Biomedical subjects

J H Hartwig

Publications and source records attributed to J H Hartwig.

At least 37 records · Page 2Linked to original sources

PubMed1999

Blood platelets are assembled principally at the ends of proplatelet processes produced by differentiated megakaryocytes.

Megakaryocytes release mature platelets in a complex process. Platelets are known to be released from intermediate structures, designated proplatelets, which are long, tubelike extensions of the megakaryocyte cytoplasm. We have resolved the ultrastructure of the megakaryocyte cytoskeleton at specific stages of proplatelet morphogenesis and correlated these structures with cytoplasmic remodeling events defined by video microscopy. Platelet production begins with the extension of large pseudopodia that use unique cortical bundles of microtubules to elongate and form thin proplatelet processes with bulbous ends; these contain a peripheral bundle of microtubules that loops upon itself and forms a teardrop-shaped structure. Contrary to prior observations and assumptions, time-lapse microscopy reveals proplatelet processes to be extremely dynamic structures that interconvert reversibly between spread and tubular forms. Microtubule coils similar to those observed in blood platelets are detected only at the ends of proplatelets and not within the platelet-sized beads found along the length of proplatelet extensions. Growth and extension of proplatelet processes is associated with repeated bending and bifurcation, which results in considerable amplification of free ends. These aspects are inhibited by cytochalasin B and, therefore, are dependent on actin. We propose that mature platelets are assembled de novo and released only at the ends of proplatelets, and that the complex bending and branching observed during proplatelet morphogenesis represents an elegant mechanism to increase the numbers of proplatelet ends.

Actins↗

PubMed1999

The small GTPase RalA targets filamin to induce filopodia.

The Ras-related small GTPases Rac, Rho, Cdc42, and RalA bind filamin, an actin filament-crosslinking protein that also links membrane and other intracellular proteins to actin. Of these GTPases only RalA binds filamin in a GTP-specific manner, and GTP-RalA elicits actin-rich filopods on surfaces of Swiss 3T3 cells and recruits filamin into the filopodial cytoskeleton. Either a dominant negative RalA construct or the RalA-binding domain of filamin 1 specifically block Cdc42-induced filopod formation, but a Cdc42 inhibitor does not impair RalA's effects, which, unlike Cdc42, are Rac independent. RalA does not generate filopodia in filamin-deficient human melanoma cells, whereas transfection of filamin 1 restores the functional response. RalA therefore is a downstream intermediate in Cdc42-mediated filopod production and uses filamin in this pathway.

3T3 Cells↗

PubMed1999

Cell crawling two decades after Abercrombie.

In response to extracellular signals, cells remodel actin networks. Monomeric actin subunits at the cell's leading edge assemble into linear polymers that are cross-linked by accessory proteins into three-dimensional structures that are contracted by myosins to generate hydraulic force; elsewhere in the cell, actin networks dismantle. Actin subunit sequestering proteins prevent spontaneous actin nucleation, but not the growth of actin sub-units on to fast-growing filament ('barbed') ends, and at least half of the actin in most cells is filamentous. Therefore regulation of cellular actin assembly also requires proteins that block ('cap') actin filament barbed ends. Members of the capping protein gelsolin family also sever actin filaments mechanically. Calcium and protons activate gelsolin for severing and capping. Phosphoinositides reverse such capping, and a pathway has been defined in which receptor perturbation operates through GTP-Rac1 to stimulate the synthesis of endogenous phosphoinositides that uncap actin filaments. Other GTPases (and other signalling pathways) target phosphoinositide synthesis where other protrusions (e.g. filopodia) emerge. Cells maintain adequate, albeit compromised, locomotion in the absence of some, but not all, important machine parts. For example, gelsolin-null fibroblasts crawl using predominantly filopodia rather than lamellae. However, ABP-280 (actin-binding protein of 280 kDa), which promotes orthogonal branching of short actin filaments, seems to be necessary for membrane stability and translational locomotion. ABP-null cells hardly crawl at all, although they are viable and engage in surface movements.

Actins↗

PubMed1999

The elegant platelet: signals controlling actin assembly.

In summary, the actin-based cytoskeleton of the human platelet has been assembled to serve two primary purposes. The first allows the platelet to circulate throughout the blood vascular system in near proximity to the endothelial cells. To accomplish this feat, a small discoid fragment has been meticulously assembled in the megakaryocyte. This released platelet is maintained in the discoid shape using a cytoplasmic scaffolding composed of rigidly crosslinked actin filament laminated to both a spectrin membrane skeleton and the cell surface by the alpha-chain of the vWFR. The small size and discoid shape of the platelet causes it to be pushed to the endothelial surface of the blood vascular system during blood flow. The second purpose of the cytoskeleton is to allow for a massive cell spreading to plug wounds. To accomplish this task, the cytoskeleton is quickly taken apart and reassembled. The temporal nature of this reaction makes the platelet one of the most useful cells in which to understand both the proteins and the signaling pathways that initiate cell shape changes mediated by the actin filament system of cells.

Actins↗

PubMed1998

Measuring actin dynamics in endothelial cells.

Cytoplasmic actin distributes between monomeric and filamentous phases in cells. As cells crawl, actin polymerizes near the plasma membrane of expanding peripheral cytoplasm and depolymerizes elsewhere. Thus, the finite actin filament lifetime, the diffusivity of actin monomer, and the distribution of actin between the polymer and monomer phases are key parameters in cell motility. The dynamics of cellular actin can be determined by following the evolution of fluorescence in the techniques of photoactivated fluorescence (PAF) or fluorescence recovery after photobleaching (FRAP) of microinjected actin derivatives. A mathematical model is discussed that measures monomer diffusion coefficients, filament turnover rates, and the fraction of actin polymerized from measurements of the evolution of fluorescence from a photoactivated band [Tardy et al. (1995) Biophys. J., 69:1674-1682; McGrath et al. (1998) Biophys. J., in press]. Applying this model to subconfluent endothelial cells shows that approximately 40% of the actin is polymer and that these filaments turn over on average every 6 minutes. This report discusses how PAF and FRAP can be combined with more traditional biochemistry to probe actin cytoskeleton remodeling in endothelial cells.

Actins↗

PubMed1998

Gelsolin is a downstream effector of rac for fibroblast motility.

Rac, a member of the rho family of GTPases, when activated transmits signals leading to actin-based membrane ruffling in fibroblasts. Compared with wild-type fibroblasts, gelsolin null (Gsn-) dermal fibroblasts have a markedly reduced ruffling response to serum or EGF stimulation, which signal through rac. Bradykinin-induced filopodial formation, attributable to activation of cdc42, is similar in both cell types. Wild-type fibroblasts exhibit typical lamellipodial extension during translational locomotion, whereas Gsn- cells move 50% slower using structures resembling filopodia. Multiple Gsn- tissues as well as Gsn- fibroblasts overexpress rac, but not cdc42 or rho, 5-fold. Re-expression of gelsolin in Gsn- fibroblasts by stable transfection or adenovirus reverts the ruffling response, translational motility and rac expression to normal. Rac migrates to the cell membrane following EGF stimulation in both cell types. Gelsolin is an essential effector of rac-mediated actin dynamics, acting downstream of rac recruitment to the membrane.

Actins↗

PubMed1998

Simultaneous measurements of actin filament turnover, filament fraction, and monomer diffusion in endothelial cells.

The analogous techniques of photoactivation of fluorescence (PAF) and fluorescence recovery after photobleaching (FRAP) have been applied previously to the study of actin dynamics in living cells. Traditionally, separate experiments estimate the mobility of actin monomer or the lifetime of actin filaments. A mathematical description of the dynamics of the actin cytoskeleton, however, predicts that the evolution of fluorescence in PAF and FRAP experiments depends simultaneously on the diffusion coefficient of actin monomer, D, the fraction of actin in filaments, FF, and the lifetime of actin filaments, tau (, Biophys. J. 69:1674-1682). Here we report the application of this mathematical model to the interpretation of PAF and FRAP experiments in subconfluent bovine aortic endothelial cells (BAECs). The following parameters apply for actin in the bulk cytoskeleton of subconfluent BAECs. PAF: D = 3.1 +/- 0.4 x 10(-8) cm2/s, FF = 0.36 +/- 0.04, tau = 7.5 +/- 2.0 min. FRAP: D = 5.8 +/- 1.2 x 10(-8) cm2/s, FF = 0.5 +/- 0.04, tau = 4.8 +/- 0.97 min. Differences in the parameters are attributed to differences in the actin derivatives employed in the two studies and not to inherent differences in the PAF and FRAP techniques. Control experiments confirm the modeling assumption that the evolution of fluorescence is dominated by the diffusion of actin monomer, and the cyclic turnover of actin filaments, but not by filament diffusion. The work establishes the dynamic state of actin in subconfluent endothelial cells and provides an improved framework for future applications of PAF and FRAP.

Actins↗

PubMed1998

Caspase-3-induced gelsolin fragmentation contributes to actin cytoskeletal collapse, nucleolysis, and apoptosis of vascular smooth muscle cells exposed to proinflammatory cytokines.

Gelsolin, an 80 kDa actin-severing protein, has been recently identified as a substrate for the cell death-promoting cysteinyl protease caspase-3 (CPP32/apopain/YAMA). We investigated the role of gelsolin and its cleavage product in apoptosis of vascular smooth muscle cells (SMC) induced by the proinflammatory cytokines interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha). Treatment with a combination of IFN-gamma and TNF-alpha reduced viability of SMC in a time- and concentration-dependent manner. Immunoblotting revealed that SMC treated with the cytokines generated a 41 kDa gelsolin fragment. The gelsolin fragmentation required activation of caspase-3, as the caspase-3 inhibitor diminished cytokine-induced cell death as well as the fragmentation. Gelsolin cleavage was accompanied by a reduction in F-actin content and by a marked disruption of cell structure. Adenovirus-mediated transfection of this N-terminal gelsolin fragment into SMC altered cell morphology, reduced cell viability, increased the number of TUNEL-positive cells, and promoted internucleosomal DNA fragmentation. Compared to wild-type cells, gelsolin-deficient SMC showed resistance to apoptosis induced by the inflammatory cytokines. These results suggest a mechanistic role for gelsolin cleavage during SMC apoptosis, a process implicated in vessel development as well as stability of atherosclerotic plaque.

Actins↗

PubMed1997

Cytoskeletal protein ABP-280 directs the intracellular trafficking of furin and modulates proprotein processing in the endocytic pathway.

Furin catalyzes the proteolytic maturation of many proproteins within the trans-Golgi network (TGN)/endosomal system. Furin's cytosolic domain (cd) directs both the compartmentalization to and transit between its manifold processing compartments (i.e., TGN/biosynthetic pathway, cell surface, and endosomes). Here we report the identification of the first furin cd sorting protein, ABP-280 (nonmuscle filamin), an actin gelation protein. The furin cd was used as bait in a yeast two-hybrid screen to identify ABP-280 as a furin-binding protein. Binding analyses in vitro and coimmunoprecipitation studies in vivo showed that furin and ABP-280 interact directly and that ABP-280 tethers furin molecules to the cell surface. Quantitative analysis of both ABP-280-deficient and genetically replete cells showed that ABP-280 modulates the rate of internalization of furin but not of the transferrin receptor, a cycling receptor. However, although ABP-280 directs the rate of furin internalization, the efficiency of sorting of the endoprotease from the cell surface to early endosomes is independent of expression of ABP-280. By contrast, efficient sorting of furin from early endosomes to the TGN requires expression of ABP-280. In addition, ABP-280 is also required for the correct localization of late endosomes (dextran bead uptake) and lysosomes (LAMP-1 staining), demonstrating a pleiotropic role for this actin binding protein in the organization of cellular compartments and directing protein traffic. Finally, and consistent with the trafficking studies on furin, we showed that ABP-280 modulates the processing of furin substrates in the endocytic but not the biosynthetic pathways. The novel roles of ABP-280 and the cytoskeleton in the sorting of furin in the TGN/ endosomal system and the formation of proprotein processing compartments are discussed.

Amino Acid Sequence↗

PubMed1997

WIP, a protein associated with wiskott-aldrich syndrome protein, induces actin polymerization and redistribution in lymphoid cells.

Wiskott-Aldrich syndrome (WAS) is an X-linked immunodeficiency caused by mutations that affect the WAS protein (WASP) and characterized by cytoskeletal abnormalities in hematopoietic cells. By using the yeast two-hybrid system we have identified a proline-rich WASP-interacting protein (WIP), which coimmunoprecipitated with WASP from lymphocytes. WIP binds to WASP at a site distinct from the Cdc42 binding site and has actin as well as profilin binding motifs. Expression of WIP in human B cells, but not of a WIP truncation mutant that lacks the actin binding motif, increased polymerized actin content and induced the appearance of actin-containing cerebriform projections on the cell surface. These results suggest that WIP plays a role in cortical actin assembly that may be important for lymphocyte function.

Actins↗

PubMed1997

The lipid products of phosphoinositide 3-kinase increase cell motility through protein kinase C.

Phosphoinositide 3-kinase has been implicated as an activator of cell motility in a variety of recent studies, yet the role of its lipid product, phosphatidylinositol 1,4,5-trisphosphate (PtdIns-3,4,5-P3), has yet to be elucidated. In this study, three independent preparations of PtdIns-3,4,5-P3 were found to increase the motility of NIH 3T3 cells when examined utilizing a microchemotaxis chamber. Dipalmitoyl L-alpha-phosphatidyl-D-myo-inositol 3,4,5-triphosphate (Di-C16-PtdIns-3,4,5-P3) also produced actin reorganization and membrane ruffling. Cells pretreated with 12-O-tetradecanoylphorbol-13-acetate to cause down-regulation of protein kinase C (PKC) exhibited complete inhibition of cell motility induced by Di-C16-PtdIns-3,4,5-P3. These results are consistent with previous observations that PtdIns-3,4,5-P3 activates Ca2+-independent PKC isoforms in vitro and in vivo and provide the first demonstration of an in vivo role for the lipid products of the phosphoinositide 3-kinase. PtdIns-3,4,5-P3 appears to directly initiate cellular motility via activation of a PKC family member.

3T3 Cells↗

PubMed1997

Identification of the region in actin-binding protein that binds to the cytoplasmic domain of glycoprotein IBalpha.

Actin-binding protein (ABP-280) is a component of the submembranous cytoskeleton and interacts with the glycoprotein (GP) Ibalpha subunit of the GP Ib-IX complex in platelets. In the present studies, we have identified the binding site for GP Ibalpha in ABP-280. A melanoma cell line lacking ABP-280 was stably transfected with the cDNAs coding for GP Ib-IX, then transiently transfected with cDNA coding for various carboxyl-truncates of ABP-280. Immunocapture assays and co-immunoprecipitation experiments from detergent-lysed cells showed that deletion of the carboxyl-terminal repeats 20-24 of ABP-280 had no effect on GP Ib-IX binding, but deletion of residues 2099 through 2136 within repeat 19 abolished binding. In the yeast two-hybrid system, an ABP-280 fragment comprising repeats 17-19 bound GP Ibalpha. Deletion from either end abolished binding. Individual or multiple repeats of ABP-280 were expressed as fusion protein in bacteria and purified; structural folding was evaluated, and binding to GP Ib-IX was assessed. Binding depended on the presence of repeats 17-19. None of the individual repeats were able to bind to GP Ib-IX. These findings demonstrate that residues 1850-2136 comprising repeats 17-19 contain the binding site for GP Ib-IX.

Binding Sites↗

PubMed1997

Immunoelectron microscopy shows a clustered distribution of NADPH oxidase components in the human neutrophil plasma membrane.

The NADPH oxidase that produces superoxide in professional phagocytic cells is a flavocytochrome b electron transport chain in the membrane, a heterodimer of gp91phox and p22phox, that is activated by a number of cytosolic proteins, including p47phox, p67phox, and the small GTP-binding protein p21rac, which translocate to the membrane and attach to the flavocytochrome on activation. The components of this oxidase were localized on the cytoplasmic surface of the plasma membrane of adherent unroofed neutrophils by immunolabeling. Components of the NADPH oxidase and p21rac were found together in punctate clusters occupying 0.03-0.1 microm2 of the cytoplasmic surface of the plasma membrane where the density of labeling of the cytosolic components was increased after stimulation with phorbol myristate acetate.

Antibodies, Monoclonal↗

PubMed1997

Signal transduction pathways involving the small G proteins rac and Cdc42 and phosphoinositide kinases.

We found that rac specifically binds to a type I PtdIns-4-P 5-kinase and that both rac and Cdc42 in the activated forms associate with PI 3-kinase. The association of PI 3-kinase with rac was stimulated by PDGF in vivo. Rac is constitutively associated with a PtdIns-4-P 5-kinase and stimulates PtdIns-4,5-P2 production in permeabilized platelets. These data suggest a model in which the initial step in the activation of rac is release from rho GDI (Fig. 7). Rac in the GDP bound form can associate with the PtdIns-4-P 5-kinase and also interact with an exchange factor. GTP bound rac may then localize to sites of actin reorganization, bringing the PtdIns-4-P 5-kinase with it. Locally synthesized PtdIns-4,5-P2 binds to actin capping proteins, leading to their release and the production of actin free ends. Actin polymerization can then occur from the free ends. Many other factors must be involved to regulate the type and extent of actin polymerization that is necessary in such complex processes as cell movement and membrane ruffling. The rac-associated PtdIns-4-P 5-kinase and its product PtdIns-4,5-P2 may act at a crucial regulatory point that permits polymerization to begin.

1-Phosphatidylinositol 4-Kinase↗

PubMed1997

Polyphosphoinositide synthesis and platelet shape change.

Polyphosphoinositides both integrate signaling pathways at the cytoplasmic surface of the plasma membrane and serve as the final messenger of signaling pathways. They have a well-defined role in the release of calcium through the degradative pathway. Recent work, however, has highlighted the role of the polyphosphoinositide synthetic pathways in activated cells. This review emphasizes the role of polyphosphoinositides in recruiting signaling proteins that have pleckstrin homology domains and in directly regulating actin assembly in the human blood platelet.

1-Phosphatidylinositol 4-Kinase↗

PubMed1997

Mechanical remodeling of the endothelial surface and actin cytoskeleton induced by fluid flow.

OBJECTIVE: The mechanism by which cultured endothelial cells respond to shear stress is controversial. The cell surface and cytoskeleton are involved, but their roles are undefined. In this study, previously unknown changes in the surface detail and actin cytoskeleton of bovine aortic endothelial cells were identified. METHODS: Actin filament content and filament number in resting and flow-oriented cells were determined by biochemical assays. The three-dimensional organization of the actin cytoskeleton in cells was defined in the confocal microscope and in the electron microscope after rapid-freezing, freeze-drying, and metal coating of detergent-permeabilized cells. RESULTS: Endothelial cells have smooth apical membranes in situ. However, cultured cells exhibit surface microvilli which increase the apical surface area, exposing the ruffled surface to forces from fluid flow and potentially enhancing cell interactions with blood-borne white cells. Stereoscopic micrographs show that stress fibers are integrated into a complex distributed cytoplasmic structural actin network (DCSA). This lattice is formed by actin filaments that frequently cross and connect to each other, stress fibers, and microfilaments and microtubules. The cytoskeletons of cells cultured in static media lack apparent order when compared to cytoskeletons from cells which have been exposed to 24 hours of laminar flow. CONCLUSIONS: The DCSA physically connects the apical and basal cell membranes and fills the volume between nucleus and membrane, providing a mechanism for transmitting mechanical forces across cells and a signaling pathway from membrane to nucleus. Stress fibers increase the mechanical modulus of the DCSA, although this increase is probably unnecessary to withstand the increase in shear stress caused by blood flow in vivo. This implies that actin rearrangements are not required for mechanical integrity, but serve an alternate function.

Actins↗

PubMed1996

D3 phosphoinositides and outside-in integrin signaling by glycoprotein IIb-IIIa mediate platelet actin assembly and filopodial extension induced by phorbol 12-myristate 13-acetate.

Phorbol 12-myristate 13-acetate (PMA) uncaps a small number of the fast-growing (barbed) ends of actin filaments, thereby eliciting slow actin assembly and extension of filopodia in human blood platelets. These reactions, which also occur in response to immunologic perturbation of the integrin glycoprotein (GP) IIb-IIIa, are sensitive to the phosphoinositide 3-kinase inhibitor wortmannin. Platelets deficient in GPIIb-IIIa integrins or with GPIIb-IIIa function inhibited by calcium chelation or the peptide RGDS have diminished PMA responsiveness. The effects of PMA contrast with thrombin receptor stimulation by >/=5 microM thrombin receptor-activating peptide (TRAP), which causes rapid and massive wortmannin-insensitive actin assembly and lamellar and filopodial extension. However, we show here that wortmannin can inhibit filopod formation if the thrombin receptor is ligated using suboptimal doses (<1 microM) of TRAP. Phosphatidylinositol 3,4-bisphosphate inhibits actin filament severing and capping by human gelsolin in vitro. The findings implicate D3 polyphosphoinositides and integrin signaling in PMA-mediated platelet stimulation and implicate D3 containing phosphoinositides generated in response to protein kinase C activation and GPIIb-IIIa signaling as late-acting intermediates leading to filopodial actin assembly.

Actin Cytoskeleton↗

PubMed1996

Phosphorylation of actin-binding protein 280 by growth factors is mediated by p90 ribosomal protein S6 kinase.

Although Ras-related small GTPases are believed to control cell proliferation and motility through activation of protein kinase cascades, little is known about the intracellular protein targets of activated kinases. Here we show that the p90 ribosomal S6 kinase 2 (RSK2) phosphorylates actin-binding protein (ABP-280) in intact rat 3Y1 fibroblasts. Growth factors such as fetal calf serum, epidermal growth factor, phorbol 12-myristate 13-acetate, and lysophosphatidic acid stimulate the phosphorylation of serine residues in ABP-280 in quiescent 3Y1 cells. Extracts from 3Y1 cells prepared after stimulation by lysophosphatidic acid, fetal calf serum, and epidermal growth factor retain activated protein kinase activity(s) toward ABP-280 in vitro. ABP kinase activities in lysates from lysophosphatidic acid-stimulated 3Y1 cells can be fractionated by MonoQ anion exchange column chromatography into three peaks having ABP kinase activities. One (ABP kinase peak 1) coelutes with the peak of RSK2 as judged by immunoblotting and S6 peptide kinase assays. Two-dimensional phosphopeptide maps show RSK2 phosphorylated ABP-280 to be phosphorylated at the same site(s) as those stimulated by growth factors in vivo. Incubation of ABP kinase peak 1 fractionated from unstimulated cells with activated ERK2 activates latent ABP kinase activity. These results show RSK2 to phosphorylate ABP-280 in vivo.

Animals↗