Association of influenza virus NP and M1 proteins with cellular cytoskeletal elements in influenza virus-infected cells

We have investigated the association of the influenza virus matrix (M1) and nucleoprotein (NP) with the host cell cytoskeletal elements in influenza virus-infected MDCK and MDBK cells. At 6.5 h postinfection, the newly synthesized M1 was Triton X-100 (TX-100) extractable but became resistant to TX-100 extraction during the chase with a t1/2 of 20 min. NP, on the other hand, acquired TX-100 resistance immediately after synthesis. Significant fractions of both M1 and NP remained resistant to differential detergent (Triton X-114, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate [CHAPS], octylglucoside) extraction, suggesting that M1 and NP were interacting with the cytoskeletal elements. However, the high-molecular-weight form of the viral transmembrane protein hemagglutinin (HA), which had undergone complex glycosylation, also became resistant to TX-100 extraction but was sensitive to octylglucoside detergent extraction, indicating that HA, unlike M1 or NP, was interacting with TX-100-insoluble lipids and not with cytoskeletal elements. Morphological analysis with cytoskeletal disrupting agents demonstrated that M1 and NP were associated with microfilaments in virus-infected cells. However, M1, expressed alone in MDCK or HeLa cells from cloned cDNA or coexpressed with NP, did not become resistant to TX-100 extraction even after a long chase. NP, on the other hand, became TX-100 insoluble as in the virus-infected cells. M1 also did not acquire TX-100 insolubility in ts 56 (a temperature-sensitive mutant with a defect in NP protein)-infected cells at the nonpermissive temperature. Furthermore, early in the infectious cycle in WSN-infected cells, M1 acquired TX-100 resistance very slowly after a long chase and did not acquire TX-100 resistance at all when chased in the presence of cycloheximide. On the other hand, late in the infectious cycle, M1 acquired TX-100 resistance when chased in either the presence or absence of cycloheximide. Taken together, these results demonstrate that M1 and NP interact with host microfilaments in virus-infected cells and that M1 requires other viral proteins or subviral components (possibly viral ribonucleoprotein) for interaction with host cytoskeletal components. The implication of these results for viral morphogenesis is discussed.     

In memoriam Frank Morrell: a man ahead of his time. June 4, 1926-October 22, 1996

Membranes obtained from whole-cell lysates of Methylococcus capsulatus (Bath) were separated by Triton X-100 extraction. The resulting insoluble fraction was enriched in outer membranes as assessed by electron microscopy and by the content of beta-hydroxy palmitic acid and particulate methane monooxygenase. Major proteins with molecular masses of approximately 27, 40, 46, 59, and 66 kDa were detected by SDS-PAGE of the Triton-X-100-insoluble membranes. MopA, MopB, MopC, MopD, and MopE (Methylococcus outer membrane protein) are proposed to designate these proteins. Several of the Mop proteins exhibited heat-modifiable properties in SDS-PAGE and were influenced by the presence of 2-mercaptoethanol in the sample buffer. The 46- and 59-kDa bands migrated as a single high-molecular-mass 95-kDa oligomer under mild denaturing conditions. When reconstituted into black lipid membranes, this oligomer was shown to serve as a channel with an estimated single-channel conductance of 1.4 nS in 1 M KCl.     

The active oligomeric state of the minimalistic influenza virus M2 ion channel is a tetramer

The influenza A virus M2 integral membrane protein is an ion channel that permits protons to enter virus particles during uncoating of virions in endosomes and also modulates the pH of the trans-Golgi network in virus-infected cells. The M2 protein is a homo-oligomer of 97 residues, and analysis by chemical cross-linking and SDS/PAGE indicates M2 forms a tetramer. However, a higher order molecular form is sometimes observed and, thus, it is necessary to determine the active form of the molecule. This was done by studying the currents of oocytes that expressed mixtures of the wild-type M2 protein (epitope tagged) and the mutant protein M2-V27S, which is resistant to the inhibitor amantadine. The composition of mixed oligomers of the two proteins expressed at the plasma membrane of individual oocytes was quantified after antibody capture of the cell surface expressed molecules and it was found that the subunits mixed freely. When the ratio of wild-type to mutant protein subunits was 0. 85:0.15, the amantadine sensitivity was reduced to 50% and for a ratio of 0.71:0.29 to 20%. These results are consistent with the amantadine-resistant mutant being dominant and the oligomeric state being a tetramer.     

Two-dimensional separation of erythrocyte membrane proteins

1). Erythrocyte membrane proteins eluted with Triton X-100 or dilute EDTA have been separated two-dimensionally by isoelectric focusing in polyacrylamide gels containing 1 percent Triton X-100 plus 8 M urea, followed by electrophoresis using sodium dodecyl sulfate. Characteristic patterns, consistent among 40 healthy donors, were obtained. 2. The resulting patterns contain at least 30 components. The "spectrin" components (sodium dodecyl sulfate Bands 1 and 2) focus in the same pH range. Other membrane components giving single bands in sodium dodecyl sulfate electrophoresis appear to be heterogeneous. 3. Triton X-100, but not EDTA, extracts the principal membrane glycoproteins and the major "intrinsic" protein. Otherwise, proteins preferentially eluted by EDTA extract poorly with Triton X-100 and vice versa. 4. Membrane glycoproteins migrate anodally during electrofocusing and can be purified in a simple, one-step procedure.     

Functional diversity of GABA-activated Cl- currents in Purkinje versus granule neurons in rat cerebellar slices

M2, an integral membrane protein of influenza A virus, was purified from either influenza A virus-infected CV-1 cells or from Spodoptera frugiperda (Sf9) cells infected with a recombinant-M2 baculovirus. The purified protein, when incorporated into phospholipid bilayer membranes, produced ion-permeable channels with the following characteristics: (1) The channels appeared in bursts during which unit conductances of diverse magnitudes (25-500 pS) were observed. (2) The most probable open state was usually the lowest unit conductance (25-90 pS). (3) The channels were selective for cations; tNa = 0.75 when 150 mM NaCl bathed both sides of the membrane. (4) Amantadine reduced the probablity of opening of the high conductance state and also the conductance of the most probable state. (5) Reducing pH increased the mean current through the open channel as well as the conductance of the most probable state. (6) The sequence of selectivity for group IA monovalent cations was Rb > K > Cs approximately Na > Li. The pH activation, amantadine block and ion selectivity of the M2 protein ion channel in bilayers are consistent with those observed on expression of the M2 protein in oocytes of Xenopus laevis as well as for those predicted for the proposed role of an ion channel in the uncoating process of influenza virus. The finding that the M2 protein has intrinsic ion channel activity supports the hypothesis that it has ion channel activity in the influenza virus particle.     

Association of Engrailed homeoproteins with vesicles presenting caveolae-like properties

We report here that the homeoproteins Engrailed-1 and Engrailed-2 are present in specific non-nuclear subcellular compartments. Using electron microscopy, we observed that chick-Engrailed-2 expressed in COS-7 cells associates with membrane fractions that are characterized as caveolae. This characterization is based on morphological, biochemical and immunological criteria such as, in particular, the absence of clathrin coat and the presence of caveolin and cholera toxin-binding sites. These data are fully confirmed by subcellular fractionation experiments, which demonstrate that transfected chick-Engrailed-2 is present in low density membrane fractions that are resistant to Triton X-100, enriched in caveolin and solubilized by the addition of a cholesterol-binding detergent, a set of properties highly characteristic of caveolae. The association of Engrailed-2 with specific membrane fractions observed after transfection in COS-7 cells is also observed for endogenous Engrailed-1 and Engrailed-2 expressed at late embryonic stages in the cerebellum and posterior mesencephalon of the rodent. Indeed, the two proteins are present in membrane fractions that bear all the characteristics of microdomains or caveolae-like domains, i.e. Triton X-100 resistance, saponin solubilization, low density on sucrose gradients, enrichment in glycosphingolipid GM1, absence of transmembrane Neural Cell Adhesion Molecule, presence of the glypiated (GPI-anchored) glycoprotein F3/F11 and of the acylated growth-associated protein GAP-43. Finally we demonstrate that part of the membrane-associated Engrailed, either expressed in COS-7 cells or endogenously present in neural tissues, is not accessible to proteolytic enzymes unless the membranes have been permeabilized with detergent. This study suggests that, in addition to their well-known presence in the nucleus, Engrailed proteins are also associated with caveolae-like vesicles that are primarily transported anterogradely into the axon, and that they can get access to a compartment compatible with secretion.     

Isolation of cytochrome b556 from the membranes of Micrococcus lysodeikticus bacteria

Proteins and cytochrome b556 were solubilized from Micrococcus lysodeikticus membranes using Triton X-100 treatment. Passing of this preparation through DEAE cellulose column in the presence of Triton X-100 made possible to isolate cytochrome b556 from other membrane cytochromes and to purify it up to the content of 2.3 nmol per mg of protein. The prostetic group of cytochrome b556 is determined to be protoheme for the spectrum of alkaline pyridinehemochrome.     

Characterization of the geranylgeranyl transferase type I from Schizosaccharomyces pombe

Transport from the TGN to the basolateral surface involves a rab/N-ethylmaleimide-sensitive fusion protein (NSF)/soluble NSF attachment protein (SNAP)/SNAP receptor (SNARE) mechanism. Apical transport instead is thought to be mediated by detergent-insoluble sphingolipid-cholesterol rafts. By reducing the cholesterol level of living cells by 60-70% with lovastatin and methyl-beta-cyclodextrin, we show that the TGN-to-surface transport of the apical marker protein influenza virus hemagglutinin was slowed down, whereas the transport of the basolateral marker vesicular stomatitis virus glycoprotein as well as the ER-to-Golgi transport of both membrane proteins was not affected. Reduction of transport of hemagglutinin was accompanied by increased solubility in the detergent Triton X-100 and by significant missorting of hemagglutinin to the basolateral membrane. In addition, depletion of cellular cholesterol by lovastatin and methyl-beta-cyclodextrin led to missorting of the apical secretory glycoprotein gp-80, suggesting that gp-80 uses a raft-dependent mechanism for apical sorting. Our data provide for the first time direct evidence for the functional significance of cholesterol in the sorting of apical membrane proteins as well as of apically secreted glycoproteins.     

Studies of the cell surface of Paramecium. Ciliary membrane proteins and immobilization antigens

We have developed a procedure to isolate the ciliary membranes of Paramecium and have analysed the membrane proteins by electrophoresis on polyacrylamide gels containing either Triton X-100 or sodium dodecyl sulphate. The electrophoretic pattern on gels containing sodium dodecyl sulphate showed 12-15 minor bands of mol.wt. 25 000-150 000 and on major band of mol.wt. 200 000-300 000 that contained approximately three-quarters of the total membrane protein. 2. We present evidence that the major membrane protein is related to, but not identical with, the immobilization antigen (i-antigen), which is a large (250 000 mol.w.), soluble, surface protein of Paramecium. The similarity of the i-antigen and the major membrane protein was shown by immunodiffusion and by the electrophoretic mobilities in sodium dodecyl sulphate of these two proteins from Paramecium of serotypes A and B. The non-identity of these two proteins was shown by their different electrophoretic mobilities on Triton X-100 containing gels and their different solubilities. 3. We propose that the major membrane protein and the i-antigen have a precursor-product relationship.     

Sensory and motor neuron-derived factor is a transmembrane heregulin that is expressed on the plasma membrane with the active domain exposed to the extracellular environment

The beta-heregulin sensory and motor neuron-derived factor (SMDF) has been suggested to be an important regulator of Schwann cell development and proliferation. In the present study, human SMDF was expressed in cultured cell lines. The cells and the recombinant protein were used to examine the membrane association and biological activity of the growth factor. Transfection of cells with SMDF cDNA constructs bearing FLAG epitope tags at either the amino- or carboxyl-terminal ends of the polypeptide resulted in expression of anti-FLAG immunoreactive polypeptides of approximately 44 and 83 kDa. The 83-kDa polypeptide was the major form expressed on the cell surface, as demonstrated by sensitivity to proteolysis in intact cells and surface biotinylation. SMDF was tightly associated with membranes isolated from transfected cells but was solubilized by Triton X-100. Immunofluorescent staining and immunoprecipitation experiments using cells expressing amino- or carboxyl-terminal tagged SMDF revealed that only the carboxyl-terminal end of the protein is exposed on the cell surface. Membranes from SMDF-transfected cells stimulated tyrosine phosphorylation of the beta-heregulin receptor ErbB3 in Schwann cells. Conditioned medium from transfected cells contained a similar activity, suggesting that SMDF is subject to proteolytic release from the plasma membrane. In contrast with other beta-heregulin isoforms, SMDF failed to bind heparin. Stimulation of Schwann cell ErbB3 receptor phosphorylation by SMDF was not affected by inhibition of Schwann cell heparan sulfate proteoglycan synthesis. These results demonstrate that SMDF is a type II transmembrane protein. This orientation places the active epidermal growth factor homology domain, which is located near the carboxyl-terminal end of the polypeptide, on the cell surface, where it can function as a membrane-anchored growth factor.     

Detergent solubility defines an alternative itinerary for a subpopulation of PDGF beta receptors

Current models of platelet-derived growth factor (PDGF) beta receptor itinerary are based upon the properties of receptors recovered from nonionic detergent-solubilized cellular extracts. Comparing several commonly used cell extraction procedures, we have determined that up to 50% of immunoreactive PDGF beta receptors, reside in a Triton X-100 insoluble pool in a wide distribution of cultured cell lines, including Balb/c-3T3, NIH 3T3, and Swiss fibroblasts, primary murine and human fibroblasts, and primary human glial cells. Many properties of Triton insoluble receptors are distinct from the well-characterized PDGF beta receptors, including 1) delayed arrival of newly synthesized receptors into the Triton insoluble fraction, 2) prolonged half-life in the presence of PDGF, 3) increased abundance with increasing cell density, 4) inaccessibility to modification by extracellular compartment enzymes, 5) cofractionation with cytoskeletal proteins, and 6) a higher basal tyrosine phosphorylation state. PDGF stimulates accumulation of tyrosine phosphorylated PDGF beta receptors in the Triton X-100 insoluble fraction. Cell surface PDGF beta receptors modified by enzymatic desialylation redistribute to the insoluble fraction. These findings distinguish the itinerary of a large subpopulation of PDGF beta receptors from those characterized previously. Receptors in this fraction represent a long-lived tyrosine phosphorylated population that may effect responses for extended periods following ligand activation.     

The Vpu protein of human immunodeficiency virus type 1 forms cation-selective ion channels

Vpu is a small phosphorylated integral membrane protein encoded by the human immunodeficiency virus type 1 genome and found in the endoplasmic reticulum and Golgi membranes of infected cells. It has been linked to roles in virus particle budding and degradation of CD4 in the endoplasmic reticulum. However, the molecular mechanisms employed by Vpu in performance of these functions are unknown. Structural similarities between Vpu and the M2 protein of influenza A virus have raised the question of whether the two proteins are functionally analogous: M2 has been demonstrated to form cation-selective ion channels in phospholipid membranes. In this paper we provide evidence that Vpu, purified after expression in Escherichia coli, also forms ion channels in planar lipid bilayers. The channels are approximately five- to sixfold more permeable to sodium and potassium cations than to chloride or phosphate anions. A bacterial cross-feeding assay was used to demonstrate that Vpu can also form sodium-permeable channels in vivo in the E. coli plasma membrane.     

Identification and characterization of a novel protein (p137) which transcytoses bidirectionally in Caco-2 cells

Antisera raised against detergent-extracted membrane fractions from the human intestinal epithelial cell line Caco-2 were used to screen a human colon cDNA library in a bacteriophage expression vector. This led to the identification, molecular cloning, and sequencing of a novel plasma membrane protein (p137) which was present in approximately equal amounts on the basolateral and apical surfaces of the cell. The pattern of extraction of p137 from membranes by Triton X-114 and its release from membranes after incubation with phosphatidylinositol-specific phospholipase C were consistent with it being a glycosylphosphatidylinositol-anchored membrane protein. Using antibodies raised against bacterial fusion proteins, it was shown that p137 was present on the cell surface as a reducible homodimer of 137 kDa subunits. There was constitutive release of p137 into the culture medium as a non-reducible 280-kDa entity. Pulse-chase experiments showed that newly synthesized p137 appeared at the basolateral side of a Caco-2 cell layer before appearing at the apical domain. Domain-specific surface biotinylation of Caco-2 cells at 4 degrees C, followed by chasing at 37 degrees C, demonstrated that p137 is capable of transcytosing in both directions across Caco-2 cells. The unusual plasma membrane domain distribution of this glycosylphosphatidylinositol-linked protein and its transcytosis characteristics demonstrate the existence of a previously uncharacterized apical to basolateral transcytotic pathway in Caco-2 cells.     

The characterization of plasma membrane-bound tubulin of cauliflower using Triton X-114 fractionation

The cortical microtubules determine how cellulose microfibrils are deposited in the plant cell wall and are thus important for the control of cell expansion. To understand how microtubules can control microfibril deposition, the components that link the microtubules to the plasma membrane (PM) of plant cells must be isolated. To obtain information on the properties of the tubulin-membrane associations, cauliflower (Brassica oleracea) PM was subjected to Triton X-114 fractionation, and the distribution of alpha- and beta-tubulin was analyzed using immunoblotting. Approximately one-half of the PM-associated tubulin was solubilized by Triton X-114 and 10 to 15% of both alpha- and beta-tubulin was recovered in the detergent phase (indicative of hydrophobic properties) and 30 to 40% was recovered in the aqueous phase. The hydrophobic tubulin could be released from the membrane by high pH extraction with preserved hydrophobicity. A large part of the PM-associated tubulin was found in the Triton-insoluble fraction. When this insoluble material was extracted a second time, a substantial amount of hydrophobic tubulin was released if the salt concentration was increased, suggesting that the hydrophobic tubulin was linked to a high-salt-sensitive protein aggregate that probably includes other components of the cytoskeleton. The hydrophobicity of a fraction of PM-associated tubulin could reflect a direct or indirect interaction of this tubulin with the lipid bilayer or with an integral membrane protein and may represent the anchoring of the cortical microtubules to the PM, a key element in the regulation of cell expansion.     

Comparative study of membranes of Streptococcus faecalis and Micrococcus lysodeikticus

A comparative study of ultrastructure and IR-spectroscopy of osmotic shock membranes from cells of glycolyzing (Streptococcus faecalis) and respiring (Micrococcus lysodeikticus) bacteria, was made. The S. faecalis and M. lysodeikticus membranes differ in their cross-section. Treatment of the preliminary washed membranes of S. faecalis and M. lysodeikticus with a low ionic strength solution removes 40% and 70% of their proteins respectively, decreases the membrane cross-section but does not change their fracture faces. Pre-cooling of the membrane suspensions within the temperature range of +5 degrees-10 degrees results in the appearance of large smooth areas on S. faecalis membrane fracture faces, but does not affect the ones of M. lysodeikticus membrane. Treatment of the washed suspensions with Triton X-100 results in the appearance of drastic changes of S. faecalis membrane fracture faces and does not change the fracture faces of M. lysodeikticus membranes; treatment by the detergent does not alter the IR-spectroscopy of membranes of both bacteria. Treatment of S. faecalis and M. lysodeikticus membranes with high temperature irreversibly changes the structure of 20% and 40% of protein components respectively,, but does not affect the distribution of the subparticles on their fracture faces. It is assumed that the differences found are determined by the composition of lipid components of the membranes studied and that the amount of proteins closely bound with lipids in the membranes of S. faecalis is likely to be greater than that of M. lysodeikticus membranes.     

The isolation and partial characterization of transferrin binding components of the rabbit reticulocyte plasma membrane

An affinity chromatograpy method utilising transferrin liganded agarose has been developed for the partial purification of transferrin binding components from Triton X-100 solubilised rabbit reticulocyte plasma membranes. A protein of molecular weight 30-35 000, shown to be located at the reticulocyte extra-cellular surface by lactoperoxidase 125I labelling, was isolated by the affinity method. The protein appeared to form a dimer of molecular weight 65-70 000 in Triton X-100 solution and was shown to associate with both 125I-labelled and unlabelled rabbit transferrin to form a high molecular weight complex in the same solution. N-[14C]Ethylmaleimide appeared to disrupt this association with transferrin and inhibit the formation of the dimer in Triton X-100 by binding to the protein. The protein appeared as a broad band of molecular weight 40 000 on sodium dodecyl sulphate polyacrylamide gel electrophoresis.     

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.     

Nuf2, a spindle pole body-associated protein required for nuclear division in yeast

The NUF2 gene of the yeast Saccharomyces cerevisiae encodes an essential 53-kd protein with a high content of potential coiled-coil structure similar to myosin. Nuf2 is associated with the spindle pole body (SPB) as determined by coimmunofluorescence with known SPB proteins. Nuf2 appears to be localized to the intranuclear region and is a candidate for a protein involved in SPB separation. The nuclear association of Nuf2 can be disrupted, in part, by 1 M salt but not by the detergent Triton X-100. All Nuf2 can be removed from nuclei by 8 M urea extraction. In this regard, Nuf2 is similar to other SPB-associated proteins including Nufl/SPC110, also a coiled-coil protein. Temperature-sensitive alleles of NUF2 were generated within the coiled-coil region of Nuf2 and such NUF2 mutant cells rapidly arrest after temperature shift with a single undivided or partially divided nucleus in the bud neck, a shortened mitotic spindle and their DNA fully replicated. In sum, Nuf2 is a protein associated with the SPB that is critical for nuclear division. Anti-Nuf2 antibodies also recognize a mammalian 73-kd protein and display centrosome staining of mammalian tissue culture cells suggesting the presence of a protein with similar function.     

The cytoplasmic tail of CD44 is required for basolateral localization in epithelial MDCK cells but does not mediate association with the detergent-insoluble cytoskeleton of fibroblasts

A number of recent reports on the trafficking of receptor proteins in MDCK epithelial cells have provided evidence that delivery to the basolateral domain requires a specific targeting sequence and that deletion of this sequence results in constitutive expression on the apical surface. To date, these studies have concentrated on receptors which are competent for internalization via the clathrin coated pits. We have examined the localization of a resident plasma membrane protein by transfecting human CD44 into MDCK cells. Using human specific and cross-species reactive antibodies, we show that in MDCK cells both the endogenous and transfected wild-type CD44 are found on the basolateral surface where they are restricted to the lateral domain. Deletion of the CD44 cytoplasmic tail reduces the half life of this mutant protein and causes it to be expressed both on the apical surface and to a significant extent within the cell. We have also used biochemical and morphological analysis to investigate the interaction of CD44 with the cytoskeleton in detergent extracted cells. Strikingly different extraction results were obtained between epithelial and fibroblast cells. However, there is no difference in the Triton X-100 solubility of the transfected wild-type and tail-less CD44 in fibroblasts and both forms of the protein remain associated with the cortical cytoskeleton after Triton X-100 extraction. These results demonstrate that the sequence present in the cytoplasmic domain of CD44 responsible for its distribution in epithelial cells is functionally and spatially separate from the ability of this protein to associate with the cytoskeleton.