Nanostructured Self-Assembly of Inverted Formin 2 (INF2) and F-Actin–INF2 Complexes Revealed by Atomic Force Microscopy
- Shivani Sharma ,
- Elena E. Grintsevich ,
- JungReem Woo ,
- Pinar S. Gurel ,
- Henry N. Higgs ,
- Emil Reisler , and
- James K. Gimzewski
Abstract

Self-organization of cytoskeletal proteins such as actin and tubulin into filaments and microtubules is frequently assisted by the proteins binding to them. Formins are regulatory proteins that nucleate the formation of new filaments and are essential for a wide range of cellular functions. The vertebrate inverted formin 2 (INF2) has both actin filament nucleating and severing/depolymerizing activities connected to its ability to encircle actin filaments. Using atomic force microscopy, we report that a formin homology 2 (FH2) domain-containing construct of INF2 (INF2-FH1-FH2-C or INF2-FFC) self-assembles into nanoscale ringlike oligomeric structures in the absence of actin filaments, demonstrating an inherent ability to reorganize from a dimeric to an oligomeric state. A construct lacking the C-terminal region (INF2-FH1-FH2 or INF2-FF) also oligomerizes, confirming the dominant role of FH2-mediated interactions. Moreover, INF2-FFC domains were observed to organize into ringlike structures around single actin filaments. This is the first demonstration that formin FH2 domains can self-assemble into oligomers in the absence of filaments and has important implications for observing unaveraged decoration and/or remodeling of filaments by actin binding proteins.
Introduction
Figure 1

Figure 1. Schematic representation of the domain organization of inverted formin 2 (INF2) showing regions important for actin binding and protein–protein interactions. Formin constructs used in this study include INF2-FH1-FH2-C, also termed INF2-FFC, and INF2-FH1-FH2 (INF2-FF). N and C denote the N- and C-termini, respectively, of protein sequences. Other abbreviations are DID (diaphanous inhibitory domain), FH1 (formin homology 1), FH2 (formin homology 2), and DAD (diaphanous autoregulatory domain).
Experimental Section
Protein Preparation
AFM Imaging of Formin and Actin–Formin Complexes
Results and Discussion
Structure of Free INF2-FFC Homodimers and Oligomers
Figure 2

Figure 2. Structural evidence of self-assembly of INF2-FFC at various concentrations. At low concentrations (0.02 μM), INF2-FFC forms ring-shaped homodimers. Representative AFM images of free INF2-FFC showing self-assembled round structures likely formed by dimerization of FH2 domains of INF2-FFC. Schematic representations are shown in the insets. (a) Single INF2-FFC dimer. (b) Compact and closed round dimers were observed also without a hollow core. (c) The central core region attached to the dimeric ring periphery via two small extensions (diamond arrows), which corresponds potentially to the unstructured FH1 domain. (d) An example of the INF2-FFC dimer in the open configuration. The FH2 domain within the INF2-FFC dimer can potentially dissociate (open) and reassociate (close) to form trimers as well as oligomers [at a higher surface concentration (0.2 μM)]. (e) AFM image of INF2-FFC in oligomeric configurations (0.2 μM INF2-FFC). Representative images of the INF2-FFC dimer, trimer, and tetramer (f–h, respectively) and their corresponding schematic representations (i–iii, respectively). At higher concentrations (2 μM), INF2-FFC assembles into tubular ringlike structures, as marked with a white arrow in part i. Other close-up views are shown in parts i–iv of panel j.
| INF2-FFC | INF2-FF | ||||
|---|---|---|---|---|---|
| concentration (μM) | 0.02 | 0.2 | 2 | 0.02 | 0.2 |
| organization | dimers | oligomers | oligomers or stacked dimers | dimers | oligomers |
| width (nm) | 22 ± 0.1 (closed) | 45 ± 2 | – | 22 ± 0.1 (closed) | 44 ± 1 to 56 ± 2 |
| 27 ± 0.4 (open) | 54.6 ± 0.8 | 26 ± 1.6 (open) | |||
| cross-section height (nm) | 0.2 | 0.3 | ≥0.3 | 0.2 | 0.3 |
Structure of Free INF2-FF Homodimers and Oligomers
Figure 3

Figure 3. AFM images showing free INF2-FF self-organization configurations. The central core region observed as a dense mass (dashed white arrows) within the oligomeric ring periphery in panel a–c. The central dense core appears to be similar to that observed for INF2-FFC dimers and oligomers, suggesting that it comes from the unstructured FH1 domain.
Figure 4

Figure 4. Structure of INF2-FFC and F-actin complexes. AFM image of INF2-FFC and F-actin complexes (at a 1:1 molar ratio) revealing ringlike binding around actin filaments. (a) Close-up of the INF2-FFC–F-actin complex displaying detailed orientations of adjacent INF2-FFC homodimer rings assembled around F-actin. Notably, there is 5–10° occasional bending of adjacent units, indicating flexibility or strain across the bound complex. (b) 3D side view (500×, 500 nm) showing the stacklike arrangement of INF2-FFC over F-actin. (c) Tubular structure spacing shown schematically.
Structure of F-Actin–INF2-FH1-FH2-C (INF2-FFC) Complexes
Figure 5

Figure 5. Structure of INF2-FF and F-actin complexes. AFM image of a 1:1 ratio of INF2-FF and F-actin complexes revealing ringlike binding around actin filaments similar to that of INF2-FFC and F-actin complexes.
Figure 6

Figure 6. Schematic representation of plausible orientations of INF2-FFC homodimers over F-actin. (a) Adjacent INF2-FFC dimeric rings interact in an end-to-end manner to form a spiral coil structure. (b) Neighboring homodimers make longitudinal contact with adjacent F-actin-bound dimers to form continuous structure without any gaps or with small angle gaps and/or openings. (c) Combinations of regions as in panels a and b, along the length of F-actin. Alternatively, dimers may reassociate to form individual dimer rings stacked along the length of the filament with or without gap openings.
Conclusions
Supporting Information
AFM imaging data. This material is available free of charge via the Internet at http://pubs.acs.org.
Terms & Conditions
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Acknowledgment
Support for this work from MEXT WPI Program International Center for Materials Nanoarchitectonics (MANA), Japan, is acknowledged. P.S.G. and H.N.H. were supported by National Institutes of Health (NIH) Grant R01 GM069818, and E.E.G. and E.R. were supported by NIH Grant R01 GM077190. We acknowledge the use of the NPC core facility supported by CNSI at UCLA.
References
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- 28Kuznetsov, Y. G.; Victoria, J. G.; Robinson, W. E.; McPherson, A. Atomic force microscopy investigation of human immunodeficiency virus (HIV) and HIV-Infected lymphocytes J. Virol. 2003, 77 (22) 11896– 11909[Crossref], [PubMed], [CAS], Google Scholar28https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXovVansLo%253D&md5=9b88c7e8aa28d09d07b47f3a853c499eAtomic force microscopy investigation of human immunodeficiency virus (HIV) and HIV-infected lymphocytesKuznetsov, Y. G.; Victoria, J. G.; Robinson, W. E., Jr.; McPherson, A.Journal of Virology (2003), 77 (22), 11896-11909CODEN: JOVIAM; ISSN:0022-538X. (American Society for Microbiology)Isolated human immunodeficiency virus (HIV) and HIV-infected human lymphocytes in culture have been imaged for the first time by at. force microscopy (AFM). Purified virus particles spread on glass substrates are roughly spherical, reasonably uniform, though pleomorphic in appearance, and have diams. of about 120 nm. Similar particles are also seen on infected cell surfaces, but morphologies and sizes are considerably more varied, possibly a reflection of the budding process. The surfaces of HIV particles exhibit "tufts" of protein, presumably gp120, which do not phys. resemble spikes. The protein tufts, which no. about 100 per particle, have av. diams. of about 200 Å, but with a large variance. They likely consist of arbitrary assocns. of small nos. of gp120 monomers on the surface. In examg. several hundred virus particles, we found no evidence that the gp120 monomers form threefold sym. trimers. Although >95% of HIV-infected H9 lymphocytic cells were producing HIV antigens by immunofluorescent assay, most lymphocytes displayed few or no virus on their surfaces, while others were almost covered by a hundred or more viruses, suggesting a dependence on cell cycle or physiol. HIV-infected cells treated with a viral protease inhibitor and their progeny viruses were also imaged by AFM and were indistinguishable from untreated virions. Isolated HIV virions were disrupted by exposure to mild neutral detergents (Tween 20 and CHAPS) at concns. from 0.25 to 2.0%. Among the products obsd. were intact virions, the remnants of completely degraded virions, and partially disrupted particles that lacked sectors of surface proteins as well as virions that were split or broken open to reveal their empty interiors. Capsids contg. nucleic acid were not seen, suggesting that the capsids were even more fragile than the envelope and were totally degraded and lost. From these images, a good est. of the thickness of the envelope protein-membrane-matrix protein outer shell of the virion was obtained. Treatment with even low concns. (<0.1%) of sodium dodecyl sulfate completely destroyed all virions but produced many interesting products, including aggregates of viral proteins with strands of nucleic acid.
- 29Ikai, A.; Yoshimura, K.; Arisaka, F.; Ritani, A.; Imai, K. Atomic-Force Microscopy of Bacteriophage-T4 and Its Tube-Baseplate Complex FEBS Lett. 1993, 326 (1-3) 39– 41Google ScholarThere is no corresponding record for this reference.
- 30McPherson, A.; Kuznetsov, Y. G. Atomic Force Microscopy Investigation of Viruses Methods Mol. Biol. 2011, 736, 171– 195[Crossref], [PubMed], [CAS], Google Scholar30https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XktV2rs7s%253D&md5=4baa9d6cdae939598742ed659eedd300Atomic force microscopy investigation of virusesMcPherson, Alexander; Kuznetsov, Yurii G.Methods in Molecular Biology (New York, NY, United States) (2011), 736 (Atomic Force Microscopy in Biomedical Research), 171-195CODEN: MMBIED; ISSN:1064-3745. (Springer)Atomic force microscopy (AFM) has proven to be a valuable approach to delineate the architectures and detailed structural features of a wide variety of viruses. These have ranged from small plant satellite viruses of only 17 nm to the giant mimivirus of 750 nm diam., and they have included diverse morphologies such as those represented by HIV, icosahedral particles, vaccinia, and bacteriophages. Because it is a surface technique, it provides images and information that are distinct from those obtained by electron microscopy, and in some cases, at even higher resoln. By enzymic and chem. dissection of virions, internal structures can be revealed, as well as DNA and RNA. The method is relatively rapid and can be carried out on both fixed and unfixed samples in either air or fluids, including culture media. It is nondestructive and even non-perturbing. It can be applied to individual isolated virus, as well as to infected cells. AFM is still in its early development and holds great promise for further investigation of biol. systems at the nanometer scale.
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- 32Sharma, S.; Grintsevich, E. E.; Phillips, M. L.; Reisler, E.; Gimzewski, J. K. Atomic force microscopy reveals drebrin induced remodeling of F-actin with subnanometer resolution Nano Lett. 2011, 11 (2) 825– 827
- 33Campellone, K. G.; Welch, M. D. A nucleator arms race: Cellular control of actin assembly Nat. Rev. Mol. Cell Biol. 2010, 11 (4) 237– 251[Crossref], [PubMed], [CAS], Google Scholar33https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXjsFSqu7g%253D&md5=9dd227594dd64ca685418148de0abab0A nucleator arms race: cellular control of actin assemblyCampellone, Kenneth G.; Welch, Matthew D.Nature Reviews Molecular Cell Biology (2010), 11 (4), 237-251CODEN: NRMCBP; ISSN:1471-0072. (Nature Publishing Group)A review. For over a decade, the actin-related protein 2/3 (ARP2/3) complex, a handful of nucleation-promoting factors, and formins were the only mols. known to directly nucleate actin filament formation de novo. However, the past several years have seen a surge in the discovery of mammalian proteins with roles in actin nucleation and dynamics. Newly recognized nucleation-promoting factors, such as WASP and SCAR homolog (WASH), WASP homolog assocd. with actin, membranes and microtubules (WHAMM), and junction-mediating regulatory protein (JMY), stimulate ARP2/3 activity at distinct cellular locations. Formin nucleators with addnl. biochem. and cellular activities have also been uncovered. Finally, the Spire, cordon-bleu and leiomodin nucleators have revealed new ways of overcoming the kinetic barriers to actin polymn.
- 34Chesarone, M. A.; DuPage, A. G.; Goode, B. L. Unleashing formins to remodel the actin and microtubule cytoskeletons Nat. Rev. Mol. Cell Biol. 2010, 11 (1) 62– 74[Crossref], [PubMed], [CAS], Google Scholar34https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXhsFaqtL3J&md5=09c1038262a4eb65bb4a505669c849ceUnleashing formins to remodel the actin and microtubule cytoskeletonsChesarone, Melissa A.; DuPage, Amy Grace; Goode, Bruce L.Nature Reviews Molecular Cell Biology (2010), 11 (1), 62-74CODEN: NRMCBP; ISSN:1471-0072. (Nature Publishing Group)A review. Formins are highly conserved proteins that have essential roles in remodeling the actin and microtubule cytoskeletons to influence eukaryotic cell shape and behavior. Recent work has identified numerous cellular factors that locally recruit, activate, or inactivate formins to bridle and unleash their potent effects on actin nucleation and elongation. The effects of formins on microtubules have also begun to be described, which places formins in a prime position to coordinate actin and microtubule dynamics. The emerging complexity in the mechanisms governing formins mirrors the wide range of essential functions that they perform in cell motility, cell division, and cell and tissue morphogenesis.
- 35DeWard, A. D.; Eisenmann, K. M.; Matheson, S. F.; Alberts, A. S. The role of formins in human disease Biochim. Biophys. Acta 2010, 1803 (2) 226– 233Google ScholarThere is no corresponding record for this reference.
- 36Otomo, T.; Tomchick, D. R.; Otomo, C.; Panchal, S. C.; Machius, M.; Rosen, M. K. Structural basis of actin filament nucleation and processive capping by a formin homology 2 domain Nature 2005, 433 (7025) 488– 494Google ScholarThere is no corresponding record for this reference.
- 37Higgs, H. N. Formin proteins: A domain-based approach Trends Biochem. Sci. 2005, 30 (6) 342– 353Google ScholarThere is no corresponding record for this reference.
- 38Goode, B. L.; Eck, M. J. Mechanism and function of formins in the control of actin assembly Annu. Rev. Biochem. 2007, 76, 593– 627[Crossref], [PubMed], [CAS], Google Scholar38https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXhtVehtbzO&md5=70232c55f6173c228781c5f7fc0c17d7Mechanism and function of formins in the control of actin assemblyGoode, Bruce L.; Eck, Michael J.Annual Review of Biochemistry (2007), 76 (), 593-627CODEN: ARBOAW; ISSN:0066-4154. (Annual Reviews Inc.)A review. Formins are a widely expressed family of proteins that govern cell shape, adhesion, cytokinesis, and morphogenesis by remodeling the actin and microtubule cytoskeletons. These large multidomain proteins assoc. with a variety of other cellular factors and directly nucleate actin polymn. through a novel mechanism. The signature formin homol. 2 (FH2) domain initiates filament assembly and remains persistently assocd. with the fast-growing barbed end, enabling rapid insertion of actin subunits while protecting the end from capping proteins. On the basis of structural and mechanistic work, an integrated model is presented for FH2 processive motion. The adjacent FH1 domain recruits profilin-actin complexes and accelerates filament elongation. The most predominantly expressed formins in animals and fungi are autoinhibited through intramol. interactions and appear to be activated by Rho GTPases and addnl. factors. Other classes of formins lack the autoinhibitory and/or Rho-binding domains and thus are likely to be controlled by alternative mechanisms.
- 39Chhabra, E. S.; Higgs, H. N. INF2 is a WASP homology 2 motif-containing formin that severs actin filaments and accelerates both polymerization and depolymerization J. Biol. Chem. 2006, 281 (36) 26754– 26767Google ScholarThere is no corresponding record for this reference.
- 40Gurel, P. S.; Ge, P.; Grintsevich, E. E.; Shu, R.; Blanchoin, L.; Zhou, Z. H.; Reisler, E.; Higgs, H. N. INF2-Mediated Severing through Actin Filament Encirclement and Disruption Curr. Biol. 2014, 24, 156– 164[Crossref], [PubMed], [CAS], Google Scholar40https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXnsFymug%253D%253D&md5=04247c09532977380cc90245c4664fddINF2-Mediated Severing through Actin Filament Encirclement and DisruptionGurel, Pinar S.; Ge, Peng; Grintsevich, Elena E.; Shu, Rui; Blanchoin, Laurent; Zhou, Z. Hong; Reisler, Emil; Higgs, Henry N.Current Biology (2014), 24 (2), 156-164CODEN: CUBLE2; ISSN:0960-9822. (Cell Press)INF2 is a formin protein with the unique ability to accelerate both actin polymn. and depolymn., the latter requiring filament severing. Mutations in INF2 lead to the kidney disease focal segmental glomerulosclerosis (FSGS) and the neurol. disorder Charcot-Marie Tooth disease (CMTD).Here, we compare the severing mechanism of INF2 with that of the well-studied severing protein cofilin. INF2, like cofilin, binds stoichiometrically to filament sides and severs in a manner that requires phosphate release from the filament. In contrast to cofilin, however, INF2 binds ADP and ADP-Pi filaments equally well. Furthermore, two-color total internal reflection fluorescence (TIRF) microscopy reveals that a low no. of INF2 mols., as few as a single INF2 dimer, are capable of severing, while measurable cofilin-mediated severing requires more extensive binding. Hence, INF2 is a more potent severing protein than cofilin. While a construct contg. the FH1 and FH2 domains alone has some severing activity, addn. of the C-terminal region increases severing potency by 40-fold, and we show that the WH2-resembling DAD motif is responsible for this increase. Helical 3D reconstruction from electron micrographs at 20 Å resoln. provides a structure of filament-bound INF2, showing that the FH2 domain encircles the filament. We propose a severing model in which FH2 binding and phosphate release causes local filament deformation, allowing the DAD to bind adjacent actin protomers, further disrupting filament structure.
- 41Xu, Y.; Moseley, J. B.; Sagot, I.; Poy, F.; Pellman, D.; Goode, B. L.; Eck, M. J. Crystal structures of a formin homology-2 domain reveal a tethered dimer architecture Cell 2004, 116 (5) 711– 723Google ScholarThere is no corresponding record for this reference.
- 42Thompson, M. E.; Heimsath, E. G.; Gauvin, T. J.; Higgs, H. N.; Kull, F. J. FMNL3 FH2-actin structure gives insight into formin-mediated actin nucleation and elongation Nat. Struct. Mol. Biol. 2013, 20 (1) 111– 118[Crossref], [PubMed], [CAS], Google Scholar42https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38Xhsl2ltLfF&md5=24e08449812c304ef9da9375ca5fcc14FMNL3 FH2-actin structure gives insight into formin-mediated actin nucleation and elongationThompson, Morgan E.; Heimsath, Ernest G.; Gauvin, Timothy J.; Higgs, Henry N.; Kull, F. JonNature Structural & Molecular Biology (2013), 20 (1), 111-118CODEN: NSMBCU; ISSN:1545-9993. (Nature Publishing Group)Formins are actin-assembly factors that act in a variety of actin-based processes. The conserved formin homol. 2 (FH2) domain promotes filament nucleation and influences elongation through interaction with the barbed end. FMNL3 is a formin that induces assembly of filopodia but whose FH2 domain is a poor nucleator. The 3.4-Å structure of a mouse FMNL3 FH2 dimer in complex with tetramethylrhodamine-actin uncovers details of formin-regulated actin elongation. We observe distinct FH2 actin-binding regions; interactions in the knob and coiled-coil subdomains are necessary for actin binding, whereas those in the lasso-post interface are important for the stepping mechanism. Biochem. and cellular expts. test the importance of individual residues for function. This structure provides details for FH2-mediated filament elongation by processive capping and supports a model in which C-terminal non-FH2 residues of FMNL3 are required to stabilize the filament nucleus.
- 43Yamashita, M.; Higashi, T.; Suetsugu, S.; Sato, Y.; Ikeda, T.; Shirakawa, R.; Kita, T.; Takenawa, T.; Horiuchi, H.; Fukai, S.; Nureki, O. Crystal structure of human DAAM1 formin homology 2 domain Genes Cells 2007, 12 (11) 1255– 1265[Crossref], [PubMed], [CAS], Google Scholar43https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXhtl2jtLnN&md5=4de4bd46d1ff5e27861b766c59cfd8d3Crystal structure of human DAAM1 formin homology 2 domainYamashita, Masami; Higashi, Tomohito; Suetsugu, Shiro; Sato, Yusuke; Ikeda, Tomoyuki; Shirakawa, Ryutaro; Kita, Toru; Takenawa, Tadaomi; Horiuchi, Hisanori; Fukai, Shuya; Nureki, OsamuGenes to Cells (2007), 12 (11), 1255-1265CODEN: GECEFL; ISSN:1356-9597. (Blackwell Publishing Ltd.)Reorganization of the actin filament is an essential process for cell motility, cell-cell attachment and intracellular transport. Formin proteins promote nucleation and elongation of the actin filament, and thus are key regulators for this process. The formin homol. 2 (FH2) domain forms a head-to-tail ring-shaped dimer, and processively moves towards the barbed end. Dishevelled-assocd. activator of morphogenesis (DAAM) is a Rho-regulated formin implicated in neuronal development. Here, the authors present the crystal structure of human DAAM1 FH2 dimer at 2.8 Å resoln. This is the first dimeric structure of the mammalian formin. The core structure of human DAAM1 is similar to those of mouse mDia1 and yeast Bni1p, whereas the orientations of the FH2 dimeric rings are different between human DAAM1 and yeast Bni1p, despite their similar dimer interactions. This difference supports the previous prediction that the dimer architecture of the formin is highly flexible in the actin-free state. The results of the actin assembly assays using the DAAM1 mutants demonstrated that the length of the linker connecting the N-terminal domain and the core region is crucial for the activity.
- 44Moseley, J. B.; Sagot, I.; Manning, A. L.; Xu, Y.; Eck, M. J.; Pellman, D.; Goode, B. L. A conserved mechanism for Bni1- and mDia1-induced actin assembly and dual regulation of Bni1 by Bud6 and profilin Mol. Biol. Cell 2004, 15 (2) 896– 907[Crossref], [PubMed], [CAS], Google Scholar44https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXhtlGlsrs%253D&md5=351e9ad3f2168ce1a9b40e287d58a3f6A conserved mechanism for Bni1- and mDia1-induced actin assembly and dual regulation of Bni1 by Bud6 and profilinMoseley, James B.; Sagot, Isabelle; Manning, Amity L.; Xu, Yingwu; Eck, Michael J.; Pellman, David; Goode, Bruce L.Molecular Biology of the Cell (2004), 15 (2), 896-907CODEN: MBCEEV; ISSN:1059-1524. (American Society for Cell Biology)Formins have conserved roles in cell polarity and cytokinesis and directly nucleate actin filament assembly through their FH2 domain. Here, we define the active region of the yeast formin Bni1 FH2 domain and show that it dimerizes. Mutations that disrupt dimerization abolish actin assembly activity, suggesting that dimers are the active state of FH2 domains. The Bni1 FH2 domain protects growing barbed ends of actin filaments from vast excesses of capping protein, suggesting that the dimer maintains a persistent assocn. during elongation. This is not a species-specific mechanism, as the activities of purified mammalian formin mDia1 are identical to those of Bni1. Further, mDia1 partially complements BNI1 function in vivo, and expression of a dominant active mDia1 construct in yeast causes similar phenotypes to dominant active Bni1 constructs. In addn., we purified the Bni1-interacting half of the cell polarity factor Bud6 and found that it binds specifically to actin monomers and, like profilin, promotes rapid nucleotide exchange on actin. Bud6 and profilin show additive stimulatory effects on Bni1 activity and have a synthetic lethal genetic interaction in vivo. From these results, we propose a model in which Bni1 FH2 dimers nucleate and processively cap the elongating barbed end of the actin filament, and Bud6 and profilin generate a local flux of ATP-actin monomers to promote actin assembly.
- 45Harris, E. S.; Higgs, H. N. Biochemical analysis of mammalian formin effects on actin dynamics Methods Enzymol. 2006, 406, 190– 214Google ScholarThere is no corresponding record for this reference.
- 46Paul, A. S.; Pollard, T. D. Review of the mechanism of processive actin filament elongation by formins Cell Motil. Cytoskeleton 2009, 66 (8) 606– 617[Crossref], [PubMed], [CAS], Google Scholar46https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXhtVKntr%252FK&md5=df0066c1e3a6409c92210e2968e6b519Review of the mechanism of processive actin filament elongation by forminsPaul, Aditya S.; Pollard, Thomas D.Cell Motility and the Cytoskeleton (2009), 66 (8), 606-617CODEN: CMCYEO; ISSN:0886-1544. (Wiley-Liss, Inc.)A review. We review recent structural and biophys. studies of the mechanism of action of formins, proteins that direct the assembly of unbranched actin filaments for cytokinetic contractile rings and other cellular structures. Formins use free actin monomers to nucleate filaments and then remain bound to the barbed ends of these filaments as they elongate. In addn. to variable regulatory domains, formins typically have formin homol. 1 (FH1) and formin homol. 2 (FH2) domains. FH1 domains have multiple binding sites for profilin, an abundant actin monomer binding protein. FH2 homodimers encircle the barbed end of a filament. Most FH2 domains inhibit actin filament elongation, but FH1 domains conc. multiple profilin-actin complexes near the end of the filament. FH1 domains transfer actin very rapidly onto the barbed end of the filament, allowing elongation at rates that exceed the rate of elongation by the addn. of free actin monomers diffusing in soln. Binding of actin to the end of the filament provides the energy for the highly processive movement of the FH2 as a filament adds thousands of actin subunits. These biophys. insights provide the context to understand how formins contribute to actin assembly in cells.
- 47Spudich, J. A.; Watt, S. The regulation of rabbit skeletal muscle contraction. I. Biochemical studies of the interaction of the tropomyosin-troponin complex with actin and the proteolytic fragments of myosin J. Biol. Chem. 1971, 246 (15) 4866– 4871Google ScholarThere is no corresponding record for this reference.
- 48Harris, E. S.; Li, F.; Higgs, H. N. The mouse formin, FRLα, slows actin filament barbed end elongation, competes with capping protein, accelerates polymerization from monomers, and severs filaments J. Biol. Chem. 2004, 279 (19) 20076– 20087Google ScholarThere is no corresponding record for this reference.
- 49Li, F.; Higgs, H. N. Dissecting requirements for auto-inhibition of actin nucleation by the formin, mDia1 J. Biol. Chem. 2005, 280 (8) 6986– 6992Google ScholarThere is no corresponding record for this reference.
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- 51Maiti, S.; Michelot, A.; Gould, C.; Blanchoin, L.; Sokolova, O.; Goode, B. L. Structure and activity of full-length formin mDia1 Cytoskeleton 2012, 69 (6) 393– 405Google ScholarThere is no corresponding record for this reference.
- 52Gould, C. J.; Maiti, S.; Michelot, A.; Graziano, B. R.; Blanchoin, L.; Goode, B. L. The formin DAD domain plays dual roles in autoinhibition and actin nucleation Curr. Biol. 2011, 21 (5) 384– 390[Crossref], [PubMed], [CAS], Google Scholar52https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXjslChsL4%253D&md5=47dd64e85d08423f4e596f461b80b628The formin DAD domain plays dual roles in autoinhibition and actin nucleationGould, Christopher J.; Maiti, Sankar; Michelot, Alphee; Graziano, Brian R.; Blanchoin, Laurent; Goode, Bruce L.Current Biology (2011), 21 (5), 384-390CODEN: CUBLE2; ISSN:0960-9822. (Cell Press)Formins are a large family of actin assembly-promoting proteins with many important biol. roles. However, it has remained unclear how formins nucleate actin polymn. All other nucleators are known to recruit actin monomers as a central part of their mechanisms. However, the actin-nucleating FH2 domain of formins lacks appreciable affinity for monomeric actin. Here, we found that yeast and mammalian formins bind actin monomers but that this activity requires their C-terminal DAD domains. Furthermore, we obsd. that the DAD works in concert with the FH2 to enhance nucleation without affecting the rate of filament elongation. We dissected this mechanism in mDia1, mapped nucleation activity to conserved residues in the DAD, and demonstrated that DAD roles in nucleation and autoinhibition are separable. Furthermore, DAD enhancement of nucleation was independent of contributions from the FH1 domain to nucleation. Together, our data show that (1) the DAD has dual functions in autoinhibition and nucleation; (2) the FH1, FH2, and DAD domains form a tripartite nucleation machine; and (3) formins nucleate by recruiting actin monomers and therefore are more similar to other nucleators than previously thought.
- 53Heimsath, E. G., Jr.; Higgs, H. N. The C terminus of formin FMNL3 accelerates actin polymerization and contains a WH2 domain-like sequence that binds both monomers and filament barbed ends J. Biol. Chem. 2012, 287 (5) 3087– 3098Google ScholarThere is no corresponding record for this reference.
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- 55Sharma, S.; Zhu, H.; Grintsevich, E. E.; Reisler, E.; Gimzewski, J. K. Correlative nanoscale imaging of actin filaments and their complexes Nanoscale 2013, 5, 5692– 5702Google ScholarThere is no corresponding record for this reference.
- 56Sharma, S.; Grintsevich, E. E.; Hsueh, C.; Reisler, E.; Gimzewski, J. K. Molecular cooperativity of drebrin(1–300) binding and structural remodeling of F-actin Biophys. J. 2012, 103 (2) 275– 283Google ScholarThere is no corresponding record for this reference.
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Abstract

Figure 1

Figure 1. Schematic representation of the domain organization of inverted formin 2 (INF2) showing regions important for actin binding and protein–protein interactions. Formin constructs used in this study include INF2-FH1-FH2-C, also termed INF2-FFC, and INF2-FH1-FH2 (INF2-FF). N and C denote the N- and C-termini, respectively, of protein sequences. Other abbreviations are DID (diaphanous inhibitory domain), FH1 (formin homology 1), FH2 (formin homology 2), and DAD (diaphanous autoregulatory domain).
Figure 2

Figure 2. Structural evidence of self-assembly of INF2-FFC at various concentrations. At low concentrations (0.02 μM), INF2-FFC forms ring-shaped homodimers. Representative AFM images of free INF2-FFC showing self-assembled round structures likely formed by dimerization of FH2 domains of INF2-FFC. Schematic representations are shown in the insets. (a) Single INF2-FFC dimer. (b) Compact and closed round dimers were observed also without a hollow core. (c) The central core region attached to the dimeric ring periphery via two small extensions (diamond arrows), which corresponds potentially to the unstructured FH1 domain. (d) An example of the INF2-FFC dimer in the open configuration. The FH2 domain within the INF2-FFC dimer can potentially dissociate (open) and reassociate (close) to form trimers as well as oligomers [at a higher surface concentration (0.2 μM)]. (e) AFM image of INF2-FFC in oligomeric configurations (0.2 μM INF2-FFC). Representative images of the INF2-FFC dimer, trimer, and tetramer (f–h, respectively) and their corresponding schematic representations (i–iii, respectively). At higher concentrations (2 μM), INF2-FFC assembles into tubular ringlike structures, as marked with a white arrow in part i. Other close-up views are shown in parts i–iv of panel j.
Figure 3

Figure 3. AFM images showing free INF2-FF self-organization configurations. The central core region observed as a dense mass (dashed white arrows) within the oligomeric ring periphery in panel a–c. The central dense core appears to be similar to that observed for INF2-FFC dimers and oligomers, suggesting that it comes from the unstructured FH1 domain.
Figure 4

Figure 4. Structure of INF2-FFC and F-actin complexes. AFM image of INF2-FFC and F-actin complexes (at a 1:1 molar ratio) revealing ringlike binding around actin filaments. (a) Close-up of the INF2-FFC–F-actin complex displaying detailed orientations of adjacent INF2-FFC homodimer rings assembled around F-actin. Notably, there is 5–10° occasional bending of adjacent units, indicating flexibility or strain across the bound complex. (b) 3D side view (500×, 500 nm) showing the stacklike arrangement of INF2-FFC over F-actin. (c) Tubular structure spacing shown schematically.
Figure 5

Figure 5. Structure of INF2-FF and F-actin complexes. AFM image of a 1:1 ratio of INF2-FF and F-actin complexes revealing ringlike binding around actin filaments similar to that of INF2-FFC and F-actin complexes.
Figure 6

Figure 6. Schematic representation of plausible orientations of INF2-FFC homodimers over F-actin. (a) Adjacent INF2-FFC dimeric rings interact in an end-to-end manner to form a spiral coil structure. (b) Neighboring homodimers make longitudinal contact with adjacent F-actin-bound dimers to form continuous structure without any gaps or with small angle gaps and/or openings. (c) Combinations of regions as in panels a and b, along the length of F-actin. Alternatively, dimers may reassociate to form individual dimer rings stacked along the length of the filament with or without gap openings.
References
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- 30McPherson, A.; Kuznetsov, Y. G. Atomic Force Microscopy Investigation of Viruses Methods Mol. Biol. 2011, 736, 171– 195[Crossref], [PubMed], [CAS], Google Scholar30https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XktV2rs7s%253D&md5=4baa9d6cdae939598742ed659eedd300Atomic force microscopy investigation of virusesMcPherson, Alexander; Kuznetsov, Yurii G.Methods in Molecular Biology (New York, NY, United States) (2011), 736 (Atomic Force Microscopy in Biomedical Research), 171-195CODEN: MMBIED; ISSN:1064-3745. (Springer)Atomic force microscopy (AFM) has proven to be a valuable approach to delineate the architectures and detailed structural features of a wide variety of viruses. These have ranged from small plant satellite viruses of only 17 nm to the giant mimivirus of 750 nm diam., and they have included diverse morphologies such as those represented by HIV, icosahedral particles, vaccinia, and bacteriophages. Because it is a surface technique, it provides images and information that are distinct from those obtained by electron microscopy, and in some cases, at even higher resoln. By enzymic and chem. dissection of virions, internal structures can be revealed, as well as DNA and RNA. The method is relatively rapid and can be carried out on both fixed and unfixed samples in either air or fluids, including culture media. It is nondestructive and even non-perturbing. It can be applied to individual isolated virus, as well as to infected cells. AFM is still in its early development and holds great promise for further investigation of biol. systems at the nanometer scale.
- 31Shao, Z. F.; Shi, D.; Somlyo, A. V. Cryoatomic force microscopy of filamentous actin Biophys. J. 2000, 78 (2) 950– 958Google ScholarThere is no corresponding record for this reference.
- 32Sharma, S.; Grintsevich, E. E.; Phillips, M. L.; Reisler, E.; Gimzewski, J. K. Atomic force microscopy reveals drebrin induced remodeling of F-actin with subnanometer resolution Nano Lett. 2011, 11 (2) 825– 827
- 33Campellone, K. G.; Welch, M. D. A nucleator arms race: Cellular control of actin assembly Nat. Rev. Mol. Cell Biol. 2010, 11 (4) 237– 251[Crossref], [PubMed], [CAS], Google Scholar33https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXjsFSqu7g%253D&md5=9dd227594dd64ca685418148de0abab0A nucleator arms race: cellular control of actin assemblyCampellone, Kenneth G.; Welch, Matthew D.Nature Reviews Molecular Cell Biology (2010), 11 (4), 237-251CODEN: NRMCBP; ISSN:1471-0072. (Nature Publishing Group)A review. For over a decade, the actin-related protein 2/3 (ARP2/3) complex, a handful of nucleation-promoting factors, and formins were the only mols. known to directly nucleate actin filament formation de novo. However, the past several years have seen a surge in the discovery of mammalian proteins with roles in actin nucleation and dynamics. Newly recognized nucleation-promoting factors, such as WASP and SCAR homolog (WASH), WASP homolog assocd. with actin, membranes and microtubules (WHAMM), and junction-mediating regulatory protein (JMY), stimulate ARP2/3 activity at distinct cellular locations. Formin nucleators with addnl. biochem. and cellular activities have also been uncovered. Finally, the Spire, cordon-bleu and leiomodin nucleators have revealed new ways of overcoming the kinetic barriers to actin polymn.
- 34Chesarone, M. A.; DuPage, A. G.; Goode, B. L. Unleashing formins to remodel the actin and microtubule cytoskeletons Nat. Rev. Mol. Cell Biol. 2010, 11 (1) 62– 74[Crossref], [PubMed], [CAS], Google Scholar34https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXhsFaqtL3J&md5=09c1038262a4eb65bb4a505669c849ceUnleashing formins to remodel the actin and microtubule cytoskeletonsChesarone, Melissa A.; DuPage, Amy Grace; Goode, Bruce L.Nature Reviews Molecular Cell Biology (2010), 11 (1), 62-74CODEN: NRMCBP; ISSN:1471-0072. (Nature Publishing Group)A review. Formins are highly conserved proteins that have essential roles in remodeling the actin and microtubule cytoskeletons to influence eukaryotic cell shape and behavior. Recent work has identified numerous cellular factors that locally recruit, activate, or inactivate formins to bridle and unleash their potent effects on actin nucleation and elongation. The effects of formins on microtubules have also begun to be described, which places formins in a prime position to coordinate actin and microtubule dynamics. The emerging complexity in the mechanisms governing formins mirrors the wide range of essential functions that they perform in cell motility, cell division, and cell and tissue morphogenesis.
- 35DeWard, A. D.; Eisenmann, K. M.; Matheson, S. F.; Alberts, A. S. The role of formins in human disease Biochim. Biophys. Acta 2010, 1803 (2) 226– 233Google ScholarThere is no corresponding record for this reference.
- 36Otomo, T.; Tomchick, D. R.; Otomo, C.; Panchal, S. C.; Machius, M.; Rosen, M. K. Structural basis of actin filament nucleation and processive capping by a formin homology 2 domain Nature 2005, 433 (7025) 488– 494Google ScholarThere is no corresponding record for this reference.
- 37Higgs, H. N. Formin proteins: A domain-based approach Trends Biochem. Sci. 2005, 30 (6) 342– 353Google ScholarThere is no corresponding record for this reference.
- 38Goode, B. L.; Eck, M. J. Mechanism and function of formins in the control of actin assembly Annu. Rev. Biochem. 2007, 76, 593– 627[Crossref], [PubMed], [CAS], Google Scholar38https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXhtVehtbzO&md5=70232c55f6173c228781c5f7fc0c17d7Mechanism and function of formins in the control of actin assemblyGoode, Bruce L.; Eck, Michael J.Annual Review of Biochemistry (2007), 76 (), 593-627CODEN: ARBOAW; ISSN:0066-4154. (Annual Reviews Inc.)A review. Formins are a widely expressed family of proteins that govern cell shape, adhesion, cytokinesis, and morphogenesis by remodeling the actin and microtubule cytoskeletons. These large multidomain proteins assoc. with a variety of other cellular factors and directly nucleate actin polymn. through a novel mechanism. The signature formin homol. 2 (FH2) domain initiates filament assembly and remains persistently assocd. with the fast-growing barbed end, enabling rapid insertion of actin subunits while protecting the end from capping proteins. On the basis of structural and mechanistic work, an integrated model is presented for FH2 processive motion. The adjacent FH1 domain recruits profilin-actin complexes and accelerates filament elongation. The most predominantly expressed formins in animals and fungi are autoinhibited through intramol. interactions and appear to be activated by Rho GTPases and addnl. factors. Other classes of formins lack the autoinhibitory and/or Rho-binding domains and thus are likely to be controlled by alternative mechanisms.
- 39Chhabra, E. S.; Higgs, H. N. INF2 is a WASP homology 2 motif-containing formin that severs actin filaments and accelerates both polymerization and depolymerization J. Biol. Chem. 2006, 281 (36) 26754– 26767Google ScholarThere is no corresponding record for this reference.
- 40Gurel, P. S.; Ge, P.; Grintsevich, E. E.; Shu, R.; Blanchoin, L.; Zhou, Z. H.; Reisler, E.; Higgs, H. N. INF2-Mediated Severing through Actin Filament Encirclement and Disruption Curr. Biol. 2014, 24, 156– 164[Crossref], [PubMed], [CAS], Google Scholar40https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXnsFymug%253D%253D&md5=04247c09532977380cc90245c4664fddINF2-Mediated Severing through Actin Filament Encirclement and DisruptionGurel, Pinar S.; Ge, Peng; Grintsevich, Elena E.; Shu, Rui; Blanchoin, Laurent; Zhou, Z. Hong; Reisler, Emil; Higgs, Henry N.Current Biology (2014), 24 (2), 156-164CODEN: CUBLE2; ISSN:0960-9822. (Cell Press)INF2 is a formin protein with the unique ability to accelerate both actin polymn. and depolymn., the latter requiring filament severing. Mutations in INF2 lead to the kidney disease focal segmental glomerulosclerosis (FSGS) and the neurol. disorder Charcot-Marie Tooth disease (CMTD).Here, we compare the severing mechanism of INF2 with that of the well-studied severing protein cofilin. INF2, like cofilin, binds stoichiometrically to filament sides and severs in a manner that requires phosphate release from the filament. In contrast to cofilin, however, INF2 binds ADP and ADP-Pi filaments equally well. Furthermore, two-color total internal reflection fluorescence (TIRF) microscopy reveals that a low no. of INF2 mols., as few as a single INF2 dimer, are capable of severing, while measurable cofilin-mediated severing requires more extensive binding. Hence, INF2 is a more potent severing protein than cofilin. While a construct contg. the FH1 and FH2 domains alone has some severing activity, addn. of the C-terminal region increases severing potency by 40-fold, and we show that the WH2-resembling DAD motif is responsible for this increase. Helical 3D reconstruction from electron micrographs at 20 Å resoln. provides a structure of filament-bound INF2, showing that the FH2 domain encircles the filament. We propose a severing model in which FH2 binding and phosphate release causes local filament deformation, allowing the DAD to bind adjacent actin protomers, further disrupting filament structure.
- 41Xu, Y.; Moseley, J. B.; Sagot, I.; Poy, F.; Pellman, D.; Goode, B. L.; Eck, M. J. Crystal structures of a formin homology-2 domain reveal a tethered dimer architecture Cell 2004, 116 (5) 711– 723Google ScholarThere is no corresponding record for this reference.
- 42Thompson, M. E.; Heimsath, E. G.; Gauvin, T. J.; Higgs, H. N.; Kull, F. J. FMNL3 FH2-actin structure gives insight into formin-mediated actin nucleation and elongation Nat. Struct. Mol. Biol. 2013, 20 (1) 111– 118[Crossref], [PubMed], [CAS], Google Scholar42https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38Xhsl2ltLfF&md5=24e08449812c304ef9da9375ca5fcc14FMNL3 FH2-actin structure gives insight into formin-mediated actin nucleation and elongationThompson, Morgan E.; Heimsath, Ernest G.; Gauvin, Timothy J.; Higgs, Henry N.; Kull, F. JonNature Structural & Molecular Biology (2013), 20 (1), 111-118CODEN: NSMBCU; ISSN:1545-9993. (Nature Publishing Group)Formins are actin-assembly factors that act in a variety of actin-based processes. The conserved formin homol. 2 (FH2) domain promotes filament nucleation and influences elongation through interaction with the barbed end. FMNL3 is a formin that induces assembly of filopodia but whose FH2 domain is a poor nucleator. The 3.4-Å structure of a mouse FMNL3 FH2 dimer in complex with tetramethylrhodamine-actin uncovers details of formin-regulated actin elongation. We observe distinct FH2 actin-binding regions; interactions in the knob and coiled-coil subdomains are necessary for actin binding, whereas those in the lasso-post interface are important for the stepping mechanism. Biochem. and cellular expts. test the importance of individual residues for function. This structure provides details for FH2-mediated filament elongation by processive capping and supports a model in which C-terminal non-FH2 residues of FMNL3 are required to stabilize the filament nucleus.
- 43Yamashita, M.; Higashi, T.; Suetsugu, S.; Sato, Y.; Ikeda, T.; Shirakawa, R.; Kita, T.; Takenawa, T.; Horiuchi, H.; Fukai, S.; Nureki, O. Crystal structure of human DAAM1 formin homology 2 domain Genes Cells 2007, 12 (11) 1255– 1265[Crossref], [PubMed], [CAS], Google Scholar43https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXhtl2jtLnN&md5=4de4bd46d1ff5e27861b766c59cfd8d3Crystal structure of human DAAM1 formin homology 2 domainYamashita, Masami; Higashi, Tomohito; Suetsugu, Shiro; Sato, Yusuke; Ikeda, Tomoyuki; Shirakawa, Ryutaro; Kita, Toru; Takenawa, Tadaomi; Horiuchi, Hisanori; Fukai, Shuya; Nureki, OsamuGenes to Cells (2007), 12 (11), 1255-1265CODEN: GECEFL; ISSN:1356-9597. (Blackwell Publishing Ltd.)Reorganization of the actin filament is an essential process for cell motility, cell-cell attachment and intracellular transport. Formin proteins promote nucleation and elongation of the actin filament, and thus are key regulators for this process. The formin homol. 2 (FH2) domain forms a head-to-tail ring-shaped dimer, and processively moves towards the barbed end. Dishevelled-assocd. activator of morphogenesis (DAAM) is a Rho-regulated formin implicated in neuronal development. Here, the authors present the crystal structure of human DAAM1 FH2 dimer at 2.8 Å resoln. This is the first dimeric structure of the mammalian formin. The core structure of human DAAM1 is similar to those of mouse mDia1 and yeast Bni1p, whereas the orientations of the FH2 dimeric rings are different between human DAAM1 and yeast Bni1p, despite their similar dimer interactions. This difference supports the previous prediction that the dimer architecture of the formin is highly flexible in the actin-free state. The results of the actin assembly assays using the DAAM1 mutants demonstrated that the length of the linker connecting the N-terminal domain and the core region is crucial for the activity.
- 44Moseley, J. B.; Sagot, I.; Manning, A. L.; Xu, Y.; Eck, M. J.; Pellman, D.; Goode, B. L. A conserved mechanism for Bni1- and mDia1-induced actin assembly and dual regulation of Bni1 by Bud6 and profilin Mol. Biol. Cell 2004, 15 (2) 896– 907[Crossref], [PubMed], [CAS], Google Scholar44https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXhtlGlsrs%253D&md5=351e9ad3f2168ce1a9b40e287d58a3f6A conserved mechanism for Bni1- and mDia1-induced actin assembly and dual regulation of Bni1 by Bud6 and profilinMoseley, James B.; Sagot, Isabelle; Manning, Amity L.; Xu, Yingwu; Eck, Michael J.; Pellman, David; Goode, Bruce L.Molecular Biology of the Cell (2004), 15 (2), 896-907CODEN: MBCEEV; ISSN:1059-1524. (American Society for Cell Biology)Formins have conserved roles in cell polarity and cytokinesis and directly nucleate actin filament assembly through their FH2 domain. Here, we define the active region of the yeast formin Bni1 FH2 domain and show that it dimerizes. Mutations that disrupt dimerization abolish actin assembly activity, suggesting that dimers are the active state of FH2 domains. The Bni1 FH2 domain protects growing barbed ends of actin filaments from vast excesses of capping protein, suggesting that the dimer maintains a persistent assocn. during elongation. This is not a species-specific mechanism, as the activities of purified mammalian formin mDia1 are identical to those of Bni1. Further, mDia1 partially complements BNI1 function in vivo, and expression of a dominant active mDia1 construct in yeast causes similar phenotypes to dominant active Bni1 constructs. In addn., we purified the Bni1-interacting half of the cell polarity factor Bud6 and found that it binds specifically to actin monomers and, like profilin, promotes rapid nucleotide exchange on actin. Bud6 and profilin show additive stimulatory effects on Bni1 activity and have a synthetic lethal genetic interaction in vivo. From these results, we propose a model in which Bni1 FH2 dimers nucleate and processively cap the elongating barbed end of the actin filament, and Bud6 and profilin generate a local flux of ATP-actin monomers to promote actin assembly.
- 45Harris, E. S.; Higgs, H. N. Biochemical analysis of mammalian formin effects on actin dynamics Methods Enzymol. 2006, 406, 190– 214Google ScholarThere is no corresponding record for this reference.
- 46Paul, A. S.; Pollard, T. D. Review of the mechanism of processive actin filament elongation by formins Cell Motil. Cytoskeleton 2009, 66 (8) 606– 617[Crossref], [PubMed], [CAS], Google Scholar46https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXhtVKntr%252FK&md5=df0066c1e3a6409c92210e2968e6b519Review of the mechanism of processive actin filament elongation by forminsPaul, Aditya S.; Pollard, Thomas D.Cell Motility and the Cytoskeleton (2009), 66 (8), 606-617CODEN: CMCYEO; ISSN:0886-1544. (Wiley-Liss, Inc.)A review. We review recent structural and biophys. studies of the mechanism of action of formins, proteins that direct the assembly of unbranched actin filaments for cytokinetic contractile rings and other cellular structures. Formins use free actin monomers to nucleate filaments and then remain bound to the barbed ends of these filaments as they elongate. In addn. to variable regulatory domains, formins typically have formin homol. 1 (FH1) and formin homol. 2 (FH2) domains. FH1 domains have multiple binding sites for profilin, an abundant actin monomer binding protein. FH2 homodimers encircle the barbed end of a filament. Most FH2 domains inhibit actin filament elongation, but FH1 domains conc. multiple profilin-actin complexes near the end of the filament. FH1 domains transfer actin very rapidly onto the barbed end of the filament, allowing elongation at rates that exceed the rate of elongation by the addn. of free actin monomers diffusing in soln. Binding of actin to the end of the filament provides the energy for the highly processive movement of the FH2 as a filament adds thousands of actin subunits. These biophys. insights provide the context to understand how formins contribute to actin assembly in cells.
- 47Spudich, J. A.; Watt, S. The regulation of rabbit skeletal muscle contraction. I. Biochemical studies of the interaction of the tropomyosin-troponin complex with actin and the proteolytic fragments of myosin J. Biol. Chem. 1971, 246 (15) 4866– 4871Google ScholarThere is no corresponding record for this reference.
- 48Harris, E. S.; Li, F.; Higgs, H. N. The mouse formin, FRLα, slows actin filament barbed end elongation, competes with capping protein, accelerates polymerization from monomers, and severs filaments J. Biol. Chem. 2004, 279 (19) 20076– 20087Google ScholarThere is no corresponding record for this reference.
- 49Li, F.; Higgs, H. N. Dissecting requirements for auto-inhibition of actin nucleation by the formin, mDia1 J. Biol. Chem. 2005, 280 (8) 6986– 6992Google ScholarThere is no corresponding record for this reference.
- 50Lu, J.; Meng, W.; Poy, F.; Maiti, S.; Goode, B. L.; Eck, M. J. Structure of the FH2 domain of Daam1: Implications for formin regulation of actin assembly J. Mol. Biol. 2007, 369 (5) 1258– 1269Google ScholarThere is no corresponding record for this reference.
- 51Maiti, S.; Michelot, A.; Gould, C.; Blanchoin, L.; Sokolova, O.; Goode, B. L. Structure and activity of full-length formin mDia1 Cytoskeleton 2012, 69 (6) 393– 405Google ScholarThere is no corresponding record for this reference.
- 52Gould, C. J.; Maiti, S.; Michelot, A.; Graziano, B. R.; Blanchoin, L.; Goode, B. L. The formin DAD domain plays dual roles in autoinhibition and actin nucleation Curr. Biol. 2011, 21 (5) 384– 390[Crossref], [PubMed], [CAS], Google Scholar52https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXjslChsL4%253D&md5=47dd64e85d08423f4e596f461b80b628The formin DAD domain plays dual roles in autoinhibition and actin nucleationGould, Christopher J.; Maiti, Sankar; Michelot, Alphee; Graziano, Brian R.; Blanchoin, Laurent; Goode, Bruce L.Current Biology (2011), 21 (5), 384-390CODEN: CUBLE2; ISSN:0960-9822. (Cell Press)Formins are a large family of actin assembly-promoting proteins with many important biol. roles. However, it has remained unclear how formins nucleate actin polymn. All other nucleators are known to recruit actin monomers as a central part of their mechanisms. However, the actin-nucleating FH2 domain of formins lacks appreciable affinity for monomeric actin. Here, we found that yeast and mammalian formins bind actin monomers but that this activity requires their C-terminal DAD domains. Furthermore, we obsd. that the DAD works in concert with the FH2 to enhance nucleation without affecting the rate of filament elongation. We dissected this mechanism in mDia1, mapped nucleation activity to conserved residues in the DAD, and demonstrated that DAD roles in nucleation and autoinhibition are separable. Furthermore, DAD enhancement of nucleation was independent of contributions from the FH1 domain to nucleation. Together, our data show that (1) the DAD has dual functions in autoinhibition and nucleation; (2) the FH1, FH2, and DAD domains form a tripartite nucleation machine; and (3) formins nucleate by recruiting actin monomers and therefore are more similar to other nucleators than previously thought.
- 53Heimsath, E. G., Jr.; Higgs, H. N. The C terminus of formin FMNL3 accelerates actin polymerization and contains a WH2 domain-like sequence that binds both monomers and filament barbed ends J. Biol. Chem. 2012, 287 (5) 3087– 3098Google ScholarThere is no corresponding record for this reference.
- 54Ramabhadran, V.; Gurel, P. S.; Higgs, H. N. Mutations to the formin homology 2 domain of INF2 protein have unexpected effects on actin polymerization and severing J. Biol. Chem. 2012, 287 (41) 34234– 34245Google ScholarThere is no corresponding record for this reference.
- 55Sharma, S.; Zhu, H.; Grintsevich, E. E.; Reisler, E.; Gimzewski, J. K. Correlative nanoscale imaging of actin filaments and their complexes Nanoscale 2013, 5, 5692– 5702Google ScholarThere is no corresponding record for this reference.
- 56Sharma, S.; Grintsevich, E. E.; Hsueh, C.; Reisler, E.; Gimzewski, J. K. Molecular cooperativity of drebrin(1–300) binding and structural remodeling of F-actin Biophys. J. 2012, 103 (2) 275– 283Google ScholarThere is no corresponding record for this reference.
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