siRNAs in 3D angiogenic sprouting assay. Sprouts were stained for Phalloidin (actin) and then depth encoded such that cooler colors are further in the Z-plane and warmer colors are closer in the Z-plane. Scale bar, 100 µm. (B) Quantification of average sprout length of 3D angiogenic sprouts shown in (A). n=42 beads (non-targeting [NT]) and 43 beads (SUN1 knockdown [KD]) compiled from five replicates. ****, p<0.0001 by Student’s two-tailed unpaired t-test. (C) Quantification of branches/mm of 3D angiogenic sprouts shown in (A). n=41 beads (NT) and 43 beads (SUN1 KD) compiled from five replicates. ****, p<0.0001 by Student’s two-tailed unpaired t-test. (D) Stills from Video 1 and Video 2 showing sprouting dynamics of HUVEC with indicated siRNAs over 50 hr. Scale bar, 50 µm. (E) Quantification of HUVEC sprout extensions and retractions shown in (D). n=101 sprouts (NT) and 77 sprouts (SUN1 KD) compiled from three replicates. p<0.001 by χ2 analysis. (F) Representative images of HUVEC with indicated siRNAs and stained with indicated antibodies in the 3D sprouting angiogenesis assay. Endothelial cells were stained for DAPI (cyan, DNA), Phalloidin (green, actin), and VE-cadherin (white, junctions). Arrows indicate normal junctions; arrowheads indicate abnormal junctions. Scale bar, 20 µm. (G) Quantification of disorganized VE-cadherin as shown in (F). n=32 junctions (NT) and 30 junctions (SUN1 KD) compiled from two replicates. ****, p<0.0001 by Student’s two-tailed unpaired t-test. For all graphs, boxes represent the upper quartile, lower quartile, and median; whiskers represent the minimum and maximum values.
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(A) Representative images of zebrafish embryos at 34 hpf (hours post fertilization) with indicated morpholino treatments; anterior to left. Tg(fli:LifeAct-GFP) (green, vessels). Insets show inter-segmental vessels (ISVs) with filopodia, outlines highlighted to show filopodia. Scale bar, 20 µm. (B) Quantification of filopodia number shown in (A). n=39 ROIs (15 fish, non-targeting [NT] morpholino [MO]) and 56 ROIs (20 fish, sun1b MO) compiled from three replicates. **, p<0.01 by Student’s two-tailed unpaired t-test. (C) Quantification of average filopodia length shown in (A). n=39 ROIs (15 fish, NT MO) and 56 ROIs (20 fish, sun1b MO) compiled from three replicates. ****, p<0.0001 by Student’s two-tailed unpaired t-test. (D) Representative images of zebrafish embryos at 34 hpf with indicated genotypes; anterior to left. Tg(fli:LifeAct-GFP) (green, vessels). Insets show ISVs with filopodia, outlines highlighted to show filopodia. Scale bar, 20 µm. (E) Quantification of filopodia number shown in (D). n=27 ROIs (9 fish, sun1b+/+) and 30 ROIs (10 fish, sun1b sa33109) compiled from two replicates. ns, not significant by Student’s two-tailed unpaired t-test. (F) Quantification of average filopodia length shown in (D). n=27 ROIs (9 fish, sun1b +/+) and 30 ROIs (10 fish, sun1b sa33109) compiled from two replicates. **, p<0.01 by Student’s two-tailed unpaired t-test. (G) Stills from Video 3 and Video 4 showing ISV sprouting from 26 to 36 hpf in zebrafish embryos with indicated morpholino treatment; anterior to left. Tg(fli:LifeAct-GFP) (green, vessels). White arrowhead points to ISV that does not extend or connect to dorsal longitudinal anastomotic vessel (DLAV). Yellow arrow points to ISV that extends but does not connect to DLAV. Scale bar, 20 µm. (H) Quantification of ISV connection to DLAV shown in (G). n=32 ISVs (6 fish, NT MO) and 36 ISVs (6 fish, sun1b MO) compiled from two replicates. p<0.05 by χ2 analysis. (I) Representative images of zebrafish embryos at 34 hpf with indicated morpholino treatments; anterior to left. Tg(fli:LifeAct-GFP) (green, vessels); ZO-1 (white junctions). Outlines highlighted to show junction shapes. Scale bar, 10 µm. (J) Quantification of junction morphology shown in (I). n=80 junctions that reach DLAV and 68 junctions that do not reach DLAV (21 fish, NT MO) and 47 junctions that reach DLAV and 37 junctions that do not reach DLAV junctions (22 fish, sun1b MO). ns, not significant; *, p<0.05; **, p<0.01 by two-way ANOVA with Tukey’s multiple comparisons test. For all graphs, boxes represent the upper quartile, lower quartile, and median; whiskers represent the minimum and maximum values.
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(A) Representative images of human umbilical vein endothelial cells (HUVEC) with indicated knockdowns (KD) in monolayers. Endothelial cells were stained for DAPI (cyan, DNA) and VE-cadherin (white, junctions). Insets show junctions. Scale bar, 10 µm. (B) Representative graph of impedance measured by real time cell analysis (RTCA). (C) Quantification of % change in cell index for RTCA measured at 5 hr. Normalized to non-targeting (NT) cell index. n=5 replicates. *, p<0.05 by Student’s two-tailed unpaired t-test. (D) Representative images of HUVEC with indicated siRNAs plated on biotinylated fibronectin and exposed to 15 dyn/cm2 shear stress for 72 hr then treated with streptavidin. Endothelial cells were stained for DAPI (cyan, DNA), streptavidin (green), and VE-cadherin (white, junctions). Arrow indicates flow direction. Insets show junctions. Scale bar, 20 µm. (E) Quantification of cell alignment shown in (D). n=59 cells (NT) and 73 cells (SUN1 KD) compiled from three replicates. (F) Quantification of streptavidin area shown in (D). n=15 ROIs (NT) and 15 ROIS (SUN1 KD) compiled from three replicates. (G) Representative images of HUVEC with indicated siRNAs showing adherens following EDTA washout. Endothelial cells were stained for DAPI (cyan, DNA) and VE-cadherin (white, junctions). Insets show junctions. Scale bar, 20 µm. (H) Quantification of VE-cadherin line scans at 20, 40, and 60 min post EDTA washout in (G). 20 min: n=31 junctions (NT) and 23 junctions (SUN1 KD); 40 min: n=49 junctions (NT) and 33 junctions (SUN1 KD); 60 min: n=33 junctions (NT) and 33 junctions (SUN1 KD) compiled from three replicates. ns, not significant; ****, p<0.0001 by Student’s two-tailed unpaired t-test. For C, error bars represent standard deviation. For E, F, and H, boxes represent the upper quartile, lower quartile, and median; whiskers represent the minimum and maximum values.
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(A) Representative images of human umbilical vein endothelial cells (HUVEC) with indicated siRNAs and indicated treatments. Endothelial cells were stained for DAPI (cyan, DNA) and VE-cadherin (white, junctions). Insets show junctions. Scale bar, 20 µm. (B) Quantification of VE-cadherin line scans for treatments shown in (A). n=106 junctions (non-targeting [NT], vehicle), 101 junctions (NT, Nocodazole), 105 junctions (SUN1 knockdown [KD], vehicle), and 96 junctions (SUN1 KD, Nocodazole) compiled from three replicates. ns, not significant; ****, p<0.0001 by two-way ANOVA with Tukey’s multiple comparisons test. (C) Representative images of HUVEC with indicated siRNAs. Endothelial cells were stained for α-tubulin (cyan, microtubules) and VE-cadherin (red, junctions). Red insets show α-tubulin at the MTOC (microtubule organizing center), yellow insets show α-tubulin contacts at junctions. Arrows denote contact sites. Scale bar, 20 µm. (D) Quantification of α-tubulin area at the MTOC shown in (C). n=19 cells (NT) and 10 cells (SUN1 KD) compiled from three replicates. ns, not significant by Student’s two-tailed unpaired t-test. (E) Quantification of peripheral α-tubulin area shown in (C). n=39 cells (NT) and 46 cells (SUN1 KD) compiled from three replicates. ****, p<0.0001 by Student’s two-tailed unpaired t-test. (F) Quantification of contacts between α-tubulin and VE-cadherin shown in (C). n=75 junctions (NT) and 48 junctions (SUN1 KD) compiled from three replicates. ***, p<0.001 by Student’s two-tailed unpaired t-test. (G) Stills from Video 5 and Video 6 showing microtubule growth in EB3-mCherry labeled HUVEC. White dot indicates start of track. Yellow line indicates growth, red line indicates shrinkage. Scale bar, 10 µm. (H) Quantification of microtubule growth rate from EB3-mCherry microtubule tracking. N=120 microtubules (12 cells, NT) and 117 microtubules (12 cells, SUN1 KD) compiled from two replicates. ns, not significant by Student’s two-tailed unpaired t-test. (I) Quantification of microtubule shrink rate from EB3-mCherry microtubule tracking. n=120 microtubules (12 cells, NT) and 117 microtubules (12 cells, SUN1 KD) compiled from two replicates. **, p<0.01 by Student’s two-tailed unpaired t-test. (J) Quantification of catastrophe rate from EB3-mCherry microtubule tracking. n=120 microtubules (12 cells, NT) and 117 microtubules (12 cells, SUN1 KD) compiled from two replicates. ****, p<0.0001 by Student’s two-tailed unpaired t-test. (K) Quantification of percent of time spent shrinking from EB3-mCherry microtubule tracking. n=120 microtubules (12 cells, NT) and 117 microtubules (12 cells, SUN1 KD) compiled from two replicates. ****, p<0.0001 by Student’s two-tailed unpaired t-test. For all graphs, boxes represent the upper quartile, lower quartile, and median; whiskers represent the minimum and maximum values.
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(A) Representative images of human umbilical vein endothelial cells (HUVEC) with indicated siRNAs and indicated treatments. Endothelial cells were stained for DAPI (cyan, DNA) and VE-cadherin (white, junctions). Insets show junctions. Scale bar, 20 µm. (B) Quantification of VE-cadherin line scans for treatments shown in (A). n=159 junctions (non-targeting [NT], vehicle), 154 junctions (NT, blebbistatin), 151 junctions (SUN1 knockdown [KD], vehicle), and 149 junctions (SUN1 KD, blebbistatin) compiled from three replicates. ns, not significant; ****, p<0.0001 by two-way ANOVA with Tukey’s multiple comparisons test. (C) Representative images of HUVEC with indicated siRNAs and indicated treatments. Endothelial cells were stained for DAPI (cyan, DNA) and VE-cadherin (white, junctions). Insets show junctions. Scale bar, 20 µm. (D) Quantification of VE-cadherin line scans for treatments shown in (C). n=75 junctions (NT, vehicle), 70 junctions (NT, thrombin), 71 junctions (SUN1 KD, vehicle), and 73 junctions (SUN1 KD, thrombin) compiled from three replicates. ns, not significant; ****, p<0.0001 by two-way ANOVA with Tukey’s multiple comparisons test. (E) Representative images of HUVEC with indicated siRNAs. Endothelial cells were stained for DAPI (blue, DNA), α-tubulin (red, microtubules), and GEF-H1 (cyan). Insets show α-tubulin and GEF-H1 colocalization. Scale bar, 20 µm. (F) Quantification of free GEF-H1 normalized to α-tubulin associated GEF-H1 shown in (E). n=30 cells (NT) and 30 cells (SUN1 KD) compiled from three replicates. **, p<0.01 by Student’s two-tailed unpaired t-test. (G) Representative images of HUVEC with indicated siRNAs and indicated treatments. Endothelial cells were stained for DAPI (cyan, DNA) and VE-cadherin (white, junctions). Insets show junctions. Scale bar, 20 µm. (H) Quantification of VE-cadherin line scans from KD shown in (G). n=169 junctions (NT), 166 junctions (SUN1 KD), 170 junctions (SUN1/GEF-H1 KD) compiled from three replicates. ns, not significant; ****, p<0.0001 by one-way ANOVA with Tukey’s multiple comparisons test. (I) Representative images of HUVEC with indicated siRNAs cultured on biotinylated fibronectin and treated with streptavidin upon confluence. Endothelial cells were stained for DAPI (cyan, DNA), streptavidin (green), and VE-cadherin (white, junctions). Insets show junctions. Scale bar, 20 µm. (J) Quantification of streptavidin area shown in (I). n=15 ROIs (NT), 15 ROIs (SUN1 KD), 15 ROIs (GEF-H1 KD), and 15 ROIs (SUN1/GEF-H1 KD) compiled from three replicates. ns, not significant; ****, p<0.0001 by one-way ANOVA with Tukey’s multiple comparisons test. For all graphs, boxes represent the upper quartile, lower quartile, and median; whiskers represent the minimum and maximum values.
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(A) Representative images of human umbilical vein endothelial cells (HUVEC) with indicated siRNAs cultured on biotinylated fibronectin and treated with streptavidin upon confluence. Endothelial cells were stained for DAPI (cyan, DNA), streptavidin (green), and VE-cadherin (white, junctions). Insets show junctions. Scale bar, 20 µm. (B) Representative images of HUVEC with indicated siRNAs. Endothelial cells were stained for α-tubulin (cyan, microtubules) and VE-cadherin (red, junctions). Insets show α-tubulin contacts at junctions. Scale bar, 20 µm. (C) Representative images of HUVEC with indicated siRNAs. Endothelial cells were stained for DAPI (blue, DNA), α-tubulin (red, microtubules), and GEF-H1 (cyan). Insets show α-tubulin and GEF-H1 colocalization. Scale bar, 20 µm. (D) Quantification of streptavidin area shown in (A). n=22 ROIs (non-targeting [NT]), 22 ROIs (SUN1 knockdown [KD]), and 22 ROIs (SUN1/nesprin-1 KD) compiled from four replicates. ns, not significant; ***, p<0.001; ****, p<0.0001 by one-way ANOVA with Tukey’s multiple comparisons test. (E) Quantification of peripheral α-tubulin area shown in (B). n=52 cells (NT), 52 cells (SUN1 KD), and 52 cells (SUN1/nesprin-1 KD) compiled from three replicates. ns, not significant; ***, p<0.001; ****, p<0.0001 by one-way ANOVA with Tukey’s multiple comparisons test. (F) Quantification of free GEF-H1 normalized to α-tubulin associated GEF-H1 shown in (C). n=32 cells (NT), 32 cells (SUN1 KD), and 32 cells (SUN1/nesprin-1 KD) compiled from three replicates. ns, not significant; **, p<0.01; ****, p<0.0001 by one- way ANOVA with Tukey’s multiple comparisons test. For all graphs, boxes represent the upper quartile, lower quartile, and median; whiskers represent the minimum and maximum values.
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Model describing proposed role of SUN1 in angiogenic sprouting and endothelial cell junction stabilization. |