Biophysical Society Thematic Meeting | Singapore

Mechanobiology of Disease

Tuesday Speaker Abstracts

Mechanical Properties of Nonmuscle Myosin-2 Filaments James R. Sellers , Luca Melli, Neil Billington, Yasuharu Takagi, Attila Nagy, Sarah M. Heissler. National Heart, Lung and Blood Institute, NIH, Bethesda, MD, USA. There are three nonmuscle myosin-2 (NM2) paralogs in humans which participate in many cellular phenomena. Mutations in NM2A are associated with thrombocytopenia, deafness and kidney disease. Mutations in NM2C are associated with deafness. Each NM2 paralog forms 310nm bipolar filaments containing either 30 (NM2A/NM2B) or 16 myosins (NM2C). The three paralogs are slow enzymatically and mechanically compared to other myosins, but have distinct kinetic signatures with NM2B having the highest duty ratio. Neither NM2A or NM2B processively interact with actin in the optical trap as single molecules. In contrast, NM2B bipolar filaments show robust processive movements in “single filament in-vitro motility assays”. EM of myosin filaments in the presence of actin and ATP show that multiple motors from a single side of a myosin filament can interact with a single actin filament or with multiple actin filaments. Motors from opposite ends of a bipolar filament can also interact with different actin filaments forming sarcomeric-type attachments. Myosins from a single filament end contact actin subunits covering about 100nm of actin filament length. NM2B molecules co-assemble with headless RFP-myosin rods, reducing the number of motor domains in a bipolar filament. About 5 NM2B motors/half filament are required for processive movements with the run-length, but not the velocity decreasing as the number of motors decrease. Surprisingly, under the same buffer conditions, NM2A filaments do not move processively. Processive movements with NM2A can be achieved by including 0.5% methylcellulose in the assay to increase the viscosity or by forming co-polymeric filaments with NM2B. Myosin filaments can associate laterally to form stacks which can dynamically gain or lose bipolar filament units when interacting with actin filaments in the presence of ATP. In-vitro assays give a model for how myosin stacks might be formed.

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