Role of fibronectin in the organisation of the cytoskeleton during the spreading of rat mammary epithelial cells.

The correlation between the extracellular deposition of fibronectin and the development of the actin-containing cytoskeleton was studied during the attachment and spreading of the rat mammary epithelial cell line Rama 25. During the initial phase of cell spreading, actin is localised in peripheral microfilament bundles. As cell spreading increases, the peripheral ring is displaced towards the perinuclear region. Fibronectin, deposited beneath the basal surface, co-localises with the actin-containing peripheral ring. The peripheral ring subsequently disappears and is replaced by a system of radial microfilaments that extend from the perinuclear region to the cell periphery. At this stage, there is no correlation between the distribution of fibronectin and actin. As cells form colonies, radial microfilament bundles are replaced by peripheral microfilament bundles which do not co-localise with fibronectin. Cells at the edges of colonies extend lamellae that contain microfilament stress fibres. In these structures there is co-localisation of actin, fibronectin and the a5 beta 1-integrin fibronectin receptor.     

Cytoarchitecture of Kirsten sarcoma virus-transformed rat kidney fibroblasts: butyrate-induced reorganization within the actin microfilament network

Murine sarcoma virus-transformed rat fibroblasts (KNRK cells) undergo marked cytoarchitectural reorganization during in vitro exposure to sodium-n-butyrate (NaB) resulting in restoration of (1) a more typical fibroblastoid morphology, (2) proper cell-to-cell orientation, and (3) substratum adherence. Augmented cell spreading, involving greater than 90% of the population, was a function of culture density and time of exposure to NaB (2 mM final concentration). Induced cell spreading reflected a 2.5- to 3.0-fold increase in both total cellular actin content and deposition of actin into the detergent-resistant cytoskeleton. Cytoskeletal actin deposition in response to NaB was accompanied by the formation of occasionally dense, parallel alignments of F-actin-containing microfilaments and by a dramatic increase in the size and incidence of actin-enriched membrane ruffles. Long-term NaB-treated cells exhibited parallel orientations of microfilaments similar to those found in untransformed fibroblasts. Increased cytoskeletal actin occurred within 24 hr of NaB exposure, correlating with the initial reorganization of actin-containing microfilaments detected microscopically, and reflected concomitant 3-fold increases in cellular alpha-actinin and fibronectin content. In contrast, the amount of vimentin, tropomyosin, and tubulin in NaB-treated cells was significantly decreased. NaB-induced morphologic restructuring of sarcoma virus-transformed fibroblasts, thus, impacts on all three basic cytoskeletal systems. Selective increases, however, were evident in particular cytoskeletal proteins (actin, alpha-actinin, fibronectin) implicated in microfilament networking and cell spreading.     

Microtubule-associated Protein 2c Reorganizes Both Microtubules and Microfilaments into Distinct Cytological Structures in an Actin-binding Protein-280–deficient Melanoma Cell Line

The emergence of processes from cells often involves interactions between microtubules and microfilaments. Interactions between these two cytoskeletal systems are particularly apparent in neuronal growth cones. The juvenile isoform of the neuronal microtubule-associated protein 2 (MAP2c) is present in growth cones, where we hypothesize it mediates interactions between microfilaments and microtubules. To approach this problem in vivo, we used the human melanoma cell, M2, which lacks actin-binding protein-280 (ABP-280) and forms membrane blebs, which are not seen in wild-type or ABP-transfected cells. The microinjection of tau or mature MAP2 rescued the blebbing phenotype; MAP2c not only caused cessation of blebbing but also induced the formation of two distinct cellular structures. These were actin-rich lamellae, which often included membrane ruffles, and microtubule-bearing processes. The lamellae collapsed after treatment with cytochalasin D, and the processes retracted after treatment with colchicine. MAP2c was immunocytochemically visualized in zones of the cell that were devoid of tubulin, such as regions within the lamellae and in association with membrane ruffles. In vitro rheometry confirmed that MAP2c is an efficient actin gelation protein capable of organizing actin filaments into an isotropic array at very low concentrations; tau and mature MAP2 do not share this rheologic property. These results suggest that MAP2c engages in functionally specific interactions not only with microtubules but also with microfilaments.     

Tumor promoters induce changes in the chick embryo fibroblast cytoskeleton

We have examined the effect of the tumor promoter, 12-0-tetradecanoyl phorbol-13-acetate (TPA), on the actin-containing elements of the cytoskeleton of chick embryo fibroblasts (CEF). TPA at concentrations as low as 7.3 times 10-10M indices a reversible change in the cytoskeleton as visualized by indirect immunofluorescence using anti-actin antibodies. Cells incubated with TPA lose the ordered actin-containing structures found in normal cells and resemble Rous sarcoma virus-transformed cells in that the immunofluorescent actin pattern is diffuse. The TPA effects are both dose-and time-dependent. Analogs of TPA which are inactive as tumor promoters do not induce cytoskeletal changes at the concentrations tested, while a second tumor promoter, PDD, is also able to cause alterations in actin-containing structures. The action of TPA requires de novo synthesis of both RNA and protein. The direct cytoskeletal changes are neither plasmin-dependent nor subject to inhibition by incubating the cells with high levels of protease inhibitors during the exposure to TPA. However, plasminogen does increase the sensitivity of cells to TPA.     

Characterization of human palladin, a microfilament-associated protein

Actin-containing microfilaments control cell shape, adhesion, and contraction. In striated muscle, alpha-actinin and other Z-disk proteins coordinate the organization and functions of actin filaments. In smooth muscle and nonmuscle cells, periodic structures termed dense bodies and dense regions, respectively, are thought to serve functions analogous to Z-discs. We describe here identification and characterization of human palladin, a protein expressed mainly in smooth muscle and nonmuscle and distributed along microfilaments in a periodic manner consistent with dense regions/bodies. Palladin contains three Ig-domains most homologous to the sarcomeric Z-disk protein myotilin. The N terminus includes an FPPPP motif recognized by the Ena-Vasp homology domain 1 domain in Ena/vasodilatator-stimulated phosphoprotein (VASP)/Wiscott-Aldrich syndrome protein (WASP) protein family. Cytoskeletal proteins with FPPPP motif target Ena/VASP/WASP proteins to sites of actin modulation. We identified palladin in a yeast two-hybrid search as an ezrin-associated protein. An interaction between palladin and ezrin was further verified by affinity precipitation and blot overlay assays. The interaction was mediated by the alpha-helical domain of ezrin and by Ig-domains 2-3 of palladin. Ezrin is typically a component of the cortical cytoskeleton, but in smooth muscle cells it is localized along microfilaments. These cells express palladin abundantly and thus palladin may be involved in the microfilament localization of ezrin. Palladin expression was up-regulated in differentiating dendritic cells (DCs), coinciding with major cytoskeletal and morphological alterations. In immature DCs, palladin localized in actin-containing podosomes and in mature DCs along actin filaments. The regulated expression and localization suggest a role for palladin in the assembly of DC cytoskeleton.     

Attachment of A-431 cells on immobilized antibodies to the EGF receptor promotes cell spreading and reorganization of the microfilament system

EGF-like sequences, inherent in a number of extracellular matrix proteins, participate in cell adhesion. It is possible that interactions of these sequences with EGF receptors (EGFR) affect actin filament organization. It was shown previously [Khrebtukova et al., 1991: Exp. Cell Res. 194:48-55] that antibodies specific to EGFR induce capping of these receptors and redistribution of cytoskeletal proteins in A-431 cells. Here we report that A-431 cells attach and spread on solid substrata coated with antibodies to EGFR, even in the absence of serum. Thus, EGFR can act as an adhesion protein and promote microfilament reorganization. Binding of the cells to the EGFR-antibody resulted in the formation of a unique cell shape characterized by numerous, actin-based filopodia radiating from the cell body, but without membrane ruffles. There was also a conspicuous circular belt of actin-containing fibers inside the cell margin, and many irregular actin aggregates in the perinuclear area. The morphologies and actin distributions in A-431 cells spread on fibronectin or laminin 2/4 were very different. On fibronectin, cells had polygonal shapes with numerous stress-fibers and thick actin-containing fibers along the cell edges. On laminin-covered substrata, the cells became fusiform and acquired broad leading lamellae with ruffles. In these cells, there were also a few bundles of filaments running the whole length of the cell body, and shorter bundles extending through the leading lamellae towards the membrane ruffles in the cell edge. These effects and those seen with immobilized EGF suggest that different ligand/receptor complexes induce specific reorganizations of the microfilament system.     

A hyaluronate binding protein transiently codistributes with p21 in cultured cell lines

Hyaluronate (HA) affects the migratory and adhesive properties of cells. HABP, one of the sites which bind HA, localizes in the ruffling lamellae of normal migrating fibroblasts. Similarly, p21 in K-ras oncogene-transformed cells appears enhanced in membrane ruffles. To investigate the possibility that p21 and HABP are functionally linked, their subcellular distribution in two K-ras-transformed lines was examined by double label immunofluorescence and correlated with motility. In both lines, the majority of cells were p21^k-ras and HABP positive at 24 h after subculture. However, immunofluorescence for HABP both decreased and relocated, from ruffles and cell processes to cell bodies, with time whereas the intensity and distribution of staining for p21 remained constant. In doubly positive cells, HABP and p21 colocalized in the ruffles at 24 h, but not at 72 h after subculture. The times after subculture at which changes in the immunofluorescent pattern of HABP occurred differed with cell type and correlated with their migratory rate. Thus, the migratory rate of KNRK cells, which was less than in the K-C3H-10T1/2 cells, correlated with both an earlier decrease in HABP and an earlier loss of codistribution between HABP and p21 compared to K-C3H-10T1/2 cells. Further evidence of a functional link between HABP and p21^k-ras was suggested by the ability of hyaluronic acid, which induces ruffling in K-C3H-10T1/2 cells, to promote the coassociation of p21^k-ras and HABP. These results demonstrate a transient codistribution of p21 and HABP, in ruffles, that is possibly related to migratory activity and/or cell-surface changes following subculture.     

Differential effects of calcium deprivation on the cytoskeleton of non-tumorigenic and tumorigenic rat liver cells in culture

Normal rat liver T51B epithelial cells and Morris no. 7795 hepatoma cells growing exponentially were exposed for 24 h to standard medium containing low (0.02 mM) calcium, a concentration which drastically reduces the proliferation of normal but not tumour cells. Cell surface morphology was examined by scanning electron microscopy (SEM); and the distribution and organization of microtubules, cytokeratin and vimentin filaments, and microfilaments were analysed by indirect immunofluorescence microscopy using specific antibodies. Calcium deprivation caused the loss of intercellular cohesion in both cell types and the appearance of some microvilli and blebs, particularly on tumour cells. However, marked differential (normal vs tumour cells) effects on the organizational integrity of the cytoskeleton fibrillar network were observed. Extracellular calcium deprivation led to a particular rearrangement of microtubules, and a perinuclear accumulation of cytokeratin and vimentin filaments in normal, but not in tumour cells. A massive concentration of actin-containing microfilaments was observed in the cell periphery and blebs of hepatoma cells. In the light of the possible involvement of calcium in controlling cytoskeleton assembly, the differing cytoskeletal changes of the two cell types may be linked to their diffferent proliferative capabilities in low-calcium medium.     

The cytoskeletal and contractile apparatus of smooth muscle: contraction bands and segmentation of the contractile elements

Confocal laser scanning microscopy of isolated and antibody-labeled avian gizzard smooth muscle cells has revealed the global organization of the contractile and cytoskeletal elements. The cytoskeleton, marked by antibodies to desmin and filamin is composed of a mainly longitudinal, meandering and branched system of fibrils that contrasts with the plait-like, interdigitating arrangement of linear fibrils of the contractile apparatus, labeled with antibodies to myosin and tropomyosin. Although desmin and filamin were colocalized in the body of the cell, filamin antibodies labeled additionally the vinculin- containing surface plaques. In confocal optical sections the contractile fibrils showed a continuous label for myosin for at least 5 microns along their length: there was no obvious or regular interruption of label as might be expected for registered myosin filaments. The cytoplasmic dense bodies, labeled with antibodies to alpha-actinin exhibited a regular, diagonal arrangement in both extended cells and in cells shortened in solution to one-fifth of their extended length: after the same shortening, the fibrils of the cytoskeleton that showed colocalization with the dense bodies in extended cells became crumpled and disordered. It is concluded that the dense bodies serve as coupling elements between the cytoskeletal and contractile systems. After extraction with Triton X-100, isolated cells bound so firmly to a glass substrate that they were unable to shorten as a whole when exposed to exogenous Mg ATP. Instead, they contracted internally, producing integral of 10 regularly spaced contraction nodes along their length. On the basis of differences of actin distribution two types of nodes could be distinguished: actin-positive nodes, in which actin straddled the node, and actin-negative nodes, characterized by an actin-free center flanked by actin fringes of 4.5 microns minimum length on either side. Myosin was concentrated in the center of the node in both cases. The differences in node morphology could be correlated with different degrees of coupling of the contractile with the cytoskeletal elements, effected by a preparation-dependent variability of proteolysis of the cells. The nodes were shown to be closely related to the supercontracted cell fragments shown in the accompanying paper (Small et al., 1990) and furnished further evidence for long actin filaments in smooth muscle. Further, the segmentation of the contractile elements pointed to a hierarchial organization of the myofilaments governed by as yet undetected elements.     

Cytochalasin B binding and actin content of endoplasmic reticulum subfractions isolated from L-929 cells

Heavy rough (HR) endoplasmic reticulum (ER) membranes and a dense fraction of light rough (LR) membranes (LR I) of L-929 cells bind H-cytochalasin B extremely poorly in comparison to smooth (S) membranes and a fraction of LR membranes of low density (LR II). The LR and S subfractions of ER are apparently heterogeneous membrane populations with respect to cytochalasin B binding. The separation of proteins in HR and LR subfractions by electrophoresis followed by immunoblotting with monoclonal antibodies against actin showed that actin was not present in the former membranes while there were large amounts in the LR subfraction. It is concluded that membranes in the LR II fraction of ER are associated with actin-containing microfilaments of the cytoskeleton, but that HR membranes are not.     

Cytoskeleton as a target in menadione-induced oxidative stress in cultured mammalian cells. I. Biochemical and immunocytochemical features

Cytoskeletal abnormalities occurring during oxidative stress generated by the metabolism of the redox cycling compound 2-methyl-1,4-naphtoquinone (menadione) have been investigated in different mammalian cells in culture. Extraction of the whole cytoskeleton as well as the intermediate filament- and the microtubule-enriched fractions from menadione-treated cells revealed a marked depletion of protein sulfhydryl groups. The analysis of the whole cytoskeletal fraction by PAGE showed a menadione-dependent and thiol-sensitive oxidation of actin, leading to the formation of high-molecular-weight aggregates. In addition, the extraction of this fraction with high concentrations of KCl entailed only a partial solubilization of actin. The comparative cytochemical analysis performed on treated cells showed a menadione-dependent clustering of actin microfilaments. The metabolism of menadione induced microtubule depolymerization and inhibition of GTP-induced microtubule assembly from soluble cytosolic components. The latter phenomenon was prevented by previously treating the cytosolic fraction with thiol reductants such as dithiothreitol. Menadione increased the protein content of the intermediate-size filament fraction, partially purified by one or more cycles of disassembly/assembly, and particularly enriched in polypeptides reacting with antikeratin antibodies. Furthermore, a reversible and oxidation-dependent change of the electrophoretic mobility of some polypeptides in this fraction was detected. The immunocytochemical investigation of intermediate-size filament distribution in menadione-treated cells, however, revealed only minor modifications mainly consisting of perinuclear condensation of cytokeratin structures. These findings suggest that cytoskeletal structures (actin microfilaments, microtubules, and intermediate-size filaments) are actually significant targets in quinone-induced oxidative stress.     

PLC-gamma1 signaling pathway and villin activation are involved in actin cytoskeleton reorganization induced by Na+/Pi cotransport up-regulation

BACKGROUND: The brief incubation of opossum kidney (OK) cells with low P(i) results in Na+/P(i) cotransport up-regulation and in substantial, but transient, cytoskeletal reorganization. In this study, we examined signaling events involved in the depolymerization of microfilaments. RESULTS: Confocal laser scanning microscopy, immunoblot and immunoprecipitation experiments revealed villin co-localization with mainly actin short filaments and monomers, indicating that under the conditions used, villin acted as an actin-severing protein. Further analysis revealed that low concentrations of extracellular phosphate resulted in phospholipase Cgammal (PLC-gammal) translocation to the actin cytoskeleton, without increases in its tyrosine phosphorylation. Additionally, tyrosine phosphorylation of a portion of insoluble villin was increased; whereas, only tyrosine phosphorylated villin associated with PLC-gammal. Although, tyrosine phosphorylation of PLC-gammal was not observed during Na+/P(i) cotransport up-regulation, genistein treatment abolished the enzyme's translocation to the actin cytoskeleton, as well as its association with villin. In addition, villin was found to associate with the 85-KDa subunit (p85) of phosphatidylinositol (PI)-3 kinase, concomitant with PLC-gammal, in the cytoskeletal fraction of Na+/P(i) cotransport up-regulated cells. CONCLUSIONS: Our observations suggest a signaling mechanism linking low ambient P(i) levels to the acute up-regulation of its cotransport with sodium and the depolymerization of the subcortical actin cytoskeleton.     

An epidermal plakin that integrates actin and microtubule networks at cellular junctions

Plakins are cytoskeletal linker proteins initially thought to interact exclusively with intermediate filaments (IFs), but recently were found to associate additionally with actin and microtubule networks. Here, we report on ACF7, a mammalian orthologue of the Drosophila kakapo plakin genetically involved in epidermal-muscle adhesion and neuromuscular junctions. While ACF7/kakapo is divergent from other plakins in its IF-binding domain, it has at least one actin (K(d) = 0.35 microM) and one microtubule (K(d) approximately 6 microM) binding domain. Similar to its fly counterpart, ACF7 is expressed in the epidermis. In well spread epidermal keratinocytes, ACF7 discontinuously decorates the cytoskeleton at the cell periphery, including microtubules (MTs) and actin filaments (AFs) that are aligned in parallel converging at focal contacts. Upon calcium induction of intercellular adhesion, ACF7 and the cytoskeleton reorganize at cell-cell borders but with different kinetics from adherens junctions and desmosomes. Treatments with cytoskeletal depolymerizing drugs reveal that ACF7's cytoskeletal association is dependent upon the microtubule network, but ACF7 also appears to stabilize actin at sites where microtubules and microfilaments meet. We posit that ACF7 may function in microtubule dynamics to facilitate actin-microtubule interactions at the cell periphery and to couple the microtubule network to cellular junctions. These attributes provide a clear explanation for the kakapo mutant phenotype in flies.     

Role of the cytoskeleton in the spreading process of epithelial cells

The role of microtubules and actin filaments in spreading of the IAR-2 cells isolated from the rat liver was studied. At the glass surface in the standard medium the cells rapidly assumed a discoidal form soon after inoculation. In the colcemid-containing medium the spreading is disturbed and delayed. In the cytochalasin D-containing medium the cells form two or more long processes. The effects of these drugs are reversible. It is supposed that microtubules are essential for sending cytoplasmic processes and stabilizing those processes and lamellae which have no numerous and stable contacts with the substrate, e.g., the processes which form at the early stages of spreading or the elongated processes of polarized cells. Bundles of actin microfilaments are essential, in particular, to ensure the discoidal form of epithelial cells. Microtubules appear to prevent the actin cytoskeleton contraction.     

Cytoskeletal changes in mouse embryonic fibroblasts exposed to colcemid. Electron microscopic research on platinum replicas

Cytoskeletons of colcemid-treated mouse embryo fibroblasts were studied using platinum replica technique. In the control cells, cytoskeletal components were oriented along direction of cell polarization. Structure of the control cytoskeleton changed regularly from the cell active edge to its centre forming several zones. Distribution of microtubules by colcemid led to significant changes in the organization of actin cytoskeleton. Both orientation and zonal differentiation of cytoskeleton disappeared in colcemid-treated fibroblasts. Changes in the fine structure of microfilament sheath were most prominent. Control sheath was composed of stretched tightly packed microfilaments. Colcemid treatment transformed it into fine microfilament meshwork, normally characteristic only for ruffle zone. Alterations of the fine structure of focal contacts and ruffles were also observed in treated cells. The role of microtubules in the organization of intracellular tensions and in the distribution of sites of actin polymerization is discussed.     

IpaC induces actin polymerization and filopodia formation during Shigella entry into epithelial cells.

Shigella proteins that are targeted to host cells by a type III secretion apparatus are essential for reorganization of the cytoskeleton during cell invasion. We have developed a semi-permeabilized cell assay that tests the effects of bacterial proteins on the actin cytoskeleton. The Shigella IpaC protein was found to induce the formation of filopodial and lamellipodial extensions in these semi-permeabilized cells. Microinjection of IpaC into cells, or cellular expression of IpaC also led to the formation of filopodial structures. Monoclonal antibodies (mAbs) directed against the C-terminus of IpaC inhibited the IpaC-induced extensions, whereas an anti-N-terminal IpaC mAb stimulated extensive lamellae formation. Shigella induced foci of actin polymerization in the permeabilized cells and these were inhibited by anti-C-terminal IpaC mAbs. Consistent with a role for IpaC in Shigella-induced cytoskeletal rearrangements during entry, stable transfectants expressing IpaC challenged with Shigella showed increased bacterial internalization. IpaC-induced extensions were inhibited by a dominant-interfering form of Cdc42 or the Cdc42-binding domain of WASP, whereas a dominant-interfering form of Rac resulted in inhibition of lamellae formation. We conclude that IpaC leads to activation of Cdc42 which in turn, causes activation of Rac, both GTPases being required for Shigella entry.     

Epithelial cell shape: cadherins and small GTPases

Cadherins are cell-cell adhesion receptors that are essential for the establishment of the epithelial cell shape and maintenance of the differentiated epithelial phenotype. In order to show efficient adhesion, cadherin receptors require an association with actin filaments and the activity of RHO proteins. The RHO family of small GTPases is primarily involved in the reorganization of the cytoskeleton. In different cell types, each member of the family can induce specific types of organization of actin filaments: stress fibers (Rho), lamellae/ruffles (Rac), or filopodia (Cdc42). This review focuses on how the function of small GTPases may impinge on the regulation of cadherin-dependent adhesion. In particular, it discusses the impact that the above cytoskeletal structures induced by RHO proteins have on the development of epithelial morphology. Finally, the participation of small GTPase-interacting proteins is considered during the remodeling of cell shape that follows cell-cell contact formation.     

Transmembrane modulation of the concanavalin A inhibition of 5'-nucleotidase is not due to a direct association of the enzyme with the cytoskeleton

The inhibition of the cell surface enzyme 5'-nucleotidase by concanavalin A is being studied as a model for understanding transmembrane modulation of cell surface functions. Nucleotidase of 13762 MAT-C1 ascites rat mammary adenocarcinoma cells is inhibited by concanavalin A in a noncooperative process. When cells are treated with the cytoplasmic effectors cytochalasins, colchicine, energy poisons, calcium plus ionophore or hypotonic buffers, the concanavalin A inhibition of the enzyme becomes cooperative. 5'-Nucleotidase of isolated MAT-C1 microvilli is also inhibited by concanavalin A in a noncooperative process; however, treatment of the microvilli with the same cytoplasmic effectors does not induce cooperativity. Since previous studies in several systems have suggested an association of nucleotidase with actin-containing microfilaments or the cell cytoskeleton, one explanation for the cooperativity changes is that they result from a change in the association of the enzyme with the cytoskeleton. However, Triton X-100 extractability of nucleotidase is the same for MAT-C1 cells exhibiting cooperative or noncooperative concanavalin A inhibition. Moreover, enzyme from cells exhibiting cooperative inhibition can be extracted into the zwitterionic detergent Zwittergent in a cooperative form, while enzyme exhibiting noncooperative behavior can be extracted into Zwittergent in a noncooperative form. Gel filtration and rate-zonal sucrose density gradient centrifugation showed little discernible size or sedimentation difference between enzyme samples exhibiting noncooperative and cooperative inhibition. These results indicate that changes in the cooperativity of the concanavalin A inhibition of nucleotidase are not a result of changes in the association of the enzyme with the cytoskeleton. These studies emphasize the caution which must be exercised in interpreting the effects of cytoskeletal perturbants on cell surface functions.     

Transmembrane modulation of the concanavalin A inhibition of 5′-nucleotidase is not due to a direct association of the enzyme with the cytoskeleton

The inhibition of the cell surface enzyme 5′-nucleotidase by concanavalin A is being studied as a model for understanding transmembrane modulation of cell surface functions. Nucleotidase of 13762 MAT-C1 ascites rat mammary adenocarcinoma cells is inhibited by concanavalin A in a noncooperative process. When cells are treated with the cytoplasmic effectors cytochalasins, colchicine, energy poisons, calcium plus ionophore or hypotonic buffers, the concanavalin A inhibition of the enzyme becomes cooperative. 5′-Nucleotidase of isolated MAT-C1 microvilli is also inhibited by concanavalin A in a noncooperative process; however, treatment of the microvilli with the same cytoplasmic effectors does not induce cooperativity. Since previous studies in several systems have suggested an association of nucleotidase with actin-containing microfilaments or the cell cytoskeleton, one explanation for the cooperativity changes is that they result from a change in the association of the enzyme with the cytoskeleton. However, Triton X-100 extractability of nucleotidase is the same for MAT-C1 cells exhibiting cooperative or noncooperative concanavalin A inhibition. Moreover, enzyme from cells exhibiting cooperative inhibition can be extracted into the zwitterionic detergent Zwittergent in a cooperative form, while enzyme exhibiting noncooperative behavior can be extracted into Zwittergent in a noncooperative form. Gel filtration and rate-zonal sucrose density gradient centrifugation showed little discernible size or sedimentation difference between enzyme samples exhibiting noncooperative and cooperative inhibition. These results indicate that changes in the cooperativity of the concanavalin A inhibition of nucleotidase are not a result of changes in the association of the enzyme with the cytoskeleton. These studies emphasize the caution which must be exercised in interpreting the effects of cytoskeletal perturbants on cell surface functions.