The Journal of Neuroscience, March 30, 2005, ():
Adherens Junctions in Myelinating Schwann Cells Stabilize Schmidt-Lanterman Incisures via Recruitment of p120 Catenin to E-Cadherin
J. Neurosci. Tricaud et al.
25: 3259
Supplemental data
Files in this Data Supplement:
- supplemental material
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Supplemental Figure S1 Quantification of the co-localization between introduced E-cadherin constructs and endogenous p120ctn
Nerve samples were immunostained with antibodies against IL2R or FLAG and p120ctn. Confocal images figuring cells expressing the constructs (detected with anti-IL2R or anti-FLAG antibodies) in red channel and p120ctn in blue channel were taken and these images were further analyzed using the ImageJ software (NIH) and its plugin RG2B co-localization. Using this plugin, the image was autothresholded and the co-localization criteria was set to the maximum of red and blue. The co-localized areas were displayed in a 8bit images and this image was tresholded to isolate clusters. The infected cell was delimited by a ROI (region of interest) and the number of particles above 5 pixels was determined in this ROI to obtain the number of clusters that are co-localized with p120ctn in infected cells.
We next determined the total number of clusters present in the infected cells of the same image. In the original image we selected only the red channel that shows the E-cadherin constructs clusters and converted this image in 8 bit image. The threshold was adjusted in the same way to isolate the clusters from the background and the same ROI delimited the infected cells. The number of particles was analyzed in this ROI using the same cut off of 5 pixels to obtain the total number of clusters.
The Figure S1A displays the percentage of E-cadherin constructs clusters that co-localize with p120ctn in infected cells.
Number of cells counted : IL2R-EcadCYTO, 11; IL2R-EcadJMD, 8; Ecad, 10; Ecad?P120, 8; IL2R-EcadCYTO?P755,756, 10.
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Supplemental Figure S2 Quantification of the E-cadherin clusters remaining in cells expressing IL2R-Ecad constructs
Nerve samples were immunostained with antibodies against IL2R and E-cadherin. Confocal images figuring both cells expressing the construct (detected with anti-IL2R antibody) and cells that do not express the construct were taken and the images picturing the E-cadherin clusters were further analyzed using the ImageJ software (NIH). First the size of the cells pictured on the images was normalized using the scales. The images were converted in 8 bits gray format and the intensity threshold was adjusted to isolate the clusters from the background. A region of interest (ROI) was delimited around the cells, the surface of this ROI was determined and then the number of clusters above 5 pixels was determined inside this ROI. The mean and maximum size of these clusters was also determined. From these data we measured the number of clusters per ?m2 for infected cells and for non-infected cells on the same image. The number of cluster per ?m2 in infected cells normalized to the number of cluster in non-infected cells is presented in Fig. S2A. The mean size of clusters in infected cells normalized to the mean size of clusters in non-infected cells is shown in Fig. S2B. Graphs show that cells expressing IL2R-EcadJMD have a number of E-cadherin clusters and a mean cluster size that are close to 1 and therefore close to the normal non-infected cells. At the contrary cells expressing IL2R-EcadCYTO or IL2R-EcadCYTO?P755,756 contain less E-cadherin clusters and smaller clusters than non-expressing cells (ratio 0.125 and 0.138 respectively in Fig. S2A and 0.39 for both constructs in Fig. S2B). The effects of these two constructs on the disappearance of E-cadherin clusters are statistically identical (t-test, p=0.67 in S2A and 0.96 in S2B). Number of cells counted: IL2R-EcadJMD, 3; IL2R-EcadCYTO, 5; IL2R-EcadCYTO?P755,756, 6.
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Supplemental Figure S3 Cytoplasmic distribution of p120ctn does not affect the incisures
(A) Mouse p120ctn clone that had its third armadillo repeat deleted (P120?arm3) to block its binding to cadherin, and its C-terminus Flag-tagged. (Nter, N-terminal domain; PD, phosphorylation domain; Cter, C-terminal domain.
(B) CHO cells expressing p120ctn (P120), P120?arm3, or a negative control construct in which amino acids 622 to 628 are deleted in p120ctn so that it is unable to affect cell shape(Anastasiadis et al., 2000). Cells were stained for F-actin stress fibers (F-actin, red) and phosphotyrosine (pTyr, green) which are primarily localized at focal adhesion contacts. Over-expression of P120 and P120?arm3 constructs led to a reduction in actin stress fibers and focal adhesions as previously shown (Noren et al., 2000; Grosheva et al., 2001). Scale bar = 10?m.
(C) P120?arm3 (red) does not co-localize with E-cadherin (green). Scale bar = 5?m.
(D) P120?arm3 (green) has a diffuse distribution in Schwann cells. Scale bar = 5?m.
(E) Detail of GFP distribution in a Schwann cell infected with GFP adenovirus (upper panel) and P120?arm3 adenovirus (lower panel). Arrows indicate incisures. Scale bars = 5?m.
(F) Percentage of cells displaying incisures determined after infection with viruses expressing GFP and P120?arm3. All the cells expressing P120?arm3 had incisures. Number of cells scored: GFP,15; P120?arm3, 18.
