spacer gif spacer gif spacer gif spacer gif spacer gif
QUICK SEARCH:   [advanced]


spacer gif
     Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents    

First published online 15 January 2008
doi: 10.1242/jcs.021485

Journal of Cell Science 121, 298-305 (2008)
Published by The Company of Biologists 2008
This Article
Right arrow Summary Freely available
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Supplementary Material
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Related articles in JCS
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Van Itallie, C. M.
Right arrow Articles by Anderson, J. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Van Itallie, C. M.
Right arrow Articles by Anderson, J. M.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati   Add to Twitter  
What's this?

The density of small tight junction pores varies among cell types and is increased by expression of claudin-2

Christina M. Van Itallie1,*, Jennifer Holmes2, Arlene Bridges3, Jody L. Gookin4, Maria R. Coccaro4, William Proctor3, Oscar R. Colegio5 and James M. Anderson2

1 Department of Medicine, University of North Carolina, Chapel Hill, NC 27599, USA
2 Cell and Molecular Physiology, University of North Carolina, Chapel Hill, NC 27599, USA
3 School of Pharmacy, University of North Carolina, Chapel Hill, NC 27599, USA
4 Department of Molecular Biomedical Sciences, North Carolina State University, Raleigh, NC 27695, USA
5 Department of Dermatology, Yale University School of Medicine, New Haven, CT 06510, USA


Figure 1
View larger version (25K):
[in this window]
[in a new window]

  		Fig. 1. Profile of PEG oligomers after flux across a Caco-2 monolayer reveals a sharp size discrimination. A mixture of continuous PEG oligomers (dimer to 28mer) was added to the apical compartment of monolayers cultured on semipermeable filters. Aliquots were removed from the donor (A) and acceptor (B) compartments after 90 minutes, derivatized with 1-NIC and subjected to HPLC and detected by fluorescence emission. Each peak represents a single ethylene glycol adduct size, and retention times are proportional to molecular mass. The peak visible at 31 minutes is pure PEG28 added as an internal recovery standard after the flux protocol.

 

Figure 2
View larger version (24K):
[in this window]
[in a new window]

  		Fig. 2. Papp as a function of PEG radius in Caco-2, MDCK II and MDCK C7 monolayers and ex vivo pig ileum. All cell types show a size-restrictive pore calculated to be of radius ~4 Å. Ileum appears to have an additional pore cut-off at ~6.5 Å. In contrast to their similar size pore, the number of pores (reflected in the Papp<4 Å) is highly variable. Caco-2 cells have the largest numbers of pores as well as the greatest permeation through the size-independent pathway; MDCKII cells have an intermediate number and MDCK C7 cells have few pores and little permeation through the second pathway. T84 cells (data not shown) had a variable pore number, depending on the source of the cell line. The relative pore number in pig ileum cannot be compared with that of cell lines because of the difference in the amplified surface area in intact tissue compared with the flat cultured cell monolayers.

 

Figure 3
View larger version (68K):
[in this window]
[in a new window]

  		Fig. 3. Lack of correlation between intercellular junction length, pore number and TER. (A) Immunofluorescent localization of ZO-1 in cells grown on filters and used for TER was used to determine the length of tight junction contacts per monolayer area. Caco-2 cells are the smallest and thus have the most potential junction contact length to leak through. Bar, 12 µm. (B) Duplicate cell fields were photographed and measurements of perimeter lengths were determined using imaging software. The perimeter lengths differed by less than 30% among the three cell lines; Caco-2 monolayers contain the most and MDCK C7 the least. TER (C) was not necessarily inversely related to junction length (B) nor to pore number, as determined by Papp of the 3.5 Å PEG species (D).

 

Figure 4
View larger version (21K):
[in this window]
[in a new window]

  		Fig. 4. Comparison of claudin protein expression profiles in MDCK II and MDCK C7 cells. Comparative immunoblotting was performed to determine whether the difference in TER and pore number between MDCK II and C7 cell lines could result from different claudin expression profiles. Duplicate cell monolayer filters that were used for immunofluorescent analysis (Fig. 3) were processed for immunoblots with a panel of antibodies against claudins and other junction proteins to compare relative protein levels; quantification was performed using Licor Odyssey software. Levels in MDCK C7 cells are presented relative to MDCK II levels. Most claudins, ZO-1, occludin and E-cadherin are expressed at similar levels, whereas, in C7 cells, claudin-2 is much less prevalent and claudin-4 much more prevalent.

 

Figure 5
View larger version (35K):
[in this window]
[in a new window]

  		Fig. 5. Expression of claudin-2, but not claudin-4, in MDCK monolayers increases the number of pores. (A) Representative immunoblots from cells used for TER and Papp studies demonstrate at least fourfold higher levels of claudin after induction (i) compared with those in uninduced (u) cells; claudin-2 expression in knockdown (KD) cells was approximately 2-10% of control cell values; occludin (occ) levels are unchanged. Approximate molecular masses: occludin, 60 kDa; claudin-2, 22 kDa, claudin-7, 20 kDa; claudin-4, 17 kDa. (B) Induction (filled bars) of claudin-2 compared with uninduced (unfilled bars) MDCK II monolayers results in a small increase in TER, and a large drop in C7 cells. Induction (filled bars) of claudin-4 in MDCK II monolayers causes a twofold increase in TER relative to uninduced (unfilled bars) monolayers; knockdown of claudin-2 results in a threefold increase in TER (filled bars) over parental cell values (unfilled bars). (C) Induction of claudin-2 (unfilled circles) in MDCK II monolayers results in a significant increase in the Papp specifically for PEG sizes that are of radius <4 Å, compared with uninduced monolayers (filled circles). Means and s.e. of four separate clones. (D) Corrected Papp of the 3.5 Å PEG5 species reveals increases in pore number after inducing claudin-2 (filled bars) relative to uninduced (unfilled bars) in both MDCK II and MDCK C7 cells, but no change in MDCK II cells expressing claudin-4 or knockdown cells with decreased claudin-2 expression (means and s.e. from at least four determinations). (E) [3H]-Mannitol flux [disintegrations per minute (DPM) versus time] is unaffected by induction of claudin-2 (unfilled circles) compared with uninduced (filled circles) MDCK II monolayers.

 

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati   Add to Twitter Twitter    What's this?



© The Company of Biologists Ltd 2008