Addendum to the main text (i) ATE1-/-embryos from ATE1 +/-intercrosses were present at the expected (~25%) frequency up to ~E13.5, but virtually no ATE1 -/-embryos were recovered alive by E17. Specifically, no ATE1 -/-mice were recovered amongst either 954 F 2 -generation pups of the C57BL/6J-129SvEv (mixed) background or 267 F 2 -generation pups of the 129SvEv (inbred) background. Timed intercrosses of ATE1 +/-mice were used to determine that ATE1 -/-embryos were present at approximately the expected (25%) frequency up to ~E13.5, but no ATE1 -/-embryos were recovered alive by E17.Until E12.5, ATE1 -/-embryos appeared to be morphologically normal; however, their growth stopped during E13.5-E15.5. By E14.5-E15.5, ~50% of ATE1 -/-embryos were still alive, but growth-retarded. Live E14.5-E15.5 embryos were capable of opening their mouths and flexing their bodies, suggesting the absence of gross neuromuscular defects. Sections through E13.5 ATE1-/-embryos indicated the presence and apparently normal appearance of major organs, except for the phenotypes described in the main text and below.(ii) We examined the expression of ATE1 mRNA during embryogenesis using Northern hybridization with total RNA from +/+ embryos ranging in age from E4.5 to E18.5. ATE1 mRNA was present at least as early as E4.5, and a strong spike of ATE1 expression was observed during E7.5-9.5 (Fig. S1E). The ~2 kb ATE1 transcript detected in adult mouse testis (1) was also clearly present during the spike of ATE1 expression in E7.5-E9.5 embryos (Fig. S1E). The ATE1 -allele was marked with NLS-β-galactosidase (hereafter βgal), expressed from the ATE1 promoter

Chromosome segregation during mitosis depends on the action of the mitotic spindle, a self-organizing, bipolar protein machine which uses microtubules (MTs) and their associated motors. Members of the BimC subfamily of kinesin-related MT-motor proteins are believed to be essential for the formation and functioning of a normal bipolar spindle. Here we report that KRP130, a homotetrameric BimC-related kinesin purified from Drosophila melanogaster embryos, has an unusual ultrastructure. It consists of four kinesin-related polypeptides assembled into a bipolar aggregate with motor domains at opposite ends, analogous to a miniature myosin filament. Such a bipolar 'minifilament' could crosslink spindle MTs and slide them relative to one another. We do not know of any other MT motors that have a bipolar structure.

Posttranslational arginylation is critical for mouse embryogenesis, cardiovascular development, and angiogenesis, but its molecular effects and the identity of proteins arginylated in vivo are unknown. We found that beta-actin was arginylated in vivo to regulate actin filament properties, beta-actin localization, and lamella formation in motile cells. Arginylation of beta-actin apparently represents a critical step in the actin N-terminal processing needed for actin functioning in vivo. Thus, posttranslational arginylation of a single protein target can regulate its intracellular function, inducing global changes on the cellular level, and may contribute to cardiovascular development and angiogenesis.