Cell-cycle regulation of formin-mediated actin cable assembly

Abstract

Assembly of appropriately oriented actin cables nucleated by formin proteins is necessary for many biological processes in diverse eukaryotes. However, compared with knowledge of how nucleation of dendritic actin filament arrays by the actin-related protein-2/3 complex is regulated, the in vivo regulatory mechanisms for actin cable formation are less clear. To gain insights into mechanisms for regulating actin cable assembly, we reconstituted the assembly process in vitro by introducing microspheres functionalized with the C terminus of the budding yeast formin Bni1 into extracts prepared from yeast cells at different cell-cycle stages. EM studies showed that unbranched actin filament bundles were reconstituted successfully in the yeast extracts. Only extracts enriched in the mitotic cyclin Clb2 were competent for actin cable assembly, and cyclin-dependent kinase 1 activity was indispensible. Cyclin-dependent kinase 1 activity also was found to regulate cable assembly in vivo. Here we present evidence that formin cellcycle regulation is conserved in vertebrates. The use of the cablereconstitution system to test roles for the key actin-binding proteins tropomyosin, capping protein, and cofilin provided important insights into assembly regulation. Furthermore, using mass spectrometry, we identified components of the actin cables formed in yeast extracts, providing the basis for comprehensive understanding of cable assembly and regulation.Cdk1 | three-dimensional structured-illumination microscopy | actin nucleation E ukaryotic cells contain populations of actin structures with distinct architectures and protein compositions, which mediate varied cellular processes (1). Understanding how F-actin polymerization is regulated in time and space is critical to understanding how actin structures provide mechanical forces for corresponding biological processes. Branched actin filament arrays, which concentrate at sites of clathrin-mediated endocytosis (2, 3) and at the leading edge of motile cells (4), are nucleated by the actin-related protein-2/3 (Arp2/3) complex. In contrast, bundles of unbranched actin filaments, which sometimes mediate vesicle trafficking or form myosin-containing contractile bundles, often are nucleated by formin proteins (5-14).Much has been learned about how branched actin filaments are polymerized by the Arp2/3 complex and how these filaments function in processes such as endocytosis (2, 15). In contrast, relatively little is known about how actin cables are assembled under physiological conditions. In previous studies, branched actin filaments derived from the Arp2/3 complex have been reconstituted using purified proteins (16)(17)(18)(19) or cellular extracts (20-25). When microbeads were coated with nucleation-promoting factors for the Arp2/3 complex and then were incubated in cell extracts, actin comet tails were formed by sequential actin nucleation, symmetry breaking, and tail elongation. Importantly, the motility behavior of F-actin assembled by the Arp2/3 complex using defined, purified pr...

Deconstructing formin-dependent actin cable assembly

“…We now return to the finding of Miao et al (2), that only extracts from mitotically arrested cells can drive cable formation from Bni1 FH1-COOH-coated beads. Supporting this finding, inhibition of the master cellcycle regulator Cdk1 completely abolished the ability of mitotic extracts to assemble actin cables.…”

“…Finally, Miao et al (2) show that their results may be generally applicable: beads coated with the FH1-COOH region of mouse formin mDia2 can drive cable assembly in extracts made from Xenopus cells arrested in mitosis, but not when made from interphase cells. Thus, the door to a detailed biochemical analysis of factors regulating formin-dependent actin assembly has been thrown open, and we can look forward to how this approach, together with in vivo analyses, will shed light on the factors that work with these important actin nucleators.…”

“…Formins are present in all eukaryoteshumans have 15 different ones-so understanding their biology is of significant importance. Miao et al (2) have now established a formin-dependent actin cable assembly reaction using yeast cell extracts that will permit a dissection of how formin activity is regulated in vivo.…”

“…The FH1-COOH region of Bni1 both nucleates actin assembly and elongates the nascent filament; therefore, Miao et al (2) attached this region to beads and added yeast extracts in the expectation of driving formin-dependent actin assembly (Fig. 1C).…”

Reconstituting the actin cytoskeleton at or near surfaces in vitro

“…Use of cell extracts also permitted other exciting developments such as the reconstruction of complex actin structures like the cleavage furrow in cytokinesis [34]. Recent advances make possible the production of mutant extracts to study individual proteins while retaining the complexity of the cell cytosol and the preparation of staged extracts to examine how actin assembly varies with the cell cycle [35,36].…”