Parallel actin monomers in the 8S complex of actin–INF2

Sanchaita Das, Zixin Zhang, Saichandra Kalvakota, Raul Soto, Martin L. Phillips, Jonathan R. Terman, Emil Reisler

Research output: Contribution to journalArticlepeer-review

Abstract

Polymerization and depolymerization of actin play an essential role in eukaryotic cells. Actin exists in cells in both monomeric (G-actin) and filamentous (polymer, F-actin) forms. Actin binding proteins (ABPs) facilitate the transition between these two states, and their interactions with these two states of actin are critical for actin-based cellular processes. Rapid depolymerization of actin is assisted in the brain and/or other cells by its oxidation by the enzyme Mical (yielding Mox-actin), and/or by the binding of Inverted Formin 2 (INF2)–which can also accelerate filaments formation. At their stoichiometric molar ratio INF2 and actin yield the 8S complex (consisting of 4 actin monomers: 2 INF2 dimer molecules). Using biochemical and biophysical methods, we investigate the structural arrangement of actin in the 8S particles and the interaction of INF2 with actin and Mox-actin. To that end, we show 2 D class averages of 8S particles obtained by negative staining electron microscopy. We also show that: (i) 8S particles can seed rapid actin assembly; (ii) Mox-actin and INF2 form 8S particles at proteins ratios similar to those of unoxidized actin; (iii) chemical crosslinkings suggest that actin monomers are in a parallel orientation in the 8S particles of both actin and Mox-actin; and (iv) INF2 accelerates the disassembly of Mox-F-actin. Our results provide better understanding of actin's arrangement in the 8S particles formed during actin depolymerization and in the early polymerization stages of both actin and Mox-actin. Communicated by Ramaswamy H. Sarma.

Original languageEnglish (US)
JournalJournal of Biomolecular Structure and Dynamics
DOIs
StateAccepted/In press - 2022
Externally publishedYes

Keywords

  • 8S (8 Svedberg sedimentation coefficient units)
  • Actin
  • actin-INF2 8S particles
  • analytical ultracentrifugation
  • fluorescence assays
  • inverted formin 2 (INF2)
  • Mical-oxidized actin (Mox-actin)
  • protein crosslinking
  • rabbit skeletal actin

ASJC Scopus subject areas

  • Structural Biology
  • Molecular Biology

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