Myo10 tail is crucial for promoting long filopodia.

Filopodia are slender cellular protrusions containing parallel actin bundles involved in environmental sensing and signaling, cell adhesion and migration, and growth cone guidance and extension. Myosin 10 (Myo10), an unconventional actin-based motor protein, was reported to induce filopodial initiation with its motor domain. However, the roles of the multifunctional tail domain of Myo10 in filopodial formation and elongation remain elusive. Herein, we generated several constructs of Myo10-full-length Myo10, Myo10 with a truncated tail (Myo10 HMM), and Myo10 containing four mutations to disrupt its coiled-coil domain (Myo10 CC mutant). We found that the truncation of the tail domain decreased filopodial formation and filopodial length, while four mutations in the coiled-coil domain disrupted the motion of Myo10 toward filopodial tips and the elongation of filopodia. Furthermore, we found that filopodia elongated through multiple elongation cycles, which was supported by the Myo10 tail. These findings suggest that Myo10 tail is crucial for promoting long filopodia.

Characterization of the PLP-dependent transaminase initiating azasugar biosynthesis.

Biosynthesis of the azasugar 1-deoxynojirimycin (DNJ) critically involves a transamination in the first committed step. Here, we identify the azasugar biosynthetic cluster signature in Paenibacillus polymyxa SC2 (Ppo), homologous to that reported in Bacillus amyloliquefaciens FZB42 (Bam), and report the characterization of the aminotransferase GabT1 (named from Bam). GabT1 from Ppo exhibits a specific activity of 4.9 nmol/min/mg at 30°C (pH 7.5), a somewhat promiscuous amino donor selectivity, and curvilinear steady-state kinetics that do not reflect the predicted ping-pong behavior typical of aminotransferases. Analysis of the first half reaction with l-glutamate in the absence of the acceptor fructose 6-phosphate revealed that it was capable of catalyzing multiple turnovers of glutamate. Kinetic modeling of steady-state initial velocity data was consistent with a novel hybrid branching kinetic mechanism which included dissociation of PMP after the first half reaction to generate the apoenzyme which could bind PLP for another catalytic deamination event. Based on comparative sequence analyses, we identified an uncommon His-Val dyad in the PLP-binding pocket which we hypothesized was responsible for the unusual kinetics. Restoration of the conserved PLP-binding site motif via the mutant H119F restored classic ping-pong kinetic behavior.