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H L Yin

Publications and source records attributed to H L Yin.

At least 73 records · Page 4Linked to original sources
PubMed1990

Severin is a gelsolin prototype.

A number of Ca2(+)-activated actin filament severing proteins have been identified in eukaryotic cells of diverse lineages. Gelsolin and villin, with molecular mass of about 80-90 kDa, and severin and fragmin, with molecular mass of about 40 kDa, have been isolated from vertebrates and invertebrates, respectively. We report here a direct comparison of the functional properties of gelsolin and severin, and the finding that the actin filament severing activity of severin, like that of gelsolin, is inhibited by polyphosphoinositides. However, severin does not nucleate actin filament assembly as well as gelsolin. These characteristics are very similar to those ascribed to the NH2-terminal half of gelsolin, supporting the idea that they are evolutionarily related. Regulation of severin by polyphospholipids raises the possibility that it may participate in agonist-stimulated regulation of the actin cytoskeleton in Dictyostelium discoideum.

Actins↗
PubMed1989

End-label fingerprintings show that the N- and C-termini of actin are in the contact site with gelsolin.

Gelsolin was cleaved by chymotrypsin or thermolysin into an N-terminal Mr 45,000 fragment (45N) and a C-terminal Mr 38,000 fragment (38C). The N-terminal half was further cleaved into two fragments with Mr 17,000 (17N) and Mr 28,000 (28N). These fragments were complexed with actin and cross-linked with 1-ethyl-3-[3-(dimethylamino)prophyl]carbodiimide (EDC) to introduce covalent bonds into their contact sites. The location of these bonds was mapped along the actin sequence by end-label fingerprinting with highly sensitive probes for the N- and C-termini of actin. The mapping studies revealed that two gelsolin N-terminal fragments (17N and 28N) were cross-linked with the actin C-terminal segment. The result indicates that the actin N- and C-terminal segments are in the binding site of gelsolin.

Actins↗
PubMed1989

Identification of critical functional and regulatory domains in gelsolin.

Gelsolin can sever actin filaments, nucleate actin filament assembly, and cap the fast-growing end of actin filaments. These functions are activated by Ca2+ and inhibited by polyphosphoinositides (PPI). We report here studies designed to delineate critical domains within gelsolin by deletional mutagenesis, using COS cells to secrete truncated plasma gelsolin after DNA transfection. Deletion of 11% of gelsolin from the COOH terminus resulted in a major loss of its ability to promote the nucleation step in actin filament assembly, suggesting that a COOH-terminal domain is important in this function. In contrast, derivatives with deletion of 79% of the gelsolin sequence exhibited normal PPI-regulated actin filament-severing activity. Combined with previous results using proteolytic fragments, we deduce that an 11-amino acid sequence in the COOH terminus of the smallest severing gelsolin derivative identified here mediates PPI-regulated binding of gelsolin to the sides of actin filaments before severing. Deletion of only 3% of gelsolin at the COOH terminus, including a dicarboxylic acid sequence similar to that found on the NH2 terminus of actin, resulted in a loss of Ca2+-requirement for filament severing and monomer binding. Since these residues in actin have been implicated as potential binding sites for gelsolin, our results raise the possibility that the analogous sequence at the COOH terminus of gelsolin may act as a Ca2+-regulated pseudosubstrate. However, derivatives with deletion of 69-79% of the COOH-terminal residues of gelsolin exhibited normal Ca2+ regulation of severing activity, establishing the intrinsic Ca2+ regulation of the NH2-terminal region. One or both mechanisms of Ca2+ regulation may occur in members of the gelsolin family of actin-severing proteins.

Actins↗
PubMed1988

Muscle is the major source of plasma gelsolin.

Gelsolin, a Ca2+- and polyphosphoinositide-regulated actin-binding protein, is unique among vertebrate proteins in being both cytoplasmic and secreted. Plasma gelsolin, present at greater than 200 micrograms/ml in human plasma, may have a protective function by promoting the clearance of actin filaments released during tissue injury. Although there is evidence that smooth muscle tissues and HepG2 cells synthesize plasma gelsolin, the predominant secretory source is hitherto unknown. We report here that skeletal, cardiac, and smooth muscles have large amounts of plasma gelsolin mRNA and devote 0.5-3% of their biosynthetic activity to plasma gelsolin, whereas liver makes relatively little. Since skeletal muscle accounts for a large fraction of body mass and total protein synthesis, it is the major source of plasma gelsolin.

Animals↗
PubMed1988

The organization and regulation of the macrophage actin skeleton.

To move, leukocytes extend portions of their cortical cytoplasm as pseudopods. These pseudopods are filled with a three-dimensional actin filament skeleton, the reversible assembly of which in response to receptor stimulation is thought to play a major role in providing the mechanical force for these protrusive movements. The organization of this actin skeleton occurs at different levels within the cell, and a number of macrophage proteins have been isolated and shown to affect the architecture, assembly, stability, and length of actin filaments in vitro. The architecture of cytoplasmic actin is regulated by proteins that cross-link filaments in higher-order structures. Actin-binding protein plays a major role in defining network structure by cross-linking actin filaments into orthogonal networks. Gelsolin may have a central role in regulating network structure. It binds to the sides of actin filaments and severs them, and binds the "barbed" filament end, thereby blocking monomer addition at this end. Gelsolin is activated to bind actin filaments by microM calcium. Dissociation of gelsolin bound on filament ends occurs in the presence of the polyphosphoinositides, PIP and PIP2. Calcium and PIP2 have been shown to be intracellular messengers of cell stimulation.

Actins↗
PubMed1988

Genomic organization and biosynthesis of secreted and cytoplasmic forms of gelsolin.

Gelsolin is an actin regulatory protein which is unique among vertebrates in that it is found as both an intrinsic cytoplasmic protein and as a secreted plasma protein. We demonstrate that plasma and cytoplasmic gelsolins are derived by alternative transcriptional initiation sites and message processing from a single gene 70 kb long, containing at least 14 exons. Their message and amino acid sequences are identical except at the 5' end/NH2 termini. The cytoplasmic-specific 5' sequence is derived from two exons that encode untranslated sequence, while the plasma message-specific 5' sequence is derived from a single exon that encodes untranslated sequence, the signal peptide, and the first 21 residues of the plasma protein. The two transcriptional initiation sites are separated by greater than or equal to 32 kb. Biosynthetic and RNase protection studies indicate that a number of cell types make both plasma and cytoplasmic gelsolin in widely varying amounts and ratios.

Base Sequence↗
PubMed1988

Identification of a polyphosphoinositide-modulated domain in gelsolin which binds to the sides of actin filaments.

Gelsolin is a Ca2+- and polyphosphoinositide-modulated actin-binding protein which severs actin filaments, nucleates actin assembly, and caps the "barbed" end of actin filaments. Proteolytic cleavage analysis of human plasma gelsolin has shown that the NH2-terminal half of the molecule severs actin filaments almost as effectively as native gelsolin in a Ca2+-insensitive but polyphosphoinositide-inhibited manner. Further proteolysis of the NH2-terminal half generates two unique fragments (CT14N and CT28N), which have minimal severing activity. Under physiological salt conditions, CT14N binds monomeric actin coupled to Sepharose but CT28N does not. In this paper, we show that CT28N binds stoichiometrically and with high affinity to actin subunits in filaments, suggesting that it preferentially recognizes the conformation of polymerized actin. Analysis of the binding data shows that actin filaments have one class of CT28N binding sites with Kd = 2.0 X 10(-7) M, which saturates at a CT28N/actin subunit ratio of 0.8. Binding of CT28N to actin filaments is inhibited by phosphatidylinositol 4,5-bisphosphate micelles. In contrast, neither CT14N nor another actin-binding domain located in the COOH-terminal half of gelsolin form stable stoichiometric complexes with actin along the filaments, and their binding to actin monomers is not inhibited by PIP2. Based on these observations, we propose that CT28N is the polyphosphoinositide-regulated actin-binding domain which allows gelsolin to bind to actin subunits within a filament before serving.

Actin Cytoskeleton↗
PubMed1988

The structure of the macrophage actin skeleton.

The actin skeleton of the macrophage consists of a three-dimensional network of actin filaments and associated proteins. The organization of this multiprotein structure is regulated at several levels in cells. Receptor stimulation induces a massive actin polymerization at the cell cortex, changes in cell shape and active cellular movements. Gelsolin may have a pivotal role in restructuring the actin skeleton in response to agonist stimulation, as the activity of this potent actin-modulating protein is regulated by both Ca2+ and polyphosphoinositides. Micromolar concentrations of Ca2+ activate gelsolin to bind to the sides of actin filaments, sever, and cap the filament end. Polyphosphoinositides, in particular PIP and PIP2, release gelsolin from the filament ends. A structure-function analysis of gelsolin indicates that its N-terminal half is primarily responsible for severing actin filaments, and elucidates mechanisms by which Ca2+ and phospholipid may regulate gelsolin functions. The ultrastructure of actin filaments in the macrophage cortical cytoplasm is regulated, to a large extent, by the actin cross-linking protein, actin-binding protein (ABP) which defines filament orthogonality.

Actins↗
PubMed1987

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.

Actins↗
PubMed1987

Reversibility of gelsolin/actin interaction in macrophages. Evidence of Ca2+-dependent and Ca2+-independent pathways.

We have developed an immunoadsorption technique for quantitating EGTA-resistant gelsolin/actin complexes in macrophages extracted with Triton X-100. We report here that the proportion of gelsolin complexed irreversibly to actin is low in freshly harvested macrophages. The amount of the EGTA-resistant complex increases spontaneously during incubation of the cells in suspension at 37 degrees C, or after exposure to the Ca2+ ionophore ionomycin. On the other hand, exposure of suspended cells to the chemotactic oligopeptide, FMLP, or plating of the cells onto tissue culture dishes causes the EGTA-resistant complex to dissociate rapidly. Plating even prevents Ca2+ ionomycin-treated cells with elevated intracellular Ca2+ from inducing this complex. Therefore, our results suggest that macrophages possess a mechanism, not directly involving Ca2+, for dissociating actin/gelsolin EGTA-resistant complexes. This mechanism may be a Ca2+-independent signal for leukocyte activation.

Actins↗
PubMed1987

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.

Actins↗
PubMed1986

The actin filament-severing domain of plasma gelsolin.

Gelsolin, a multifunctional actin-modulating protein, has two actin-binding sites which may interact cooperatively. Native gelsolin requires micromolar Ca2+ for optimal binding of actin to both sites, and for expression of its actin filament-severing function. Recent work has shown that an NH2-terminal chymotryptic 17-kD fragment of human plasma gelsolin contains one of the actin-binding sites, and that this fragment binds to and severs actin filaments weakly irrespective of whether Ca2+ is present. The other binding site is Ca2+ sensitive, and is found in a chymotryptic peptide derived from the COOH-terminal two-thirds of plasma gelsolin; this fragment does not sever F-actin or accelerate the polymerization of actin. This paper documents that larger thermolysin-derived fragments encompassing the NH2-terminal half of gelsolin sever actin filaments as effectively as native plasma gelsolin, although in a Ca2+-insensitive manner. This result indicates that the NH2-terminal half of gelsolin is the actin-severing domain. The stringent Ca2+ requirement for actin severing found in intact gelsolin is not due to a direct effect of Ca2+ on the severing domain, but indirectly through an effect on domains in the COOH-terminal half of the molecule to allow exposure of both actin-binding sites.

Actin Cytoskeleton↗
PubMed1985

Isolation and properties of two actin-binding domains in gelsolin.

Gelsolin is a Ca2+-sensitive 90-kDa protein which regulates actin filament length. A molecular variant of gelsolin is present in plasma as a 93-kDa protein. Functional studies have shown that gelsolin contains two actin-binding sites which are distinct in that after Ca2+-mediated binding, removal of free Ca2+ releases actin from one site but not from the other. We have partially cleaved human plasma gelsolin with alpha-chymotrypsin and identified two distinct actin-binding domains. Peptides CT17 and CT15, which contain one of the actin-binding domains, bind to actin independently of Ca2+; peptides CT54 and CT47, which contain the other domain, bind to actin reversibly in response to changes in Ca2+ concentration. These peptides sequester actin monomers inhibiting polymerization. Unlike intact gelsolin, neither group of peptides nucleates actin assembly or forms stable filament end caps. CT17 and CT15 can however sever actin filaments. Amino acid sequence analyses place CT17 at the NH2 terminus of gelsolin and CT47 at the carboxyl-terminal two-thirds of gelsolin. Circular dichroism measurements show that Ca2+ induces an increase in the alpha-helical content of CT47. These studies provide a structural basis for understanding the interaction of gelsolin with actin and allow comparison with other Ca2+-dependent actin filament severing proteins.

Actins↗
PubMed1985

Effects of semi-dilute actin solutions on the mobility of fibrin protofibrils during clot formation.

Low concentrations of actin filaments (F-actin) inhibit the rate and extent of turbidity developed during polymerization of purified fibrinogen by thrombin. Actin incorporates into the fibrin clot in a concentration-dependent manner that does not reach saturation, indicating nonspecific trapping of actin filaments in the fibrin network. Actin does not retard activation of fibrinogen by thrombin, but rather the alignment of fibrin protofibrils into bundles which constitute the coarse clot. In contrast, equivalent F-actin concentrations have little or no effect on the turbidity of plasma clots. The difference is attributed to the presence of a plasma protein, gelsolin, that severs actin filaments. Purified gelsolin greatly reduces the effect of F-actin on the turbidity of a pure fibrin clot and decreases the fraction of actin incorporated by the clot. A calculation of the extent to which the gelsolin concentrations used in these experiments reduce the fraction of actin filaments which are long enough to impede each other's rotational diffusion indicates that it is the overlapping actin filaments which retard the association of fibrin protofibrils. The findings suggest that one role for the F-actin depolymerizing and particularly actin severing activities in blood is to prevent actin filaments released by tissue injury from interfering with the formation of coarse fibrin clots.

Actins↗