The actin cytoskeleton is one of the most important players in cell motility, adhesion, division, and functioning. The regulation of specific microfilament formation largely determines cellular functions. The main actin-binding protein in animal cells is tropomyosin (Tpm). The unique structural and functional diversity of microfilaments is achieved through the diversity of Tpm isoforms. In our work, we studied the properties of the cytoplasmic isoforms Tpm1.8 and Tpm1.9. The results showed that these isoforms are highly thermostable and differ in the stability of their central and C-terminal fragments. The properties of these isoforms were largely determined by the 6th exons. Thus, the strength of the end-to-end interactions, as well as the affinity of the Tpm molecule for F-actin, differed between the Tpm1.8 and Tpm1.9 isoforms. They were determined by whether an alternative internal exon, 6a or 6b, was included in the Tpm isoform structure. The strong interactions of the Tpm1.8 and Tpm1.9 isoforms with F-actin led to the formation of rigid actin filaments, the stiffness of which was measured using an optical trap. It is quite possible that the structural and functional features of the Tpm isoforms largely determine the appearance of these isoforms in the rigid actin structures of the cell cortex.

About half of the mutations that lead to hypertrophic cardiomyopathy (HCM) occur in the MYBPC3 gene. However, the molecular mechanisms of pathogenicity of point mutations in cardiac myosin-binding protein C (cMyBP-C) remain poorly understood. In this study, we examined the effects of the D75N and P161S substitutions in the C0 and C1 domains of cMyBP-C on the structural and functional properties of the C0-C1-m-C2 fragment (C0-C2). Differential scanning calorimetry revealed that these mutations disorder the tertiary structure of the C0-C2 molecule. Functionally, the D75N mutation reduced the maximum sliding velocity of regulated thin filaments in an in vitro motility assay, while the P161S mutation increased it. Both mutations significantly reduced the calcium sensitivity of the actin–myosin interaction and impaired thin filament activation by cross-bridges. D75N and P161S C0-C2 fragments substantially decreased the sliding velocity of the F-actin-tropomyosin filament. ADP dose-dependently reduced filament sliding velocity in the presence of WT and P161S fragments, but the velocity remained unchanged with the D75N fragment. We suppose that the D75N mutation alters nucleotide exchange kinetics by decreasing ADP affinity to the ATPase pocket and slowing the myosin cycle. Our molecular dynamics simulations mean that the D75N mutation affects myosin S1 function. Both mutations impair cardiac contractility by disrupting thin filament activation. The results offer new insights into the HCM pathogenesis caused by missense mutations in N-terminal domains of cMyBP-C, highlighting the distinct effects of D75N and P161S mutations on cardiac contractile function.

Tropomodulin (Tmod) is an actin-binding protein that interacts with tropomyosin and the actin filament at the pointed end. The influence of Tmod on the thin filament activation in the myocardium is not clear. We studied the interactions of Tmod1 and Tmod4 with the cardiac tropomyosin isoforms Tpm1.1 and Tpm1.2 using size-exclusion chromatography, a pull-down assay, and cross-linking with glutaraldehyde. We found that Tmod1 and Tmod4 form complexes with both Tpm1.1 and Tpm1.2, indicating durable interactions between these proteins. The effects of both Tmods on the actin–myosin interaction were studied using an in vitro motility assay. Tmod did not affect the sliding velocity of bare F-actin. Tmod1 slightly dose-dependently decreased the sliding velocity of F-actin–Tpm1.1 filaments and had no effect on the velocity of F-actin–Tpm1.2 filaments. With ventricular myosin, Tmod1 reduced the calcium sensitivity of the sliding velocity of thin filaments containing Tpm1.1 but did not affect it with filaments containing Tpm1.2. With atrial myosin, Tmod1 decreased the calcium sensitivity of the sliding velocities of thin filaments containing both Tpm1.1 and Tpm1.2. We can conclude that Tmod takes part in the regulation of actin–myosin interactions in the myocardium through interactions with Tpm. The effect of Tmod on the activation of thin filaments depends on the protein isoforms.

Meiotic errors during oocyte maturation are the primary cause of embryonic aneuploidy. Coordinated functioning of the cytoskeleton and its associated contractile proteins is essential for karyokinesis and cytokinesis. This review summarizes studies investigating the roles of actin, myosin, and kinesin in mouse and human oocyte maturation. Mechanisms underlying the involvement of contractile proteins in oocyte maturation are described. Specifically, actin participates in spindle assembly during the first and second meiotic divisions by stabilizing K-fibers. Cytoplasmic actin, together with myosin-5b and myosin-2, mediates the migration and orientation of the meiotic spindle to the periphery, driving asymmetry in human oocytes; in mouse oocytes, actin and myosin-5b are also responsible for central positioning of the nucleus in prophase I. Polar body extrusion occurs via contraction of the actomyosin contractile ring. Kinesins are also widely represented in oocyte maturation processes. In mouse oocytes, Kif11, Kif16A, and Kif16B contribute to spindle formation and stabilization, whereas KifC1 stabilizes kinetochore fibers (K-fibers). Additionally, Kif16A and Kif16B regulate cell cycle progression by modulating spindle assembly checkpoint activation and the G2–M meiotic transition, respectively. In human oocytes, HSET (kinesin-14) is responsible for spindle pole determination, and Kid (kinesin-10) is involved in chromosome alignment at the metaphase plate.