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 27 January 2009
doi: 10.1242/jcs.036970

Journal of Cell Science 122, 554-562 (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 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 Google Scholar
Google Scholar
Right arrow Articles by Simek, J.
Right arrow Articles by Laird, D. W.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Simek, J.
Right arrow Articles by Laird, D. W.
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?

Cx43 has distinct mobility within plasma-membrane domains, indicative of progressive formation of gap-junction plaques

Jamie Simek, Jared Churko, Qing Shao and Dale W. Laird*

Department of Anatomy and Cell Biology, University of Western Ontario, London, Ontario N6A 5C1, Canada


Figure 1
View larger version (103K):
[in this window]
[in a new window]

  		Fig. 1. GFP-tagged Cx43 localizes to multiple cell-surface domains. (A,B) Cx43-GFP was localized to GJ-like clusters and non-GJ membranes in untreated cells (A) but, when BICR-M1Rk cells were treated with BFA for 6 hours, GJ-like clusters were not evident (B). (C) Following BFA-washout and recovery for 1 hour, GJ-like clusters were again evident, as was Cx43 at cell-surface areas where no apposing cell was apparent (arrows). (D-F) When expressed in BICR-M1Rk cells, GFP-Cx43 (E) or GFP-Cx43{Delta}2 (F) was localized to four distinct areas of the plasma membrane, as illustrated in the schematic model (D). The fluorescent images presented in E and F were overlaid on DIC images to denote contacting cells. (G-I) GFP-Cx43{Delta}2 (G) and Cx43-RFP (H) colocalized (I, note yellow), demonstrating that these Cx43 variants have similar distribution profiles. Nuclei are stained with Hoechst (A-C). Scale bars: 10 µm.

 

Figure 2
View larger version (94K):
[in this window]
[in a new window]

  		Fig. 2. Pleiomorphic vesicles carrying GFP-Cx43{Delta}2 were observed to reach the cell surface in non-GJ regions and generate bright fluorescent sites. (A-C) Live cells expressing GFP-Cx43{Delta}2 (A) were superimposed on a DIC image (B) to generate an overlay image (C). (D-G) A region of interest (boxed region in C) was selected and imaged at 0, 20, 60 and 100 seconds (D-G, respectively) to show pleiomorphic vesicles apparently merging with the plasma membrane to generate bright fluorescent sites (arrows). See supplementary material Movie 1. Scale bars: 2 µm.

 

Figure 3
View larger version (86K):
[in this window]
[in a new window]

  		Fig. 3. Cx43 localizes to membrane protrusions. Rapid time-lapse imaging of live cells revealed that GFP-Cx43{Delta}2 (A-H) and Cx43-GFP (I-N) localize to cell surfaces that are not in contact with other cells. Two time series (D-H and J-N) reveal GFP-Cx43{Delta}2 and Cx43-GFP, respectively, at the cell surface, including within dynamic membrane protrusions (white and yellow arrows). Fluorescence images were superimposed over a DIC background to depict cell boundaries (B-H). See supplementary material Movie 2. Scale bar: 2 µm.

 

Figure 4
View larger version (46K):
[in this window]
[in a new window]

  		Fig. 4. Cx43 has a variety of mobilities at the cell surface and Cx43 GJ-like clusters can be categorized into two general populations in BICR-M1Rk cells. (A) FRAP analysis of GFP-Cx43{Delta}2-expressing BICR-M1Rk cells revealed that the mobility of Cx43 in GJ-like clusters was significantly less than Cx43 in all other plasma-membrane locations (*P<0.05). (B-E) Cells expressing Cx43-GFP (B,D,E), GFP-Cx43 (B) or Cx43T244-GFP (C) were subjected to FRAP, and fluorescence recovery into the photobleached area was monitored over a period of 300 seconds. Cx43H represents GJ-like clusters that exhibited a highly mobile Cx43 fraction, whereas Cx43L represents GJ-like clusters that exhibited a low-mobility Cx43 fraction. Note that the Cx43-GFP FRAP results presented in B were originally presented as one combined pool by Thomas et al. (Thomas et al., 2005Go). Scale bars: 2 µm.

 

Figure 5
View larger version (62K):
[in this window]
[in a new window]

  		Fig. 5. GFP-tagged Cx43 and truncated Cx43 partitioned into both low- and high-mobility GJ-like clusters in HeLa cells lacking endogenous Cx43. (A-C) GFP fluorescence and immunolabeling revealed GFP-Cx43 in GJ-like clusters in HeLa cells. (D,E) FRAP revealed that Cx43 (D) and truncated Cx43 (T244; E) exhibited both high and low mobility within GJ-like clusters in HeLa cells, similar to the GJ-like clusters that assembled in BICR-M1Rk cells that express endogenous Cx43. Similar ratios of GJ-like clusters with both high- and low-mobility fractions were assembled in HeLa cells expressing Cx43 or truncated Cx43. For direct comparison, the dotted curves presented in this figure are the same data as presented in Fig. 4. (E) Asterisks represent statistical significance between the high-mobility fractions of the Cx43H-T244-GFP variant when expressed in HeLa or BICR-M1Rk cells. P<0.05. (F) Histograms show that the proportion of GJ clusters in a highly mobile state do not change upon the truncation of the Cx43 C-terminal tail. Scale bar: 10 µm.

 

Figure 6
View larger version (24K):
[in this window]
[in a new window]

  		Fig. 6. The proportion of GJ-like clusters exhibiting low Cx43 mobility was partially dependent on the C-terminal domain of Cx43 but independent of cluster size. (A) BICR-M1Rk cells expressing Cx43-GFP, Cx43T244-GFP or GFP-Cx43 were assessed by FRAP to determine the percentage of GJ-like clusters that exhibited either a high or low Cx43 mobile fraction. (B) GJ-like clusters assessed in cells expressing Cx43 variants were measured to determine the cluster size. No significant difference was observed in the size of the clusters assessed. n=13-22 per Cx43 variant.

 

Figure 7
View larger version (66K):
[in this window]
[in a new window]

  		Fig. 7. GFP-tagged Cx43 fluorescent aggregates fail to recover from photobleaching. (A-D) MDCK cells expressing Cx43T244-GFP that harbors two additional point mutations was subjected to FRAP (see boxed area in B). Note that fluorescence recovery was limited to the lace-like endoplasmic reticulum, whereas the bright fluorescent aggregates revealed no recovery of fluorescence even after 45 minutes (D). Scale bar: 10 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 2009