Adducin promotes micrometer-scale organization of beta2-spectrin in lateral membranes of bronchial epithelial cells.
Adducin promotes assembly of spectrin-actin complexes, and is a target for regulation by calmodulin, protein kinase C, and rho kinase. We demonstrate here that adducin is required to stabilize preformed lateral membranes of human bronchial epithelial (HBE) cells through interaction with beta2-spectrin. We use a Tet-on regulated inducible small interfering RNA (siRNA) system to deplete alpha-adducin from confluent HBE cells. Depletion of alpha-adducin resulted in increased detergent solubility of spectrin after normal membrane biogenesis during mitosis. Conversely, depletion of beta2-spectrin resulted in loss of adducin from the lateral membrane. siRNA-resistant alpha-adducin prevented loss of lateral membrane, but only if alpha-adducin retained the MARCKS domain that mediates spectrin-actin interactions. Phospho-mimetic versions of adducin with S/D substitutions at protein kinase C phosphorylation sites in the MARCKS domain were not active in rescue. We find that adducin modulates long-range organization of the lateral membrane based on several criteria. First, the lateral membrane of adducin-depleted cells exhibited reduced height, increased curvature, and expansion into the basal surface. Moreover, E-cadherin-GFP, which normally is restricted in lateral mobility, rapidly diffuses over distances up to 10 microm. We conclude that adducin acting through spectrin provides a novel mechanism to regulate global properties of the lateral membrane of bronchial epithelial cells.
Abdi
Khadar M
KM
Howard Hughes Medical Institute and the Department of Cell Biology, Duke University Medical Center, Durham, NC 27710, USA.
Bennett
Vann
V
D000595:Amino Acid Sequence; D001980:Bronchi / Q000166:cytology*; D015820:Cadherins / Q000378:metabolism; D002148:Calmodulin-Binding Proteins / Q000737:chemistry / Q000172:deficiency / Q000378:metabolism*; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism*; D048430:Cell Shape / Q000187:drug effects; D002478:Cells, Cultured; D004847:Epithelial Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D017830:Octoxynol / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects; D021381:Protein Transport / Q000187:drug effects; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013049:Spectrin / Q000378:metabolism*; D000099291:Adducins
D015820:Cadherins; D002148:Calmodulin-Binding Proteins; D049452:Green Fluorescent Proteins; D017830:Octoxynol; D011993:Recombinant Fusion Proteins; D013049:Spectrin; D000099291:Adducins; C452439:SPTBN1 protein, human