Data supporting the analysis of lymphatic endothelial cell junctions and shape

Data in support of: 

Dynamic cytoskeletal regulation of cell shape supports resilience of lymphatic endothelium

Hans Schoofs1#, Nina Daubel1#, Sarah Schnabellehner1, Max Grönloh2, Sebastián Palacios Martínez3, Aleksi Halme4, Amanda M. Marks1, Marie Jeansson1, Sara Barcos5, Cord Brakebusch6, Rui Benedito7, Britta Engelhardt5, Dietmar Vestweber8, Konstantin Gängel1, Fabian Linsenmeier9, Sebastian Schürmann9, Pipsa Saharinen4,10, Jaap D. van Buul2,3,11, Oliver Friedrich9, Richard S. Smith12, Mateusz Majda13, and Taija Mäkinen1,4,10*

1Uppsala University, Department of Immunology, Genetics and Pathology, Dag Hammarskjölds väg 20, 751 85 Uppsala, Sweden.

2Department of Medical Biochemistry at the Amsterdam UMC, location AMC, The Netherlands.

3Department of Molecular Cytology, Leeuwenhoek Centre for Advanced Microscopy at Swammerdam Institute for Life Sciences at the University of Amsterdam, The Netherlands.

4Translational Cancer Medicine Program and Department of Biochemistry and Developmental Biology, University of Helsinki, Haartmaninkatu 8, 00014 Helsinki, Finland.

5Theodor Kocher Institute, University of Bern, Bern, Switzerland.

6Biotech Research and Innovation Center, University of Copenhagen, Ole Maaløes Vej 5, 2200 Denmark.

7Centro Nacional de Investigaciones Cardiovasculares, Melchor Fernández Almagro 3, E-28029 Madrid, Spain.

8Max Planck Institute for Molecular Biomedicine, Münster, Germany.

9Institute of Medical Biotechnology, Department of Chemical and Biological Engineering, Friedrich-Alexander-University, Erlangen-Nürnberg, Paul-Gordan-Str.3, 91052 Erlangen, Germany.

10Wihuri Research Institute, Haartmaninkatu 8, 00290 Helsinki, Finland.

11Amsterdam UMC, Sanquin Research and Landsteiner Laboratory, The Netherlands.

12John Innes Centre, Norwich Research Park, Norwich NR4 7UH, UK.

13Department of Plant Molecular Biology, University of Lausanne, CH-1015 Lausanne, Switzerland.

#These authors contributed equally.

*Corresponding author: Taija Mäkinen, E-mail: [e-mail], [e-mail]

DATASET A: Annotated cell-cell junction types in lymphatic capillaries of wild type mouse ear skin at different ages __________________________________________________________________________________________________________

Contents

SOURCE DATA Fig1 FINAL. xlsx

3w

animal 1

animal 2

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sprouts

5w

animal 1

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animal 3

animal 4

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diaphragm

Overview of diaphragm and high mag. of different capillary ends

trachea

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animal 5

diaphragm

trachea

File legends

C1 images: inverted LYVE1 signal (.tif)C2 images: inverted VE-cadherin signal (.tif)MAX images: RGB merge of LYVE1 (cyan) and VE-cadherin (red) (.tif)"NAME".roi: Regions of interest (ROI) of annotated junctions can be imported in ImageJ

Methods

Junctional classification: Analysis of junction morphology was done on blunt-ended initial lymphatic capillaries in the segment between the intial tip and the first valve. Junction types were quantified in Z-stack projection by numbering of individual lobes of LYVE1 and VE-cadherin-stained LECs and subsequent categorizing of lobe-associated junctions based on VE-cadherin signal.

Four categories were defined:

1) Button junction – a punctate VE-cadherin+ deposit at the neck of LYVE1+ lobe/overlap, with no detectable VE-cadherin at the borders of the overlap,

2) Curvilinear junction – unsegmented(continous) or segmented (discontinuous) distribution of VE-cadherin within one border of LYVE1+ lobe/cellular overlap,

3) Double junction – unsegmented(continous) or segmented (discontinuous) distribution of VE-cadherin within both borders of LYVE1+ lobe/cellular overlap, and

4) LYVE1- curvilineair junction – unsegmented(continous) linear VE-cadherin distribution at cell-cell contacts in the absence of LYVE1.

Wild-type C57BL/6J mice were used for analysis of junction types, and 4-5 blunt ended vessels per mouse from five mice per age group and condition were analysed; in total 1785 junctions were annoted

Imaging:Confocal images were obtained using a Leica Stellaris 5 confocal microscope equipped with 405 nm and white light lasers, 63x/1.3 HC PL APO CORR CS2 Glycerol immersion objective, and Leica LAS X software. Images were aquired at 1.51 digital zoom using a 2048x2048 resolution

Tissue processing and staining:  Tissues were fixed in 4% paraformaldehyde for 2 h at RT and permeabilized in 0.3% Triton X-100 in PBS (PBST) for 10 min. After blocking in PBST with 2% bovine serum albumin, 1% FBS for 2 h, tissues were incubated with primary antibodies in blocking buffer overnight, followed by PBST washing and incubation with fluorescent dye-conjugated secondary antibodies for 2 h. All incubation steps were carried out at RT. Prior to mounting in Mowiol, samples were repeatedly washed in PBST and water. Antibodies used: Goat anti-mouse VE-cadherin (R&D Systems, AF1002; 1:200), Rat anti-mouse LYVE1 (R&D Systems, MAB2125; 1:200)

DATASET B: Finite element method (FEM) simulations of cellular stresses_______________________________________________________________

The FEM simulations were performed with MorphoMechanX using available models adapted from Sapala et al, eLife 7, e32794 (2018). A regular cylindrical grid 45 µm wide and 200 µm long was created and outlines from the cells of a lymphatic vessel were projected onto it and smoothed. These cells were then extruded inward to make 3D volumetric cells with a depth of 2 µm and triangulated using a threshold area of 4 µm.  The template was then used as the reference configuration for triangular 3 node membrane elements which were given a thickness of 0.1um. An isotropic St. Venant material model (linear, large deformation) was used with the Young's modulus set to 100 kPa to match a 10 kPa cell level Young's modulus estimated from the literature (ignoring the cell ends, the 2 x 0.1 µm membrane thickness occupied roughly 1/10th the cross-sectional area of the cell that were 2 µm deep). A uniform internal pressure was applied normal to the inside faces of the elements, which cancels out on the shared walls between cells. For simulations with a lower pressure inside the vessel, the inside faces were assigned a higher pressure. Stresses were visualized as the trace of the stress tensor.