Competition between Tropomyosin, Fimbrin, and ADF/Cofilin drives their sorting to distinct actin filament networks

Abstract

The fission yeast actin cytoskeleton is an ideal, simplified system to investigate fundamental mechanisms behind cellular self-organization. By focusing on the stabilizing protein tropomyosin Cdc8, bundling protein fimbrin Fim1, and severing protein coffin Adf1, we examined how their pairwise and collective interactions with actin filaments regulate their activity and segregation to functionally diverse F-actin networks. Utilizing multi-color TIRF microscopy of in vitro reconstituted F-actin networks, we observed and characterized two distinct Cdc8 cables loading and spreading cooperatively on individual actin filaments. Furthermore, Cdc8, Fim1, and Adf1 all compete for association with F-actin by different mechanisms, and their cooperative association with actin filaments affects their ability to compete. Finally, competition between Fim1 and Adf1 for F-actin synergizes their activities, promoting rapid displacement of Cdc8 from a dense F-actin network. Our findings reveal that competitive and cooperative interactions between actin binding proteins help define their associations with different F-actin networks.DOI: http://dx.doi.org/10.7554/eLife.23152.001

Actin filament oxidation by MICAL1 suppresses protections from cofilin-induced disassembly

“…We first observed that some cofilin-1 domains could still assemble on Tpm-saturated filaments, and we confirmed that the mCherry-cofilin-1 and neonGreen-Tpm1.8 fluorescence signals never overlap, as previously observed (Christensen et al, 2017;Gateva et al, 2017;Jansen and Goode, 2019): cofilin-1 binding induces the unbinding of Tpm. However, the majority of cofilin-1 domains nucleate from the free barbed end (Figure 4a, central kymograph).…”

“…This phosphorylation strongly and persistently inhibits cofilin-1 as it reduces its affinity for actin filaments and slows down subsequent severing (Elam et al, 2017). Additionally, cofilin-1 activity can be restrained by tropomyosins (Tpm) (Brayford et al, 2016;Christensen et al, 2017;Gateva et al, 2017;Jansen and Goode, 2019). Tpm is a long dimeric protein which binds and saturates most actin filaments in cells (Meiring et al, 2018).…”

Capping protein insulates Arp2/3-assembled actin patches from formins

“…When CP is absent from patches, these acquire characteristics normally specific to linear actin structures: they are decorated by myosin V Myo51 and tropomyosin, which normally preferentially associates with actin cables, ring and focus [44,51,52] [7,43,[47][48][49]; they also accumulate the myosin V Myo52, which erroneously transports its cargoes to these locations. The coincidence of tropomyosin and fimbrin at patches devoid of CP is particularly striking given the previous findings that competition between these two actinbinding proteins is sufficient for their specific association with formin-and Arp2/3-assembled structure in vitro [9][10][11]. We conclude that, in absence of CP, actin patches acquire a double identity.…”

“…Finally, tropomyosin Cdc8, which stabilizes actin cables and is largely absent from actin patches in WT cells, formed foci that colocalized with Fim1 in acp2Δ cells ( Figure 6D; Movie S4). The coincidence of fimbrin and tropomyosin is particularly remarkable given recent data showing that competition between these two proteins drives their sorting to distinct Arp2/3 and forminnucleated networks, respectively [9]. Thus, actin patches assume a dual identity in absence of CP both during mating and during vegetative growth.…”

“…Third, self-assembly principles likely govern the segregation of specific actin-binding proteins to diverse structures. For instance, competition between fimbrin and tropomyosin for F-actin binding, together with their individual cooperative loading on actin filament, drives their association to distinct actin structures [9]. This is manifested in vivo by preferential association of tropomyosin to formin-assembled structures and fimbrin to Arp2/3-nucleated actin patches [10,11].…”