Glycosphingolipids with extended sugar chain have specialized functions in development and behavior of Drosophila

Glycosphingolipids (GSL) are glycosylated polar lipids in cell membranes essential for development of vertebrates as well as Drosophila. Mutants that impair enzymes involved in biosynthesis of GSL sugar chains provide a means to assess the functions of the sugar chains in vivo. The Drosophila glycosyltransferases Egghead and Brainiac are responsible for the 2nd and 3rd steps of GSL sugar chain elongation. Mutants lacking these enzymes are lethal and the nature of the defects that occur has suggested that GSL might impact on signaling by the Notch and EGFR pathways. Here we report on characterization of enzymes involved in the 4th and 5th steps of GSL sugar chain elongation in vitro and explore the biological consequences of removing the enzymes involved in step 4 in vivo. Two beta4-N-Acetylgalactosyltransferase enzymes can carry out step 4 (beta4GalNAcTA and beta4GalNAcTB), and while they may have overlapping activity, the mutants produce distinct phenotypes. The beta4GalNAcTA mutant displays behavioral defects, which are also observed in viable brainiac mutants, suggesting that proper locomotion and coordination primarily depend on GSL elongation. beta4GalNAcTB mutant animal shows ventralization of ovarian follicle cells, which is caused by defective EGFR signaling between the oocyte and the dorsal follicle cells to specify dorsal fate. GSL sequentially elongated by Egh, Brn and beta4GalNAcTB in the oocyte contribute to this signaling pathway. Despite the similar enzymatic activity, we provide evidence that the two enzymes are not functionally redundant in vivo, but direct distinct developmental functions of GSL.     

Activation of ROCK by RhoA is regulated by cell adhesion, shape, and cytoskeletal tension

Adhesion to the extracellular matrix regulates numerous changes in the actin cytoskeleton by regulating the activity of the Rho family of small GTPases. Here, we report that adhesion and the associated changes in cell shape and cytoskeletal tension are all required for GTP-bound RhoA to activate its downstream effector, ROCK. Using an in vitro kinase assay for endogenous ROCK, we found that cells in suspension, attached on substrates coated with low density fibronectin, or on spreading-restrictive micropatterned islands all exhibited low ROCK activity and correspondingly low myosin light chain phosphorylation, in the face of high levels of GTP-bound RhoA. In contrast, allowing cells to spread against substrates rescued ROCK and myosin activity. Interestingly, inhibition of tension with cytochalasin D or blebbistatin also inhibited ROCK activity within 20 min. The abrogation of ROCK activity by cell detachment or inhibition of tension could not be rescued by constitutively active RhoA-V14. These results suggest the existence of a feedback loop between cytoskeletal tension, adhesion maturation, and ROCK signaling that likely contributes to numerous mechanochemical processes.     

Interaction between single molecules of Mac-1 and ICAM-1 in living cells: an atomic force microscopy study

The interaction between integrin macrophage differentiation antigen associated with complement three receptor function (Mac-1) and intercellular adhesion molecule-1 (ICAM-1), which is controlled tightly by the ligand-binding activity of Mac-1, is central to the regulation of neutrophil adhesion in host defense. Several "inside-out" signals and extracellular metal ions or antibodies have been found to activate Mac-1, resulting in an increased adhesiveness of Mac-1 to its ligands. However, the molecular basis for Mac-1 activation is not well understood yet. In this work, we have carried out a single-molecule study of Mac-1/ICAM-1 interaction force in living cells by atomic force microscopy (AFM). Our results showed that the binding probability and adhesion force of Mac-1 with ICAM-1 increased upon Mac-1 activation. Moreover, by comparing the dynamic force spectra of different Mac-1 mutants, we expected that Mac-1 activation is governed by the downward movement of its alpha7 helix.     

Epidermal growth factor and transforming growth factor-beta1 enhance HK-2 cell migration through a synergistic increase of matrix metalloproteinase and sustained activation of ERK signaling pathway

Epidermal growth factor (EGF) and transforming growth factor-beta1 (TGF-beta1), upregulated in renal diseases, have a combinational effect on epithelial-mesenchymal transformation (EMT) of renal proximal tubular cells. The aim of this study was to examine the mechanism regarding the combinational effect of EGF and TGF-beta1 on cell migration following EMT. The results demonstrated that EGF (10 ng/ml) and TGF-beta1 (3 ng/ml) synergistically increased cell migration, accompanied by an increase in matrix metalloproteinase-9 (MMP-9) gene expression, production and activity. Inhibition of MMP-9 production and activity by an MMP-2/MMP-9-specific inhibitor blocked the synergistic effect of EGF and TGF-beta1 on cell migration. The kinetic profile of extracellular signal-regulated kinase (ERK) signals demonstrated that ERK1/2 activation was rapidly and strongly induced by EGF but delayed and less marked by TGF-beta1 stimulation. In contrast, co-administration of EGF and TGF-beta1 caused an early pronounced and persistent ERK1/2 activation. Inhibition of the ERK1/2 activity by PD98059 abrogated the synergistic effect of EGF and TGF-beta1 on cell migration, MMP-9 production and activity, indicating that EGF and TGF-beta1 converged at the ERK signaling pathway to mediate cell migration. This study demonstrates that EGF and TGF-beta1 synergistically stimulate proximal tubular cell migration through the increased MMP-9 function and enhanced ERK1/2 activation.     

Regulation of cell differentiation by hNUDC via a Mpl-dependent mechanism in NIH 3T3 cells

Thrombopoietin receptor (Mpl) belongs to the cytokine receptor surperfamily with a large extracellular N-terminal portion responsible for cytokine recognition and binding. Thrombopoietin (TPO) has so far been the only widely studied cytokine for Mpl. However we have recently identified human NUDC (hNUDC), previously described as a human homolog of a fungal nuclear migration protein, as another putative binding partner of Mpl. The purpose of this study is to test the extent of the functioning of hNUDC by identifying protein-protein interactions with Mpl in mammalian cells. The full-length cDNAs encoding Mpl and hNUDC were cloned into pEGFP-N1 and pDsRed2-N1 respectively which were subsequently expressed as Mpl-EGFP (green) and hNUDC-DsRed (red) fusion proteins. Using ELISA and immunofluorescence studies, we have demonstrated the direct binding of hNUDC to cell surface-captured Mpl. We also observed that hNUDC induced significant changes in cellular morphology in NIH 3T3 cells stably transfected with pMpl-EGFP. Interestingly, these morphological changes were characteristic of cells undergoing megakaryocyte differentiation. Extracellular-signal-regulated protein kinases 1 and 2 (ERK1/2) have been shown to mediate such megakaryocyte-like differentiation. In addition, co-expression of Mpl-EGFP and hNUDC-DsRed led to the release of hNUDC-DsRed into the culture medium.     

Neurexin-1 is required for synapse formation and larvae associative learning in Drosophila

Neurexins are highly polymorphic cell-surface adhesive molecules in neurons. In cultured mammalian cell system, they were found to be involved in synaptogenesis. Here, we report for the first time that Drosophila neurexin is required for synapse formation and associative learning in larvae. Drosophila genome encodes a single functional neurexin (CG7050; Neurexin-1 or Nrx-1), which is a homolog of vertebrate alpha-neurexin. Neurexin-1 is expressed in central nervous system and highly enriched in synaptic regions of the ventral ganglion and brain. Neurexin-1 null mutants are viable and fertile, but have shortened lifespan. The synapse number is decreased in central nervous system in Neurexin-1 null mutants. In addition, Neurexin-1 null mutants exhibit associative learning defect in larvae.     

The joint allele-frequency spectrum in closely related species

We develop the theory for computing the joint frequency spectra of alleles in two closely related species. We allow for arbitrary population growth in both species after they had a common ancestor. We focus on the case in which a single chromosome is sequenced from one of the species. We use classical diffusion theory to show that, if the ancestral species was at equilibrium under mutation and drift and a chromosome from one of the descendant species carries the derived allele, the frequency spectrum in the other species is uniform, independently of the demographic history of both species. We also predict the expected densities of segregating and fixed sites when the chromosome from the other species carries the ancestral allele. We compare the predictions of our model with the site-frequency spectra of SNPs in the four HapMap populations of humans when the nucleotide present in the Neanderthal DNA sequence is ancestral or derived, using the chimp genome as the outgroup.     

Cell-cell fusion

Cell-cell fusion is a highly regulated and dramatic cellular event that is required for development and homeostasis. Fusion may also play a role in the development of cancer and in tissue repair by stem cells. While virus-cell fusion and the fusion of intracellular membranes have been the subject of intense investigation during the past decade, cell-cell fusion remains poorly understood. Given the importance of this cell-biological phenomenon, a number of investigators have begun analyses of the molecular mechanisms that mediate the specialized fusion events of a variety of cell types and species. We discuss recent genetic and biochemical studies that are beginning to yield exciting insights into the fusion mechanisms of Saccharomyces cerevisiae mating pairs, Caenorhabditis elegans epithelial cells and gametes, Drosophila melanogaster and mammalian myoblasts, and mammalian macrophages.