Coordinated regulation of platelet actin filament barbed ends by gelsolin and capping protein

Exposure of cryptic actin filament fast growing ends (barbed ends) initiates actin polymerization in stimulated human and mouse platelets. Gelsolin amplifies platelet actin assembly by severing F-actin and increasing the number of barbed ends. Actin filaments in stimulated platelets from transgenic gelsolin-null mice elongate their actin without severing. F-actin barbed end capping activity persists in human platelet extracts, depleted of gelsolin, and the heterodimeric capping protein (CP) accounts for this residual activity. 35% of the approximately 5 microM CP is associated with the insoluble actin cytoskeleton of the resting platelet. Since resting platelets have an F- actin barbed end concentration of approximately 0.5 microM, sufficient CP is bound to cap these ends. CP is released from OG-permeabilized platelets by treatment with phosphatidylinositol 4,5-bisphosphate or through activation of the thrombin receptor. However, the fraction of CP bound to the actin cytoskeleton of thrombin-stimulated mouse and human platelets increases rapidly to approximately 60% within 30 s. In resting platelets from transgenic mice lacking gelsolin, which have 33% more F-actin than gelsolin-positive cells, there is a corresponding increase in the amount of CP associated with the resting cytoskeleton but no change with stimulation. These findings demonstrate an interaction between the two major F-actin barbed end capping proteins of the platelet: gelsolin-dependent severing produces barbed ends that are capped by CP. Phosphatidylinositol 4,5-bisphosphate release of gelsolin and CP from platelet cytoskeleton provides a mechanism for mediating barbed end exposure. After actin assembly, CP reassociates with the new actin cytoskeleton.     

Polarization of gelsolin and actin binding protein in kidney epithelial cells

Vasopressin regulates transepithelial osmotic water permeability in the kidney collecting duct and in target cells in other tissues. In the presence of hormone, water channels are inserted into an otherwise impermeable apical plasma membrane and the apical surface of these cells is dramatically remodelled. Because cytochalasin B and D greatly reduce the response of these cells to vasopressin, actin filaments are believed to participate in the events leading to an increase in transepithelial water permeability. Modulation of the actin filamentous network requires the concerted action of specific actin regulatory proteins, and in the present study we used protein A-gold immunocytochemistry to localize two important molecules, gelsolin and actin binding protein (ABP), in epithelial cells of the kidney inner medulla. Gelsolin and, to a lesser extent, ABP were concentrated in clusters in the apical cell web of principal cells of the collecting duct. Aggregates of gold particles were often associated with the cytoplasmic side of plasma membrane regions forming surface extensions or microvilli. The basolateral plasma membrane was labeled to a much lesser extent than the apical plasma membrane. In the thin limbs of Henle, ABP was localized over the apical plasma membrane in ascending limbs, but gelsolin labeling was weak in these cells. In thin descending limbs, the pattern of labeling was completely reversed, with abundant apical gelsolin labeling but only weak ABP immunolabeling. Although the significance of the distribution of actin regulatory proteins in thin limbs is unknown, the abundance and the predominantly apical polarization of both ABP and gelsolin in principal cells of the collecting duct is consistent with a role of the actin cytoskeleton in the mechanism of vasopressin actin.     

Three-dimensional reconstruction of the membrane skeleton at the plasma membrane interface by electron tomography

Three-dimensional images of the undercoat structure on the cytoplasmic surface of the upper cell membrane of normal rat kidney fibroblast (NRK) cells and fetal rat skin keratinocytes were reconstructed by electron tomography, with 0.85-nm-thick consecutive sections made approximately 100 nm from the cytoplasmic surface using rapidly frozen, deeply etched, platinum-replicated plasma membranes. The membrane skeleton (MSK) primarily consists of actin filaments and associated proteins. The MSK covers the entire cytoplasmic surface and is closely linked to clathrin-coated pits and caveolae. The actin filaments that are closely apposed to the cytoplasmic surface of the plasma membrane (within 10.2 nm) are likely to form the boundaries of the membrane compartments responsible for the temporary confinement of membrane molecules, thus partitioning the plasma membrane with regard to their lateral diffusion. The distribution of the MSK mesh size as determined by electron tomography and that of the compartment size as determined from high speed single-particle tracking of phospholipid diffusion agree well in both cell types, supporting the MSK fence and MSK-anchored protein picket models.     

Microinjection of gelsolin into living cells

Gelsolins are actin-binding proteins that cap, nucleate, and sever actin filaments. Microinjection of cytoplasmic or plasma gelsolin into living fibroblasts and macrophages did not affect the shape, actin distribution, deformability, or ruffling activity of the cells. Gelsolin requires calcium for activity, but the NH2-terminal half is active without calcium. Microinjection of this proteolytic fragment had marked effects: the cells rounded up, stopped ruffling, became soft, and stress fibers disappeared. These changes are similar to those seen with cytochalasin, which also caps barbed ends of actin filaments. Attempts to raise the cytoplasmic calcium concentration and thereby activate the injected gelsolin were unsuccessful, but the increases in calcium concentration were minimal or transient and may not have been sufficient. Our interpretation of these results is that at the low calcium concentrations normally found in cells, gelsolin does not express the activities observed in vitro at higher calcium concentrations. We presume that gelsolin may be active at certain times or places if the calcium concentration is elevated to a sufficient level, but we cannot exclude the existence of another molecule that inhibits gelsolin. Microinjection of a 1:1 gelsolin/actin complex had no effect on the cells. This complex is stable in the absence of calcium and has capping activity but no severing and less nucleation activity as compared with either gelsolin in calcium or the NH2-terminal fragment. The NH2-terminal fragment-actin complex also has capping and nucleating activity but no severing activity. On microinjection it had the same effects as the fragment alone. The basis for the difference between the two complexes is unknown. The native molecular weight of rabbit plasma gelsolin is 82,500, and the extinction coefficient at 280 nm is 1.68 cm2/mg. A new simple procedure for purification of plasma gelsolin is described.     

Mechanisms of actin rearrangements mediating platelet activation.

The detergent-insoluble cytoskeleton of the resting human blood platelet contains approximately 2,000 actin filaments approximately 1 micron in length crosslinked at high angles by actin-binding protein and which bind to a spectrin-rich submembrane lamina (Fox, J., J. Boyles, M. Berndt, P. Steffen, and L. Anderson. 1988. J. Cell Biol. 106:1525-1538; Hartwig, J., and M. DeSisto. 1991. J. Cell Biol. 112:407-425). Activation of the platelets by contact with glass results within 30 s in a doubling of the polymerized actin content of the cytoskeleton and the appearance of two distinct new actin structures: bundles of long filaments within filopodia that end at the filopodial tips (filopodial bundles) and a circumferential zone of orthogonally arrayed short filaments within lamellipodia (lamellipodial network). Neither of these structures appears in cells exposed to glass with cytochalasin B present; instead the cytoskeletons have numerous 0.1-0.3-microns-long actin filament fragments attached to the membrane lamina. With the same time course as the glass-induced morphological changes, cytochalasin-sensitive actin nucleating activity, initially low in cytoskeletons of resting platelets, increases 10-fold in cytoskeletons of thrombin-activated platelets. This activity decays with a time course consistent with depolymerization of 0.1-0.3-microns-long actin filaments, and phalloidin inhibits this decay. Cytochalasin-insensitive and calcium-dependent nucleation activity also increases markedly in platelet extracts after thrombin activation of the cells. Prevention of the rise in cytosolic Ca2+ normally associated with platelet activation with the permeant Ca2+ chelator, Quin-2, inhibits formation of lamellipodial networks but not filopodial bundles after glass contact and reduces the cytochalasin B-sensitive nucleation activity by 60% after thrombin treatment. The filopodial bundles, however, are abnormal in that they do not end at the filopodial tips but form loops and return to the cell body. Addition of calcium to chelated cells restores lamellipodial networks, and calcium plus A23187 results in cytoskeletons with highly fragmented actin filaments within seconds. Immunogold labeling with antibodies against gelsolin reveals gelsolin molecules at the ends of filaments attached to the submembrane lamina of resting cytoskeletons and at the ends of some filaments in the lamellipodial networks and filopodial bundles of activated cytoskeletons. Addition of monomeric actin to myosin subfragment 1-labeled activated cytoskeletons leads to new (undecorated) filament growth off the ends of filaments in the filopodial bundles and the lamellipodial network. The simplest explanation for these findings is that gelsolin caps the barbed ends of the filaments in the resting platelet. Uncapping some of these filaments after activation leads to filopodial bundles.(ABSTRACT TRUNCATED AT 400 WORDS)     

Annexin V binds to the actin-based cytoskeleton at the plasma membrane of activated platelets.

Immunocytochemical studies demonstrate that annexin V relocates to the plasma membranes of intact stimulated blood platelets. Anti-annexin V antibodies label the cytoplasmic side of the substrate-adherent plasma membrane of mechanically unroofed, glass-activated platelets and colocalize with actin. In addition, crosslinking experiments using detergent-solubilized membranes of activated platelets have identified an 85-kDa complex containing annexin V. The 85-kDa complex is also recognized by antibodies against actin, suggesting that annexin V interacts with actin. In addition, annexin V was found to associate with filamentous actin in the presence of millimolar Ca(2+). Annexin V was also shown by immunofluorescence microscopy to be associated with platelet cytoskeletons, colocalizing with actin in the presence of micromolar Ca(2+). These findings provide the first evidence for annexin V binding to the plasma membrane and to the actin-based cytoskeleton in activated platelets and indicate that annexin V may function in both cytoskeletal and membrane domains.     

Immunocytochemical evidence for the translocation of alpha-granule membrane glycoprotein IIb/IIIa (integrin alpha IIb beta 3) of human platelets to the surface membrane during the release reaction.

The localization of glycoprotein (GP) IIb/IIIa (integrin alpha IIb beta 3) in both resting and thrombin-activated platelets was studied immunocytochemically. By the preembedding method where only the GP IIb/IIIa molecules on the surface of platelets were immunostained, the distribution of protein A-colloidal gold label was randomly distributed along the surface membrane of resting platelets at a density of 18.0 +/- 2.7 gold particles/microns of membrane. At 15 s after stimulation by 0.1 U/ml of thrombin in an unstirred platelet suspension, the spheroid-shaped platelets with pseudopodia still had normal numbers of alpha-granules, and the density of gold particles was 19.7 +/- 3.6 particles/microns. At 5 min, the alpha-granules were no longer present because of the release reaction, and the density of gold particles significantly increased (27.0 +/- 3.7 particles/microns; p less than 0.01). In immuno-stained ultra-thin frozen sections, the gold particles were detected not only on the surface membrane, including the open canalicular system (OCS), but also on the alpha-granule membranes of resting platelets. At 30 s after thrombin stimulation the alpha-granules fused with the OCS, resulting in the formation of a swollen OCS, which still had gold particles on its membrane. At 5 min, the gold particles were detected on the membrane of the swollen OCS located near the surface membrane, while very few gold particles were present on the membrane of the OCS in the central part of the platelets. These results demonstrate that alpha-granule membrane GPIIb/IIIa translocates to the surface membrane through the membrane of the OCS.(ABSTRACT TRUNCATED AT 250 WORDS)     

凝溶胶蛋白及其生物医学作用

gelsolin(凝溶胶蛋白)有胞浆和血清二型,分别存在于哺乳动物各类细胞和血清中。该蛋白的基本生物功能是以钙依赖的方式通过切断、封端肌动蛋白丝,或使肌动蛋白聚集成核等方式控制肌动蛋白的结构。大量动物实验和临床研究均表明,gelsolin的结构、功能及调节与烧伤和急性挫伤的诊断与治疗,以及某些炎症、肿瘤和淀粉样变等多种疾病的病理过程密切相关。作为一种急症治疗药,该蛋白的工程化产品已在美国进行Ⅱ期临床实验。最近发现正常细胞经电离辐射后,gelsolin的表达水平发生显著改变,其在辐射效应中的作用和意义值得关注。     

Gelsolin affects the migratory ability of human colon adenocarcinoma and melanoma cells

AimsFormation of different protrusive structures by migrating cells is driven by actin polymerization at the plasma membrane region. Gelsolin is an actin binding protein controlling the length of actin filaments by its severing and capping activity. The main goal of this study was to determine the effect of gelsolin expression on the migration of human colon adenocarcinoma LS180 and melanoma A375 cells.Main methodsColon adenocarcinoma cell line LS180 was stably transfected with plasmid containing human cytoplasmic gelsolin cDNA tagged to enhanced green fluorescence protein (EGFP). Melanoma A375 cells were transfected with siRNAs directed against gelsolin. Real-time PCR and Western blotting were used to determine the level of gelsolin. The ability of actin to inhibit DNase I activity was used to quantify monomeric and total actin level and calculate the state of actin polymerization. Fluorescence confocal microscopy was applied to observe gelsolin and vinculin distribution along with actin cytoskeleton organization.Key findingsIncreased level of gelsolin expression leads to its accumulation at the submembranous region of the cell accompanied by distinct changes in the state of actin polymerization and an increase in the migration of LS180 cells. In addition, LS180 cells overexpressing gelsolin form podosome-like structures as indicated by vinculin redistribution and its colocalization with gelsolin and actin. Downregulation of gelsolin expression in melanoma A375 cells significantly reduces their migratory potential.SignificanceOur experimental data indicate that alterations in the expression level of gelsolin and its subcellular distribution may be directly responsible for determining migration capacity of human cancer cells.     

Phosphatidylinositol cycle and its possible involvement in the regulation of cytoskeleton-membrane interactions

Receptor-mediated activation of many cells, including blood platelets, leads to changes at the cytoplasmic side of the membrane. In platelets, phospholipases, such as phospholipase C and phospholipase A2, have been shown to become activated. From phospholipids they generate the second messengers diacylglycerol and inositol phosphate(s) and fatty acids, respectively. At the same time, actin polymerization and reorganization of actin filaments into bundles and networks occurs. Here, the association of lipids, radiolabeled either with saturated (palmitic acid) or unsaturated (arachidonic acid) fatty acids, with the cytoskeletons of resting and activated human blood platelets was studied. The relative binding of lipid components to the cytoskeleton of activated platelets labeled with palmitic acid is six times higher than that of platelets labeled with arachidonic acid. Analysis of lipids associated with isolated cytoskeletons of resting and activated platelets (labeled with palmitic acid) showed a 30-fold increase in the binding of labeled lipids to the cytoskeletal structures during activation. Both diacylglycerol and fatty acids were found to be associated with the cytoskeleton of activated platelets. Gel filtration, chromatofocusing, and immunoprecipitation studies demonstrated tight binding of these lipids to alpha-actinin. alpha-Actinin is one of the proteins that rapidly becomes associated with the cytoskeleton during platelet aggregation; it is also one of the molecules proposed to act as an actin-membrane linker. The results reported indicate a possible participation of alpha-actinin, fatty acids, and the phosphoinositide-derived second messenger diacylglycerol in the regulation of cytoskeleton-membrane interactions. Together with the results of others they suggest a possible involvement of the phosphatidylinositol cycle in the assembly of actin filaments and their anchoring to membranes.     

Subcellular localization of sea urchin egg spectrin: evidence for assembly of the membrane-skeleton on unique classes of vesicles in eggs and embryos

A recent study from our laboratory on the sea urchin egg suggested that spectrin was not solely restricted to the plasma membrane, but instead had a more widespread distribution on the surface of a variety of membranous inclusions. (E. M. Bonder et al., 1989, Dev. Biol. 134, 327-341). In this report we extend our initial findings and provide experimental and ultrastructural evidence for the presence of spectrin on three distinct classes of cytoplasmic vesicles. Immunoblot analysis of membrane fractions prepared from egg homogenates establishes that spectrin coisolates with vesicle-enriched fractions, while indirect immunofluorescence microscopy on cryosections of centrifugally stratified eggs demonstrates that spectrin specifically associates with cortical granules, acidic vesicles, and yolk platelets in vivo. Immunogold ultrastructural localization of spectrin on cortices isolated from eggs and early embryos details the striking distribution of spectrin on the cytoplasmic surface of the plasma membrane and the membranes of cortical granules, acidic vesicles, and yolk platelets, while quantitative studies show that relatively equivalent amounts of spectrin are present on the different membrane surfaces both before and after fertilization. These data, in combination with the localization of numerous spectrin crosslinks between actin filaments in surface microvilli, suggest that spectrin plays a pivotal role in structuring the cortical membrane-cytoskeletal complex of the egg and the embryo.     

Annexin V relocates to the periphery of activated platelets following thrombin activation: an ultrastructural immunohistochemical approach

We have previously shown biochemically that the physiological agonist thrombin can cause translocation of endogenous annexin V to a fraction containing all platelet membranes. This paper reports ultrastructural immunohistochemical data revealing that annexin V molecules localize with plasma membranes of blood platelets following thrombin activation. When ultrathin sections of resting platelets were examined by immunogold staining, annexin V was found to be cytosolic, having a generalized distribution throughout the platelet. After thrombin activation, annexin V became peripheral in location and plasmalemma association increased. Morphometric analysis of gold particles shows that annexin V relocates specifically to the plasma membrane and its underlying cytoskeleton following treatment with thrombin. In control platelets 6.1% +/- 0.78 of annexin V is present at the plasma membrane and 15.0% +/- 0.82 in the region corresponding to the membrane cytoskeleton (10-80 nm); after stimulation with 0.5 unit/ml thrombin for 2 min this increased to 16.7% +/- 0.22 and 40.4% +/- 0.53, respectively.     

Mobility of GPIIb-IIIa receptors within membranes of surface- and suspension-activated platelets does not depend on assembly and contraction of cytoplasmic actin

Investigations using fibrinogen coupled to colloidal gold (Fgn/Au) have shown that glycoprotein IIb-IIIa (GPIIb-IIIa) receptors are mobile and undergo centripetal reorganization on spread platelets following surface activation. The assembly of cytoplasmic actin and its constriction into an inner filamentous zone have been proposed as the mechanism driving the Fgn/Au receptor complexes across the plasma membrane. The present study has used cytochalasin B (CB), an agent known to inhibit actin assembly and cause breakdown of newly formed actin filaments to test that hypothesis. At a concentration which inhibited pseudopod formation and spreading, CB did not block movement of Fgn/Au receptor complexes toward platelet centers or into the open canalicular system (OCS). Channels of the OCS filled with Fgn/Au receptor complexes were evident in over 90% of the CB-treated, surface-activated platelets. The findings do not support the concept that assembly of cytoplasmic actin and its contraction move receptor-ligand complexes on the platelet plasma membrane.     

Molecular biology of gelsolin, a calcium-regulated actin filament severing protein

Gelsolin is a Ca2+-binding protein of mammalian leukocytes, platelets and other cells which has multiple and closely regulated powerful effects on actin. In the presence of micromolar Ca2+, gelsolin severs actin filaments, causing profound changes in the consistency of actin polymer networks. A variant of gelsolin containing a 25-amino acid extension at the NH2-terminus is present in plasma where it may be involved in the clearance of actin filaments released during tissue damage. Gelsolin has two sites which bind actin cooperatively. These sites have been localized using proteolytic cleavage and monoclonal antibody mapping techniques. The NH2-terminal half of the molecule contains a Ca2+-insensitive actin severing domain while the COOH-terminal half contains a Ca2+-sensitive actin binding domain which does not sever filaments. These data suggest that the NH2-terminal severing domain in intact gelsolin is influenced by the Ca2+-regulated COOH-terminal half of the molecule. The primary structure of gelsolin, deduced from human plasma gelsolin cDNA clones, supports the existence of actin binding domains and suggests that these may have arisen from a gene duplication event, and diverged subsequently to adopt their respective unique functions. The plasma and cytoplasmic forms of gelsolin are encoded by a single gene, and preliminary results indicate that separate mRNAs code for the two forms. Further application of molecular biological techniques will allow exploration into the structural basis for the multifunctionality of gelsolin, as well as the molecular basis for the genesis of the cytoplasmic and secreted forms of gelsolin.     

Molecular cloning of human macrophage capping protein cDNA. A unique member of the gelsolin/villin family expressed primarily in macrophages.

Macrophage capping protein (MCP) is a Ca(2+)-sensitive protein which reversibly blocks the barbed ends of actin filaments but does not sever preformed actin filaments. The human cDNA for MCP has been cloned and sequenced. The derived amino acid sequence predicts a polypeptide of 38.4 kDa. Human MCP expressed in Escherichia coli using a pET12a vector was functionally identical to the native protein purified from rabbit alveolar macrophages with respect to Ca2+ sensitivity and ability to block monomer exchange at the barbed end of actin filaments. Sequence comparison with other actin-binding protein sequences indicates that MCP is a member of the gelsolin/villin family of barbed end blocking proteins. Unlike gelsolin, this protein has a limited tissue distribution being detected primarily in macrophages where it was abundant, representing 0.9-1% of the total cytoplasmic protein. Northern blot analysis of U937 and HL60 cells differentiated to macrophage-like cells demonstrated that MCP message increases to 2.6 and greater than 7 times initial levels, respectively. Human MCP displays a 93% amino acid sequence identity with two recently described mouse proteins, gCap39 and Mbh1. Its abundance in macrophages and the corresponding increases in mRNA levels upon promyelocyte and monocyte development into macrophages indicate that MCP may play an important role in macrophage function.     

The Actin-Binding Proteins Eps8 and Gelsolin Have Complementary Roles in Regulating the Growth and Stability of Mechanosensory Hair Bundles of Mammalian Cochlear Outer Hair Cells

Sound transduction depends upon mechanosensitive channels localized on the hair-like bundles that project from the apical surface of cochlear hair cells. Hair bundles show a stair-case structure composed of rows of stereocilia, and each stereocilium contains a core of tightly-packed and uniformly-polarized actin filaments. The growth and maintenance of the stereociliary actin core are dynamically regulated. Recently, it was shown that the actin-binding protein gelsolin is expressed in the stereocilia of outer hair cells (OHCs) and in its absence they become long and straggly. Gelsolin is part of a whirlin scaffolding protein complex at the stereocilia tip, which has been shown to interact with other actin regulatory molecules such as Eps8. Here we investigated the physiological effects associated with the absence of gelsolin and its possible overlapping role with Eps8. We found that, in contrast to Eps8, gelsolin does not affect mechanoelectrical transduction during immature stages of development. Moreover, OHCs from gelsolin knockout mice were able to mature into fully functional sensory receptors as judged by the normal resting membrane potential and basolateral membrane currents. Mechanoelectrical transducer current in gelsolin-Eps8 double knockout mice showed a profile similar to that observed in the single mutants for Eps8. We propose that gelsolin has a non-overlapping role with Eps8. While Eps8 is mainly involved in the initial growth of stereocilia in both inner hair cells (IHCs) and OHCs, gelsolin is required for the maintenance of mature hair bundles of low-frequency OHCs after the onset of hearing.     

Ultrastructural localization of concanavalin A receptors in the plasma membrane: association with underlying actin filaments

Whole-mount cell preparations of cultured rat 3Y1 cells were examined by stereo electron microscopy to identify the ultrastructural localization of concanavalin A (Con A) receptors in the plasma membrane, and to clarify the relationship between Con A receptors and cytoskeletal components. Well spread monolayer cells were extracted with saponin, briefly fixed, and then partially broken open with shearing force to facilitate the introduction of antibodies for identification of actin filaments. Stereo electron microscopy of such treated cells revealed a 3-dimensional image of filamentous structures such as fine filaments, microtubules (MT) and endoplasmic reticulum (ER) in the flattened areas of each cell. Just beneath the plasma membrane were meshworks of actin-containing fine filaments, as identified by an immunogold staining method. Microtubules and ER were observed to be either directly or indirectly associated with this meshwork. The broken open part of each cell exhibited a meshwork of filaments which were associated with the cytoplasmic surface of the plasma membrane. Some of the filaments were connected to the plasma membrane either by their ends or by their lateral surfaces. The localization of Con A receptors was examined by binding colloidal gold-labelled Con A to the surface of fixed, saponin-extracted cells. Virtually all gold particles bound externally at the same membrane sites where intracellular actin filaments attached internally. The observations strongly suggest that the distribution of Con A receptors was regulated by the underlying meshwork of actin filaments.     

Actin assembly induced by polylysine beads or purified phagosomes: quantitation by a new flow cytometry assay

BACKGROUND: Actin assembly on biological membranes is a poorly understood process. We have previously shown that phagosomal membranes could induce actin assembly in the presence of thymosin beta4 (an actin sequestering protein that inhibits nonspecific nucleation), via the barbed ends of actin filaments. METHODS: Here, we have developed an in vitro system based on fluorescein-labeled G (monomeric) actin and flow cytometry analysis, which allowed us to quantify de novo actin assembly on the cytoplasmic side of purified phagosomes. To standardize the system, we also used latex beads covalently coupled with polylysine, which efficiently promote actin nucleation. RESULTS: Flow cytometry analysis showed that the percentage of polylysine beads positive for F-actin filaments increased in a time- and G-actin concentration-dependent manner. Incubation of phagosomes with reagents affecting actin dynamics allowed us to extend our previous data showing that the phagosomal membranes assemble actin filaments de novo. Finally, our results pin-point a potential role for gelsolin as a positive regulator of actin assembly on the phagosomal membrane. CONCLUSIONS: We propose that our system could facilitate the development of other in vitro assays for the analysis of actin assembly and its links to signaling in cells.     

凝溶胶蛋白与相关疾病的关系

凝溶胶蛋白（gelsolin,GSN）是一种在机体内普遍存在的,对细胞结构和代谢功能具有多种调节作用的蛋白。GSN作为凝溶胶蛋白超家族的成员之一,是一种重要的肌动蛋白（actin）结合蛋白,可通过切断、封闭肌动蛋白丝,或使actin聚集成核等方式来调控actin的结构与代谢功能.GSN不仅能在重组的肌动蛋白细丝（F-actin）中发挥作用,而且在细胞运动、细胞凋亡等细胞活动中也发挥着重要的作用。GSN有血浆型（plasma gelsolin,pGSN）和细胞质型（cytoplasmic gelsolin,cGSN）两种亚型,它们在淀粉样变性、炎症、癌症、心血管疾病、阿茨海默病（AD）及肾脏疾病中都起着重要的作用,GSN可能成为多种疾病的一个新的生物标记物或者治疗靶点。本文将就GSN与相关疾病的关系的研究进展做一综述。     

Polyphosphoinositide micelles and polyphosphoinositide-containing vesicles dissociate endogenous gelsolin-actin complexes and promote actin assembly from the fast-growing end of actin filaments blocked by gelsolin

The Ca2+-activated actin-binding protein gelsolin regulates actin filament length by severing preformed filaments and by binding actin monomers, stabilizing nuclei for their assembly into filaments. Gelsolin binds to phosphatidylinositol 4,5-bisphosphate (PIP2), with consequent inhibition of its filament severing activity and dissociation of EGTA-resistant complexes made with rabbit macrophage or human plasma gelsolin and rabbit muscle actin. This study provides evidence for an interaction of gelsolin with phosphatidylinositol monophosphate (PIP) as well as PIP2 and further describes their effects on gelsolin's function. Both phosphoinositides completely dissociate EGTA-insensitive rabbit macrophage cytoplasmic gelsolin-actin complexes and inhibit gelsolin's severing activity. The magnitude of inhibition depends strongly on the physical state of the phosphoinositides, being maximal in preparations that contain small micelles of either purified PIP or PIP2. Aggregation of PIP or PIP2 micelles by divalent cations or insufficient sonication or their incorporation into vesicles containing other phospholipids decreases but does not eliminate the inhibitory properties of the polyphosphoinositides. The presence of gelsolin partly inhibits the divalent cation-induced aggregation of PIP2 micelles. PIP2 in combination with EGTA inactivates gelsolin molecules that block the fast-growing end of actin filaments, thereby accelerating actin polymerization. Regulation of gelsolin by the intracellular messengers Ca2+ and polyphosphoinositides allows for the formation of several different gelsolin-actin intermediates with distinct functional properties that may be involved in changes in the state of cytoplasmic actin following cell stimulation.