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The CellBorderTracker, a novel tool to quantitatively analyze spatiotemporal endothelial junction dynamics at the subcellular level

Abstract

Endothelial junctions are dynamic structures organized by multi-protein complexes that control monolayer integrity, homeostasis, inflammation, cell migration and angiogenesis. Newly developed methods for both the genetic manipulation of endothelium and microscopy permit time-lapse recordings of fluorescent proteins over long periods of time. Quantitative data analyses require automated methods. We developed a software package, the CellBorderTracker, allowing quantitative analysis of fluorescent-tagged cell junction protein dynamics in time-lapse sequences. The CellBorderTracker consists of the CellBorderExtractor that segments cells and identifies cell boundaries and mapping tools for data extraction. The tool is illustrated by analyzing fluorescent-tagged VE-cadherin the backbone of adherence junctions in endothelium. VE-cadherin displays high dynamics that is forced by junction-associated intermittent lamellipodia (JAIL) that are actin driven and WASP/ARP2/3 complex controlled. The manual segmentation and the automatic one agree to 90 %, a value that indicates high reliability. Based on segmentations, different maps were generated allowing more detailed data extraction. This includes the quantification of protein distribution pattern, the generation of regions of interest, junction displacements, cell shape changes, migration velocities and the visualization of junction dynamics over many hours. Furthermore, we demonstrate an advanced kymograph, the J-kymograph that steadily follows irregular cell junction dynamics in time-lapse sequences for individual junctions at the subcellular level. By using the CellBorderTracker, we demonstrate that VE-cadherin dynamics is quickly arrested upon thrombin stimulation, a phenomenon that was largely due to transient inhibition of JAIL and display a very heterogeneous subcellular and divers VE-cadherin dynamics during intercellular gap formation and resealing.

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Abbreviations

CBT:

CellBorderTracker

CBE:

CellBorderExtractor

CM:

CellMapper

ID-maps:

Identification maps

HUVEC:

Human umbilical vein endothelial cell

Junc-ID:

Junction identification number

Cell-ID:

Cell identification number

Border-ID:

Border identification number

EGFP:

Enhanced green fluorescence protein

ARP2/3:

Actin-related protein complex 2/3

p20:

Protein 20; a subunit of the ARP 2/3 complex

VE-cadherin:

Vascular endothelial cadherin

E-cadherin:

Epithelial cadherin

FRAP:

Fluorescence recovery after photobleaching

ROI:

Region of interest

References

  1. Ayollo DV, Zhitnyak IY, Vasiliev JM, Gloushankova NA (2009) Rearrangements of the actin cytoskeleton and E-cadherin-based adherens junctions caused by neoplasic transformation change cell-cell interactions. PLoS One 4:e8027

  2. Barrett WA, Mortensen EN (1996) Fast, accurate, and reproducible live-wire boundary extraction. Vis Biomed Comput 1131:183–192

  3. Barrett WA, Mortensen EN (1997) Interactive live-wire boundary extraction. Med Image Anal 1:331–341

  4. Baum B, Georgiou M (2011) Dynamics of adherens junctions in epithelial establishment, maintenance, and remodeling. J Cell Biol 192:907–917

  5. Bertocchi C, Vaman Rao M, Zaidel-Bar R (2012) Regulation of adherens junction dynamics by phosphorylation switches. J Signal Transduct 2012:125295

  6. Coutu DL, Schroeder T (2013) Probing cellular processes by long-term live imaging–historic problems and current solutions. J Cell Sci 126:3805–3815

  7. Curry FR, Adamson RH (2010) Vascular permeability modulation at the cell, microvessel, or whole organ level: towards closing gaps in our knowledge. Cardiovasc Res 87:218–229

  8. de Beco S, Gueudry C, Amblard F, Coscoy S (2009) Endocytosis is required for E-cadherin redistribution at mature adherens junctions. Proc Natl Acad Sci USA 106:7010–7015

  9. Dejana E (2004) Endothelial cell–cell junctions: happy together. Nat Rev Mol Cell Biol 5:261–270

  10. Dejana E, Tournier-Lasserve E, Weinstein BM (2009) The control of vascular integrity by endothelial cell junctions: molecular basis and pathological implications. Dev Cell 16:209–221

  11. Farber M, Ehrhardt J, Handels H (2007) Live-wire-based segmentation using similarities between corresponding image structures. Comput Med Imaging Graph 31:549–560

  12. Friedl P, Gilmour D (2009) Collective cell migration in morphogenesis, regeneration and cancer. Nat Rev Mol Cell Biol 10:445–457

  13. Frigault MM, Lacoste J, Swift JL, Brown CM (2009) Live-cell microscopy—tips and tools. J Cell Sci 122:753–767

  14. Garcia JG (2009) Concepts in microvascular endothelial barrier regulation in health and disease. Microvasc Res 77:1–3

  15. Gavard J, Gutkind JS (2006) VEGF controls endothelial-cell permeability by promoting the beta-arrestin-dependent endocytosis of VE-cadherin. Nat Cell Biol 8:1223–1234

  16. Geyer H, Geyer R, Odenthal-Schnittler M, Schnittler HJ (1999) Characterization of human vascular endothelial cadherin glycans. Glycobiology 9:915–925

  17. Harris ES, Nelson WJ (2010) VE-cadherin: at the front, center, and sides of endothelial cell organization and function. Curr Opin Cell Biol 22:651–658

  18. Huveneers S, Oldenburg J, Spanjaard E, van der Krogt G, Grigoriev I, Akhmanova A, Rehmann H, de Rooij J (2012) Vinculin associates with endothelial VE-cadherin junctions to control force-dependent remodeling. J Cell Biol 196:641–652

  19. Ishikawa-Ankerhold HC, Ankerhold R, Drummen GP (2012) Advanced fluorescence microscopy techniques—FRAP, FLIP, FLAP, FRET and FLIM. Molecules 17:4047–4132

  20. Kametani Y, Takeichi M (2007) Basal-to-apical cadherin flow at cell junctions. Nat Cell Biol 9:92–98

  21. Komarova Y, Malik AB (2010) Regulation of endothelial permeability via paracellular and transcellular transport pathways. Annu Rev Physiol 72:463–493

  22. Kowalczyk AP, Reynolds AB (2004) Protecting your tail: regulation of cadherin degradation by p120-catenin. Curr Opin Cell Biol 16:522–527

  23. Kronstein R, Seebach J, Grossklaus S, Minten C, Engelhardt B, Drab M, Liebner S, Arsenijevic Y, Taha AA, Afanasieva T, Schnittler HJ (2012) Caveolin-1 opens endothelial cell junctions by targeting catenins. Cardiovasc Res 93:130–140

  24. Lampugnani MG, Resnati M, Raiteri M, Pigott R, Pisacane A, Houen G, Ruco LP, Dejana E (1992) A novel endothelial-specific membrane protein is a marker of cell–cell contacts. J Cell Biol 118:1511–1522

  25. Lampugnani MG, Corada M, Caveda L, Breviario F, Ayalon O, Geiger B, Dejana E (1995) The molecular organization of endothelial cell to cell junctions: differential association of plakoglobin, beta-catenin, and alpha-catenin with vascular endothelial cadherin (VE-cadherin). J Cell Biol 129:203–217

  26. Laugsch M, Seebach J, Schnittler H, Jessberger R (2013) Imbalance of SMC1 and SMC3 cohesins causes specific and distinct effects. PLoS One 8:e65149

  27. Levayer R, Lecuit T (2013) Oscillation and polarity of E-cadherin asymmetries control actomyosin flow patterns during morphogenesis. Dev Cell 26:162–175

  28. Lindemann D, Schnittler H (2009) Genetic manipulation of endothelial cells by viral vectors. Thromb Haemost 102:1135–1143

  29. Mashburn DN, Lynch HE, Ma X, Hutson MS (2012) Enabling user-guided segmentation and tracking of surface-labeled cells in time-lapse image sets of living tissues. Cytometry Part A 81:409–418

  30. Mishra A, Wong A, Zhang W, Clausi D, Fieguth P (2008) Improved interactive medical image segmentation using Enhanced Intelligent Scissors (EIS). In: Conference proceedings: annual international conference of the IEEE engineering in medicine and biology society IEEE engineering in medicine and biology society conference 2008, pp. 3083–3086

  31. Mortensen EN, Barrett WA (1998) Interactive segmentation with intelligent scissors. Graph Models Image Process 60:349–384

  32. Pariente N, Mao SH, Morizono K, Chen IS (2008) Efficient targeted transduction of primary human endothelial cells with dual-targeted lentiviral vectors. J Gene Med 10:242–248

  33. Pereira AJ, Maiato H (2010) Improved kymography tools and its applications to mitosis. Methods 51:214–219

  34. Rabiet MJ, Plantier JL, Rival Y, Genoux Y, Lampugnani MG, Dejana E (1996) Thrombin-induced increase in endothelial permeability is associated with changes in cell-to-cell junction organization. Atertioscler Thromb Vasc Biol 16:488–496

  35. Schroeder T (2011) Long-term single-cell imaging of mammalian stem cells. Nat Methods 8:S30–S35

  36. Seebach J, Madler HJ, Wojciak-Stothard B, Schnittler HJ (2005) Tyrosine phosphorylation and the small GTPase rac cross-talk in regulation of endothelial barrier function. Thromb Haemost 94:620–629

  37. Seebach J, Donnert G, Kronstein R, Werth S, Wojciak-Stothard B, Falzarano D, Mrowietz C, Hell SW, Schnittler HJ (2007) Regulation of endothelial barrier function during flow-induced conversion to an arterial phenotype. Cardiovasc Res 75:596–607

  38. Smith MB, Karatekin E, Gohlke A, Mizuno H, Watanabe N, Vavylonis D (2011) Interactive, computer-assisted tracking of speckle trajectories in fluorescence microscopy: application to actin polymerization and membrane fusion. Biophys J 101:1794–1804

  39. Suzuki S, Sano K, Tanihara H (1991) Diversity of the cadherin family: evidence for eight new cadherins in nervous tissue. Cell Regul 2:261–270

  40. Szulcek R, Beckers CM, Hodzic J, de Wit J, Chen Z, Grob T, Musters RJ, Minshall RD, van Hinsbergh VW, van Nieuw Amerongen GP (2013) Localized RhoA GTPase activity regulates dynamics of endothelial monolayer integrity. Cardiovasc Res 99:471–482

  41. Taha AA, Taha M, Seebach J, Schnittler HJ (2014) ARP2/3-mediated junction-associated lamellipodia control VE-cadherin-based cell junction dynamics and maintain monolayer integrity. Mol Biol Cell 25:245–256

  42. Truong Quang BA, Mani M, Markova O, Lecuit T, Lenne PF (2013) Principles of E-cadherin supramolecular organization in vivo. Curr Biol 23:2197–2207

  43. Vandenbroucke E, Mehta D, Minshall R, Malik AB (2008) Regulation of endothelial junctional permeability. Ann N Y Acad Sci 1123:134–145

  44. Vestweber D (2000) Molecular mechanisms that control endothelial cell contacts. J Pathol 190:281–291

  45. Vestweber D, Winderlich M, Cagna G, Nottebaum AF (2009) Cell adhesion dynamics at endothelial junctions: VE-cadherin as a major player. Trends Cell Biol 19:8–15

  46. Vincent L (1991) Exact Euclidean distance function by chain propagations. In: IEEE computer society conference on computer vision and pattern recognition, 1991 proceedings CVPR ‘91, pp. 520–525

  47. Vincent L, Soille P (1991) Watersheds in digital spaces—an efficient algorithm based on immersion simulations. IEEE Trans Pattern Anal Mach Intell 13:583–598

  48. Wieclawek W, Pietka E (2012) Fuzzy clustering in intelligent scissors. Comput Med Imaging Graph 36:396–409

  49. Wojciak-Stothard B, Entwistle A, Garg R, Ridley AJ (1998) Regulation of TNF-alpha-induced reorganization of the actin cytoskeleton and cell-cell junctions by Rho, Rac, and Cdc42 in human endothelial cells. J Cell Physiol 176:150–165

  50. Xiao K, Allison DF, Kottke MD, Summers S, Sorescu GP, Faundez V, Kowalczyk AP (2003) Mechanisms of VE-cadherin processing and degradation in microvascular endothelial cells. J Biol Chem 278:19199–19208

  51. Yamada S, Nelson WJ (2007) Localized zones of Rho and Rac activities drive initiation and expansion of epithelial cell-cell adhesion. J Cell Biol 178:517–527

  52. Zobel T, Brinkmann K, Koch N, Schneider K, Seemann E, Fleige A, Qualmann B, Kessels MM, Bogdan S (2014) Cooperative functions of the two F-BAR proteins Cip4 and Nostrin in regulating E-cadherin in epithelial morphogenesis. J Cell Sci 128:499–515

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Acknowledgments

We gratefully thank Masatoshi Takeichi for providing the VE-cadherin–EGFP adenovirus vector. We further thank Martin Muermann for editing the manuscript. The work was supported by the Deutsche Forschungs-Gemeinschaft, DFG, INST 2105/24-1 and SCHN 430/6-2 to HS and from the cluster of excellence ‘Cells in Motion.’

Authors contributions

J.S. acquired the time-lapse movie using VE-cadherin–EGFP, designed and implemented the algorithms, analyzed the data made the figures and animations under supervision of H.S. A.A.T. acquired the time-lapse movies of VE-cadherin–mCherry and EGFP-p20 in HUVEC under supervision of H.S.. J.L. did the FRAP experiments and analyzed them together with JS. X.J. contributed with algorithm discussion. N.L. acquired the VE-cadherin immunofluorescence images. S.B. and K.B. provided the time-lapse sequence of fruit fly embryo. H.S. raised the topic, supervised the entire work and wrote together with J.S. the MS.

Author information

Correspondence to Hans-Joachim Schnittler.

Ethics declarations

Conflict of interest

The authors declare that they have no competing interests.

Additional information

For questions to program applications: Jochen Seebach.

Electronic supplementary material

Below is the link to the electronic supplementary material.

The tutorial explains the interactive segmentation of a subconfluent cell layer expressing VE-cadherin-EGFP by the CBE (MP4 3135 kb)

The time-lapse movie shows a movie of a confluent HUVEC layer expressing VE-cadherin-mCherry (left) with the segmentation generated by the CBE (right, white lines) (MP4 5050 kb)

Online resource 1: Algorithm CBE (.pdf)

Description of the CBE algorithm. In particular the used cost function and the automated generation of appropriate seeding is explained in detail (PDF 864 kb)

Online resource 2: Movie tutorial (.mp4)

The tutorial explains the interactive segmentation of a subconfluent cell layer expressing VE-cadherin-EGFP by the CBE (MP4 3135 kb)

Online resource 3: J-kymograph (.pdf)

Illustration of the generation of a junctional kymograph from a fluorescent image and a ID-map stack (PDF 775 kb)

Online resource 4: Movie of confluent cells (.mp4)

The time-lapse movie shows a movie of a confluent HUVEC layer expressing VE-cadherin-mCherry (left) with the segmentation generated by the CBE (right, white lines) (MP4 5050 kb)

Online resource 5: Movie thrombin (.mp4)

Time-lapse movie of a HUVEC layer expressing VE-cadherin-mCherry (red) and EGFP-p20 (green) stimulated with thrombin (2U/ml) (MP4 16270 kb)

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Seebach, J., Taha, A.A., Lenk, J. et al. The CellBorderTracker, a novel tool to quantitatively analyze spatiotemporal endothelial junction dynamics at the subcellular level. Histochem Cell Biol 144, 517–532 (2015). https://doi.org/10.1007/s00418-015-1357-8

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Keywords

  • Endothelial junction dynamics
  • Image analysis
  • Fluorescence live cell imaging
  • VE-cadherin
  • ARP2/3 complex
  • Inflammation