Formin proteins nucleate actin filaments, remaining processively associated with the fast-growing barbed ends. Although formins possess common features, the diversity of functions and biochemical activities raised the possibility that formins differ in fundamental ways. Further, a recent study suggested that profilin and ATP hydrolysis are both required for processive elongation mediated by the formin mDia1. We used total internal reflection fluorescence microscopy to observe directly individual actin filament polymerization in the presence of two mammalian formins (mDia1 and mDia2) and two yeast formins (Bni1p and Cdc12p). We show that these diverse formins have the same basic properties: movement is processive in the absence or presence of profilin; profilin accelerates elongation; and actin ATP hydrolysis is not required for processivity. These results suggest that diverse formins are mechanistically similar, but the rates of particular assembly steps vary.
Microscopy of fluorescent fusion proteins and genetic dependencies show that fission yeast assemble and constrict a cytokinetic contractile ring in a precisely timed, sequential order. More than 90 min prior to separation of the spindle pole bodies (SPB), the anillin-like protein (Mid1p) migrates from the nucleus and specifies a broad band of cortex around the equator as the division site. Between 10 min before and 2 min after SPB separation, conventional myosin-II (Myo2p), IQGAP (Rng2p), PCH protein (Cdc15p), and formin (Cdc12p) join the broad band independent of actin filaments. Over the subsequent 10 min prior to anaphase B, this broad band of proteins condenses into a contractile ring including actin, tropomyosin (Cdc8p), and alpha-actinin (Ain1p). During anaphase B, unconventional myosin-II (Myp2p) joins the ring followed by the septin (Spn1p). Ring contraction and disassembly begin 37 min after SPB separation. This spatial and temporal hierarchy provides the framework for analysis of molecular mechanisms.
Formins are large multidomain proteins required for assembly of actin cables that contribute to the polarity and division of animal and fungal cells. Formin homology-1 (FH1) domains bind profilin, and highly conserved FH2 domains nucleate actin filaments. We characterized the effects of two formins, budding yeast Bni1p and fission yeast Cdc12p, on actin assembly. We used evanescent wave fluorescence microscopy to observe assembly of actin filaments (i) nucleated by soluble formin FH1FH2 domains and (ii) associated with formin FH1FH2 domains immobilized on microscope slides. Bni1p(FH1FH2)p and Cdc12p(FH1FH2)p nucleated new actin filaments or captured the barbed ends of preformed actin filaments that grew by insertion of subunits between the immobilized formin and the barbed end of the filament. Both formins remained bound to growing actin filament barbed ends for >1,000 sec. Elongation of a filament between an immobilized formin and a second anchor point buckled filament segments as short as 0.7 m, demonstrating that polymerization of single actin filaments produces forces of >1 piconewton, close to the theoretical maximum. After buckling, further growth produced long loops that did not supercoil, suggesting that formins do not stair step along the two subunits exposed on the growing barbed end. In agreement, Arp2͞3 complex branched filaments did not rotate as they grew from formins attached to the slide surface. Formins are not mechanistically identical because barbed end elongation from Cdc12(FH1FH2)p, but not Bni1(FH1FH2)p, requires profilin. However, profilin increased the rate of Bni1(FH1FH2)p-mediated barbed end elongation from 75% to 100% of full-speed. Many cellular structures composed of actin filaments depend on formins, so the discovery that the FH2 domain of budding yeast Bni1p stimulates spontaneous polymerization of pure actin was a major advance (1, 2). Biochemical experiments in those pioneering papers and subsequent work (3-5) suggest that Bni1(FH1FH2)p nucleates filaments and then allows both the fast growing barbed end and slowly growing pointed end of the filaments to elongate.Several lines of evidence suggest that formins bind actin filament barbed ends and remain in place as filaments grow. Although Bni1(FH1FH2)p does not raise the critical concentration like a barbed end capping protein, it was observed to bind near barbed ends by immunoelectron microscopy (1) and inhibits association of capping protein with barbed ends (4, 5). The FH1FH2 domains of fission yeast formin Cdc12p tightly cap actin filament barbed ends and nucleate short filaments that grow slowly from their pointed ends (6). The actin-monomer binding protein profilin allows filaments nucleated by Cdc12(FH1FH2)p to grow at their barbed ends (6). Because Cdc12p inhibits end to end annealing of actin filaments even in the presence of profilin (6), profilin might not simply dissociate Cdc12p from barbed ends. Indeed, Higashida et al. (7) recently showed that aggregates of the mammalian formin mDia1 persistently associate with elon...
We observed live fission yeast expressing pairs of functional fluorescent fusion proteins to test the popular model that the cytokinetic contractile ring assembles from a single myosin II progenitor or a Cdc12p-Cdc15p spot. Under our conditions, the anillin-like protein Mid1p establishes a broad band of small dots or nodes in the cortex near the nucleus. These nodes mature by the addition of conventional myosin II (Myo2p, Cdc4p, and Rlc1p), IQGAP (Rng2p), pombe Cdc15 homology protein (Cdc15p), and formin (Cdc12p). The nodes coalesce laterally into a compact ring when Cdc12p and profilin Cdc3p stimulate actin polymerization. We did not observe assembly of contractile rings by extension of a leading cable from a single spot or progenitor. Arp2/3 complex and its activators accumulate in patches near the contractile ring early in anaphase B, but are not concentrated in the contractile ring and are not required for assembly of the contractile ring. Their absence delays late steps in cytokinesis, including septum formation and cell separation.
Cytokinesis in most eukaryotes requires the assembly and contraction of a ring of actin filaments and myosin II. The fission yeast Schizosaccharomyces pombe requires the formin Cdc12p and profilin (Cdc3p) early in the assembly of the contractile ring. The proline-rich formin homology (FH) 1 domain binds profilin, and the FH2 domain binds actin. Expression of a construct consisting of the Cdc12 FH1 and FH2 domains complements a conditional mutant of Cdc12 at the restrictive temperature, but arrests cells at the permissive temperature. Cells overexpressing Cdc12(FH1FH2)p stop growing with excessive actin cables but no contractile rings. Like capping protein, purified Cdc12(FH1FH2)p caps the barbed end of actin filaments, preventing subunit addition and dissociation, inhibits end to end annealing of filaments, and nucleates filaments that grow exclusively from their pointed ends. The maximum yield is one filament pointed end per six formin polypeptides. Profilins that bind both actin and poly-l-proline inhibit nucleation by Cdc12(FH1FH2)p, but polymerization of monomeric actin is faster, because the filaments grow from their barbed ends at the same rate as uncapped filaments. On the other hand, Cdc12(FH1FH2)p blocks annealing even in the presence of profilin. Thus, formins are profilin-gated barbed end capping proteins with the ability to initiate actin filaments from actin monomers bound to profilin. These properties explain why contractile ring assembly requires both formin and profilin and why viability depends on the ability of profilin to bind both actin and poly-l-proline.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.