DFG Research Training Group "TJ-Train" (GRK 2318)
For many years there had been a dispute regarding the contribution and even existence of paracellular water transport. A major breakthrough was the finding, that tight junction protein claudin-2 does not only form a channel for cations (13) but that this channel also conducts water (9, 6). However, the ion permeability of other claudins is not necessarily coupled to water permeability (1): the cation channel claudin-10b (9) and the anion channel claudin-17 (8) proved to be not water permeable.
A highly specialized and thus most interesting part ot the overall TJ is formed by the tricellular TJ, the site where three cells meet (independent review: 14). Its major components are tricellulin, a TJ protein belonging to the TAMP family, and angulin-1 (LSR) and -2 (ILDR1). Tricellulin seals the tricellular TJ against medium-sized and large molecules (10). The angulins play a regulative role and act as precursors in tTJ formation. Most exciting, in pilot experiments we found that overexpression of tricellulin in epithelial cells with low endogenous tricellulin content seals against paracellular water flux. This would be of clinical importance, as tricellulin is downregulated in ulcerative colitis (4).
Hypothesis (a) In cell lines and tissues featuring high tricellulin expression, the tricellular TJ is impermeable to water and vice versa. (b) Downregulation of tricellulin is found under pathologic conditions and possibly increases water permeability of the intestinal epithelia and thus contributes to leak-flux diarrhea.
Aim of the doctoral project is to clarify the contribution of tricellulin and angulin-1 and -2 to epithelial water permeability which may lead to new insights concerning overall transport characteristics in different tissues. With these findings we want to resolve pathological mechanisms associated with increased epithelial water permeability due to changed expression of tricellulin. Our studies will be performed in collaboration with project C2 (Krug & Fromm).
Methods For this, tricellulin overexpression and knock-down clones will be generated using cell lines with low or high endogenous tricellulin expression, respectively. Moreover, specific stimulations will be used to modulate tricellulin abundance. Angulins will be altered accordingly or by applying angubindin-1 (3). Standard molecular biological techniques and confocal microscopy as well as two-path impedance spectroscopy will be applied to analyze expression and localization of transfected/regulated TJ proteins and the effect on ion conductance. Water transport will be measured in clones with unchanged expression of other transport-relevant proteins.
In brief, the specific aim of this thesis project for a PhD student is to clarify the role of tricellulin in transepithelial water permeability.
Suggested reading: See Project-related publications, especially Refs. 1, 3, 6, 9, and 14
PhD doctoral student
Participation with project A3
Krug SM, Hayaishi T, Iguchi D, Watari A, Takahashi A, Fromm M, Nagahama M, Takeda H, Okada Y, Sawasaki T, Doi T, Yagi K, Kondoh M (2017) Angubindin-1, a novel paracellular absorption enhancer acting at the tricellular tight junction. J. Contr. Release 260: 1-11 [PubMed] [WebPage] [PDF] [Supplement]
Krug SM, Bojarski C, Fromm A, Lee IM, Dames P, Richter JF, Turner JR, Fromm M*, Schulzke JD* (*shared last authorship) (2017) Tricellulin is regulated via interleukin-13-receptor α2, affects macromolecule uptake, and is decreased in ulcerative colitis. Mucosal Immunol. ###: ###-### [PubMed] [WebPage] [PDF] [Supplement] [Supplementary Movie]
Conrad MP*, Piontek J* (*shared first authorship), Günzel D, Fromm M, Krug SM (2016) Molecular basis of claudin-17 anion selectivity. Cell. Mol. Life Sci. 73(1): 185-200 [PubMed] [WebPage] [PDF] [Supplement]
Krug SM, Amasheh M, Dittmann I, Christoffel I, Fromm M, Amasheh S (2013) Sodium caprate as an enhancer of macromolecule permeation across tricellular tight junctions of intestinal cells. Biomaterials 34(1): 275-282 [PubMed] [WebPage] [PDF]
Krug SM, Günzel D, Conrad MP, Rosenthal R, Fromm A, Amasheh S, Schulzke JD, Fromm M (2012) Claudin-17 forms tight junction channels with distinct anion selectivity. Cell. Mol. Life Sci. 69(16): 2765-2778 [PubMed] [WebPage] [PDF] [Supplement]
Rosenthal R, Milatz S, Krug SM, Oelrich B, Schulzke JD, Amasheh S, Günzel D, Fromm M (2010) Claudin-2, a component of the tight junction, forms a paracellular water channel. J. Cell Sci. 123(11): 1913-1921 [PubMed] [WebPage] [PDF] [Supplement]
Krug SM, Amasheh S, Richter JF, Milatz S, Günzel D, Westphal JK, Huber O, Schulzke JD, Fromm M (2009) Tricellulin forms a barrier to macromolecules in tricellular tight junctions without affecting ion permeability. Mol. Biol. Cell 20: 3713-3724 [PubMed] [WebPage] [PDF] [Supplement text] [Supplement video]
Krug SM, Fromm M, Günzel D (2009) Two-path impedance spectroscopy for measuring paracellular and transcellular epithelial resistance. Biophys. J. 97(8): 2202-2211 [PubMed] [WebPage] [PDF] [Supplement]
Yu ASL, Cheng MH, Angelow S, Günzel D, Kanzawa SA, Schneeberger EE, Fromm M, Coalson RD (2009) Molecular basis for cation selectivity in claudin-2-based paracellular pores: Identification of an electrostatic interaction site. J. Gen. Physiol. 133(1): 111-127 [PubMed] [WebPage] [PDF]
Amasheh S, Meiri N, Gitter AH, Schöneberg T, Mankertz J, Schulzke JD, Fromm M (2002) Claudin-2 expression induces cation-selective channels in tight junctions of epithelial cells. J. Cell Sci. 115(24): 4969-4976 [PubMed] [WebPage] [PDF]