Syd/JIP3 and JNK signaling are required for myonuclear positioning and muscle function.

Highlighting the importance of proper intracellular organization, many muscle diseases are characterized by mispositioned myonuclei. Proper positioning of myonuclei is dependent upon the microtubule motor proteins, Kinesin-1 and cytoplasmic Dynein, and there are at least two distinct mechanisms by which Kinesin and Dynein move myonuclei. The motors exert forces both directly on the nuclear surface and from the cell cortex via microtubules. How these activities are spatially segregated yet coordinated to position myonuclei is unknown. Using Drosophila melanogaster, we identified that Sunday Driver (Syd), a homolog of mammalian JNK-interacting protein 3 (JIP3), specifically regulates Kinesin- and Dynein-dependent cortical pulling of myonuclei without affecting motor activity near the nucleus. Specifically, Syd mediates Kinesin-dependent localization of Dynein to the muscle ends, where cortically anchored Dynein then pulls microtubules and the attached myonuclei into place. Proper localization of Dynein also requires activation of the JNK signaling cascade. Furthermore, Syd functions downstream of JNK signaling because without Syd, JNK signaling is insufficient to promote Kinesin-dependent localization of Dynein to the muscle ends. The significance of Syd-dependent myonuclear positioning is illustrated by muscle-specific depletion of Syd, which impairs muscle function. Moreover, both myonuclear spacing and locomotive defects in syd mutants can be rescued by expression of mammalian JIP3 in Drosophila muscle tissue, indicating an evolutionarily conserved role for JIP3 in myonuclear movement and highlighting the utility of Drosophila as a model for studying mammalian development. Collectively, we implicate Syd/JIP3 as a novel regulator of myogenesis that is required for proper intracellular organization and tissue function.

ccm2-like is required for cardiovascular development as a novel component of the Heg-CCM pathway.

The Heart of Glass-Cerebral Cavernous Malformation (Heg-CCM) pathway is essential for normal cardiovascular development in zebrafish and mouse. In zebrafish, the Heg-CCM pathway mutants santa(ccm1/san), valentine (ccm2/vtn), and heart of glass (heg) exhibit severely dilated hearts and inflow tracts and a complete absence of blood circulation. We identified a novel gene based on its sequence identity with ccm2, which we have named ccm2-like (ccm2l), and characterized its role in cardiovascular development. Disruption of ccm2l by morpholino injection causes dilation of the atrium and inflow tract and compromised blood circulation. Morpholino co-injection experiments identify ccm2l as an enhancer of the characteristic Heg-CCM dilated heart phenotype, and we find that ccm2 overexpression can partially rescue ccm2l morphant defects. Finally, we show that Ccm2l binds Ccm1 and perform deletion and mutational analyses to define the regions of Ccm1 that mediate its binding to Ccm2l and its previously established interactors Ccm2 and Heg. These genetic and biochemical data argue that ccm2l is a necessary component of the Heg-CCM pathway.

The Drosophila Ninein homologue Bsg25D cooperates with Ensconsin in myonuclear positioning.

Skeletal muscle consists of multinucleated cells in which the myonuclei are evenly spaced throughout the cell. In Drosophila, this pattern is established in embryonic myotubes, where myonuclei move via microtubules (MTs) and the MT-associated protein Ensconsin (Ens)/MAP7, to achieve their distribution. Ens regulates multiple aspects of MT biology, but little is known about how Ens itself is regulated. We find that Ens physically interacts and colocalizes with Bsg25D, the Drosophila homologue of the centrosomal protein Ninein. Bsg25D loss enhances myonuclear positioning defects in embryos sensitized by partial Ens loss. Bsg25D overexpression causes severe positioning defects in immature myotubes and fully differentiated myofibers, where it forms ectopic MT organizing centers, disrupts perinuclear MT arrays, reduces muscle stiffness, and decreases larval crawling velocity. These studies define a novel relationship between Ens and Bsg25D. At endogenous levels, Bsg25D positively regulates Ens activity during myonuclear positioning, but excess Bsg25D disrupts Ens localization and MT organization, with disastrous consequences for myonuclear positioning and muscle function.

Myofibrils put the squeeze on nuclei.

During muscle development, nuclei travel from the centre of the myofibre to the periphery, a process defective in certain diseases. A new study reveals that this movement is due to centripetal forces imposed on nuclei by the crosslinking and contraction of myofibrils.

Microinjection of zebrafish embryos to analyze gene function.

One of the advantages of studying zebrafish is the ease and speed of manipulating protein levels in the embryo. Morpholinos, which are synthetic oligonucleotides with antisense complementarity to target RNAs, can be added to the embryo to reduce the expression of a particular gene product. Conversely, processed mRNA can be added to the embryo to increase levels of a gene product. The vehicle for adding either mRNA or morpholino to an embryo is microinjection. Microinjection is efficient and rapid, allowing for the injection of hundreds of embryos per hour. This video shows all the steps involved in microinjection. Briefly, eggs are collected immediately after being laid and lined up against a microscope slide in a Petri dish. Next, a fine-tipped needle loaded with injection material is connected to a microinjector and an air source, and the microinjector controls are adjusted to produce a desirable injection volume. Finally, the needle is plunged into the embryo's yolk and the morpholino or mRNA is expelled.