For the first time, researchers at the University of Chicago describe in detail how neighboring skin cells adhere to one another to form sealed barriers. Without tight seals, the skin could not perform its primary function as a continuous barrier that keeps germs out and essential body fluids in. Their findings were published in the January 21 issue of the journal Cell.
"We wanted to understand the molecular mechanisms that enable epithelial cells to bind to each other in order to form an impermeable barrier," said Valeri Vasioukhin, Ph.D., a postdoctoral fellow in the lab of Elaine Fuchs, Ph.D., Amgen Professor of molecular genetics & cell biology and biochemistry & molecular biology. Fuchs is an expert on epithelial cells and skin diseases.
The researchers used keratinocytes, a type of skin cell, to study the earliest stages of cell-cell binding. The first step to a tight bond between cells is the formation of what scientists call adherence junctions. These can be seen in fluorescence microscopy images as yellow dots on the outer membranes of adjacent cells. Scientists call these dots "puncta."
Because cell-cell adhesion is dependent on calcium concentration, Vasioukhin was able to capture cells in the act of forming puncta. Low levels of calcium inhibit cell-cell adhesion, enabling the scientists to monitor the adhesion process after adding calcium.
"What we saw as we raised the calcium concentration was the formation of two perfect rows of dots, or puncta, along the cell membranes between two adjacent cells," said Vasioukhin. Puncta on adjacent cells lined up perfectly with one another. The researchers named the matched puncta rows the "adhesion zipper."
Several hours after the calcium concentration was raised, Vasioukhin observed that some of the puncta in the rows fused into one dot. "It is as if the puncta are the teeth of a zipper, and when an adherence junction starts to form, the zipper closes to form a single continuous sealed barrier between cells," explained Vasioukhin. "The genesis of adherence junctions, which is what we observed so clearly in our fluorescence studies, has never been described before," Vasioukhin said.
The researchers also found that without calcium, adherence junctions don't form at all, further boosting the theory that adherence junctions are calcium dependent.
Next, Fuchs and her colleagues looked more closely at the puncta. Christoph Bauer, a postdoctoral associate in Fuchs' lab and co-author of the paper, noticed that in response to calcium, the skin cells send out finger-like projections called filopodia. Filopodia from opposing cells touch and slide past each other to form a "zig-zag" seal.
"These fingers are packed with actin fibers-filamentous structural proteins. Remarkably, the rapid polymerization of these fibers at the finger tips generates the force to project them into the membranes of adjacent cells, like fingers poking into a rubber balloon. Puncta form at the tips of these fingers," Fuchs explains.
After puncta form, actin filaments begin to grow near the puncta, enmeshing the fingers ever more tightly together and encouraging cell-cell adhesion. When Vasioukhin disrupted the actin filaments with chemicals, the adhesion zippers and adhesion junctions were unable to form.
Researchers have known that puncta are composed of the proteins E-cadherin and catenin, but when Vasioukhin looked more closely at the composition of the puncta, he found that they also contained VASP and Mena, two proteins important in directing actin filament growth. When the researchers disturbed the normal function of VASP and Mena, adherence junctions could not form properly.
Mei Yin, a research technician was also an author on the paper.
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