Characterization of Cytokinetic F-BARs and Other Membrane-Binding Proteins

McDonald, Nathan A.; Gould, Kathleen L.

doi:10.1007/978-1-4939-3145-3_13

Cited by 3 publications

(3 citation statements)

References 13 publications

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“…For Figure 5A, lipids were obtained from Sigma-Aldrich (B1502, Brain extract from bovine brain, Type I, Folch Fraction). In vitro liposome preparation was performed as previously described (McDonald and Gould, 2016). For liposome co-pelleting assay, 10 μg of Flag-Cdc15(E30KE152K) was incubated for 30 min at 30°C with lambda phosphatase (New England Biolabs, P0753) and 1 mM MnCl 2 .…”

Section: Lipid Binding Assaymentioning

confidence: 99%

DYRK kinase Pom1 drives F-BAR protein Cdc15 from the membrane to promote medial division

Bhattacharjee

Mangione

Woś

et al. 2020

MBoC

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Section: Lipid Binding Assaymentioning

confidence: 99%

DYRK kinase Pom1 drives F-BAR protein Cdc15 from the membrane to promote medial division

Bhattacharjee

Mangione

Woś

et al. 2020

MBoC

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“…To prepare liposomes, lipids were obtained from Sigma-Aldrich (B1502, brain extract from bovine brain, type I, Folch fraction). In vitro liposome preparation was performed as previously described (McDonald and Gould, 2016a). Radioactive in vitro kinase assays using recombinant His-Imp2 protein as substrate were performed either in Cdk1 buffer (20 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM DTT, 10 mM MgCl 2 and 100 µM ATP) or in Hhp1 buffer [1× PMP buffer (New England Biolabs), 10 mM MgCl 2 and 100 µM ATP] with 1 µCi γ-32 P-ATP (PerkinElmer BLU002250UC).…”

Section: Liposome Co-pelleting Assaymentioning

confidence: 99%

Phosphorylation in the intrinsically disordered region of F-BAR protein Imp2 regulates its contractile ring recruitment

Willet

Igarashi

Chen

et al. 2021

Journal of Cell Science

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The F-BAR protein Imp2 is an important contributor to cytokinesis in the fission yeast, Schizosaccharomyces pombe. Because cell cycle regulated phosphorylation of the central intrinsically disordered region (IDR) of the Imp2 paralog, Cdc15, controls Cdc15 oligomerization state, localization, and ability to bind protein partners, we investigated whether Imp2 is similarly phosphoregulated. We found that Imp2 is endogenously phosphorylated on 28 sites within its IDR with the bulk of phosphorylation being constitutive. In vitro, casein kinase 1 (CK1) Hhp1 and Hhp2 can phosphorylate 17 sites and Cdk1 the remaining 11 sites. Mutations that prevent Cdk1 phosphorylation result in precocious Imp2 recruitment to the cell division site, and mutations designed to mimic these phosphorylation events delay Imp2 CR accumulation. Mutations that eliminated CK1 phosphorylation sites allowed CR sliding, and phosphomimetic substitutions at these sites reduced Imp2 protein levels and slowed CR constriction. Thus, like Cdc15, the Imp2 IDR is phosphorylated at many sites by multiple kinases. In contrast to Cdc15, for which phosphorylation plays a major cell cycle regulatory role, Imp2 phosphorylation is primarily constitutive with milder effects on localization and function.

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“…Giant unilamellar vesicles were formed as previously described (McDonald and Gould, 2016a). Chloroform lipid stocks of the desired composition were evaporated on ITO-coated glass coverslides.…”

Section: Liposome Co-pelleting Assaysmentioning

confidence: 99%

The F-BAR Domain of Rga7 Relies on a Cooperative Mechanism of Membrane Binding with a Partner Protein during Fission Yeast Cytokinesis

Liu¹,

McDonald²,

Naegele³

et al. 2019

Cell Reports

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SUMMARYF-BAR proteins bind the plasma membrane (PM) to scaffold and organize the actin cytoskeleton. To understand how F-BAR proteins achieve their PM association, we studied the localization of a Schizosaccharomyces pombe F-BAR protein Rga7, which requires the coiled-coil protein Rng10 for targeting to the division site during cytokinesis. We find that the Rga7 F-BAR domain directly binds a motif in Rng10 simultaneously with the PM, and that an adjacent Rng10 motif independently binds the PM. Together, these multivalent interactions significantly enhance Rga7 F-BAR avidity for membranes at physiological protein concentrations, ensuring the division site localization of Rga7. Moreover, the requirement for the F-BAR domain in Rga7 localization and function in cytokinesis is bypassed by tethering an Rga7 construct lacking its F-BAR to Rng10, indicating that at least some F-BAR domains are necessary but not sufficient for PM targeting and are stably localized to specific cortical positions through adaptor proteins.

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Characterization of Cytokinetic F-BARs and Other Membrane-Binding Proteins

Cited by 3 publications

References 13 publications

DYRK kinase Pom1 drives F-BAR protein Cdc15 from the membrane to promote medial division

DYRK kinase Pom1 drives F-BAR protein Cdc15 from the membrane to promote medial division

Phosphorylation in the intrinsically disordered region of F-BAR protein Imp2 regulates its contractile ring recruitment

The F-BAR Domain of Rga7 Relies on a Cooperative Mechanism of Membrane Binding with a Partner Protein during Fission Yeast Cytokinesis

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