Dynamic behavior of paired claudin strands within apposing plasma membranes

Abstract

The tight junction (TJ) strand is a linear proteinaceous polymer spanning plasma membranes, and each TJ strand associates laterally with another TJ strand in the apposing membranes of adjacent cells to form ''paired'' TJ strands. Claudins have been identified as the major constituents of TJ strands, and when exogenously expressed in L fibroblasts, they polymerize into paired strands, which are morphologically similar to paired TJ strands in epithelia. Here, we show that a fusion protein of GFP with claudin-1 can also form similar paired strands in L fibroblasts, allowing us to directly observe individual paired claudin strands in live cells in real time. These paired strands showed more dynamic behavior than expected; they were occasionally broken and annealed, and dynamically associated with each other in both an end-to-side and side-to-side manner. Through this behavior of individual paired claudin strands, the network of strands was reorganized dynamically. Furthermore, fluorescence recovery after photobleaching analyses revealed that claudin molecules were not mobile within paired strands. Although these observations are not necessarily representative of TJ strands per se in epithelial cells, they provide important information on the structural and kinetic properties of TJ strands in situ with significant implications for barrier function of TJs. T he tight junction (TJ) is one mode of cell-to-cell adhesion in epithelial and endothelial cells. TJs seal the cells to create a primary barrier to the diffusion of solutes across the cellular sheet, and also function as a boundary between the apical and basolateral membrane domains to produce their polarization (1-5). On ultrathin section electron microscopy, TJs appear as a series of discrete sites of apparent fusion, involving the outer leaflets of the plasma membranes of adjacent cells (6). On freeze-fracture electron microscopy, TJs appear as a set of continuous, anastomosing intramembranous particle strands (TJ strands; ref. 7). These observations led to our current understanding of the three-dimensional structure of TJs; each TJ strand associates laterally with another TJ strand in apposing membranes of adjacent cells to form ''paired'' TJ strands, where the intercellular space is completely obliterated (reviewed in ref. 5).To date, three distinct types of integral membrane proteins have been shown to be localized at TJs; occludin (8, 9), junctional adhesion molecule (10), and claudins (11). Occludin, an Ϸ65-kDa integral membrane protein with four transmembrane domains, was identified as the first component of TJ strands. However, several studies including gene knockout analyses revealed that TJ strands can be formed without occludin (12-15). JAM with a single transmembrane domain was recently shown to associate laterally with TJ strands, but not to constitute the strands per se (16). In contrast, claudin is now believed to be a major constituent of TJ strands (reviewed in refs. 5 and 17). Claudins with molecular masses of Ϸ23 kDa comprise a multigene family...

Lecture

“…37 Co-culture of HeLa cells expressing different claudin genes revealed that while CLDN1 and CLDN5 were heterotypically interacting with CLDN3, they would not heterotypically bind to CLDN4, demonstrating considerable selectivity in heterotypic claudin-claudin interactions. 38 39 FRAP studies suggest that claudin molecules assembled in tight junctions have limited mobility, 40 consistent with their known heteromeric interactions with scaffold proteins in the tight junction.…”

Selective Decrease in Paracellular Conductance of Tight Junctions: Role of the First Extracellular Domain of Claudin-5

“…Furthermore, it is not clear that the same molecular pathway is utilized by ions as opposed to larger molecules. Interestingly, when claudins are exogenously expressed in fibroblasts, the dynamic behavior of the polymerized paired strands can be observed (49). Biochemical analysis of claudin-4 produced in insect cells revealed a labile nature of the claudin oligomer, implying a dynamic state (39).…”

Matrix metalloproteinases in early diabetic retinopathy and their role in alteration of the blood–retinal barrier

“…As compared to occludin, the endothelial-specific the tight junction protein claudin-5 did not appear to be susceptible to degradation by MMP-2 or MMP-9 under the conditions of these experiments. Although an exact role for occludin in the function of tight junctions has not been fully established, it is postulated that occludin has some role in stabilizing claudin oligomers 11 as well as regulating the activity of the cytoskeleton with various signaling pathways in the cell. 4 A recent study has demonstrated that the addition of peptides corresponding to a portion of the occludin extracellular domain caused increased permeability in cells as measured by TER.…”