Non diaphanous formin delphilin acts as a barbed end capping protein

Dutta, Priyanka; Das, Soumyajit; Maiti, Sankar

doi:10.1016/j.yexcr.2017.05.014

Cited by 12 publications

(20 citation statements)

References 36 publications

(40 reference statements)

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“…Consistent with weak side binding, at saturation only one mDelFFC dimer was bound per 20 actin monomers. Another group reported much tighter binding ( Dutta et al. , 2017 ).…”

Section: Resultsmentioning

confidence: 99%

The neuron-specific formin Delphilin nucleates nonmuscle actin but does not enhance elongation

Silkworth

Kunes

Nickel

et al. 2018

MBoC

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The formin Delphilin binds the glutamate receptor, GluRδ2, in dendritic spines of Purkinje cells. Both proteins play a role in learning. To understand how Delphilin functions in neurons, we studied the actin assembly properties of this formin. Formins have a conserved formin homology 2 domain, which nucleates and associates with the fast-growing end of actin filaments, influencing filament growth together with the formin homology 1 (FH1) domain. The strength of nucleation and elongation varies widely across formins. Additionally, most formins have conserved domains that regulate actin assembly through an intramolecular interaction. Delphilin is distinct from other formins in several ways: its expression is limited to Purkinje cells, it lacks classical autoinhibitory domains, and its FH1 domain has minimal proline-rich sequence. We found that Delphilin is an actin nucleator that does not accelerate elongation, although it binds to the barbed end of filaments. In addition, Delphilin exhibits a preference for actin isoforms, nucleating nonmuscle actin but not muscle actin, which has not been described or systematically studied in other formins. Finally, Delphilin is the first formin studied that is not regulated by intramolecular interactions. We speculate how the activity we observe is consistent with its localization in the small dendritic spines.

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“…Consistent with weak side binding, at saturation only one mDelFFC dimer was bound per 20 actin monomers. Another group reported much tighter binding ( Dutta et al. , 2017 ).…”

Section: Resultsmentioning

confidence: 99%

The neuron-specific formin Delphilin nucleates nonmuscle actin but does not enhance elongation

Silkworth

Kunes

Nickel

et al. 2018

MBoC

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“…However, in control experiments without actin, LmForminA and LmForminB proteins remained in the supernatant fraction (fig.3A, B, respectively). Results indicate that LmForminA and LmForminB protein have an F-actin binding ability in the in vitro condition with the FH2 domain similar to other well-characterized formins(Shimada et al, 2004;Dutta et al, 2017).The dissociation constant of LmForminA and LmForminB binds with F-actin actin were determined by analyzing the SDS-PAGE gel of co-sedimentation assay. The fraction of bound LmFormins with F-actin curve fitting shows the KD value 1.84 µM and 0.2 µM for LmForminA and LmForminB, respectively (fig.S5C, D).…”

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confidence: 84%

“…Post-harvest cells were resuspended in the lysis buffer (50 mM Tris-Cl pH 8.0, 100 mM NaCl, 30 mM Imidazole pH 8.0, 1 mM DTT, 0.2% IGEPAL (Sigma-Aldrich), 0.2% Thesit (Sigma-Aldrich), protease inhibitor cocktail followed by sonication (1 minute’s pulse with 5 second interval) and centrifuged at 12,000 rpm for 15 minutes. 50% Ni-NTA beads (Qiagen) were added with the supernatant, then washed with wash buffer (50 mM Tris-Cl pH 8.0, 100 mM NaCl, 30 mM imidazole pH 8.0) and eluted with elution buffer (50 mM Tris-Cl pH 8.0, 100 mM NaCl, 300Mm imidazole pH 8.0 pH 8.0 and 5% glycerol (Sigma-Aldrich) (Dutta et al, 2017). Purified protein dialysis in TNEG5 buffer (20 mM Tris pH 8.0, 100 mM NaCl, 1 mM EDTA, 5% Glycerol) at 4 °C.…”

Section: Experimental Methodsmentioning

confidence: 99%

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Formins play important role in Leishmania physiology by acting as cytosolic actin bundlers

Kushwaha

Seth

Jijumon

et al. 2021

Preprint

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Formin proteins regulate actin dynamics, are conserved throughout the eukaryotes cells. They play an important role in cell adhesion, motility, vesicular trafficking, and cytokinesis. Formins from class Kinetoplastida which includes infective organisms such as Leishmania and Trypanosoma not characterized to date, even though they are shown to be important in other protozoan parasites. The protozoan parasite Leishmania major (Lm) has two homologous formin proteins; LmForminA and LmForminB. Our study showed that LmForminA and LmForminB are expressed at RNA and protein levels in L. major cells. LmForminA and LmForminB are localized in the cytosol in patchy distribution patterns. LmForminA and LmForminB puncta also colocalize with the actin patches. The biochemical properties of L. major formins divulge that both formins are potent actin nucleator. LmForminA and LmForminB bind with the actin filament and have actin-bundling activity. We have also observed that formin inhibitor SMIFH2 influences the growth and physiology of L. major cells indicating formins are important for the Leishmania parasite.

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“…The rate of subunit addition mediated by a formin is thus dictated by the probability that an incoming actin monomer will find the FH2-barbed end complex in an open conformation, a property known as the Bgating factor ( Vavylonis et al 2006). The extent to which an FH2 dimer gates elongation varies widely among formin isoforms: FH2 dimers of the Schizosaccharomyces pombe formin Cdc12p and the mammalian formin Delphilin inhibit elongation by approximately 99% (Dutta et al 2017;Kovar et al 2003Kovar et al , 2006Silkworth et al 2018), suggesting that these formins strongly favor a closed conformation, whereas mouse mDia1 slows elongation only modestly (i.e., by about 5-10%), indicating that its FH2 domain frequently populates an open conformation (Kovar et al 2006). The equilibrium between open and closed conformations is formin-specific, and FH2 dimers from other formins produce intermediate effects on elongation (Gurel et al 2015;Kovar et al 2006;Thompson et al 2013).…”

Section: Actin Filament Assembly Mediated By Fh2 Domainsmentioning

confidence: 99%

Mechanisms of formin-mediated actin assembly and dynamics

2018

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Cellular viability requires tight regulation of actin cytoskeletal dynamics. Distinct families of nucleation-promoting factors enable the rapid assembly of filament nuclei that elongate and are incorporated into diverse and specialized actin-based structures. In addition to promoting filament nucleation, the formin family of proteins directs the elongation of unbranched actin filaments. Processive association of formins with growing filament ends is achieved through continuous barbed end binding of the highly conserved, dimeric formin homology (FH) 2 domain. In cooperation with the FH1 domain and C-terminal tail region, FH2 dimers mediate actin subunit addition at speeds that can dramatically exceed the rate of spontaneous assembly. Here, I review recent biophysical, structural, and computational studies that have provided insight into the mechanisms of formin-mediated actin assembly and dynamics.

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Non diaphanous formin delphilin acts as a barbed end capping protein

Cited by 12 publications

References 36 publications

The neuron-specific formin Delphilin nucleates nonmuscle actin but does not enhance elongation

The neuron-specific formin Delphilin nucleates nonmuscle actin but does not enhance elongation

Formins play important role in Leishmania physiology by acting as cytosolic actin bundlers

Mechanisms of formin-mediated actin assembly and dynamics

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