Capping protein terminates but does not initiate chemoattractant-induced actin assembly in Dictyostelium.

The first step in the directed movement of cells toward a chemotactic source involves the extension of pseudopods initiated by the focal nucleation and polymerization of actin at the leading edge of the cell. We have previously isolated a chemoattractant-regulated barbed-end capping activity from Dictyostelium that is uniquely associated with capping protein, also known as cap32/34. Although uncapping of barbed ends by capping protein has been proposed as a mechanism for the generation of free barbed ends after stimulation, in vitro and in situ analysis of the association of capping protein with the actin cytoskeleton after stimulation reveals that capping protein enters, but does not exit, the cytoskeleton during the initiation of actin polymerization. Increased association of capping protein with regions of the cell containing free barbed ends as visualized by exogenous rhodamine-labeled G-actin is also observed after stimulation. An approximate threefold increase in the number of filaments with free barbed ends is accompanied by increases in absolute filament number, whereas the average filament length remains constant. Therefore, a mechanism in which preexisting filaments are uncapped by capping protein, in response to stimulation leading to the generation of free barbed ends and filament elongation, is not supported. A model for actin assembly after stimulation, whereby free barbed ends are generated by either filament severing or de novo nucleation is proposed. In this model, exposure of free barbed ends results in actin assembly, followed by entry of free capping protein into the actin cytoskeleton, which acts to terminate, not initiate, the actin polymerization transient.

Cytoskeleton-dependent membrane domain segregation during neutrophil polarization.

On treatment with chemoattractant, the neutrophil plasma membrane becomes organized into detergent-resistant membrane domains (DRMs), the distribution of which is intimately correlated with cell polarization. Plasma membrane at the front of polarized cells is susceptible to extraction by cold Triton X-100, whereas membrane at the rear is resistant to extraction. After cold Triton X-100 extraction, DRM components, including the transmembrane proteins CD44 and CD43, the GPI-linked CD16, and the lipid analog, DiIC(16), are retained within uropods and cell bodies. Furthermore, CD44 and CD43 interact concomitantly with DRMs and with the F-actin cytoskeleton, suggesting a mechanism for the formation and stabilization of DRMs. By tracking the distribution of DRMs during polarization, we demonstrate that DRMs progress from a uniform distribution in unstimulated cells to small, discrete patches immediately after activation. Within 1 min, DRMs form a large cap comprising the cell body and uropod. This process is dependent on myosin in that an inhibitor of myosin light chain kinase can arrest DRM reorganization and cell polarization. Colabeling DRMs and F-actin revealed a correlation between DRM distribution and F-actin remodeling, suggesting that plasma membrane organization may orient signaling events that control cytoskeletal rearrangements and, consequently, cell polarity.

A major agonist-regulated capping activity in Dictyostelium is due to the capping protein, cap32/34.

Stimulation of starved Dictyostelium amoebae with the chemoattractant cAMP produces a rapid increase in actin nucleation activity at 5 seconds which is cotemporal with an increase in actin assembly and a decrease in Ca(2+)-insensitive capping activity [1]. Further characterization of this capping activity, called aginactin, led to the isolation of an Hsc70 [2]. Here, we demonstrate that purified aginactin contains both Hsc70 and the heterodimeric barbed-end capping protein, cap32/34. Immunoprecipitation of cap32/34 from purified aginactin removes all capping activity while immunoprecipitation of Hsc70 does not, indicating that the capping activity of aginactin is an intrinsic property of cap32/34. Gel filtration and immunoprecipitation assays fail to demonstrate the existence of a stable, high affinity complex between Hsc70 and cap32/34 in either lysate supernatants or aginactin pools but indicate the presence of a transient, ATP-sensitive interaction in cell lysates. Reconstitution experiments with purified Hsc70 and cap32/34 demonstrate that Hsc70 neither stimulates nor inhibits the capping activity of native cap32/34. Furthermore, we measured a Kd of approx. 0.8 nM for the binding of cap32/34 to barbed ends of actin filaments in the absence or presence of Hsc70, in agreement with Kd values measured for purified capping protein from other sources. We conclude, therefore, that cap32/34 is responsible for the capping activity called aginactin and that Hsc70 is not a regulatory cofactor for cap32/34 in Dictyostelium but may function as a chaperone during assembly of the cap32/34 heterodimer.

Cost-effectiveness of CT scanning compared with mediastinoscopy in the preoperative staging of lung cancer.

In lung cancer, evaluation of the mediastinum is a critical factor in the determination of surgical resectability. Mediastinoscopy with nodal biopsy has been the preferred method of assessing the mediastinum. An alternate approach is to first perform computed tomography (CT) and then decide on the need for further tests. The present study is a cost-effectiveness analysis of these two diagnostic strategies. A decision tree was constructed incorporating the two approaches. Costs were determined in a rigorous manner by calculating a fully allocated unit price for all relevant hospital services. These costs include both direct costs and an appropriate share of support and overhead expenses. The frequency of complications with the tests, the need for further tests, and the expected outcomes for the patients were derived from published data. Hospital charts were reviewed to determine the amount of services used by patients who had actually experienced the outcomes or complications of interest. The comparative costs of the two strategies were then determined by multiplying the fully allocated unit costs by the amount of services that a cohort of patients could be expected to use following one or the other pathway. The strategy employing CT scanning and selective mediastinoscopy resulted in a reduction of costs and in hospital days ranging from 10% to 30%. CT scanning and selective mediastinoscopy are, therefore, more cost-effective than mediastinoscopy alone in the preoperative staging of lung cancer.

Aginactin, an agonist-regulated F-actin capping activity is associated with an Hsc70 in Dictyostelium.

We have previously isolated an agonist-regulated actin filament capping activity, called aginactin, that is associated with a 70-kDa protein (Sauterer, R. A., Eddy, R. J., Hall, A. L., and Coneelis, J. S. (1991) J. Biol. Chem. 266, 24533-24539). A 2.0-kilobase clone isolated from a Dictyostelium lambda gt11 cDNA library screened with affinity-purified aginactin antibodies displays an overall sequence identity of 73% to the 70-kDa heat shock cognate protein, Hsc70, from various species. Aginactin capping activity and the 70-kDa protein bind to ATP-agarose columns and are quantitatively depleted from the load, indicating that an Hsc70 is associated with aginactin activity. Moderate stringency Southern blots indicate the presence of no fewer than six Hsc70-related sequences. Immunofluorescent staining of vegetative Dictyostelium AX3 cells with aginactin antibodies reveals a colocalization of aginactin-associated Hsc70 in F-actin-rich regions of the cell cortex and cell protrusions. Nuclei and organelles lacked positive staining indicating that the aginactin-associated Hsc70 is cytosolic. The levels of cytoskeletal-associated Hsc70 correlate with the loss of barbed end capping activity following cAMP stimulation, suggesting that the uncapping of barbed filament ends through an Hsc70-associated process may account for the increase in nucleation activity observed at 5 s following agonist stimulation.

Evidence for the nonmuscle nature of the "myofibroblast" of granulation tissue and hypertropic scar. An immunofluorescence study.

Contraction is an important phenomenon in wound repair and hypertrophic scarring. Studies indicate that wound contraction involves a specialized cell known as the myofibroblast, which has morphologic characteristics of both smooth muscle and fibroblastic cells. In order to better characterize the myofibroblast, the authors have examined its cytoskeleton and surrounding extracellular matrix (ECM) in human burn granulation tissue, human hypertrophic scar, and rat granulation tissue by indirect immunofluorescence. Primary antibodies used in this study were directed against 1) smooth muscle myosin and 2) nonmuscle myosin, components of the cytoskeleton in smooth muscle and nonmuscle cells, respectively, and 3) laminin and 4) fibronectin, extracellular glycoproteins mediating cell-matrix attachment in smooth muscle and nonmuscle cells, respectively. Myofibroblasts can be identified by their intense staining of actin bundles with either anti-actin antibody or NBD-phallacidin. Myofibroblasts in all tissues stained for nonmuscle but not smooth muscle myosin. In addition, nonmuscle myosin was localized as intracellular fibrils, which suggests their similarity to stress fibers in cultured fibroblasts. The ECM around myofibroblasts stains intensely for fibronectin but lacks laminin, which suggests that a true basal lamina is not present. The immunocytochemical findings suggest that the myofibroblast is a specialized nonmuscle type of cell, not a smooth muscle cell.

Oriented endocytic recycling of alpha5beta1 in motile neutrophils.

During cell migration, integrin attachments to the substratum provide the means to generate the traction and force necessary to achieve locomotion. Once the cell has moved over these attachments, however, it is equally important that integrins detach from the substratum. The fate of integrins after detachment may include release from the cell, lateral diffusion across the cell surface, or endocytosis and redelivery to the cell surface. Polymorphonuclear neutrophils (PMNs) become stuck on the extracellular matrix proteins fibronectin and vitronectin when their intracellular free calcium concentration ([Ca(++)]i) is buffered. Taking advantage of this feature of PMN migration, we investigated the fate of integrins to differentiate among various models of migration. We demonstrate that alpha5beta1, one of the fibronectin-binding integrins, is responsible for immobilization of [Ca(++)](i)-buffered PMNs on fibronectin. We find that alpha5 and beta1 are in endocytic vesicles in PMNs and that alpha5 colocalizes with a marker for an endocytic recycling compartment. When [Ca(++)](i) is buffered, alpha5 and beta1 become concentrated in clusters in the rear of the adherent cells, suggesting that [Ca(++)](i) transients are required for alpha5beta1 detachment from the substratum. Inhibition of alpha5beta1 detachment by buffering [Ca(++)](i) results in the depletion of alpha5 from both endocytic vesicles and the recycling compartment, providing compelling evidence that integrins are normally recycled by way of endocytosis and intracellular trafficking during cell migration. This model is further refined by our demonstration that the endocytic recycling compartment reorients to retain its localization just behind the leading lamella as PMNs migrate, indicating that membrane recycling during neutrophil migration has directionality. (Blood. 2000;95:2471-2480)

Purification and characterization of aginactin, a newly identified agonist-regulated actin-capping protein from Dictyostelium amoebae.

Amoeboid chemotaxis involves a regulated increase in actin nucleation activity that is correlated with an increase in actin polymerization occurring seconds after chemotactic stimulation (Carson, M., Weber, A., and Zigmond, S. H. (1986) J. Cell Biol. 103, 2707-2714; Hall, A. L., Warren, V., Dharmawardhane, S., and Condeelis, J. (1989) J. Cell Biol. 109, 2207-2213). We report the isolation and characterization of an agonist-regulated capping protein, aginactin, from Dictyostelium that may regulate these changes in actin nucleation activity. Aginactin is isolated from low speed supernatants of starved amoebae by sequential anion exchange, hydrophobic interaction, fast protein liquid chromatography anion exchange, and hydroxyapatite chromatography. Aginactin migrates with an apparent molecular weight of 70,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and gel filtration columns, suggesting that it is a globular monomer. Aginactin is a barbed-end capping protein by several criteria. It inhibits the rate and final extent of actin polymerization and increases the apparent critical concentration at substoichiometric ratios to actin. It also inhibits depolymerization of F-actin and inhibits polymerization at the barbed end of Limulus acrosomal bundles. Aginactin is unaffected by micromolar Ca2+, and it neither severs F-actin nor nucleates actin polymerization in either the presence or absence of Ca2+. Aginactin binds to and cosediments with F-actin and has an apparent Kd for capping F-actin of 2.7 nM.

Ca2+-dependent myosin II activation is required for uropod retraction during neutrophil migration.

Buffering of intracellular Ca2+ transients in human neutrophils leads to reduced motility due to defective uropod detachment on fibronectin and vitronectin-coated surfaces. Since one potential target of a rise in [Ca2+]i is the activation of myosin II, we characterized the role of myosin II during motility. Treatment of neutrophils with a myosin inhibitor (2,3-butanedione monoxime), or myosin light chain kinase inhibitors (ML-7, ML-9, or KT5926) resulted in impaired uropod retraction and a dose-dependent decrease in chemokinesis following stimulation with N-formyl-Met-Leu-Phe (fMLP). Treatment with ML-9 resulted in a redistribution of F-actin and talin to the non-retracted uropods, mimicking the redistribution observed during [Ca2+]i buffering. Impairment of uropod retraction and redistribution of F-actin and talin by myosin II inhibition was only observed on adhesive substrates such as fibronectin and not on poorly adhesive substrates such as human serum-coated glass. At higher concentrations of ML-9, cell polarization was inhibited and pseudopod extension occurred radially. Using an antibody specific for serine 19-phosphorylated regulatory light chain of myosin II, regions of activated myosin II were found at the leading edge as well as the uropod in motile fMLP-stimulated cells. [Ca2+]i depletion caused a 50% decrease in the level of serine 19-phosphorylated myosin II suggesting that activation of myosin II by intracellular Ca2+ transients may be an essential step in establishing a polarized pseudopod and providing the force required for uropod retraction during PMN motility on adhesive surfaces.

Fibroblast contraction occurs on release of tension in attached collagen lattices: dependency on an organized actin cytoskeleton and serum.

The generation of tension in granulation tissue undergoing contraction is believed to be a cell-mediated event. In this study we used attached collagen lattices as a model system for studying the cellular mechanisms of tension generation by fibroblasts in an extracellular matrix. Fibroblasts in attached collagen lattices developed stress fibers, surface associated fibronectin fibrils, and a fibronexus-like transmembrane association interconnecting the two structural components. Release of the attached collagen lattice from its points of attachment resulted in a rapid, symmetrical contraction of the collagen lattice. Rapid contraction occurred within the first 10 minutes after release of the lattice from the substratum, with greater than 70% of the contraction occurring within the first 2 minutes. Rapid contraction resulted in a shortening of the elongate fibroblasts and compaction of the stress fibers with their subsequent disappearance from the cell. Cytochalasin D treatment prior to release disrupted the actin cytoskeleton and completely inhibited rapid contraction. The removal of serum prior to release inhibited rapid contraction, while the re-addition of serum restored rapid contraction. These results demonstrate that fibroblasts can develop tension in an attached collagen lattice and that upon release of tension the fibroblasts undergo contraction resulting in a rapid contraction of the collagen lattice. Fibroblast contraction is dependent upon an organized actin cytoskeleton and is promoted by the presence of serum.

The uptake and degradation of matrix-bound lipoproteins by macrophages require an intact actin Cytoskeleton, Rho family GTPases, and myosin ATPase activity.

A key cellular event in atherogenesis is the interaction of macrophages with lipoproteins in the subendothelium. In vivo, these lipoproteins are bound to matrix and often aggregated, yet most cell-culture experiments explore these events using soluble monomeric lipoproteins. We hypothesized that the internalization and degradation of matrix-retained and aggregated low density lipoprotein (LDL) by macrophages may involve the actin-myosin cytoskeleton in a manner that distinguishes this process from the endocytosis of soluble LDL. To explore these ideas, we plated macrophages on sphingomyelinase-aggregated LDL bound to smooth muscle cell-derived matrix in the presence of lipoprotein lipase. The macrophages internalized and degraded the LDL, which was mediated partially by the LDL receptor-related protein. Cytochalasin D and latrunculin A, which block actin polymerization, markedly inhibited the uptake and degradation of matrix-retained LDL but not soluble LDL. Inhibition of Rho family GTPases by Clostridium difficile toxin B blocked the degradation of matrix-retained and aggregated LDL by >90% without any inhibition of soluble LDL degradation. However, specific inhibition of Rho had no effect, suggesting the importance of Rac1 and Cdc42. Degradation of matrix-retained, but not soluble, LDL was also blocked by inhibitors of tyrosine kinase, phosphatidylinositol 3-kinase, and myosin ATPase. These findings define fundamental cytoskeletal pathways that may be involved in macrophage foam cell formation in vivo but have been missed by the use of previous cell culture models.