Structure-Function Studies on Nonmuscle Myosin-2
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Date
2025-05
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The Ohio State University
Abstract
Background: C.elegans is a powerful model organism for studying actomyosin-dependent processes such as development, cell division, and migration and has been used in numerous studies to decipher the role of nonmuscle myosin-2 in this simplified system.
Nonmuscle myosin-2 is an ATP-dependent actin-based molecular motor that moves and shapes cells by pulling on actin filaments. However, unlike mammalian nonmuscle myosin-2 motors, the functional capacities and limitations of the C.elegans proteins are unknown, creating a roadblock for computational and functional studies.
Methods: The FLAG-tagged minimal functional unit of C.elegans nonmuscle myosin-2 was recombinantly produced using the baculovirus/Sf9 insect cell expression system and a purification strategy was devised. Rabbit skeletal muscle actin was purified from native sources. The complex between nonmuscle myosin-2 and actin was assembled and subjected to negative stain and cryo-electron microscopy analyses. Cryo-electron microscopy data were processed using cryoSPARC to reveal the three-dimension structure of the actomyosin complex.
Results: Negative stain electron microscopy experiments showed that the motor itself undergoes conformational changes in the presence of different nucleotides, indicating that C.elegans nonmuscle myosin-2 is a proper, enzymatically active motor. The high-resolution cryo-electron microscopy structure showed that myosin binds actin in a typical arrowhead conformation and that myosin undergoes conformation changes when bound to actin. Further, the data revealed the structural details of nonmuscle myosin-2 that explain how it interacts with actin to power fundamental biological processes.
Conclusions: In summary, the three-dimensional structure of the protein will contribute to defining the physiological capacities and limitations of the proteins, which will be used to inform cell biological and computational studies of nonmuscle myosin-2 in C.elegans.
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Keywords
Nonmuscle myosin-2, Actin, Caenorhabditis elegans (C.elegans), Cryo electron microscopy, Structural biology