The unconventional myosin-VIIa associates with lysosomes

Mutations in the myosin-VIIa (MYO7a) gene cause human Usher disease, characterized by hearing impairment and progressive retinal degeneration. In the retina, myosin-VIIa is highly expressed in the retinal pigment epithelium, where it plays a role in the positioning of melanosomes and other digestion organelles. Using a human cultured retinal pigmented epithelia cell line, ARPE-19, as a model system, we have found that a population of myosin-VIIa is associated with cathepsin D- and Rab7-positive lysosomes. Association of myosin-VIIa with lysosomes was Rab7 independent, as dominant negative and dominant active versions of Rab7 did not disrupt myosin-VIIa recruitment to lysosomes. Association of myosin-VIIa with lysosomes was also independent of the actin and microtubule cytoskeleton. Myosin-VIIa copurified with lysosomes on density gradients, and fractionation and extraction experiments suggested that it was tightly associated with the lysosome surface. These studies suggest that myosin-VIIa is a lysosome motor.     

Molecular characterization of KLHL3, a human homologue of the Drosophila kelch gene

The Drosophila kelch protein is a structural component of ring canals and is required for oocyte maturation. Here, we report the cloning and genomic structure of a new human homologue of kelch, KLHL3. At the amino acid level, KLHL3 shares 77% similarity with Drosophila kelch and 89% similarity with Mayven (KLHL2), another human kelch homolog. The approximately 6.5-kb mRNA has a single open reading frame encoding a protein of 587 amino acids with a predicted molecular mass of 650 kDa. Like kelch and KLHL2, the KLHL3 protein contains a poxvirus and zinc finger domain at the N-terminus and six tandem repeats (kelch repeats) at the C-terminus. At least three isoforms, which differ in the length of the N-terminus, are produced and may be the result of alternative promoter usage. We also identified alternative polyadenylation sites and alternative splicing; thus, as many as 12 mRNA variants and six putative protein isoforms could be produced. The KLHL3 gene is mapped to human chromosome 5, band q31, contains 17 exons, and spans approximately 120 kb of genomic DNA. KLHL3 maps within the smallest commonly deleted segment in myeloid leukemias characterized by a deletion of 5q; however, we detected no inactivating mutations of KLHL3 in malignant myeloid disorders with loss of 5q.     

hDKIR, a human homologue of the Drosophila kelch protein, involved in a ring-like structure

We have previously purified and cloned an apoptosis-inducing protein (AIP) derived from fish infected with the anisakis simplex. Recently, we identified a series of AIP-responsive genes in the HL-60 cell line using a subtractive hybridization method. Here we report the molecular cloning and characterization of one of these genes, which encodes a novel human kelch protein containing 568 amino acid residues, termed hDKIR. The Drosophila Kelch protein localizes to a ring canal structure, which is required for oocyte development. When hDKIR was expressed in cultured-mammalian cells, hDKIR localized to a ring-like structure. Furthermore, when coexpressed with Mayven or Keap1, hDKIR bound to Mayven and recruited Mayven into ring-like structures perfectly. This indicates that kelch homologues can interact with each other in a specific manner and such interaction can affect the subcellular localization of kelch proteins. Finally, domain analysis revealed that both the N-terminal POZ (poxviruses and zinc fingers) and intervening region (IVR) domains of hDKIR are essential for ring-like structure activity, suggesting that the development of the ring-like structure is independent of the ability to bind actin.     

GAKIN, a novel kinesin-like protein associates with the human homologue of the Drosophila discs large tumor suppressor in T lymphocytes

Reorganization of the cortical cytoskeleton is a hallmark of T lymphocyte activation. Upon binding to antigen presenting cells, the T cells rapidly undergo cytoskeletal re-organization thus forming a cap at the cell-cell contact site leading to receptor clustering, protein segregation, and cellular polarization. Previously, we reported cloning of the human lymphocyte homologue of the Drosophila Discs Large tumor suppressor protein (hDlg). Here we show that a novel protein termed GAKIN binds to the guanylate kinase-like domain of hDlg. Affinity protein purification, peptide sequencing, and cloning of GAKIN cDNA from Jurkat J77 lymphocytes identified GAKIN as a novel member of the kinesin superfamily of motor proteins. GAKIN mRNA is ubiquitously expressed, and the predicted amino acid sequence shares significant sequence similarity with the Drosophila kinesin-73 motor protein. GAKIN sequence contains a motor domain at the NH(2) terminus, a central stalk domain, and a putative microtubule-interacting sequence called the CAP-Gly domain at the COOH terminus. Among the MAGUK superfamily of proteins examined, GAKIN binds to the guanylate kinase-like domain of PSD-95 but not of p55. The hDlg and GAKIN are localized mainly in the cytoplasm of resting T lymphocytes, however, upon CD2 receptor cross-linking the hDlg can translocate to the lymphocyte cap. We propose that the GAKIN-hDlg interaction lays the foundation for a general paradigm of coupling MAGUKs to the microtubule-based cytoskeleton, and that this interaction may be functionally important for the intracellular trafficking of MAGUKs and associated protein complexes in vivo.     

A homologue of Drosophila aurora kinase is oncogenic and amplified in human colorectal cancers.

Genetic and biochemical studies in lower eukaryotes have identified several proteins that ensure accurate segregation of chromosomes. These include the Drosophila aurora and yeast Ipl1 kinases that are required for centrosome maturation and chromosome segregation. We have identified two human homologues of these genes, termed aurora1 and aurora2, that encode cell-cycle-regulated serine/threonine kinases. Here we demonstrate that the aurora2 gene maps to chromosome 20q13, a region amplified in a variety of human cancers, including a significant number of colorectal malignancies. We propose that aurora2 may be a target of this amplicon since its DNA is amplified and its RNA overexpressed, in more than 50% of primary colorectal cancers. Furthermore, overexpression of aurora2 transforms rodent fibroblasts. These observations implicate aurora2 as a potential oncogene in many colon, breast and other solid tumors, and identify centrosome-associated proteins as novel targets for cancer therapy.     

Vinculin associates with endothelial VE-cadherin junctions to control force-dependent remodeling

To remodel endothelial cell-cell adhesion, inflammatory cytokine- and angiogenic growth factor-induced signals impinge on the vascular endothelial cadherin (VE-cadherin) complex, the central component of endothelial adherens junctions. This study demonstrates that junction remodeling takes place at a molecularly and phenotypically distinct subset of VE-cadherin adhesions, defined here as focal adherens junctions (FAJs). FAJs are attached to radial F-actin bundles and marked by the mechanosensory protein Vinculin. We show that endothelial hormones vascular endothelial growth factor, tumor necrosis factor α, and most prominently thrombin induced the transformation of stable junctions into FAJs. The actin cytoskeleton generated pulling forces specifically on FAJs, and inhibition of Rho-Rock-actomyosin contractility prevented the formation of FAJs and junction remodeling. FAJs formed normally in cells expressing a Vinculin binding-deficient mutant of α-catenin, showing that Vinculin recruitment is not required for adherens junction formation. Comparing Vinculin-devoid FAJs to wild-type FAJs revealed that Vinculin protects VE-cadherin junctions from opening during their force-dependent remodeling. These findings implicate Vinculin-dependent cadherin mechanosensing in endothelial processes such as leukocyte extravasation and angiogenesis.     

A new Drosophila APC homologue associated with adhesive zones of epithelial cells

Adenomatous polyposis coli protein (APC) is an important tumour suppressor in the human colon epithelium. In a complex with glycogen synthase kinase-3 (GSK-3), APC binds to and destabilizes cytoplasmic ('free') beta-catenin. Here, using a yeast two-hybrid screen for proteins that bind to the Drosophila beta-catenin homologue, Armadillo, we identify a new Drosophila APC homologue, E-APC. E-APC also binds to Shaggy, the Drosophila GSK-3 homologue. Interference with E-APC function produces embryonic phenotypes like those of shaggy mutants. Interestingly, E-APC is concentrated in apicolateral adhesive zones of epithelial cells, along with Armadillo and E-cadherin, which are both integral components of the adherens junctions in these zones. Various mutant conditions that cause dissociation of E-APC from these zones also obliterate the segmental modulation of free Armadillo levels that is normally induced by Wingless signalling. We propose that the Armadillo-destabilizing protein complex, consisting of E-APC, Shaggy, and a third protein, Axin, is anchored in adhesive zones, and that Wingless signalling may inhibit the activity of this complex by causing dissociation of E-APC from these zones.     

A cell adhesion protein from the crayfish Pacifastacus leniusculus, a serine proteinase homologue similar to Drosophila masquerade

A cDNA encoding a protein resembling masquerade, a serine proteinase homologue expressed during embryogenesis, larval, and pupal development in Drosophila melanogaster, was identified in hemocytes of the adult freshwater crayfish, Pacifastacus leniusculus. The crayfish protein is similar to Drosophila masquerade in the following aspects: (a) overall sequence of the serine proteinase domain, such as the position of three putative disulfide bridges, glycine in the place of the catalytic serine residue, and the presence of a substrate-lining pocket typical for trypsins; (b) the presence of several copies of a disulfide-knotted motif in the putative propeptide. This masquerade-like protein is cleaved into a 27-kDa fragment, which could be detected by immunoblot analysis using an affinity-purified antibody against a synthetic peptide in the C-terminal domain of the protein. The 27-kDa protein could be immunoaffinity-purified from hemocyte lysate supernatant and exhibited cell adhesion activity in vitro, indicating that the C-terminal domain of the crayfish masquerade-like protein mediates cell adhesion.     

DCC regulates cell adhesion in human colon cancer derived HT-29 cells and associates with ezrin

The deleted in colorectal cancer (DCC) gene encodes a 170- to 190-kDa protein of the Immunoglobulin superfamily. Firstly identified as a tumor suppressor gene in human colorectal carcinomas, the main function for DCC has been described in the nervous system as part of a receptor complex for netrin-1. Moreover, roles in mucosecretory cell differentiation and as inducer of apoptosis have also been reported. DCC knockout mice supported a crucial role for this gene in axonal migration, yet questioned its implication in tumor suppression and mucosecretory differentiation. The work presented here demonstrates that a DCC-transfected HT-29 colonic human cell line (HT-29/DCC) displays an increase in cell-cell adhesion to the detriment of cell-matrix interactions: HT-29/DCC cells exhibit more and better-structured desmosomes while focal adhesions and hemidesmosomes are disrupted. HT-29/DCC cells show no changes in adherent junctions but upon treatment with TPA, HT-29/DCC cells show resistance to scattering, and maintain E-cadherin in the membrane. In addition, the actin cytoskeleton is affected in HT-29/DCC cells: stress fibers are disrupted while cortical actin remains intact. We identified a putative ERM-M (ezrin/radixin/moesin and merlin) binding domain in the juxtamembrane region of the DCC protein. In vitro pull-down assays demonstrate the interaction of the DCC cytoplasmic domain with the N-terminal region of ezrin and merlin, and co-immunoprecipitation assays in transiently DCC-transfected COS-1 cells showed that the interaction between DCC and ezrin also takes place in vivo. Altogether, our results suggest that DCC could regulate cell adhesion and migration through its association with ERM-M proteins.     

Cyclin A associates with the fusome during germline cyst formation in the Drosophila ovary

Regulated changes in the cell cycle underlie many aspects of growth and differentiation. Prior to meiosis, germ cell cycles in many organisms become accelerated, synchronized, and modified to lack cytokinesis. These changes cause cysts of interconnected germ cells to form that typically contain 2(n) cells. In Drosophila, developing germ cells during this period contain a distinctive organelle, the fusome, that is required for normal cyst formation. We find that the cell cycle regulator Cyclin A transiently associates with the fusome during the cystocyte cell cycles, suggesting that fusome-associated Cyclin A drives the interconnected cells within each cyst synchronously into mitosis. In the presence of a normal fusome, overexpression of Cyclin A forces cysts through an extra round of cell division to produce cysts with 32 germline cells. Female sterile mutations in UbcD1, encoding an E2 ubiquitin-conjugating enzyme, have a similar effect. Our observations suggest that programmed changes in the expression and cytoplasmic localization of key cell cycle regulatory proteins control germline cyst production.     

Adherens junctions in Drosophila retinal morphogenesis

Adherens junctions and their core molecular components, classic cadherins, make major contributions to animal morphogenesis. Although the significance of cadherins in development is generally accepted, the mechanisms regulating adherens junction function during morphogenesis remain a subject of intense research. Adherens junctions are involved in the organization of simple cellular patterns, and more complex cell shape changes and cell movements that depend on the dynamic modulation of adherens junctions.     

Cell adhesion molecule Echinoid associates with unconventional myosin VI/Jaguar motor to regulate cell morphology during dorsal closure in Drosophila

Echinoid (Ed) is a homophilic immunoglobulin domain-containing cell adhesion molecule (CAM) that localizes to adherens junctions (AJs) and cooperates with Drosophila melanogaster epithelial (DE)-cadherin to mediate cell adhesion. Here we show that Ed takes part in many processes of dorsal closure, a morphogenetic movement driven by coordinated cell shape changes and migration of epidermal cells to cover the underlying amnioserosa. Ed is differentially expressed, appearing in epidermis but not in amnioserosa cells. Ed functions independently from the JNK signaling pathway and is required to regulate cell morphology, and for assembly of actomyosin cable, filopodial protrusion and coordinated cell migration in dorsal-most epidermal cells. The effect of Ed on cell morphology requires the presence of the intracellular domain (Ed^intra). Interestingly, Ed forms homodimers in vivo and Ed^intra monomer directly associates with unconventional myosin VI/Jaguar (Jar) motor protein. We further show that ed genetically interacts with jar to control cell morphology. It has previously been shown that myosin VI is monomeric in vitro and that its dimeric form can associate with and travel processively along actin filaments. Thus, we propose that Ed mediates the dimerization of myosin VI/Jar in vivo which in turn regulates the reorganization and/or contraction of actin filaments to control changes in cell shape. Consistent with this, we found that ectopic ed expression in the amnioserosa induces myosin VI/Jar-dependent apical constriction of this tissue.     

A Syd-1 homologue regulates pre- and postsynaptic maturation in Drosophila

Active zones (AZs) are presynaptic membrane domains mediating synaptic vesicle fusion opposite postsynaptic densities (PSDs). At the Drosophila neuromuscular junction, the ELKS family member Bruchpilot (BRP) is essential for dense body formation and functional maturation of AZs. Using a proteomics approach, we identified Drosophila Syd-1 (DSyd-1) as a BRP binding partner. In vivo imaging shows that DSyd-1 arrives early at nascent AZs together with DLiprin-α, and both proteins localize to the AZ edge as the AZ matures. Mutants in dsyd-1 form smaller terminals with fewer release sites, and release less neurotransmitter. The remaining AZs are often large and misshapen, and ectopic, electron-dense accumulations of BRP form in boutons and axons. Furthermore, glutamate receptor content at PSDs increases because of excessive DGluRIIA accumulation. The AZ protein DSyd-1 is needed to properly localize DLiprin-α at AZs, and seems to control effective nucleation of newly forming AZs together with DLiprin-α. DSyd-1 also organizes trans-synaptic signaling to control maturation of PSD composition independently of DLiprin-α.