Fesselin, an intrinsically disordered smooth muscle protein, organizes and stabilizes actin-myosin and myosin

ECU Author/Contributor (non-ECU co-authors, if there are any, appear on document)

Nathaniel Kingsbury (Creator)

Institution

East Carolina University (ECU )

Web Site: http://www.ecu.edu/lib/

Abstract: Fesselin is an intrinsically disordered protein that is known to bind a large variety of cytoskeletal proteins. The proteins fesselin is known to bind include: actin (Leinweber et al. 1999), [alpha]-actinin (Pham et al. 2006), calmodulin (Schroeter et al. 2004), filamin (Weins et al. 2001), and smooth myosin (Schroeter et al. 2005). The binding of fessilin to smooth myosin is of particular interest because unphosphorylated smooth muscle myosin filaments are unstable in the presence of ATP (Trybus et al. 1982, Ikebe et al. 1983, Suzuki et al. 1978). However, in smooth muscle cells unphosphorylated myosin filaments are maintained (Milton et al. 2011). Several proteins have been identified that stabilize myosin filaments and actin-myosin filament interactions. Our experiments show that fesselin may be one such protein. The organization of F-actin and myosin filaments by fesselin was observed by monitoring the rate of dissociation of actin-myosin by ATP in a stopped-flow device. Actin-myosin dissociation was measured by light scattering (a measure of particle size) and by pyrene-actin or acrylodan-tropomyosin fluorescence (a measure of myosin-actin bond breaking). The stopped-flow studies were further supported with electron microscopy analysis. These experiments showed that fesselin was able to tether actin and myosin filaments together without significantly impacting the rate of the actin-myosin bond breaking. The stabilization of myosin filaments by fesselin was tested using a similar method. First, stopped-flow rapid kinetics were used to measure the rate of ATP induced myosin filament break down. Next, electron microscopy was used to support the stopped-flow data and observe the effects of fesselin on myosin filament organization. Through the stopped-flow and electron microscopy experiments it was found that fesselin stabilizes myosin filaments. The electron microscopy experiments further revealed that fesselin enhanced myosin filament size and organized them into bundles. Previously published results showed that the interaction between fesselin and actin is regulated by calmodulin (Schroeter et al. 2004). The final set of experiments presented here examined the possibility that calmodulin regulates the interactions between fessilin and myosin. The calmodulin regulation of fesselin myosin interactions would greatly expand the role that fesselin has within smooth muscle by placing it within the calcium signaling pathway. The regulation of fesselin-myosin interactions by calmodulin was tested using pyrene labeled actin as well as N,N'-Dimethyl-N-(Iodoacetyl)-N'-(7-Nitrobenz-2-Oxa-1,3-Diazol-4-yl)Ethylenediamine labeled fesselin (IANBD-fesselin). These experiments showed that the interactions between fesselin and myosin are regulated by calmodulin. Overall, our results support that fesselin plays a critical role within smooth muscle to organize contractile elements.

Additional Information

Publication

Thesis

Language: English

Date: 2023

Subjects

Biochemistry;Molecular biology;Actin;Fesselin;Myosin;Rapid kinetics;Synaptopodin-2

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