Formation of syncytial muscle fibers involves repeated rounds of cell fusion between growing myotubes and neighboring myoblasts. We have established that Wsp, the Drosophila homolog of the WASp family of microfilament nucleation-promoting factors, is an essential facilitator of myoblast fusion in Drosophila embryos. D-WIP, a homolog of the conserved Verprolin/WASp Interacting Protein family of WASp-binding proteins, performs a key mediating role in this context. D-WIP, which is expressed specifically in myoblasts, associates with both the WASp-Arp2/3 system and with the myoblast adhesion molecules Dumbfounded and Sticks and Stones, thereby recruiting the actin-polymerization machinery to sites of myoblast attachment and fusion. Our analysis demonstrates that this recruitment is normally required late in the fusion process, for enlargement of nascent fusion pores and breakdown of the apposed cell membranes. These observations identify cellular and developmental roles for the WASp-Arp2/3 pathway, and provide a link between force-generating actin polymerization and cell fusion.

Intracellular trafficking of the precursor of Spitz (Spi), the major Drosophila EGF receptor (EGFR) ligand, is facilitated by the chaperone Star, a type II transmembrane protein. This study identifies a novel mechanism for modulating the activity of Star, thereby influencing the levels of active Spi ligand produced. We demonstrate that Star can efficiently traffic Spi even when present at sub-stoichiometric levels, and that in Drosophila S 2 R þ cells, Spi is trafficked from the endoplasmic reticulum to the late endosome compartment, also enriched for Rhomboid, an intramembrane protease. Rhomboid, which cleaves the Spi precursor, is now shown to also cleave Star within its transmembrane domain both in cell culture and in flies, expanding the repertoire of known Rhomboid substrates to include both type I and type II transmembrane proteins. Cleavage of Star restricts the amount of Spi that is trafficked, and may explain the exceptional dosage sensitivity of the Star locus in flies.

Nir2, like its Drosophila homolog retinal degeneration B (RdgB), contains an N-terminal phosphatidylinositol-transfer protein (PI-TP)-like domain. Previous studies have suggested that RdgB plays an important role in the fly phototransduction cascade and that its PI-transfer domain is critical for this function. In this domain, a specific mutation, T59E, induces a dominant retinal degeneration phenotype. Here we show that a similar mutation, T59E in the human Nir2 protein, targets Nir2 to spherical cytosolic structures identified as lipid droplets by the lipophilic dye Nile red. A truncated Nir2T59E mutant consisting of only the PI-transfer domain was also targeted to lipid droplets, whereas neither the wild-type Nir2 nor the Nir2T59A mutant was associated with lipid droplets under regular growth conditions. However, oleic-acid treatment caused translocation of wild-type Nir2, but not translocation of the T59A mutant, to lipid droplets. This treatment also induced partial targeting of endogenous Nir2, which is mainly associated with the Golgi apparatus, to lipid droplets. Targeting of Nir2 to lipid droplets was attributed to its enhanced threonine phosphorylation. These results suggest that a specific threonine within the PI-transfer domain of Nir2 provides a regulatory site for targeting to lipid droplets. In conjunction with the role of PI-TPs in lipid transport, this targeting may affect intracellular lipid trafficking and distribution and may provide the molecular basis underlying the dominant effect of the RdgB-T59E mutant on retinal degeneration.

Cytokinesis, the final stage of eukaryotic cell division, ensures the production of two daughter cells. It requires fine coordination between the plasma membrane and cytoskeletal networks, and it is known to be regulated by several intracellular proteins, including the small GTPase Rho and its effectors. In this study we provide evidence that the protein Nir2 is essential for cytokinesis. Microinjection of anti-Nir2 antibodies into interphase cells blocks cytokinesis, as it results in the production of multinucleate cells. Immunolocalization studies revealed that Nir2 is mainly localized in the Golgi apparatus in interphase cells, but it is recruited to the cleavage furrow and the midbody during cytokinesis. Nir2 colocalizes with the small GTPase RhoA in the cleavage furrow and the midbody, and it associates with RhoA in mitotic cells. Its N-terminal region, which contains a phosphatidylinositol transfer domain and a novel Rho-inhibitory domain (Rid), is required for normal cytokinesis, as overexpression of an N-terminal-truncated mutant blocks cytokinesis completion. Time-lapse videomicroscopy revealed that this mutant normally initiates cytokinesis but fails to complete it, due to cleavage furrow regression, while Rid markedly affects cytokinesis due to abnormal contractility. Rid-expressing cells exhibit aberrant ingression and ectopic cleavage sites; the cells fail to segregate into daughter cells and they form a long unseparated bridge-like cytoplasmic structure. These results provide new insight into the cellular functions of Nir2 and introduce it as a novel regulator of cytokinesis.

Chronic neuroleptic treatment induces an increase in the density of D2 dopamine receptors in the striatum. The effect of prolonged administration of haloperidol on mRNA levels of the short (D,J and long (DzL) isoforms of the D, dopamine receptor and the D, dopamine receptor in different brain regions was examined. Haloperidol caused a significant rise in both D,, and D,, mRNA levels in the striatum, but had no effect on D, mRNA levels. This rise was restricted to the striatum, showing that the effect of haloperidol on dopamine receptor mRNA is both subtype-and tissue-specific.