First published online 20 May 2003
doi: 10.1242/jcs.00482
The type 3 inositol 1,4,5-trisphosphate receptor is concentrated at the tight junction level in polarized MDCK cells
Pascal Colosetti*,
,1,
Richard E. A. Tunwell,2,
Caroline Cruttwell1,
Jean-Pierre Arsanto3,
Jean-Pierre Mauger1,
and
Doris Cassio1
1 INSERM U-442, Signalisation cellulaire et calcium, Bât 443,
Université Paris-Sud, 91405 Orsay Cedex, France
2 Department of Anatomy & Developmental Biology, University College London,
Gower Street Campus, London WC1E 6BT, UK
3 Laboratoire de Neurogenése et Morphogenése dans le
Développement et chez l'Adulte (NMDA, Unité Mixte de Recherche
6156), Institut de Biologie du Développement de Marseille,
Faculté des Sciences de Luminy, Université de la
Méditerranée, case 907, 13288 Marseille Cedex 09, France
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Fig. 1. Expression and localization of InsP3R-3 during the polarization
of MDCK cells. MDCK cells, plated at 104 cells/cm2 were
cultured over a period of 12 days. (A) Western blot: 50 µg of protein of
cell lysates, corresponding to 1.2-1.4x105 cells from
cultures of 3, 6 and 10 days, were loaded on each lane; the last lane was
loaded, as a positive control, with a sample of HeLa cell lysate. Proteins
were subjected to 5% (w/v) SDS-PAGE and the western blotting was carried out
using a primary antibody directed against the type 3 InsP3R. (B)
TER. (C) Localization of InsP3R-3 and proteins of the endoplasmic
reticulum (ER) in MDCK cells cultured for 3, 6 and 10 days; in each case the
corresponding phase contrast images are shown on the left. (D) Localization of
another marker of the endoplasmic reticulum, the protein PDI, in cultures of 3
and 10 days, with the corresponding phase contrast image on the left. Bar, 10
µm.
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Fig. 7. Analysis of stable transfectant MDCK cells expressing
InsP3R-1-EGFP. (A) Western blot: 50 µg of protein of cell
lysates from parental MDCK, clone 10B and clone 1E were loaded on a gradient
4-10% polyacrylamide minigel (BioRad MiniPROTEAN system). The gradient minigel
allowed both the small molecular weight of the GFP and the high molecular
weight of the InsP3R to be visualized on the same blot. After
transfer, western blotting was performed using antibodies against
InsP3R-1, GFP and N-terminus InsP3R-3, respectively. (B)
Localization of InsP3R-3 and InsP3R-1-EGFP in MDCK clone
10B, which stably expressed InsP3R-1-EGFP. The tagged
InsP3R-1 is expressed by most cells of this clone, except a few
(arrows), whereas all cells express InsP3R-3. Bar, 10 µm.
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Fig. 2. Localization of InsP3R-3 in polarized MDCK cells using two
different specific antibodies: confocal analysis. MDCK plated at
104 cells/cm2 were cultured for 13 days and double
stained using a monoclonal antibody (A) and a polyclonal antibody (B) specific
for InsP3R-3. The images shown correspond to a complete compiled z
series of 30 xy sections taken in 0.2 µm steps. Note the patchy staining
along the plasma membrane with, in some cases, a double row of patches
(arrow). Bar, 10 µm.
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Fig. 3. Double staining of InsP3R-3 and polarity markers in polarized
MDCK cells: confocal analysis. MDCK plated at 104
cells/cm2 were cultured for 10-12 days and double stained using a
monoclonal antibody anti-InsP3R-3 and antibodies directed against
the tight junction associated protein ZO-1 (A), the tight junction protein
occludin (B), the lateral marker ErbB-2 (C) and the apical protein gp-114 (D).
The immunolocalization obtained for each protein is presented, in addition to
the merged image. In A and C, two xy sections taken at the apex and at the
base of the cell layer were shown with an xz section taken at the level
indicated by the arrow on the corresponding xy sections. In B and D, one
apical xy section was shown with an xz section taken at the level indicated by
the arrow on the corresponding xy section. The top and the bottom of the cell
layer shown in the xz sections are indicated by asterisks (*). In each case a
restricted distribution of InsP3R-3 was observed in the tight
junction zone, which corresponds to the frontier between the lateral and the
apical pole. This restricted distribution is particularly visible in the xy
and xz sections stained for InsP3R-3 and the lateral marker ErbB-2
shown in C. Bar, 10 µm.
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Fig. 4. Immunoelectron microscopy analysis. InsP3R-3 was immunostained
(6 nm gold particles, arrows) in ultrathin cryosections of polarized MDCK
cells. Panel A shows a section passing through the different junctions present
along the contact zone between two adjacent cells. The image shown in the
inset B is centered on the upper part of the cells, at the limit between the
apical pole and the lateral one. InsP3R-3 labeling was nearly
exclusively seen in the vicinity of the tight junction (TJ). AJ, adherens
junction; DS, desmosome; ap, apical surface; mv, microvilli. Bar, 100 nm.
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Fig. 5. InsP3R-3 in non-polarized ras transformed MDCK f3 cells. (A) f3
cells were plated at 104 cells/cm2 and examined for the
localization of InsP3R-3 and ZO-1 or E-cadherin after 3 and 10
days, respectively. (B) After 10 days in culture, cells were treated with the
MEK inhibitor U0126 (25 µM): TER was measured and cells were double stained
for InsP3R-3 and ZO-1 or InsP3R-3 and E-cadherin, at
regular intervals during this treatment. After 1 day with U0126, ZO-1 was
detected at the PM of most cells; E-cadherin was re-expressed in only a
fraction of treated cells and in a small minority of those cells
InsP3R-3 was present not only in the cytoplasm, but also at the
plasma membrane (*). Bar, 10 µm.
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Fig. 6. Changes in InsP3R-3 and ZO-1 localization in MDCK cells during
calcium switch. Polarized MDCK cells were incubated for 2 hours in medium
deprived of calcium and then incubated in a calcium-containing medium for 24
hours. (A) TER measurement during this treatment; the arrow indicates the time
when the cells were renewed with calcium-containing medium. (B)
InsP3R-3 and ZO-1 localization. Bar, 10 µm.
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Fig. 8. Confocal analysis of MDCK 10B cells expressing InsP3R-1-EGFP.
(A) Localization of InsP3R-1-EGFP and proteins of the endoplasmic
reticulum in non-polarized (3 days) and polarized (12 days) 10B cells. For the
3-day culture an xy section taken in the middle of the cell layer is
presented; note the more intense perinuclear staining of
InsP3R-1-EGFP, particularly visible in the merged image. For the
12-day culture a complete compiled z series of 27 xy sections taken in 0.3
µm steps is shown. (B) Localization of both types of InsP3-R in
polarized 10B cells. The staining obtained for each protein is presented in
addition to the merged images. The images shown correspond to a complete
compiled z series of 30 xy sections taken in 0.3 µm steps with an xz
section taken at the plane indicated by the arrow on the corresponding
compilation. (C) Localization of InsP3R-1-EGFP and ER proteins in
polarized 10B cells: an xz section of cells stained with a polyclonal antibody
directed against a bulk of ER proteins is shown. The staining for each protein
and the merge image are presented. InsP3R-1-EGFP is mostly present
at the upper part of the lateral pole, whereas ER proteins are widely
distributed around the nucleus, in the whole cytoplasm. (D) Localization of
InsP3R-1-EGFP and cadherin(s) in polarized 10B cells: an xz section
of cells stained with anti-pan cadherin is shown, the staining obtained for
each protein being presented in addition to the merged image. Note the
restricted distribution of InsP3-R-1-EGFP at the top of the lateral
pole. (E) Localization of InsP3R-1-EGFP and sec6 in
polarized 10B cells: an xz section is shown with the staining for each protein
and the merged image. (F) Localization of InsP3R-1-EGFP in living
polarized 10B cells. An xy section and an xz one taken through the cells at
the plane indicated by the arrow are presented. The top and the bottom of the
cell layer shown in all the xz sections are indicated by asterisks (*). Bars,
10 µm.
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© The Company of Biologists Ltd 2003
