Dephosphorylation of neural wiring protein shootin1 by PP1 phosphatase regulates netrin-1-induced axon guidance.

Axon pathfinding is an essential step in neuronal network formation. Shootin1a is a clutch-linker molecule that is mechanically involved in axon outgrowth and guidance. It was previously shown that concentration gradients of axon guidance molecule netrin-1 in the extracellular environment elicit asymmetrically localized Pak1 kinase-mediated phosphorylation of shootin1a within axonal growth cones, which is higher on the netrin-1 source side. This asymmetric phosphorylation promotes shootin1a-mediated local actin-adhesion coupling within growth cones, thereby generating directional forces for turning the growth cone toward the netrin-1 source. However, how the spatial differences in netrin-1 concentration are transduced into the asymmetrically localized signaling within growth cones remains unclear. Moreover, the protein phosphatases that dephosphorylate shootin1a remain unidentified. Here, we report that protein phosphatase-1 (PP1) dephosphorylates shootin1a in growth cones. We found that PP1 overexpression abolished the netrin-1-induced asymmetric localization of phosphorylated shootin1a as well as axon turning. In addition, we show PP1 inhibition reversed the asymmetrically localized shootin1a phosphorylation within growth cones under netrin-1 gradient, thereby changing the netrin-1-induced growth cone turning from attraction to repulsion. These data indicate that PP1-mediated shootin1a dephosphorylation plays a key role in organizing asymmetrically localized phosphorylated shootin1a within growth cones, which regulates netrin-1-induced axon guidance.

Shrimp miRNAs regulate innate immune response against white spot syndrome virus infection.

MicroRNAs are short noncoding RNAs of RNA interference pathways that regulate gene expression through partial complementary base-pairing to target mRNAs. In this study, miRNAs that are expressed in white spot syndrome virus (WSSV)-infected Penaeus monodon, were identified using next generation sequencing. Forty-six miRNA homologs were identified from WSSV-infected shrimp hemocyte. Stem-loop real-time RT-PCR analysis showed that 11 out of 16 selected miRNAs were differentially expressed upon WSSV infection. Of those, pmo-miR-315 and pmo-miR-750 were highly responsive miRNAs. miRNA target prediction revealed that the miRNAs were targeted at 5'UTR, ORF, and 3'UTR of several immune-related genes such as genes encoding antimicrobial peptides, signaling transduction proteins, heat shock proteins, oxidative stress proteins, proteinases or proteinase inhibitors, proteins in blood clotting system, apoptosis-related proteins, proteins in prophenoloxidase system, pattern recognition proteins and other immune molecules. The highly conserved miRNA homolog, pmo-bantam, was characterized for its function in shrimp. The pmo-bantam was predicted to target the 3'UTR of Kunitz-type serine protease inhibitor (KuSPI). Binding of pmo-bantam to the target sequence of KuSPI gene was analyzed by luciferase reporter assay. Correlation of pmo-bantam and KuSPI expression was observed in lymphoid organ of WSSV-infected shrimp. These results implied that miRNAs might play roles as immune gene regulators in shrimp antiviral response.