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1.  Adducins Regulate Remodeling of Apical Junctions in Human Epithelial Cells 
Molecular Biology of the Cell  2010;21(20):3506-3517.
This article identifies membrane skeleton proteins, adducins, as important regulators of epithelial cell–cell adhesions that promote assembly and antagonize stimulus-induced disassembly of adherens and tight junctions.
Epithelial adherens junctions (AJs) and tight junctions (TJs) are dynamic structures that readily undergo disintegration and reassembly. Remodeling of the AJs and TJs depends on the orchestrated dynamics of the plasma membrane with its underlying F-actin cytoskeleton, and the membrane–cytoskeleton interface may play a key role in junctional regulation. Spectrin–adducin–ankyrin complexes link membranes to the actin cytoskeleton where adducins mediate specrtrin–actin interactions. This study elucidates roles of adducins in the remodeling of epithelial junctions in human SK-CO15 colonic and HPAF-II pancreatic epithelial cell monolayers. These cells expressed the α and γ isoforms of adducin that positively regulated each others protein level and colocalized with E-cadherin and β-catenin at mature, internalized and newly assembled AJs. Small interfering RNA-mediated down-regulation of α- or γ-adducin expression significantly attenuated calcium-dependent AJ and TJ assembly and accelerated junctional disassembly triggered by activation of protein kinase C. Two mechanisms were found to mediate the impaired AJ and TJ assembly in adducin-depleted cells. One mechanism involved diminished expression and junctional recruitment of βII-spectrin, and the other mechanism involved the decrease in the amount of cellular F-actin and impaired assembly of perijunctional actin bundles. These findings suggest novel roles for adducins in stabilization of epithelial junctions and regulation of junctional remodeling.
doi:10.1091/mbc.E10-03-0259
PMCID: PMC2954116  PMID: 20810786
2.  Adducin: Ca++-dependent association with sites of cell-cell contact 
The Journal of Cell Biology  1989;109(2):557-569.
Adducin is a protein recently purified from erythrocytes and brain that has properties in in vitro assays suggesting a role in assembly of a spectrin-actin lattice. This report describes the localization of adducin to plasma membranes of a variety of tissues and the discovery that adducin is concentrated at sites of cell-cell contact in the epithelial tissues where it is expressed. Adducin in tissues and cultured cells always was observed in association with spectrin and actin, although spectrin and actin were evident in the absence of adducin. In sections of intestinal epithelial cells spectrin was present on all plasma membrane surfaces while adducin was restricted to the lateral cell borders. Adducin also was not detected in association with actin stress fibers in cultured cells. The presence of adducin at cell-cell contact sites of cultured epithelial cells requires extracellular Ca++ and occurs within 15 min of addition of 0.3 mM Ca++. Redistribution of adducin after addition of extracellular Ca++ is independent of formation of desmosomal and adherens junctions since assembly of adducin at contact sites requires lower concentrations of Ca++ and occurs more rapidly than redistribution of desmoplakin or vinculin. Treatment of keratinocytes and MDCK cells with nanomolar concentrations of 12-O-tetradecanoylphorbol-13-acetate (TPA) induces redistribution of adducin away from contact sites. The effect of TPA may be a direct consequence of phosphorylation of adducin, since adducin is phosphorylated in TPA-treated cells and the phosphorylation of adducin occurs before disassembly of adducin from sites of cell-cell contact. Spectrin and adducin are both present in a detergent-insoluble form at cell-cell contact sites of cultured cells. These observations are consistent with the idea that adducin recognizes and associates with specific "receptors" localized at regions of cell-cell contact and promotes assembly of spectrin into a more stable structure, perhaps analogous to the highly organized spectrin-actin network of erythrocyte membranes.
PMCID: PMC2115715  PMID: 2503523
3.  Targeted Deletion of the γ-Adducin Gene (Add3) in Mice Reveals Differences in α-Adducin Interactions in Erythroid and Nonerythroid Cells 
American journal of hematology  2009;84(6):354-361.
In red blood cells (RBCs) adducin heterotetramers localize to the spectrin-actin junction of the peripheral membrane skeleton. We previously reported that deletion of β-adducin results in osmotically fragile, microcytic RBCs and a phenotype of hereditary spherocytosis (HS). Notably, α-adducin was significantly reduced, while γ-adducin, normally present in limited amounts, was increased ~5-fold, suggesting that α-adducin requires a heterologous binding partner for stability and function, and that γ-adducin can partially substitute for the absence of β-adducin. To test these assumptions we generated γ-adducin null mice. γ-adducin null RBCs appear normal on Wright’s stained peripheral blood smears and by scanning electron microscopy. All membrane skeleton proteins examined are present in normal amounts, and all hematological parameters measured are normal. Despite a loss of ~70% of α-adducin in γ-adducin null platelets, no bleeding defect is observed and platelet structure appears normal. Moreover, systemic blood pressure and pulse are normal in γ-adducin null mice. γ- and β-adducin null mice were intercrossed to generate double null mice. Loss of γ-adducin does not exacerbate the β-adducin null HS phenotype although the amount α-adducin is reduced to barely detectable levels. The stability of α-adducin in the absence of a heterologous binding partner varies considerably in various tissues. The amount of α-adducin is modestly reduced (~15%) in the kidney, while in the spleen and brain is reduced by ~50% with the loss of a heterologous β- or γ-adducin binding partner. These results suggest that the structural properties of adducin differ significantly between erythroid and various nonerythroid cell types.
doi:10.1002/ajh.21427
PMCID: PMC2827150  PMID: 19425068
4.  α-Adducin dissociates from F-actin and spectrin during platelet activation 
The Journal of Cell Biology  2003;161(3):557-570.
Aspectrin-based skeleton uniformly underlies and supports the plasma membrane of the resting platelet, but remodels and centralizes in the activated platelet. α-Adducin, a phosphoprotein that forms a ternary complex with F-actin and spectrin, is dephosphorylated and mostly bound to spectrin in the membrane skeleton of the resting platelet at sites where actin filaments attach to the ends of spectrin molecules. Platelets activated through protease-activated receptor 1, FcγRIIA, or by treatment with PMA phosphorylate adducin at Ser726. Phosphoadducin releases from the membrane skeleton concomitant with its dissociation from spectrin and actin. Inhibition of PKC blunts adducin phosphorylation and release from spectrin and actin, preventing the centralization of spectrin that normally follows cell activation. We conclude that adducin targets actin filament ends to spectrin to complete the assembly of the resting membrane skeleton. Dissociation of phosphoadducin releases spectrin from actin, facilitating centralization of spectrin, and leads to the exposure of barbed actin filament ends that may then participate in converting the resting platelet's disc shape into its active form.
doi:10.1083/jcb.200211122
PMCID: PMC2172952  PMID: 12743105
spectrin; adducin; actin; platelet; cell motility
5.  Phosphorylation of Adducin by Rho-Kinase Plays a Crucial Role in Cell Motility  
The Journal of Cell Biology  1999;145(2):347-361.
Adducin is a membrane skeletal protein that binds to actin filaments (F-actin) and thereby promotes the association of spectrin with F-actin to form a spectrin-actin meshwork beneath plasma membranes such as ruffling membranes. Rho-associated kinase (Rho- kinase), which is activated by the small guanosine triphosphatase Rho, phosphorylates α-adducin and thereby enhances the F-actin–binding activity of α-adducin in vitro. Here we identified the sites of phosphorylation of α-adducin by Rho-kinase as Thr445 and Thr480. We prepared antibody that specifically recognized α-adducin phosphorylated at Thr445, and found by use of this antibody that Rho-kinase phosphorylated α-adducin at Thr445 in COS7 cells in a Rho-dependent manner. Phosphorylated α-adducin accumulated in the membrane ruffling area of Madin-Darby canine kidney (MDCK) epithelial cells and the leading edge of scattering cells during the action of tetradecanoylphorbol-13-acetate (TPA) or hepatocyte growth factor (HGF). The microinjection of Botulinum C3 ADP-ribosyl-transferase, dominant negative Rho-kinase, or α-adducinT445A,T480A (substitution of Thr445 and Thr480 by Ala) inhibited the TPA-induced membrane ruffling in MDCK cells and wound-induced migra- tion in NRK49F cells. α-AdducinT445D,T480D (substi- tution of Thr445 and Thr480 by Asp), but not α-adducinT445A,T480A, counteracted the inhibitory effect of the dominant negative Rho-kinase on the TPA-induced membrane ruffling in MDCK cells. Taken together, these results indicate that Rho-kinase phosphorylates α-adducin downstream of Rho in vivo, and that the phosphorylation of adducin by Rho-kinase plays a crucial role in the regulation of membrane ruffling and cell motility.
PMCID: PMC2133101  PMID: 10209029
Rho; Rho-kinase; adducin; membrane ruffling; cell motility
6.  Erythrocyte adducin: A structural regulator of the red blood cell membrane 
Adducin is an α, β heterotetramer that performs multiple important functions in the human erythrocyte membrane. First, adducin forms a bridge that connects the spectrin–actin junctional complex to band 3, the major membrane-spanning protein in the bilayer. Rupture of this bridge leads to membrane instability and spontaneous fragmentation. Second, adducin caps the fast growing (barbed) end of actin filaments, preventing the tetradecameric protofilaments from elongating into macroscopic F-actin microfilaments. Third, adducin stabilizes the association between actin and spectrin, assuring that the junctional complex remains intact during the mechanical distortions experienced by the circulating cell. And finally, adducin responds to stimuli that may be important in regulating the global properties of the cell, possibly including cation transport, cell morphology and membrane deformability. The text below summarizes the structural properties of adducin, its multiple functions in erythrocytes, and the consequences of engineered deletions of each of adducin subunits in transgenic mice.
doi:10.1016/j.tracli.2010.05.008
PMCID: PMC3385930  PMID: 20655268
Erythrocyte adducin; Erythrocyte membrane; β-adducin; α-adducin; γ-adducin; Adducin’s function; Adducin’s regulation
7.  Adducin Is an In Vivo Substrate for Protein Kinase C: Phosphorylation in the MARCKS-related Domain Inhibits Activity in Promoting Spectrin–Actin Complexes and Occurs in Many Cells, Including Dendritic Spines of Neurons  
The Journal of Cell Biology  1998;142(2):485-497.
Adducin is a heteromeric protein with subunits containing a COOH-terminal myristoylated alanine-rich C kinase substrate (MARCKS)-related domain that caps and preferentially recruits spectrin to the fast-growing ends of actin filaments. The basic MARCKS-related domain, present in α, β, and γ adducin subunits, binds calmodulin and contains the major phosphorylation site for protein kinase C (PKC). This report presents the first evidence that phosphorylation of the MARCKS-related domain modifies in vitro and in vivo activities of adducin involving actin and spectrin, and we demonstrate that adducin is a prominent in vivo substrate for PKC or other phorbol 12-myristate 13-acetate (PMA)-activated kinases in multiple cell types, including neurons. PKC phosphorylation of native and recombinant adducin inhibited actin capping measured using pyrene-actin polymerization and abolished activity of adducin in recruiting spectrin to ends and sides of actin filaments. A polyclonal antibody specific to the phosphorylated state of the RTPS-serine, which is the major PKC phosphorylation site in the MARCKS-related domain, was used to evaluate phosphorylation of adducin in cells. Reactivity with phosphoadducin antibody in immunoblots increased twofold in rat hippocampal slices, eight- to ninefold in human embryonal kidney (HEK 293) cells, threefold in MDCK cells, and greater than 10-fold in human erythrocytes after treatments with PMA, but not with forskolin. Thus, the RTPS-serine of adducin is an in vivo phosphorylation site for PKC or other PMA-activated kinases but not for cAMP-dependent protein kinase in a variety of cell types. Physiological consequences of the two PKC phosphorylation sites in the MARCKS-related domain were investigated by stably transfecting MDCK cells with either wild-type or PKC-unphosphorylatable S716A/S726A mutant α adducin. The mutant α adducin was no longer concentrated at the cell membrane at sites of cell–cell contact, and instead it was distributed as a cytoplasmic punctate pattern. Moreover, the cells expressing the mutant α adducin exhibited increased levels of cytoplasmic spectrin, which was colocalized with the mutant α adducin in a punctate pattern. Immunofluorescence with the phosphoadducin-specific antibody revealed the RTPS-serine phosphorylation of adducin in postsynaptic areas in the developing rat hippocampus. High levels of the phosphoadducin were detected in the dendritic spines of cultured hippocampal neurons. Spectrin also was a component of dendritic spines, although at distinct sites from the ones containing phosphoadducin. These data demonstrate that adducin is a significant in vivo substrate for PKC or other PMA-activated kinases in a variety of cells, and that phosphorylation of adducin occurs in dendritic spines that are believed to respond to external signals by changes in morphology and reorganization of cytoskeletal structures.
PMCID: PMC2133059  PMID: 9679146
membrane skeleton; cytoskeleton; actin binding protein; synapse; synaptic plasticity
8.  Disruption of Spectrin-Like Cytoskeleton in Differentiating Keratinocytes by PKCδ Activation Is Associated with Phosphorylated Adducin 
PLoS ONE  2011;6(12):e28267.
Spectrin is a central component of the cytoskeletal protein network in a variety of erythroid and non-erythroid cells. In keratinocytes, this protein has been shown to be pericytoplasmic and plasma membrane associated, but its characteristics and function have not been established in these cells. Here we demonstrate that spectrin increases dramatically in amount and is assembled into the cytoskeleton during differentiation in mouse and human keratinocytes. The spectrin-like cytoskeleton was predominantly organized in the granular and cornified layers of the epidermis and disrupted by actin filament inhibitors, but not by anti-mitotic drugs. When the cytoskeleton was disrupted PKCδ was activated by phosphorylation on Thr505. Specific inhibition of PKCδ(Thr505) activation with rottlerin prevented disruption of the spectrin-like cytoskeleton and the associated morphological changes that accompany differentiation. Rottlerin also inhibited specific phosphorylation of the PKCδ substrate adducin, a cytoskeletal protein. Furthermore, knock-down of endogenous adducin affected not only expression of adducin, but also spectrin and PKCδ, and severely disrupted organization of the spectrin-like cytoskeleton and cytoskeletal distribution of both adducin and PKCδ. These results demonstrate that organization of a spectrin-like cytoskeleton is associated with keratinocytes differentiation, and disruption of this cytoskeleton is mediated by either PKCδ(Thr505) phosphorylation associated with phosphorylated adducin or due to reduction of endogenous adducin, which normally connects and stabilizes the spectrin-actin complex.
doi:10.1371/journal.pone.0028267
PMCID: PMC3233558  PMID: 22163289
9.  Adducin Promotes Micrometer-Scale Organization of β2-Spectrin in Lateral Membranes of Bronchial Epithelial Cells 
Molecular Biology of the Cell  2008;19(2):536-545.
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 α-adducin from confluent HBE cells. Depletion of α-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 α-adducin prevented loss of lateral membrane, but only if α-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.
doi:10.1091/mbc.E07-08-0818
PMCID: PMC2230604  PMID: 18003973
10.  Erythrocyte adducin: a calmodulin-regulated actin-bundling protein that stimulates spectrin-actin binding 
The Journal of Cell Biology  1987;105(6):2837-2845.
Adducin is an erythrocyte membrane skeletal phosphoprotein comprised of two related subunits of 105,000 and 100,000 Mr. These peptides form a functional heterodimer, and the smaller of the two binds calmodulin in a calcium-dependent fashion. Although this protein has been physicochemically characterized, its function remains unknown. We have examined the interaction of human adducin with actin and with human erythrocyte spectrin using sedimentation, electrophoretic, and morphologic techniques. Purified adducin binds actin at physiologic ionic strength and bundles it into arrays of laterally arranged filaments, the adducin forming cross-bridges between the filaments at 35.2 /- 3.8 (2 SD) nm intervals. The stoichiometry of high affinity adducin binding to actin at saturation is 1:7, corresponding to a dimer of adducin for every actin helical unit. Adducin also promotes the binding of spectrin to actin independently of protein 4.1. At saturation, each adducin promotes the association of one spectrin heterodimer. The formation of this ternary spectrin-actin-adducin complex is independent of the assembly path, and the complex exists in a readily reversible equilibrium with the free components. The binding of adducin to actin and its ability to stimulate spectrin-actin binding is down-regulated by calmodulin in a calcium-dependent fashion. These results thus identify a putative role for adducin, and define a calcium- and calmodulin-dependent mechanism whereby higher states of actin association and its interaction with spectrin in the erythrocyte may be controlled.
PMCID: PMC2114693  PMID: 3693401
11.  The membrane cytoskeletal protein adducin is phosphorylated by protein kinase C in D1 neurons of the nucleus accumbens and dorsal striatum following cocaine administration 
Journal of neurochemistry  2009;111(5):1129-1137.
Repeated cocaine administration results in persistent changes in synaptic function in the mesolimbic dopamine system that are thought to be critical for the transition to addiction. Cytoskeletal rearrangement and actin dynamics are essential for this drug-dependent plasticity. Cocaine administration increases levels of F-actin in the nucleus accumbens and is associated with changes in the phosphorylation state of actin binding proteins. The adducins constitute a family of proteins that interact with actin and spectrin to maintain cellular architecture. The interaction of adducin with these cytoskeletal proteins is regulated by phosphorylation, and it is therefore expected that phosphorylation of adducin may be involved in morphological changes underlying synaptic responses to drugs of abuse including cocaine. In the current study, we characterized the regulation of adducin phosphorylation in the nucleus accumbens and dorsal striatum in response to various regimen of cocaine. Our results demonstrate that adducin is phosphorylated by PKC in medium spiny neurons that express the dopamine D1 receptor. These data indicate that adducin phosphorylation is a signaling event regulated by cocaine administration and further suggest that adducin may be involved in remodeling of neuronal cytoskeleton in response to cocaine administration.
doi:10.1111/j.1471-4159.2009.06405.x
PMCID: PMC2810345  PMID: 19780900
addiction; signaling; dopamine; D1; chelerythrine
12.  Hematopoietic Protein-1 Regulates the Actin Membrane Skeleton and Membrane Stability in Murine Erythrocytes 
PLoS ONE  2013;8(2):e54902.
Hematopoietic protein-1 (Hem-1) is a hematopoietic cell specific member of the WAVE (Wiskott-Aldrich syndrome verprolin-homologous protein) complex, which regulates filamentous actin (F-actin) polymerization in many cell types including immune cells. However, the roles of Hem-1 and the WAVE complex in erythrocyte biology are not known. In this study, we utilized mice lacking Hem-1 expression due to a non-coding point mutation in the Hem1 gene to show that absence of Hem-1 results in microcytic, hypochromic anemia characterized by abnormally shaped erythrocytes with aberrant F-actin foci and decreased lifespan. We find that Hem-1 and members of the associated WAVE complex are normally expressed in wildtype erythrocyte progenitors and mature erythrocytes. Using mass spectrometry and global proteomics, Coomassie staining, and immunoblotting, we find that the absence of Hem-1 results in decreased representation of essential erythrocyte membrane skeletal proteins including α- and β- spectrin, dematin, p55, adducin, ankyrin, tropomodulin 1, band 3, and band 4.1. Hem1−/− erythrocytes exhibit increased protein kinase C-dependent phosphorylation of adducin at Ser724, which targets adducin family members for dissociation from spectrin and actin, and subsequent proteolysis. Increased adducin Ser724 phosphorylation in Hem1−/− erythrocytes correlates with decreased protein expression of the regulatory subunit of protein phosphatase 2A (PP2A), which is required for PP2A-dependent dephosphorylation of PKC targets. These results reveal a novel, critical role for Hem-1 in the homeostasis of structural proteins required for formation and stability of the actin membrane skeleton in erythrocytes.
doi:10.1371/journal.pone.0054902
PMCID: PMC3570531  PMID: 23424621
13.  Expression of functional domains of beta G-spectrin disrupts epithelial morphology in cultured cells 
The Journal of Cell Biology  1995;128(6):1069-1080.
Spectrin is a major structural protein associated with the cytoplasmic surface of plasma membranes of many types of cells. To study the functions of spectrin, we transfected Caco-2 intestinal epithelial cells with a plasmid conferring neomycin resistance and encoding either actin-binding or ankyrin-binding domains of beta G-spectrin fused with beta-galactosidase. These polypeptides, in principle, could interfere with the interaction of spectrin with actin or ankyrin, as well as block normal assembly of alpha- and beta-spectrin subunits. Cells expressing the fusion proteins represented only a small fraction of neomycin-resistant cells, but they could be detected based on expression of beta-galactosidase. Cells expressing spectrin domains exhibited a progressive decrease in amounts of endogenous beta G- spectrin, although alpha-spectrin was still present. Beta G-spectrin- deficient cells lost epithelial cell morphology, became multinucleated, and eventually disappeared after 10-14 d in culture. Spectrin- associated membrane proteins, ankyrin and adducin, as well as the Na+,K(+)-ATPase, which binds to ankyrin, exhibited altered distributions in cells transfected with beta G-spectrin domains. E- cadherin and F-actin, in contrast to ankyrin, adducin, and the Na+,K(+)- ATPase, were expressed, and they exhibited unaltered distribution in beta G-spectrin-deficient cells. Cells transfected with the same plasmid encoding beta-galactosidase alone survived in culture as the major population of neomycin-resistant cells, and they exhibited no change in morphology or in the distribution of spectrin-associated membrane proteins. These results establish that beta G-spectrin is essential for the normal morphology of epithelial cells, as well as for their maintenance in monolayer culture.
PMCID: PMC2120414  PMID: 7896872
14.  Primary structure and domain organization of human alpha and beta adducin 
The Journal of Cell Biology  1991;115(3):665-675.
Adducin is a membrane-skeletal protein which is a candidate to promote assembly of a spectrin-actin network in erythrocytes and at sites of cell-cell contact in epithelial tissues. The complete sequence of both subunits of human adducin, alpha (737 amino acids), and beta (726 amino acids) has been deduced by analysis of the cDNAs. The two subunits have strikingly conserved amino acid sequences with 49% identity and 66% similarity, suggesting evolution by gene duplication. Each adducin subunit has three distinct domains: a 39-kD NH2-terminal globular protease-resistant domain, connected by a 9-kD domain to a 33-kD COOH- terminal protease-sensitive tail comprised almost entirely of hydrophilic amino acids. The tail is responsible for the high frictional ratio of adducin noted previously, and was visualized by EM. The head domains of both adducin subunits exhibit a limited sequence similarity with the NH2-terminal actin-binding motif present in members of the spectrin superfamily and actin gelation proteins. The COOH- termini of both subunits contain an identical, highly basic stretch of 22 amino acids with sequence similarity to the MARCKS protein. Predicted sites of phosphorylation by protein kinase C include the COOH- terminus and sites at the junction of the head and tail. Northern blot analysis of mRNA from rat tissues, K562 erythroleukemia cells and reticulocytes has shown that alpha adducin is expressed in all the tissues tested as a single message size of 4 kb. In contrast, beta adducin shows tissue specific variability in size of mRNA and level of expression. A striking divergence between alpha and beta mRNAs was noted in reticulocytes, where alpha adducin mRNA is present in at least 20-fold higher levels than that of beta adducin. The beta subunit thus is a candidate to perform a limiting role in assembly of functional adducin molecules.
PMCID: PMC2289184  PMID: 1840603
15.  Shigella flexneri utilize the spectrin cytoskeleton during invasion and comet tail generation 
BMC Microbiology  2012;12:36.
Background
The spectrin cytoskeleton is emerging as an important host cell target of enteric bacterial pathogens. Recent studies have identified a crucial role for spectrin and its associated proteins during key pathogenic processes of Listeria monocytogenes and Salmonella Typhimurium infections. Here we investigate the involvement of spectrin cytoskeletal components during the pathogenesis of the invasive pathogen Shigella flexneri.
Results
Immunofluorescent microscopy reveals that protein 4.1 (p4.1), but not adducin or spectrin, is robustly recruited to sites of S. flexneri membrane ruffling during epithelial cell invasion. Through siRNA-mediated knockdowns, we identify an important role for spectrin and the associated proteins adducin and p4.1 during S. flexneri invasion. Following internalization, all three proteins are recruited to the internalized bacteria, however upon generation of actin-rich comet tails, we observed spectrin recruitment to those structures in the absence of adducin or p4.1.
Conclusion
These findings highlight the importance of the spectrin cytoskeletal network during S. flexneri pathogenesis and further demonstrate that pathogenic events that were once thought to exclusively recruit the actin cytoskeletal system require additional cytoskeletal networks.
doi:10.1186/1471-2180-12-36
PMCID: PMC3384245  PMID: 22424399
16.  Nonredundant roles of cytoplasmic β- and γ-actin isoforms in regulation of epithelial apical junctions 
Molecular Biology of the Cell  2012;23(18):3542-3553.
The functional effects of cytoplasmic actins on epithelial junctions are examined by using isoform-specific siRNAs and cell-permeable inhibitory peptides. Unique roles of cytoplasmic actin isoforms in regulating structure and remodeling of adherens and tight junctions are revealed.
Association with the actin cytoskeleton is critical for normal architecture and dynamics of epithelial tight junctions (TJs) and adherens junctions (AJs). Epithelial cells express β-cytoplasmic (β-CYA) and γ-cytoplasmic (γ-CYA) actins, which have different cellular localization and functions. This study elucidates the roles of cytoplasmic actins in regulating structure and remodeling of AJs and TJs in model intestinal epithelia. Immunofluorescence labeling and latrunculin B treatment reveal affiliation of dynamic β-CYA filaments with newly assembled and mature AJs, whereas an apical γ-CYA pool is composed of stable perijunctional bundles and rapidly turning-over nonjunctional filaments. The functional effects of cytoplasmic actins on epithelial junctions are examined by using isoform-specific small interfering RNAs and cell-permeable inhibitory peptides. These experiments demonstrate unique roles of β-CYA and γ-CYA in regulating the steady-state integrity of AJs and TJs, respectively. Furthermore, β-CYA is selectively involved in establishment of apicobasal cell polarity. Both actin isoforms are essential for normal barrier function of epithelial monolayers, rapid AJ/TJ reassembly, and formation of three-dimensional cysts. Cytoplasmic actin isoforms play unique roles in regulating structure and permeability of epithelial junctions.
doi:10.1091/mbc.E12-02-0162
PMCID: PMC3442403  PMID: 22855531
17.  Polarization of membrane associated proteins in the choroid plexus epithelium from normal and slc4a10 knockout mice 
The choroid plexus epithelium (CPE) has served as a model-epithelium for cell polarization and transport studies and plays a crucial role for cerebrospinal fluid (CSF) production. The normal luminal membrane expression of Na+,K+-ATPase, aquaporin-1 and Na+/H+ exchanger 1 in the choroid plexus is severely affected by deletion of the slc4a10 gene that encodes the bicarbonate transporting protein Ncbe/NBCn2. The causes for these deviations from normal epithelial polarization and redistribution following specific gene knockout are unknown, but may be significant for basic epithelial cell biology. Therefore, a more comprehensive analysis of cell polarization in the choroid plexus is warranted. We find that the cytoskeleton in the choroid plexus contains αI-, αII-, βI-, and βII-spectrin isoforms along with the anchoring protein ankyrin-3, most of which are mainly localized in the luminal membrane domain. Furthermore, we find α-adducin localized near the plasma membranes globally, but with only faint expression in the luminal membrane domain. In slc4a10 knockout mice, the abundance of β1 Na+,K+-ATPase subunits in the luminal membrane is markedly reduced. Anion exchanger 2 abundance is increased in slc4a10 knockout and its anchor protein, α-adducin is almost exclusively found near the basolateral domain. The αI- and βI-spectrin abundances are also decreased in the slc4a10 knockout, where the basolateral domain expression of αI-spectrin is exchanged for a strictly luminal domain localization. E-cadherin expression is unchanged in the slc4a10 knockout, while small decreases in abundance are observed for its probable adaptor proteins, the catenins. Interestingly, the abundance of the tight junction protein claudin-2 is significantly reduced in the slc4a10 knockouts, which may critically affect paracellular transport in this epithelium. The observations allow the generation of new hypotheses on basic cell biological paradigms that can be tested experimentally in future studies.
doi:10.3389/fphys.2013.00344
PMCID: PMC3842056  PMID: 24348423
sodium hydrogen exchanger; sodium bicarbonate cotransporter; epithelial polarization; cytoskeleton; choroid plexus; cerebrospinal fluid
18.  Visualization of the hexagonal lattice in the erythrocyte membrane skeleton 
The Journal of Cell Biology  1987;104(3):527-536.
The isolated membrane skeleton of human erythrocytes was studied by high resolution negative staining electron microscopy. When the skeletal meshwork is spread onto a thin carbon film, clear images of a primarily hexagonal lattice of junctional F-actin complexes crosslinked by spectrin filaments are obtained. The regularly ordered network extends over the entire membrane skeleton. Some of the junctional complexes are arranged in the form of pentagons and septagons, approximately 3 and 8%, respectively. At least five forms of spectrin crosslinks are detected in the spread skeleton including a single spectrin tetramer linking two junctional complexes, three-armed Y- shaped spectrin molecules linking three junctional complexes, three- armed spectrin molecules connecting two junctional complexes with two arms bound to one complex and the third arm bound to the adjacent complex, double spectrin filaments linking two junctional complexes, and four-armed spectrin molecules linking two junctional complexes. Of these, the crosslinks of single spectrin tetramers and three-armed molecules are the most abundant and represent 84 and 11% of the total crosslinks, respectively. These observations are compatible with the presence of spectrin tetramers and oligomers in the erythrocyte membrane skeleton. Globular structures (9-12 nm in diameter) are attached to the majority of the spectrin tetramers or higher order oligomer-like molecules, approximately 80 nm from the distal ends of the spectrin tetramers. These globular structures are ankyrinor ankyrin/band 3-containing complexes, since they are absent when ankyrin and residual band 3 are extracted from the skeleton under hypertonic conditions.
PMCID: PMC2114560  PMID: 2434513
19.  Hts/Adducin Controls Synaptic Elaboration and Elimination 
Neuron  2011;69(6):1114-1131.
Neural development requires both synapse elaboration and elimination, yet relatively little is known about how these opposing activities are coordinated. Here we provide evidence Hts/Adducin can serve this function. We show that Drosophila Hts/Adducin is enriched both pre- and postsynaptically at the NMJ. We then demonstrate that presynaptic Hts/Adducin is necessary and sufficient to control two opposing processes associated with synapse remodeling: 1) synapse stabilization as determined by light level, ultrastructural and electrophysiological assays and 2) the elaboration of actin-based, filopodia-like protrusions that drive synaptogenesis and growth. Synapse remodeling is sensitive to Hts/Adducin levels and we provide evidence that the synaptic localization of Hts/Adducin is controlled via phosphorylation. Mechanistically, Drosophila Hts/Adducin protein has actin-capping activity. We propose that phosphorylation-dependent regulation of Hts/Adducin controls the level, localization and activity of Hts/Adducin, influencing actin-based synapse elaboration and spectrin-based synapse stabilization. Hts/Adducin may define a mechanism to switch between synapse stability and dynamics.
doi:10.1016/j.neuron.2011.02.007
PMCID: PMC3073818  PMID: 21435557
20.  γ-Adducin Promotes Process Outgrowth and Secretory Protein Exit from the Golgi Apparatus 
α, β, and γ adducins mediate F-actin remodeling of plasma membrane structures as heterotetramers. Here, we present two new functions of γ-adducin. (1) Overexpression of γ-adducin promoted formation of neurite-like processes in non-neuronal fibroblast COS7 cells. Conversely, overexpression of the C-terminal 38 amino acids of γ-adducin (γAddC38) acting as a dominant negative inhibited formation of neurites/processes in Neuro2A cells and anterior pituitary AtT20 cells. (2) γ-Adducin appears to facilitate pro-opiomelanocortin (POMC) exit from the trans-Golgi network (TGN) by re-organizing the actin network around the Golgi complex. Filamentous actins (F-actins) which formed puncti around the Golgi complex in control cells were dispersed in AtT20 cells stably transfected with γAddC38. Furthermore, γAddC38-transfectants showed significant accumulation of POMC/adrenocorticotropin (ACTH) in the Golgi complex and diminished POMC/ACTH vesicles in the cell processes. The C-terminal 38 amino acids of γ-adducin interacted with F-actins around the Golgi complex, to facilitate F-actin-mediated budding of POMC/ACTH vesicles from the TGN. Thus, we propose that γ-adducin, via its interaction with F-actins, plays a critical role in actin remodeling to facilitate process/neurite outgrowth, as well as budding of POMC/ACTH vesicles from the TGN via its interaction with peri-Golgi F-actins.
doi:10.1007/s12031-012-9827-0
PMCID: PMC3681813  PMID: 22706708
γ-Adducin; F-actins; Neurite outgrowth; Vesicle budding; TGN; POMC/ACTH
21.  Brain-specific promoter and polyadenylation sites of the β-adducin pre-mRNA generate an unusually long 3′-UTR 
Nucleic Acids Research  2006;34(1):243-253.
Adducins are a family of membrane skeleton proteins composed of α-, β- and γ-subunits that promote actin and spectrin association in erythrocytes. The α- and γ-subunits are expressed ubiquitously, while the β-subunit is found in brain and erythropoietic tissues. The brain β-adducin protein is similar in size to that of spleen, but the mRNA transcript is a brain-specific one that has not been yet characterized, having an estimated length of 8–9 kb instead of the 3–4 kb of spleen mRNA. Here, we show the molecular basis for these differences by determining the structure of the brain-specific β-adducin transcript in rats, mice and humans. We identified a brain-specific promoter in rodents that, apparently, was not conserved in humans. In addition, we present evidence that the brain-mRNAs are formed by a common mechanism consisting in the tissue-specific use of alternative polyadenylation sites generating unusually long 3′-untranslated region of up to 6.6 kb. This hypothesis is supported by the presence of highly-conserved regions flanking the brain-specific polyadenylation site that suggest the involvement of these sequences in the translational regulation, stability and/or subcellular localization of the β-adducin transcript in the brain.
doi:10.1093/nar/gkj425
PMCID: PMC1326019  PMID: 16414955
22.  Small GTPases of the Ras superfamily regulate intestinal epithelial homeostasis and barrier function via common and unique mechanisms 
Tissue Barriers  2013;1(5):e26938.
The intestinal epithelium forms a stable barrier protecting underlying tissues from pathogens in the gut lumen. This is achieved by specialized integral membrane structures such as tight and adherens junctions that connect neighboring cells and provide stabilizing links to the cytoskeleton. Junctions are constantly remodeled to respond to extracellular stimuli. Assembly and disassembly of junctions is regulated by interplay of actin remodeling, endocytotic recycling of junctional proteins, and various signaling pathways. Accumulating evidence implicate small G proteins of the Ras superfamily as important signaling molecules for the regulation of epithelial junctions. They function as molecular switches circling between an inactive GDP-bound and an active GTP-bound state. Once activated, they bind different effector molecules to control cellular processes required for correct junction assembly, maintenance and remodelling. Here, we review recent advances in understanding how GTPases of the Rho, Ras, Rab and Arf families contribute to intestinal epithelial homeostasis.
doi:10.4161/tisb.26938
PMCID: PMC3942330  PMID: 24868497
Rho; Rac; cdc42; Ras; Rap; Arf; inflammatory bowel disease; colitis; tight junction; adherens junction; actin cytoskeleton
23.  Impaired synaptic plasticity and learning in mice lacking β-adducin, an actin-regulating protein 
The adducin family of proteins interacts with the actin cytoskeleton and the plasma membrane in a calcium- and cAMP-dependent fashion. Thus, adducins may be involved in changes in cytoskeletal organization resulting from synaptic stimulation. β-adducin knockout mice were examined in physiological and behavioral paradigms related to synaptic plasticity to elucidate the role the adducin family plays in processes underlying learning and memory. In situ hybridization for α- and β-adducin demonstrates that these mRNAs are found throughout the brain, with high levels of expression in the hippocampus. Schaffer-collateral-CA1 tetanic LTP decayed rapidly in acute hippocampal slices from β-adducin knockout mice, although baseline spine morphology and PSD density were normal. Interestingly, the input-output relationship was significantly increased in hippocampal slices from β-adducin knockout mice. Further, β-adducin knockout mice were impaired in performance of fear conditioning and the water maze paradigm. The current results indicate that β-adducin may play an important role in the cellular mechanisms underlying activity-dependent synaptic plasticity associated with learning and memory.
doi:10.1523/JNEUROSCI.3530-04.2005
PMCID: PMC1352335  PMID: 15728854
cytoskeleton; memory; LTP; Morris water maze; fear conditioning; dendritic spines
24.  Actin, spectrin and associated proteins form a periodic cytoskeletal structure in axons 
Science (New York, N.Y.)  2012;339(6118):10.1126/science.1232251.
Actin and spectrin play important roles in neurons, but their organization in axons and dendrites remains unclear. We used stochastic optical reconstruction microscopy (STORM) to study the organization of actin, spectrin and associated proteins in neurons. Actin formed ring-like structures that wrapped around the circumference of axons and evenly spaced along axonal shafts with a periodicity of ~180–190 nm. This periodic structure was not observed in dendrites, which instead contained long actin filaments running along dendritic shafts. Adducin, an actin-capping protein, colocalized with the actin rings. Spectrin exhibited periodic structures alternating with those of actin and adducin, and the distance between adjacent actin-adducin rings was comparable to the length of a spectrin tetramer. Sodium channels in axons were distributed in a periodic pattern coordinated with the underlying actin-spectrin-based cytoskeleton.
doi:10.1126/science.1232251
PMCID: PMC3815867  PMID: 23239625
25.  Tmod1 and CP49 Synergize to Control the Fiber Cell Geometry, Transparency, and Mechanical Stiffness of the Mouse Lens 
PLoS ONE  2012;7(11):e48734.
The basis for mammalian lens fiber cell organization, transparency, and biomechanical properties has contributions from two specialized cytoskeletal systems: the spectrin-actin membrane skeleton and beaded filament cytoskeleton. The spectrin-actin membrane skeleton predominantly consists of α2β2-spectrin strands interconnecting short, tropomyosin-coated actin filaments, which are stabilized by pointed-end capping by tropomodulin 1 (Tmod1) and structurally disrupted in the absence of Tmod1. The beaded filament cytoskeleton consists of the intermediate filament proteins CP49 and filensin, which require CP49 for assembly and contribute to lens transparency and biomechanics. To assess the simultaneous physiological contributions of these cytoskeletal networks and uncover potential functional synergy between them, we subjected lenses from mice lacking Tmod1, CP49, or both to a battery of structural and physiological assays to analyze fiber cell disorder, light scattering, and compressive biomechanical properties. Findings show that deletion of Tmod1 and/or CP49 increases lens fiber cell disorder and light scattering while impairing compressive load-bearing, with the double mutant exhibiting a distinct phenotype compared to either single mutant. Moreover, Tmod1 is in a protein complex with CP49 and filensin, indicating that the spectrin-actin network and beaded filament cytoskeleton are biochemically linked. These experiments reveal that the spectrin-actin membrane skeleton and beaded filament cytoskeleton establish a novel functional synergy critical for regulating lens fiber cell geometry, transparency, and mechanical stiffness.
doi:10.1371/journal.pone.0048734
PMCID: PMC3492431  PMID: 23144950

Results 1-25 (599035)