Actin Cytoskeleton: Formins Lead the Way Dispatch

Actin Cytoskeleton: Formins Lead the Dispatch Way

E.S. Harris and H.N. Higgs The FH1 domain is proline-rich and binds to the actin monomer binding protein profilin, in addition to signaling molecules [2]. In the in vitro experiments, the authors Formins are eukaryotic proteins that potently used time-lapse microscopy to follow the growth of influence actin polymerization dynamics. Recent actin filaments (labeled with Oregon green-phalloidin) evidence strongly suggests that these proteins move from aggregates of a GST–mDia1∆N3 fusion protein, the processively with the elongating barbed ends of actin GST being labeled with an Alexa594-coupled antibody filaments. (Figure 1A,B). In this experimental set up, profilin inhibits pointed-end growth, so the actin filaments grow almost exclusively from their barbed ends. During a special sub-group session on formin proteins Higashida et al. [1] found that a mass of filaments at last December’s meeting of the American Society for often grew from a GST–mDia1∆N3 aggregate, but those Cell Biology, the buzz was all about how formins stay cases where only one filament emanated from the attached to the barbed end of an elongating actin fila- formin were the most revealing, because these could ment, apparently by processively moving with the end. be more easily resolved. Imaging of these single fila- Many of us were very pleased with ourselves and our in ments by speckle microscopy — in which uneven fluo- vitro biochemical data which suggested that formins rescent labeling of filaments enables one to track have this property. Then, a voice came from the back of filament growth — showed that the actin elongates the room which cooled enthusiasm somewhat. In her away from the GST–mDia1∆N3, indicating that characteristically forthright way, Clare Waterman-Storer monomers add on to the barbed end despite the bound said: “You guys really think this proves processive formin. If GST–mDia1∆N3 molecules were associating movement? I won’t believe it until I see it happening”. and dissociating from the actin filaments during elon- Now, however, a paper by Higashida et al. [1] has gone gation, the Alexa594-labeled spot would rapidly diffuse a long way towards assuaging these doubts. away from the elongating end. Formin proteins are widely expressed in eukaryotes, For the in vivo experiments, Higashida et al. [1] with most species having multiple formin genes [2,3]. expressed an EGFP–mDia1∆N3 fusion protein at low While formins are generally large proteins, of more than levels in Xenopus fibroblasts and observed its 1000 amino acids, the crucial region for effects on actin movement by live cell imaging. This construct moves is the formin homology 2 (FH2) domain, of about 400 rapidly and in a highly directional manner within amino acids, which appears dimeric in all formins fibroblasts, often directed toward the cell periphery examined so far [4–7]. Depending on the formin, the (Figure 1C,D). The movement is not simply a result of overall effect on actin polymerization in vitro can vary normal actin network flow, as the EGFP–mDia1∆N3 greatly (see below). But all FH2 domains studied bio- spots were seen to move at speeds two orders of chemically have two common features: they slow either magnitude faster than lamellar flow — 2.0 µm sec–1 association or dissociation of actin monomers from versus 0.025 µm sec–1. EGFP–mDia1∆N3 spots actin filament barbed ends; and they potently block accumulated at the tips of filopodia-like protrusions, complete capping of the barbed end by capping towards which the barbed ends of actin filaments are protein [4,5,7]. oriented. Interestingly, a construct lacking the FH1 These two characteristics suggest that formins bind domain was found to have a much slower rate of at or very near the barbed end of an actin filament. Fur- movement (0.13 µm sec–1). thermore, they must be able to move with a barbed end Treatment with the actin-filament-binding drug as it elongates, because in most cases actin monomers cytochalasin D abruptly stopped the movement of can still add to a barbed end, even though the effect of EGFP–mDia1∆N3 spots. Cytochalasin D binds the the the formin on elongation rate or capping protein is con- barbed end of an actin filament, preventing elongation. stant. Various in vitro kinetic results suggest that formins These results suggest that the observed movement of move processively with the barbed end, as opposed to mDia1∆N3 is due to its association with the elongating dissociating and re-associating every time an actin filament, because when barbed end elongation is pre- monomer adds to the filament. Still, one is always a little vented, mDia1∆N3 movement halts. The fact that spots uneasy without direct visual evidence. Higashida et al. stop moving completely, and do not switch to Brown- [1] have now provided such evidence with two different ian motion, suggests that the mDia1∆N3 molecules experiments, one in vitro and the other in cells. remain attached to the agent that causes their directed For these experiments, Higashida et al. [1] used a movement — presumably actin filaments. In contrast, construct containing the FH1 and FH2 domains of the treatment of the cells with the actin-monomer-seques- mammalian formin mDia1, which they called mDia1∆N3. tering molecule Latrunculin A causes a gradual decrease in the rate of directed movement of Department of Biochemistry, Dartmouth Medical School, EGFP–mDia1∆N3 spots. The interpretation of this is Hanover, New Hampshire 03755, USA. that the drug causes a gradual decrease in the level of E-mail: [email protected] polymerization-competent monomers. Current Biology R521

Figure 1. The formin mDia1 associates with the barbed end of an actin filament. AB (A) Schematic of in vitro assay. Direction of GST–mDia1∆N3, bound indirectly to fluo-

YYYY growth rescent antibodies, stays at the barbed YYY end of an elongating actin filament. (B) GST–mDia1∆N3 was labeled using an Coverslip anti-GST antibody and Alexa594-sec- ondary antibody. Labeled GST–mDia1∆N3 (red) was mixed with actin (green) and profilin and absorbed to poly-L-lysine coated C D coverslips. Filament growth was observed by time-lapse microscopy (scale bar 5 µm, time indicated in minutes). (C) Schematic of mDia1’s interaction with actin filaments in cells. mDia1 binds at the barbed end of Cell actin filaments, protecting the barbed end membrane from the inhibitory effects of capping protein. (D) XTC cell transiently expressing EGFP–mDia1∆N3. Cells were fixed after mDia1 absorption on poly-L-lysine glass cover- GST slips and stained with Texas Red phal- Capping protein loidin. mDia1∆N3 (green) accumulates both Goat anti-GST Y at filopodia tips and on actin filaments (red) Alexa594 anti-Goat Y µ Barbed within the body of the cell. Scale bar 5 m. Actin filament (Images B and D kindly provided by end Chiharu Higashida and Naoki Watanabe.) Current Biology

The overall picture drawn from these experiments is domains project in the barbed end direction. Complete that mDia1∆N3 stays associated with the barbed end of inhibition of capping protein is due to the fact that the an elongating actin filament. Even brief dissociation from major interaction between capping protein and the the filament would be expected to result in rapid diffu- barbed end appears to be at the interface between the sion away from the filament. Given the similar basic two barbed end actin subunits [8], which is probably on properties of formins — their common ability to inhibit the filament side. capping protein and to modify the barbed end elongation The second question is: do all formins have the same rate — it is probable that most or all formins share this overall effect on actin polymerization? Current results ability to move processively at the barbed end. suggest not. Of the four formins for which published These findings also raise a number of questions, two biochemical results are available, mDia1 is a potent of which will be addressed here. First, what is the mech- nucleator, capable of assembling filaments de novo anism of this processive barbed end movement? The general model that has been floated by several groups A End binding is that the dimeric nature of the FH2 domain allows formins to ‘walk’ with the elongating barbed end, such that one monomer of the FH2 dimer can release and re- bind further toward the barbed end, while the other stays tightly bound [4–7]. The question then arises: in what manner is the FH2 domain bound at the barbed end? This question is complicated by the fact that different formins have different effects on the rate of barbed B end elongation. Of the four formins studied biochemi- End-side binding cally, fission yeast Cdc12 completely blocks barbed end elongation in the absence of profilin, mouse FRLα slows elongation by 80%, budding yeast Bni1p slows elongation by 25–50%, and mouse mDia1 does not slow elongation at all. Where measured, this inhibition of elongation is a high affinity effect. At the same time, all of these formins appear to competitively block access of capping protein to barbed ends with high affinity. Thus, there is a disconnect between formin’s effect Current Biology on monomer addition and its effect on capping protein. Our proposal is that FH2 domains bind to the sides of Figure 2. Models for the binding of formins to the barbed end actin subunits at the filament barbed end, as opposed of an actin filament. (A) End binding model. A formin dimer (red oval) binds to the to binding the barbed end surface itself (Figure 2). Dif- exposed terminal surface of the actin filament’s barbed end ferential occlusion of the barbed end to monomers (green). (B) End-side binding model. A formin dimer binds to the results from different degrees to which specific FH2 sides of barbed end subunits. Dispatch R522

from monomers [9]. Bni1p and Cdc12 appear capable 12. Kovar, D.R., Kuhn, J.R., Tichy, A.L., and Pollard, T.D. (2003). The of nucleation as well, albeit perhaps not as potently as fission yeast cytokinesis formin Cdc12p is a barbed end actin α filament capping protein gated by profilin. J. Cell Biol. 161, 875-887. mDia1 [10–12]. FRL appears to be a different kettle of 13. Pollard, T.D., Blanchoin, L., and Mullins, R.D. (2000). Molecular fish altogether: nucleation by FRLα is weak at best, and mechanisms controlling actin filament dynamics in nonmuscle cells. its ability to accelerate polymerization from monomers Annu. Rev. Biophys. Biomol. Struct. 29, 545-576. is thought to be due to its severing ability, a property 14. Feierbach, B., and Chang, F. (2001). Roles of the fission yeast formin for3p in cell polarity, actin cable formation, and symmetric not shown by mDia1 [7]. So while all formins might bind cell division. Curr. Biol. 11, 1656-1665. barbed ends, their actual effects on actin polymeriza- 15. Evangelista, M., Pruyne, D., Amberg, D.C., Boone, C., and Bretscher, tion may vary. Our phylogenetic analysis suggests that A. (2002). Formins direct Arp2/3-independent actin filament assembly to polarize cell growth in yeast. Nat. Cell Biol. 4, 32-41. mammals have 15 genes for proteins with FH2 domains, which fall into seven groups, and our initial biochemical analysis of one novel FH2 domain shows it to be similar to mDia1 in that it is a potent nucleator (unpublished observations). Despite these differences, inhibition of capping protein seems a constant feature of formins. This property might be most important to cells. The high cellular abundance of capping protein, and its high affinity for barbed ends, means that a cellular actin filament is capped in less than one second in the absence of mechanisms to prevent capping [13]. This short window for elongation means that filaments over a few hundred nanometers in length should be very rare. Yet, many cells have populations of filaments several microns in length, some of the most notable being filopodia and microvilli, as well as actin cables in yeast. The formins Bni1p (budding yeast) and For3p (fission yeast) are nec- essary for actin cable formation, and Bni1p localizes to cable barbed ends [14,15]. Thus, a common cellular role for formins might be their ability to allow extended elongation in the presence of capping protein. In any case, the results of Higashida et al. [1] provide compelling evidence for processive movement of formins with filament barbed ends. At least, that is, until the next American Society for Cell Biology meeting. References 1. Higashida, C., Miyoshi, T., Fujita, A., Oceguera-Yanez, F., Monypenny, J., Andou, Y., Narumiya, S., and Watanabe, N. (2004). Actin polymerization-driven molecular movement of mDia1 in living cells. Science 303, 2007-2010. 2. Wallar, B.J., and Alberts, A.S. (2003). The formins: active scaffolds that remodel the cytoskeleton. Trends Cell Biol. 13, 435-446. 3. Zigmond, S.H. (2004). Formin-induced nucleation of actin filaments. Curr. Opin. Cell Biol. 16, 99-105. 4. Zigmond, S.H., Evangelista, M., Boone, C., Yang, C., Dar, A.C., Sicheri, F., Forkey, J., and Pring, M. (2003). Formin leaky cap allows elongation in the presence of tight capping proteins. Curr. Biol. 13, 1820-1823. 5. Moseley, J.B., Sagot, I., Manning, A.L., Xu, Y., Eck, M.J., Pellman, D., and Goode, B.L. (2004). A conserved mechanism for Bni1- and mDia1- induced actin assembly and dual regulation of Bni1 by Bud6 and profilin. Mol. Biol. Cell 16, 896-907. 6. Xu, Y., Moseley, J., Sagot, I., Poy, F., Pellman, D., Goode, B.L., and Eck, M.J. (2004). Crystal Structures of a formin homology-2 domain reveal a tethered dimer architecture. Cell 116, 711-723. 7. Harris, E.S., Li, F., and Higgs, H.N. (2004). The mouse formin, FRLa, slows actin filament barbed end elongation, competes with capping protein, accelerates polymerization from monomers, and severs filaments. J. Biol. Chem. 279, 20076-20087. 8. Wear, M.A., Yamashita, A., Kim, K., Maeda, Y., and Cooper, J.A. (2003). How capping protein bind the barbed end of the actin filament. Curr. Biol. 13, 1531-1537. 9. Li, F., and Higgs, H.N. (2003). The mouse formin mDia1 is a potent actin nucleation factor regulated by autoinhibition. Curr. Biol. 13, 1335-1340. 10. Pruyne, D., Evangelista, M., Yang, C., Bi, E., Zigmond, S., Bretscher, A., and Boone, C. (2002). Role of formins in actin assembly: nucleation and barbed-end association. Science 297, 612-615. 11. Sagot, I., Rodal, A.A., Moseley, J., Goode, B.L., and Pellman, D. (2002). An actin nucleation mechanism mediated by Bni1 and profilin. Nat. Cell Biol. 8, 626-631.