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First published online 18 October 2005
doi: 10.1242/jcs.02631

Journal of Cell Science 118, 5109-5118 (2005)
Published by The Company of Biologists 2005
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Paracellin-1 and the modulation of ion selectivity of tight junctions

Jianghui Hou1, David L. Paul2 and Daniel A. Goodenough1,*

1 Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA
2 Department of Neurobiology, Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115, USA



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  		Fig. 1. Translational start site of paracellin-1. (A) The structure of paracellin-1. (B) Comparison of amino acid sequence of paracellin-1 across the species of mouse, rat and human. Note that the human sequence possesses two in-frame methionines with the second methionine highly conserved throughout the species. (C) A series of retroviral constructs for expression of the paracellin-1 gene. (D) Western immunoblots of MDCK cells infected with retrovirus expressing the constructs in C. Note that both methionines (M1 and M71) in human paracellin-1 initiate translation, suggestive of an internal ribosomal entry site (IRES) downstream of the ATG (encoding M1) in the mRNA transcripts. Positions of molecular mass markers in kDa are indicated. (E) Top panels, paracellin-1 subcellular localization. Note that the full-length paracellin-1 is mis-targeted to lysosomes (arrowheads). In contrast, paracellin-1 {Delta}70 is localized at cell-cell junctions (arrows). Bottom panels, paracellin-1 {Delta}70 colocalizes with occludin at tight junctions. Red, occludin staining; green, paracellin-1 staining; yellow to orange, colocalization of occludin and paracellin-1 in the merged panel. Bar, 10 µm.

 


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  		Fig. 2. Expression and localization of paracellin-1. (A) Constitutive expression of paracellin-1 in MDCK-II and LLC-PK1 cells. Paracellin-1 migrates as a 27 kDa band (*). (B) Confocal microscopy reveals the colocalization of paracellin-1 with occludin at the tight junction. Red, occludin staining; green, paracellin-1 staining; yellow to orange, colocalization of occludin and paracellin-1 on the merged panel. Bar, 10 µm.

 


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  		Fig. 3. Function of paracellin-1. (A) Effects of paracellin-1 on the permeability of Na+ and Cl- in LLC-PK1 cells. (B) Ratio of PNa to PCl and diffusion potential (bottom) across a LLC-PK1 cell monolayer. (C) TER across an LLC-PK1 cell monolayer over a period of 12 days in cells expressing paracellin-1 and control cells. (D) Summary of the effects of paracellin-1 upon permeability of various cations in LLC-PK1 cells.

 


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  		Fig. 4. Expression of paracellin-1 mutants. (A) Amino acid sequence of the first extracellular loop of paracellin-1. Negatively charged amino acids are labeled in bold and underlined; positively charged amino acids are in bold italics. (B-D) Protein immunoblots of expression of paracellin-1 mutants. (B) Mutations to replace the negatively charged amino acids (D or E) with S or T. Names of mutants are shown underneath the blot, followed by the positions of mutations. (C) Mutations to replace the positively charged amino acids (K or R) with S or T. (D) Mutations found in human patients with FHHNC. (E) Gallery of epifluorescence images showing mis-targeted localization of paracellin-1 mutants in LLC-PK1 cells. D97S, R149T, R149L and S235P in the ER; G239R in the Golgi apparatus. The ER shows a reticular cytoplasmic and perinuclear distribution and the Golgi apparatus tubular structures close to the periphery of the nucleus. Bar, 10 µm.

 


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  		Fig. 5. Structural requirements of paracellin-1 function. (A) Mutations in paracellin-1. Yellow dots, negatively charged amino acids; red dots, mutations found in patients having FHHNC. (B) and (D) Effects of paracellin-1 and its mutants upon dilution potential across LLC-PK1 cell monolayers. (C and E) Effects of paracellin-1 and its mutants upon the ratio of PNa to PCl (correlated to the values of diffusion potential in B and D respectively). *P<0.001 and **P<0.01 compared to ratio in the WT.

 

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© The Company of Biologists Ltd 2005