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 April 1, 2009
doi: 10.1242/10.1242/jcs.037317

Journal of Cell Science 122, 1229-1237 (2009)
Published by The Company of Biologists 2009
This Article
Right arrow Summary Freely available
Right arrow Full Text
Right arrow Full Text (PDF)
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 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 Google Scholar
Google Scholar
Right arrow Articles by Mohammad-Panah, R.
Right arrow Articles by Bear, C. E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Mohammad-Panah, R.
Right arrow Articles by Bear, C. E.
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?

An essential role for ClC-4 in transferrin receptor function revealed in studies of fibroblasts derived from Clcn4-null mice

Raha Mohammad-Panah1,*, Leigh Wellhauser1,2,*, Benjamin E. Steinberg3, Yanchun Wang1, Ling Jun Huan1, Xiang-Dong Liu4 and Christine E. Bear1,2,{ddagger}

1 Programme in Molecular Structure and Function, Hospital for Sick Children, 555 University Avenue, Toronto, Canada
2 Department of Biochemistry, Faculty of Medicine, University of Toronto, Canada
3 Programme in Cell Biology, Hospital for Sick Children, 555 University Avenue, Toronto, Canada
4 Department of Genetics, Hospital for Sick Children, 555 University Avenue, Toronto, Canada


Figure 1
View larger version (33K):
[in this window]
[in a new window]

  		Fig. 1. Elimination of ClC-4 expression in ClC-4-null mice maintains wild-type levels of ClC-3 and ClC-5 protein expression. (A) Left panel: ClC-4 antibody ({alpha}ClC-4) recognizes ClC-4 protein as a 90 kDa band in brain tissue from wild-type (Wt) mice. This band is absent in ClC-4-null mice brain tissue, as expected. Immunoreactivity to β-actin was assessed as a sample loading control. Middle panel: the polyclonal antibody against ClC-3 ({alpha}ClC-3, Chemicon) recognizes two specific bands of 90 and 100 kDa in both wild-type and ClC-4-null mice brain tissues, but not in brain tissue obtained from four Clcn3 knockout (KO) mice. Right panel: the polyclonal antibody against ClC-5 ({alpha}ClC-5) recognizes ClC-5 protein as a 85-90 kDa band present in both wild-type and ClC-4-null mice kidney tissues and absent in immunoblots of kidney tissue obtained from four Clcn5 KO mice (right panel). (B) ClC-4 protein expression is absent in Clcn4-null mice; ClC-3 and ClC-5 protein expression is not significantly altered in ClC-4-null mice (n=4, P=0.50 and P=0.21, respectively). All bar graphs indicate the mean±s.e.m. of signal intensity normalized to β-actin.

 

Figure 2
View larger version (31K):
[in this window]
[in a new window]

  		Fig. 2. Fluid phase endocytosis is not defective in ClC-4-null renal fibroblasts. (A) Confocal images showing the uptake of 1 mg/ml BODIPY-dextran (green) in wild-type and ClC-4-null fibroblasts for 30 minutes at 37°C, immunolabeled for ClC-4 (red). Partial colocalization between ClC-4- and dextran-containing compartments in wild-type renal fibroblasts are observed. The distribution of BODIPY-dextran is similar to wild type in ClC-4-null fibroblasts. Scale bar: 10 µm. (B) Quantification of BODIPY-dextran uptake at 30 minutes revealed no significant difference in the amount of accumulation between wild-type and ClC-4-null fibroblasts (n=23 and n=20, respectively; P=0.3054).

 

Figure 3
View larger version (63K):
[in this window]
[in a new window]

  		Fig. 3. ClC-4-null fibroblasts exhibit defective endocytosis that can be rescued by reconstitution of ClC-4 expression. (A,B) Transferrin accumulation is reduced in primary renal fibroblast cultures obtained from Clcn4-null mice. (A) Confocal images showing ClC-4 specific staining of punctuate structures (green) and Rhd-Fe2+-Tfn taken up by wild-type fibroblasts, 20 minutes after incubation at 37°C (red), the merge of these images is shown in yellow. Scale bar: 10 µm. Inset shows higher magnification of the merged image. Scale bar: 2 µm. (B) Confocal images showing the lack of specific punctuate ClC-4 staining (green) and demonstrating the reduced Rhd-Fe2+-Tfn uptake in ClC-4-null renal primary fibroblasts. Scale bar: 10 µm. (C,D) Defect in Rhd-Tfn uptake in ClC-4-null fibroblasts is rescued by reconstitution of wild-type ClC-4. (C) Confocal images show that transfection and expression of recombinant hClC-4 (left panel) leads to the rescue of Rhd-Tfn uptake (middle panel). The merged image shown in the right panel shows that the expressed ClC-4 protein colocalizes with Rhd-Tfn-bearing compartments and these structures appear somewhat swollen relative to the Rhd-Tfn bearing compartments in the wild-type fibroblasts. Scale bar: 10 µm. (D) Mean±s.e.m. of quantified immunofluorescence, representing transferrin accumulation (Rhd-Tfn uptake) in wild type (open bar, n=30), untransfected ClC-4-null (black bar, n=13) primary cells and ClC-4-null primary cells transfected with ClC-4 (grey bar, n=22). Rhd-Tfn uptake was significantly enhanced in ClC-4-null fibroblasts by transfection with recombinant ClC-4 (*, P<0.0001).

 

Figure 4
View larger version (13K):
[in this window]
[in a new window]

  		Fig. 4. ClC-4-null fibroblasts exhibit defective acidification in early and sorting endosomes. Wild-type and ClC-4-null fibroblasts were allowed to accumulate FITC-dextran for 5 minutes at 37°C. The pH of FITC dextran-containing endosomes was quantified using fluorescence ratio imaging as described in the Materials and Methods. (A) A representative calibration curve. (B) Bar graph representing the mean±s.e.m. of endocytic pH collected from wild-type (white bar, 750 endosomes) and ClC-4-null (black bar, 556 endosomes) fibroblasts compiled from eight independent experiments. An unpaired t-test reveals a significantly higher pH in the endosomes of ClC-4-null cells in comparison with the endosomes of wild-type fibroblasts (5.60±0.013 and 6.07±0.10, respectively; *P=0.0063).

 

Figure 5
View larger version (27K):
[in this window]
[in a new window]

  		Fig. 5. ClC-4-null fibroblasts show enhanced surface expression of the Tfn receptor but exhibit defective Tfn binding to these receptors. (A) Western blots of the cell surface biotinylation of the Tfn receptor performed in wild-type and ClC-4-null fibroblasts. (B) Quantification of the surface expression of the Tfn receptor relative to whole cell expression revealed a significant enhancement of the Tfn receptor expression at the cell surface in ClC-4-null fibroblasts (1.00±0.01 and 1.07±0.03, *P=0.015). (C) Western blots demonstrating the binding of biotinylated-Tfn (Tfn-Btn) to the surface Tfn receptors of iced wild-type or ClC-4-null fibroblasts with and without a desoxiferramine treatment to remove pre-bound diferric-Tfn. The accessibility of surface Tfn receptors for ligand binding was accessed through the quantification of the amount of Tfn-Btn bound at the surface in these cell lines. Values were normalized to β-actin for each sample. (D) Statistical analysis indicated a significant reduction in surface binding of Tfn-Btn to ClC-4-null fibroblasts relative to the wild type, suggesting Tfn receptors in the absence of ClC-4 exhibit reduced accessibility for ligand binding (0.68±0.036 and 1.00±0.039, respectively; *P=0.0040). However, upon treatment, ClC-4-null fibroblasts exhibited a significant enhancement of surface Tfn-Btn binding relative to the amount bound in untreated cells and, in fact, treatment to remove diferric-Tfn restored Tfn-Btn binding in ClC-4-null fibroblasts to wild-type levels (1.09±0.02 and 1.00±0.05, P=0.1633).

 

Figure 6
View larger version (23K):
[in this window]
[in a new window]

  		Fig. 6. The reduced binding of Tfn to receptors accounts for the majority of the endocytic defect observed in ClC-4-null fibroblasts. (A) Time course of Tfn-Btn uptake in wild-type (squares) and ClC-4-null fibroblasts (triangles) normalized relative to β-actin. Mean±s.e.m. are shown. The wild-type and ClC-4-null data were fit using the Michaelis Menton binding algorithm (r2=0.8661 and 0.7230, respectively). Total accumulation after 20 minutes was significantly reduced in ClC-4-null fibroblasts (*P=0.004). (B) Time course of Tfn-Btn uptake in wild-type and ClC-4-null fibroblasts normalized relative to the amount of accessible Tfn receptors. Mean±s.e.m. is shown. The wild-type and ClC-4-null data were fit using the Michaelis Menton binding algorithm (r2=0.8670 and 0.7961, respectively). There was no significant difference in uptake normalized to initial binding, measured at 20 minutes, between the two different types of cells (P=0.1801). (C) Time course of Tfn-Btn recycling in wild-type and ClC-4-null fibroblasts after a 20-minute accumulation of Tfn-Btn. There is no significant difference in the amount of recycled Tfn-Btn between wild-type and ClC-4-null fibroblasts after 20 minutes of recycling (0.32±0.11 and 0.16±0.044 densitometry units, P=0.259). The time for half-maximal recycling is also similar in the presence and absence of ClC-4 (t1/2=6.62 minutes and t1/2=6.13 minutes, respectively).

 

Figure 7
View larger version (25K):
[in this window]
[in a new window]

  		Fig. 7. ClC-4-null fibroblasts exhibit normal lysosomal degradation of the EGFR. (A) Primary wild-type (upper panels) and ClC-4-null fibroblasts (lower panels) were exposed to 100 ng/ml EGF (0, 15, 60, 120, 180 minutes) after being serum starved for 1 hour in the presence of 10 µg/ml cycloheximide. Cells were lysed and analyzed by SDS-PAGE and western blotting against EGFR and β-actin. (B) The time courses for EGFR degradation were compared in studies of wild-type (n=3 different cultures, solid square) and ClC-4-null primary cell cultures (n=3, solid triangle). There was no significant difference in the relative amount of EGFR remaining (% initial EGFR/actin) between wild-type and ClC-4-null fibroblasts at 180 minutes (P=0.425).

 

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 2009