Immunofluorescent patterns of spectrin in lymphocyte cell lines

Spectrin, a membrane-associated cytoskeletal protein, has been observed in all of 45 lymphoid and myeloid cell lines examined. For these experiments, formalin-fixed cells from randomly selected lines propagated by using conventional tissue culture procedures were examined by immunofluorescence, using an antibody directed against chicken erythrocyte alpha-spectrin. Two distinct immunofluorescent patterns of spectrin distribution were identified. In most lines examined (16 mouse and 18 human lymphoid or myeloid lines), spectrin was symmetrically distributed near the submembranous region of the plasma membrane. In the remainder of the cell lines examined, a second pattern was observed; in these cultures, the cells contain a polar submembranous aggregate of spectrin with little staining at the rest of the plasma membrane. Long-term T lymphocyte cell lines in which greater than 60% of the cells expressed a polar submembranous aggregate of spectrin (PSA-S) include mouse cell lines EL-4, LBRM-33, CT-6X, NIXT, 22CM-37, and 7ON-2 and human lines JM and PEER. Other established cultures in which PSA-S were observed included the human macrophage-like line U-937 and gibbon T cell line MLA-144. Phorbol myristate acetate or mezerin caused a reversible alteration in the distribution of spectrin in these cell lines. These drugs, which increase membrane fluidity, caused a complete but temporary symmetrical redistribution of the spectrin aggregate. Our results indicate that the pattern of spectrin distribution, either aggregated or evenly dispersed, is a stable characteristic (but one that can be altered) in various cell lines, and that because similar variations in pattern have been noted in situ, it is likely that the pattern present in any given cell line reflects a characteristic associated with a particular stage of a cell's maturation. It is anticipated that these cell lines, positive and negative for the expression of natural polarity of spectrin distribution, will provide useful models for future studies to define further the role of spectrin in lymphocyte plasma membrane functions.     

Dynamic properties of ankyrin in T lymphocytes: colocalization with spectrin and protein kinase C beta

Ankyrin is a well characterized membrane skeletal protein which has been implicated in the anchorage of specific integral membrane proteins to the spectrin-based membrane skeleton in a number of systems. In this study, the organization of ankyrin was examined in lymphocytes in relation to T cell function. Light and electron microscope immunolocalization studies revealed extensive heterogeneity in the subcellular distribution of ankyrin in murine tissue-derived lymphocytes. While ankyrin can be localized at the lymphocyte plasma membrane, it can also be accumulated at some distance from the cell periphery, in small patches or in a single discrete, nonmembrane-bound structure. Double immunofluorescence studies demonstrated that ankyrin colocalizes with spectrin and with the signal transducing molecule protein kinase C beta (PKC beta) in tissue-derived lymphocytes, suggesting a functional association between these molecules in the lymphocyte cytoplasm. In addition, T lymphocyte activation-related signals and phorbol ester treatment, both of which lead to PKC activation, cause a rapid translocation of ankyrin, together with spectrin and PKC beta, to a single Triton X-100-insoluble aggregate in the cytoplasm. This finding suggests a mechanism for the reported appearance of PKC in the particulate fraction of cells after activation: activated lymphocyte PKC beta may interact with insoluble cytoskeletal elements like ankyrin and spectrin. Further evidence for a link between the subcellular organization of these proteins and PKC activity is provided by the observation that inhibitors of PKC activity cause their concomitant redistribution to the cell periphery. The dynamic nature of lymphocyte ankyrin and its ability to accumulate at sites distant from the plasma membrane are properties which may be unique to the lymphocyte form of the molecule. Its colocalization with PKC beta in the lymphocyte cytoplasm, together with its redistribution in response to physiological signals, suggests that structural protein(s) may play a role in signal transduction pathways in this cell type. Our data support the conclusion that ankyrin is not solely involved in anchorage of proteins at the plasma membrane in lymphoid cells.     

Effects of hyperthermia on spectrin expression patterns of murine lymphocytes

In this study the influence of whole-body hyperthermia on the distribution of spectrin in murine lymphocytes isolated from various lymphoid tissues is examined. Lymphocytes normally vary in terms of the pattern of spectrin distribution within the cell. In certain populations of lymphocytes, spectrin is distributed into a dense submembranous aggregate that can be easily identified by immunofluorescence microscopy. In these lymphocytes, little or no spectrin is seen at the plasma membrane region in the rest of the cell. Other lymphocytes have no such cytoplasmic aggregates, and the protein is seen at the region of the plasma membrane. Following whole-body hyperthermia (40.5 degrees C for 90 min) there is a 100% increase in cells exhibiting polar spectrin aggregates in the spleen, while lymphocytes from the thymus show no alteration in the number of cells showing such aggregates. The increase in the percentage of splenic cells that express aggregated spectrin is a result of increases occurring in both T- and B-cell subsets. This increase gradually returns to control levels by 48 h post-heating. During recovery to control levels this phenomenon is resistant to additional changes when a second heat treatment is applied. The effects described above are not observed when the experiments are performed in vitro; therefore, it is likely that the in vivo heat-induced alteration in the splenic lymphocyte population reflects the physiological response of lymphocytes to stimuli during a natural fever. The role that spectrin may play in the modulation of lymphocyte membrane properties is discussed.     

HSP70 Translocates into a cytoplasmic aggregate during lymphocyte activation

The percentage of T and B lymphocytes expressing a distinct cytoplasmic aggregate enriched in spectrin, ankyrin, and in several other proteins including protein kinase C greatly increases following various activation protocols. Members of the 70 kDa family of heat shock proteins (hsp70) temporarily bind to and stabilize unfolded segments of other proteins, a function apparently required for proper protein folding and assembly. Considering the multiprotein and dynamic nature of the lymphocyte aggregate, the possibility that hsp70 also might be associated with componets of this structure is considered here. Double immunofluorescence analysis indicates that hsp70 is a component of the lymphocyte aggregate and is coincident with spectrin in a subpopulation of freshly isolated, untreated lymphocytes from various murine tissues and in a T-lymphocyte hybridoma. When cell lysates of lymph node T cells are immunoprecipitated using an antibody against hsp70 or spectrin and then analyzed by Western blot utilizing the alternate antibody, it was found that hsp70 and spectrin coprecipitated with one another. Moreover, this coprecipitation could be abolished by addition of ATP. This latter observation was extended to lymphoid cells using a transient permeabilization procedure, and it was shown that addition of exogenous ATP results in the dissipation of the aggregate structure itself. Finally, conditions that result in T-cell activation and aggregate formation, i.e., treatment with the phorbol ester PMA or T-cell receptor cross-linking, also lead to the repositioning of hsp70 into the aggregate from a membrane/cytosolic locale in congruence with spectrin. These data suggest that hsp70 is an active component of the aggregate and that it may function in the interactions believed to occur in this unique activation-associated organelle. © 1995 Wiley-Liss, Inc.     

Relationship between membrane lipid mobility and spectrin distribution in lymphocytes.

We have previously established that T and B lymphocytes in situ are remarkably heterogeneous with respect to the cytoskeletal protein spectrin. Since in erythrocytes spectrin is known to play an important role in the regulation of membrane fluidity, lipid organization and lateral mobility of membrane proteins, we have sought to determine if the heterogeneous patterns of spectrin distribution that we have observed are related to possible differences in membrane lipid organization in these various subsets. To this end, we have utilized a fluorescent pyrene-labelled phospholipid as a probe of the lipid lateral mobility and have examined two related T cell systems maintained in vitro, DO.11.10 cells and a spontaneously arising variant, DO.11.10V. In these (and other cloned in vitro systems) we have previously observed that the cells homogeneously express one of the kinds of spectrin distribution patterns observed in situ. Thus the uniformity of staining of these systems permits us to address whether the various patterns of spectrin distribution may be predictive of differences in membrane lipid properties. Here we show that in cells in which there is little or nor spectrin at the plasma membrane (DO.11.10) that the lipids in the plasma membrane are considerably less mobile than in its related variant in which spectrin is diffusely distributed within the cell and at the plasma membrane. From this and previous results, we conclude that differences in the distribution of the cytoskeletal protein spectrin among lymphocytes may be a useful parameter in helping to predict the status of membrane lipid organization.     

Relation between the organization of spectrin and of membrane lipids in lymphocytes

In lymphocytes, the cytoskeletal protein spectrin exhibits two organizational states. Because the plasma membrane lipids of lymphocytes also display two organizational states, it was asked whether there is a relation between the organization of spectrin and of membrane lipids. When mouse thymocytes were stained with merocyanine 540 (MC540), a fluorescent lipophilic probe that binds preferentially to loosely packed, disorganized lipid bilayers, some cells fluoresced brightly and some only dimly or not at all. When the same population was stained for spectrin by indirect immunofluorescence, the spectrin in some cells was uniformly distributed, while in others it was concentrated in a unipolar aggregate. Techniques enriching for mature thymocytes selected for cells displaying low MC540 fluorescence and aggregated spectrin, the same characteristics found in peripheral blood lymphocytes. Flow cytometric sorting of thymocytes based on MC540 phenotype simultaneously sorted them by spectrin phenotype. Finally, treatment with agents that alter the distribution of spectrin caused mature lymphocytes to display high MC540 fluorescence and uniform spectrin. Thus, a relation exists between the organizational states of spectrin and of membrane lipids in lymphocytes: aggregated spectrin is found in cells with tightly organized membrane lipids, uniform spectrin in those with loosely organized lipids. Spectrin may thus be involved in modulating membrane lipid organization in lymphocytes as it is in erythrocytes. Since loosely organized lipids may promote adhesion of blood cells to reticuloendothelial cells, spectrin may thereby be involved in transducing an internally generated adhesion signal to the lymphocyte surface.     

Distribution of HSP70, protein kinase C,and spectrin is altered in lymphocytes during a fever-like hyperthermia exposure

Many B and T lymphocytes display a significant heterogeneity with respect to the subcellular distribution of the cytoskeletal protein spectrin and protein kinase C (PKC), both of which often can be found in a large cytoplasmic aggregate in these cell types. In addition to spectrin and PKC, we recently have reported that HSP70 is also a component of this lymphocyte aggregate. Moreover, these three proteins can undergo dynamic and reversible changes in their localization causing “assembly” of the aggregate in response to various conditions associated with lymphocyte activation, indicating that this naturally occurring aggregate structure is sensitive to activation status. We show here that the same changes in HSP70/spectrin/PKC localization induced by PKC activation also can be caused, in vitro and in vivo, by a mild hyperthermia exposure, as occurs during a natural fever (39.5–40°C, 2–12 hr). This mild heat exposure also triggers the activation of PKC, a major heat shock response, and lymphocyte proliferation. The increase in PKC activity, HSP70-spectrin-PKC aggregate formation, and heat shock protein expression resulting from exposure to fever-like hyperthermia are all inhibited by calphostin C, a specific inhibitor of PKC. These data demonstrate that changes observed during lymphocyte activation could be induced by a mild hyperthermia exposure occurring during a normal febrile episode. J. Cell. Physiol. 172:44–54, 1997. © 1997 Wiley-Liss, Inc.     

Drosophila spectrin: the membrane skeleton during embryogenesis

The distribution of alpha-spectrin in Drosophila embryos was determined by immunofluorescence using affinity-purified polyclonal or monoclonal antibodies. During early development, spectrin is concentrated near the inner surface of the plasma membrane, in cytoplasmic islands around the syncytial nuclei, and, at lower concentrations, throughout the remainder of the cytoplasm of preblastoderm embryos. As embryogenesis proceeds, the distribution of spectrin shifts with the migrating nuclei toward the embryo surface so that, by nuclear cycle 9, a larger proportion of the spectrin is concentrated near the plasma membrane. During nuclear cycles 9 and 10, as the nuclei reach the cell surface, the plasma membrane-associated spectrin becomes concentrated into caps above the somatic nuclei. Concurrent with the mitotic events of the syncytial blastoderm period, the spectrin caps elongate at interphase and prophase, and divide as metaphase and anaphase progress. During cellularization, the regions of spectrin concentration appear to shift: spectrin increases near the growing furrow canal and concomitantly increases at the embryo surface. In the final phase of furrow growth, the shift in spectrin concentration is reversed: spectrin decreases near the furrow canal and concomitantly increases at the embryo surface. In gastrulae, spectrin accumulates near the embryo surface, especially at the forming amnioproctodeal invagination and cephalic furrow. During the germband elongation stage, the total amount of spectrin in the embryo increases significantly and becomes uniformly distributed at the plasma membrane of almost all cell types. The highest levels of spectrin are in the respiratory tract cells; the lowest levels are in parts of the forming gut. The spatial and temporal changes in spectrin localization suggest that this protein plays a role in stabilizing rather than initiating changes in structural organization in the embryo.     

Subcellular localization of sea urchin egg spectrin: evidence for assembly of the membrane-skeleton on unique classes of vesicles in eggs and embryos

A recent study from our laboratory on the sea urchin egg suggested that spectrin was not solely restricted to the plasma membrane, but instead had a more widespread distribution on the surface of a variety of membranous inclusions. (E. M. Bonder et al., 1989, Dev. Biol. 134, 327-341). In this report we extend our initial findings and provide experimental and ultrastructural evidence for the presence of spectrin on three distinct classes of cytoplasmic vesicles. Immunoblot analysis of membrane fractions prepared from egg homogenates establishes that spectrin coisolates with vesicle-enriched fractions, while indirect immunofluorescence microscopy on cryosections of centrifugally stratified eggs demonstrates that spectrin specifically associates with cortical granules, acidic vesicles, and yolk platelets in vivo. Immunogold ultrastructural localization of spectrin on cortices isolated from eggs and early embryos details the striking distribution of spectrin on the cytoplasmic surface of the plasma membrane and the membranes of cortical granules, acidic vesicles, and yolk platelets, while quantitative studies show that relatively equivalent amounts of spectrin are present on the different membrane surfaces both before and after fertilization. These data, in combination with the localization of numerous spectrin crosslinks between actin filaments in surface microvilli, suggest that spectrin plays a pivotal role in structuring the cortical membrane-cytoskeletal complex of the egg and the embryo.     

alpha-Spectrin has a stage-specific asymmetrical localization during Xenopus oogenesis

Xenopus oocyte organization largely depends upon the cytoskeleton distribution, which is dynamically regulated during oogenesis. An actin-based cytoskeleton is present in the cortex starting from stage 1. At stages 4-6, a complex and polarized cytoskeleton network forms in the cytoplasm. In this paper, we studied the distribution of spectrin, a molecule that has binding sites for several cytoskeletal proteins and is responsible for the determination of regionalized membrane territories. The localization of alpha-spectrin mRNA was analyzed during Xenopus oogenesis by in situ hybridization on both whole mount and sections, utilizing a cDNA probe encoding a portion of Xenopus alpha-spectrin. Furthermore, an antibody against mammalian alpha-spectrin was used to localize the protein. Our results showed a stage-dependent mRNA localization and suggested that spectrin may participate in the formation of specific domains in oocytes at stages 1 and 2 and 4-6. Mol. Reprod. Dev. 55:229-239, 2000.     

Drosophila development requires spectrin network formation

The head-end associations of spectrin give rise to tetramers and make it possible for the molecule to form networks. We analyzed the head-end associations of Drosophila spectrin in vitro and in vivo. Immunoprecipitation assays using protein fragments synthesized in vitro from recombinant DNA showed that interchain binding at the head end was mediated by segment 0-1 of alpha-spectrin and segment 18 of beta- spectrin. Point mutations equivalent to erythroid spectrin mutations that are responsible for human hemolytic anemias diminished Drosophila spectrin head-end interchain binding in vitro. To test the in vivo consequence of deficient head-end interchain binding, we introduced constructs expressing head-end interchain binding mutant alpha-spectrin into the Drosophila genome and tested for rescue of an alpha-spectrin null mutation. An alpha-spectrin minigene lacking the codons for head- end interchain binding failed to rescue the lethality of the null mutant, whereas a minigene with a point mutation in these codons overcame the lethality of the null mutant in a temperature-dependent manner. The rescued flies were viable and fertile at 25 degrees C, but they became sterile because of defects in oogenesis when shifted to 29 degrees C. At 29 degrees C, egg chamber tissue disruption and cell shape changes were evident, even though the mutant spectrin remained stably associated with cell membranes. Our results show that spectrin's capacity to form a network is a crucial aspect of its function in nonerythroid cells.     

Alteration of alpha-spectrin ubiquitination due to age-dependent changes in the erythrocyte membrane.

Mammalian red blood cell alpha-spectrin is ubiquitinated in vitro and in vivo [Corsi, D., Galluzzi, L., Crinelli, R., Magnani, M. (1995) J. Biol. Chem. 270, 8928-8935]. This process shows a cell age-dependent decrease, with senescent red blood cells having approximately one third of the amount of ubiquitinated alpha-spectrin found in young cells. In-vitro ubiquitination of alpha-spectrin was dependent on the source of the red cell membranes (those from older cells are less susceptible to ubiquitination than those from younger cells), on the source of ubiquitin-conjugating enzymes (those from older cells catalyze the process at a reduced rate compared to those from younger cells) and on the ubiquitin isopeptidase activity (which decreases during red cell ageing). However, once alpha-spectrin has been extracted from the membranes of young or old red blood cells, it is susceptible to ubiquitination to a similar extent regardless of source. This suggests that it is the membrane architecture, and not spectrin itself, that is responsible for the age-dependent decline in ubiquitination. Furthermore, spectrin oligomers, tetramers and dimers are also equally susceptible to ubiquitination. As spectrin ubiquitination occurs on domains alphaIII and alphaV of alpha-spectrin, and domain alphaV contains the nucleation site for the association of the alpha- and beta-spectrin chains, alterations in ubiquitination during red cell ageing could affect the stability and deformability of the erythrocyte membrane.     

Ubiquitination of spectrin regulates the erythrocyte spectrin-protein-4.1-actin ternary complex dissociation: implications for the sickle cell membrane skeleton.

It has been demonstrated by our laboratory that the irreversibly sickled cell (ISC) spectrin-4.1-actin complex dissociates slowly as compared to ternary complexes formed out of control (AA) and reversibly sickle cell (RSCs) core skeletons. These studies indicated that the molecular basis for the inability of irreversibly sickled cells (ISCs) to change shape is a skeleton that disassembles, and therefore reassembles, very slowly. The present study is based on the following observations: a) alpha-spectrin repeats 20 and 21 contain ubiquitination sites, and b) The spectrin repeats beta-1 and beta-2 are in direct contact with spectrin repeats alpha-20 and alpha-21 during spectrin heterodimer formation, and contain the protein 4.1 binding domain. We demonstrate here that alpha-spectrin ubiquitination at repeats 20 and 21 increases the dissociation of the spectrin-protein-4.1-actin ternary complex thereby regulating protein 4.1's ability to stimulate the spectrin-actin interaction. Performing in vitro ternary complex dissociation assays with AA control and sickle cell SS spectrin (isolated from high-density sickle cells), we further demonstrate that reduced ubiquitination of alpha-spectrin is, in part, responsible for the locked membrane skeleton in sickle cell disease.     

Ubiquitination of erythrocyte spectrin regulates the dissociation of the spectrin-adducin-f-actin ternary complex in vitro.

We demonstrate that ubiquitinated red blood cell (RBC) spectrin dissociates more rapidly from the spectrin-adducin-actin ternary complex, than non-ubiquitinated spectrin. Homozygous (SS) sickle cell spectrin has substantially diminished ubiquitination of alpha-spectrin resulting in slower dissociation from the spectrin-adducin-actin ternary complex, than normal (AA) cell spectrin. These results supply a partial explanation of the slow dissociation of the irreversible sickle cell (ISC) membrane skeleton, which leads to the inability of the ISC to change shape.     

Caspase remodeling of the spectrin membrane skeleton during lens development and aging

Terminal differentiation of lens fiber cells resembles the apoptotic process in that organelles are lost, DNA is fragmented, and changes in membrane morphology occur. However, unlike classically apoptotic cells, which are disintegrated by membrane blebbing and vesiculation, aging lens fiber cells are compressed into the center of the lens, where they undergo cell-cell fusion and the formation of specialized membrane interdigitations. In classically apoptotic cells, caspase cleavage of the cytoskeletal protein alpha-spectrin to approximately 150-kDa fragments is believed to be important for membrane blebbing. We report that caspase(s) cleave alpha-spectrin to approximately 150-kDa fragments and beta-spectrin to approximately 120- and approximately 80-kDa fragments during late embryonic chick lens development. These fragments continue to accumulate with age so that in the oldest fiber cells of the adult lens, most, if not all, of the spectrin is cleaved to discrete fragments. Thus, unlike classical apoptosis, where caspase-cleaved spectrin is short lived, lens fiber cells contain spectrin fragments that appear to be stable for the lifetime of the organism. Moreover, fragmentation of spectrin results in reduced membrane association and thus may lead to permanent remodeling of the membrane skeleton. Partial and specific proteolysis of membrane skeleton components by caspases may be important for age-related membrane changes in the lens.     

The effect of free fatty acids on spectrin organization in lymphocytes.

Previous studies have shown that cis unsaturated free fatty acids (uFFAs) are able to cause alterations in the normal distribution pattern of certain cytoskeletal proteins in lymphocytes, including tubulin, actin, alpha-actinin, and myosin. The cytoskeletal protein spectrin naturally possesses a marked heterogeneity of distribution among resting T and B lymphocytes isolated from all murine lymphoid organs. In some cells, spectrin is observed in a ring-like staining pattern at the periphery of the cell, reflecting a likely association with the cell membrane; in other cells, spectrin is found within the cytoplasm as a large single aggregate or in several smaller aggregates. Addition of uFFA to freshly isolated murine lymphocytes causes disruption in the latter pattern of spectrin organization. Following short-term incubation (15 min) of tissue-derived lymphocytes (from spleen, thymus, and lymph node) and 1 microgram/mL uFFA (oleic [18:1 cis], linoleic [18:2 cis, cis], arachidonic [20:4], or elaidic [18:1 trans] acid) there is a loss of cytoplasmic aggregates of spectrin and a concomitant increase in cells in which spectrin is diffusely distributed. This effect is not seen when two saturated FFAs (sFFAs) were used. When using DO11.10 cells, a T-cell hybridoma in which nearly all cells constitutively express a cytoplasmic aggregate of spectrin, a similar effect was observed, but greater concentrations (10-20 micrograms/mL) of FFA were needed to obtain the same effect. Addition of calcium to the incubation buffer substantially blocks spectrin reorganization. In several disease states, serum levels of FFA are observed to be excessively high; our data support the hypothesis that cytoskeletal reorganization in lymphocytes may be related to the altered immune function frequently observed in these conditions.     

The 34 kd pp60src substrate is located at the inner face of the plasma membrane

The subcellular localization of the 34 kd protein substrate of the pp60src kinase was investigated by immunofluorescence microscopy and subcellular fractionation. When permeabilized fibroblasts were stained with a monoclonal anti-34 kd protein antibody, a diffuse reticular pattern was observed. The 34 kd protein was not exposed on the outside surface of the cell. Double immunofluorescence staining experiments established that the 34 kd protein distribution was similar to that of the membrane-associated protein alpha-spectrin. The 34 kd protein was found in cell sections to be concentrated along the cell edges. Taken together, these results suggested that the 34 kd pp60src substrate was associated with the inside surface of the plasma membrane. This conclusion was supported by subcellular fractionation experiments in which the 34 kd protein was observed to fractionate with the plasma membrane. These localization studies support further the hypothesis that many of the primary effects of the pp60src kinase occur at the plasma membrane.     

An SH3 binding region in the epithelial Na+ channel (alpha rENaC) mediates its localization at the apical membrane.

The amiloride-sensitive Na+ channel constitutes the rate-limiting step for Na+ transport in epithelia. Immunolocalization and electrophysiological studies have demonstrated that this channel is localized at the apical membrane of polarized epithelial cells. This localization is essential for proper channel function in Na+ transporting epithelia. In addition, the channel has been shown to associate with the cytoskeletal proteins ankyrin and alpha-spectrin in renal epithelia. However, the molecular mechanisms underlying the cytoskeletal interactions and apical membrane localization of this channel are largely unknown. In this study we show that the putative pore forming subunit of the rat epithelial (amiloride-sensitive) Na+ channel (alpha ENaC) binds to alpha-spectrin in vivo, as determined by co-immunoprecipitation. This binding is mediated by the SH3 domain of alpha-spectrin which binds to a unique proline-rich sequence within the C-terminal region of alpha rENaC. Accordingly, the C-terminal region is sufficient to mediate binding to intact alpha-spectrin from alveolar epithelial cell lysate. When microinjected into the cytoplasm of polarized primary rat alveolar epithelial cells, a recombinant fusion protein containing the C-terminal proline-rich region of alpha rENaC localized exclusively to the apical area of the plasma membrane, as determined by confocal microscopy. This localization paralleled that of alpha-spectrin. In contrast, microinjected fusion protein containing the N-terminal (control) protein of alpha rENaC remained diffuse within the cytoplasm. These results suggest that an SH3 binding region in alpha rENaC mediates the apical localization of the Na+ channel. Thus, cytoskeletal interactions via SH3 domains may provide a novel mechanism for retaining proteins in specific membranes of polarized epithelial cells.     

Rapid capping in alpha-spectrin-deficient MEL cells from mice afflicted with hereditary hemolytic anemia

A spectrin-based membrane skeleton is important for the stability and organization of the erythrocyte. To study the role of spectrin in cells that possess complex cytoskeletons, we have generated alpha-spectrin- deficient erythroleukemia cell lines from sph/sph mice. These cells contain beta-spectrin, but lack alpha-spectrin as determined by immunoblot and Northern blot analyses. The effects of alpha-spectrin deficiency are apparent in the cells' irregular shape and fragility in culture. Capping of membrane glycoproteins by fluorescent lectin or antibodies occurs more rapidly in sph/sph than in wild-type erythroleukemia cells, and the caps appear more concentrated. The data support the idea that spectrin plays an important role in organizing membrane structure and limiting the lateral mobility of integral membrane glycoproteins in cells other than mature erythrocytes.     

Biogenesis of the avian erythroid membrane skeleton: receptor-mediated assembly and stabilization of ankyrin (goblin) and spectrin

Ankyrin is an extrinsic membrane protein in human erythrocytes that links the alpha beta-spectrin-based extrinsic membrane skeleton to the membrane by binding simultaneously to the beta-spectrin subunit and to the transmembrane anion transporter. To analyse the temporal and spatial regulation of assembly of this membrane skeleton, we investigated the kinetics of synthesis and assembly of ankyrin ( goblin ) with respect to those of spectrin in chicken embryo erythroid cells. Electrophoretic analysis of Triton X-100 soluble and cytoskeletal fractions show that at steady state both ankyrin and spectrin are detected exclusively in the cytoskeleton. In contrast, continuous labeling of erythroid cells with [35S]methionine, and immunoprecipitation of ankyrin and alpha- and beta-spectrin, reveals that newly synthesized ankyrin and spectrin are partitioned into both the cytoskeletal and Triton X-100 soluble fractions. The soluble pools of ankyrin and beta-spectrin reach a plateau of labeling within 1 h, whereas the soluble pool of alpha-spectrin is substantially larger and reaches a plateau more slowly, reflecting an approximately 3:1 ratio of synthesis of alpha- to beta-spectrin. Ankyrin and beta-spectrin enter the cytoskeletal fraction within 10 min of labeling, and the amount assembled into the cytoskeletal fraction exceeds the amount present in their respective soluble pools within 1 h of labeling. Although alpha-spectrin enters the cytoskeletal fraction with similar kinetics to beta-spectrin and ankyrin, and in amounts equimolar to beta-spectrin, the amount of cytoskeletal alpha-spectrin does not exceed the amount of soluble alpha-spectrin even after 3 h of labeling. Pulse-chase labeling experiments reveal that ankyrin and alpha- and beta-spectrin assembled into the cytoskeleton exhibit no detectable turnover, whereas the Triton X-100 soluble polypeptides are rapidly catabolized, suggesting that stable assembly of the three polypeptides is dependent upon their association with their respective membrane receptor(s). The existence in the detergent-soluble compartment of newly synthesized ankyrin and alpha- and beta-spectrin that are catabolized, rather than assembled, suggests that ankyrin and spectrin are synthesized in excess of available respective membrane binding sites, and that the assembly of these polypeptides, while rapid, is not tightly coupled to their synthesis. We hypothesize that the availability of the high affinity receptor(s) localized on the membrane mediates posttranslationally the extent of assembly of the three cytoskeletal proteins in the correct stoichiometry, their stability, and their spatial localization.