The DIAPH3 linker specifies a Beta-actin network that maintains RhoA and Myosin-II at the cytokinetic furrow

The diverse functions of actin are regulated through a combination of unique actin isoforms, distinct actin-binding proteins, spatial regulation, and post-translation modifications; for example, during cell division β-actin and γ-actin perform unique functions and have discrete localization patterns.  During these cytokinesis events the formins DIAPH3 and DIAPH1 interact with β-actin and γ-actin, respectively, to generate unique networks that assist in remodeling the plasma membrane.  Both actin networks appear important for cytokinesis; however, whether these mechanisms overlap or can substitute for each other is unknown.  Recently, Shah et al. performed in-depth studies of these actin isoform networks to understand the interplay and overlap that may exist.  Initial studies of fusion proteins containing the FH2 domains of either DIAPH3 or DIAPH1 fused to a mitochondrial localizing signal showed that the FH2 of these formins are sufficient to regulate β-actin and γ-actin respectively.  The group then utilized purified beta and gamma actin to perform in vitro co-sedimentation assays and western blots to ensure other cellular factors were not contributing to the FH2-dependent effects.  In this assay, they expressed GST-tagged DIAPH1 and DIAPH3, as well as chimeras that included DIAPH1-3F (DIAPH1 with the DIAPH3 FH2 domain) and DIAPH3-1F (DIAPH3 with the DIAPH1 FH2 domain).  These studies confirmed that the FH2 domain of DIAPH3 sufficiently regulates β-actin polymerization, while the FH2 domain of DIAPH1 generated both β-actin and γ-actin filaments. Further dissection of the DIAPH3 FH2 domain identified that the linker region controls β-actin isoform specificity.  Chimeria fusion proteins where only the linker region was swapped sufficiently re-localized actin networks during cytokinesis.  However, these relocalized actin networks were not sufficient to perform effective cytokinesis, and the replacement of the β-actin network with the gamma actin network due to DIAPH3-1L expression in DIAPH3 siRNA treated cells (only targets wild-type DIAPH3) led to roughly 50% of these cells being multi-nucleated.  Ultimately, β-actin networks that regulate myosin II and RhoA are important for furrowing and proper cytokinesis. Cytoskeleton Inc.’s purified platelet actin (Cat. # APHL99) and chicken gizzard actin (Cat. # AS99) were critical tools to investigate the role of the DIAPH1 and DIAPH3 FH2 domains in this study.