Purification of tropomyosin Br‐3 and 5NM1 and characterization of their interactions with actin

Abstract

Tropomyosins were first identified in neuronal systems in 1973. Although numerous isoforms were found and described since then, many aspects of their function and interactions remained unknown. Tropomyosin isoforms show different sorting pattern in neurogenesis. As one example, TM5NM1/2 is present in developing axons, but it is replaced by TMBr‐3 in mature neurons, suggesting that these tropomyosin isoforms contribute differently to the establishment of the functional features of the neuronal actin networks. We developed a method for the efficient purification of TMBr‐3 and TM5NM1 as recombinant proteins using bacterial expression system and investigated their interactions with actin. We found that both isoforms bind actin filaments, however, the binding of TM5NM1 was much stronger than that of TMBr‐3. TMBr‐3 and TM5NM1 modestly affected actin assembly kinetics, in an opposite manner. Consistently with the higher affinity of TM5NM1 it inhibited actin filament disassembly more efficiently than TMBr‐3. Similarly to other previously studied tropomyosins TM5NM1 inhibited the Arp2/3 complex‐mediated actin assembly. Notably, TMBr‐3 did not influence the Arp2/3 complex‐mediated polymerization. This is a unique feature of TMBr‐3, since so far it is the only known tropomyosin supporting the activity of the Arp2/3 complex, indicating that TMBr‐3 may colocalize and work simultaneously with Arp2/3 complex in neuronal cells. © 2013 Wiley Periodicals, Inc.

Tropomyosin isoforms differentially modulate the regulation of actin filament polymerization and depolymerization by cofilins

“…As expected, muscle Tpm1.1 and Tpm1.1/Tpm2.2 reduced the initial rate, whereas Tpm3.1 had no significant effect [31,44,45]. Using tropomyosins in combination with cofilins complicated the findings.…”

“…Because the lack of this modification reduces the actin affinity of Tpm1.1, we applied the N-terminal Ala-Ser extension to compensate for this defect [30]. Tpm3.1, which belongs to the class of low molecular weight isoforms, was recently shown to bind well to F-actin [31,32]; therefore, it was not modified additionally. Tpm2.2 was the component of the native tropomyosin isolated from rabbit skeletal muscle known to form heterodimers with Tpm1.1 [33].…”

“…Depending on the isoform, tropomyosin can either slow down or accelerate spontaneous actin assembly [31,44,45]. To gain more insight into the mechanisms of filament dynamics regulation, the effects of tropomyosin and cofilin isoforms on actin polymerization were analyzed by monitoring salt-induced changes in turbidity.…”

Biochemical Activities of the Wiskott-Aldrich Syndrome Homology Region 2 Domains of Sarcomere Length Short (SALS) Protein

“…5E). Tropomyosin maintains filament end dynamics (52,53), and hence it does not influence the accumulation of unassembled actin due to sequestration. Considering this, we propose that tropomyosin can inhibit only one of the two main effects of SALS-WH2 on actin dynamics.…”

Actin regulation by tropomodulin and tropomyosin in neuronal morphogenesis and function

“…As Tmods, Tpms also have isoform-specific impacts on actin polymerization rates (Janco et al, 2016; Kis-Bicskei et al, 2013; Robaszkiewicz et al, 2016) and either increase or decrease the rate. Tpms regulate actin branching by ARP2/3 (Blanchoin et al, 2001) in an isoform-specific manner (Brayford et al, 2016; Kis-Bicskei et al, 2013).…”