JCS045203 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Diffusion coefficient of GFP in fiber cell cytoplasm determined by fluorescence correlation spectroscopy. (A) Normalized autocorrelation functions for GFP diffusion in aqueous solution (green), superficial fiber cells (red), and fiber cells located 200 µm below the lens surface (blue), with corresponding fits (χ²<1×10^-6) shown in dashed lines. (B) Depth dependence of GFP diffusion coefficient (D) in the lens. Each point represents a measurement from an individual cell located at a specific depth below the lens surface. Average values are indicated by the horizontal bars. Data from three such experiments were used to calculate mean values for D (see text).

• Supplemental Figure S2 -

  Fig. S2. The LMDP is a property of the lateral membrane. (A) A maximum intensity projection of the anterior hemisphere of a Cre-ER^TM;Z/EG lens 19 weeks after tamoxifen treatment reveals scattered epithelial cells (arrows) overlying diffusely labeled bands of fiber cells (*). The position and orientation of the Y-shaped anterior suture is indicated. Intercellular diffusion of GFP does not occur across the suture branches. (B) A higher magnification view of the posterior suture confirms the lack of transfer across the suture branches (arrows). Scale bars: 500 µm (A); 250 µm (B).

• Supplemental Figure S3 -

  Fig. S3. Distribution of GFP-expressing cells in the cortex of a 7-month-old lens from a Lim2^Gt/Gt;Cre-ER^TM;Z/EG mouse. Cryosections were cut in the equatorial plane and immunostained with anti-Aqp0 (red) to visualize the membrane architecture. Cut in this orientation, cells are seen in cross section. Clusters (A-D) of discretely labeled GFP-positive cells (green) are present in the lens cortex (see also Fig. 6C, which shows a similar image of an intact, living lens). Cell clusters are located 114 µm (A), 143 µm (B), 196 µm (C) and 276 µm (D) from the lens surface. Scale bar: 25 µm.

• Supplemental Figure S4 -

  Fig. S4. Gap junction-mediated cell-cell communication remains patent in the lenses of Lim2^Gt/Gt mice. Lenses from 2-day-old wild-type (A) or Lim2^Gt/Gt mice (B) were injected with neurobiotin. One hour after injection, lenses were fixed and the distribution of neurobiotin was visualized using a fluorescent steptavidin (green). In both cases, neurobiotin diffused throughout the tissue, indicating that gap junction-mediated communication persisted in the absence of Lim2. Scale bar: 250 µm.

• Supplemental Figure S5 -

  Fig. S5. Distribution of Lim2 in mid-cortical fiber cells of 1-month-old lenses. Cryosections were cut in the equatorial plane, revealing an array of cross-sectioned lens fiber cells. In wild-type lenses (A), Lim2 immunofluorescence is abundant in both the narrow (arrowheads) and broad (arrows) faces of the hexagonal membrane profile. In lenses from age-matched Lim2^Gt/Gt mice (B), immunofluorescence is reduced to a low, background level. Scale bar: 10 µm.

• Movie 1 -

  Movie 1. X-Z image of the cortical region of a Cre-ER^TM;Z/EG lens from a 6-week-old mouse, 3 weeks after tamoxifen treatment. In this orientation, lens fiber cells are seen in cross section. Two individual GFP-expressing cells (*) are present near the lens surface. Further into the lens are two groups of cells (A, B) in which GFP appears to have spread between adjacent fibers. Over the 14 hour observation period, GFP is observed to diffuse from one labeled cell (open triangle) into a neighboring, previously non-fluorescent cell (filled triangle). The transfer of GFP suggests that the two cells became linked by the LMDP during the culture period. Scale bar: 25 µm.