Triton-insoluble Cytoskeleton Research Articles

Biochemical and immunocytochemical characterization of coronins in platelets

Rapid reorganization of the actin cytoskeleton in response to receptor-mediated signaling cascades allows platelets to transition from a discoid shape to a flat spread shape upon adhesion to damaged vessel walls. Coronins are conserved regulators of the actin cytoskeleton turnover but they also participate in signaling events. To gain a better picture of their functions in platelets we have undertaken a biochemical and immunocytochemical investigation with a focus on Coro1. We found that class I coronins Coro1, 2 and 3 are abundant in human and mouse platelets whereas little Coro7 can be detected. Coro1 is mainly cytosolic, but a significant amount associates with membranes in an actin-independent manner and does not translocate from or to the membrane fraction upon exposure to thrombin, collagen or prostacyclin. Coro1 rapidly translocates to the Triton insoluble cytoskeleton upon platelet stimulation with thrombin or collagen. Coro1, 2 and 3 show a diffuse cytoplasmic localization with discontinuous accumulation at the cell cortex and actin nodules of human platelets, where all three coronins colocalize. Our data are consistent with a role of coronins as integrators of extracellular signals with actin remodeling and suggests a high extent of functional overlap among class I coronins in platelets.

Translocation of GPIb and Fc receptor γ‐chain to cytoskeleton in mucetin‐activated platelets

Recent studies have implied that GPIb-IX-V as well as functioning as an adhesion receptor may also induce signaling to mediate binding of platelets to damaged vessel wall to prevent bleeding. Reorganization of the cytoskeleton and redistribution of platelet structural proteins and signaling molecules are thought to be important in this early activation process, though the molecular mechanisms remain to be fully defined. In this study, we have used mucetin, a snake venom lectin protein that activates platelets via GPIb, to study the redistribution of GPIb in platelets. In unstimulated platelets, a minor portion of GPIb localized to Triton-insoluble cytoskeleton fractions (TIC). This portion increased considerably after platelet activation by mucetin. We also find increased contents of the FcRgamma chain in TIC. Anti-GPIb antibodies, mocarhagin or cytochalasin D completely inhibited the cytoskeletal translocation. In addition, BAPTA-AM, a cytoplasmic calcium chelator, strongly inhibited this process. On the other hand, inhibitors of alphaIIbbeta3, PLCgamma, PKC, tyrosine kinases, ADP receptor, PI3-kinase or EDTA are effective in preventing GPIb relocation in convulxin- but not in mucetin-activated platelets. We propose that cytoskeletal translocation of GPIb is upstream of alphaIIbbeta3 activation and cross-linking of GPIb is sufficient to induce this event in mucetin-activated platelets.

Villidin, a novel WD-repeat and villin-related protein from Dictyostelium, is associated with membranes and the cytoskeleton.

Villidin is a novel multidomain protein (190 kDa) from Dictyostelium amoebae containing WD repeats at its N-terminus, three PH domains in the middle of the molecule, and five gelsolin-like segments at the C-terminus, followed by a villin-like headpiece. Villidin mRNA and protein are present in low amounts during growth and early aggregation, but increase during development and reach their highest levels at the tipped mound stage. The protein is present in the cytosol as well as in the cytoskeletal and membrane fractions. GFP-tagged full-length villidin exhibits a similar distribution as native villidin, including a distinct colocalization with Golgi structures. Interestingly, GFP fusions with the gelsolin/villin-like region are uniformly dispersed in the cytoplasm, whereas GFP fusions of the N-terminal WD repeats codistribute with F-actin and are associated with the Triton-insoluble cytoskeleton. Strains lacking villidin because of targeted deletion of its gene grow normally and can develop into fruiting bodies. However, cell motility is reduced during aggregation and phototaxis is impaired in the mutant strains. We conclude that villidin harbors a major F-actin binding site in the N-terminal domain and not in the villin-like region as expected; association of villidin with vesicular membranes suggests that the protein functions as a linker between membranes and the actin cytoskeleton.

Expression of junctional proteins in human platelets

Platelets play a major role in thrombosis and hemostasis by binding the sub-endothelial matrix at sites of injury, but also participate in vascular pathologies such as atherosclerosis. Recently, junctional proteins like PECAM-1 and JAM-family members have been recovered from platelets, therefore we examined what other junctional molecules may be present in platelets. We observed immunoreactivity for APC (147 kD), β-catenin (92 kD), E-cadherin (120 and 84 kD) and occludin (70-85 kD) by western blotting. Additionally, β-catenin, pan-reactive cadherins, E-cadherin and occludin were seen to partition with the triton insoluble cytoskeleton in platelets. These proteins were also found in a megakaryocyte ( platelet precursor) line, MEG-01. Our data suggest that conventional junctional molecules are expressed in platelets and could possibly participate in aggregation, clot formation and wound healing.

Renal Alpha-Actinin-4: Purification and Puromycin Aminonucleoside-Binding Property

Mutations in the gene encoding nonmuscle alpha-actinin-4 (actinin-4), an actin cross-linking protein, lead to congenital nephrosis. This suggests that actinin-4 is an essential component of the glomerular filtration barrier. In the present study, we attempted to purify actinin-4 from the mammalian kidney. We also examined an interaction of the protein with puromycin aminonucleoside (PAN), which can induce nephrosis in animals. A 100-kD protein reactive with antibody against muscle alpha-actinin was purified from the Triton-insoluble cytoskeleton of porcine kidney, by MgCl₂ treatment, ammonium sulfate fractionation, and subsequent DEAE-cellulose chromatography and hydroxyapatite chromatography. Its partial amino acid sequence was then determined. A filamentous actin (F-actin)-binding activity of the purified protein was examined by a cosedimentation assay. Interactions of the purified protein and its fragments with PAN were analyzed by an affinity assay using PAN-Sepharose. Determined 134 amino acid sequences of the purified porcine renal 100-kD protein were completely identical with those deduced from nucleotide sequence of the cDNA encoding human actinin-4. The purified protein possessed the known function of alpha-actinin, the F-actin-binding activity, and was tightly bound to PAN. The PAN-binding site was mapped within a central rod domain of the protein, which is a possible interaction site for various cytoskeletal and transmembrane proteins. We have established an efficient purification method for renal actinin-4. Moreover, our findings indicate that the central rod domain of actinin-4 has a high affinity to PAN. In the PAN nephrosis animal model, actinin-4 might be a target protein from PAN nephrotoxicity.

Bruton’s tyrosine kinase is a substrate of calpain in human platelets

Platelet-associated Bruton’s tyrosine kinase (Btk) was completely cleaved if treated with calcium ionophore A23187 with appearance of a proteolytic product of 27 kDa size. Aggregation with thrombin also induced about 10% degradation of Btk after 30 min. Calpain inhibitors prevented Btk degradation in both. The proteolytic products of the Wiskott–Aldrich syndrome protein (WASP), a calpain and Btk substrate, and the 27 kDa degradation product of Btk did not redistribute to the Triton-insoluble cytoskeleton in thrombin-aggregated platelets, in contrast to the uncleaved proteins. The degradation of Btk and WASP was independent of their tyrosine phosphorylation status. These results indicate that Btk is an endogenous substrate for calpain, the cleavage of which may have functional consequences in long-term post-aggregation events in platelets.

Leiomodin and tropomodulin in smooth muscle

Evidence is accumulating to suggest that actin filament remodeling is critical for smooth muscle contraction, which implicates actin filament ends as important sites for regulation of contraction. Tropomodulin (Tmod) and smooth muscle leiomodin (SM-Lmod) have been found in many tissues containing smooth muscle by protein immunoblot and immunofluorescence microscopy. Both proteins cofractionate with tropomyosin in the Triton-insoluble cytoskeleton of rabbit stomach smooth muscle and are solubilized by high salt. SM-Lmod binds muscle tropomyosin, a biochemical activity characteristic of Tmod proteins. SM-Lmod staining is present along the length of actin filaments in rat intestinal smooth muscle, while Tmod stains in a punctate pattern distinct from that of actin filaments or the dense body marker alpha-actinin. After smooth muscle is hypercontracted by treatment with 10 mM Ca(2+), both SM-Lmod and Tmod are found near alpha-actinin at the periphery of actin-rich contraction bands. These data suggest that SM-Lmod is a novel component of the smooth muscle actin cytoskeleton and, furthermore, that the pointed ends of actin filaments in smooth muscle may be capped by Tmod in localized clusters.

Actomyosin cross-linking by caldesmon in non-muscle cells

The role of myosin-binding in cytoskeletal arrangement of non-muscle low molecular weight caldesmon ( l-caldesmon) was studied. The N-terminal myosin-binding domain of caldesmon (N152) colocalized with myosin in transiently transfected chicken fibroblasts. When added exogenously to the Triton-insoluble cytoskeleton, N152 enhanced l-caldesmon displacement by exogenous C-terminal actin-binding fragment (H1). Thus, a significant fraction of l-caldesmon cross-links actin and myosin. In contrast, in epithelioid HeLa cells most of l-caldesmon was only actin-bound as H1 alone was enough for its displacement. Phosphorylation by mitogen-activated protein kinase reduced the capability of H1 to displace endogenous l-caldesmon, suggesting it may represent a regulatory mechanism for actin–caldesmon interaction in vivo.

Unique sequence of a high molecular weight myosin light chain kinase is involved in interaction with actin cytoskeleton

Myosin light chain kinase (MLCK) is the key regulator of cell motility and smooth muscle contraction in higher vertebrates. We searched for the features of the high molecular weight MLCK (MLCK-210) associated with its unique N-terminal sequence not found in a more ubiquitous lower molecular weight MLCK (MLCK-108). MLCK-210 demonstrates stronger association with the Triton-insoluble cytoskeletons than MLCK-108, suggesting the role for this sequence in subcellular targeting. Indeed, the expressed unique domain of MLCK-210 binds and bundles F-actin in vitro and colocalises with the microfilaments in transfected cells reproducing endogenous MLCK-210 distribution. Thus, MLCK-210 features an extensive actin binding interface and, perhaps, acts as an actin cytoskeleton stabiliser.

N-terminally truncated Vav induces the formation of depolymerization-resistant actin filaments in NIH 3T3 cells

The Dbl family proto-oncogene vav is a guanine nucleotide exchange factor (GEF) for Rho family GTPases. Deletion of the N-terminus of Vav, harboring the single calponin homology (CH) domain, activates Vav’s transforming potential, suggesting an important role of the CH domain in influencing Vav function. Since calponin binds actin, it has been suggested that the CH domain may mediate association with the actin cytoskeleton. In this study we have analyzed the subcellular localization and investigated the putative actin association of the Vav protein using enhanced green fluorescent protein (EGFP) fusion constructs. Our data show that both EGFP-tagged full length Vav and the CH domain-depleted EGFPvav 143–845 construct localize throughout the cytoplasm but fail to colocalize with F-actin. However, the latter construct of Vav was more strongly retained in the Triton-insoluble cytoskeleton fraction than full length Vav. Whereas removal of the CH domain had no apparent influence on the subcellular localization of Vav, deletion of the SH domains caused nuclear localization, indicating that Vav contains a functional nuclear localization signal. Expression of N-terminally truncated Vav constructs caused depolarization of fibroblasts and triggered the bundling of actin stress fibers into parallel arrays in NIH 3T3 cells. Notably, the parallel actin bundles showed prolonged resistance to the actin polymerization antagonists cytochalasin B and latrunculin B. These data point towards a regulatory role for the CH domain in Vav and suggest an actin cross-linking or bundling protein as a downstream effector molecule of vav-mediated signalling pathways.

α6 Integrin Is Regulated with Lens Cell Differentiation by Linkage to the Cytoskeleton and Isoform Switching

The developing chicken embryo lens provides a unique model for examining the relationship between α6 integrin expression and cell differentiation, since multiple stages of differentiation are expressed concurrently at one stage of development. We demonstrate that α6 integrin is likely to mediate the inductive effects of laminin on lens differentiation as well as to function in a matrix-independent manner along the cell–cell interfaces of the differentiating cortical lens fiber cells. Both α6 isoform expression and its linkage to the cytoskeleton were regulated in a differentiation-specific manner. The association of α6 integrin with the Triton-insoluble cytoskeleton increased as the lens cells differentiated, reaching its highest levels in the cortical fiber region where the lens fiber cells are formed. In this region of the lens α6 integrin was uniquely localized along the cell–cell borders of the differentiating fiber cells, similar to β1. α6β4, the primary transmembrane protein of hemidesmosomes, is also expressed in the lens, but in the absence of hemidesmosomes. Differential expression of α6A and α6B isoforms with lens cell differentiation was seen at both the mRNA and the protein levels. RT-PCR studies demonstrated that α6B was the predominant isoform expressed both early in development, embryonic day 4, and in the epithelial regions of the day 10 embryonic lens. Isoform switching, with α6A now the predominant isoform, occurred in the fiber cell zones. Immunoprecipitation studies showed that α6B, which is characteristic of undifferentiated cells, was expressed by the lens epithelial cells but was dramatically reduced in the lens fiber zones. Expression of α6B began to drop as the cells initiated their differentiation and then dropped precipitously in the cortical fiber zone. In contrast, expression of the α6A isoform remained high until the cells became terminally differentiated. α6A was the predominant isoform expressed in the cortical fiber region. The down-regulation of α6B relative to α6A provides a developmental switch in the process of lens fiber cell differentiation.

A 26–30 kDa developmentally-regulated tauisoform localized within nuclei of mitotic humanneuroblastoma cells

Tau isoforms migrating at 46–68 and 97–115 kDa were prominent within heat-stableTriton-soluble material, and were present in lesser concentration within Triton-insolublecytoskeletons, derived from undifferentiated SH-SY-5Y human neuroblastoma cells. Conversely,a 26–30 kDa tau isoform was enriched in the cytoskeleton and detected at relatively minor levelswithin cytosolic fractions. Pulse labeling with 35S-methionine indicated that this 26–30kDa small tau did not represent a breakdown product of larger isoforms. Since the nucleus isretained within the Triton-insoluble cytoskeleton, additional cultures were fractionated ontosucrose to obtain purified nuclei. The vast majority of small tau was recovered within purifiednuclei. Small tau was reactive with tau antibodies directed towards N-terminal, C-terminal andcentral epitopes, further confirming that this small isoform was not derived from proteolyticcleavage of larger tau isoforms. Small tau demonstrated alkaline phosphatase-sensitive reactivitywith multiple phospho-dependent tau antibodies. Small tau was depleted within 3 days of retinoicacid-induced differentiation, suggesting that the putative function of this isoform may be obsoletefollowing terminal differentiation of neurons.

Aluminum inhibits neurofilament assembly, cytoskeletal incorporation, and axonal transport. Dynamic nature of aluminum-induced perikaryal neurofilament accumulations as revealed by subunit turnover.

The mechanism by which aluminum induces formation of perikaryal neurofilament (NF) inclusions remains unclear. Aluminum treatment inhibits: 1. The incorporation of newly synthesized NF subunits into Triton-insoluble cytoskeleton of axonal neurites; 2. Their degradation and dephosphorylation; 3. Their translocation into axonal neurites. It also fosters the accumulation of phosphorylated NFs within perikarya. In the present study, we addressed the relationship among these effects. Aluminum reduced the assembly of newly synthesized NF subunits into NFs. During examination of those subunits that did assemble in the presence of aluminum, it was revealed that aluminum also interfered with transport of newly assembled NFs into axonal neurites. Similarly, a delay in axonal transport of microinjected biotinylated NF-H was observed in aluminum-treated cells. Aluminum also inhibited the incorporation of newly synthesized and microinjected subunits into the Triton-insoluble cytoskeleton within both perikarya and neurites. Once incorporated into Triton-insoluble cytoskeletons, however, biotinylated subunits were retained within perikarya of aluminum-treated cells to a greater extent than within untreated cells. Notably, these subunits were depleted in the presence and absence of aluminum within 48 h, despite the persistence of the aluminum-induced perikaryal accumulation itself, suggesting that individual NF subunits undergo turnover even within aluminum-induced perikaryal accumulations. These findings demonstrate that aluminum interferes with multiple aspects of neurofilament dynamics and furthermore leaves open the possibility that aluminum-induced perikaryal NF whorls may not represent permanent structures, but rather may require continued recruitment of cytoskeletal constituents.

Actin Polymerisation Regulates Integrin-Mediated Adhesion as Well as Rigidity of Neutrophils

Activation of adherent neutrophils causes them to convert from selectin-mediated rolling to integrin-mediated immobilisation and migration. Migration is known to depend on formation and redistribution of filamentous (F) actin, but although immobilisation in seconds parallels early cortical actin polymerisation, no link has been proven. We tested the effect of the actin-polymerising agent jasplakinolide (10μM) on adhesive and mechanical properties of neutrophils. Pretreated cells were able to adhere and roll on immobilised platelets in a flow-based adhesion assay, but whereas untreated rolling cells became immobilised in seconds when chemotactic formyl peptide (fMLP, 0.1μM) was superfused over them, the cells treated with jasplakinolide continued rolling. Pretreatment with jasplakinolide also blockedde novoexpression of integrin CD11b and shape change which otherwise occurred in minutes after treatment with fMLP. Jasplakinolide directly caused actin polymerisation within neutrophils, evidenced by a marked increase in rigidity (resistance to aspiration into a 5μm micropipette) and increase in association of actin with the Triton-insoluble cytoskeleton. These results indicate that rearrangement of the actin cytoskeleton regulates integrin-mediated adhesion of activated neutrophils, as well as their migration and mechanical properties.

Stimulation of Intracellular Ca2+ Levels in Human Neutrophils by Soluble Immune Complexes

Soluble immune complexes bind to unprimed neutrophils and generate intracellular Ca2+ transients but fail to activate the NADPH oxidase. Following priming of the neutrophils with either tumor necrosis factor alpha or granulocyte-macrophage colony-stimulating factor, stimulation of the cells with the soluble immune complexes leads to an enhanced Ca2+ signal and significant secretion of reactive oxidants. The enhanced Ca2+ signal observed in primed neutrophils results from the influx of Ca2+ from the external environment and is partly sensitive to tyrosine kinase inhibitors. This is in contrast to the Ca2+ signal observed in unprimed neutrophils, which arises from the mobilization of intracellular stores. When the surface expression of FcgammaRIIIb on primed neutrophils was decreased either through incubation with Pronase or phosphoinositide-specific phospholipase C, the extra enhanced Ca2+ mobilization seen in primed cells was significantly lowered, while the initial rise in intracellular Ca2+ was unaffected. Depletion of FcgammaRIIIb had no significant effect on the Ca2+ transients in unprimed neutrophils. Cross-linking FcgammaRII, but not FcgammaRIIIb, induced increases in intracellular Ca2+ in unprimed neutrophils, while cross-linking either of these receptors increased Ca2+ levels in primed neutrophils. The FcgammaRII-dependent intracellular Ca2+ rise in primed cells was unaffected by incubation in Ca2+-free medium, whereas the FcgammaRIIIb-dependent transient was significantly decreased when Ca2+ influx was prevented in Ca2+-free medium supplemented with EGTA. Cross-linking either FcgammaRII or FcgammaRIIIb in primed or unprimed cells failed to stimulate substantial levels of inositol 1,4,5-trisphosphate production. These results indicate that following stimulation of primed neutrophils with soluble immune complexes the enhanced Ca2+ mobilization observed is the result of a functional activation of the glycosylphosphatidylinositol-linked FcgammaRIIIb.

Shear stress-induced myosin association with cytoskeleton and phosphorylation in human platelets.

Upon platelet activation by a high shear stress (108 dyne/cm2), actin and actin-binding protein increased rapidly into the Triton-insoluble cytoskeleton, whereas the association of myosin increased gradually. The amounts of cytoskeleton-associated myosin depended on the extent of aggregation. Preceding the maximal aggregation and ATP secretion, the 20 kDa light chain of myosin (MLC) is rapidly phosphorylated to approx. 45% of 20 kDa MLC and is then dephosphorylated. Cytoskeletal association of myosin and phosphorylation of 20 kDa MLC was inhibited by OP-41483, a prostaglandin I2 analog, which inhibited the full aggregation response to shear stress. Exposure to high shear stress resulted in an increased association of myosin light chain kinase and protein phosphatase types 1 and 2A with the cytoskeleton, while the cytoskeletal association of protein kinase C was not evident. These results indicate that 20 kDa MLC phosphorylation is involved in shear stress-induced platelet activation, and that cytoskeletal association of protein phosphatases may regulate the phosphorylation level of cytoskeletal elements such as myosin together with myosin light chain kinase.

Differential cytoprotection by glycine against oxidant damage to proximal tubule cells

Tert-butyl hydroperoxide (tBHP) injured freshly isolated proximal tubules in an Fe-dependent fashion that was ameliorated by a lipophilic antioxidant, diphenyl-p-phenylenediamine (DPPD), but was only minimally affected by glycine. Menadione-induced injury was Fe-independent and was unaffected by DPPD, but was strongly blocked by glycine. Fe was highly toxic when intracellular loading was facilitated by concomitant treatment with hydroxyquinoline (HQ). This toxicity was blocked by DPPD or chelating the Fe, but not by glycine. All of the lesions were characterized by severe depletion of glutathione and other soluble thiols. Menadione induced large increases in protein associated with the Triton-insoluble cytoskeleton and decreases in protein thiol content, consistent with extensive cross linking, but did not increase thiobarbituric acid reactive substances (TBARS). tBHP and HQ + Fe had either no effect or only moderate, delayed effects on cytoskeletal proteins, but induced substantial increases of TBARS. Glycine did not the alter changes in cytoskeletal proteins, thiols, or TBARS produced by any of the agents. Protection against tBHP toxicity by deferoxamine and DPPD was accompanied by substantial suppression of TBARS accumulation. Superimposition of hypoxia during tBHP exposure reduced TBARS accumulation and restored cytoprotective activity to glycine. Thus, in contrast to its consistently strong cytoprotection against a number of other insults, glycine is only variably cytoprotective against oxidant lesions in freshly isolated proximal tubules. Extensive oxidative crosslinking of proteins is compatible with maintenance of glycine cytoprotection against lethal membrane damage. Fe-induced injury to proximal tubules associated with lipid peroxidation as manifested by TBARS formation is a relatively glycine-insensitive insult.

Relative changes in F‐actin during the first cell cycle: Evidence for two distinct pools of F‐actin in the sea urchin egg

The cortical actin cytoskeleton undergoes dramatic rearrangements during fertilization of sea urchin eggs. To characterize these changes further, we quantified the relative changes in filamentous actin (F-actin) during fertilization and the first cell cycle in both intact eggs and in isolated cortices by quantitative fluorescence microscopy. The level of F-actin in the intact egg decreased after fertilization and continued to decrease throughout the first cell cycle. By 60 min after fertilization, the level of F-actin had decreased to 50% of the unfertilized sea urchin egg. By cytokinesis, the level of F-actin had decreased to 30% of the unfertilized egg. After completion of cell division, individual blastomeres had 10% of the F-actin in the unfertilized egg. In contrast, there was an increase in cortical F-actin to 370% of the level in the unfertilized egg after fertilization. This increase corresponded to the formation of microvilli. There was little change in the level of cortical F-actin during the first cell cycle. We draw parallels to other systems that increase the amount of F-actin in the Triton-insoluble cytoskeleton by recruiting actin from a Triton-soluble pool of F-actin. © 1996 Wiley-Liss, Inc.

Differential Association of Protein Ser/Thr Phosphatase Types 1 and 2A with the Cytoskeleton upon Platelet Activation

The association of protein Ser/Thr phosphatase type 1(PP1) and type 2A (PP2A) with the cytoskeleton (Triton X-100 insoluble residue) during human platelet activation was investigated. In unstimulated platelets, 40% of total PP1-like activity was present in the Triton-insoluble cytoskeleton, while only 10% of the total PP2A-like activity was present in this fraction. Stimulation with 1 U/ml thrombin produced a 1.8-fold increase in PP1-like activity and a 7-fold increase in PP2A-like activity, respectively, in the cytoskeletal fraction, under aggregating conditions. Immunoblot analysis revealed that thrombin treatment increased association of PP1 catalytic subunit isozymes (PP1 alpha, PP1 gamma, PP1 delta) and PP2A catalytic subunit with the cytoskeleton, with concomitant decrease of these enzymes in Triton-soluble fractions. The amounts of cytoskeleton-associated PP1 and PP2A depended on the dose of thrombin which could activate platelets. Agonist-induced redistribution of PP1 and PP2A into the cytoskeleton was inhibited by OP-41483 (a prostaglandin I2 analog). Interaction of PP2A with cytoskeletal proteins strongly correlates with aggregation, whereas the association of PP1 with cytoskeleton can be detected upon platelet activation, even in the absence of aggregation. Co-extraction of protein kinase C and myosin light chain kinase with the cytoskeleton eventually translocated to the cytoskeleton, but only during aggregation. These results suggest that differential translocation of PP1 and PP2A to the cytoskeleton is involved in platelet activation, and their association with cytoskeletal proteins may regulate phosphorylation levels together with protein kinases in platelets.

Stimulation of catecholamine secretion from adrenal chromaffin cells by 14-3-3 proteins is due to reorganisation of the cortical actin network

Catecholamine release from digitonin-permeabilized adrenal chromaffin cells is increased by exogenous 14-3-3 proteins. In order to determine how 14-3-3 proteins stimulate exocytosis their effect on the cortical actin network was examined. Increased amounts of β and γ isoforms of 14-3-3 proteins were associated with the Triton-insoluble cytoskeleton of chromaffin cells following incubation with exogenous 14-3-3 proteins. The stimulation of catecholamine release by 14-3-3 proteins was abolished by prior incubation with the actin filament stabilising drug phalloidin. Rhodamine phalloidin staining showed that the cortical actin network was disassembled and actin reorganised into intracellular foci following treatment with 14-3-3 proteins. These data suggest that 14-3-3 proteins enhance catecholamine release in permeabilized chromaffin cells by reorganisation of the cortical actin barrier to allow increased availability of secretory vesicles for exocytosis.