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

Abstract

Actin nucleators Cappuccino and Spire collaborate to build an actin mesh in oocytes. Data demonstrate that the collaboration leads to synergistic actin nucleation, as opposed to elongation.Further, Spire binds both ends of polar, actin filaments, resolving a long-outstanding question. AbstractAn actin mesh fills both mouse and fly oocytes. The meshes are built by a conserved mechanism and used to establish polarity. Two actin nucleators, Spire and Cappuccino, collaborate to build actin filaments that connect vesicles and the cortex. Direct interaction between Spire and Cappuccino is required for in vitro synergistic actin assembly; however, we understand little about why the interaction is necessary. To mimic the geometry of Spire and Cappuccino in vivo, we immobilized Spire on beads. We found that increased nucleation is a major part of synergy and that Spire alone binds both barbed-and pointed-ends of actin filaments. We identified Spire's barbed-end binding domain. Partial rescue of fertility by a loss-of-function mutant indicates that barbed-end binding is not necessary for Spire's in vivo function, but that it may play a role under normal circumstances. We propose that Spire stimulates nucleation by Cappuccino in a manner similar to the collaboration between APC and mDia1.

Identification of Maternal-Effect Genes in Zebrafish using Maternal Crispants

“…We did not identify maternal-effect phenotypes for two targeted genes, the spire- family gene zgc:114123 and the S subfamily potassium channel kcns3b, in spite of efficient production of INDELS in somatic cells of injected F0 embryos. Considering the expression pattern of zgc:114123 and role of Spire in oogenesis in other systems (Bradley et al, 2020; Leader et al, 2002; Manseau and Schüpbach, 1989; Pfender et al, 2011), it is possible that a loss of function for zgc:114123 affects oocyte development, precluding obtaining maternal crispant embryos in F1 clutches. The number of maternal transcripts for kcns3b is significantly less than other targets in our studies, so that the absence of maternal crispants for this gene may reflect a minor or non-essential role for this gene during development.…”

Alterations to the broad-spectrum formin inhibitor SMIFH2 improve potency

“…28 Profilin from Schizosaccharomyces pombe was expressed in a pET plasmid in BL21-DE3* cells and was purified as described. 41 Actin was purified from rabbit skeletal muscle and labeled with pyrene-iodoacetamide as described. 42 For pyrene-labeled actin, the pyrene concentration was calculated using an extinction coefficient of 21,978 M −1 cm −1 at 344 nm, and the actin concentration was calculated using the correction factor of 0.127 at 290 nm ([actin] = (A 290 −0.127 × A 344 ) × 38 μM).…”