Membrane skeleton in cultured chick cardiac myocytes revealed by high resolution immunocytochemistry

Distribution of cytoskeletal proteins with emphasis on the membrane-cytoskeleton interface was examined in cultured cardiac myocytes. Using specific antibodies recognizing α-sarcomeric actin, desmin, β-tubulin, spectrin/α-fodrin and ankyrin, respectively, the cellular localization of these cytoskeletal proteins was detected by laser scanning confocal microscopy. In addition, the fine filamentous structure of these proteins was identified by combining silver-enhanced immunogold labelling with electron microscopy. The latter technique employed the sequence of quick-freezing, deep-etching and rotary shadowing of the specimens. Conventional transmission electron microscopy of the spherical cardiac myocytes revealed a filamentous submembranous layer, approximately 100 nm thick. Specific immunolabelling of α-sarcomeric actin and spectrin/α-fodrin as well as ankyrin was seen beneath the plasmalemma. A three-dimensional meshwork of spectrin/α-fodrin was shown. Numerous desmin filaments that exhibited a tortuous course throughout the cells were also observed running in parallel with the surface in the submembranous area, whereas β-tubulin was infrequently detected in these areas. In conclusion, the present study shows that spherical cardiac myocytes contain a distinct and complex three-dimensional membrane skeleton. Major constituents of this distinct submembranous layer were spectrin/α-fodrin fibres as well as actin and desmin filaments. Accepted: 28 July 1999     

Neuroglian-mediated cell adhesion induces assembly of the membrane skeleton at cell contact sites

The protein ankyrin links integral membrane proteins to the spectrin- based membrane skeleton. Ankyrin is often concentrated within restricted membrane domains of polarized epithelia and neurons, but the mechanisms responsible for membrane targeting and its segregation within a continuous lipid bilayer remain unexplained. We provide evidence that neuroglian, a cell adhesion molecule related to L1 and neurofascin, can transmit positional information directly to ankyrin and thereby polarize its distribution in Drosophila S2 tissue culture cells. Ankyrin was not normally associated with the plasma membrane of these cells. Upon expression of an inducible neuroglian minigene, however, cells aggregated into large clusters and ankyrin became concentrated at sites of cell-cell contact. Spectrin was also recruited to sites of cell contact in response to neuroglian expression. The accumulation of ankyrin at cell contacts required the presence of the cytoplasmic domain of neuroglian since a glycosyl phosphatidylinositol- linked form of neuroglian failed to recruit ankyrin to sites of cell- cell contact. Double-labeling experiments revealed that, whereas ankyrin was strictly associated with sites of cell-cell contact, neuroglian was more broadly distributed over the cell surface. A direct interaction between neuroglian and ankyrin was demonstrated using yeast two-hybrid analysis. Thus, neuroglian appears to be activated by extracellular adhesion so that ankyrin and the membrane skeleton selectively associate with sites of cell contact and not with other regions of the plasma membrane.     

Low intracellular pH induces redistribution of fodrin and instabilization of lateral walls in MDCK cells.

We have studied the effect of intracellular pH on the establishment and maintenance of the cellular polarity in MDCK cells by utilizing nigericin which causes lowering of the cytoplasmic pH. At pH below 6.5, MDCK cells lost their polarized morphology and became roundish, with an increased apical area and shortened and unstable lateral walls. The lateral wall marker proteins uvomorulin and Na,K-ATPase remained segregated to the lateral walls in the acidified cells, as shown by immunofluorescence microscopy. Fodrin, on the other hand, was released from its normal basolateral residence and was found in the cytoplasm. Actin, which normally co-localizes with fodrin along the basolateral walls, showed a dotty distribution in the cytoplasm of acidified cells, while stress fibers remained intact. Microtubular network appeared flattened, but the Golgi complex retained its apical position. The pH change-induced alterations were readily reversible, as the normal basal-apical polarity (columnar shape, distinct apical and lateral domains with apposing and stiff lateral membranes) was reformed within 10 minutes after restoring the normal pH gradient across the cell membrane. This coincided with the translocation of fodrin from the cytoplasm to the lateral walls. The results show that lowering of intracellular pH leads to temporary segregation of fodrin from the other components of the membrane skeleton assembly, and that association of fodrin with the lateral walls seems to be a prerequisite for their close apposition and for the maintenance of normal basal-axial polarity.     

Calcium-dependent peripheral localization of 4.1-like proteins and fodrin in cultured human keratinocytes

Summary— Recently, several proteins immunologically related to erythrocyte membrane skeletal proteins, such as protein 4.1 and fodrin (non-erythroid spectrin), have been found in keratinocytes. In the present study, in order to investigate the roles of these proteins in cell-cell contact, we analyzed the distribution of non-erythroid protein 4.1, β-fodrin and actin in cultured human keratinocytes at low (0.15 mM) and standard (1.85 mM) Ca²⁺ concentrations. Immunofluorescence microscopy revealed that immunoreactive forms of protein 4.1, β-fodrin and actin filaments were present in the cytoplasm of cells cultured in low Ca²⁺ medium, while in cells in the standard Ca²⁺ medium, these proteins were localized at the cell boundary and partially in the cytoplasm. When cells in the low-Ca²⁺ medium were treated with 100 nM 12-O-tetradecanoylphorbol-13-acetate (TPA) for 1 h, these proteins were also present at the cell boundary. Increasing extracellular Ca²⁺ concentration from low to standard in the medium induces cell-cell contact among the cultured human keratinocytes, accompanied by the translocation of protein 4.1 and β-fodrin from the cytoplasm to the membrane. On the basis of the present study, movement of membrane skeletal proteins from the cytosol to the membrane suggests that either these proteins or the membrane skeletal lattice plays an important role in the regulation of cell-cell intergigitations in response to changes in the Ca²⁺ concentrations in culture medium, and that phosphorylation of these skeletal proteins might be involved in the regulation of the membrane skeletal proteins of keratinocytes in response to Ca²⁺.     

Protein 4.1N is required for the formation of the lateral membrane domain in human bronchial epithelial cells

The membrane skeleton forms a scaffold on the cytoplasmic side of the plasma membrane. The erythrocyte membrane represents an archetype of such structural organization. It has been documented that a similar membrane skeleton also exits in the Golgi complex. It has been previously shown that βII spectrin and ankyrin G are localized at the lateral membrane of human bronchial epithelial cells. Here we show that protein 4.1N is also located at the lateral membrane where it associates E-cadherin, β-catenin and βII spectrin. Importantly, depletion of 4.1N by RNAi in human bronchial epithelial cells resulted in decreased height of lateral membrane, which was reversed following re-expression of mouse 4.1N. Furthermore, although the initial phase of lateral membrane biogenesis proceeded normally in 4.1N-depleted cells, the final height of the lateral membrane of 4.1N-depleted cells was shorter compared to that of control cells. Our findings together with previous findings imply that 4.1N, βII spectrin and ankyrin G are structural components of the lateral membrane skeleton and that this skeleton plays an essential role in the assembly of a fully functional lateral membrane.     

Membrane domains of intestinal epithelial cells: distribution of Na+,K+- ATPase and the membrane skeleton in adult rat intestine during fetal development and after epithelial isolation

The organization of the basolateral membrane domain of highly polarized intestinal absorptive cells was studied in adult rat intestinal mucosa, during development of polarity in fetal intestine, and in isolated epithelial sheets. Semi-thin frozen sections of these tissues were stained with a monoclonal antibody (mAb 4C4) directed against Na+,K+-ATPase, and with other reagents to visualize distributions of the membrane skeleton (fodrin), an epithelial cell adhesion molecule (uvomorulin), an apical membrane enzyme (aminopeptidase), and filamentous actin. In intact adult epithelium, Na+,K+-ATPase, membrane-associated fodrin, and uvomorulin were concentrated in the lateral, but not basal, subdomain. In the stratified epithelium of fetal intestine, both fodrin and uvomorulin were localized in areas of cell-cell contact at 16 and 17 d gestation, a stage when Na+,K+-ATPase was not yet expressed. These molecules were excluded from apical domains and from cell surfaces in contact with basal lamina. When Na+,K+-ATPase appeared at 18-19 d, it was codistributed with fodrin. Detachment of epithelial sheets from adult intestinal mucosa did not disrupt intercellular junctions or lateral cell contacts, but cytoplasmic blebs appeared at basal cell surfaces, and a diffuse pool of fodrin and actin accumulated in them. At the same time, Na+,K+-ATPase moved into the basal membrane subdomain, and extensive endocytosis of basolateral membrane, including Na+,K+-ATPase, occurred. Endocytosis of uvomorulin was not detected and no fodrin was associated with endocytic vesicles. Uvomorulin, along with some membrane-associated fodrin and some Na+,K+-ATPase, remained in the lateral membrane as long as intercellular contacts were maintained. Thus, in this polarized epithelium, interaction of lateral cell-cell adhesion molecules as well as basal cell-substrate interactions are required for maintaining the stability of the lateral membrane skeleton and the position of resident membrane proteins concentrated in the lateral membrane domain.     

αE-catenin regulates cell-cell adhesion and membrane blebbing during zebrafish epiboly

αE-catenin is an actin-binding protein associated with the E-cadherin-based adherens junction that regulates cell-cell adhesion. Recent studies identified additional E-cadherin-independent roles of αE-catenin in regulating plasma membrane dynamics and cell migration. However, little is known about the roles of αE-catenin in these different cellular processes in vivo during early vertebrate development. Here, we examined the functions of αE-catenin in cell-cell adhesion, cell migration and plasma membrane dynamics during morphogenetic processes that drive epiboly in early Danio rerio (zebrafish) development. We show that depletion of αE-catenin caused a defect in radial intercalation that was associated with decreased cell-cell adhesion, in a similar manner to E-cadherin depletion. Depletion of αE-catenin also caused deep cells to have protracted plasma membrane blebbing, and a defect in plasma membrane recruitment of ERM proteins that are involved in controlling membrane-to-cortex attachment and membrane blebbing. Significantly, depletion of both E-cadherin and αE-catenin suppressed plasma membrane blebbing. We suggest that during radial intercalation the activities of E-cadherin and αE-catenin in the maintenance of membrane-to-cortex attachment are balanced, resulting in stabilization of cell-cell adhesion and suppression of membrane blebbing, thereby enabling proper radial intercalation.     

Polarized distribution of Mr 210,000 and 190,000 analogs of erythrocyte ankyrin along the plasma membrane of transporting epithelia, neurons and photoreceptors

In the present study we have examined several types of nucleated cells with respect to the occurrence and subcellular distribution of ankyrin. In red blood cells ankyrin links and integral membrane protein, the anion channel (band 3), to the subplasmalemmal cytoskeleton which is comprised largely of spectrin and actin. Since nucleated cells also contain spectrin and other constituents of the erythrocyte membrane skeleton it is possible that in nonerythroid cells ankyrin is also important for connecting membrane proteins to the cytoskeleton. We show here that membrane fractions of rat brain and various types of rat epithelial cells contain analogs of ankyrin at Mr 210,000 and 190,000 that are immunologically related to human erythrocyte ankyrin. In transporting epithelial cells, such as epithelia of the intestine, pancreas, prostate or kidney (various species) the analogs of ankyrin are confined to the basolateral plasma membrane and are absent from the apical membrane. In neurons of the central and peripheral nervous system and in photoreceptors of the retina, ankyrin was found restricted to the membrane of the cell body and axons and was not detected by immunostaining along the afferent processes (dendrites, photoreceptor inner and outer segments). Linkage of integral membrane proteins via ankyrin to the spectrin-based membrane cytoskeleton may provide a molecular basis for restricting the lateral mobility of certain membrane proteins and localizing them in a nonrandom or polarized fashion at specialized domains of the plasma membrane.     

Cadherin/Catenin Complexes in Murine Epidermal Keratinocytes: E-Cadherin Complexes Containing either β-Catenin or Plakoglobin Contribute to Stable Cell-Cell Contacts

The cadherin/catenin complexes expressed by a murine epidermal keratinocyte cell line PDV, expressing E- and P-cadherin, have been analysed using a combination of biochemical and confocal microscopy analysis. Two types of E-cadherin complexes, containing β-catenin or plakoglobin and α-catenin, were detected in PDV cells as in other cell types, while β-cadherin was mainly detected in complexes containing β-catenin and α-catenin in PDV and other murine epidermal keratinocytes. Bio tin-labelling studies have shown that both types of E-cadherin complexes are present at the surface of confluent cells. Furthermore, confocal microscopy analysis indicated that E-cadherin/ plakoglobin complexes are located in stable cell-cell contacts at the middle lateral membranes and associated with α-catenin and the actin cytoskeleton, with a similar distribution to that of the E-cadherin/β-catenin complexes. In addition, E-cadherin/ plakoglobin complexes not associated with α-catenin or the actin cytoskeleton were detected in lower planes of the lateral contacting membranes as well as E-cadherin non-associated with catenins in the more basal planes. These studies support that in murine epidermal keratinocytes both β-catenin- and plakoglobin-containing E-cadherin complexes contribute to the maintenance of stable cell-cell contacts and suggest a differential role of the plakoglobin containing complexes in different epithelial cell types.     

Ankyrin-G is a molecular partner of E-cadherin in epithelial cells and early embryos

E-cadherin is a ubiquitous component of lateral membranes in epithelial tissues and is required to form the first lateral membrane domains in development. Here, we identify ankyrin-G as a molecular partner of E-cadherin and demonstrate that ankyrin-G and beta-2-spectrin are required for accumulation of E-cadherin at the lateral membrane in both epithelial cells and early embryos. Ankyrin-G binds to the cytoplasmic domain of E-cadherin at a conserved site distinct from that of beta-catenin. Ankyrin-G also recruits beta-2-spectrin to E-cadherin-beta-catenin complexes, thus providing a direct connection between E-cadherin and the spectrin/actin skeleton. In addition to restricting the membrane mobility of E-cadherin, ankyrin-G and beta-2-spectrin also are required for exit of E-cadherin from the trans-Golgi network in a microtubule-dependent pathway. Ankyrin-G and beta-2-spectrin co-localize with E-cadherin in preimplantation mouse embryos. Moreover, knockdown of either ankyrin-G or beta-2-spectrin in one cell of a two-cell embryo blocks accumulation of E-cadherin at sites of cell-cell contact. E-cadherin thus requires both ankyrin-G and beta-2-spectrin for its cellular localization in early embryos as well as cultured epithelial cells. We have recently reported that ankyrin-G and beta-2-spectrin collaborate in biogenesis of the lateral membrane ( Kizhatil, K., Yoon, W., Mohler, P. J., Davis, L. H., Hoffman, J. A., and Bennett, V. (2007) J. Biol. Chem. 282, 2029-2037 ). Together with the current findings, these data suggest a ankyrin/spectrin-based mechanism for coordinating membrane assembly with extracellular interactions of E-cadherin at sites of cell-cell contact.     

Tankyrase recruitment to the lateral membrane in polarized epithelial cells: regulation by cell-cell contact and protein poly(ADP-ribosyl)ation

PARsylation [poly(ADP-ribosyl)ation] of proteins is implicated in the regulation of diverse physiological processes. Tankyrase is a molecular scaffold with this catalytic activity and has been proposed as a regulator of vesicular trafficking on the basis, in part, of its Golgi localization in non-polarized cells. Little is known about tankyrase localization in polarized epithelial cells. Using MDCK (Madin-Darby canine kidney) cells as a model, we found that E-cadherin-mediated intercellular adhesion recruits tankyrase from the cytoplasm to the lateral membrane (including the tight junction), where it stably associates with detergent-insoluble structures. This recruitment is mostly completed within 8 h of calcium-induced formation of cell-cell contact. Conversely, when intercellular adhesion is disrupted by calcium deprivation, tankyrase returns from the lateral membrane to the cytoplasm and becomes more soluble in detergents. The PARsylating activity of tankyrase promotes its dissociation from the lateral membrane as well as its ubiquitination and proteasome-mediated degradation, resulting in an apparent protein half-life of approximately 2 h. Inhibition of tankyrase autoPARsylation using H2O2-induced NAD+ depletion or PJ34 [N-(6-oxo-5,6-dihydrophenanthridin-2-yl)-N,N-dimethylacetamide hydrochloride] treatment results in tankyrase stabilization and accumulation at the lateral membrane. By contrast, stabilization through proteasome inhibition results in tankyrase accumulation in the cytoplasm. These data suggest that cell-cell contact promotes tankyrase association with the lateral membrane, whereas PARsylating activity promotes translocation to the cytosol, which is followed by ubiquitination and proteasome-mediated degradation. Since the lateral membrane is a sorting station that ensures domain-specific delivery of basolateral membrane proteins, the regulated tankyrase recruitment to this site is consistent with a role in polarized protein targeting in epithelial cells.     

Posterior Midgut Epithelial Cells Differ in Their Organization of the Membrane Skeleton from Other Drosophila Epithelia

In epithelial cells, the various components of the membrane skeleton are segregated within specialized subregions of the plasma membrane, thus contributing to the development and stabilization of cell surface polarity. It has previously been shown that, in various Drosophila epithelia, the membrane skeleton components ankyrin and alphabeta-spectrin reside at the lateral surface, whereas alphabeta(H)-spectrin is restricted to the apical domain. By use of confocal immunofluorescence microscopy, the present study characterizes the membrane skeleton of epithelial cells in the posterior midgut, leading to a number of unexpected results. First, ankyrin and alphabeta-spectrin are not detected on the entire lateral surface but appear to be restricted to the apicolateral area, codistributing with fasciclin III at smooth septate junctions. The presumptive ankyrin-binding proteins neuroglian and Na(+),K(+)-ATPase, however, do not colocalize with ankyrin. Second, alphabeta(H)-spectrin is enriched at the apical domain but is also present in lower amounts on the entire lateral surface, colocalizing apicolaterally with ankyrin/alphabeta-spectrin. Finally, despite the absence of zonulae adherentes, F-actin, beta(H)-spectrin, and nonmuscle myosin-II are enriched in the midlateral region. Thus, the model established for the organization of the membrane skeleton in Drosophila epithelia does not hold for the posterior midgut, and there is quite some variability between the different epithelia with respect to the organization of the membrane skeleton.     

The AE2 anion exchanger is necessary for the structural integrity of the Golgi apparatus in mammalian cells

The structural integrity of the Golgi apparatus is known to be dependent on multiple factors, including the organizational status of microtubules, actin and the ankyrin/spectrin-based Golgi membrane skeleton, as well as vesicular trafficking and pH homeostasis. In this respect, our recently identified Golgi-associated anion exchanger, AE2, may also be of importance, since it potentially acts as a Golgi pH regulator and as a novel membrane anchor for the spectrin-based Golgi membrane skeleton. Here, we show that inhibition (>75%) of AE2 expression by antisense oligonucleotides in COS-7 cells results in the fragmentation of the juxtanuclear Golgi apparatus and in structural disorganization of the Golgi stacks, the cisternae becoming generally shorter, distorted, vesiculated and/or swollen. These structural changes occurred without apparent dissociation of the Golgi membrane skeletal protein Ankyrin(195), but were accompanied by the disappearance of the well-focused microtubule-organizing center (MTOC), suggesting the involvement of microtubule reorganization. Similar changes in Golgi structure and assembly of the MTOC were also observed upon transient overexpression of the EGFP-AE2 fusion protein. These data implicate a clear structural role for the AE2 protein in the Golgi and in its cytological positioning around the MTOC.     

Different organizational states of fodrin in cultured MDCK cells are induced by treatment with low pH, calmodulin antagonist TFP, and tumor promoter PMA.

We have investigated the molecular mechanisms underlying dynamic organization of the fodrin network by treating the epithelial MDCK cells with various agents affecting intracellular pH, intracellular calcium ion concentration, intracellular calmodulin, and protein kinase C (PKC) activity. Elevation of intracellular calcium level by A23187 or treatment with trifluoperazine (TFP), a calmodulin inhibitor, did not have any drastic effect on the fodrin distribution as judged by immunofluorescence microscopy. A long-term incubation with phorbol-12-myristate-13-acetate (PMA), a protein kinase C activator, in contrast, released fodrin from the lateral walls of the MDCK cells, leading to a diffuse cytoplasmic distribution. TFP, along with PMA, accelerated destabilization of the fodrin skeleton. Treatment with TFP alone rapidly released the cells from the substratum, which, however, could be prevented by PMA. We have previously shown that lowering of intracellular pH (< 6.5) leads to a removal of fodrin from its basolateral residence (Eskelinen et al., 1992) and that this translocation is reversed upon returning normal pH. We now show that the rebuilding of the membrane skeleton can be prevented if TFP is added to the acidified cells. Moreover, in TFP-treated acidified cells, fodrin shows a clusterlike organization similar to that observed in resting lymphocytes. We also noticed that interconversions between these different organizational states of fodrin are independent of the intracellular calcium concentration. Thus manipulation of the intracellular pH and treatment with TFP and PMA reveals different organizational states of the fodrin skeleton. This suggests that fodrin may participate in PMA-, TFP- and pH-sensitive signal transduction pathways.     

Plasmodium falciparum histidine-rich protein 1 associates with the band 3 binding domain of ankyrin in the infected red cell membrane

Infection of erythrocytes by the malaria parasite Plasmodium falciparum results in the export of several parasite proteins into the erythrocyte cytoplasm. Changes occur in the infected erythrocyte due to altered phosphorylation of proteins and to novel interactions between host and parasite proteins, particularly at the membrane skeleton. In erythrocytes, the spectrin based red cell membrane skeleton is linked to the erythrocyte plasma membrane through interactions of ankyrin with spectrin and band 3. Here we report an association between the P. falciparum histidine-rich protein (PfHRP1) and phosphorylated proteolytic fragments of red cell ankyrin. Immunochemical, biochemical and biophysical studies indicate that the 89 kDa band 3 binding domain and the 62 kDa spectrin-binding domain of ankyrin are co-precipitated by mAb 89 against PfHRP1, and that native and recombinant ankyrin fragments bind to the 5' repeat region of PfHRP1. PfHRP1 is responsible for anchoring the parasite cytoadherence ligand to the erythrocyte membrane skeleton, and this additional interaction with ankyrin would strengthen the ability of PfEMP1 to resist shear stress.     

A Nup133-dependent NPC-anchored network tethers centrosomes to the nuclear envelope in prophase

Maintenance of stable E-cadherin-dependent adhesion is essential for epithelial function. The small GTPase Rac is activated by initial cadherin clustering, but the precise mechanisms underlying Rac-dependent junction stabilization are not well understood. Ajuba, a LIM domain protein, colocalizes with cadherins, yet Ajuba function at junctions is unknown. We show that, in Ajuba-depleted cells, Rac activation and actin accumulation at cadherin receptors was impaired, and junctions did not sustain mechanical stress. The Rac effector PAK1 was also transiently activated upon cell-cell adhesion and directly phosphorylated Ajuba (Thr172). Interestingly, similar to Ajuba depletion, blocking PAK1 activation perturbed junction maintenance and actin recruitment. Expression of phosphomimetic Ajuba rescued the effects of PAK1 inhibition. Ajuba bound directly to Rac·GDP or Rac·GTP, but phosphorylated Ajuba interacted preferentially with active Rac. Rather than facilitating Rac recruitment to junctions, Ajuba modulated Rac dynamics at contacts depending on its phosphorylation status. Thus, a Rac-PAK1-Ajuba feedback loop integrates spatiotemporal signaling with actin remodeling at cell-cell contacts and stabilizes preassembled cadherin complexes.     

The role of the beta1 subunit of the Na,K-ATPase and its glycosylation in cell-cell adhesion

Based on recent data showing that overexpression of the Na,K-ATPase beta(1) subunit increased cell-cell adhesion of nonpolarized cells, we hypothesized that the beta(1) subunit can also be involved in the formation of cell-cell contacts in highly polarized epithelial cells. In support of this hypothesis, in Madin-Darby canine kidney (MDCK) cells, the Na,K-ATPase alpha(1) and beta(1) subunits were detected as precisely co-localized with adherens junctions in all stages of the monolayer formation starting from the initiation of cell-cell contact. The Na,K-ATPase and adherens junction protein, beta-catenin, stayed partially co-localized even after their internalization upon disruption of intercellular contacts by Ca(2+) depletion of the medium. The Na,K-ATPase subunits remained co-localized with the adherens junctions after detergent treatment of the cells. In contrast, the heterodimer formed by expressed unglycosylated Na,K-ATPase beta(1) subunit and the endogenous alpha(1) subunit was easily dissociated from the adherens junctions and cytoskeleton by the detergent extraction. The MDCK cell line in which half of the endogenous beta(1) subunits in the lateral membrane were substituted by unglycosylated beta(1) subunits displayed a decreased ability to form cell-to-cell contacts. Incubation of surface-attached MDCK cells with an antibody against the extracellular domain of the Na,K-ATPase beta(1) subunit specifically inhibited cell-cell contact formation. We conclude that the Na,K-ATPase beta(1) subunit is involved in the process of intercellular adhesion and is necessary for association of the heterodimeric Na,K-ATPase with the adherens junctions. Further, normal glycosylation of the Na,K-ATPase beta(1) subunit is essential for the stable association of the pump with the adherens junctions and plays an important role in cell-cell contact formation.     

A single point mutation in the V3 region affects protein kinase Calpha targeting and accumulation at cell-cell contacts

Given the importance of intercellular adhesion for many regulatory processes, we have investigated the control of protein kinase Calpha (PKCalpha) targeting to the cell-cell contacts. We have previously shown that, upon treatment of the pituitary cell line GH3B6 with thyrotropin-releasing hormone (TRH) or phorbol 12-myristate 13-acetate (PMA), human PKCalpha (hPKCalpha) is selectively targeted to the cell-cell contacts (42). Here we show that the D294G mutation of hPKCalpha, previously identified in a subpopulation of human tumors, induces the loss of this selective targeting. The D294G mutant is instead targeted to the entire plasma membrane, including the cell-cell contacts, and the duration of the first rapid and transient translocation induced by TRH (42) is longer than that of the wild-type enzyme (93.3 versus 22.5 s), coinciding with the duration of the [Ca(2+)](i) increase. We found that in the presence or absence of PMA, RACK1 is never localized at the cell-cell contacts nor was it coimmunoprecipitated with hPKCalpha wild type or the D294G mutant. In contrast, PMA treatment or long-term TRH stimulation resulted in the presence of F-actin and beta-catenin at the cell-cell contacts and their exclusion from the rest of the plasma membrane. Upon disruption of the F-actin network with phalloidin or cytochalasin D, wild-type hPKCalpha translocates but did not accumulate at the plasma membrane and beta-catenin did not accumulate at the cell-cell contacts. In contrast, the disruption of the F-actin network affected neither translocation nor accumulation of the D294G mutant. These results show that the presence of PKCalpha at the cell-cell contacts is a regulated process which depends upon the integrity of both PKCalpha and the actin microfilament network.     

Integral protein linkage and the bilayer-skeletal separation energy in red blood cells

Stabilization of the lipid bilayer membrane in red blood cells by its association with an underlying membrane-associated cytoskeleton has long been recognized as critical for proper red blood cell function. One of the principal connections between skeleton and bilayer is via linkages between band 3, the integral membrane protein that transports anions across the cell surface, and membrane skeletal elements including ankyrin, adducin, spectrin, and the junctional complex of the skeleton. Here, we use membrane tether formation coupled with fluorescent labeling of membrane components to examine the importance of band 3 in stabilizing the bilayer-skeletal association. In membranes from a patient deficient in band 3, the energy associated with the bilayer skeleton is approximately zero, whereas when band 3 is immobilized by ligation with the monoclonal antibody R10, the energy of association approximately doubles. Fluorescence images of tethers reveal that ∼40% of the band 3 on the normal cell surface can be pulled into the tether, confirming a lateral segregation of membrane components during tether formation. These results validate a critical role for band 3 in stabilizing the bilayer-skeletal association in red cells.     

α-Adducin translocates to the nucleus upon loss of cell-cell adhesions

The F-actin binding protein adducin plays an important role in plasma membrane stability, cell motility and cell-cell junctions. In this study, we demonstrate that α-adducin is mainly localized in the nucleus of sparsely cultured epithelial cells, whereas it is localized at cell-cell junctions when the cells are grown to confluence. Disruption of cell-cell adhesions induces a nuclear translocation of α-adducin. Conversely, α-adducin is redistributed to the cytoplasm and cell-cell junctions in the process of establishing cell-cell adhesions. We identify that α-adducin contains a bipartite nuclear localization signal (NLS) in its COOH-terminal tail domain and a nuclear export signal in its neck region. The phosphorylation of α-adducin at Ser716 that is immediately adjacent to the NLS appears to antagonize the function of the NLS. Moreover, we show that depletion of α-adducin has adverse effects on cell-cell adhesions and, to our surprise, cell proliferation. The impaired cell proliferation is associated with mitotic defects characterized by disorganized mitotic spindles, aberrant chromosomal congregation/segregation and abnormal centrosomes. Taken together, our results not only reveal the mechanism for α-adducin to shuttle between the cytoplasm and nucleus, but also highlight a potential role for α-adducin in mitosis.