Mechanochemical properties of human myosin-1C are modulated by isoform-specific differences in the N-terminal extension

Abstract

Myosin-1C is a single-headed, short-tailed member of the myosin class I subfamily that supports a variety of actin-based functions in the cytosol and nucleus. In vertebrates, alternative splicing of the MYO1C gene leads to the production of three isoforms, myosin-1C0, myosin-1C16 and myosin-1C35, that carry N-terminal extensions of different length. However, it is not clear how these extensions affect the chemomechanical coupling of human myosin-1C isoforms. Here, we report on the motor activity of the different myosin-1C isoforms measuring the unloaded velocities of constructs lacking the C-terminal lipid binding domain on nitrocellulose-coated glass surfaces and full-length constructs on reconstituted, supported lipid bilayers. The higher yields of purified protein obtained with constructs lacking the lipid binding domain allowed a detailed characterization of the individual kinetic steps of human myosin-1C isoforms in their productive interaction with nucleotides and filamentous actin. Isoform-specific differences include 18-fold changes in the maximum power output per myosin-1C motor and 4-fold changes in the velocity and the resistive force at which maximum power output occurs. Our results support a model in which the isoform-specific N-terminal extensions affect chemomechanical coupling by combined steric and allosteric effects, thereby reducing both the length of the working stroke and the rate of ADP release in the absence of external loads by a factor of two for myosin-1C35. As the large change in maximum power output shows, the functional differences between the isoforms are further amplified by the presence of external loads.

Distinct actin–tropomyosin cofilament populations drive the functional diversification of cytoskeletal myosin motor complexes

“…Binding of surface-attached a-actinin to A-Tpm iScience Article cofilaments leads to a reduction in the filament sliding velocity as the external load increases with the concentration a-actinin and impedes the driving force of myosin (Figure 4C). We have previously observed that the load-dependent changes in the sliding velocities of Myo1C 0 -DTH1 are best described by a tensionsensing mechanism that includes a force-dependent and a force-independent transition (Giese et al, 2020). The resulting force-velocity dependences (Figure 4D) indicate a marked reduction in motive power generation in the presence of actin cofilaments containing Tpm1.7 or Tpm3.1.…”

“…6 iScience 25, 104484, July 15, 2022 iScience Article to 0.07 G 0.01 s À1 in the presence of cofilaments containing either Tpm1.7 or Tpm3.1. We have previously shown that in the presence of bare F-actin k cat , the maximum value of ATP turnover in the presence of saturating actin concentrations, is reduced from $0.37 s À1 at 37 C to 0.09 G 0.02 s À1 at 20 C (Giese et al, 2020). Thus, in the absence and presence of Tpm isoforms, the observed differences between the rate limiting step k +4 and k cat are primarily owing to the different temperatures at which the transient and steady-state kinetics experiments were performed.…”

“…The steady-state ATPase rates of NM-2A and myosin-5A were measured using an NADHcoupled assay (Furch et al, 1998) in ATPase buffer-I containing 0.5-mM NM-2A-HMM or 0.1 mM myosin-5A, 20 mM MOPS pH 7.3, 50 KCl, 5 mM MgCl 2 , 1-mM ATP, 0.8-mM NADH, 0.5-mM phosphoenolpyruvate, 20 mg/mL lactate dehydrogenase, 50 mg/mL pyruvate kinase at 30 C. Steady-state ATP turnover by myosin-1C 0 was measured in ATPase buffer-II containing 0.1 mM Myo1C 0 -DTH1, 25 mM HEPES pH 7.5, 50 mM KCl, 5 mM MgCl 2 , 0.5 mM DTT, 1 mM ATP, 0.4 mM NADH, 0.5 mM phosphoenolpyruvate, 20 mg/mL lactate dehydrogenase and 50 mg/mL pyruvate kinase at 37 C. [A] or [A-Tpm] was varied over the range from 0 to 50 mM. The change in absorption at 340 nm was recorded in 96-well plates on a CLARIOstar Plus microplate reader (BMG Labtech, Ortenberg, Germany) (Giese et al, 2020). All combinations were tested with and without the addition of myosin, and the actin ATPase rate was then subtracted from the myosin ATPase rates.…”

“…The data for the ratio of slow to fast phase amplitudes (A slow /A fast ) plotted against the ATP concentration are best-fitted to a hyperbola, where the plateau value defines K a . The dependence of k obs,slow on ATP concentration are best-fitted with a hyperbola, where the plateau values define k +a (Giese et al, 2020). Kinetic Studio software (TgK Scientific Ltd., Bradford on Avon, UK) was used for initial data inspection and analysis of transient kinetic data.…”

“…Considering the essential functions of actomyosin complexes in non-muscle cells and the almost complete association of cytoskeletal F-actin with Tpm cables (Meiring et al, 2018), it is of paramount importance to elucidate the contribution of Tpm cables to contractile processes by investigating the influence of their isoform composition, exon usage, and N-terminal acetylation (Arnesen et al, 2009;Silva and Martinho, 2015). Here, we describe how the activities of the cytoskeletal myosin isoforms Myo1C 0 (Adamek et al, 2008;Giese et al, 2020;Zattelman et al, 2017), NM-2A (Kova ´cs et al, 2003;Mu ¨ller et al, 2013), and myosin-5A (De La Cruz et al, 1999;Mehta et al, 1999;Rock et al, 2000), which exert distinct enzymatic properties and cellular functions, are regulated by different Tpm isoforms and how N-terminal Tpm acetylation influences the functional properties of these myosin isoforms. We show by biochemical analysis of reconstituted A-Tpm-M complexes that the motor activity of human Myo1C 0 , NM-2A, and myosin-5A are each regulated in different ways by Tpm isoforms, and that N-terminal acetylation of Tpm alters this regulation to a significant extent and in a manner specific to each myosin isoform.…”