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

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

Keywords:

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

Acknowledgements

We thank Dr. Wong Hoi Hui and Dr. Peng Ge from the California NanoSystems Institute at UCLA (CNSI) EM facility for their assistance with electron microscopy experiments. Supported by grants from the NIH (NS073968) and the Welch Foundation (I-1749) to JRT and NIH (GM077190) to ER.

Disclosure statement

The authors declare that they have no conflicts of interest with the contents of this article.

Additional information

Funding

Supported by grants from the NIH (NS073968) and the Welch Foundation (I-1749) to JRT and NIH (GM077190) to ER.