Multicomponent regulation of actin barbed end assembly by twinfilin, formin and capping protein

Abstract

Cells control actin assembly by regulating reactions at actin filament barbed ends. Formins accelerate elongation, capping protein (CP) arrests growth and twinfilin promotes depolymerization at barbed ends. How these distinct activities get integrated within a shared cytoplasm is unclear. Using microfluidics-assisted TIRF microscopy, we find that formin, CP and twinfilin can simultaneously bind filament barbed ends. Three‑color, single-molecule experiments reveal that twinfilin cannot bind barbed ends occupied by formin unless CP is present. This trimeric complex is short-lived (~1 s), and results in dissociation of CP by twinfilin, promoting formin-based elongation. Thus, the depolymerase twinfilin acts as a pro-formin pro-polymerization factor when both CP and formin are present. While one twinfilin binding event is sufficient to displace CP from the barbed-end trimeric complex, ~31 twinfilin binding events are required to remove CP from a CP-capped barbed end. Our findings establish a paradigm where polymerases, depolymerases and cappers together tune actin assembly.

Cyclase-associated protein is a pro-formin anti-capping processive depolymerase of actin barbed and pointed ends

“…A SNAP-tagged construct of mouse capping protein (SNAP-CP) was expressed and labeled with benzylguanine functionalized green-excitable (549-CP) fluorescent dye. As seen previously, SNAP-tagging and labelling did not alter CP’s interactions with the barbed-end [40, 41]. Alexa-488 labeled actin filaments with free barbed ends were transiently exposed to 549-CP in presence of profilin and G-actin (Fig.…”

“…We then asked if Srv2/CAP altered barbed-end processivity of formin. Since processivity is influenced by actin labelling fraction, elongation rate and presence of profilin [42], we employed an alternative strategy in (absence of profilin and G-actin) which we and others have previously used to study processivity of formin (Fig 3E) [38, 40, 42]. Actin filaments were nucleated by exposing coverslip-anchored formins to a solution containing fluorescent actin monomers and profilin (Fig.…”

“…Thus, CAP alters barbed-end residence time of elongators (formin), blockers (capping protein) and depolymerases (twinfilin). In future, it will be interesting to investigate if CAP might also co-localize with formin-CP-twinfilin complex and influence the dynamics of this multiprotein barbed-end complex [40].…”

Understanding the Role of Filamentous Actin in Food Quality: From Structure to Application

“…Meanwhile, in order to prevent further polymerization or depolymerization of actin monomers, the barbed end and pointed end of microfilaments can combine with different capping proteins respectively to keep the length of microfilaments unchanged. 29,30 This phenomenon of which is called "treadmilling". 26 The structural differences and specific interactions between actin monomers at the barbed and pointed ends are crucial for regulating the dynamics of actin polymerization and filament formation.…”

“…At the same time, the length of the microfilament basically remains unchanged, that is, the equilibration phase. Meanwhile, in order to prevent further polymerization or depolymerization of actin monomers, the barbed end and pointed end of microfilaments can combine with different capping proteins respectively to keep the length of microfilaments unchanged. , This phenomenon of which is called “treadmilling” …”