The F-BAR Protein Syp1 Negatively Regulates WASp-Arp2/3 Complex Activity during Endocytic Patch Formation

Douglas R. Boettner, Jessica L. D'Agostino, Onaidy Teresa Torres, Karen Daugherty-Clarke, Aysu Uygur, Amanda Reider, Beverly Wendland, Sandra Lemmon, Bruce L. Goode

Research output: Contribution to journalArticle

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Abstract

Background: Actin polymerization by Arp2/3 complex must be tightly regulated to promote clathrin-mediated endocytosis. Although many Arp2/3 complex activators have been identified, mechanisms for its negative regulation have remained more elusive. To address this, we analyzed the yeast arp2-7 allele, which is biochemically unique in causing unregulated actin assembly in vitro in the absence of Arp2/3 activators. Results: We examined endocytosis in arp2-7 mutants by live-cell imaging of Sla1-GFP, a coat marker, and Abp1-RFP, which marks the later actin phase of endocytosis. Sla1-GFP and Abp1-RFP lifetimes were accelerated in arp2-7 mutants, which is opposite to actin nucleation-impaired arp2 alleles or deletions of Arp2/3 activators. We performed a screen for multicopy suppressors of arp2-7 and identified SYP1, an FCHO1 homolog, which contains F-BAR and AP-2μ homology domains. Overexpression of SYP1 in arp2-7 cells slowed Sla1-GFP lifetimes closer to wild-type cells. Further, purified Syp1 directly inhibited Las17/WASp stimulation of Arp2/3 complex-mediated actin assembly in vitro. This activity was mapped to a fragment of Syp1 located between its F-BAR and AP-2μ homology domains and depends on sequences in Las17/WASp outside of the VCA domain. Conclusions: Together, these data identify Syp1 as a novel negative regulator of WASp-Arp2/3 complex that helps choreograph the precise timing of actin assembly during endocytosis.

Original languageEnglish
Pages (from-to)1979-1987
Number of pages9
JournalCurrent Biology
Volume19
Issue number23
DOIs
StatePublished - Dec 15 2009

Fingerprint

Actin-Related Protein 2-3 Complex
actin
Actins
endocytosis
Endocytosis
Proteins
proteins
sequence homology
Alleles
alleles
clathrin
mutants
Clathrin
cells
Polymerization
polymerization
Yeast
Nucleation
Yeasts
image analysis

Keywords

  • CELLBIO

ASJC Scopus subject areas

  • Agricultural and Biological Sciences(all)
  • Biochemistry, Genetics and Molecular Biology(all)

Cite this

Boettner, D. R., D'Agostino, J. L., Torres, O. T., Daugherty-Clarke, K., Uygur, A., Reider, A., ... Goode, B. L. (2009). The F-BAR Protein Syp1 Negatively Regulates WASp-Arp2/3 Complex Activity during Endocytic Patch Formation. Current Biology, 19(23), 1979-1987. https://doi.org/10.1016/j.cub.2009.10.062

The F-BAR Protein Syp1 Negatively Regulates WASp-Arp2/3 Complex Activity during Endocytic Patch Formation. / Boettner, Douglas R.; D'Agostino, Jessica L.; Torres, Onaidy Teresa; Daugherty-Clarke, Karen; Uygur, Aysu; Reider, Amanda; Wendland, Beverly; Lemmon, Sandra; Goode, Bruce L.

In: Current Biology, Vol. 19, No. 23, 15.12.2009, p. 1979-1987.

Research output: Contribution to journalArticle

Boettner, DR, D'Agostino, JL, Torres, OT, Daugherty-Clarke, K, Uygur, A, Reider, A, Wendland, B, Lemmon, S & Goode, BL 2009, 'The F-BAR Protein Syp1 Negatively Regulates WASp-Arp2/3 Complex Activity during Endocytic Patch Formation', Current Biology, vol. 19, no. 23, pp. 1979-1987. https://doi.org/10.1016/j.cub.2009.10.062
Boettner DR, D'Agostino JL, Torres OT, Daugherty-Clarke K, Uygur A, Reider A et al. The F-BAR Protein Syp1 Negatively Regulates WASp-Arp2/3 Complex Activity during Endocytic Patch Formation. Current Biology. 2009 Dec 15;19(23):1979-1987. https://doi.org/10.1016/j.cub.2009.10.062
Boettner, Douglas R. ; D'Agostino, Jessica L. ; Torres, Onaidy Teresa ; Daugherty-Clarke, Karen ; Uygur, Aysu ; Reider, Amanda ; Wendland, Beverly ; Lemmon, Sandra ; Goode, Bruce L. / The F-BAR Protein Syp1 Negatively Regulates WASp-Arp2/3 Complex Activity during Endocytic Patch Formation. In: Current Biology. 2009 ; Vol. 19, No. 23. pp. 1979-1987.
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abstract = "Background: Actin polymerization by Arp2/3 complex must be tightly regulated to promote clathrin-mediated endocytosis. Although many Arp2/3 complex activators have been identified, mechanisms for its negative regulation have remained more elusive. To address this, we analyzed the yeast arp2-7 allele, which is biochemically unique in causing unregulated actin assembly in vitro in the absence of Arp2/3 activators. Results: We examined endocytosis in arp2-7 mutants by live-cell imaging of Sla1-GFP, a coat marker, and Abp1-RFP, which marks the later actin phase of endocytosis. Sla1-GFP and Abp1-RFP lifetimes were accelerated in arp2-7 mutants, which is opposite to actin nucleation-impaired arp2 alleles or deletions of Arp2/3 activators. We performed a screen for multicopy suppressors of arp2-7 and identified SYP1, an FCHO1 homolog, which contains F-BAR and AP-2μ homology domains. Overexpression of SYP1 in arp2-7 cells slowed Sla1-GFP lifetimes closer to wild-type cells. Further, purified Syp1 directly inhibited Las17/WASp stimulation of Arp2/3 complex-mediated actin assembly in vitro. This activity was mapped to a fragment of Syp1 located between its F-BAR and AP-2μ homology domains and depends on sequences in Las17/WASp outside of the VCA domain. Conclusions: Together, these data identify Syp1 as a novel negative regulator of WASp-Arp2/3 complex that helps choreograph the precise timing of actin assembly during endocytosis.",
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