Figure 2. LAD-1 is localized to the plasma membrane at sites of cell-cell contact in all cells throughout development. (A, i) An immunoblot of total C. elegans lysates reveals four major LAD-1 polypeptides (200, 185, 120, and 65 kD) that were detected by a rabbit polyclonal antibody raised against the LAD-1 cytoplasmic domain (lane 2). The lower bands are not LAD-1 related, since they are also recognized by 125 I-labeled protein A (lane 3). (Lane 1) Coomassie blue staining of total C. elegans lysate. Equivalent lysate amounts. (ii) nDf41 homozygous embryos, which contains the
lad-1 gene, show LAD-1 immunodetection (green) unlike stDf7 homozygous embryos, which lack the
lad-1 gene; only control antibody staining of JAM-1 is seen in red (iii). B and C show immunodetection of LAD-1 in embryos. (B) LAD-1 is localized to sites of cell-cell contact, including the partial plasma membrane of a developing two-cell staged embryo (arrow). A one-cell embryo, which lacks contact sites, does not show LAD-1 localization (arrowhead). (C) LAD-1 is found in the developing nervous system (arrow) with robust LAD-1 staining in the nerve ring, the major C. elegans neuropil (arrowhead). D and E show LAD-1 expression in multiple tissues in a larva and adult, respectively. LAD-1 is present in the nerve ring and ventral nerve cord (D, arrowhead) and in the hypodermis (skin cells) (D, arrows), the plasma membrane of body wall muscles (F, arrow), neuronal cell bodies (G, long arrow), dendrites (G, arrowhead), and the pharynx (G, short arrow). LAD-1 is also present in the syncytial germline where its localization at contact sites is maintained. A schematic of the C. elegans germline is shown in H: arrowheads point to oocytes and arrows point to germ nuclei. The cross section of the cylindrical gonad reveals a single layer of germ nuclei around a cytoplasmic core with T-shaped membranes between each nucleus. In a grazing section of the germline shown in I, LAD-1 is localized at oocyte-oocyte contact (arrowhead) and only the vertical part of the T-shaped membrane, which divides two neighboring germline nuclei (arrows). The top of the T does not show LAD-1 localization. Bars: (A) 20 um; (B-I) 25 um.