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PubMed1995

Dynamics of triton-insoluble and triton-soluble F-actin pools in calcium-activated human polymorphonuclear leukocytes: evidence for regulation by gelsolin.

Gelsolin, a Ca++ activated, 90 kd actin binding protein, can regulate actin polymerization in polymorphonuclear leukocytes (PMNs) via severing of filaments to dissolve gels or by capping of filament ends to limit polymerization. In Triton-lysed PMNs, 30% of gelsolin is bound to the Triton-soluble F-actin (TSF) pool and none is bound to the Triton-insoluble F-actin (TIF) pool. Calcium-activated PMNs exhibit concurrent temporal and quantitative TIF growth and TSF and total F-actin loss. To determine if gelsolin plays a role in regulating TSF pool size, we monitored gelsolin-actin interactions and TIF, TSF and G-actin content at 5 second intervals in PMNs activated with the calcium ionophore, ionomycin. Actin pools were measured by NBDphallacidin binding and by gel scans and expressed relative to basal; gelsolin-actin interactions were measured as change in the amount of EGTA-resistant gelsolin:actin (G:A) complexes and by immunoblot quantification of gelsolin in actin pools. In basal PMNs, 33% of PMN gelsolin is bound in 1:1 EGTA-resistant G:A complexes and TSF and TIF retain 30% and 0% of PMN gelsolin, respectively. By 20 seconds after ionomycin addition, TSF decreases, TIF increases and a fraction of gelsolin repartitions from the TSF to the TIF pool. At maximum change (60 seconds), total F-actin (TIF + TSF) and TSF decrease and TIF increases by 25%; gelsolin is bound to both TSF and TIF (35% of total gelsolin in each pool), and 1:1 EGTA-resistant G:A complexes increase from 33% to 70%. No changes occur in cells activated by ionomycin in the absence of Ca++. The data show Ca++ activated TIF growth and TSF loss are temporally and quantitatively associated with an increase in the percent of gelsolin bound to actin and the translocation of gelsolin from TSF to TIF. This is unique, since no other PMN activator is known to repartition gelsolin into TIF actin. Further, the Ca++ activated initial increase in TIF concurrent with a fall in TSF without a change in total F-actin or G-actin content suggest that TIF grows initially only by TSF annealing/cross-linking to TIF. Gelsolin may regulate these events.

Actins↗
PubMed1997

A novel gelsolin isoform expressed by oligodendrocytes in the central nervous system.

Two isoforms of the Ca2+-sensitive, actin-binding protein gelsolin have been identified thus far; one is an intracellular protein, cytoplasmic gelsolin, and the other is a secretory protein called plasma gelsolin. Gelsolin expression in the mammalian CNS appears to be localized mainly to oligodendrocytes where it is presumed that the cytoplasmic isoform predominates. Here, we show that oligodendrocytes not only contain cytoplasmic gelsolin, but they also express a novel gelsolin isoform that we have named gelsolin-3. Cytoplasmic gelsolin, plasma gelsolin, and gelsolin-3 arise by alternative splicing from the same gene. The N-terminal amino acid sequence unique to gelsolin-3 is shown to be encoded by a single exon in a region previously thought to be an intron in the human gelsolin gene. In situ hybridization analysis confirmed that gelsolin-3 mRNA is localized primarily to oligodendrocytes in rat brain. In other tissues, gelsolin-3 shows a more restricted pattern of expression than cytoplasmic gelsolin. These data support the view that the gelsolin isoforms have differential roles in the regulation of the actin cytoskeleton.

Amino Acid Sequence↗
PubMed1998

Frequent loss of gelsolin expression in non-small cell lung cancers of heavy smokers.

Most lung and bladder cancers have been shown to be associated with smoking. We have previously demonstrated the frequent loss of gelsolin expression and its tumor suppressor activity in bladder cancer (M. Tanaka et al., Cancer Res., 55: 3228-3232, 1995). Here, we examined gelsolin expression in 12 cultured non-small cell lung cancer (NSCLC) cell lines. Furthermore, we analyzed gelsolin expression in relation to patients' smoking habits in 88 surgically resected NSCLCs to investigate whether gelsolin could be a molecular target for tobacco-induced carcinogenesis of lung cancer. All 12 NSCLC cell lines showed low-to-undetectable expression of the gelsolin gene, compared to that in normal lung tissue, by Northern blot analysis. On the other hand, Southern blot analysis of genomic DNA did not show any gross rearrangements or deletions of the gene in the NSCLC cell lines. Western blot analysis of gelsolin expression showed low-to-undetectable gelsolin expression in all 12 NSCLC cell lines, compared to normal lung tissue. Immunocytochemical analysis of gelsolin expression in NSCLC cell lines showed results that were consistent with those obtained by Western blot analysis, using normal bronchial epithelial cells as a positive control: two cell lines with lower gelsolin expression by Western blot analysis had reduced but positive cytoplasmic immunostaining of gelsolin, compared with primary normal bronchial epithelial cells, whereas no such immunostaining was observed in two cell lines with much lower or undetectable gelsolin expression by Western blot analysis. Therefore, gelsolin expression was analyzed in surgically resected NSCLCs by immunohistochemistry. Reduced or undetectable gelsolin expression was observed in 48 of 88 (55 %) resected NSCLCs. Such altered gelsolin expression significantly correlated with heavy smoking of patients (> or =20 pack-years; P = 0.008 by the chi2 test and P = 0.03 by multivariate logistic regression analysis), whereas there was no significant correlation between gelsolin expression and histological type, pathological tumor-node-metastasis (pTNM) stage, or survival. These findings suggest that the frequent loss of gelsolin expression may be involved in the development of NSCLCs as a potential molecular target of tobacco-induced carcinogenesis.

Aged↗
PubMed1998

Gelsolin, a protein that caps the barbed ends and severs actin filaments, enhances the actin-based motility of Listeria monocytogenes in host cells.

The actin-based motility of Listeria monocytogenes requires the addition of actin monomers to the barbed or plus ends of actin filaments. Immunofluorescence micrographs have demonstrated that gelsolin, a protein that both caps barbed ends and severs actin filaments, is concentrated directly behind motile bacteria at the junction between the actin filament rocket tail and the bacterium. In contrast, CapG, a protein that strictly caps actin filaments, fails to localize near intracellular Listeria. To explore the effect of increasing concentrations of gelsolin on bacterial motility, NIH 3T3 fibroblasts stably transfected with gelsolin cDNA were infected with Listeria. The C5 cell line containing 2.25 times control levels of gelsolin supported significantly higher velocities of bacterial movement than did control fibroblasts (mean +/- standard error of the mean, 0.09 +/- 0.003 micro(m)/s [n = 176] versus 0.05 +/- 0.003 micro(m)/s [n = 65]). The rate of disassembly of the Listeria-induced actin filament rocket tail was found to be independent of gelsolin content. Therefore, if increases in gelsolin content result in increases in Listeria-induced rocket tail assembly rates, a positive correlation between gelsolin content and tail length would be expected. BODIPY-phalloidin staining of four different stably transfected NIH 3T3 fibroblast cell lines confirmed this expectation (r = 0.92). Rocket tails were significantly longer in cells with a high gelsolin content. Microinjection of gelsolin 1/2 (consisting of the amino-terminal half of native gelsolin) also increased bacterial velocity by more than 2.2 times. Microinjection of CapG had no effect on bacterial movement. Cultured skin fibroblasts derived from gelsolin-null mice were capable of supporting intracellular Listeria motility at velocities comparable to those supported by wild-type skin fibroblasts. These experiments demonstrated that the surface of Listeria contains a polymerization zone that can block the barbed-end-capping activity of both gelsolin and CapG. The ability of Listeria to uncap actin filaments combined with the severing activity of gelsolin can accelerate actin-based motility. However, gelsolin is not absolutely required for the actin-based intracellular movement of Listeria because its function can be replaced by other actin regulatory proteins in gelsolin-null cells, demonstrating the functional redundancy of the actin system.

3T3 Cells↗
PubMed1988

Decreased plasma gelsolin levels in patients with Plasmodium falciparum malaria: a consequence of hemolysis?

Mammalian plasma contains a high-affinity actin-binding protein, plasma gelsolin, that severs actin filaments. Destruction of erythrocytes could result in the release of erythrocyte cytoskeletal actin into the plasma where it could bind to gelsolin. If the clearance of actin-gelsolin complexes exceeds its synthesis, lowering of the plasma gelsolin concentration might follow. To test this hypothesis, we measured plasma gelsolin levels in patients with falciparum malaria, a disease where at least part of the hemolysis takes place in the intravascular space and that is usually not accompanied by dysfunction of other organs. Two functional gelsolin assays showed that the mean plasma gelsolin concentration of 18 Nigerian children with Plasmodium falciparum malaria was less than 50% (P less than .001) of healthy Nigerian control subjects tested at the same time. Patients with pneumonia and febrile seizures also had depressed gelsolin levels, which indicates that factors other than hemolysis can lower gelsolin concentrations. Gelsolin levels were measured in 11 patients from The Gambia with P falciparum malaria before and approximately 3 weeks after treatment. In all cases the gelsolin level increased after treatment. To confirm the hypothesis that hemolysis can result in a lowering of plasma gelsolin levels, hemolysis was induced in rabbits, either acutely (by the injection of human serum) or subacutely (by the administration of phenylhydrazine). A fall in plasma gelsolin levels was seen, the rate of fall differing with the extent of hemolysis. Affinity adsorption of plasma from animals undergoing acute hemolysis with Sepharose beads coupled to the actin-binding protein DNase I, followed by immunoblotting of adherent proteins with antiactin antiserum demonstrated the presence of actin in circulating rabbit plasma. These studies suggest that under some conditions components of the red cell cytoskeleton are exposed to plasma proteins and that accelerated clearance of actin-gelsolin complexes may explain in part the depressed plasma gelsolin levels seen in patients with falciparum malaria.

Actins↗
PubMed1999

A role for gelsolin in stress fiber-dependent cell contraction.

Gelsolin is an abundant actin binding protein that mediates the rapid remodeling of cortical actin filaments through severing, capping, and nucleating activities. Most of the attention on the intracellular function of gelsolin has focused on the remodeling of the cortical actin meshwork but the localization of gelsolin to other regions of the cell suggests that it may have other important functions elsewhere. In cultured fibroblasts, gelsolin is heavily enriched in stress fibers, where its function in these contractile organelles is unknown. To study gelsolin function during stress fiber formation and cell contraction, we first assessed gelsolin levels in stress fiber preparations from lysophosphatidic acid (LPA)-treated human fibroblasts. LPA induced a large, time-dependent, calcium-independent increase of actin, gelsolin, alpha-actinin, and tropomyosin in stress fiber preparations. A microinjected gelsolin antibody that inhibits severing by gelsolin reduced stress fibers. Anti-sense-transfected gelsolin-depleted 3T3 cell lines treated with LPA after serum starvation required approximately 6 h to form stress fibers and focal adhesions, in contrast to control lines transfected with vector only, which formed stress fibers 15 min after addition of LPA. In cells microinjected with the gelsolin antibody that inhibits severing, Mg-ATP-induced cell contraction was greatly reduced in approximately 90% of injected cells compared to cells injected with an irrelevant antibody. Gelsolin-depleted cells were incapable of collagen gel contraction and showed no apparent Mg-ATP-induced cell contraction compared to cell lines transfected with vector only. The involvement of gelsolin in cell contraction and remodeling of collagen gels suggests a novel role for gelsolin in stress fiber-dependent cell function.

3T3 Cells↗
PubMed1995

Functions of [His321]gelsolin isolated from a flat revertant of ras-transformed cells.

A mutant gelsolin, [His321]gelsolin, was isolated from R1, a flat revertant of human activated c-Ha-ras oncogene-transformed NIH/3T3 cells (EJ-NIH/3T3) produced by ethylmethanesulfonate treatment. [His321]Gelsolin has a histidine instead of a proline residue at position 321 and suppresses the tumorigenicity of EJ-NIH/3T3 cells when it is constitutively expressed [Müllauer, L., Fujita, H., Ishizaki, A. & Kuzumaki, N. (1993) Oncogene 8, 2531-2536]. To investigate the biochemical consequences of the amino acid substitution of His321, we expressed the [His321]gelsolin and wild-type gelsolin in Escherichia coli, purified them, and analyzed their effects on actin, polyphosphoinositol lipids and phospholipase C. [His321]Gelsolin has decreased actin-filament-severing activity and increased nucleating activity compared with wild-type gelsolin in vitro. Furthermore, compared to wild-type gelsolin both nucleation and severing by [His321]gelsolin are inhibited more strongly by the phosphoinositol lipids phosphatidylinositol 4-phosphate (PtdInsP) and phosphatidylinositol 4,5-bisphosphate (PtdInsP2). In addition, [His321]gelsolin inhibits PtdInsP2 hydrolysis by phospholipase C gamma 1 more strongly than wild-type gelsolin in vitro because of its higher binding capacity for phosphoinositol lipid. Gelsolin has six homologous amino acid repeats called S1-S6. Our results suggest that the segment S3 which contains the mutation is functionally relevant for regulation of gelsolin's activities even though the relevant actin-binding domains are in segments 1, 2, and 4-6, and that the region around the residue 321 may contain a phosphoinositol-lipid-binding site. Altered functions of [His321]gelsolin might be important for the loss of tumorigenicity of the ras-transformed cells.

3T3 Cells↗
PubMed1981

Ca2+ control of actin filament length. Effects of macrophage gelsolin on actin polymerization.

Gelsolin complexes with calcium (gelsolin-Ca2+) binds to the ends of actin filaments to which monomers add preferentially during elongation. It forms a stable complex with actin in a low ionic strength solution which does not normally favor the polymerization of actin. Gelsolin-Ca2+ increases the rate of nucleation of actin which precedes polymerization, but decreases the rate of elongation of the filaments. The final average length of filaments formed in the presence of gelsolin-Ca2+ is shorter and the equilibrium monomer concentration increases relative to actin polymerized in the absence of gelsolin-Ca2+. Gelsolin-Ca2+ also increases the number of actin filaments because the magnitude of the increase in monomer concentration is disproportionately small compared with the reduction in polymer length. In these respects, the population of actin filaments formed during polymerization in the presence of gelsolin-Ca2+ is similar to that resulting from the action of gelsolin on previously assembled actin filaments (Yin, H. L., Zaner, K. S., and Stossel, T. P. (1980) J. Biol. Chem. 255, 9494-9500). The calcium-dependent shortening of ects, the population of actin filaments formed during polymerization in the presence of gelsolin-Ca2+ is similar to that resulting from the action of gelsolin on previously assembled actin filaments (Yin, H. L., Zaner, K. S., and Stossel, T. P. (1980) J. Biol. Chem. 255, 9494-9500). The calcium-dependent shortening of ects, the population of actin filaments formed during polymerization in the presence of gelsolin-Ca2+ is similar to that resulting from the action of gelsolin on previously assembled actin filaments (Yin, H. L., Zaner, K. S., and Stossel, T. P. (1980) J. Biol. Chem. 255, 9494-9500). The calcium-dependent shortening of actin filaments is the primary mechanism for the dissolution of an actin gel by gelsolin. Therefore, the ability of gelsolin to produce short filaments irrespective of the initial state of assembly of the actin offers flexibility for controlling the network structure of the cytoplasm in which either the monomeric or polymeric form of actin molecules might predominate at different times.

Actins↗
PubMed1984

Structure and biosynthesis of cytoplasmic and secreted variants of gelsolin.

Gelsolin is an actin-fragmenting cytoplasmic protein. A functionally similar protein has also been identified in plasma. We have compared the structure of the cytoplasmic and plasma forms of gelsolin and examined their biosynthetic relationships. Plasma gelsolin is larger than cytoplasmic gelsolin (Mr 93,000 versus 90,000, respectively) and is more positively charged. Partial amino acid sequencing analyses show that the two gelsolins share a common 29 amino acid sequence which lies at the NH2-terminal end of cytoplasmic gelsolin and spans residues 26-55 of plasma gelsolin. Compared with cytoplasmic gelsolin, plasma gelsolin contains an additional peptide of 25 amino acids at its NH2 terminus. The human hepatoma-derived cell line, HepG2, synthesizes both the 90-kDa and the 93-kDa gelsolins but secretes only the 93-kDa form. Pulse-chase experiments demonstrate that the rate of disappearance of the 93-kDa gelsolin from the cells corresponds with the rate of appearance of the 93-kDa gelsolin in the medium, whereas the rate of disappearance of the 90-kDa gelsolin is independent of and slower than that of the secreted plasma protein. We conclude that cytoplasmic and plasma gelsolins are structurally similar but not identical, that after synthesis these proteins are processed independently, and that the fate of each is distinct.

Amino Acid Sequence↗
PubMed2005

Loss of Gelsolin expression in human ovarian carcinomas.

The ubiquitously expressed actin-binding protein, gelsolin, is known to play a role in the modulation of the actin network and in the regulation of cell growth and cell motility. In the present study, we analysed the expression of gelsolin in 241 matched cDNA pairs from human normal and tumour tissues using a Cancer Profiling Array. We found a decreased expression of gelsolin in cancer tissue from female reproductive organs, including the ovary. On a protein level, we examined the expression of gelsolin in human ovarian cancer cell lines and in a set of 110 cases of human benign and malignant ovarian tumours. Low levels of gelsolin protein were observed in four of six ovarian carcinoma cell lines, in contrast to its expression in normal ovarian surface epithelial cells. In addition, we found a reduced expression of gelsolin in borderline tumours and ovarian carcinomas compared with the epithelium of normal ovaries and benign adenomas. Decreased gelsolin expression was associated with poorly differentiated carcinomas (p=0.014). No significant association between gelsolin expression and other clinicopathological markers or patient survival could be established. In addition, we investigated the growth regulatory function of gelsolin in human ovarian cancer cell lines using cDNA transfections. Re-expression of gelsolin in OAW42 and ES-2 cells resulted in a suppression of tumour cell survival in vitro. To explore the mechanism responsible for the downregulation of gelsolin expression in ovarian carcinoma cells, we treated cells with inhibitors of DNA methylation and histone deacetylation. We observed an upregulation of gelsolin in ovarian cancer cells after treatment with both types of inhibitor. Our results suggest that gelsolin might be involved in the growth regulation of human ovarian cancer.

Adult↗
PubMed1997

Gelsolin activates DNase I in vitro and cystic fibrosis sputum.

Because actin can form a complex in vitro containing both gelsolin and DNase I, gelsolin and DNase I have been assumed to bind independently to actin. Although this assumption is consistent with the known crystalline structures of gelsolin with one actin and of actin with DNase I, which suggest that the binding sites on actin for both gelsolin and DNase I are distinct and separate, we propose that a second actin binding site on gelsolin competes with DNase I for actin. Since actin is an inhibitor of DNase I, competition at the second binding site results in activation of DNase I by gelsolin. Covalent cross-linking experiments confirm that DNase I prevents dimerization of actin by gelsolin, consistent with displacement of one actin from gelsolin by DNase I. Activation of DNase I by gelsolin is a novel function for a cytoskeletal protein and could have broad implications for biology, such as a role in initiating apoptosis. These results also may explain why both gelsolin and DNase I decrease sputum viscosity in cystic fibrosis (CF). While the activity of DNase I had originally been attributed to fragmentation of DNA, subsequent data suggested that both gelsolin and DNase I may affect viscosity by depolymerizing filamentous actin. The current results alternatively suggest aht dissociation of the actin-DNase I complex by gelsolin in CF sputum results in activation of the nuclease activity of constitutive DNase I. The nuclease activity of DNase I alone is therefore sufficient to explain the effects of both gelsolin and DNase I on CF sputum.

Actins↗
PubMed2000

Calcium regulation of gelsolin and adseverin: a natural test of the helix latch hypothesis.

The gelsolin family of actin filament binding proteins have highly homologous structures. Gelsolin and adseverin, also known as scinderin, are the most similar members of this family, with adseverin lacking a C-terminal helix found in gelsolin. This helix has been postulated to serve as a calcium-sensitive latch, keeping gelsolin inactive. To test this hypothesis, we have analyzed the kinetics of severing by gelsolin, adseverin, and a gelsolin truncate which lacks the C-terminal latch. We find that the relationship between severing rate and calcium ion concentration differs between gelsolin and adseverin, and suggest that calcium controls one rate-limiting step in the activation of adseverin and two in the activation of gelsolin. In contrast, both proteins are activated equally by protons, and have identical severing kinetics at pHs below 6.3. The temperature sensitivity of severing by adseverin and gelsolin is remarkably different, with gelsolin increasing its severing rate 8-fold per 10 degrees C increase in temperature and adseverin increasing its rate only 2-fold per 10 degrees C increase in temperature. Analysis of the gelsolin construct lacking the C-terminal helix demonstrates that this helix is responsible for the regulatory differences between gelsolin and adseverin. These results support the C-terminal latch hypothesis for the calcium ion activation of gelsolin.

Actins↗
PubMed2006

A direct interaction with calponin inhibits the actin-nucleating activity of gelsolin.

Gelsolin and calponin are well-characterized cytoskeletal proteins that are abundant and widely expressed in vertebrate tissues. It is also becoming apparent, however, that they are involved in cell signalling. In the present study, we show that gelsolin and calponin interact directly to form a high-affinity (K(d)=16 nM) 1:1 complex, by the use of fluorescent probes attached to both proteins, by affinity chromatography and by immunoprecipitation. These methods show that gelsolin can form high-affinity complexes with two calponin isoforms (basic h1 and acidic h3). They also show that gelsolin binds calponin through regions that have been identified previously as being calponin's actin-binding sites. Moreover, gelsolin does not interact with calponin while calponin is bound to F-actin. Reciprocal experiments to find calponin-binding sites on gelsolin show that these are in both the N- and C-terminal halves of gelsolin. Calponin has minimal effects on actin severing by gelsolin. In contrast, calponin markedly affects the nucleation activity of gelsolin. The maximum inhibition of nucleation by gelsolin was 50%, which was achieved with a ratio of two calponins for every gelsolin. Thus the interaction of calponin with gelsolin may play a regulatory role in the formation of actin filaments through modulation of gelsolin's actin-binding function and through the prevention of calponin's actin-binding activities.

Actins↗
PubMed1996

Dependence of fibroblast migration on actin severing activity of gelsolin.

Gelsolin nucleates actin filament assembly, blocks the fast-exchanging ends of actin filaments, and severs filaments, processes that may play an important role in cell motility. We studied the relationship between cell migration, gelsolin content, and actin severing activity in human gingival fibroblasts. These cells were keratin negative and desmin negative but expressed vimentin and myosin II. Cells were separated by their ability to migrate in response to a chemoattractant stimulus. Northern analysis of mRNA, [35S]methionine incorporation into immunoprecipitated gelsolin, immunoblots of cell lysates, and quantitative confocal microscopy showed 1.4-2-fold higher levels of gelsolin in nonmigrant compared with migrant cells. Because the concentration of intracellular gelsolin did not appear to be a central determinant of cell migration, we assessed its requirement for motility. Cells that were electroinjected with a gelsolin antibody that inhibits actin severing by gelsolin in vitro showed a 72% reduction of the number of migrant cells compared with controls treated with an irrelevant antibody. Cells that were electroinjected with free gelsolin exhibited a 33% increase in migration compared with cells electroinjected with bovine serum albumin. Compared with nonmigrant cells, migrant cells contained abundant free gelsolin and exhibited gelsolin-dependent F-actin severing activity, which required Ca2+. Serum stimulation of cell migration required increases in [Ca2+]i because incubation with 3 microM 1,2-bis-(o-aminophenoxy)-ethane-N,N, N',N'-tetraacetic acid tetra(acetoxymethyl)-ester blocked calcium flux and cell migration. Serum also stimulated the recruitment of gelsolin into the supernatants of lysates from migrant but not from nonmigrant cells. In fibroblasts, gelsolin concentration alone does not apparently determine migratory capacity. Instead, the Ca2+-dependent actin severing activity of free gelsolin appears to be a major determinant of cell migration.

Actin Cytoskeleton↗
PubMed2001

Serum withdrawal induces a redistribution of intracellular gelsolin towards F-actin in NIH 3T3 fibroblasts preceding apoptotic cell death.

The intracellular distribution of gelsolin in NIH 3T3 cells was examined by immunostaining using affinity-purified polyclonal gelsolin antibodies before and after induction of apoptosis by serum withdrawal. Serum deprivation induced detachment of an increasing number of NIH 3T3 cells, but also apoptosis in attached cells as verified morphologically by chromatin condensation, nuclear fragmentation and labelling of their periphery by FITC-annexin V. Ongoing apoptosis was also demonstrated by activation of caspase-3 activity and chromatin cleavage into high-molecular-mass fragments, although no internucleosomal chromatin degradation (DNA-ladder formation) was detected. When cells were maintained in the presence of 10% foetal calf serum, gelsolin immunoreactivity was evenly distributed in the cytoplasm. No obvious co-localisation of gelsolin and the actin-containing stress fibres was detected under these conditions. At day one after serum withdrawal, a redistribution of gelsolin to actin filaments was detected within a few attached cells by double fluorescence staining. The number of cells exhibiting this redistribution increased at days two to four. In addition, the stress fibres increased in thickness and their length was continuously reduced. At day four, many cells contained shortened stress fibres, which had lost their longitudinal orientation. Additionally, the cytoplasm of a number of attached cells was highly condensed around their nuclei and a homogenous distribution of both gelsolin and actin was detected in the remaining cytoplasmic rim. Up to day two, these effects were reversible after re-addition of serum to attached cells. A similar redistribution of gelsolin immunore-activity was observed after induction of apoptosis by cycloheximide, but not after initiation of necrosis by hydrogen peroxide. In NIH 3T3 cells no alteration in the expression of gelsolin at the level of protein (Western blot) or specific mRNA (Northern blot) was observed after serum withdrawal. Using Western blotting, no proteolysis of gelsolin was detected up to day 4, although caspase-3 activity was found to have increased fivefold after serum withdrawal. These results suggested that in these cells F-actin severing might occur in the absence or advance of gelsolin cleavage by caspases. Intact gelsolin on its own may be sufficient for the dissolution of the microfilaments, since micro-injection of gelsolin into primary bovine lens cells led to a transient disappearance of the stress fibres and to a reduction of their attachment area to the substratum. In NIH 3T3 cells similar effects of micro-injected gelsolin were only observed at day one after serum withdrawal.

3T3 Cells↗
PubMed1993

Horse plasma gelsolin labelled with fluorescein isothiocyanate responds to calcium and actin.

Reaction between horse plasma gelsolin and fluorescein-5-isothiocyanate (FITC) resulted in incorporation of 4.8 +/- 0.6 fluorescein groups/gelsolin molecule. The sites of modification were not clustered in any one portion of the gelsolin polypeptide chain; all major peptides produced by proteolytic digestion with alpha-chymotrypsin exhibited a fluorescence characteristic of fluorescein. FITC-gelsolin has a peptide-backbone circular dichroism spectrum at 20 degrees C that is indistinguishable from that of native gelsolin, but FITC-gelsolin is considerably more resistant than native gelsolin to thermally induced precipitation. FITC-gelsolin is fully able to carry out severing of F-actin filaments, the prime function of gelsolin in plasma. An opening up of the structure of gelsolin on binding Ca2+ is evident from an increased susceptibility of FITC-gelsolin to quenching by I-. Ca2+ dependence of the interaction between gelsolin and actin is evident in titrations both of intensity and polarization of the fluorescence of FITC-gelsolin solutions. A Ca(2+)-sensitive interaction between gelsolin and tropomyosin also is observed.

Actins↗
PubMed2003

Fibrillogenesis in gelsolin-related familial amyloidosis.

To clarify the mechanisms involved in amyloid formation in Finnish-type familial amyloidosis (FAF), we have tested the in vitro fibrillogenicity of synthetic wild-type and mutated gelsolin peptide analogs and studied the fragmentation patterns of gelsolin in the circulation of FAF patients with the Asn-187 or Tyr-187 gelsolin mutation. Fibril formation of synthetic peptides having sequence homology with wild-type or mutant gelsolins was monitored by Congo-red staining and polarization microscopy, negative staining electron microscopy and quantitative thioflavine-T fluorometry. Immunoblotting with anti-gelsolin and amyloid-specific antibodies and sequence analyses were used to study the fragmentation pattern of gelsolin. Ultrastructurally amyloid-like fibrils were formed from mutant Asn-187 and Tyr-187 gelsolin peptides. Fluorometric analysis revealed highly accelerated fibril formation from the mutant peptides as compared with the corresponding wild-type peptides. Addition of mercaptoethanol alone or in combination with dithiotreitol tended to enhance fibril formation of the 9-mer and 11-mer Asn peptides. Blocking of the C-terminal carboxyl of the mutant Asn-187 gelsolin182-192 peptide by amidation increased amyloidogenicity. The Tyr-187 gelsolin mutation, corresponding to the naturally occurring mutation in the Danish subtype of FAF, required acidic conditions to form fibrils meeting the criteria of amyloid. In FAF patients, in addition to the full-sized gelsolin, a series of lower-molecular mass C-terminal fragments of gelsolin (70,000-45,000 Da) was found in the circulation. In homozygous FAF(Asn-187) the 65-kDa fragment containing the amyloid forming region and the 55-kDa fragment, devoid of that region, was the major gelsolin species in the plasma. The results indicate that the 65-kDa gelsolin fragment derived by alpha-gelsolinase cleavage at the mutation-induced novel proteolysis site Arg172-Ala173 represents the putative circulating precursor protein of tissue amyloid in FAF and that the Asp187Asn/Tyr substitution in gelsolin creates a conformation that is highly fibrillogenic.

Amino Acid Sequence↗
PubMed1987

Quantitative measurement of plasma gelsolin and its incorporation into fibrin clots.

Gelsolin is a newly recognized actin-binding protein of plasma that severs actin filaments. The concentration of plasma gelsolin was measured by three independent methods: an enzyme-linked immunosorbent assay (ELISA) and two functional assays based on the ability of gelsolin to accelerate the polymerization of actin (a "nucleating" assay) or to sever preformed actin filaments (a "cutting" assay). The gelsolin level in 56 samples of human plasma ranged between 100 and 330 micrograms/ml. The mean plasma concentrations measured by the different assays were ELISA, 207 micrograms/ml; nucleating assay, 233 micrograms/ml; cutting assay, 247 micrograms/ml. The mean serum level of gelsolin was found to be 24% lower than that of plasma when paired samples were examined (183 micrograms/ml). The difference between plasma and serum gelsolin concentrations can be accounted for by a direct interaction between gelsolin and fibrin as shown by the binding of radiolabeled gelsolin to clots made from purified fibrinogen. Further, unlabeled gelsolin inhibits the binding of the labeled species to fibrin, but hemoglobin and albumin do not. Fibrin oligomers, but not fibrinogen, alter the sedimentation characteristics of radiolabeled gelsolin in sucrose gradients. The amount of gelsolin incorporated into the clot is increased if the clots are made in the presence of actin filaments or fibronectin. Thus, serum levels of gelsolin are lower than plasma levels because of an interaction between gelsolin and fibrin clots.

Actins↗