Spire stimulates nucleation by Cappuccino and binds both ends of actin filaments

Mol Biol Cell. 2020 Feb 15;31(4):273-286. doi: 10.1091/mbc.E19-09-0550. Epub 2019 Dec 26.

Abstract

The actin nucleators Spire and Cappuccino synergize to promote actin assembly, but the mechanism of their synergy is controversial. Together these proteins promote the formation of actin meshes, which are conserved structures that regulate the establishment of oocyte polarity. Direct interaction between Spire and Cappuccino is required for oogenesis and for in vitro synergistic actin assembly. This synergy is proposed to be driven by elongation and the formation of a ternary complex at filament barbed ends, or by nucleation and interaction at filament pointed ends. To mimic the geometry of Spire and Cappuccino in vivo, we immobilized Spire on beads and added Cappuccino and actin. Barbed ends, protected by Cappuccino, grow away from the beads while pointed ends are retained, as expected for nucleation-driven synergy. We found that Spire is sufficient to bind barbed ends and retain pointed ends of actin filaments near beads and we identified Spire's barbed-end binding domain. Loss of barbed-end binding increases nucleation by Spire and synergy with Cappuccino in bulk pyrene assays and on beads. Importantly, genetic rescue by the loss-of-function mutant indicates that barbed-end binding is not necessary for oogenesis. Thus, increased nucleation is a critical element of synergy both in vitro and in vivo.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Actin Cytoskeleton / metabolism*
  • Actin Cytoskeleton / ultrastructure
  • Amino Acid Sequence
  • Animals
  • Biotin / chemistry
  • Drosophila Proteins / genetics*
  • Drosophila Proteins / metabolism
  • Drosophila melanogaster / genetics*
  • Drosophila melanogaster / growth & development
  • Drosophila melanogaster / metabolism
  • Female
  • Gene Expression Regulation, Developmental
  • Immobilized Proteins
  • Microfilament Proteins / genetics*
  • Microfilament Proteins / metabolism
  • Microspheres
  • Mutation
  • Oocytes / cytology
  • Oocytes / growth & development
  • Oocytes / metabolism*
  • Oogenesis / genetics*
  • Profilins / genetics
  • Profilins / metabolism
  • Protein Domains
  • Streptavidin / chemistry

Substances

  • Drosophila Proteins
  • Immobilized Proteins
  • Microfilament Proteins
  • Profilins
  • capu protein, Drosophila
  • spir protein, Drosophila
  • Biotin
  • Streptavidin