Actin dynamics are precisely regulated by a large number of actin binding proteins which collectively alter the rates of actin filament assembly and disassembly. Spectrin, an actin cross-linking protein, forms lateral filamentous networks that are linked to the plasma membrane and are required for membrane stability and resistance to mechanical stress. Adducin binds to spectrin-actin complexes, recruiting additional spectrin molecules, thereby further stabilising the membrane. In addition, adducin can bundle and cap actin filaments, and its actions have been implicated in cytoskeletal rearrangement in a variety of cell types. In vascular smooth muscle there is evidence that rearrangement of the actin cytoskeleton is involved in contraction and transmission of force to the extracellular matrix which leads to tissue remodelling. In addition, cytoskeletal dynamics are involved in vascular smooth muscle cell migration, proliferation and membrane dynamics. Protein kinase C (PKC), Rho-kinase, calmodulin and myosin light chain phosphatase are signalling proteins that are involved in these processes in vascular smooth muscle, and adducin is regulated by these signalling proteins in platelets and epithelial cells. The current study provides evidence for regulation of the actin cytoskeleton by α-adducin in vascular smooth muscle. Both α-adducin and spectrin are associated with the cytoskeleton in vascular smooth muscle cells of rat mesenteric small arteries. In response to activation by noradrenaline (NA), α-adducin becomes rapidly phosphorylated on Ser 724, a site specific for PKC, and dissociates from the actin cytoskeleton and spectrin in a PKC-dependent manner. Longer exposure of vessels to NA results in dephosphorylation of α-adducin on Ser 724 and its Rho-kinase-dependent reassociation with the actin cytoskeleton. Concurrent with this reassociation is enhanced association between the two proteins and an increase in the proportion of spectrin associated with the actin cytoskeleton. In addition, a rise in filamentous actin is observed, which can be blocked by inhibition of PKC or Rho-kinase and also by delivery of the α-adducin antibody into vessels in order to inhibit the function of endogenous a-adducin. These data provide evidence for a model in which α-adducin functions as an actin capping protein in resting vascular smooth muscle cells. Upon vasoconstrictor activation α-adducin becomes phosphorylated by PKC, inducing its dissociation from the actin cytoskeleton allowing elongation of actin filaments and further rearrangement of the actin cytoskeleton. Following this reorganisation, α-adducin re-associates with the actin cytoskeleton, possibly in response to phosphorylation by Rho-kinase, and recruits additional spectrin molecules, thus strengthening the newly formed actin filament network. These data provide further insight into the regulation of the actin cytoskeleton in vascular smooth muscle.

Adherens junctions (AJs) and tight junctions (TJs) are characteristic features of differentiated epithelial cells and are critical for regulation of epithelial barriers and cell polarity. Integrity and remodeling of epithelial junctions depend on their interactions with underlying actomyosin cytoskeleton. Anillin is a multifunctional scaffold able to interact with different cytoskeletal proteins including F-actin and Myosin II. This project aimed to investigate roles of anillin in regulating epithelial AJs and TJs. Using A549 human lung epithelial and DU145 human prostate epithelial cells, we demonstrated the anillin depletion-induced loss of AJs and TJs. This was accompanied by disorganization of perijunctional actomyosin belt and disruption of the adducin-based membrane skeleton that links actin filaments to the plasma membrane and epithelial junctions. Depletion of anillin decreased protein levels of γ-adducin and downregulation of γ-adducin mimicked effects anillin knockdown on AJ and TJ integrity. These findings suggest a novel role for anillin in the assembly of epithelial junctions.