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Vol. 19, Issue 2, 536-545, February 2008

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Adducin Promotes Micrometer-Scale Organization of β2-Spectrin in Lateral Membranes of Bronchial Epithelial Cells

Howard Hughes Medical Institute and the Department of Cell Biology, Duke University Medical Center, Durham, NC 27710

Submitted August 23, 2007; Revised October 29, 2007; Accepted November 6, 2007
Monitoring Editor: Ben Margolis

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 β2-spectrin. We use a Tet-on regulated inducible small interfering RNA (siRNA) system to deplete -adducin from confluent HBE cells. Depletion of -adducin resulted in increased detergent solubility of spectrin after normal membrane biogenesis during mitosis. Conversely, depletion of β2-spectrin resulted in loss of adducin from the lateral membrane. siRNA–resistant -adducin prevented loss of lateral membrane, but only if -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 µm. We conclude that adducin acting through spectrin provides a novel mechanism to regulate global properties of the lateral membrane of bronchial epithelial cells.

Address correspondence to: Vann Bennett (v.bennett{at}cellbio.duke.edu)

Abbreviations used: HBE, human bronchial epithelial cell; PKC, protein kinase C; CTL, control; ADD1, adducin siRNA 1; FRAP, fluorescence recovery after photobleaching; FLIP, fluorescence loss in photobleaching.