Drosophila laminin: characterization and localization.

Abstract

Abstract. Drosophila laminin was isolated from the medium of Drosophila K~ cell cultures. It was purified by velocity sedimentation, gel filtration, and chromatography. Drosophila laminin is a disulfide-linked molecule consisting of three chains with apparent molecular masses of 400, 215, and 185 kD. In electron micrographs, it has the cross-shaped appearance with globular domains characteristic of vertebrate laminin with closely similar dimensions. The amino acid composition and lectin-binding properties of Drosophila laminin are given.Polyclonal antibodies to Drosophila laminin were prepared and their specificity was established. In developing embryos immunofluorescence staining was detected between 6 and 8 h of development; and in sections of 8-9-h and older embryos immunostaining was seen at sites where basement membranes are present surrounding internal organs, muscles, underlying the hypodermal epithelium, and in the nervous system. Basement membrane staining was also seen in larva and adults. Cells from Drosophila embryos dissociated at the cellular blastoderm stage were grown in culture and some specific, differentiated cells synthesized laminin after several hours of culture as shown by immunofluorescence. The significance of the evolutionary conservation of the structure of this basement membrane component is discussed.T HE extracellular protein laminin is a component of vertebrate basement membranes (7,44). It is a multidomain protein which facilitates growth and migration of cultured cells (22,23,42,43) and promotes the extension of growth cones by neurites (4,12,15,19,25). The fly Drosophila melanogaster provides a unique experimental system for investigating the control of expression and functioning of gene products during development. To learn more about the assembly of basement membranes, and the roles of these structures in development, we initiated a search for their component materials in Drosophila (18). Here we report the isolation and characterization of a molecule with properties of a Drosophila laminin, and the production of antibodies which enable us to study the early appearance and distribution of this material during Drosophila embryogenesis.Molecules of vertebrate laminin have a characteristic, unique appearance of a cross in the electron microscope after spraying onto a flat surface and rotary metal shadowing (14). After reduction, three polypeptide chains have been obtained from vertebrate laminin: A, B1, mad B2. The glycosylated chains migrate in SDS-PAGE with apparent molecular masses of 400, 220, and 210 kD, respectively. A model of the molecule has been suggested in which the carboxyl portions of one copy of each chain participate in a coiled-coil a-helix in the long arm of the cross, while each short arm of the cross is made up of only one chain. The model is supported by biophysical measurements and a rapidly progressing knowledge of the amino acid and nucleotide coding sequences of the three chains (3,32,38,48).The primary sources of vertebrate laminin have been extra-embryo...

Dystroglycan is required for polarizing the epithelial cells and the oocyte inDrosophila

“…The following antibodies were used: rabbit anti-Dlg (1:500) (Woods and Bryant, 1991); mouse anti-Orb (1:20) (Lantz et al, 1994); rabbit anti Baz (1:500) (Wodarz et al, 2000); mouse anti-Crb (CQ4,1:20) (Tepass et al, 1990); rabbit anti DG (1:3000; this study); mouse or rabbit anti-βGal (1:5000, Sigma); mouse anti-Neurotactin (BP102; 1:20) (Hortsch et al, 1990); rabbit anti-Laminin (1:3000) (Fessler et al, 1987); ant-Dlt (1:1000) (Bhat et al, 1999); anti-β-HSpectrin (1:1000) (Thomas and Kiehart, 1994); Alexa 488, 568 or 633 goat anti-mouse (Molecular Probes); Alexa 568 or 633 Goat antiRabbit or Rat (Molecular Probes); and Alexa 568 or 633 Phalloidin (Molecular Probes). DAB staining was done with the Vectastain Kit (Vector Laboratories, CA).…”

Extrinsic and intrinsic mechanisms directing epithelial cell sheet replacement duringDrosophilametamorphosis

“…Primary antibodies used were: rabbit anti-laminin A antiserum at 1:100 dilution (Fessler et al, 1987), mouse anti-Fasciclin III (7G10-Hybridoma Bank) at 1:1000, mouse anti-GFP at 1:500 (Cell Signalling), rabbit antiactive Caspase 3 at 1:100 (Cell Signalling), mouse anti-EcRB1 at 1:10 (AD4.4 Hybridoma Bank) and mouse anti-EcRA at 1:10 (15G1A-Hybridoma Bank). Secondary antibodies were anti-mouse or anti-rabbit FITC, Cy3 or Cy5 conjugated (Molecular Probes) at 1:250 dilutions.…”

Haemocyte-derived SPARC is required for collagen-IV-dependent stability of basal laminae inDrosophilaembryos

“…Whole-mount immunostaining was carried out using standard procedures with antibodies obtained from the Developmental Studies Hybridoma Bank, developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biological Sciences, Iowa City, IA 52242 against the following targets: DN-cadherin (DN-Ex number 8), Neurotactin (BP106), Futsch (22C10), CNS axons (BP102), Crumbs (Cq4) and Repo (8D12). The following antibodies were also used: monoclonal antibody against Drosophila collagen IV and polyclonal antibodies against Drosophila SPARC (Martinek et al, 2002), Drosophila laminin (α3,5/β1/γ1) (Fessler et al, 1987), Drosophila perlecan (Friedrich et al, 1999) and hunchback (Kosman et al, 1998). Secondary antibodies conjugated to Alexa Fluor 488, Cy3, Cy5 (Molecular Probes) or HRP (Jackson Laboratories) were used.…”

“…Laminin is expressed by a broad range of tissues during embryogenesis and, similar to SPARC and collagen Journal of Cell Science 121 (10) , 1992). The laminin antibodies used in this study only recognized fully assembled, extracellular laminin (Fessler et al, 1987) and, thus, haemocyte immunostaining of laminin was not observed. Immunostaining of ES12 wild-type embryos revealed a laminin network that overlies the neuroepithelium (Fig.…”