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Elucidating the principles of the molecular organization of heteropolymeric tight junction strands

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Abstract

Paracellular barrier properties of tissues are mainly determined by the composition of claudin heteropolymers. To analyze the molecular organization of tight junctions (TJ), we investigated the ability of claudins (Cld) to form homo- and heteromers. Cld1, -2, -3, -5, and -12 expressed in cerebral barriers were investigated. TJ-strands were reconstituted by claudin-transfection of HEK293-cells. cis-Interactions and/or spatial proximity were analyzed by fluorescence resonance energy transfer inside and outside of strands and ranked: Cld5/Cld5 > Cld5/Cld1 > Cld3/Cld1 > Cld3/Cld3 > Cld3/Cld5, no Cld3/Cld2. Classic Cld1, -3, and -5 but not non-classic Cld12 showed homophilic trans-interaction. Freeze-fracture electron microscopy revealed that, in contrast to classic claudins, YFP-tagged Cld12 does not form homopolymers. Heterophilic trans-interactions were analyzed in cocultures of differently monotransfected cells. trans-Interaction of Cld3/Cld5 was less pronounced than that of Cld3/Cld1, Cld5/Cld1, Cld5/Cld5 or Cld3/Cld3. The barrier function of reconstituted TJ-strands was demonstrated by a novel imaging assay. A model of the molecular organization of TJ was generated.

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Acknowledgments

We gratefully acknowledge the help of Ria Knittel in freeze-fracturing. This work was funded by DFG BL308/7-3, 7-4 and PI 837/2-1 and NIH DK61931.

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Authors and Affiliations

  1. Leibniz-Institut für Molekulare Pharmakologie, Robert-Rössle-Str. 10, 13125, Berlin, Germany

    Jörg Piontek, Susanne Fritzsche, Jimmi Cording, Sandra Richter, Maria Walter, Claudia Gehring & Ingolf E. Blasig

  2. Max-Delbrück-Center for Molecular Medicine, Berlin, Germany

    Jens Hartwig & Hans-Peter Rahn

  3. Department of Pathology, The University of Chicago, Chicago, IL, 60637, USA

    Dan Yu & Jerrold R. Turner

  4. Institute of Pathology, University Hospital Tübingen, Tübingen, Germany

    Hartwig Wolburg

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  1. Jörg Piontek
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Correspondence to Jörg Piontek.

Electronic supplementary material

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18_2011_680_MOESM1_ESM.pdf

Supplementary material 1 The FRET-efficiency is strongly dependent on the ratio of acceptor/donor (YFP/CFP) fluorescence intensity (A, R2 = 0.51) but not on the YFP fluorescence intensity (B, R2 = 0.02) or the CFP+YFP fluorescence intensity (C, R2 = 0.02) as a measure of the expression level. Individual data points and non-linear regression curve for Cld5-CFP/Cld5-YFP. (PDF 45 kb)

18_2011_680_MOESM2_ESM.pdf

Supplementary material 2 (A) Flow cytometric expression analysis of HEK cells transfected with different constructs. Non- (mock), CFP- or YFP-transfection were used to account for auto fluorescence. CFP/YFP cotransfection and CFP-YFP tandem were used as FRET negative or positive control, respectively. Cdl5-CFP/CRFR1-YFP and Cld5-CFP/Cld5-YFP were used as pairs of transmembrane proteins shown to be FRET-negative or -positive, respectively, by conventional method (Piontek et al., 2008). On the axes the channels are given. FSC, forward scatter. (B) Visualization of FRET signal by comparison of CFP/YFP - FRET negative -, CFP-YFP tandem - FRET positive - (left panel), Cld5-CFP/CRFR1-YFP - FRET negative - and Cld5-CFP/Cld5-YFP - FRET positive - (right panel). FRET-signal is indicated by left shift. (C) Normalization of FRET and YFP values relative to CFP intensity with the corresponding exponential decay fits (left: CFP/YFP (black dots, green line) and CFP-YFP (red dots, blue line); right: Cld5-CFP/CRFR1-YFP (black dots, green line) and Cld5-CFP/Cld5-YFP (red dots, blue line). The fits were used to calculate the FRET-ratioFC as a measure of FRET- efficiency (see Methods). (PDF 455 kb)

18_2011_680_MOESM3_ESM.pdf

Supplementary material 3 FRET-analysis in intracellular compartments by confocal microscopy. For Cld5-CFP/Cld5-YFP, FRET was significant higher than for Cld3-CFP/Cld3-YFP for which FRET was still higher than for the negative control (CFP-tagged endoplasmic reticulum marker, CFP-ER (BD Biosciences) coexpressed with Cld5-YFP, CFP-ER/Cld5-YFP). 36, 34, and 19 cell–cell contacts were analyzed, respectively; p < 0.001. (PDF 35 kb)

18_2011_680_MOESM4_ESM.pdf

Supplementary material 4 In intracellular compartments claudins did not considerably colocalize with markers for endosomes, Golgi apparatus or lysosomes but partly with that for the endoplasmic reticulum (A, arrow). HEK 293 cells were cotransfected with Cld5-YFP and (A) CFP-ER (marker for endoplasmic reticulum, Clontech), (B) CFP-Endo (marker for endosomes, Clontech) or (C, left) CFP-Golgi (marker for Golgi apparatus, Clontech) and 3 days later analyzed by confocal microscopy. In addition, cells were transfected with Cld5 (C, middle) or Cld3 (C, right) and 3 days later incubated with 50 nM LysoTracker (Invitrogen) in growth medium for 30 min at 37°C, the medium exchanged by DMEM with 10 mM N-(2-hydroxyethyl)piperazine-N’(2-ethanesulfonic acid) pH 7.5 without phenol red and analyzed by confocal microscopy. Bar, 5 µm. (PDF 105 kb)

18_2011_680_MOESM5_ESM.pdf

Supplementary material 5 CellMask labels the apical and basolateral plasma membrane; reconstituted TJs delay the labeling. HEK cells stably transfected with Cld5 were labeled with 0.25 μg/ml CellMask for 5 min (A, B) or 20 min (C, D). After 5 min, CellMask labeling (red) is detected in the apical (ap) and basal (bl) part of the lateral plasma membrane but not at the Cld5-positive (green) area at cell–cell contacts (A,B). In contrast, after 20 min, CellMask (red) colocalizes with claudin-5 (green) at cell–cell contacts (C, D). Merge (A, C); CellMalsk (B, D); arrow, Cld5-enrichment at cell–cell contact; arrowhead, apical and basolateral plasma membrane adjacent to claudin-5; bar, 5 µm. (PDF 272 kb)

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Piontek, J., Fritzsche, S., Cording, J. et al. Elucidating the principles of the molecular organization of heteropolymeric tight junction strands. Cell. Mol. Life Sci. 68, 3903–3918 (2011). https://doi.org/10.1007/s00018-011-0680-z

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