Numb, an evolutionarily conserved cell fate-determining factor, plays a pivotal role in the development of Drosophila and vertebrate nervous systems. Despite lacking a transmembrane segment, Numb is associated with the cell membrane during the asymmetric cell division of Drosophila neural precursor cells and is selectively partitioned to one of the two progeny cells from a binary cell division. Numb contains an N-terminal phosphotyrosine-binding (PTB) domain that is essential for both the asymmetric localization and the fate specification function of Numb. We report here the isolation and characterization of a novel PTB domain-binding protein, NIP (Numb-interacting protein). NIP is a multipass transmembrane protein that contains two PTB domainbinding, NXXF motifs required for the interaction with Numb. In dividing Drosophila neuroblasts, NIP is colocalized to the cell membrane with Numb in a basal cortical crescent. Expression of NIP in Cos-7 cells recruited Numb from the cytosol to the plasma membrane. This recruitment of Numb to membrane by NIP was dependent on the presence of at least one NXXF site. In Drosophila Schneider 2 cells, NIP and Numb were colocalized at the plasma membrane. Inhibition of NIP expression by RNA interference released Numb to the cytosol. These results suggest that a direct protein-protein interaction between NIP and Numb is necessary and sufficient for the recruitment of Numb to the plasma membrane. Recruitment of Numb to a basal cortical crescent in a dividing neuroblast is essential for Numb to function as an intrinsic cell fate determinant.Asymmetric cell division, which can involve extrinsic and/or intrinsic factors, is a fundamental mechanism of generating cell diversity during the development of complex organisms (1, 2). Extrinsic factors such as Delta and its receptor Notch (3, 4) function in cell-cell communication to specify the fate of cells (5-7). Asymmetric determinants are intrinsic factors that are selectively segregated into one of the two daughter cells when a cell divides (2,8). Consequently, the sibling cell that inherits the asymmetric determinants adopts a different fate from the one that does not. Numb is a member of a growing family of proteins that also includes Prospero, Miranda, Inscuteable, and PON (partner of numb) (9 -14), which act as intrinsic determinants in asymmetric cell division. These proteins were identified through their requirement in the development of Drosophila peripheral and central nervous systems. The external sensory organ in Drosophila is composed of two outer (hair and socket) cells and three inner (sheath, glial, and neuron) cells, which are derived from a single sensory organ precursor through three consecutive asymmetric cell divisions. Numb is selectively partitioned to one of the two daughter cells at each binary division (15). Numb is also required for the development of the central nervous system (16 -18). During delaminating from the neuroectoderm and asymmetric division of a neuroblast, Numb, Prospero, and several other proteins...

No abstract

Lumican is a small leucine-rich proteoglycan (SLRP), which contributes to cell migration, proliferation, tissue hydration, and collagen fibrillogenesis. Whether lumican is localized in rat aortic smooth muscle cells (SMCs) and what its relationships might be to other extracellular matrix components have not yet been elucidated. In this study, using reverse transcription-polymerase chain reaction (RT-PCR), competitive RT-PCR, and western blot, lumican messenger ribonucleic acid (mRNA) was expressed in cultured rat aortic SMCs. SMCs cultured in serum-free medium showed four bands at 68, 62, 50, and 37 kD. The 68 and 62 kD bands corresponded to proteoglycan, the 50 kD band to glycoprotein, and the 37 kD band to the core protein form of lumican. The relationships of lumican to fibronectin and laminin were also investigated. The lumican mRNA level in SMCs cultured on fibronectin was highest at day 1, but it increased at day 3 in SMCs cultured on laminin. On the fibronectin or laminin-coated plates, SMCs expressed only the 68 and 62 kD bands, corresponding to proteoglycan. Pretreatment with anti-beta1 integrin receptor antibody revealed a decrease in the proteoglycan forms of lumican protein and an additional two bands at 50 and 37 kD, indicating glycoprotein and the core protein of lumican. These results show that lumican was synthesized in cultured rat aortic SMCs as proteoglycan, glycoprotein, and core protein. The extracellular matrix (ECM) affected lumican protein production and restricted the lumican protein form to proteoglycan via the beta1 integrin receptor in SMCs.

NIP/DuoxA, originally cloned as a protein capable of binding to the cell fate determinant Numb in Drosophila, was recently identified as a modulator of reactive oxygen species (ROS) production in mammalian systems. Despite biochemical and cellular studies that link NIP/DuoxA to the generation of ROS through the dual oxidase (Duox) enzyme, the in vivo function of NIP/DuoxA has not been characterized to date. Here we report a genetic and functional characterization of nip in Drosophila melanogaster. We show that nip is essential for Drosophila development as nip null mutants die at the 1st larval instar. Expression of UAS-nip, but not UAS-Duox, rescued the lethality. To understand the function of nip beyond the early larval stage, we generated GAL4 inducible UAS-RNAi transgenes. daG32-GAL4 driven, ubiquitous RNAi-mediated silencing of nip led to profound abnormality in pre-adult development, crinkled wing and markedly reduced lifespan at 29°C. Compared to wild type flies, da-GAL4 induced nip-RNAi transgenic flies exhibited significantly reduced ability to survive under oxidative stress and displayed impaired mitochondrial aconitase function. Our work provides in vivo evidence for a critical role for nip in the development and oxidative stress response in Drosophila.