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

Summary The effects of N-terminal acetylation of the high molecular weight tropomyosin isoforms Tpm1.6 and Tpm2.1 and the low molecular weight isoforms Tpm1.12, Tpm3.1, and Tpm4.2 on the actin affinity and the thermal stability of actin-tropomyosin cofilaments are described. Furthermore, we show how the exchange of cytoskeletal tropomyosin isoforms and their N-terminal acetylation affects the kinetic and chemomechanical properties of cytoskeletal actin-tropomyosin-myosin complexes. Our results reveal the extent to which the different actin-tropomyosin-myosin complexes differ in their kinetic and functional properties. The maximum sliding velocity of the actin filament as well as the optimal motor density for continuous unidirectional movement, parameters that were previously considered to be unique and invariant properties of each myosin isoform, are shown to be influenced by the exchange of the tropomyosin isoform and the N-terminal acetylation of tropomyosin.


INTRODUCTION
Tropomyosins (Tpm) form a large family of polar, double-stranded, a-helical coiled-coil actin filament binding proteins (McLachlan and Stewart, 1976;von der Ecken et al., 2015). The human Tpm genes are referred to as TPM1 through TPM4 (Geeves et al., 2015;Gunning et al., 2008;Lin et al., 1997). Variations in the DNA sequences of the coding regions of the four genes and alternative splicing of the variable exons 1, 2, 6, and 9 give rise to considerable molecular diversity ( Figure 1A). As some Tpm splice isoforms are missing variable exon 2, this results in shorter and longer isoforms with 245-248 and 284-285 amino acid residues, respectively. Individual Tpm dimers interact with actin filaments only weakly, with dissociation constants estimated to be in the millimolar range (Tobacman, 2008;Wegner, 1980;Weigt et al., 1991). High-affinity binding occurs only upon polymerization into continuous Tpm cables on the actin filament surface (Fischer et al., 2016;Heald and Hitchcock-DeGregori, 1988;Vilfan, 2001;Wegner, 1980). This process is driven by the head-to-tail association of Tpm dimers (Fischer et al., 2016;Heald and Hitchcock-DeGregori, 1988;Vilfan, 2001;Wegner, 1980), and is further enhanced by the presence of myosin motors (Eaton, 1976;Moraczewska et al., 1999;Pathan-Chhatbar et al., 2018). In general, the dynamic nature of actin-Tpm (A-Tpm) interactions is characterized by high cooperativity and has been shown to influence the competitive association of actin-binding proteins, such as fimbrin and cofilin (Christensen et al., 2017).
Whereas some Tpm isoforms are specifically associated with muscle tissues, most isoforms function within the context of cytoskeletal A-Tpm cofilaments (Gunning et al., 2005;Pittenger et al., 1994). Members of the Tpm family are thought to play a key role in coordinating the interaction of cytoskeletal actin filaments with other types of actin-binding proteins by defining the functional properties of individual actin filament populations. This idea took shape in recent years after it was recognized that almost all actin filaments of the cytoskeleton exist as cofilaments with Tpm isoforms (Meiring et al., 2018) and after various disease associations became known (Marston et al., 2013;Redwood and Robinson, 2013;Reumiller et al., 2018). Specifically, cytoskeletal Tpm isoforms have been linked to diseases affecting neurosensory functions, platelet disorders, and cancer (Brettle et al., 2016;Latham et al., 2018;Pleines et al., 2017). Transformation and cancerous growth are frequently accompanied by upregulated production of low molecular weight (LMW) Tpm isoforms, such as Tpm 3.1 and Tpm4.2, and loss of high molecular weight (HMW) Tpm isoforms (Hendricks and Weintraub, 1981; Kabbage et al., 2013;Stefen et al., 2006Stefen et al., , 2018. Thus, over-production of Tpm4.2 has been directly linked to the invasiveness of infiltrating breast cancer cells. In transformed cells, Tpm3.1 accounts for up to 70% of total Tpm, as opposed to 25% in corresponding control cells (Meiring et al., 2018). Compounds that manipulate actin function by directly targeting Tpm3.1 are therefore attractive and promising anti-cancer agents Stehn et al., 2013).
The regulation of myosin motor activity by A-Tpm cofilaments differs between sarcomeric and cytoskeletal isoforms. In the context of muscle, calcium binding to troponin triggers a series of conformational changes that shift the Tpm filament on F-actin to reveal the myosin-binding interface on the actin filament (Parry and Squire, 1973;Spudich et al., 1972;Spudich and Watt, 1971). In contrast to the muscle system, cytoskeletal actomyosin (B)-(E) Myosin family motor proteins consist of a generic motor domains (green) followed by a lever arm with light chain binding sites (orange) and the tail region (blue). (B) Class 1 myosins act in part by connecting membrane lipids with the actin cytoskeleton. They support short range vesicle transport, modulate actin assembly, and function, act as the adaptation motor in the stereocilia of the inner ear, and are implicated in transcription regulation. (C) The actomyosin ATPase cycle, which is shared by all myosin classes with the exception of class 18, includes the rigor state (state A) and progresses via the following transitions: ATP binding (A/B; K 1 ), dissociation of the actomyosin complex (B/C; K 2 ), ATP hydrolysis (C/D; K 3 ), reassociation with formation of a weak actomyosin complex (D/E, K 9 ), phosphate release, formation of a strong actomyosin complex and powerstroke (E/F; K 4 ), ADP release (F/A; K 5 ), which returns the Myo1C to the rigor state. (D) Non-muscle myosin-2 isoforms are predominantly organized into stress fibers that are able to generate contractile forces that support functions such as cell adhesion, migration and mechanotransduction. (E) Class 5 myosin dimers act in part by transporting cargo such as vesicles from the center of the cell toward the periphery. The polarity of the actin filament is indicated as + (barbed) and -(pointed) ends. iScience Article complexes function in the absence of troponin or isofunctional troponin-like proteins and the context of greater Tpm isoform diversity. Cell-based and biochemical studies support the hypothesis that the association of filamentous actin with different tropomyosin isoforms determines the identity and modulates the activity of the interacting myosin motor proteins (Gateva et al., 2017;Manstein and Mulvihill, 2016). Specifically, it has been shown that bipolar bundles of non-muscle myosin-2 (NM-2) isoforms that function as integral components of stress fibers, actin arcs, and contractile rings, and the various unconventional myosin motors that support vesicle and organelle transport, as well as discrete steps of endocytosis and exocytosis, are integrated into the appropriate functional context by distinct A-Tpm cofilaments as schematically shown in Figures 1B-1E ( Barua et al., 2018;Clayton et al., 2015;Kee et al., 2015;Meiring et al., 2019;Sckolnick et al., 2016).
Corroborating evidence includes the observation that class 1 myosin-dependent delivery of organelles to the perinuclear region is affected by the association of F-actin with Tpm1.7 but not with Tpm3.1 (Pelham et al., 1996). Cargo structures that carry myosin-1C (Myo1C) on their surface are regulated by Tpm1.6 with respect to their run initiation and run termination, which may enable effective sorting of cargo by co-regulation of the initiation and termination of kinesin-driven runs at actin/microtubule intersections (McIntosh et al., 2015). In the cortical region of adipocytes, Tpm3.1 but not Tpm1.7 was proposed to define the actin population regulating the recruitment of Myo1C and non-muscle myosin-2A (NM-2A), which are both required for GLUT4-mediated glucose uptake and blood glucose clearance (Kee et al., 2015). In stress fibers, the incorporation of the HMW splice isoforms Tpm1.6 and Tpm2.1 produces opposite effects with respect to NM-2-dependent force development, with Tpm1.6 causing an increase in intracellular pressure, whereas incorporation of Tpm2.1 decreases traction force (Sao et al., 2019). In addition, Tpm2.1 has a specific role that other Tpm isoforms cannot replace, supporting correct rigidity sensing and detection of tensile stress at the cell periphery (Wolfenson et al., 2016). The incorporation of both LMW isoforms Tpm3.1 and 4.2 into stress fibers promotes the specific recruitment of NM-2A (Bryce et al., 2003). Cofilaments formed by F-actin and Tpm3.1 or Tpm4.2 are characterized by rapid dynamic exchange and are not effectively protected from disassembly (Gateva et al., 2017). NM-2A NM-2A was shown to have increased sliding velocity and ATPase activity in complex with actin-Tpm3.1 (Barua et al., 2014) and actin-Tpm4.2 cofilaments . Moreover, the association of actin filaments with Tpm3.1 or Tpm4.2 has been shown to increase ATP turnover by NM-2A and NM-2B ATPase owing to a specific acceleration of the rate-limiting step of phosphate release. Under resisting force conditions, actin-Tpm3.1 and actin-Tpm4.2 cofilaments induce a slower release of the hydrolysis product ADP, leading to an increase in duty-cycle and processive behavior of NM-2B and NM-2A Pathan-Chhatbar et al., 2018).
In the case of cytoskeletal Tpm isoforms without N-terminal acetylation, apparent affinities for filamentous actin in the range of 0.1-5 mM have been reported for Tpm1.6, Tpm1.7, Tpm1.8, Tpm2.1, Tpm3. 1, and Tpm4.2. No clear correlation was found between affinity and size of Tpm isoforms (Carman et al., 2021;Janco et al., 2016;Moraczewska et al., 1999;Ngo et al., 2016). It is known that N-terminal acetylation is important for the ability of sarcomeric Tpm isoforms to interact with F-actin, but less critical for the binding of the cytoskeletal Tpm isoforms (Meiring et al., 2018;Palm et al., 2003). Biochemical and structural biology studies suggest that N-terminal acetylation of Tpm isoforms not only acts locally but has consequences for conformational dynamics far from the Tpm N-terminus and thus on the function and stability of A-Tpm cofilaments (East et al., 2011;Greenfield et al., 1994;Johnson et al., 2018;Monteiro et al., 1994;Palm et al., 2003). In the case of muscle-specific isoforms Tpm1.1 and Tpm1.3, it was shown that the lack of N-terminal acetylation leads to an at least 30-fold lower actin affinity (Heald and Hitchcock-DeGregori, 1988;Hitchcock-DeGregori and Heald, 1987). Results available for Tpm3. 1 / 3 .2 and Tpm4.2 show that these cytoskeletal isoforms are almost exclusively acetylated in primary and transformed human fibroblasts and epithelial cells (Meiring et al., 2018). However, the precise impact of this post-translational modification on actin binding by cytoskeletal Tpm isoforms and the functional properties of A-Tpm cofilaments has remained unresolved. Further, N-terminal extensions consisting of one to three amino acid residues including Gly (Palm et al., 2003), Gly-Cys (Greenfield et al., 1994), Ala-Ser (Monteiro et al., 1994;Palm et al., 2003), Ala-Ala-Ser (Monteiro et al., 1994), Lys-Met-Thr (Heald and Hitchcock-DeGregori, 1988;Hitchcock-DeGregori and Heald, 1987), and Ala-Ser-Arg (Urbancikova and Hitchcock-DeGregori, 1994) have been shown to restore the high-affinity actin binding of the muscle-specific isoforms Tpm1.1 and Tpm1.3 with little or no interference with self-polymerization, myosin interactions and other regulatory functions (Hitchcock-DeGregori and Heald, 1987;Maytum et al., 2000;Monteiro et al., 1994). As such, these peptide extensions, but especially the Ala-Ser dipeptide, are now widely used as N-terminal acetylation mimics for the production of recombinant muscle-specific as well as cytoskeletal Tpm constructs (Coulton et al., 2006;Palm et al., 2003).

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iScience 25, 104484, July 15, 2022 3 iScience Article 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 (Ková cs et al., 2003;Mü 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.

Expression, purification, and initial characterization of proteins for the in vitro reconstitution of physiological A-Tpm-M complexes
To characterize the functional properties of cytoskeletal A-Tpm cofilaments and their interactions with myosin motors, we produced and purified the human isoforms of b-actin, g-actin, tail truncated versions of Myo1C isoform C 0 (Myo1C 0 -DTH1), and heavy meromyosin (HMM)-like constructs of NM-2A and myosin-5A in active and soluble forms using the SF9/baculovirus system. In humans, alternative splicing of the MY O 1C gene leads to the production of three isoforms, which differ in the length of their N-terminal extensions (Ihnatovych et al., 2012;Nowak et al., 1997). Compared with Myo1C 0 , the isoforms Myo1C 16 and Myo1C 35 contain 16 and 35 additional amino acids at their N-terminus. Typically, we obtained up to 3.5 mg of the cytoskeletal actin isoforms, 1.6-mg Myo1C 0 -DTH1, 0.6 mg NM-2A-HMM, and 0.5 mg myosin-5A-HMM per 1 L of culture medium.
Human Tpm isoforms were produced in Escherichia coli BL21(DE3) for the non-acetylated isoforms and the acetylated isoforms in E. coli BL21(DE3) cells coproducing either the fission yeast NatA or NatB complex. The sequential induction of the appropriate Nat complex, followed by expression and repression of the respective Tpm isoform of interest, has been shown to result in near-complete N-terminal acetylation . Moreover, the presence of N-terminal acetylation was probed for isoforms derived from TPM1 and TPM2 using antibodies recognizing either the acetylated or non-acetylated Tpm N-terminus ( Figure 2A). Both the non-acetylated and acetylated Tpm isoforms were purified to homogeneity in yields of 15-20 mg per liter. Previous work from our laboratory has shown significant differences in the ability of isoactins to bind and stimulate the enzymatic activity of human NM-2A, -2B, -2C, and myosin-7A. In the case of NM-2A and -2B, the interaction with either cytoplasmic actin isoform results in four-fold greater stimulation of myosin ATPase activity than was observed in the presence of a-skeletal muscle actin . Significant differences were also observed in initial control experiments performed for the interaction of human myosin-5A with the different isoactins. Accordingly, all subsequently described experiments using NM-2A and myosin-5A constructs were performed with cytoskeletal b-actin. The situation is different for Myo1C, where we observed no significant change in ATP turnover and motile activity upon exchange of the actin isoform (Figures S1A and S1B). Likewise, we observed comparable maximal inhibition of 40 G 5% and IC 50 values of 3.5 G 0.3 mM for ATP turnover by acto-Myo1C 0 -DTH1 in the presence of Tpm3.1 and AcTpm3.1. Therefore, all experiments involving Myo1C 0 -DTH1 were performed using a-actin and nonacetylated Tpm isoforms.
To investigate the effects of exon usage 1a.2b in HMW-Tpm isoforms compared with 1b in LMW isoforms, as well as the importance of internal exon 6a compared to 6b, we performed cosedimentation tests with b-actin in combination with HMW-Tpm isoforms 1.6 and 2.1 or LMW isoforms 3.1 and 4.2. Myo1C motor functions were studied in detail in complex with Tpm1.7 and Tpm3.1 as in vivo and in vitro studies provide clear evidence for the role of Tpm1.7 and Tpm3.1 decorated actin cytoskeleton in the regulation of Myo1C localization at specific cell sites and motor functions (Kee et al., 2015;McIntosh et al., 2015;Pelham et al., 1996).
We observed values for half-maximal saturation of binding (K 50% ) in a narrow range between 1.3 and 2.0 mM with the HMW isoforms Tpm1.6 and Tpm2.1 and the LMW isoforms Tpm3.1, and Tpm4.2 ( Figure 2C). Each of these isoforms is prominently associated with stress fibers in a variety of human cell types (Gateva et al., 2017). Tpm isoforms included in our study that are typically not associated with stress fibers such as Tpm1.8 ll OPEN ACCESS 4 iScience 25, 104484, July 15, 2022 iScience Article and Tpm1.12 have K 50% values that lie outside this narrow range. Approximately 10-fold higher and lower apparent binding affinities were observed with Tpm1.8 and Tpm1.12, respectively (Table 1).

Impact of N-terminal Tpm acetylation on interaction with filamentous actin
Following N-terminal acetylation, we observed only minor changes in the K 50% for filamentous b-actin, with increases in apparent binding affinity for the N-acetylated forms from 1.32 to 1.23 mM for Tpm1.6, 1.50 to 1.15 mM for Tpm3.1, and 1.99 to 1.33 mM for Tpm4.2. The opposite effect was observed upon acetylation for Tpm1.12 and Tpm2.1, resulting in a decrease in apparent binding affinity from 18.2 to 28.8 mM and 1.73 to 2.29 mM, respectively (Figures 2C and Table 1). At least in the absence of other actin-binding proteins, our results support the view that there exists no direct relationship between the F-actin binding affinities of cytoskeletal Tpm isoforms and molecular weight (Carman et al., 2021;Janco et al., 2016;Moraczewska et al., 1999;Ngo et al., 2016). Our results show in addition that N-terminal acetylation has only a minor impact on the actin affinity of cytoskeletal Tpm isoforms, again without a clear correlation to size.
Next, we investigated whether the thermal stability of A-Tpm cofilaments is altered by N-terminal acetylation. In agreement with previous data obtained using smooth muscle isoforms (Levitsky et al., 2000), we show that the dissociation of cytoskeletal Tpm isoforms from filamentous b-actin is reversible and shows pronounced hysteresis, with dissociation occurring at higher temperatures than reassociation ( Figure 2D). A comparison of T diss for non-acetylated and acetylated Tpm1.6, Tpm2.1, Tpm3.1, and Tpm4.2 revealed significant isoform-specific differences in dissociation properties ( Figures 3A-3D, Table 1). Specifically,  Table 1. Four independent experiments were carried out for each isoform, each with four individual measurements. Error bars represent standard deviations. (D) Graph showing the temperature-induced dissociation (10-50 C) and reassociation (50-10 C) of A-Tpm1.6 cofilaments, monitored by recording the associated changes in light scattering intensity.

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iScience 25, 104484, July 15, 2022 5 iScience Article in the absence of N-terminal acetylation, the HMW isoforms Tpm1.6 and Tpm2.1 display higher T diss values of 41.2 and 38.0 C than the LMW isoforms Tpm3.1 and Tpm4.2 with T diss values of 35.6 and 36.5 C. In the case of the HMW isoforms, N-terminal acetylation leads to a slight reduction of T diss . In contrast, the LMW isoforms show marked stabilization of their cofilaments with F-actin by 9 C or more after N-terminal acetylation. In the case of Tpm4.2, N-terminal acetylation shifts the light scattering signal into the range where denaturation of F-actin starts to occur, the observed processes become irreversible, and dissociation cannot be fully resolved (Figures 3D and Table 1). In line with previous studies (Levitsky et al., 2000), we observed that the T diss values measured for the different acetylated and non-acetylated isoforms depend strongly on Tpm concentration. Secondary plots of T diss against the total Tpm concentration are well described by hyperbolas. N-terminal acetylation of Tpm 1.6 resulted in small increases in T diss over the entire concentration range. A much stronger stabilization of cofilaments was observed after N-terminal acetylation of Tpm3.1, evident from a marked leftward shift and an approximately 10 C higher plateau value of the hyperbola ( Figures 3E and 3F).

Modulation of actin-activated ATPase and motor activity of myosin-1C 0 by cytoskeletal Tpm isoforms
The actin-activated steady-state ATPase activities of Myo1C 0 -DTH1 were determined with 10-mM A-Tpm cofilaments composed of a-actin and non-acetylated LMW isoforms Tpm3.1, Tpm4.2, or HMW isoforms Tpm1.6, Tpm1.7. Marked reductions in ATP turnover of Myo1C 0 -DTH1 were observed in the presence of all Tpm isoforms tested ( Figure 4A). It should be noted that the ATPase rates reported in Table 2 refer to values obtained at a sub-saturating concentration of 10-mM A-Tpm cofilaments and thus cannot be directly compared with the values obtained at saturating concentrations of bare F-actin and Tpm cofilaments. The actin-activated steady-state ATPase activities of Myo1C 0 -DTH1 in the absence and presence of HMW isoform Tpm1.7 or LMW isoform Tpm3.1 were determined at actin concentrations ranging from 0 to 50 mM. Under both conditions, we observed a reduction in maximum ATP turnover of approximately 33% for Myo1C 0 -DTH1 (Table 2). We used stopped-flow kinetics to determine the impact of HMW Tpm1.7 and LMW Tpm3.1 on the binding of Myo1C 0 -DTH1 to F-actin in the absence of nucleotide, ATP-induced dissociation of Myo1C 0 -DTH1 from F-actin, ADP affinity of acto-Myo1C 0 -DTH1, and the release of the hydrolysis products phosphate and ADP. The results of these measurements are summarized in Table 3. They show a significant reduction in the rate constant for phosphate release (k +4 ) from 0.09 G 0.01 s À1 in the presence of bare F-actin 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 . 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 impact of the Tpm isoforms on the rate of ADP release from Myo1C 0 -DTH1 was determined by ATP-induced dissociation of the actomyosin complex (Geeves, 1989). The active sites of 0.5-mM Myo1C 0 -DTH1 were saturated with 20-mM ADP, so that ATP binding is rate-limited by the slow dissociation of ADP. The transients were best fitted with a single exponential function yielding rate constants for ADP release (k +5 ) that are 30 and 34% slower for cofilaments containing Tpm1.7 (1.11 G 0.09 s À1 ) and Tpm3.1 (1.05 G 0.05 s À1 ) than the rate constant obtained with bare F-actin (1.59 G 0.07 s À1 ). Finally, measurements of the ATP-induced dissociation of pyrene-labeled acto-Myo1C 0 -DTH1 revealed a To determine the influence of A-Tpm cofilaments on myosin-1C motor function, we performed in vitro motility assays. No motile activity was detectable on Myo1C 0 -DTH1-decorated surfaces, when bare actin filaments were replaced with A-Tpm cofilaments under the standard conditions of the in vitro motility assay. Titrations of the Myo1C 0 -DTH1surface density showed that the minimum motor density to achieve a stable plateau value for continuous directional movement increased from 900 motors mm À2 with bare F-actin to >4,000 motors mm -2 in the presence of A-Tpm cofilaments ( Figure S2). Compared with the plateau value of 52.1 G 4.9 nm s -1 obtained for filament sliding velocity with bare F-actin on lawns of Myo1C 0 -DTH1, approximately 5.5-fold slower values were observed in the presence of Tpm1.6, Tpm1.7, Tpm1.12, Tpm2.1, Tpm3.1, and Tpm4.2 (Table 2). Based on results obtained with class-2 myosins (Greene and Eisenberg, 1980;Ngo et al., 2016), we expected the cytoskeletal Tpm isoforms and Myo1C 0 to mutually strengthen each other's affinity for F-actin in the presence of ATP. With NM-2B, we had observed a change in the K 50% for F-actin binding by Tpm1.12 from R40 mM to <1 mM (12). However, when we replaced NM-2B with Myo1C 0 , a much smaller increase in the apparent actin affinity of Tpm1.12 (K 50% = 19.3 G 1.1 mM) was recorded. The reduced mutual cooperative enhancement of actin binding by the cytoskeletal Tpm isoforms and Myo1C 0 is clearly evident in the dose-response curves for inhibition of Myo1C 0 -supported motile iScience Article activity by the various cytoskeletal Tpm isoforms ( Figure 4B). Tpm2.1 showed a similar two-fold change in K 50% in the presence of Myo1C 0 . Smaller changes were recorded for cofilaments containing Tpm3.1 and Tpm4.2 in the presence of Myo1C 0 , with increases in K 50% of 13 and 25%, respectively (Table 1).
To analyze the influence of the distinct Tpm isoforms on Myo1C-driven force development, we determined the ability of Myo1C 0 -DTH1 to translocate A-Tpm cofilaments in the presence of an external load using frictional loading experiments (Greenberg and Moore, 2010). 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 . 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.

Modulation of actin-activated ATPase and motor activity of non-muscle myosin-2A and myosin-5A by non-acetylated cytoskeletal Tpm isoforms
The observed maximum actin-activated ATPase activity of NM-2A with bare filamentous b-actin (k cat = 0.26 G 0.02 s À1 ) is in good agreement with published data Ková cs et al., 2003). Functional assays performed with NM-2A in the presence of saturating concentrations of non-acetylated HMW tropomyosin isoforms Tpm1.6 and Tpm2.1 and LMW isoforms Tpm3.1 and Tpm4.2 show isoform-specific variations in catalytic activity. The strongest effect was observed for Tpm3.1, with a 2.5-fold increase in k cat and 63% faster velocity compared with bare F-actin. Cofilaments containing Tpm2.1 showed a 1.5-fold increase in k cat and a 12% faster velocity. In the case of Tpm1.6 and Tpm4.2, we observed a 34 and 42% increase in k cat together with smaller changes in velocity, which are within the margin of error of our assay ( Figures 5A and 5B; Table 2).  Table 2). In contrast, cofilaments containing Tpm1.12 move approximately 12% faster than bare F-actin at a myosin-5A-HMM surface density corresponding to approximately 18 double-headed motor molecules per mm 2 . ATP turnover by myosin-5A was not significantly affected by cofilaments containing Tpm1.12. With respect to the run length of filaments, we observed a significant reduction for cofilaments containing Tpm1.12 and a significant increase for those containing Tpm4.2. The run length of cofilaments containing Tpm1.6, Tpm2.1, or Tpm3.1 was unchanged compared with bare filamentous b-actin ( Figure S3). Similar protection of filaments from fragmentation was observed for all Tpm isoforms tested.

Impact of N-terminal Tpm acetylation on actin-activated ATPase and motor activity of NM-2A and myosin-5A
For the HMW isoforms Tpm1.6 and Tpm2.1, only minor differences in the k cat of NM-2A were observed between the acetylated and non-acetylated forms of the proteins. The observed changes in the concentration required for half-maximal activation of ATP turnover (K app ) are within the margin of error of our assay ( Figures 7A and 7B). Larger opposing effects were observed for NM-2A in the presence of saturating concentrations of A-Tpm cofilaments containing either the acetylated or non-acetylated forms of LMW Tpm3.1 and Tpm4.2. Cofilaments containing Tpm3.1 show a 2.5-fold increase in k cat (0.26 s À1 versus 0.64 s À1 ), whereas K app is reduced 1.4-fold and k cat /K app is more than 3.6-fold increased in the presence of Tpm3.1. These are the strongest effects observed for a non-acetylated Tpm isoform (Figure 7C). N-terminal acetylation of Tpm3.1 reduces the increase in k cat /K app to 2.2-fold, together with corresponding changes in k cat and K app . In the case of Tpm4.2, N-terminal acetylation has the opposite effect than that observed with Tpm3.1. The calculated k cat is increased 1.6-fold for AcTpm4.2 from 0.37 to 0.61 s À1 and k cat /K app increases 2.1-fold from 1.12 3 10 À2 to 2.34 3 10 À2 mM À1 s À1 in the presence of AcTpm4.2 ( Figure 7D).
AcTpm1.6, AcTpm2.1, AcTpm3.1, and AcTpm4.2 containing cofilaments move approximately 53, 21, 63, and 43% faster on lawns of NM-2A than bare F-actin ( Figure 7E). Whereas k cat is reduced by 25% in the presence of AcTpm3.1 compared to Tpm3.1, both Tpm3.1 and AcTpm3.1 containing cofilaments move on lawns of NM-2A with the same velocity and approximately 63% faster than bare F-actin. With this exception iScience Article all other cofilaments containing AcTpm isoforms move faster than cofilaments containing the non-acetylated Tpm isoforms. In the case of Tpm2.1 and Tpm4.2, the changes in motor activity observed upon N-terminal acetylation correspond to the trend observed in ATP turnover measurements. Following N-terminal acetylation, similar increases in both parameters were observed for Tpm2.1 (<8%) and Tpm4.2 (>40%). In contrast, N-terminal acetylation of Tpm1.6 results in a 35% increase in sliding velocity but a 11% decrease in k cat (Figure 7; Table 2).
In the case of myosin-5A, the differences in unloaded velocity, event frequency, and run length between acetylated and nonacetylated Tpm isoforms are mostly within the margin of error, with the following exceptions. Cofilaments containing Tpm1.12 move significantly faster on lawns of myosin-5A-HMM than bare filamentous b-actin and cofilaments containing AcTpm1.12 ( Figure 8; Table 2). The percentage of filaments showing unidirectional uniform motion on lawns of myosin-5A-HMM was equal and higher than 98% for bare filaments and all cofilaments studied. Similar protection of filaments from fragmentation was observed for the acetylated and nonacetylated Tpm isoforms. The run length of cofilaments containing AcTpm1.6 or AcTpm3.1 was unchanged relative to bare filamentous b-actin and cofilaments containing the nonacetylated Tpm isoforms. Cofilaments containing Tpm2.1 showed a small but significant increase in run length that was not observed for AcTpm2.1. The run lengths of cofilaments containing Tpm4.2 and AcTpm4.2 were 8 and 12% longer, whereas those of cofilaments containing Tpm1.12 or AcTpm1.12 were approximately 15 and 20% shorter, respectively ( Figure S3).

Actin-Tpm interactions
Myosin-induced contraction has long been studied in skeletal and cardiac muscle, where thin actin filaments move along thick myosin filaments and actomyosin-based motility is regulated by Tpm-dependent cooperative on-off switching, which is mediated by the binding of Ca 2+ to the troponin-C subunit of the troponin-Tpm complex on thin filaments (Greaseri and Gergely, 1971). The binding of Tpm to F-actin has been extensively characterized for the muscle isoforms and more recently also for individual cytoskeletal Tpm isoforms (Marchenko et al., 2021;Maytum et al., 2001;Pathan-Chhatbar et al., 2018;Schmidt et al., 2015). Emerging experimental results and molecular dynamics simulations support a form-function relationship that has been referred to as Gestalt-binding (  iScience Article Gestalt-binding allows global changes in the position of Tpm cables as well as local positional perturbations caused by troponin, myosin or other actin-binding proteins and gives the A-Tpm cofilaments sufficient flexibility to allow alternative binding modes at a low-energy cost, while promoting tight cooperative coupling that is highly sensitive to allosteric trigger events such as post-translational modifications, protein-protein interactions, ligand binding, or isoform-specific amino acid substitutions (Fischer et al., 2016;Holmes and Lehman, 2008;Li et al., 2010). This provides the cell with a toolbox of modular components consisting of cofilaments containing different Tpm isoforms with and without post-translational modifications, capable of acting as mechanical sensors, selective tracks, for myosin motors and ultrasensitive switches. As mediators between chemical and mechanical signal transduction pathways, they enable cells to carry out precisely tuned reactions. The characterization of such systems requires an approach that incorporates accurate allosteric awareness overcoming difficulties in producing the correct combinations of protein isoforms with relevant post-translational modifications for the accurate determination of rate and equilibrium constants, structural features, forces, and velocities . Therefore, we used contractile filaments composed of human cytoskeletal actin, myosin, and Tpm isoforms and produced the recombinant proteins without introducing any changes to their native sequence. Amino-terminally acetylated Tpm isoforms were produced in bacteria rather than the commonly used isoforms carrying a short N-terminal extension, such as Ala-Ser, to mimic acetylation. Phalloidin labeled with tetramethylrhodamine B isothiocyanate was used exclusively to stabilize and stain F-actin for in vitro motility assays, but omitted from all other assays. Apart from the influence of post-translational modifications, Tpm binding to actin has been reported to be primarily dependent on exon usage (Cho and Hitchcock-DeGregori, 1991;Moraczewska et al., 1999;Pathan-Chhatbar et al., 2018;Schmidt et al., 2015). Variable exons 1 and 9, which specify residues contributing to the Tpm overlap complex, have been shown to affect actin affinity in the order 1b9d > 1b9a > Ac1a9a>1a9d[1a9aS1a9cy1b9c in the context of recombinant Tpm1.3 smooth muscle variants that are identical except for the terminal regions encoded by exons 1a or 1b and exons 9a, 9c, or 9d . Exon 1a is expressed in muscle and non-muscle cells, whereas exon 1b replaces exons 1a and 2 in non-muscle cells, resulting in the production of LMW Tpm isoforms. The C-terminal exon 9a is expressed in striated muscles, endothelial cells, and in the brain, exon 9c exclusively in the brain, and exon 9d in non-muscle as well as in smooth muscle cells (Dufour et al., 1998;Schevzov et al., 2011;Vrhovski et al., 2003 Tpm2.1(a.b.a.d) shows that global sequence differences between the four TPM genes are of greater importance than variable exon 6 usage. Moreover, the differences that can be attributed to exon six usage are comparable in extent with those mediated by the presence or absence of N-terminal acetylation.
The weakest binding to F-actin was observed with the use of exon 9c by .
Our investigations of the temperature-dependent dissociation and reassociation of human cytoskeletal A-Tpm cofilaments revealed Tpm isoform-specific differences in T diss , as well as differences in the extent of hysteresis exhibited by the dissociation and reassociation reactions, and the effects of N-terminal acetylation. Whereas, in the absence of N-terminal acetylation, the T diss values of the HMW isoforms Tpm1.6 and Tpm2.1 exceeded those measured for the LMW isoforms Tpm3.1 and Tpm4.2 by 1 -6 C, the opposite was true for the isoforms with N-terminal acetylation. Compared with an increase of T diss by 10 -13 C upon acetylation of Tpm3.1 and Tpm4.2, acetylation of Tpm1.6 and Tpm2.1 leads to a reduction of T diss by 1 -2 C. In line with the kinetic binding model of Tpm molecules to actin (Bareja et al., 2020), the greater thermal stability of A-Tpm can be explained by stronger end-to-end contacts of Tpm molecules. Alternative explanations include stronger coordination of Tpm assembly on actin filaments or greater flexibility of Tpm cables. Both lead to changes in gap formation, which have been described as sites of dissociation (Orzechowski et al., 2014;Schmidt et al., 2015). The number of gaps is thought to be controlled by Tpm concentration (Schmidt et al., 2015), which is consistent with our observation that changes in Tpm concentration affect the size of cooperative units. Gap formation potentially provides an additional selectivity filter for Tpm-gated access of actinbinding proteins to cytoskeletal F-actin. This is evident from differences in the exchange of free Tpm3.1 and Tpm4.2 with Tpm isoforms bound to F-actin. The much stronger effect of N-terminal acetylation on the temperature stability of A-Tpm containing LMW-Tpm isoforms compared with HMW-Tpm isoforms suggests that changes within the overlap region of Tpm cables lead to analogous differences in allosteric communication.
A comparison of our results obtained with the acetylated and non-acetylated isoforms of Tpm1.6, Tpm1.12, and Tpm4.2 (Table 1) and the reported T diss and K 50% values for the corresponding Ala-Ser-tagged isoforms reveals minor changes (Marchenko et al., 2021). Thus, Ala-Ser labeling may have an influence on allosteric communication and cooperative coupling in A-Tpm cofilaments that remains to be confirmed, but is not required for efficient binding of these cytoskeletal Tpm isoforms to F-actin.

Functional diversification of cytoskeletal A-Tpm cofilaments and A-Tpm-M complexes
Tpm-specific regulation of cytoskeletal myosin isoforms has been described as being dependent on the myosin isoform or myosin class involved (Barua et al., 2014;Clayton et al., 2014Clayton et al., , 2015Kee et al., 2015;Pathan-Chhatbar et al., 2018). It has been proposed that A-Tpm3.1 cofilaments limit or block motor function, allowing Myo1C to perform its cellular functions only in areas containing bare actin filaments (Kee et al., 2015). iScience Article A-Tpm cofilaments where they interact productively with human Myo1C by shifting motor activity from faster movement on bare F-actin to more efficient tension sensing on A-Tpm cofilaments. Such a shift is consistent with the distinct roles of Myo1C in transport, storage, and insertion of GLUT4 vesicles into the plasma membrane (Boguslavsky et al., 2012).
Based on the event frequency, run length, and unloaded velocity, actin-Tpm3.1 and actin-Tpm1.8 cofilaments were described as equal or better tracks compared with bare actin for murine myosin 5A-HMM. In contrast, actin-Tpm4.2 was described to exclude myosin-5A-HMM from productive interactions (Sckolnick et al., 2016). Our results show more subtle changes supporting graded functional adjustments as a consequence of an exchange of Tpm isoform. Under the experimental conditions used, both the rate of ATP turnover and the sliding velocity supported by human myosin-5A show significant reductions for most tested cytoskeletal Tpm isoforms with and without N-terminal acetylation. The exception is Tpm1.12 showing a significant increase in sliding velocity. Taking also into account the observed changes in run lengths, a uniformly high event frequency for all cofilament combinations examined, and the limitations of the assays used, we conclude that our results are dominated by a cofilament-specific modulation of the myosin-5A duty ratio. Therefore, exactly how motile and contractile activities play out in a cellular environment will depend on several other parameters, including local variations in motor density, external load, spatial constraints, and the presence of actin-binding auxiliary proteins.
Unlike the two other myosin isoforms tested, NM-2A shows increased enzymatic activity in the presence of most of the A-Tpm cofilaments tested. The extent of the enhancement of ATPase activity and sliding velocity varied for the different Tpm isoforms as well as with their N-terminal acetylation status. The largest changes in ATP turnover kinetics and the fastest sliding velocities were observed for A-Tpm cofilaments containing either Tpm3.1 or AcTpm3.1. Cofilaments containing AcTpm1.6 and AcTpm4.2 moved significantly faster compared to the non-acetylated isoforms and bare F-actin. Cofilaments containing AcTpm2.1 displayed a small but significant increase in velocity only over bare F-actin. These findings provide a more nuanced picture compared with the previously reported enhanced NM-2A-dependent motor activity of A-Tpm cofilaments containing LMW Tpm3.1 and Tpm4.2 and a reduced productive interaction of cofilaments containing HMW Tpm1.6 and Tpm2.1 with NM-2A (Gateva et al., 2017). Inclusion of the N-terminally acetylated isoforms increases the physiologically relevance of our results.
Collectively, our ensemble kinetics values provide accurate descriptions of the effects of Tpm isoform exchange or changes in Tpm-acetylation status on the maximum rate of ATP turnover, the apparent affinity for the filament track in the presence of ATP, and the strength of the coupling between binding to filament tracks and the acceleration of rate-limiting steps in the actomyosin ATPase cycle. Opposing changes in the catalysis of ATP turnover and motor activity can be attributed to isoform-specific allosteric coupling between the different components of A-Tpm-M complexes that is responsive to the extent of saturation at which the solution kinetics and in vitro motility experiments are performed and to the surface density and arrangement of myosin motors in the in vitro motility assays Pertici et al., 2018;Toyoshima et al., 1989;Uyeda et al., 1990). Our results show how motor properties such as maximum velocity in the absence and presence of external loads and optimal motor density for continuous unidirectional motion, previously considered specific and invariant properties of each myosin isoform, are modulated by the presence of different Tpm isoforms as well as their post-translational modification. This provides a solid and indispensable foundation for future studies aimed at discovering small molecule therapeutics for the treatment of non-muscular myosinopathies and actinopathies.

Limitations of the study
The scope of our study is limited by the allosteric nature of the system under investigation, with the existence of a large number of possible combinations of cytoskeletal actin, myosin, and Tpm isoforms. Our study is focused on the in vitro characterization of selected A-Tpm-M combinations. The examples presented in our work reveal important key aspects for the major isoforms. In addition, they define the range over which the functional properties of A-Tpm-M complexes typically vary, and they allow certain combinations of isoforms to be clustered into groups with similar properties. Overall, our work provides a starting point and framework for quantitative modeling of motor processes at the cellular level. To realize its full potential, further studies are needed that provide insights into the subcellular localization of related motor processes, the spatial distribution, density, and concentration of the individual components, and the impact of posttranslational modifications on their functional properties.

DECLARATION OF INTERESTS
The authors declare no competing interests.

INCLUSION AND DIVERSITY
While citing references scientifically relevant for this work, we also actively worked to promote gender balance in our reference list.