Detergent-insoluble Cytoskeleton Research Articles

Anti-cytoskeleton Immunoscreening of Trypanosoma brucei Expression Library Reveals Novel Immunogenic Conserved Putative Proteins

Background: The overall shape of the trypanosome is defined by an internal cytoskeleton consisting of a network of microtubules that are cross linked both to each other and the inner face of the plasma membrane. However, the total compliment and identity of the trypanosome cytoskeleton proteins are not yet fully determined despite the fact that some of them may be good targets for diagnostics, drugs and/or vaccines discovery.
 Methods: In this study, rabbit anti-Trypanosoma brucei detergent insoluble cytoskeleton sera were produced in vivo and used to probe a T. brucei expression library. The picked plaques were made clonal by a series of library screening followed by PCR amplification, cDNA sequencing and identification of the proteins coded by these sequences using BLAST.
 Results: The previously well-known cytoskeleton proteins (paraflagella rod protein and histone H2B), putative cytoskeleton proteins (Dynein light chain and nucleoporin), conserved hypothetical protein (Tb10.61.2430) and novel cytoskeleton protein coding cDNA sequences (not in the sequenced and published T. brucei genome) were identified in this study.
 Conclusion: This approach is therefore, useable in the search for novel proteins whose utility in the design and development of diagnostics, drugs and/or vaccines can further be studied.

The Position of the GFP Tag on Actin Affects the Filament Formation in Mammalian Cells.

Actin, a major component of microfilaments, is involved in various eukaryotic cellular functions. Over the past two decades, actin fused with fluorescent protein has been used as a probe to detect the organization and dynamics of the actin cytoskeleton in living eukaryotic cells. It is generally assumed that the expression of fusion protein of fluorescent protein does not disturb the distribution of endogenous actin throughout the cell, and that the distribution of the fusion protein reflects that of endogenous actin. However, we noticed that EGFP-β-actin caused the excessive formation of microfilaments in several mammalian cell lines. To investigate whether the position of the EGFP tag on actin affected the formation of filaments, we constructed an expression vector harboring a β-actin-EGFP gene. In contrast to EGFP-β-actin, cells expressing β-actin-EGFP showed actin filaments in a high background from the monomer actin in cytosol. Additionally, the detergent insoluble assay revealed that the majority of the detergent-insoluble cytoskeleton from cells expressing EGFP-β-actin was recovered in the pellet. Furthermore, we found that the expression of EGFP-β-actin affects the migration of NBT-L2b cells and the mechanical stiffness of U2OS cells. These results indicate that EGFP fused to the N-terminus of actin tend to form excessive actin filaments. In addition, EGFP-actin affects both the cellular morphological and physiological phenotypes as compared to actin-EGFP.Key words: actin, GFP, cytoskeleton and probe.

Enoxacin Directly Inhibits Osteoclastogenesis without Inducing Apoptosis

Enoxacin has been identified as a small molecule inhibitor of binding between the B2-subunit of vacuolar H+-ATPase (V-ATPase) and microfilaments. It inhibits bone resorption by calcitriol-stimulated mouse marrow cultures. We hypothesized that enoxacin acts directly and specifically on osteoclasts by disrupting the interaction between plasma membrane-directed V-ATPases, which contain the osteoclast-selective a3-subunit of V-ATPase, and microfilaments. Consistent with this hypothesis, enoxacin dose-dependently reduced the number of multinuclear cells expressing tartrate-resistant acid phosphatase (TRAP) activity produced by RANK-L-stimulated osteoclast precursors. Enoxacin (50 μM) did not induce apoptosis as measured by TUNEL and caspase-3 assays. V-ATPases containing the a3-subunit, but not the "housekeeping" a1-subunit, were isolated bound to actin. Treatment with enoxacin reduced the association of V-ATPase subunits with the detergent-insoluble cytoskeleton. Quantitative PCR revealed that enoxacin triggered significant reductions in several osteoclast-selective mRNAs, but levels of various osteoclast proteins were not reduced, as determined by quantitative immunoblots, even when their mRNA levels were reduced. Immunoblots demonstrated that proteolytic processing of TRAP5b and the cytoskeletal protein L-plastin was altered in cells treated with 50 μM enoxacin. Flow cytometry revealed that enoxacin treatment favored the expression of high levels of DC-STAMP on the surface of osteoclasts. Our data show that enoxacin directly inhibits osteoclast formation without affecting cell viability by a novel mechanism that involves changes in posttranslational processing and trafficking of several proteins with known roles in osteoclast function. We propose that these effects are downstream to blocking the binding interaction between a3-containing V-ATPases and microfilaments.

The Neuronal Protein GAP-43 Associates with Microtubules and Lipid Rafts in Platelets and Megakaryocytes and Is Involved in Platelet Genesis

Abstract 2201GAP-43 (neuromodulin) is a membrane-associated protein involved in neurite outgrowth, pathfinding and branching during development where it regulates neurotransmitter release and endocytosis, and plays a role in cytoskeletal signal transduction at the nerve ending. We have found GAP-43 to be expressed in platelets and megakaryocytes (MK) as evidenced by immunoblot and flow cytometry analysis. Immunofluorescence shows that GAP-43 localizes intracellularly as discrete punctuate structures mostly near the marginal microtubule (MT) coil of platelets and along the MT bundles in MKs and proplatelets. In contrast to neuronal cells where GAP-43 is associated with their detergent insoluble F-actin cytoskeleton, it is mostly soluble in platelets unless the platelets are first treated with taxol, a drug that stabilizes MTs. MT disruption with nocodazole or by chilling decreases the amount of GAP-43 in the detergent-insoluble cytoskeleton and disturbs its localization in proplatelets. GAP-43 colocalizes in platelets and MKs with the glycosylphosphatidylinositol-linked lipid raft marker Thy-1 and Cherry-PH PLC-delta1 (pleckstrin homology domain of phospholipase C-delta1) that stains membrane rafts. Since GAP-43 overexpression in MKs diminishes proplatelet formation, it appears to play a negative role in thrombopoiesis. Together, the data shows that GAP-43 is associated with MT and lipid rafts and suggests a role in signaling reactions between these two components to modulate proplatelet elaboration. Disclosures:No relevant conflicts of interest to declare.

AKAP‐independent localization of type‐II protein kinase A to dynamic actin microspikes

Regulation of the cyclic AMP-dependent protein kinase (PKA) in subcellular space is required for cytoskeletal dynamics and chemotaxis. Currently, spatial regulation of PKA is thought to require the association of PKA regulatory (R) subunits with A-kinase anchoring proteins (AKAPs). Here, we show that the regulatory RIIalpha subunit of PKA associates with dynamic actin microspikes in an AKAP-independent manner. Both endogenous RIIalpha and a GFP-RIIalpha fusion protein co-localize with F-actin in microspikes within hippocampal neuron growth cones and the leading edge lamellae of NG108-15 cells. Live-cell imaging demonstrates that RIIalpha-associated microspikes are highly dynamic and that the coupling of RIIalpha to actin is tight, as the movement of both actin and RIIalpha are immediately and coincidently stopped by low-dose cytochalasin D. Importantly, co-localization of RIIalpha and actin in these structures is resistant to displacement by a cell-permeable disrupter of PKA-AKAP interactions. Biochemical fractionation confirms that a substantial pool of PKA RIIalpha is associated with the detergent-insoluble cytoskeleton and is resistant to extraction by a peptide inhibitor of AKAP interactions. Finally, mutation of the AKAP-binding domain of RIIalpha fails to disrupt its association with actin microspikes. These data provide the first demonstration of the physical association of a kinase with such dynamic actin structures, as well as the first demonstration of the ability of type-II PKA to localize to discrete subcellular structures independently of canonical AKAP function. This association is likely to be important for microfilament dynamics and cell migration and may prime the investigation of novel mechanisms for localizing PKA activity.

Effect of 50 Hz power frequency magnetic field on microfilament cytoskeleton assembly of human amnion FL cells

Investigations were carried out to understand the effect of 50 Hz power frequency magnetic field on microfilament assembly of human amniotic cells and on expression of actin and epidermal growth factor receptor. Human amnion FL cells were exposed to 0.1, 0.2, 0.3, 0.4, 0.5 mT power frequency magnetic field for 30 minutes. Microfilaments were marked using Phalloidin-TRITC, and then were observed under a fluorescence microscope. An optical method was used to detect the relative content of microfilament in cells. A scanning electron microscope was used to detect the cell shape. The content of actin and epidermal growth factor receptor in the preparation of the detergent-insoluble cytoskeleton were measured by western-blotting to analyse the potential mechanism of the change induced by magnetic field. Intracellular stress fibers were found to decrease after exposing cells to a 0.2 mT power frequency magnetic field for 30 minutes. New microfilament and filopodia bundles appeared at the cell periphery after exposure, but the detected total F-actin content per cell was not significantly changed, detected by a F-actin-specific dye. The change in the amount of microfilaments caused by the field could be recovered 2 hours later when the field was withdrawn. The mean height of microfilament cytoskeleton decreased from (12.37 +/- 1.28) microm to (9.97 +/- 0.38) microm (t = 6.96, P > 0.05) after exposure using a confocal microscope. The cell shapes became more flat and lamellipodia appeared after exposure observed by a scanning electron microscope. By using Western blotting method, the intracellular contents of epidermal growth factor receptor and of actin in the preparation of the detergent-insoluble cytoskeleton which are associated with high-affinity epidermal growth factor receptors, increased about (31.2 +/- 4.1)% (t = 17.10, P < 0.05) and (16.8 +/- 2.3)% (t = 16.68, P < 0.05) respectively, compared with that of the control. These results suggest that a short time exposure to a 0.2 mT power frequency magnetic field induces re-organization of microfilament in human amnion FL cells. These changes could be recovered by field withdraw and may have something with the clustering of epidermal growth factor receptors induced by magnetic field.

Actin Binding Activity of Subunit B of Vacuolar H+-ATPase Is Involved in Its Targeting to Ruffled Membranes of Osteoclasts

Adeno-associated virus was used to transduce primary mouse osteoclasts with the B1 isoform of vacuolar H(+)-ATPase. B1, which is not normally expressed in osteoclasts, was correctly targeted to ruffled membranes of resorbing osteoclasts. Mutant subunit B1 that lacked a functional actin-binding site did not accumulate in ruffled membranes. The B1 "kidney" and B2 "brain" isoforms of subunit B of vacuolar H(+)-ATPase (V-ATPase) have actin binding sites that mediate interactions between the intact enzyme and filamentous-actin. Accumulating data support the hypothesis that the actin binding activity in subunit B is required for targeting of V-ATPases to the ruffled plasma membrane of osteoclasts. This study was designed to directly test this hypothesis. Osteoclasts express B2, but not B1. Adeno-associated virus vectors were used to transduce mouse osteoclasts with wildtype B1 or B1(mut), a full-length B subunit that contained minor alterations that disrupted actin-binding activity. Immunofluorescence was performed using polyclonal antibodies specific for subunit E, B2, and B1 of V-ATPase. Immunoprecipitations were performed using an anti-E subunit antibody. Microfilaments were detected with phalloidin and actin rings were stained with phalloidin or anti-vinculin antibodies. Images were collected using a confocal microscope. Immunoprecipitations of transduced osteoclasts suggested that both B1 and B1(mut) assembled with endogenous V-ATPase subunits to form intact enzyme in osteoclasts. Both B1 and B1(mut) were localized like endogenous V-ATPase subunits in unactivated osteoclasts. Wildtype B1 associated with the detergent-insoluble cytoskeleton and was transported to ruffled membranes of resorbing osteoclasts. In contrast, B1(mut) failed to associate with the actin cytoskeleton and was not transported efficiently to ruffled membranes. The B1 isoform of B subunit contains the necessary information for targeting to the ruffled membranes of osteoclasts even though it is not normally expressed in osteoclasts. The actin binding activity of B1 is involved in proper ruffled membrane targeting.

Expanding the Role of the Dynein Regulatory Complex to Non‐Axonemal Functions: Association of GAS11 with the Golgi Apparatus

The mammalian GAS11 gene is a candidate tumor suppressor of unknown function that was previously identified as one of several genes upregulated upon growth arrest. Interestingly, although GAS11 homologs in Trypanosoma brucei (trypanin) and Chlamydomonas reinhardtii (PF2) are integral components of the flagellar axoneme and are necessary for regulating flagellar beat, the GAS11 gene was discovered based on its expression in cells that do not assemble a motile cilium. This suggests that GAS11 function might not be restricted to the cilium. To investigate this possibility, we generated GAS11-specific antibodies and demonstrate here that GAS11 is expressed in a variety of mammalian cells that lack a motile cilium. In COS7 cells, GAS11 is associated with the detergent-insoluble cytoskeleton and exhibits a juxtanuclear localization that overlaps with the pericentrosomal Golgi apparatus. This localization is dependent upon intact microtubules and is cell-cycle regulated, such that GAS11 is dispersed throughout the cytoplasm as cells progress through mitosis. GAS11 remains associated with Golgi fragments following depolymerization of cytoplasmic microtubules but is dispersed upon disruption of the Golgi with brefeldin A. These data suggest that GAS11 is associated with the Golgi apparatus. In support of this, recombinant GAS11 binds Golgi membranes in vitro. In growth-arrested mIMCD3 cells, GAS11 co-localizes with gamma-tubulin at the base of the primary cilium. The pericentrosomal Golgi apparatus and base of the cilium both represent convergence points for microtubule minus ends and correspond to sites where dynein regulation is required. The algal GAS11 homolog functions as part of a dynein regulatory complex (DRC) in the axoneme (Rupp and Porter. J Cell Biol 2003;162:47-57) and our findings suggest that components of this axonemal dynein regulatory system have been adapted in mammalian cells to participate in non-axonemal functions.

Mollicutes

Mollicutes

Membrane cholesterol content modulates activation of BK channels in colonic epithelia

Changes in the level of membrane cholesterol regulate a variety of signaling processes including those mediated by acylated signaling molecules that localize to lipid rafts. Recently several types of ion channels have been shown to have cholesterol-dependent activity and to localize to lipid rafts. In this study, we have investigated the role of cholesterol in the regulation of ion transport in colonic epithelial cells. We observed that methyl-β-cyclodextrin (MβCD), a cholesterol-sequestering molecule, activated transepithelial short circuit current ( I sc), but only from the basolateral side. Similar results were obtained with a cholesterol-binding agent, filipin, and with the sphingomyelin-degrading enzyme, sphingomyelinase. Experiments with ΔF508CFTR mutant mice indicated that raft disruption affected CFTR-mediated anion secretion, while pharmacological studies showed that this effect was due to activation of basolateral large conductance Ca 2+-activated K + (BK) channels. Sucrose density gradient centrifugation studies demonstrated that BK channels were normally present in the high-density fraction containing the detergent-insoluble cytoskeleton, and that following treatment with MβCD, BK channels redistributed into detergent-soluble fractions. Our evidence therefore implicates novel high-density cholesterol-enriched plasma membrane microdomains in the modulation of BK channel activation and anion secretion in colonic epithelia.

The role of caldesmon in the regulation of endothelial cytoskeleton and migration

The actin- and myosin-binding protein, caldesmon (CaD) is an essential component of the cytoskeleton in smooth muscle and non-muscle cells and is involved in the regulation of cell contractility, division, and assembly of actin filaments. CaD is abundantly present in endothelial cells (EC); however, the contribution of CaD in endothelial cytoskeletal arrangement is unclear. To examine this contribution, we generated expression constructs of l-CaD cloned from bovine endothelium. Wild-type CaD (WT-CaD) and truncated mutants lacking either the N-terminal myosin-binding site or the C-terminal domain 4b (containing actin- and calmodulin-binding sites) were transfected into human pulmonary artery EC. Cell fractionation experiments and an actin overlay assay demonstrated that deleting domain 4b, but not the N-terminal myosin-binding site, resulted in decreased affinity to both the detergent-insoluble cytoskeleton and soluble actin. Recombinant WT-CaD co-localized with acto-myosin filaments in vivo, but neither of CaD mutants did. Thus both domain 4b and the myosin-binding site are essential for proper localization of CaD in EC. Overexpression of WT-CaD led to cell rounding and formation of a thick peripheral subcortical actin rim in quiescent EC, which correlated with decreased cellular migration. Pharmacological inhibition of p38 MAPK, but not ERK MAPK, caused disassembly of this peripheral actin rim in CaD-transfected cells and decreased CaD phosphorylation at Ser531 (Ser789 in human h-CaD). These results suggest that CaD is critically involved in the regulation of the actin cytoskeleton and migration in EC, and that p38 MAPK-mediated CaD phosphorylation may be involved in endothelial cytoskeletal remodeling.

Mutational analysis of the cytoplasmic domain of beta1,4-galactosyltransferase I: influence of phosphorylation on cell surface expression.

Beta1,4-galactosyltransferase I (GalT I) exists in two subcellular compartments where it performs two distinct functions. The majority of GalT I is localized in the Golgi complex where it participates in glycoprotein biosynthesis; however, a small portion of GalT I is expressed on the cell surface where it functions as a matrix receptor by binding terminal N-acetylglucosamine residues on extracellular glycoside ligands. The GalT I polypeptide occurs in two alternate forms that differ only in the length of their cytoplasmic domains. It is thought that the longer cytoplasmic domain is responsible for GalT I function as a cell surface receptor because of its ability to associate with the detergent-insoluble cytoskeleton. In this study, we demonstrate that the long GalT I cytoplasmic and transmembrane domains are capable of targeting a reporter protein to the plasma membrane, whereas the short cytoplasmic and transmembrane domains do not have this property. The surface-localized GalT I reporter protein partitions with the detergent-insoluble pool, a portion of which co-fractionates with caveolin-containing lipid rafts. Site-directed mutagenesis of the cytoplasmic domain identified a requirement for serine and threonine residues for cell surface expression and function. Replacing either the serine or threonine with aspartic acid reduces surface expression and function, whereas substitution with neutral alanine has no effect on surface expression or function. These results suggest that phosphorylation negatively regulates GalT I function as a surface receptor. Consistent with this, phosphorylation of the endogenous, full-length GalT I inhibits its stable expression on the cell surface. Thus, the 13 amino acid extension unique to the long GalT I isoform is required for GalT I expression on the cell surface, the function of which is regulated by phosphorylation.

2P-0477 Leukocyte but not endothelial cell adhesion molecules are increased in patients with hypercholesterolemia: The effect of atorvastatin treatment

In most cases epithelial cells reveal a polarized distribution of integrin receptors in basolateral domains of the plasma membrane. To evaluate the functional state of integrin receptors in these restricted sites we were interested in the physical association of integrins with the cytoskeleton. Basically, we extracted cells with Triton X-100 to obtain detergent insoluble cytoskeleton fractions and used monoclonal antibodies for the detection of integrins linked to the cytoskeleton. We found that no permanent physical integrin–cytoskeleton associations exist in a confluent culture of the hepatocyte cell line mHepR1. However, incubation with anti-integrin antibodies and cross linking with a secondary antibody induced a physical linkage of β1 as well as of different α subunits to the cytoskeleton. The association of integrins with the cytoskeleton was also inducible in suspended cells, which was detected in flow cytometric analyses and indicates that the formation of a physical integrin–cytoskeleton connection is independent of the localization of integrins, cell shape, and adhesion on a substrate. Using the Ca2+chelators BAPTA-AM and EGTA, we found that intracellular calcium is a necessary prerequisite to induce a connection of integrins to the cytoskeleton. ATP or tauroursodeoxycholic acid, which provoke an intracellular calcium elevation, partly induced the formation of an integrin–cytoskeleton linkage. These results indicate the obvious role of intracellular calcium in integrin-dependent outside-in as well as inside-out signaling.

Regulation of leukemic cell adhesion, proliferation, and survival by β-catenin

AbstractIn epithelial cells β-catenin plays a critical role as a component of the cell-cell adhesion apparatus and as a coactivator of the TCF/LEF (T-cell transcription factor/lymphoid enhancer binding factor) family of transcription factors. Deregulation of β-catenin has been implicated in the malignant transformation of cells of epithelial origin. However, a function for β-catenin in hematologic malignancies has not been reported. β-Catenin is not detectable in normal peripheral blood T cells but is expressed in T–acute lymphoblastic leukemia cells and other tumor lines of hematopoietic origin and in primary lymphoid and myeloid leukemia cells. β-Catenin function was examined in Jurkat T–acute lymphoblastic leukemia cells. Overexpression of dominant-negative β-catenin or dominant-negative TCF reduced β-catenin nuclear signaling and inhibited Jurkat proliferation and clonogenicity. Similarly, these constructs inhibited proliferation of K562 and HUT-102 cells. Reduction of β-catenin expression with β-catenin antisense down-regulated adhesion of Jurkat cells in response to phytohemagglutinin. Incubation of Jurkat cells with anti-Fas induced caspase-dependent limited proteolysis of β-catenin N- and C-terminal regions and rapid redistribution of β-catenin to the detergent-insoluble cytoskeleton, concomitant with a marked decline in nuclear β-catenin signaling. Fas-mediated apoptosis was potentiated by inhibition of β-catenin nuclear signaling. The data suggest that β-catenin can play a significant role in promoting leukemic cell proliferation, adhesion, and survival.

Tau and HMW tau phosphorylation and compartmentalization in apoptotic neuronal PC12 cells.

In the Alzheimer disease brain, the microtubule-associated protein tau is hyperphosphorylated. There is also evidence that apoptotic-like processes may contribute to the neuronal loss in AD. In an apoptotic model that involves replating neuronal PC12 cells without serum and nerve growth factor (NGF), tau was hyperphosphorylated. During replating, however, neurites are removed. Here, differentiated cells were maintained in serum-free media before growth factor removal, thus maintaining neuritic processes during the apoptotic process and allowing for evaluation of neuritic changes. Tau phosphorylation, evaluated by immunoblotting and immunocytochemistry, was compared with various measures of cell death. Compared with control, NGF-deprived cells exhibited gradual and consistent increases of lactate dehydrogenase release over a 5-day period and a peak of caspase-3 activity at Day 2 after NGF removal. Nuclear staining demonstrated chromatin condensation in NGF-deprived cells. Apoptotic cells had thickened, tortuous, and shortened neuritic processes compared with control cells. Immunoblotting showed an increase in both tau and high molecular weight (HMW) tau phosphorylation during the apoptotic process. Immunoreactivity of both tau isoforms shifted from the detergent insoluble cytoskeleton to the detergent soluble compartment in the apoptotic cells. The microtubule binding of both tau isoforms from apoptotic cells also was impaired. Immunoblotting of purified plasma membrane showed preferential association of HMW tau with the plasma membrane during apoptosis. Also, plasma membrane-associated HMW tau was more phosphorylated during apoptosis. Immunocytochemistry demonstrated increased tau phosphorylation in most apoptotic cells, especially in the neurites. Tau was, however, dephosphorylated cells in the last stages of apoptosis.

Mechanisms of Cold-induced Platelet Actin Assembly

Various agonists but also chilling cause blood platelets to increase cytosolic calcium, polymerize actin, and change shape. We report that cold increases barbed end nucleation sites in octyl glucoside-permeabilized platelets by 3-fold, enabling analysis of the intermediates of this response. Although chilling does not change polyphosphoinositide (ppI) levels, a ppI-binding peptide completely inhibits cold-induced nucleation. The C terminus of N-WASp, which inhibits the Arp2/3 complex, blocks nucleation by 40%; GDPbetaS, N17Rac and N17Cdc42 have no effects. Some gelsolin translocates to the detergent-insoluble cytoskeleton after cooling. Chilled platelets from gelsolin-deficient mice have approximately 50% fewer new actin nuclei compared with platelets from wild-type mice. EGTA completely inhibits gelsolin translocation into the cytoskeleton, and the small amount of gelsolin initially there becomes soluble. Chilling releases adducin from the detergent-resistant cytoskeleton. We conclude that platelet actin filament assembly induced by cooling involves ppI-mediated actin filament barbed end uncapping and de novo nucleation independently of surface receptors or downstream signaling intermediates besides calcium. The actin-related changes occur in platelets at temperatures below 37 degrees C, suggesting that the platelet may be more activable at temperatures at the body surface than at core temperature, thereby favoring superficial hemostasis over internal thrombosis.

Signaling across the Platelet Adhesion Receptor Glycoprotein Ib-IX Induces αIIbβ3 Activation Both in Platelets and a Transfected Chinese Hamster Ovary Cell System

In platelets, alpha(IIb)beta(3) exists in a form that cannot bind adhesive proteins in the plasma; although it can interact with immobilized fibrinogen it cannot interact with immobilized von Willebrand factor in the vessel wall. Soluble agonists such as thrombin convert alpha(IIb)beta(3) to a form that recognizes soluble and immobilized ligands. Attempts to reconstitute alpha(IIb)beta(3) activation in a non-hematopoietic, nucleated cell system have been unsuccessful. In the present study, we have developed a transfected Chinese hamster ovary cell model in which alpha(IIb)beta(3) activation is induced by signaling across glycoprotein (GP) Ib-IX by its ligand, von Willebrand factor. GPIb-IX activates not only the transfected alpha(IIb)beta(3) but also endogenous alpha(v)beta(3). Activation of the pathways leading to integrin activation occurred even in cells transfected with GPIb-IX lacking the domain on GPIbalpha that binds 14-3-3 or that which binds actin-binding protein. These studies demonstrate that signals induced by interaction of GPIb-IX with von Willebrand factor lead to alpha(IIb)beta(3) activation and suggest that the signaling pathways by which GPIb-IX induces alpha(IIb)beta(3) activation are different to those used by thrombin. Elucidation of these differences may provide insights into therapeutic ways in which to inhibit integrin activation in selective clinical settings.

P39 activates cdk5 in neurons, and is associated with the actin cytoskeleton.

Cyclin-dependent kinase 5 (cdk5) is a small serine/threonine kinase that displays close sequence homology to the mitotically active cyclin-dependent kinases. Cdk5 has been shown to play an essential role in the development of the nervous system, including neuronal migration and neurite outgrowth. Cdk5 activation requires the presence of a regulatory activator such as p35. cdk5 -/- mice have much more extensive defects in the development of the nervous system than p35 -/- mice, leading to the speculation that other regulatory activators of cdk5 exist. Indeed, p39 is a p35 related protein isolated by sequence homology to p35. We show here that p39 associates with cdk5 in brain lysates, and that this complex is active in phosphorylation of histone H1. By extensive characterization of p39 subcellular localization in different cell types, we demonstrate the presence of p39 in lamellipodial and fillopodial structures of cells and in growth cones of neurons. We show that p39 colocalizes with actin, and cofractionates with the detergent insoluble cytoskeleton from brain. Further, p39 coimmunoprecipitates with actin in brain lysates. Finally, disruption of the actin cytoskeleton alters p39 subcellular localization as well as kinase activity of the p39/cdk5 complex. Therefore, our results reveal the existence of the p39/cdk5 complex in vivo and suggest that it might play a role in regulating actin cytoskeletal dynamics in cells.

Progressive association of a "soluble" glycolytic enzyme with the detergent-insoluble cytoskeleton during in vitro morphogenesis of MDCK epithelial cells.

In MDCK epithelial cells, cell contact at confluency initiates a protracted process of morphogenesis during which several proteins known to bind the cytoskeleton become progressively associated with the detergent-resistant cell fraction and distributed to their characteristic polarized domains. Using extraction protocols that identify this tight cytoskeletal linkage, here we show a similar but slower, time-dependent enrichment in the detergent resistant fraction of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a highly abundant glycolytic enzyme that is traditionally considered soluble. Similar enrichment did not occur for two other glycolytic enzymes, phosphoglycerate mutase or lactate dehydrogenase. Insoluble GAPDH was not homogeneously distributed in the cytoplasm but rather displayed several discrete patterns that varied within and among MDCK cells. It also localized prominently to a few nuclei in the phenotypically heterogeneous cells of late confluency cultures. Disruptors of cytoskeletal filaments were relatively ineffective in the postconfluent epithelial monolayers, although use of disrupting agents implicated actin as the cytoplasmic filament that tethers insoluble GAPDH. Catalytic activity could be demonstrated in the insoluble fraction of GAPDH from postconfluent cultures, but only after release by mechanical disruption of insoluble extracts. Treatment of postconfluent cells with agents that deplete ATP diminished the fraction of cytoskeletally associated GAPDH, and levels of insoluble GAPDH were restored with ATP repletion, suggesting that ATP levels may regulate cytoskeletal linkage and thereby local enzyme activity. We conclude that the highly abundant and ubiquitous enzyme GAPDH becomes progressively enriched in detergent stable subcellular compartments during the process of epithelial morphogenesis. The process that produces GAPDH compartments is slow, suggesting that epithelial cells just at confluency, when they are typically analyzed, have not yet maximized the organizational state that can be attained in monolayer culture.

Dynamic Association of L-Selectin with the Lymphocyte Cytoskeletal Matrix

L-selectin mediates lymphocyte extravasation into lymphoid tissues through binding to sialomucin-like receptors on the surface of high endothelial venules (HEV). This study examines the biochemical basis and regulation of interactions between L-selectin, an integral transmembrane protein, and the lymphocyte cytoskeleton. Using a detergent-based extraction procedure, constitutive associations between L-selectin and the insoluble cytoskeletal matrix could not be detected. However, engagement of the L-selectin lectin domain by Abs or by glycosylation-dependent cell adhesion molecule-1, an HEV-derived ligand for L-selectin, rapidly triggered redistribution of L-selectin to the detergent-insoluble cytoskeleton. L-selectin attachment to the cytoskeleton was not prevented by inhibitors of actin/microtubule polymerization (cytochalasin B, colchicine, or nocodozole) or serine/threonine and tyrosine kinase activity (staurosporine, calphostin C, or genistein), although L-selectin-mediated adhesion of human PBL was markedly suppressed by these agents. Exposure of human PBL or murine pre-B transfectants expressing full-length human L-selectin to fever-range hyperthermia also markedly increased L-selectin association with the cytoskeleton, directly correlating with enhanced L-selectin-mediated adhesion. In contrast, a deletion mutant of L-selectin lacking the COOH-terminal 11 amino acids failed to associate with the cytoskeletal matrix in response to Ab cross-linking or hyperthermia stimulation and did not support adhesion to HEV. These studies, when taken together with the previously demonstrated interaction between the L-selectin cytoplasmic domain and the cytoskeletal linker protein alpha-actinin, strongly implicate the actin-based cytoskeleton in dynamically controlling L-selectin adhesion.