Nuclear SUN1 stabilizes endothelial cell junctions via microtubules to regulate blood vessel formation

  1. Danielle B Buglak
  2. Pauline Bougaran
  3. Molly R Kulikauskas
  4. Ziqing Liu
  5. Elizabeth Monaghan-Benson
  6. Ariel L Gold
  7. Allison P Marvin
  8. Andrew Burciu
  9. Natalie T Tanke
  10. Morgan Oatley
  11. Shea N Ricketts
  12. Karina Kinghorn
  13. Bryan N Johnson
  14. Celia E Shiau
  15. Stephen Rogers
  16. Christophe Guilluy
  17. Victoria L Bautch  Is a corresponding author
  1. Curriculum in Cell Biology and Physiology, The University of North Carolina at Chapel Hill, United States
  2. Department of Biology, The University of North Carolina at Chapel Hill, United States
  3. Department of Molecular Biomedical Sciences, College of Veterinary Medicine, North Carolina State University, United States
  4. Department of Pathology, The University of North Carolina at Chapel Hill, United States
  5. McAllister Heart Institute, The University of North Carolina at Chapel Hill, United States
10 figures, 6 videos, 2 tables and 1 additional file

Figures

Figure 1 with 1 supplement
The nuclear LINC protein SUN1 regulates vascular development.

(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 …

Figure 1—figure supplement 1
Loss of Sun1 in the postnatal retina leads to altered sprouting and junction integrity.

(A) Schematic showing strategy for generation of the Sun1 floxed allele and subsequent Cre-mediated excision of exon 4 of the Sun1 allele. (B) Agarose ethidium bromide gel showing PCR bands specific …

Figure 1—figure supplement 1—source data 1

Agarose ethidium bromide gel showing PCR bands specific for WT or Sun1fl allele (left) or PCR band for the presence of the Cdh5-CreERT2 allele (right).

DNA was extracted from mouse tail cuts.

https://cdn.elifesciences.org/articles/83652/elife-83652-fig1-figsupp1-data1-v1.zip
Figure 1—figure supplement 1—source data 2

Agarose ethidium bromide gel showing PCR band specific for the excised Sun1 allele.

DNA was extracted from mouse lung tissue.

https://cdn.elifesciences.org/articles/83652/elife-83652-fig1-figsupp1-data2-v1.zip
Figure 2 with 1 supplement
Nuclear SUN1 is required for sprouting angiogenesis.

(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 …

Figure 2—figure supplement 1
SUN1 is nuclear localized in endothelial cells and does not regulate proliferation.

(A) Representative images of human umbilical vein endothelial cells (HUVEC) with indicated siRNAs and stained with the indicated antibodies. Endothelial cells were stained for DAPI (DNA) and SUN1. …

SUN1 regulates actin dynamics and angiogenic sprout extension in vivo.

(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 …

Figure 4 with 1 supplement
SUN1 stabilizes endothelial cell-cell junctions and regulates junction integrity.

(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, …

Figure 4—figure supplement 1
SUN1 regulates endothelial junction integrity.

(A) Representative images of human umbilical vein endothelial cells (HUVEC) with indicated siRNAs. Endothelial cells were stained for DAPI (cyan, DNA) and VE-cadherin (white, junctions). Insets show …

Figure 4—figure supplement 1—source data 1

Western blot showing four experimental replicates.

Protein was extracted from human umbilical vein endothelial cells (HUVEC) with indicated siRNAs. Blot was probed for VE-cadherin (top band) and GAPDH (bottom band) as a loading control. Red box indicates lanes used for Figure 4—figure supplement 1B.

https://cdn.elifesciences.org/articles/83652/elife-83652-fig4-figsupp1-data1-v1.zip
Figure 5 with 2 supplements
SUN1 regulates microtubule localization and dynamics in endothelial cells.

(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 …

Figure 5—figure supplement 1
SUN1 does not affect transcription in endothelial cells.

Volcano plots showing transcriptional changes in HUVEC from bulk RNASeq data in NT (non-targeting) under flow vs. NT under static conditions (left), SUN1 KD under static vs. NT under static …

Figure 5—figure supplement 2
SUN1 regulates the microtubule cytoskeleton in endothelial cells.

(A) Representative images of human umbilical vein endothelial cells (HUVEC) with indicated siRNAs showing changes in microtubules at nucleus. Endothelial cells were stained for α-tubulin (white, …

Figure 6 with 3 supplements
SUN1 regulates endothelial cell contractility and exerts its effects on junctions through the microtubule-associated GEF-H1.

(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 …

Figure 6—figure supplement 1
SUN1 regulates the actin cytoskeleton in endothelial cells.

(A) Representative images of human umbilical vein endothelial cells (HUVEC) with indicated siRNAs showing changes in actin structures at the cell periphery. Endothelial cells were stained for DAPI …

Figure 6—figure supplement 2
SUN1 acts through RhoA to affect junctions.

(A) Representative images of human umbilical vein endothelial cells (HUVEC) with indicated siRNAs. Endothelial cells were stained for DAPI (cyan, DNA) and VE-cadherin (white, junctions). Insets show …

Figure 6—figure supplement 2—source data 1

Western blots showing representative replicate.

Protein was extracted from human umbilical vein endothelial cells (HUVEC) with indicated siRNAs and treatments. Blots were probed for RhoA from whole cell lysates (Total RhoA, right blot) or following RBD pulldown (RhoA-GTP, left blot). Thrombin treatment was used as positive control and serum starvation was used as a negative control. Red boxes indicate portions of blots used for Figure 6—figure supplement 2D.

https://cdn.elifesciences.org/articles/83652/elife-83652-fig6-figsupp2-data1-v1.zip
Figure 6—figure supplement 3
GEF-H1 signaling is downstream of SUN1 and microtubules.

(A) Representative western blot showing total levels of GEF-H1 protein in human umbilical vein endothelial cells (HUVEC) with indicated siRNAs. Total protein was used as a loading control. …

Figure 6—figure supplement 3—source data 1

Western blot showing representative replicate.

Protein was extracted from human umbilical vein endothelial cells (HUVEC) with indicated siRNAs. Blot was probed for GEF-H1 (labeled GEF-H1 in source file). Total protein from transfer was used as a loading control (labeled total protein in source file). Red boxes indicate portions of blots used for Figure 6—figure supplement 3A.

https://cdn.elifesciences.org/articles/83652/elife-83652-fig6-figsupp3-data1-v1.zip
Figure 7 with 1 supplement
SUN1 regulates endothelial cell junctions through nesprin-1.

(A) Representative images of human umbilical vein endothelial cells (HUVEC) with indicated siRNAs cultured on biotinylated fibronectin and treated with streptavidin upon confluence. Endothelial …

Figure 7—figure supplement 1
Loss of nesprin-1 alone does not impact endothelial junctions or microtubules.

(A) Representative images of human umbilical vein endothelial cells (HUVEC) with indicated siRNAs and indicated antibodies. Endothelial cells were stained for DAPI (DNA) and nesprin-1. Scale bar, 20 …

Proposed role of SUN1 in angiogenic sprouting and endothelial cell junction stabilization.

Model describing proposed role of SUN1 in angiogenic sprouting and endothelial cell junction stabilization.

Author response image 1
Cdh5CreERT2 efficiently excises in endothelial cells of the mouse postnatal retina.

(A) Representative images of P7 mouse retinas with the indicated genotypes, stained for ERG (white, nucleus). tdTomato (magenta) is expressed in cells that have not undergone Cre-mediated excision, …

Author response image 2
Cdh5CreERT2 does not affect radial expansion.

Reproduced graph from Figure 1—figure supplement 1D with additional statistical comparisons. There is no significant difference in radial expansion between Cdh5CreERT2/+ mice and WT mice.

Videos

Video 1
Control endothelial cells elongate in 3D sprouting assay.

3D sprouting angiogenesis of control (non-targeting [NT]) human umbilical vein endothelial cells (HUVEC) over 60 hr, showing elongation of NT sprouts. Scale bar, 50 µm. Frames acquired every 30 min.

Video 2
SUN1-depleted endothelial cells retract in 3D sprouting assay.

3D sprouting angiogenesis of SUN1 knockdown (KD) sprouts over 60 hr, showing retraction of SUN1 KD sprouts. Scale bar, 50 µm. Frames acquired every 30 min.

Video 3
Control zebrafish have normal inter-segmental vessel (ISV) growth.

Movie taken from 26 to 36 hpf (hours post fertilization) in Tg(fli:LifeAct-GFP) zebrafish embryos injected with a non-targeting (NT) morpholino, showing elongation of ISVs and connection to the …

Video 4
Loss of SUN1 in zebrafish leads to abnormal inter-segmental vessel (ISV) growth.

Movie taken from 26 to 36 hpf in Tg(fli:LifeAct-GFP) zebrafish embryos injected with a sun1b morpholino, showing an ISV that fails to elongate and connect to the dorsal longitudinal anastomotic …

Video 5
Control endothelial cells have normal microtubule dynamics.

Movie taken for 120 s in human umbilical vein endothelial cells (HUVEC) with non-targeting (NT) siRNA labeled with EB3-mCherry. Quantified microtubule tracks are indicated. Scale bar, 5 µm. Frames …

Video 6
Loss of SUN1 in endothelial cells leads to impaired microtubule dynamics.

Movie taken for 120 s in human umbilical vein endothelial cells (HUVEC) with SUN1 siRNA labeled with EB3-mCherry. Quantified microtubule tracks are indicated. Scale bar, 5 µm. Frames acquired every …

Tables

Table 1
SUN1 depletion does not alter endothelial gene expression.
Condition# Upregulated DEG# Downregulated DEG
NT_FLOW vs. NT_STAT13231109
SUN1_STAT vs. NT_STAT01
SUN1_FLOW vs. NT_FLOW11
  1. Bold numbers indicate that single downregulated gene was SUN1.

  2. Abbreviation: NT, non-targeting; STAT, static; DEG, differentially expressed genes.

Appendix 1—key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Gene (Mus musculus)Sun1EnsemblEnsembl_ID:
ENSMUSG00000036817
Gene (Homo sapiens)SUN1EnsemblEnsemble_ID:
ENSG00000164828
Gene (Danio rerio)sun1bZFINZFIN_ID:ZDB
-GENE-050522–551
Strain, strain background (M. musculus)B6NJ;B6N-Sun1tm1a(EUCOMM)/Wtsi/
CipheOrl
European Mouse Mutant Archive (EMMA)EMMA_ID:EM:09532
Strain, strain background (M. musculus)FlpO-B6N-Albino
(Rosa26-FlpO/+)
OtherUNC Animal Models Core
Strain, strain background (M. musculus)Tg(Cdh5-cre/ERT2)1RhaSörensen et al., 2009 (PMID: 19144989)Dr Ralf Adams
Strain,strain background (D. rerio)Tg(fli:LifeAct-GFP)OtherDr Wiebke Herzog
Strain,strain background (D. rerio)sun1bsa33109Zebrafish International Resource Center (ZIRC)Cat#:ZL12625.02
Cell line (Homo sapiens)Human Umbilical Vein Endothelial Cells (HUVEC)LonzaCat#:C2519AHuman primary endothelial cells, mixed sex
Cell line (H. sapiens)Normal Human Lung Fibroblasts (NHLF)LonzaCat#:CC2512Human primary lung fibroblast cells, mixed sex
Transfected construct (H. sapiens)Non-targeting (NT) siRNALife TechnologiesCat#:4390847Silencer select
Transfected construct (H. sapiens)SUN1 siRNA #1Life TechnologiesCat#:439240;
#s23630
Silencer select
Transfected construct (H. sapiens)SUN1 siRNA #2Life TechnologiesCat#:439240; #s23629Silencer select
Transfected construct (H. sapiens)GEF-H1 siRNALife TechnologiesCat#:439240;
#s17546
Silencer select
Transfected construct (H. sapiens)Nesprin-1 siRNADharmaconCat#:
M-014039-02-0005
SMARTpool
Transfected construct (H. sapiens)EB3-mCherryKushner et al., 2014 (PMID: 25049273)Lentiviral construct
AntibodyAnti-mouseCD144 (rat monoclonal)BD PharmingenCat#:550548Primary antibody, detects VE-cadherin in mouse tissue IF
mouse (1:100)
AntibodyAnti-ERG (rabbit monoclonal)AbcamCat#:ab196149Primary antibody conjugated to AlexaFluor647, detects nuclei in endothelial cells IF mouse (1:500)
AntibodyAnti-VE-cadherin (rabbit monoclonal)Cell SignalingCat#:2500SPrimary antibody, detects human
VE-cadherin IF cells 3D(1:1000) IF cells 2D (1:500) Western (1:14000)
AntibodyAnti-ZO1 (mouse monoclonal)Thermo FisherCat#:33-9100Primary antibody, detects ZO1 in zebrafish IF zebrafish (1:500)
AntibodyAnti-SUN1 (rabbit monoclonal)AbcamCat#:ab124770Primary antibody, detects human SUN1 IF cells (1:500)
AntibodyAnti-Ki67 (rabbit polyclonal)AbcamCat#:ab15580Primary antibody IF cells (1:500)
AntibodyAnti-phospho-myosin light chain 2 (Thr18/Ser19) (rabbit polyclonal)Cell SignalingCat#:3674SPrimary antibody IF cells (1:500)
AntibodyAnti-alpha-tubulin (mouse monoclonal)Cell SignalingCat#:3873
S
Primary antibody IF cells (1:500)
AntibodyAnti-GEF-H1 (rabbit polyclonal)AbcamCat#:ab155785Primary antibody IF cells
(1:500) Western (1:1000)
AntibodyAnti-SYNE1 (rabbit polyclonal)Atlas AntibodiesCat#:HPA019113Primary antibody IF cells (1:500)
AntibodyAnti-GAPDH (mouse monoclonal)Cell SignalingCat#:97166SPrimary antibody Western (1:5000)
AntibodyAnti-VE-cadherin BV6 (mouse monoclonal)EnzoCat#:ALX-803-305C100Primary antibody, detects
the extracellular region of
VE-cadherin IF cells (1:100)
AntibodyAnti-RhoA (rabbit monoclonal)Cell SignalingCat#:2117Primary antibody Western (1:1000)
AntibodyGoat anti-mouse AlexaFluor 488 (goat polyclonal)Life TechnologiesCat#:A11029Secondary antibody
IF cells (1:500)
AntibodyGoat anti-rabbit AlexaFluor 594 (goat polyclonal)Life TechnologiesCat#:A11037Secondary antibody
IF cells (1:500)
AntibodyGoat anti-rat AlexaFluor 647 (goat polyclonal)Life TechnologiesCat#:A21247Secondary antibody IF mouse (1:500)
AntibodyGoat anti-mouse AlexaFluor 647 (goat polyclonal)Life TechnologiesCat#:A21236Secondary antibody IF zebrafish (1:1000) IF cells (1:500)
AntibodyGoat anti-rabbit AlexaFluor 647 (goat polyclonal)Life TechnologiesCat#:A21245Secondary antibody IF cells (1:500)
AntibodyDonkey anti-rabbit HRP (donkey polyclonal)Thermo FisherCat#:A16035Secondary antibody Western (1:10,000)
AntibodyGoat anti-rabbit HRP (goat polyclonal)Jackson
ImmunoResearch
Cat#:111-035-144Secondary antibody Western
Sequence-based reagentLacZ_FThis paperPCR primersACTATCCCGACCGCCTTACT
Sequence-based reagentLacZ_RThis paperPCR primersTAGCGGCTGATGTTGAACTG
Sequence-based reagentSun1fl_FThis paperPCR primersGCTCTCTGAAACATGGCTGA
Sequence-based reagentSun1fl_RThis paperPCR primersATCCGGGGTGTTTGGATTAT
Sequence-based reagentSun1excised_FThis paperPCR primersCTTTTGGGCTGCTCTGTTGT
Sequence-based reagentSun1excised_RThis paperPCR primersATCCGGGGTGTTTGGATTAT
Sequence-based reagentFlpO_FThis paperPCR primersTGAGCTTCGACATCGTGAAC
Sequence-based reagentFlpO_RThis paperPCR primersTCAGCATCTTCTTGCTGTGG
Sequence-based reagentCdh5Cre_FThis paperPCR primersTCCTGATGGTGCCTATCCTC
Sequence-based reagentCdh5Cre_RThis paperPCR primersCCTGTTTTGCACGTTCACCG
Sequence-based reagentsun1b_FThis paperPCR primersGGCTGCGTCAGACTCCATTA
Sequence-based reagentsun1b_RThis paperPCR primersTTGAGTTAAACCCAGCGCCT
Sequence-based reagentNon-targeting (NT)
morpholino (MO)
This paperCCTCTTACCTCAGTTACAATTTATA
Sequence-based reagentsun1b morpholino (MO)This paperCGCAGTTTGACCATCAGTTTCTACA
Peptide, recombinant proteinIsolectin B4 AlexaFluor 488Thermo FisherCat#:I21411IF(1:100)
Peptide, recombinant proteinAlexaFluor 488 PhalloidinLife TechnologiesCat#:A12379IF cells 3D (1:50)
Peptide, recombinant proteinStreptavidin-488InvitrogenCat#:S1122325 μg/ml
Peptide, recombinant protein10 kDa Dextran-Texas RedInvitrogenCat#:D1863100 μl injected at 5 mg/ml
Peptide, recombinant proteinFibrinogenFisherCat#:820224500 μl at 2.2 mg/ml
Peptide, recombinant proteinFibronectinSigmaCat#:F2006-2MG5 μg/ml
Peptide, recombinant proteinEZ-Link Sulfo-NHS-LC-BiotinThermo FisherCat#:A392570.5 mM
Chemical compound, drugTamoxifenSigmaCat#:T564850 μl injected at 1 mg/ml
Chemical compound, drugThrombinSigmaCat#:T7201-500UNFor bead assay: 7 μl at 50 U/ml For cell treatments: 0.5 U/ml for 10 min at 37°C
Chemical compound, drug(-) BlebbistatinSigmaB0560-1MG10 μM for 15 min at 37°C
Chemical compound, drugY-27632VWRCat#:10187-69410 μM for 30 min at 37°C
Chemical compound, drugNocodazoleSigmaCat#:M140410 μM for 20 min at 37°C
Chemical compound, drugEDTASigma-AldrichCat#:EDS-100G3 mM
Commercial assay or kitKAPA mRNA HyperPrep KitRocheCat#:7961901001
Commercial assay or kitClick-It EdU Kit 488InvitrogenCat#:C10337
Software, algorithmFijiLinkert et al., 2010 (PMID: 20513764); Schindelin et al., 2012 (PMID: 22743772)
Software, algorithmSTARDobin et al., 2013 (PMID: 23104886)
Software, algorithmHTSeq 2.0Putri et al., 2022 (PMID: 35561197)
Software, algorithmDESeq2Love et al., 2014 (PMID: 25516281)
Software, algorithmVisual Basic algorithm for tip trackingOtherDr Dan Buster
Software, algorithmPrism 9GraphPad
Software, algorithmFluoview FV31S-SWOlympus
Software, algorithmMetaMorphMetaMorph
OtherDAPISigmaCat#:10236276001DNA stain, 0.3 μM
OtherDRAQ7AbcamCat#:ab109202DNA stain, 1:1000
OtherxCELLigence Real-Time Cell AnalyzerAcea Biosciences/ Roche Applied ScienceEquipment to assess electrical resistance across cell monolayer
OtherIbidi pump systemIbidiCat#:10902Pump system to generate
laminar flow across cells

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