Multiple Forms of Spire-Actin Complexes and their Functional Consequences

Chen, Christine K.; Sawaya, M.R.; Phillips, Martin; Reisler, Emil; Quinlan, Margot E.

doi:10.1074/jbc.m111.317792

Cited by 23 publications

(23 citation statements)

References 30 publications

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“…In neither case was the change dramatic: the K d of S c3 was 0.11 µM, about twice as tight as that for S c alone ( K d (S c ) = 0.21 µM); in contrast, S 3d is slightly weaker than S d alone ( K d (S 3d ) = 0.78 µM vs. K d (S d ) = 0.62 µM). In our previously published co-crystal of S c3d with actin, we observed S d and part of Linker 3 bound to actin [9]. Linker 3 extends away from the actin monomer, suggesting that it would not contribute significantly to equilibrium binding, consistent with what we report here.…”

Section: Resultssupporting

confidence: 91%

“…The later possibility still lends itself to an interesting role in nucleation. Finally, we note that Spir WH2 domains sever filaments [9,28], which presumably required filament binding. Perhaps S c mediates this interaction.…”

Section: Discussionmentioning

confidence: 99%

“…Coverslips for TIRF elongation assays were prepared as described [9]. Briefly, they were silanized with 5% 3-aminopropyl-triethoxysilane (Sigma-Aldrich, St. Louis, MO) and PEGylated with N-hydroxylsuccinimide–functionalized polyethylene glycol (PEG-NHS; 97% methoxy-PEG-NHS and 3% biotin-PEG-NHS (JenKem Technology, Allen, TX).…”

Section: Methodsmentioning

confidence: 99%

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Filament Assembly by Spire: Key Residues and Concerted Actin Binding

Rasson

Bois

Pham

et al. 2015

Journal of Molecular Biology

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The most recently identified class of actin nucleators, WASp Homology domain 2 (WH2) – nucleators, use tandem repeats of monomeric actin-binding WH2 domains to facilitate actin nucleation. WH2 domains are involved in a wide variety of actin regulatory activities. Structurally, they are expected to clash with interprotomer contacts within the actin filament. Thus, the discovery of their role in nucleation was surprising. Here we use Drosophila Spire (Spir) as a model system to investigate both how tandem WH2 domains can nucleate actin and what differentiates nucleating WH2-containing proteins from their non-nucleating counterparts. We found that the third WH2 domain in Spir (Spir-C or Sc), plays a unique role. In the context of a short nucleation construct (containing only two WH2 domains), placement of Sc in the N-terminal position was required for the most potent nucleation. We found that the native organization of the WH2 domains with respect to each other is necessary for binding to actin with positive cooperativity. We identified two residues within Sc that are critical for its activity. Using this information we were able to convert a weak synthetic nucleator into one with activity equal to a native Spir construct. Lastly, we found evidence that Sc binds actin filaments, in addition to monomers.

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Section: Resultssupporting

confidence: 91%

Section: Discussionmentioning

confidence: 99%

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Filament Assembly by Spire: Key Residues and Concerted Actin Binding

Rasson

Bois

Pham

et al. 2015

Journal of Molecular Biology

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“…Our data are consistent with the idea that Spir nucleates de novo actin filaments in dendrites via its WH2 and KIND domains, but Spir activity could be more complex as Spir can also sequester, sever and depolymerize F-actin in vitro (Chen et al, 2012). We think it is likely that Spir contributes to specific subcellular F-actin pools or structures that promote the probability of dendrite branching at their correct positions within arbors, perhaps acting in concert with other nucleators such as the Arp2/3 complex and formins.…”

Section: Discussionsupporting

confidence: 80%

Dendrite architecture organized by transcriptional control of the F-actin nucleator Spire

Ferreira

et al. 2014

Journal of Cell Science

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The architectures of dendritic trees are crucial for the wiring and function of neuronal circuits because they determine coverage of receptive territories, as well as the nature and strength of sensory or synaptic inputs. Here, we describe a cell-intrinsic pathway sculpting dendritic arborization (da) neurons in Drosophila that requires Longitudinals Lacking (Lola), a BTB/POZ transcription factor, and its control of the F-actin cytoskeleton through Spire (Spir), an actin nucleation protein. Loss of Lola from da neurons reduced the overall length of dendritic arbors, increased the expression of Spir, and produced inappropriate F-actin-rich dendrites at positions too near the cell soma. Selective removal of Lola from only class IV da neurons decreased the evasive responses of larvae to nociception. The increased Spir expression contributed to the abnormal F-actinrich dendrites and the decreased nocifensive responses because both were suppressed by reduced dose of Spir. Thus, an important role of Lola is to limit expression of Spir to appropriate levels within da neurons. We found Spir to be expressed in dendritic arbors and to be important for their development. Removal of Spir from class IV da neurons reduced F-actin levels and total branch number, shifted the position of greatest branch density away from the cell soma, and compromised nocifensive behavior. We conclude that the Lola-Spir pathway is crucial for the spatial arrangement of branches within dendritic trees and for neural circuit function because it provides balanced control of the F-actin cytoskeleton.

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“…In addition to a series of four WH2 motifs, Spire contains an N-terminal KIND domain that mediates binding to the C-terminal tail of its cognate formin Cappuccino (21). The four WH2 domains in Spire are closely spaced and can create a linear arrangement of monomers in one strand but may also promote lateral actin subunit interactions (46). Irrespective of whether lateral interactions arise in association with a single Spire molecule, two Spire molecules can bind to one Cappuccino dimer, which could catalyze formation of a twostranded seed.…”

Section: Discussionmentioning

confidence: 99%

Structure of the formin-interaction domain of the actin nucleation-promoting factor Bud6

Graziano

Park

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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Formin proteins and their associated factors cooperate to assemble unbranched actin filaments in diverse cellular structures. The Saccharomyces cerevisiae formin Bni1 and its associated nucleation-promoting factor (NPF) Bud6 generate actin cables and mediate polarized cell growth. Bud6 binds to both the tail of the formin and G-actin, thereby recruiting monomeric actin to the formin to create a nucleation seed. Here, we structurally and functionally dissect the nucleation-promoting C-terminal region of Bud6 into a Bni1-binding “core” domain and a G-actin binding “flank” domain. The ∼2-Å resolution crystal structure of the Bud6 core domain reveals an elongated dimeric rod with a unique fold resembling a triple-helical coiled-coil. Binding and actin-assembly assays show that conserved residues on the surface of this domain mediate binding to Bni1 and are required for NPF activity. We find that the Bni1 dimer binds two Bud6 dimers and that the Bud6 flank binds a single G-actin molecule. These findings suggest a model in which a Bni1/Bud6 complex with a 2:4 subunit stoichiometry assembles a nucleation seed with Bud6 coordinating up to four actin subunits.

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Multiple Forms of Spire-Actin Complexes and their Functional Consequences

Cited by 23 publications

References 30 publications

Filament Assembly by Spire: Key Residues and Concerted Actin Binding

Filament Assembly by Spire: Key Residues and Concerted Actin Binding

Dendrite architecture organized by transcriptional control of the F-actin nucleator Spire

Structure of the formin-interaction domain of the actin nucleation-promoting factor Bud6

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