Actin Nucleation: Spire — Actin Nucleator in a Class of Its Own
2005; Elsevier BV; Volume: 15; Issue: 8 Linguagem: Inglês
10.1016/j.cub.2005.04.004
ISSN1879-0445
Autores Tópico(s)3D Printing in Biomedical Research
ResumoThe rate limiting step for actin filament polymerisation is nucleation, and two types of nucleator have been described: the Arp2/3 complex and the formins. A recent study has now identified in Spire a third class of actin nucleator. The four short WH2 repeats within Spire bind four consecutive actin monomers to form a novel single strand nucleus for ‘barbed end’ actin filament elongation. The rate limiting step for actin filament polymerisation is nucleation, and two types of nucleator have been described: the Arp2/3 complex and the formins. A recent study has now identified in Spire a third class of actin nucleator. The four short WH2 repeats within Spire bind four consecutive actin monomers to form a novel single strand nucleus for ‘barbed end’ actin filament elongation. Actin filaments help control the dynamic shape of all eukaryotic cells [1Pollard T.D. Borisy G.G. Cellular motility driven by assembly and disassembly of actin filaments.Cell. 2003; 112: 453-465Abstract Full Text Full Text PDF PubMed Scopus (3109) Google Scholar]. They grow in a polarised fashion via the addition of ATP–actin monomers to the filament ‘barbed end’. As a filament ages, ATP is rapidly hydrolysed and phosphate released. Filamentous ADP–actin is then disassembled by the removal of subunits from the polymer’s slow growing or ‘pointed end’, and the ADP moiety is exchanged for ATP to ready actin monomers for another round of polymerisation. In a eukaryotic cell, this cycle of actin filament growth and disassembly is regulated at each step by a diverse set of actin-binding proteins. As the rate-limiting step in the formation of an actin filament from purified actin–ATP monomers is the generation of dimeric and trimeric actin nuclei, actin filament nucleation is likely to be a key point of control in this process [1Pollard T.D. Borisy G.G. Cellular motility driven by assembly and disassembly of actin filaments.Cell. 2003; 112: 453-465Abstract Full Text Full Text PDF PubMed Scopus (3109) Google Scholar]. It is only recently, however, that the Arp2/3 complex and formins have been identified as distinct factors that can catalyze de novo actin filament formation. Now, Quinlan et al. [2Quinlan M.E. Heuser J.E. Kerkhoff E. Mullins R.D. Drosophila Spire is an actin nucleation factor.Nature. 2005; 433: 382-388Crossref PubMed Scopus (245) Google Scholar] have identified Spire as a third class of actin nucleator. The highly conserved, multi-subunit Arp2/3 complex was the first actin nucleation factor to be characterized [3Machesky L.M. Atkinson S.J. Ampe C. Vandekerckhove J. Pollard T.D. Purification of a cortical complex containing two unconventional actins from Acanthamoeba by affinity chromatography on profilin-agarose.J. Cell Biol. 1994; 127: 107-115Crossref PubMed Scopus (373) Google Scholar]. When activated, its two actin-like subunits, Arp2 and Arp3, serve as a template for monomer addition by mimicking the ‘barbed end’ of a growing actin filament [4Volkmann N. Amann K.J. Stoilova-McPhie S. Egile C. Winter D.C. Hazelwood L. Heuser J.E. Li R. Pollard T.D. Hanein D. Structure of Arp2/3 complex in its activated state and in actin filament branch junctions.Science. 2001; 293: 2456-2459Crossref PubMed Scopus (195) Google Scholar]. The Arp2/3 complex also interacts with the sides of existing actin filaments: this augments its nucleation activity, so that the Arp2/3 complex generates new actin filament branches at a characteristic angle of 70 degrees to the host filament. As a result of these simple biochemical properties, Arp2/3-dependent actin filament formation is auto-catalytic and generates an expanding, branched network of filaments similar to that seen in the lamellipodia of many motile cells [1Pollard T.D. Borisy G.G. Cellular motility driven by assembly and disassembly of actin filaments.Cell. 2003; 112: 453-465Abstract Full Text Full Text PDF PubMed Scopus (3109) Google Scholar]. The formins catalyze de novo actin filament formation via a completely different mechanism [5Xu 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: 711-723Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar]. Formins dimerize to form a hoop-shaped structure that acts like a ‘barbed end’ filament cap to stabilize the formation of an adjacent actin dimer. Remarkably, this nascent, formin-bound actin seed is able to elongate by the insertion of ATP–actin monomers between the formin cap and the ‘barbed end’ of the filament. Although it remains to be established exactly how this is achieved, an attractive model is that one subunit of the formin dimer binds an actin subunit at the tip of the filament ‘barbed end’, while the other subunit of the dimer catalyses addition of an ATP–actin monomer to the opposite strand of the actin filament [5Xu 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: 711-723Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar, 6Romero S. Le Clainche C. Didry D. Egile C. Pantaloni D. Carlier M.F. Formin is a processive motor that requires profilin to accelerate actin assembly and associated ATP hydrolysis.Cell. 2004; 119: 419-429Abstract Full Text Full Text PDF PubMed Scopus (408) Google Scholar]. As the filament elongates, the formin dimer will then step between staggered actin subunits at the filament tip as if climbing a growing spiral staircase. The ‘leaky’ dimeric formin cap also protects the growing filament from other ‘barbed end’ capping proteins. As a result, formin-induced actin nucleation generates long, unbranched bundles of actin filaments, like those used to construct actin rings during cytokinesis. With the actin field still buzzing over the discovery of formin-dependent nucleation, Quinlan et al. [2Quinlan M.E. Heuser J.E. Kerkhoff E. Mullins R.D. Drosophila Spire is an actin nucleation factor.Nature. 2005; 433: 382-388Crossref PubMed Scopus (245) Google Scholar] have identified a further novel mechanism of actin filament nucleation that is catalyzed by Spire. The Spire gene was first identified, together with Cappuccino, in a Drosophila screen for mutations affecting oocyte polarity [7Manseau L.J. Schupbach T. cappuccino and spire: two unique maternal-effect loci required for both the anteroposterior and dorsoventral patterns of the Drosophila embryo.Genes Dev. 1989; 3: 1437-1452Crossref PubMed Scopus (175) Google Scholar]. Although it is not clear how the oogenesis defects arise in the two mutants, aspects of this phenotype can be mirrored by loss of the actin-nucleotide exchange factor profilin, or by feeding flies with the actin poison cytochalasin D, implying that the phenotype reflects an underlying reduction in the rate of actin filament formation [8Manseau L. Calley J. Phan H. Profilin is required for posterior patterning of the Drosophila oocyte.Development. 1996; 122: 2109-2116PubMed Google Scholar]. This inference was confirmed when the corresponding genes were cloned and Cappuccino was found to encode a formin [8Manseau L. Calley J. Phan H. Profilin is required for posterior patterning of the Drosophila oocyte.Development. 1996; 122: 2109-2116PubMed Google Scholar], and Spire a conserved metazoan protein that has multiple copies of a well-characterised actin-binding domain, the WH2 motif [9Wellington A. Emmons S. James B. Calley J. Grover M. Tolias P. Manseau L. Spire contains actin binding domains and is related to ascidian posterior end mark-5.Development. 1999; 126: 5267-5274Crossref PubMed Google Scholar]. WH2-like motifs are present in a wide range of actin binding proteins [10Paunola E. Mattila P.K. Lappalainen P. WH2 domain: a small, versatile adapter for actin monomers.FEBS Lett. 2002; 513: 92-97Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar]. They fall into two broad subclasses: those related to a domain present in the Arp2/3 activator WASp, and those more similar to an actin monomer sequestering protein called thymosin β4. The two types of motif are thought to make near-identical contacts with ATP–actin via a conserved stretch of six amino acids (LKKTET) that lies extended along the ‘outer face’ of the actin subunit [11Hertzog M. van Heijenoort C. Didry D. Gaudier M. Coutant J. Gigant B. Didelot G. Preat T. Knossow M. Guittet E. et al.The beta-thymosin/WH2 domain; structural basis for the switch from inhibition to promotion of actin assembly.Cell. 2004; 117: 611-623Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar, 12Irobi E. Aguda A.H. Larsson M. Guerin C. Yin H.L. Burtnick L.D. Blanchoin L. Robinson R.C. Structural basis of actin sequestration by thymosin-beta4: implications for WH2 proteins.EMBO J. 2004; 23: 3599-3608Crossref PubMed Scopus (93) Google Scholar] (Figure 1). In WASp family proteins, this motif promotes actin filament formation by bringing ATP–actin monomers into the proximity of the Arp2/3 complex to promote the formation of the Arp2/3–actin nucleus [1Pollard T.D. Borisy G.G. Cellular motility driven by assembly and disassembly of actin filaments.Cell. 2003; 112: 453-465Abstract Full Text Full Text PDF PubMed Scopus (3109) Google Scholar], whereas the corresponding motif in thymosin β4 inhibits actin filament elongation by sequestering actin–ATP monomers [10Paunola E. Mattila P.K. Lappalainen P. WH2 domain: a small, versatile adapter for actin monomers.FEBS Lett. 2002; 513: 92-97Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar]. It is in this context that Quinlan et al. [2Quinlan M.E. Heuser J.E. Kerkhoff E. Mullins R.D. Drosophila Spire is an actin nucleation factor.Nature. 2005; 433: 382-388Crossref PubMed Scopus (245) Google Scholar] set out to study the biochemical function of Spire, perhaps intrigued by its actin-related mutant phenotype in flies and its four evenly spaced WH2 domains. Quinlan et al. [2Quinlan M.E. Heuser J.E. Kerkhoff E. Mullins R.D. Drosophila Spire is an actin nucleation factor.Nature. 2005; 433: 382-388Crossref PubMed Scopus (245) Google Scholar] began their study by confirming that over-expression of Spire is sufficient to induce actin filament formation in mammalian cells [13Otto I.M. Raabe T. Rennefahrt U.E. Bork P. Rapp U.R. Kerkhoff E. The p150-Spir protein provides a link between c-Jun N-terminal kinase function and actin reorganization.Curr. Biol. 2000; 10: 345-348Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar]. Although similar structures are formed following the expression of constitutively active WASp, through activation of Arp2/3, Quinlan et al. [2Quinlan M.E. Heuser J.E. Kerkhoff E. Mullins R.D. Drosophila Spire is an actin nucleation factor.Nature. 2005; 433: 382-388Crossref PubMed Scopus (245) Google Scholar] found that the filament clumps induced by Spire do not co-localise with the Arp2/3 complex. How then is Spire able to induce actin filament formation? To answer this question, Quinlan et al. [2Quinlan M.E. Heuser J.E. Kerkhoff E. Mullins R.D. Drosophila Spire is an actin nucleation factor.Nature. 2005; 433: 382-388Crossref PubMed Scopus (245) Google Scholar] tested the activity of Spire WH2 domains in an actin polymerisation assay. Surprisingly, they found that the amino terminus of Spire, which carries the four WH2 repeats, is sufficient to promote the formation of actin filaments from purified actin–ATP monomers, even in the absence of the Arp2/3 complex , and does so at a rate similar to that induced by the formin Cappuccino. After the new actin filament seeds are formed, they extend by rapid ‘barbed end’ elongation, while Spire caps the filament ‘pointed end’, protecting the filament from disassembly. Thus Spire represents a new class of metazoan protein that can catalyse actin filament nucleation. Quinlan et al. [2Quinlan M.E. Heuser J.E. Kerkhoff E. Mullins R.D. Drosophila Spire is an actin nucleation factor.Nature. 2005; 433: 382-388Crossref PubMed Scopus (245) Google Scholar] then carried out a molecular dissection of Spire to identify the region responsible for this novel nucleation activity. In the actin polymerization assay, actin filament nucleation was most profoundly affected by loss of the two most carboxy-terminal WH2 domains and by loss of a small linker region connecting the last two WH2 domains (even though these regions of the protein have been poorly conserved during metazoan evolution). In isolation, however, individual WH2 motifs were unable to induce efficient actin filament nucleation. Thus, the novel nucleation activity exhibited by Spire appears to depend both on the sequence of the WH2 motifs involved and on their concatamerisation. These data led Quinlan et al. to propose a model (Figure 1) for Spire-dependent nucleation. They propose that this process is initiated by the carboxy-terminal WH2 domain binding to monomeric actin. The three other, evenly-spaced WH2 domains then bring consecutive actin monomers into alignment to promote the formation of a single-stranded polymer. This acts as a template for rapid actin polymerization, while the carboxy-terminal WH2 domain caps the ‘pointed end’ of the filament. Quinlan et al. [2Quinlan M.E. Heuser J.E. Kerkhoff E. Mullins R.D. Drosophila Spire is an actin nucleation factor.Nature. 2005; 433: 382-388Crossref PubMed Scopus (245) Google Scholar] used electron microscopy to obtain concrete evidence for hypothetical intermediates in this pathway. The structures observed were of the expected size for single filament actin tetramers and, as predicted by the model, appeared to be approximately half the length when two carboxy-terminal WH2 motifs were used in the actin nucleation assay in place of all four. Because actin nuclei rapidly elongate, it seems likely that the complexes identified represent pre-nuclei which, after the recruitment of additional actin monomers, form a two stranded polymer substrate for rapid ‘barbed end’ elongation. Prior to this study [2Quinlan M.E. Heuser J.E. Kerkhoff E. Mullins R.D. Drosophila Spire is an actin nucleation factor.Nature. 2005; 433: 382-388Crossref PubMed Scopus (245) Google Scholar], two other WH2 repeat proteins had been shown to regulate actin filament dynamics [11Hertzog M. van Heijenoort C. Didry D. Gaudier M. Coutant J. Gigant B. Didelot G. Preat T. Knossow M. Guittet E. et al.The beta-thymosin/WH2 domain; structural basis for the switch from inhibition to promotion of actin assembly.Cell. 2004; 117: 611-623Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar, 14Hertzog M. Yarmola E.G. Didry D. Bubb M.R. Carlier M.F. Control of actin dynamics by proteins made of beta-thymosin repeats: the actobindin family.J. Biol. Chem. 2002; 277: 14786-14792Crossref PubMed Scopus (59) Google Scholar]. Although these proteins, Actobinin and Ciboulet, do not promote de novo actin filament formation, they facilitate ‘barbed end’ elongation and, like Spire, cap the ‘pointed ends’ of actin filaments. Interestingly, the WH2-like motifs in Actobindin and Ciboulet are evenly spaced at approximately 30–35 residue intervals, as they are in Spire. This arrangement of closely apposed WH2-like motifs will facilitate monomer–filament and monomer–monomer interactions, lowering the critical actin concentration required for nucleation and/or polymerization. Moreover, it will severely restrict the geometry of actin–WH2 complexes, driving the formation of single polymer strands like those visualized by Quinlan et al. [2Quinlan M.E. Heuser J.E. Kerkhoff E. Mullins R.D. Drosophila Spire is an actin nucleation factor.Nature. 2005; 433: 382-388Crossref PubMed Scopus (245) Google Scholar]. Thus, in spite of their distinct biochemical functions, the tandem WH2 motifs in Actobindin, Ciboulet and Spire may have a common mode of action. In fact, flexibility of function may be a general feature of WH2 domains, as Spire, or any one of its WH2 domains, can also act to sequester actin monomers [2Quinlan M.E. Heuser J.E. Kerkhoff E. Mullins R.D. Drosophila Spire is an actin nucleation factor.Nature. 2005; 433: 382-388Crossref PubMed Scopus (245) Google Scholar]. And, conversely, the classic actin monomer sequestering protein thymosin β4 can promote actin filament formation when present at very high, but physiological concentrations [15Sun H.Q. Kwiatkowska K. Yin H.L. beta-Thymosins are not simple actin monomer buffering proteins. Insights from overexpression studies.J. Biol. Chem. 1996; 271: 9223-9230Crossref PubMed Scopus (53) Google Scholar, 16Carlier M.F. Didry D. Erk I. Lepault J. Van Troys M.L. Vandekerckhove J. Perelroizen I. Yin H. Doi Y. Pantaloni D. Tbeta 4 is not a simple G-actin sequestering protein and interacts with F-actin at high concentration.J. Biol. Chem. 1996; 271: 9231-9239Crossref PubMed Scopus (66) Google Scholar]. Moreover, the actin polymers formed in the presence of thymosin β4 have a tendency to form separated individual actin strands [17Ballweber E. Hannappel E. Huff T. Stephan H. Haener M. Taschner N. Stoffler D. Aebi U. Mannherz H.G. Polymerisation of chemically cross-linked actin:thymosin beta(4) complex to filamentous actin: alteration in helical parameters and visualisation of thymosin beta(4) binding on F-actin.J. Mol. Biol. 2002; 315: 613-625Crossref PubMed Scopus (49) Google Scholar], reminiscent of those identified by Quinlan et al in mixtures of actin and Spire. These data show that the ability of a WH2 domain to influence actin filament dynamics can be profoundly affected by its local concentration, as well as by its sequence. The residues that flank the core LKKTET motif, however, are likely to be the most important factor in determining the function of a particular WH2 domain. These residues control access to the ‘barbed’ and ‘pointed’ ends of growing filaments and they differ widely between WH2 domains that promote or inhibit actin filament formation [11Hertzog M. van Heijenoort C. Didry D. Gaudier M. Coutant J. Gigant B. Didelot G. Preat T. Knossow M. Guittet E. et al.The beta-thymosin/WH2 domain; structural basis for the switch from inhibition to promotion of actin assembly.Cell. 2004; 117: 611-623Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar, 12Irobi E. Aguda A.H. Larsson M. Guerin C. Yin H.L. Burtnick L.D. Blanchoin L. Robinson R.C. Structural basis of actin sequestration by thymosin-beta4: implications for WH2 proteins.EMBO J. 2004; 23: 3599-3608Crossref PubMed Scopus (93) Google Scholar]. It has also been suggested that interactions between the amino and carboxyl termini of adjacent WH2 domains regulate capping [11Hertzog M. van Heijenoort C. Didry D. Gaudier M. Coutant J. Gigant B. Didelot G. Preat T. Knossow M. Guittet E. et al.The beta-thymosin/WH2 domain; structural basis for the switch from inhibition to promotion of actin assembly.Cell. 2004; 117: 611-623Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar]. So the WH2-linker region identified by Quinlan et al. [2Quinlan M.E. Heuser J.E. Kerkhoff E. Mullins R.D. Drosophila Spire is an actin nucleation factor.Nature. 2005; 433: 382-388Crossref PubMed Scopus (245) Google Scholar] may catalyse the release of the pointed end cap of the adjacent WH2 motif to promote the formation of a dimeric actin nucleus. In conclusion, the ability of WH2-like domains to bind actin monomers and the exposed face of actin-ATP subunits within a filament gives them an unsurpassed flexibility. This enables them to be adapted during evolution for use in actin monomer sequestration, activation of the Arp2/3 complex, actin filament elongation and, as discovered by Quinlan et al. [2Quinlan M.E. Heuser J.E. Kerkhoff E. Mullins R.D. Drosophila Spire is an actin nucleation factor.Nature. 2005; 433: 382-388Crossref PubMed Scopus (245) Google Scholar], in actin nucleation. The Arp2/3 complex generates branched filament networks, formins give rise to single filaments that are resistant to ‘barbed end’ capping [5Xu 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: 711-723Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar], and Spire generates unbranched filaments that are resistant to pointed end disassembly [2Quinlan M.E. Heuser J.E. Kerkhoff E. Mullins R.D. Drosophila Spire is an actin nucleation factor.Nature. 2005; 433: 382-388Crossref PubMed Scopus (245) Google Scholar]. It therefore seems likely that each nucleator will give rise to a distinct set of actin-based structures, helping to generate the diversity of cell forms and behaviours observed within developing and adult animals. And given the phenotypic similarities of Spire and Cappuccino mutants [7Manseau L.J. Schupbach T. cappuccino and spire: two unique maternal-effect loci required for both the anteroposterior and dorsoventral patterns of the Drosophila embryo.Genes Dev. 1989; 3: 1437-1452Crossref PubMed Scopus (175) Google Scholar], and the fact that the two nucleators appear to have an identical distribution in mouse embryos [18Schumacher N. Borawski J.M. Leberfinger C.B. Gessler M. Kerkhoff E. Overlapping expression pattern of the actin organizers Spir-1 and formin-2 in the developing mouse nervous system and the adult brain.Gene Expr. Patterns. 2004; 4: 249-255Crossref PubMed Scopus (37) Google Scholar], it is possible that they function together. Although there is no evidence for biochemical synergy between these proteins [2Quinlan M.E. Heuser J.E. Kerkhoff E. Mullins R.D. Drosophila Spire is an actin nucleation factor.Nature. 2005; 433: 382-388Crossref PubMed Scopus (245) Google Scholar], their active collaboration would be expected to generate relatively long and stable filaments that are protected at both barbed and pointed ends. Perhaps such filaments have a specific function during the development of the Drosophila oocyte. As discussed above, however, subtle changes in the concentration and context of an individual actin binding domain can dramatically alter actin filaments dynamics in a defined biochemical system. Thus, it is difficult to extrapolate from in vitro experiments to predict the role of an actin regulator in a living cell, where actin filament dynamics are orchestrated by dozens of actin regulators functioning in concert. Hence the need for a combination of biochemistry, genetics and cell biology in the study of the actin cytoskeleton.
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