QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 17 January 2006
doi: 10.1242/jcs.02770

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Lai, A. Articles by Charles, A. C. Search for Related Content PubMed PubMed Citation Articles by Lai, A. Articles by Charles, A. C. Social Bookmarking                         What's this?

Oculodentodigital dysplasia connexin43 mutations result in non-functional connexin hemichannels and gap junctions in C6 glioma cells

¹ Department of Neurology, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095, USA
² The Henry E Singleton Brain Cancer Research Program, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095, USA
³ Institute of Genetic Medicine, Department of Pediatrics, Johns Hopkins University School of Medicine, Baltimore, MD 21287, USA
⁴ Departments of Medicine and Surgery, Johns Hopkins University School of Medicine, Baltimore, MD 21287, USA

View larger version (33K): [in a new window]   Fig. 1. Schematic diagram of Cx43-eYFP indicating predicted topology, position of eYFP and locations of ODDD-associated amino acid mutations. Connexin 43 is predicted to span the plasma membrane four times and has cytoplasmically located N and C termini. Y17S is in the N-terminal domain. G21R and A40V are in the first transmembrane domain. F52dup is in the first extracellular loop. L90V is in the second transmembrane domain. I130T is in the cytoplasmic loop. Amino acids sites of other published ODDD-associated mutations have been circled. Fusion of eYFP to the C terminus of Cx43 by an eight amino acid linker segment is indicated. Mutations reported in this study are labeled, and mutations indicated by an asterisk are those producing neurological symptoms.

View larger version (69K): [in a new window]   Fig. 2. Western blot analysis of wild-type and mutant Cx43-eYFP fusion protein expression and quantitation of surface expression. Stable C6R cell lines expressing wild-type and indicated mutant Cx43-eYFP constructs were generated using the retroviral BH-RCAS expression vector. (A) Equal amounts (20 µg) of lysates prepared from uninfected control cells (C6R) or representative clones of C6R cells stably expressing wild-type Cx43-eYFP (wild type) or eGFP (eGFP) were probed with an anti-GFP polyclonal antibody. A 70 kDa band representing the full-length fusion protein was produced by wild-type cells but not C6R or eGFP cells. There was also a faint 30 kDa band detected from wild-type lysates that co-migrates with the eGFP band and represents either an independently transcribed species or a degradation product. (B) Lysates prepared from C6 cells, uninfected control C6R cells (C6R) or C6R wild-type Cx43-eYFP (WT) or mutant Cx43-eYFP constructs were also probed with an anti-Cx43 polyclonal antibody. The full-length fusion protein (70 kDa band) is again detected for wild-type cells, as well as each of the mutants tested but not detected in C6 or C6R cells. All cell lines produce a 40 kDa band that represents endogenous Cx43. Probing the same blot for actin demonstrates that equal amounts of lysates were loaded. (C,D) Surface proteins were selectively biotinylated at 4°C and isolated with streptavidin-agarose beads. Biotinylated samples were recovered from roughly 12-fold more lysate than was loaded in A. The full-length wild-type Cx43-eYFP 70 kDa band was again detected with both antibodies. No band was seen after recovery of biotinylated proteins from the eGFP lysate after probing for GFP indicating that non-specific biotinylation of cytoplasmic proteins did not occur (C). Actin bands were also not detected in the surface fraction (D). (E) Quantitation of cell-surface wild-type or mutant Cx43-eYFP fusion protein was performed by measuring eYFP fluorescence on a fluorescence microplate reader prior to loading on gel. Data shown were obtained from one to four experiments on two independent clonal cell lines for wild type and for each mutant. Relative fluorescence units/µg protein from total lysates are given.

View larger version (80K): [in a new window]   Fig. 3. Subcellular localization of Cx43-eYFP fusion proteins reveals formation of punctate structures at cell-cell junctions. Immunofluorescence microscopy of live cells growing on glass coverslips was performed on a custom laser-scanning confocal microscope equipped with a blue diode laser (475 nm) using a 63x1.3 objective. These data are representative of separate experiments on two cell lines for each construct. (A) Uninfected C6R was included as a control demonstrating lack of fluorescence signal. Mutants L90V (G) and I130T (H) showed a slightly reduced abundance of puncta, while mutants Y17S (C), G21R (D) and A40V (E) formed significantly fewer puncta than wild type. F52dup appeared to be localized at the cell membrane with only occasional formation of punctate structures at the cell surface (F). Scale bar, 10 µm.

View larger version (77K): [in a new window]   Fig. 4. Punctate structures are resistant to Triton X-100 extraction. Confocal immunofluorescence microscopy was performed on live C6R cell monolayers expressing wild-type and mutant Cx43-eYFP constructs, as indicated after incubation in HBSS (A,C,E,G,I,K,M) and after incubation for 15 minutes in HBSS containing 1% Triton X-100 (B,D,F,H,J,L,N) on the same field. Significant numbers of puncta remained after Triton X-100 extraction for the wild type and the various mutants. Although F52dup did not form plaque structures, some of the cell surface signal was resistant to extraction. Real-time monitoring of Triton X-100 extraction revealed that signal extracted was nearly instantaneously after addition of Triton. Overlays of images cannot be created because of an apparent mild deformation of cell architecture by Triton X-100 in non-fixed cells. Scale bar, 10 µm.

View larger version (15K): [in a new window]   Fig. 5. Mutant Cx43-eYFP do not form functional hemichannels as assessed by propidium iodide uptake. C6 cells, C6R control cells and clonal C6R cell lines expressing Cx43-eYFP constructs were analyzed for hemichannel function by determining the level of propidium iodide uptake after opening hemichannels by incubation of cell monolayers in 0 Ca2+. Stable expression of each construct in every cell was determined by detecting eCFP or eYFP fluorescence (A,D). A representative experiment showing PI uptake for control C6R (eCFP) (B,C) and wild-type Cx43-eYFP (E,F) after incubation in HBSS (B,E) with Ca2+ or HBSS (C,F) without Ca2+ containing PI for 15 minutes. Cells were fixed and uptake was visualized on a Nikon upright fluorescent microscope using rhodamine filter settings using a 20x objective. Digital photographs were quantitated using ImageJ software by averaging randomly selected average cell intensities for 40 cells per coverslip. (G) Quantitation of C6, C6R, wild type and each mutant is represented for HBSS with Ca2+ (white) and HBSS without Ca2+ (hatched). Data are expressed as mean±s.e.m. of several experiments on at least two cell lines for each mutant. Scale bar, 100 µm.

View larger version (112K): [in a new window]   Fig. 6. Mutant Cx43-eYFP do not form functional gap junctions, as assessed by scrape-loading analysis using sulforhodamine B. Clonal C6 cell lines expressing Cx43-eYFP constructs were analyzed for gap junctional transfer by scrape-loading of sulforhodamine B. (A,B) A representative experiment showing sulforhodamine B or rhodamine-dextran dye-transfer for control C6R(eCFP). No transfer occurs beyond initially loaded cells. (C,D) In wild-type Cx43-eYFP C6R, transfer of sulforhodamine B occurs four to six cell layers beyond initially loaded cells. (E-L) A representative experiment on the mutant Cx43-eYFP shows that all mutants behaved similarly to C6R with absence of dye transfer. Scale bar, 100 µm.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter