Gap junctions of the medial collateral ligament: structure, distribution, associations and function

Abstract

Ligaments are composed of two major components: cells and extracellular matrix. The cells express gap junction proteins and are arranged into a series of rows that traverse the tissue, suggesting that all the cells of the tissue are functionally interconnected. The results of our study demonstrate that medial collateral ligament (MCL) cells do not have a uniform fusiform morphology or placement along a row of cells as previously suggested, but rather display a complex placement and form that weaves within the collagen matrix in a manner that is far more extensive and complex than previously appreciated. Within this morphological context, we find that MCL cells in vivo contain functional gap junctions (verified using fluorescence recovery after photobleaching) that are localized to sites of close cell-cell contact, and this pattern imparts or reflects a bipolarity inherent to each cell. When we studied ligament cells in conventional tissue culture we found that this bipolarity is lost, and the placement of gap junctions and their related proteins, as well as general cell morphology, is also altered. Finally, our study demonstrates, for the first time, that in addition to gap junctions, adherens junctions and desmosomes are also expressed by MCL cells both in vivo and in vitro and map to sites of cell-cell contact.

Fig. 1

(A) Frozen section of a rabbit MCL stained with DAPI for DNA. Nuclei are arranged in rows that pass along the long axis of the tissue (right–left). Note the rows cannot be followed continuously for long distances. Scale bar = 40 µm. (B)SEM micrograph of a rabbit MCL that has been torn along the long axis of the tissue revealing rows of cells (arrow 1). The cells are large (in many instances a portion of the cell is buried in the collagen matrix; compare the two cells denoted at 1), having a complex morphology and displaying prominent cytoplasmic extensions. Portions of these extensions permeating the collagen matrix are denoted at 3. The diversion of a row into another plane within the tissue is shown at 2. Scale bar = 25 µm. (C) A region between two adjacent cells within a row illustrating the pericellular matrix, which is bordered by large collagen fibres characteristic of the ECM. The pericellular matrix is populated, with collagen fibres of varying sizes (arrows) and distribution paths (arrow) as well as abundant vesicles. Scale bar = 11 µm. (D) A vesicle-filled seam representing an extension of the pericellular matrix that extends between collagen bundles into the ECM. These seams contain cytoplasmic extensions of the ligament cells as well as vesicular material similar that that seen in the pericellular matrix adjacent to the cell. Scale bar = 0.5 µm.

Fig. 2

(A) TEM micrograph illustrating two adjacent cells connected by a gap junction (GJ), an adherens junction (AJ), and a desmosome (D) in a region were two cells and their nuclei are in close appositions. Scale bar = 50 nm. (B) High-magnification image of a region similar to that shown in A demonstrating an extensive gap junction (GJ, arrows). Scale bar = 25 nm. (C) High-magnification region of a region similar to that shown in A illustrating an adherens junction (AJ). Scale bar = 30 nm. (D) Indirect immunofluorescent image of cells reacted with DSI II, a desomosome marker (red), as well as DAPI to denote nuclei (blue). Note the prominent reactivity in the region between cells (arrow) at the site of desmosomes detected by TEM. Scale bar = 8 nm. (E) Two adjacent cells connected by a gap junction (boxed regions and shown in inset) in a region where the nuclei are far apart. Scale bar = 1.5 nm, inset = 10 nm. (F) Adjacent cells in a region lateral to a region shown in A where the surfaces of the cells are not in close apposition. Note the numerous short cytoplasmic projections. These projections often come in close apposition (arrow). Scale bar = 2 µm. (G) Immunoflourescence image showing the pattern of Cx43 staining (red) in vivo. The staining pattern corresponds to the placement of gap junctions seen by TEM. Nuclei are stained with DAPI (blue). Scale bar = 10 µm.

Fig. 3

(A) TEM micrograph taken from a region of contact between two MCL cells in culture. Note the presence of gap junctions (arrow GJ) and an adherens junction (arrow AJ). Desmosomes from a similar region of another set of cells is shown in the inset. Scale bar = 15 µm. (B) MCL cell in culture stained with Cx43 (red), beta-catenin (green) and DAPI (blue). Note that these proteins are concentrated in cytoplasmic projections and frequently co-localize. Scale bar = 20 µm. (C) Beta-catenin (red) distribution pattern and DAPI (blue) seen in MCL cells in vivo. Scale bar = 20 µm. (D) L cell in culture illustrating that Cx43 (red) and ZO-1 (green) are concentrated in cytoplasmic extensions and often co-localize. Nuclei are stained with DAPI (blue). Scale bar = 20 µm. (E) In vivo ZO-1 distribution pattern (red). Nuclei are stained with DAPI (blue). Scale bar = 20 µm.

Fig. 4

Fluorescence recovery after photobleaching (FRAP) experiments. (A) Confocal photomicrographs of MCL cells in situ. Time series showing calcein-AM fluorescence just before, just after and 15 min after photobleaching for untreated cells (top half) and for cells treated with the gap-junction disrupting agent octanol (bottom half). Scale bar = 25 µm. (B) Confocal photomicrographs of MCL cells in vitro. Time series showing calcein-AM fluorescence just before, just after and 15 min after photobleaching for untreated cells (top half) and for cells treated with the gap-junction disrupting agent octanol (bottom half). Scale bar = 25 µm.