Formins are actin polymerization factors that elongate unbranched actin filaments at the barbed end. Rho family GTPases activate Diaphanous-related formins through the relief of an autoregulatory interaction. The crystal structures of the N-terminal domains of human FMNL1 and FMNL2 in complex with active Cdc42 show that Cdc42 mediates contacts with all five armadillo repeats of the formin with specific interactions formed by the Rho-GTPase insert helix. Mutation of three residues within Rac1 results in a gain-of-function mutation for FMNL2 binding and reconstitution of the Cdc42 phenotype in vivo. Dimerization of FMNL1 through a parallel coiled coil segment leads to formation of an umbrella-shaped structure that—together with Cdc42—spans more than 15 nm in diameter. The two interacting FMNL–Cdc42 heterodimers expose six membrane interaction motifs on a convex protein surface, the assembly of which may facilitate actin filament elongation at the leading edge of lamellipodia and filopodia.
The human mutant cardiac α-actins p.A295S or p.R312H and p.E361G, correlated with hypertrophic or dilated cardiomyopathy, respectively, were expressed by the baculovirus/Sf21 insect cell system and purified to homogeneity. The purified cardiac actins maintained their native state but showed differences in Ca2+-sensitivity to stimulate the myosin-subfragment1 ATPase. Here we analyzed the interactions of these c-actins with actin-binding and -modifying proteins implicated in cardiomyocyte differentiation. We demonstrate that Arp2/3 complex and the formin mDia3 stimulated the polymerization rate and extent of the c-actins, albeit to different degrees. In addition, we tested the effect of the MICAL-1 monooxygenase, which modifies the supramolecular actin organization during development and adaptive processes. MICAL-1 oxidized these c-actin variants and induced their de-polymerization, albeit at different rates. Transfection experiments using MDCK cells demonstrated the preferable incorporation of wild type and p.A295S c-actins into their microfilament system but of p.R312H and p.E361G actins into the submembranous actin network. Transduction of neonatal rat cardiomyocytes with adenoviral constructs coding HA-tagged c-actin variants showed their incorporation into microfilaments after one day in culture and thereafter into thin filaments of nascent sarcomeric structures at their plus ends (Z-lines) except the p.E361G mutant, which preferentially incorporated at the minus ends.
Human wild type (wt) cardiac α-actin and its mutants p.A295S or p.R312H and p.E361G correlated with hypertrophic or dilated cardiomyopathy, respectively, were expressed by using the baculovirus/Sf21 insect cell system. The c-actin variants inhibited DNase I, indicating maintenance of their native state. Electron microscopy showed the formation of normal appearing actin filaments though they showed mutant specific differences in length and straightness correlating with their polymerization rates. TRITC-phalloidin staining showed that p.A295S and p.R312H exhibited reduced and the p.E361G mutant increased lengths of their formed filaments. Decoration of c-actins with cardiac tropomyosin (cTm) and troponin (cTn) conveyed Ca2+-sensitivity of the myosin-S1 ATPase stimulation, which was higher for the HCM p.A295S mutant and lower for the DCM p.R312H and p.E361G mutants than for wt c-actin. The lower Ca2+-sensitivity of myosin-S1 stimulation by both DCM actin mutants was corrected by the addition of levosimendan. Ca2+-dependency of the movement of pyrene-labeled cTm along polymerized c-actin variants decorated with cTn corresponded to the relations observed for the myosin-S1 ATPase stimulation though shifted to lower Ca2+-concentrations. The N-terminal C0C2 domain of cardiac myosin-binding protein-C increased the Ca2+-sensitivity of the pyrene-cTM movement of bovine, recombinant wt, p.A295S, and p.E361G c-actins, but not of the p.R312H mutant, suggesting decreased affinity to cTm.
The human mutant cardiac α-actins p.A295S or p.R312H (plus p.R312K) and p.E361G correlated with hypertrophic or dilative cardiomyopathy, respectively, were expressed by using the baculovirus/Sf21 insect cell system. After purification their biochemical and cell biological properties were analysed and compared to wild type (wt) cardiac actin identically obtained or conventionally isolated from bovine hearts. DNase I inhibition and their polymerization behaviour indicated that all c-α-actins had maintained their native state. Cardiomyopathy type specific differences were observed except for the p.R312K mutant, which behaved like wt c-α-actin. The extent of myosin-S1 ATPase stimulation by the c-actin variants and its Ca2+-sensitivity after decoration with tropomyosin (cTm) and troponin complex (cTn) varied being highest for the HCM p.A295S and lower for both DCM mutants. Similar Ca2+-sensitivity differences were observed by recording the fluorescence increase of pyrene-cTm in the absence or presence of myosin-S1 and/or the actin-binding N-terminal fragment of cardiac myosin binding protein C (N-cMyBP-C). Transfection experiments showed the incorporation of the c-actin variants into existing cytoskeletal elements of non-muscle cells. Wt and p.A295S c-α-actin preferably incorporated into the microfilament system and p.R312H and p.E361G into the submembranous actin network of MDCK cells. Transduction of neonatal rat cardiomyocytes with adenoviral constructs coding for HA-tagged c-α-actins showed their incorporation into thin filaments of nascent sarcomeric structures at their plus ends (Z-lines) except the p.E361G mutant, which preferably incorporated at the minus ends. Our data indicate functional differences of the c-α-actins that may be causative for the different cardiomyopathy phenotypes.
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