Myosin essential light chain 1sa decelerates actin and thin filament gliding on β-myosin molecules

Osten, Jennifer; Mohebbi, Maral; Uta, Petra; Matinmehr, Faramarz; Wang, Tianbang; Kraft, Theresia; Amrute-Nayak, Mamta; Scholz, Tim

doi:10.1085/jgp.202213149

Cited by 3 publications

(1 citation statement)

References 86 publications

(104 reference statements)

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“…However, it is important to consider, in this connection, that other studies demonstrate a dramatic effect of essential 79,80 and regulatory 81,82 light chain isoforms on both striated muscle myosin ATPase kinetic properties and actin filament gliding velocities. Moreover, when performing experiments in parallel, Tang and coworkers 55 , observed that substitutions of the mouse regulatory chain for the human ventricular regulatory light chain (without exchange of the mouse ELC; study 3.1 vs 3.2 in Table S2) demonstrated reduced K ATPase and actin filament velocity by ~ 40% and ~ 15%, respectively (see also Fig.…”

Section: Parametersmentioning

confidence: 99%

Virus-free transfection, transient expression, and purification of human cardiac myosin in mammalian muscle cells for biochemical and biophysical assays

Velayuthan

Moretto

Tågerud

et al. 2023

Sci Rep

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Myosin expression and purification is important for mechanistic insights into normal function and mutation induced changes. The latter is particularly important for striated muscle myosin II where mutations cause several debilitating diseases. However, the heavy chain of this myosin is challenging to express and the standard protocol, using C2C12 cells, relies on viral infection. This is time and work intensive and associated with infrastructural demands and biological hazards, limiting widespread use and hampering fast generation of a wide range of mutations. We here develop a virus-free method to overcome these challenges. We use this system to transfect C2C12 cells with the motor domain of the human cardiac myosin heavy chain. After optimizing cell transfection, cultivation and harvesting conditions, we functionally characterized the expressed protein, co-purified with murine essential and regulatory light chains. The gliding velocity (1.5–1.7 µm/s; 25 °C) in the in vitro motility assay as well as maximum actin activated catalytic activity (kcat; 8–9 s−1) and actin concentration for half maximal activity (KATPase; 70–80 µM) were similar to those found previously using virus based infection. The results should allow new types of studies, e.g., screening of a wide range of mutations to be selected for further characterization.

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Section: Parametersmentioning

confidence: 99%

Virus-free transfection, transient expression, and purification of human cardiac myosin in mammalian muscle cells for biochemical and biophysical assays

Velayuthan

Moretto

Tågerud

et al. 2023

Sci Rep

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Mechanical and signaling responses of unloaded rat soleus muscle to chronically elevated β-myosin activity

Sergeeva,

Tyganov,

Zaripova

et al. 2024

Archives of Biochemistry and Biophysics

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Cost-Efficient Expression of Human Cardiac Myosin Heavy Chain in C2C12 Cells with a Non-Viral Transfection Reagent

Berg,

Velayuthan,

Månsson

et al. 2024

IJMS

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Production of functional myosin heavy chain (MHC) of striated muscle myosin II for studies of isolated proteins requires mature muscle (e.g., C2C12) cells for expression. This is important both for fundamental studies of molecular mechanisms and for investigations of deleterious diseases like cardiomyopathies due to mutations in the MHC gene (MYH7). Generally, an adenovirus vector is used for transfection, but recently we demonstrated transfection by a non-viral polymer reagent, JetPrime. Due to the rather high costs of JetPrime and for the sustainability of the virus-free expression method, access to more than one transfection reagent is important. Here, we therefore evaluate such a candidate substance, GenJet. Using the human cardiac β-myosin heavy chain (β-MHC) as a model system, we found effective transfection of C2C12 cells showing a transfection efficiency nearly as good as with the JetPrime reagent. This was achieved following a protocol developed for JetPrime because a manufacturer-recommended application protocol for GenJet to transfect cells in suspension did not perform well. We demonstrate, using in vitro motility assays and single-molecule ATP turnover assays, that the protein expressed and purified from cells transfected with the GenJet reagent is functional. The purification yields reached were slightly lower than in JetPrime-based purifications, but they were achieved at a significantly lower cost. Our results demonstrate the sustainability of the virus-free method by showing that more than one polymer-based transfection reagent can generate useful amounts of active MHC. Particularly, we suggest that GenJet, due to its current ~4-fold lower cost, is useful for applications requiring larger amounts of a given MHC variant.

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Myosin essential light chain 1sa decelerates actin and thin filament gliding on β-myosin molecules

Cited by 3 publications

References 86 publications

Virus-free transfection, transient expression, and purification of human cardiac myosin in mammalian muscle cells for biochemical and biophysical assays

Virus-free transfection, transient expression, and purification of human cardiac myosin in mammalian muscle cells for biochemical and biophysical assays

Mechanical and signaling responses of unloaded rat soleus muscle to chronically elevated β-myosin activity

Cost-Efficient Expression of Human Cardiac Myosin Heavy Chain in C2C12 Cells with a Non-Viral Transfection Reagent

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