Cytoskeletal Extracts Research Articles

Subcellular RNA distribution and its change during human embryonic stem cell differentiation

The spatial localization of RNA within cells is closely related to its function and also involved in cell fate determination. However, the atlas of RNA distribution within cells and dynamic changes during the developmental process are largely unknown. In this study, five subcellular components, including cytoplasmic extract, membrane extract, soluble nuclear extract, chromatin-bound nuclear extract, and cytoskeletal extract, were isolated and the rules of subcellular RNA distribution in human embryonic stem cells (hESCs) and its change during hESC differentiation are summarized for the first time. The overall distribution patterns of coding and non-coding RNAs are revealed. Interestingly, some developmental genes are found to be transcribed but confined to the chromatin in undifferentiated hESC. Unexpectedly, alternative splicing and polyadenylation endow spatial heterogeneity among different isoforms of the same gene. Finally, the dynamic pattern of RNA distribution during hESC differentiation is characterized, which provides new clues for a comprehensive understanding hESC pluripotency and differentiation.

A particle-field approach bridges phase separation and collective motion in active matter

Whereas self-propelled hard discs undergo motility-induced phase separation, self-propelled rods exhibit a variety of nonequilibrium phenomena, including clustering, collective motion, and spatio-temporal chaos. In this work, we present a theoretical framework representing active particles by continuum fields. This concept combines the simplicity of alignment-based models, enabling analytical studies, and realistic models that incorporate the shape of self-propelled objects explicitly. By varying particle shape from circular to ellipsoidal, we show how nonequilibrium stresses acting among self-propelled rods destabilize motility-induced phase separation and facilitate orientational ordering, thereby connecting the realms of scalar and vectorial active matter. Though the interaction potential is strictly apolar, both, polar and nematic order may emerge and even coexist. Accordingly, the symmetry of ordered states is a dynamical property in active matter. The presented framework may represent various systems including bacterial colonies, cytoskeletal extracts, or shaken granular media.

Active processes make mixed lipid membranes either flat or crumpled

Whether live cell membranes show miscibility phase transitions (MPTs), and if so, how they fluctuate near the transitions remain outstanding unresolved issues in physics and biology alike. Motivated by these questions we construct a generic hydrodynamic theory for lipid membranes that are active, due for instance, to the molecular motors in the surrounding cytoskeleton, or active protein components in the membrane itself. We use this to uncover a direct correspondence between membrane fluctuations and MPTs. Several testable predictions are made: (i) generic active stiffening with orientational long range order (flat membrane) or softening with crumpling of the membrane, controlled by the active tension and (ii) for mixed lipid membranes, capturing the nature of putative MPTs by measuring the membrane conformation fluctuations. Possibilities of both first and second order MPTs in mixed active membranes are argued for. Near second order MPTs, active stiffening (softening) manifests as a super-stiff (super-soft) membrane. Our predictions are testable in a variety of in vitro systems, e.g. live cytoskeletal extracts deposited on liposomes and lipid membranes containing active proteins embedded in a passive fluid.

Active hydrodynamics of synchronization and ordering in moving oscillators.

The nature of emergent collective behaviors of moving interacting physical agents is a long-standing open issue in physical and biological systems alike. This calls for studies on the control of synchronization and the degree of order in a collection of diffusively moving noisy oscillators. We address this by constructing a generic hydrodynamic theory for active phase fluctuations in a collection of a large number of nearly-phase-coherent moving oscillators in two dimensions. Our theory describes the general situation where phase fluctuations and oscillator mobility mutually affect each other. We show that the interplay between the active effects and the mobility of the oscillators leads to a variety of phenomena, ranging from synchronization with long-range, nearly-long-range, and quasi-long-range orders to instabilities and desynchronization with short-range order of the oscillator phases. We highlight the complex dependences of synchronization on the active effects. These should be testable in wide-ranging systems, e.g., oscillating chemical reactions in the presence of different reaction inhibitors and facilitators, live oriented cytoskeletal extracts, and vertebrate segmentation clocks.

Activating membranes.

We present a general dynamical theory of a membrane coupled to an actin cortex containing polymerizing filaments with active stresses and currents, and demonstrate that active membrane dynamics [S. Ramaswamy etal., Phys. Rev. Lett. 84, 3494 (2000)] and spontaneous shape oscillations emerge from this description. We also consider membrane instabilities and patterns induced by the presence of filaments with polar orientational correlations in the tangent plane of the membrane. The dynamical features we predict should be seen in a variety of cellular contexts involving the dynamics of the membrane-cytoskeleton composite and cytoskeletal extracts coupled to synthetic vesicles.

Phase-Synchronized State of Oriented Active Fluids

We present a theory for self-driven fluids, such as motorized cytoskeletal extracts or microbial suspensions, that takes into account the underlying periodic duty cycle carried by the constituent active particles. We show that an orientationally ordered active fluid can undergo a transition to a state in which the particles synchronize their phases. This spontaneous breaking of time-translation invariance gives rise to flow instabilities distinct from those arising in phase-incoherent active matter. Our work is of relevance to the transport of fluids in living systems and makes predictions for concentrated active-particle suspensions and optically driven colloidal arrays.

Involvement of CaMK‐IIδ and gelsolin in Cd2+‐dependent cytoskeletal effects in mesangial cells

Cadmium is a toxic metal with pleiotropic effects on cell death and survival. The mesangial cell is particularly responsive to Cd's effects on kinase signaling pathways and cytoskeletal dynamics. Here we show that CaMK-II is a participant in the cytoskeletal effects of Cd(2+) . A major mesangial cell isoform, CaMK-IIδ, was identified in pellets of DNase I pull-downs and cytosolic immunoprecipitates of G-actin. CaMK-IIδ was also present in Triton X-100-insoluble cytoskeletal preparations and translocated to the cytoskeleton in a concentration-dependent manner in Cd-treated cells. Translocation was suppressed by KN93, an inhibitor of CaMK-II phosphorylation. In vitro actin polymerization studies indicated that recombinant CaMK-IIδ sequestered actin monomer. Cytoskeletal preparations from Cd-treated cells decrease the rate of polymerization, but KN93 co-treatment prevents this effect. Over-expressed CaMK-IIδ also translocated to the cytoskeleton upon Cd exposure, and this was prevented by KN93. Conversely, siRNA silencing of CaMK-IIδ increases the effect of cytoskeletal extracts on actin polymerization, and abrogates the effect of Cd. The actin capping and severing protein, gelsolin, translocates to the cytoskeleton in the presence of Cd(2+) , dependent upon the phosphorylation of CaMK-II, and is recovered together with actin and CaMK-IIδ in G-actin pull-downs and F-actin sedimentation. Translocation is accompanied by generation of a 50 kDa gelsolin fragment whose appearance is prevented by KN93 and CaMK-IIδ silencing. We conclude that cytoskeletal effects of Cd in mesangial cells are partially mediated by Cd-dependent activation of CaMK-IIδ, binding of CaMK-IIδ and gelsolin to actin filaments, and cleavage of gelsolin.

A drop of active matter

AbstractWe study theoretically the hydrodynamics of a fluid drop containing oriented filaments endowed with active contractile or extensile stresses and placed on a solid surface. The active stresses alter qualitatively the wetting properties of the drop, leading to new spreading laws and novel static drop shapes. Candidate systems for testing our predictions include cytoskeletal extracts with motors and ATP, suspensions of bacteria or pulsatile cells, or fluids laden with artificial self-propelled colloids.

Involvement of Gelsolin in Cadmium-Induced Disruption of the Mesangial Cell Cytoskeleton

Cadmium (Cd2+) is known to cause a selective disruption of the filamentous actin cytoskeleton in the smooth muscle-like renal mesangial cell. We examined the effect of Cd2+ on the distribution of the actin-severing protein, gelsolin. Over 8 h, CdCl2 (10 microM) caused a progressive shift of gelsolin from a diffuse perinuclear and cytoplasmic distribution to a pattern decorating F-actin filaments. Over this time filaments were decreased in number in many cells, and membrane ruffling was initiated. Western blotting and 125I-F-actin gel overlays demonstrated an increase in actin-binding gelsolin activity in the cytoskeletal fraction of cell extracts following Cd2+ treatment. In in vitro polymerization assays, gelsolin acted as a nucleating factor and increased the rate of polymerization. Cytosolic extracts also increased the polymerization rate. Addition of Cd2+ together with gelsolin further increased the rate of polymerization. Gelsolin enhanced depolymerization of purified actin, and Cd2+ partially suppressed this effect. However, cytoskeletal extracts from Cd2+-treated cells also markedly increased depolymerization, suggesting further that Cd2+ may activate cellular component(s) such as gelsolin for actin binding. We conclude that a major effect of Cd2+ on the mesangial cell cytoskeleton is manifest through activating the association of gelsolin with actin, with gelsolin's severing properties predominating under conditions found in Cd2+-treated cells.

Efficient tyrosine phosphorylation of proteins after activation of platelets with thrombin depends on intact glycoprotein Ib

The role of platelet glycoprotein Ib as a thrombin receptor has been often a subject of controversy. We have investigated the role of the thrombin receptors, GPIb and protease-activated receptor (PAR)-1. Tyrosine phosphorylation in whole platelet lysates and in cytoskeletal extracts was evaluated after activation with thrombin and with the thrombin receptor-activating peptide (TRAP). Different experimental approaches were applied including: (i) congenital deficiency of platelet GPIb (Bernard Soulier syndrome, BSS), (ii) antibody to GPIb (AP1), (iii) selective protease cleavage (metalloprotease), and (iv) antibody to (PAR)-1. After activation of control platelets with thrombin or TRAP, multiple proteins became tyrosine phosphorylated in platelet lysates and some of them associated with the cytoskeletal fraction. These effects were absent in BSS platelets. Presence of AP1 or metalloprotease treatment showed an inhibitory effect when platelets were activated with a low concentration of thrombin or TRAP. Blockade of PAR-1 with a specific antibody, SPAN 12, inhibited platelet response to both agonists. This study reinforces the hypothesis that GPIb is the high-affinity receptor for thrombin. The signaling mechanisms occurring through tyrosine phosphorylation of proteins triggered by thrombin seem to be dependent on intact GPIb. Moreover, our results indicate that both receptors, GPIb and PAR-1, are necessary to achieve a full platelet response to thrombin.

Increased Levels of IgA Antibodies against Desmin in Children with Coeliac Disease

Background: In addition to IgA anti-endomysial antibodies, IgA anti-smooth muscle antibodies have been observed in a number of patients with coeliac disease (CoD), but only limited data exist on the frequency and antigen specificity of IgA against cytoskeletal proteins in CoD. Methods: We evaluated the sera of 42 untreated CoD patients, follow-up sera of 26 CoD patients after treatment, and 116 control sera for IgA reactivity to cytoskeletal proteins from the human umbilical cord (HUC) by immunoblotting, and compared the results with anti-tissue transglutaminase IgA (anti-tTG IgA) ELISA and immunofluorescence results. Results: Serum IgA from CoD patients most frequently recognized a 57-kD antigen in HUC cytoskeletal extract, identified as desmin using mass spectrometry and internal peptide sequencing. Increased IgA reactivity to the human desmin band was detected in 22 children with untreated CoD (52.4%), in 4 treated CoD patients (15.4%), and in 12 control subjects (10.3%) (p < 0.01); similar results were obtained with anti-chicken desmin IgA ELISA. Anti-tTG IgA levels (increased in 71.4% of untreated CoD patients) correlated significantly with anti-desmin IgA levels in untreated CoD, and both autoantibody reactivities decreased in response to a gluten-free diet. Pre-adsorption experiments and affinity purification of anti-desmin IgA antibodies further confirmed that desmin is an IgA autoantigen in CoD and is recognized by antibodies which are not cross-reactive with tTG. Conclusions: Anti-desmin IgA antibodies are frequently occurring if not the predominant cytoskeletal antibodies in children with untreated CoD and could be related to the disease process in the small intestine.

The Catalog of Human Hair Keratins: II. EXPRESSION OF THE SIX TYPE II MEMBERS IN THE HAIR FOLLICLE AND THE COMBINED CATALOG OF HUMAN TYPE I AND II KERATINS

The human type II hair keratin subfamily consists of six individual members and can be divided into two groups. The group A members hHb1, hHb3, and hHb6 are structurally related, whereas group C members hHb2, hHb4, and hHb5 are rather distinct. Specific antisera against the individual hair keratins were used to establish the two-dimensional catalog of human type II hair keratins. In this catalog, hHb5 showed up as a series of isoelectric variants, well separated from a lower, more acidic, and complex protein streak containing isoelectric variants of hair keratins hHb1, hHb2, hHb3, and hHb6. Both in situ hybridization and immunohistochemistry on anagen hair follicles showed that hHb5 and hHb2 defined early stages of hair differentiation in the matrix (hHb5) and cuticle (hHb5 and hHb2), respectively. Although cuticular differentiation proceeded without the expression of further type II hair keratins, cortex cells simultaneously expressed hHb1, hHb3, and hHb6 at an advanced stage of differentiation. In contrast, hHb4, which is undetectable in hair follicle extracts and sections, could be identified as the largest and most alkaline member of this subfamily in cytoskeletal extracts of dorsal tongue. This hair keratin was localized in the posterior compartment of the tongue filiform papillae. Comparative analysis of type II with the previously published type I hair keratin expression profiles suggested specific, but more likely, random keratin-pairing principles during trichocyte differentiation. Finally, by combining the previously published type I hair keratin catalog with the type II hair keratin catalog and integrating both into the existing catalog of human epithelial keratins, we present a two-dimensional compilation of the presently known human keratins.

Phosphodiesterase 4D and Protein Kinase A Type II Constitute a Signaling Unit in the Centrosomal Area

The mediation of cAMP effects by specific pools of protein kinase A (PKA) targeted to distinct subcellular domains raises the question of how inactivation of the cAMP signal is achieved locally and whether similar targeting of phosphodiesterases (PDEs) to sites of cAMP/PKA action could be observed. Here, we demonstrate that Sertoli cells of the testis contain an insoluble PDE4D3 isoform, which is shown by immunofluorescence to target to centrosomes. Staining of PDE4D and PKA shows co-localization of PDE4D with PKA-RIIalpha and RIIbeta in the centrosomal region. Co-precipitation of RII subunits and PDE4D3 from cytoskeletal extracts indicates a physical association of the two proteins. Distribution of PDE4D overlaps with that of the centrosomal PKA-anchoring protein, AKAP450, and AKAP450, PDE4D3, and PKA-RIIalpha co-immunoprecipitate. Finally, both PDE4D3 and PKA co-precipitate with a soluble fragment of AKAP450 encompassing amino acids 1710 to 2872 when co-expressed in 293T cells. Thus, a centrosomal complex that includes PDE4D and PKA constitutes a novel signaling unit that may provide accurate spatio-temporal modulation of cAMP signals.

Identification of a Novel Taxol-Sensitive Kinase Activity Associated with the Cytoskeleton

The microtubule-targeted drug, taxol, enhances assembly of αβ tubulin dimers into microtubules. Recent work has established that taxol also elicits diverse effects on intracellular signaling. In-gel kinase assays with myelin basic protein as substrate revealed that taxol treatment significantly (P ≤ 0.05) reduced the activity of a 55 kD kinase present in cytoskeletal extracts from CV-1 cells. In vitro phosphorylation of myelin basic protein by tubulin immunoprecipitates revealed a comparable activity, consistent with the association of this kinase activity with microtubules. This novel kinase activity was detected in the cytoskeletal fraction of several other cell types including 10T12 fibroblasts and PC-3 prostate carcinoma cells, but was not detected in cytoskeletal fractions from HeLa cells. This taxol-sensitive kinase activity may participate in conveying information about taxol-induced structural changes in microtubules to changes in intracellular signaling.

Localization of gamma-tubulin in interphase and mitotic cells of a unicellular eukaryote, Giardia intestinalis

Giardia intestinalis, a bi-nucleated amitochondrial flagellate, possesses a complex cytoskeleton based on several microtubular systems (flagella, adhesive disk, median body, funis, mitotic spindles). MTOCs of the individual systems have not been fully defined. By using monoclonal antibodies against a conserved synthetic peptide from the C-terminus of human gamma-tubulin we investigated occurrence and distribution of gamma-tubulin in interphase and mitotic Giardia cells. On the immunoblots of Giardia cytoskeletal extracts the antibodies bound to a single polypeptide of approximately 50 kDa. Immunostaining of the interphase cell demonstrated gamma-tubulin as four bright spots at the basis of four out of eight flagella. Gamma-tubulin label was associated with perikinetosomal areas of the ventral and posterolateral pairs of flagella which are formed de novo during cell division. Basal body regions of the anterolateral and caudal pairs of flagella which persist during the division and are integrated into the flagellar systems of the daughter cells did not show gamma-tubulin staining. At early mitosis, gamma-tubulin spots disappeared reappearing again at late mitosis in accord with reorientation of parent flagella and reorganization of flagellar apparatus during cell division. The antibody-detectable gamma-tubulin epitope was absent at the poles of both mitotic spindles. Albendazole-treated Giardia, in which spindle assembly was completely inhibited, showed the same gamma-tubulin staining pattern thus confirming that the fluorescent label is exclusively located in the basal body regions. Our results point to a role of gamma-tubulin in nucleation of microtubules of newly formed flagella and indicate unusual mitotic spindle assembly. Moreover, the demonstration of gamma-tubulin in Giardia shows ubiquity of this protein through the evolutionary history of eukaryotes.

In vivo and in vitro phosphorylation and subcellular localization of trypanosomatid cytoskeletal giant proteins.

Promastigote forms of Phytomonas serpens, Leptomonas samueli, and Leishmania tarentolae express cytoskeletal giant proteins with apparent molecular masses of 3,500 kDa (Ps 3500), 2,500 kDa (Ls 2500), and 1,200 kDa (Lt 1200), respectively. Polyclonal antibodies to Lt 1200 and to Ps 3500 specifically recognize similar polypeptides of the same genera of parasite. In addition to reacting with giant polypeptides of the Leptomonas species, anti-Ls 2500 also cross reacts with Ps 3500, and with a 500-kDa polypeptide of Leishmania. Confocal immunofluorescence and immunogold electron microscopy showed major differences in topological distribution of these three proteins, though they partially share a common localization at the anterior end of the cell body skeleton. Furthermore, Ps 3500, Ls 2500, and Lt 1200 are in vivo phosphorylated at serine and threonine residues, whereas, in vitro phosphorylation of cytoskeletal fractions reveal that only Ps 3500 and Ls 2500 are phosphorylated. Heat treatment (100 degrees C) of high salt cytoskeletal extracts demonstrates that Ps 3500 and Ls 2500 remain stable in solution, whereas Lt 1200 is denatured. Kinase assays with immunocomplexes of heat-treated giant proteins show that only Ps 3500 and Ls 2500 are phosphorylated. These results demonstrate the existence of a novel class of megadalton phosphoproteins in promastigote forms of trypanosomatids that appear to be genera specific with distinct cytoskeletal functions. In addition, there is also evidence that Ps 3500 and Ls 2500, in contrast to Lt 1200, seem to be autophosphorylating serine and threonine protein kinases, suggesting that they might play regulatory roles in the cytoskeletal organization.

A novel healing filament in ciliate regeneration.

The interphase cells of the hypotrich ciliate Paraurostyla weissei possess a complex fibrillar system surrounding basal bodies in the compound ciliary assemblages, cirri and membranelles. During replacement of the ciliature at cell division, transient filaments precede and accompany the development of ciliary primordia and participate in the formation of the fission furrow. Both fibrillar systems are recognized by monoclonal antibody FXXXIX 12G9. We studied regeneration of cellular fragments after transection employing the mAb 12G9 and found a new cytoskeletal structure involved in healing of the excisional wound. The healing filament is formed at the wound edge, distally and in connection with the bases of cirri closest to the wound. It is visible 5 min after transection. Concomitant with development of new ciliary primordia, the healing filament shrinks and finally disappears together with other transient fibers formed in this process. Ultrastructural analysis of immunolabeled regenerating cells revealed that structures recognized by mAb 12G9 contain fine filaments whose packing and arrangement depends on accompanying cytoplasmic elements and the developmental status of a fragment. Assembly of the healing fiber does not depend on microtubules and microfilaments since it develops in cellular fragments exposed to cold, nocodazole, and Cytochalasin D. On Western blots of whole cell and cytoskeletal extracts of P. weissei the 12G9 antibody identified one protein band whose molecular weight corresponds to 60 kDa.

Cultured human sole-derived keratinocyte grafts re-express site-specific differentiation after transplantation

Cultured epithelial autografts (CEA) derived from sole skin were transplanted to full-thickness wounds excised to muscle fascia over a variety of diverse body sites in 12 pediatric patients treated for acute burns or giant congenital nevi. The skin regenerated from the grafts was biopsied from 7 days to 6 years after grafting. The resultant epidermal phenotype was analyzed histologically and by immunohistochemical localization of keratin 9 (K9) as objective evidence of sole-type site-specific differentiation. Expression of K9 was also verified by one-dimensional gel electrophoresis of epidermal cytoskeletal extracts and K9 immunoblot analysis. Grafts prepared from epidermis of axilla, groin or foreskin and transplanted to wounds of comparable depth in an identical manner in the same patients served as controls of postgrafting differentiation. Biopsies of sole skin from amputation specimens from patients of comparable age served as normal positive controls, and biopsies of nonsole skin from patients of comparable age served as normal negative controls. As early as 2 weeks postgrafting, the histologic appearance of sole-derived CEA differed substantively from that of axilla- or groin-derived CEA controls and displayed a phenotype characteristic of sole skin with a thick compact stratum corneum, a thick stratum granulosum, and a distinct stratum lucidum. In sole-derived grafts rete ridges regenerated within 2 months postgrafting, whereas nonsole-derived grafts required 4–6 months for rete ridge regeneration. Once acquired, the sole skin phenotype was maintained long-term by all sole-derived CEA. In vitro, sole-derived keratinocytes synthesized little, if any, K9. However, within 7 days after grafting, K9 synthesis by multiple suprabasal keratinocytes was seen within the epidermis regenerated from sole-derived CEA. Protein of K9 appeared progressively more diffuse throughout the suprabasal layers, attaining a confluent pattern of expression comparable to normal controls of sole skin by 6 to 12 months postgrafting, and the confluent pattern of suprabasal K9 synthesis was maintained long-term. The results demonstrate that site-specific differentiation is an intrinsic property of postnatal human keratinocytes and can be expressed and maintained in a permissive environment in the absence of dermal tissue.

A novel taxol-induced vimentin phosphorylation and stabilization revealed by studies on stable microtubules and vimentin intermediate filaments.

To understand how protein phosphorylation modulates cytoskeletal organization, we used immunofluorescence microscopy to examine the effects of okadaic acid, a serine/threonine protein phosphatase inhibitor, and taxol, a microtubule-stabilizing agent, on stable (acetylated and detyrosinated) microtubules, vimentin intermediate filaments and other cytoskeletal elements in CV-1 cells. Okadaic acid caused major changes in both stable microtubules and vimentin intermediate filaments, but through independent mechanisms. At 300 nM, okadaic acid caused apparent fragmentation and loss of stable microtubules which was not prevented by prior exposure to K252a. In contrast, major reorganization of vimentin intermediate filaments elicited at 750 nM okadaic acid was prevented by prior exposure to K252a. Taxol pretreatment blocked the effects of okadaic acid on stable microtubules and vimentin intermediate filaments. Recent reports have revealed that taxol can activate cellular signal transduction pathways in addition to its known ability to promote microtubule stabilization, so the possibility that taxol-induced resistance of vimentin intermediate filaments to okadaic acid was through a microtubule-independent mechanism involving direct phosphorylation of intermediate filament proteins was explored. Vimentin immunoprecipitation from cytoskeletal extracts from 32P-labeled cells revealed that taxol (4 microM, 1 or 2 hours) caused about a 2-fold increase in vimentin phosphorylation. This phosphorylation was recovered exclusively in cytoskeletal vimentin, in contrast to the increased phosphorylation of soluble and cytoskeletal vimentin caused by exposure to 750 nM okadaic acid. Phosphorylation of soluble and cytoskeletal vimentin from cells exposed to taxol (4 microM, 1 hour) then okadaic acid (750 nM, 1 hour) was comparable to taxol-treatment alone. These findings demonstrate a novel new activity of taxol, induction of vimentin phosphorylation, that may impact on vimentin organization and stability.

Differential inactivation of postsynaptic density-associated and soluble Ca2+/calmodulin-dependent protein kinase II by protein phosphatases 1 and 2A.

Autophosphorylation of Ca2+/calmodulin-dependent protein kinase II (CaMKII) at Thr286 generates Ca2+-independent activity. As an initial step toward understanding CaMKII inactivation, protein phosphatase classes (PP1, PP2A, PP2B, or PP2C) responsible for dephosphorylation of Thr286 in rat forebrain subcellular fractions were identified using phosphatase inhibitors/activators, by fractionation using ion exchange chromatography and by immunoblotting. PP2A-like enzymes account for >70% of activity toward exogenous soluble Thr286-autophosphorylated CaMKII in crude cytosol, membrane, and cytoskeletal extracts; PP1 and PP2C account for the remaining activity. CaMKII is present in particulate fractions, specifically associated with postsynaptic densities (PSDs); each protein phosphatase is also present in isolated PSDs, but only PP1 is enriched during PSD isolation. When isolated PSDs dephosphorylated exogenous soluble Thr286-autophosphorylated CaMKII, PP2A again made the major contribution. However, CaMKII endogenous to PSDs (32P autophosphorylated in the presence of Ca2+/calmodulin) was predominantly dephosphorylated by PP1. In addition, dephosphorylation of soluble and PSD-associated CaMKII in whole forebrain extracts was catalyzed predominantly by PP2A and PP1, respectively. Thus, soluble and PSD-associated forms of CaMKII appear to be dephosphorylated by distinct enzymes, suggesting that Ca2+-independent activity of CaMKII is differentially regulated by protein phosphatases in distinct subcellular compartments.