To get its job done, each cell in the human body must constantly change its inner skeleton and therefore its outer shape. This skeleton also serves as a vast network of "tracks," which grow and shrink and move in different directions as needed to transport proteins and other materials within the cell and to organize cells within a tissue or organ.
But until now, no one understood how these individual rail lines were connected into an integrated system of three different types of moving "tracks." Proteins called microtubules, which are the conveyer belts inside the cells, and actin and intermediate filament proteins, which make up the inner skeleton, form the tracks that linearly crisscross a cell.
In the Oct. 31 issue of the journal Cell, researchers at Rockefeller University and the Howard Hughes Medical Institute report that a protein called ACF7 (actin crosslinking family 7) accumulates along growing microtubules and connects them to the actin filament network. This connection helps to guide the microtubule to its proper destination within the cell
"Not only do we now know a protein that links microtubules to actin, but we discovered this protein is really important, crucial to the organizational life of a cell," says lead investigator Elaine Fuchs, Ph.D., professor and head of the Laboratory of Mammalian Cell Biology and Development at Rockefeller and an investigator at HHMI.
The protein is one of the largest ever found in any organism, and because the protein is so important to development, Fuchs and her colleagues had to employ a special technique — culturing the "knockout cells" from the mouse embryo when it was less than the size of a pinhead — to find out what the protein does.
"With the ACF7 protein, microtubules move in straight, purposeful lines along polarized actin bundles. Without it, they wander aimlessly, often in loops," says Fuchs.
To tease out medical implications, the scientists plan additional research into ACF7 , but Fuchs says, "the features we've uncovered so far for this protein will make it very interesting in the future to examine its role in normal cell repair processes, like wound healing, and in diseases marked by cellular disarray, such as cancer."
The study's first author, Atsuko Kodama, M.D., Ph.D., an associate in the Fuchs lab, says that ACF7 acts like a "molecular reinforcer, or vice clamp" to strongly hold microtubule and actin proteins together and directionally position the microtubule. But it likely does much more than that, she adds, since only a very small part of the protein is involved in binding the two cytoskeletal proteins.
"This giant protein has plenty of potential for as-yet unidentified functions that we surmise must be important to the life of a cell," says Kodama.
ACF7 belongs to a family of molecules called spectraplakin proteins, which have been found in cells ranging from fruit flies and worms to mice and humans, says Fuchs. Because her lab specializes in processes that lead to development of skin and hair, Fuchs first studied a spectraplakin protein called BPAG1 (bullous pemphigoid antigen 1) that was known to exist in skin cells. Knocking out the BPAG1 gene in mice produced a mild blistering defect in skin, as well as rapid degeneration in sensory neurons in the mice, leading to a loss of control of the limbs of the animals. In an article published this week in the Journal of Cell Biology, Fuchs' former associate Yanmin Yang, M.D., Ph.D., now at Stanford University, reported in a collaboration with Fuchs that the BPAG1 protein in neurons functions in movement of proteins from the nerve endings to the body of the neuron.
The Fuchs team has turned to ACF7 because ACF7 is more broadly expressed by cells, suggesting a more universal function for the protein. The ACF7 gene was so enormous — much larger than the BPAG1 found in epidermis— that it took researchers around the world years to clone it, given limitations in technology, Fuchs says. Once cloned, we created antibodies to the protein in order to find out where ACF7 "localized" in skin cells. In that way, they discovered it accumulates along microtubules and also has the ability to bind to actin filaments.
But that insight didn't describe what role the protein played once bound to the cytoskeletal proteins, so the research team set out to create "knockout" mice lacking the gene that produces ACF7 protein. The embryos did not survive past a very early stage of development — the embryos were only blastocysts, a few hundred cells large. So, Fuchs lab associates and co-author Iakowos Karakesisoglou, Ph.D., created a "knockout cell" by growing a cell from the blastocysts that did not express ACF7. This cell is a precursor to the yolk sac, the tissue that surrounds the embryo.
To determine what ACF7 proteins do, Kodama introduced green fluorescent proteins that attached to microtubules in both the knockout cells and normal cells, and then she made video microscopy movies of both cell lines as they moved in the culture dish. "Without the ACF7 protein, the actin cytoskeleton looked normal, but the microtubules did not know where to go. They could not tether to the actin, and went in all directions, sometimes in loops," says Kodama. The normal cells, by contrast, formed straight microtubules, she says.
"This is a clean demonstration of what can functionally happen if you knock out one protein in a cell," says co-author Alec Vaezi, M.D.
Further experimentation showed that if a scratch is introduced into the monolayer of knockout cells, the knockout cells could not repair their damage in a normal manner. "If you wound normal cells, they will reorganize their microtubule and actin cytoskeleton in the direction of the wound, and start to march to the "wound site" and fill it in," says Fuchs. "In the absence of ACF7, the cells start to reorganize their actin cytoskeleton, but their microtubules don't reorganize. They start to wander and sometimes head in the other direction," she says.
"Epithelial cells are always organized. They know which side is up, which side is down, who their neighbors are and what their orientation within a tissue is," says Fuchs. "ACF7 is the first example of a protein that can help coordinate the functioning and directionality of the actin-microtubule cytoskeletons, and we are now looking to see how it is regulated within a cell."
This research was supported by Howard Hughes Medical Institute and by a grant from the National Institutes of Health. Senior research specialist Ellen Wong also participated in the study.
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