Without nuts and bolts, bridges and buildings would be nothing more than piles of beams and girders. Like skyscrapers, cells have internal support systems, but instead of steel, cells use a variety of rod-like protein fibers for their microscopic cytoskeletons.
At the University of Chicago, scientists interested in skin disorders caused by a weakening of the cytoskeleton have long known that a group of bolt-like proteins called plakins, brace the intermediate filaments. Of the three filamentous systems of the cytoskeleton, intermediate filaments have a diameter that places them between the microfilaments (think of them as high tension wires) and microtubules (the I-beam of the cell).
Now, researchers led by Elaine Fuchs, PhD, Amgen Professor of Molecular Genetics & Cell Biology and Howard Hughes Investigator, report in the July 23 issue of Cell, that plakins bind to all three components of the cytoskeleton, providing an integrated bracing system that gives cells their strength and flexibility.
This important finding sheds new light on how nerve axons, which in humans can reach as much as a meter in length for a single neuron, maintain their rigidity and allow for the transport of neurotransmitter-filled vesicles from one end to the other.
"We have known for several years that plakins give intermediate filaments their mechanical strength by anchoring them to the actin cytoskeleton just inside the cellular membrane, and that plakins also can stabilize intercellular junctions by linking them to the cytoskeleton," says Fuchs "But now we are seeing how truly versatile these fascinating plakin proteins are in that some of them can bind to and stabilize microtubules."
An important component of the cytoskeleton, microtubules serve as the cellular highway for the transport of vesicles and other proteins. "Stabilizing microtubules is especially important for cells like neurons which need to transport vesicles over very long distances," Fuchs explains.
Scientists became curious about new roles for plakins when they found them in fruit flies, which lack intermediate filaments.
"Intermediate filaments are thought to have evolved in animals without exoskeletons or other protective armor," says Fuchs. "In mammals, intermediate filaments are needed to provide a supportive framework to cells at the skin surface that are exposed to the physical stresses of the environment. When we found plakins in fruit flies, we knew that they couldn't interact just with intermediate filaments."
Further hints that plakins don't bind exclusively to IFs came from mouse knockout studies. In mice lacking the gene coding for BPAG1 plakins, neurons exhibit severe perturbations in their neuronal intermediate filaments (neurofilaments) and microtubules. The mutant axons also contain swellings that appear to be filled with what look like traffic jams of vesicles, grid-locked on their way down the axon.
When Fuchs and her team looked more closely at the BPAG1 plakin proteins, they discovered a new type of neuronal plakin. Instead of binding intermediate filaments to the actin cytoskeleton, this new plakin bound primarily to microtubules in the axon.
Yanmin Yang, PhD, a member of Fuchs' research team, showed that by adding the new BPAG1 plakin to cells with very sensitive microtubules, the cells became more resistant to cold and colchicine. Exposure to cold temperatures or the chemical colchicine normally cause microtubules to depolymerize, or fall apart, within minutes. With the addition of the new plakin, microtubules were stable over much longer time periods.
Next, Yang removed the BPAG1 plakin gene from neurons, which have the most stable mictrotubule networks of all cells of the body. When she treated them with cold and colchicine, the microtubules depolymerized within minutes. In addition, the microtubules from neurons lacking BPAG1 were dysfunctional, unable to direct traffic of proteins and vesicles.
"Our studies clearly show that by expressing BPAG1 plakins, neurons can stabilize their microtubules. This is crucial to the neurons that specialize in transporting cargo over long distances," Fuchs says. "The plakins we found in neurons may not be necessary in other cell types that are only 20 to 30 microns in diameter."
Fuchs thinks it is likely that certain humans with severe congenital neurodegenerative disorders may have mutations in the BPAG1 gene. Intriguingly, people with mutations in the gene coding for plectin, another kind of plakin protein, develop a rare form of muscular dystrophy associated with skin blistering. "Learning more about the plakin proteins and their functions may help us understand the nature of some severe neurodegenerative diseases better," explains Fuchs.
Other University of Chicago researchers on the paper include Christoph Bauer and Geraldine Strasser from the department of molecular genetics & cell biology; Robert Wollman, professor in the departments of pathology and neurology; and Jean-Pierre Julien form the Center for Research in Neuroscience at McGill University in Montreal.
Journal
Cell