2023-06-10 16:25:43
It has long been known that actin filaments – protein structures essential for the living movement of individual cells into animals – have a polarity associated with their physical characteristics, with growing and shrinking “sharp” ends. The ends of the filament are also different in how they interact with other proteins in cells. However, the mechanism that determines these differences has never been entirely clear to scientists. Now, researchers from the Perelman School of Medicine at the University of Pennsylvania have revealed the key atomic structures of actin filament ends through the use of a technique called cryo-electron microscopy (cryo-EM). The study, published in Scienceprovides fundamental information that can help provide details regarding disorders affecting certain muscle, bone, cardiac, neurological, and immune disorders resulting from actin defects or deficiencies.
Actin is the most abundant protein inside the cells of higher organisms, such as animals. It serves as the building block for long, thin structures called filaments, which provide key structural support as part of the cellular ‘cytoskeleton’, the system that gives cells their shape and polarity. Rapid changes in actin filaments underlie key cellular events such as movement along surfaces, cell-to-cell contact, and cell division. Actin filaments are also major components of muscle fibers.
“The results of our study provide a mechanistic understanding of a process that we have known for more than 40 years, called the filament treadmill, and impacts how we perceive the cellular roles of actin in health and disease,” said the study’s lead author. Roberto Dominguez, PhD, William Maul Measey Presidential Professor of Physiology at Penn.
Actin filament dynamics are largely governed by the “conveyor belt” process, whereby individual actin proteins are removed from one end of the filament, known as the pointed end, and added to the other end barbed. Actin filaments can be stabilized by distinct so-called “capping” proteins that bind to the ends of filaments to stop the addition or loss of individual actin proteins. Many other proteins also bind to the barbed and pointed ends of the actin filament. But the structural details determining the specificity of these interactions – the details that explain why these two ends work so differently – have remained obscure.
In their study, the researchers, including two Penn students — Peter Carman, PhD, a recent graduate student in Dominguez’s lab, and Kyle Barrie, PhD, a graduate student currently in the lab who served as co-first authors — analyzed actin filaments using cryo-EM. With this high-resolution imaging technique, a researcher obtains several thousand snapshots of a target molecule, aligns them computationally, and then averages them to reduce random image “noise,” resulting in a reconstruction 3D of the molecule which can be sharp. enough to visualize the individual atoms.
With the help of artificial intelligence (AI), the researchers were able to focus on the ends of the filaments instead of their middles, as had previously been the norm in similar research. In doing so, they identified hundreds of thousands of filament end views, allowing them to obtain near-atomic-scale reconstructions. These revealed a “flat” actin shape, or conformation, at the uncapped barbed end, compared to a “twisted” conformation at the uncapped pointed end.
The data also detailed the structural changes induced by two actin filament capping proteins, CapZ at the barbed end and tropomodulin at the pointed end. These are the two proteins present at the filament ends of skeletal and cardiac muscles, playing a critical role in stabilizing actin filaments in muscle fibers, and without these proteins our muscles would collapse.
The results of this study provide crucial mechanistic details for a deeper understanding of actin biology as a whole. The researchers believe that this study information should also be useful in understanding and ultimately treating disorders caused by actin dysfunction.
Funding was provided by the National Institutes of Health (R01 GM073791, F31 HL156431).
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