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


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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Identification maps


Human umbilical vein endothelial cell


Junction identification number


Cell identification number


Border identification number


Enhanced green fluorescence protein


Actin-related protein complex 2/3


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


Vascular endothelial cadherin


Epithelial cadherin


Fluorescence recovery after photobleaching


Region of interest


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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.

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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).

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