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 August 17, 2004
doi: 10.1242/10.1242/jcs.01301

Journal of Cell Science 117, 4169-4177 (2004)
Published by The Company of Biologists 2004
This Article
Right arrow Summary Freely available
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Supplemental Data
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 de Pavert, S. A.
Right arrow Articles by Wijnholds, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by van de Pavert, S. A.
Right arrow Articles by Wijnholds, J.
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?

Crumbs homologue 1 is required for maintenance of photoreceptor cell polarization and adhesion during light exposure

Serge A. van de Pavert1,*, Albena Kantardzhieva1,*, Anna Malysheva1, Jan Meuleman1, Inge Versteeg1, Christiaan Levelt1, Jan Klooster1, Sylvia Geiger2, Mathias W. Seeliger2, Penny Rashbass3, Andre Le Bivic4 and Jan Wijnholds1,{ddagger}

1 The Netherlands Ophthalmic Research Institute, Royal Netherlands Academy of Arts and Sciences, Meibergdreef 47, 1105 BA Amsterdam, The Netherlands
2 Retinal Electrodiagnostics Research Group, Department of Ophthalmology, University of Tübingen, Schleichstr. 12-16, 72076 Tübingen, Germany
3 Centre for Developmental Genetics, Department of Biomedical Science, University of Sheffield, Western Bank, Sheffield, S10 2TN, UK
4 Laboratoire de Neurogenèse et Morphogenèse au cours du Développement et chez l'Adulte (NMDA), UMR 6156 CNRS, IBDM, Université de la Méditerranée, Campus de Luminy, case 907, 13288 Marseille CEDEX 09, France



View larger version (39K):

[in a new window]
  		Fig. 1. Confocal images of 3-month-old wild-type mouse and human retinas. These images are high power insets of the OLM. (A) Crb1 is confined to the SAR, whereas ß-catenin localized more basally at the AJ. (B) Crb1 localized at the basal part of the F-actin localization in the PRC inner segments. (C) Mpp4, (D) Pals1, (E) Mupp1 and (F) Patj localized at the SAR, compared to the location of ß-catenin at the AJ. (G) aPKC co-localized with Crb1 to the SAR. (H-I) Crb2 and Crb3 localized to the SAR. A similar staining was detected in Crb1-/- retina. (J) CD44 localized in the MGC apical villi, but did not co-localize with Crb1 in the SAR. Par6, moesin or ZO-3 were not detected in the OLM (data not shown). Claudin-1 to -5 and occludin were not detected in the retina, but claudin-2 and occludin were detected in the retinal pigment epithelium (RPE). (K) CRB1 localized at the SAR of the human retina, apical to ß-catenin in the AJ. (L) MUPP1 localized at the SAR of the human retina. (M) Schematic diagram of the localization for the different proteins at the SAR or AJ. Scale bar: 2.5 µm.

 


View larger version (115K):

[in a new window]
  		Fig. 5. Retinal phenotype of 3-month-old Crb1-/- mice exposed to 3000 lux for 72 hours. (A) Representative experiment with wild-type and Crb1-/- retinas indicating number of protrusions, ingressions and total amount in cycled light (12 hours dark/12 hours 100 lux) versus 72 hours of 3000 lux. Error bars represent s.e.m. Asterisks indicate statistical difference (P<0.02) between the groups exposed to cycled light and 72 hours of 3000 lux. (B) Numerous ingression areas through the OPL (arrow) and protrusions through a distorted OLM (arrowhead) in the Crb1-/- retina. (C) OLM present in rosette (arrow), no segments are present on the disorganized PRCs (arrowhead). (D-J) Fluorescence microscopy images of degenerated areas. Nuclei are stained with Hoechst (blue). Note that in unaffected areas, adjacent to the ingression areas, localization of the proteins is normal. GFAP localized near the inner limiting membrane, at the MGC end-feet and in horizontally radiating MGC rootlets in the OPL (data not shown) in wild-type mice exposed to both cycled light and 72 hours of 3000 lux. (E) Detail of D, strong staining of GFAP in the ONL and through OLM. (F) Areas of protrusions (arrowheads) and ingressions (arrows) where Mpp4 is lost at the OLM and OPL as well as mislocalized into the ONL (asterisk). (G) ß-Catenin mislocalization throughout the ONL in ingression areas (arrows). (H) ZO-2 localization perturbed in a protrusion and ingression area. (I,J) Mislocalization and loss of Patj or Mupp1 in affected areas (arrowheads). (K) Apoptotic cells are rarely present in wild-type retinas. (L) Slightly increased apoptosis is apparent in Crb1-/- mice around ingression areas. Scale bars: 50 µm. (M) SLO image (514.5 nm) of a 3-month-old Crb1-/- mouse fundus after exposure for 72 hours to 3000 lux. The multiple dots (arrowhead) indicate areas of rosette formation in the inferior temporal quadrant of the retina. Scale bars: 50 µm.

 


View larger version (50K):

[in a new window]
  		Fig. 2. Mupp1 immunoprecipitation (IP) co-precipitates Crb1, Mpp4 and Pals1. Crb1 was co-immunoprecipitated from retinal lysates of wild-type but not Crb1-/- mice. Patj was not found in the precipitated protein complex but was present only in the total retinal lysate (L). Anti-Pals1 detected a Pals1 doublet (Roh et al., 2002Go). In the control (Pre), mouse IgGs were used for immunoprecipitation.

 


View larger version (86K):

[in a new window]
  		Fig. 3. Generation of Crb1-/- mice and confocal images of mouse retinas. (A) Crb1 disrupted by insertion of the targeting vector. E1, exon 1; E2, Exon 2; pA, polyadenylation signal; PGK, phosphoglycerate kinase promoter; B, BamHI; RV, EcoRV; Bg, BglII. (B,C) Deletion of the exon encoding the N-terminal signal peptide prevents the production of Crb1 protein with C-terminal transmembrane and intracellular domains. (B) EcoRV Southern blot analysis using a 750 bp BglII-AccI fragment probe in the 3' flanking region. (C) Immunoprecipitation of Crb1, with AK7, from lysate of wild-type but not of the Crb1-/- retina. As positive control 293/CRB1 cell lysates were used. Crb1 was stained using AK2. Asterisks indicate cross-reacting bands with AK2 in 293 cells. (D-G) Localization of Crb1 (red) in the OLM and staining of cone segments and pedicles by peanut agglutinin (PNA; green) in the retinas of wild-type (D) and Crb1-/- (F) mice. Detail of the localization of Crb1 at the SAR for the wild-type (E) and Crb1-/- (G) retina. Scale bars: 30 µm.

 


View larger version (172K):

[in a new window]
  		Fig. 4. Retinal phenotype of Crb1-/- mice exposed to cycled light (12 hours dark/12 hours 100 lux). (A-C) Different stages of degeneration in 3-month-old Crb1-/- retinas. (A) Start of degeneration in which some PRCs protrude through the OLM and ingress into the OPL. (B) More PRCs are involved in larger ingression areas and PRCs ingressed onto the INL. (C) Ingressed PRCs re-aggregate into half rosette structures, and form new inner segments and an OLM (arrow). (D,E) Morphology of 6-month-old Crb1-/- retinas. (D) Formation of a giant half rosette of PRCs with outgrown inner segments. Note the presence of cells from the INL, possibly MGC nuclei, close to the edges of and inside this structure (arrowheads). (E) Complete degeneration of the ONL, presence of ghost structure (arrow) and ingression of RPE into the retina (arrowheads). Scale bars: 100 µm.

 

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 2004