Crystal structures of the vitamin D-binding protein and its complex with actin: structural basis of the actin-scavenger system

Actin is the most abundant protein in eukaryotic cells, but its release from cells into blood vessels can be lethal, being associated with clinical situations including hepatic necrosis and septic shock. A homeostatic mechanism, termed the actin-scavenger system, is responsible for the depolymerization and removal of actin from the circulation. During the first phase of this mechanism, gelsolin severs the actin filaments. In the second phase, the vitamin D-binding protein (DBP) traps the actin monomers, which accelerates their clearance. We have determined the crystal structures of DBP by itself and complexed with actin to 2.1 A resolution. Similar to its homologue serum albumin, DBP consists of three related domains. Yet, in DBP a strikingly different organization of the domains gives rise to a large actin-binding cavity. After complex formation the three domains of DBP move slightly to "clamp" onto actin subdomain 3 and to a lesser extent subdomain 1. Contacts between actin and DBP throughout their extensive 3,454-A(2) intermolecular interface involve a mixture of hydrophobic, electrostatic, and solvent-mediated interactions. The area of actin covered by DBP within the complex approximately equals the sum of those covered by gelsolin and profilin. Moreover, certain interactions of DBP with actin mirror those observed in the actin-gelsolin complex, which may explain how DBP can compete effectively with gelsolin for actin binding. Formation of the strong actin-DBP complex proceeds with limited conformational changes to both proteins, demonstrating how DBP has evolved to become an effective actin-scavenger protein.     

Coordinated inhibition of actin-induced platelet aggregation by plasma gelsolin and vitamin D-binding protein

Actin is an abundant intracellular protein that is released into the blood during tissue injury and its injection into rats causes microthrombi to form in the vasculature. This report and others have shown that actin filaments are able to aggregate platelets in an adenosine diphosphate (ADP)-dependent manner. The effects on this process of two plasma actin-binding proteins, vitamin D-binding protein (DBP) and gelsolin, were examined separately and together. The addition of DBP, a monomer-binding protein, to actin filaments did not affect their ability to induce platelet aggregation. However, severing of actin filaments with gelsolin resulted in an increased degree of platelet aggregation. Preincubation of F-actin with both gelsolin and DBP resulted in a significant inhibition of aggregation. The effects of DBP and gelsolin on actin-induced aggregation paralleled their effects on exchange of actin-bound adenine nucleotides. DBP inhibited 1, N6- ethenoadenosine 5' triphosphate (epsilon-ATP) exchange with G-actin but not with F-actin. Gelsolin increased epsilon-ATP exchange with F-actin, which was largely abrogated by the addition of DBP. These results suggest that gelsolin's severing (and subsequent capping) of actin filaments not only results in an increase in the number of pointed filament ends but also in the dissociation of actin monomers containing ADP. Phalloidin, which stabilizes actin filaments while decreasing both monomer and nucleotide exchange, inhibited actin-induced aggregation, as well, indicating that depolymerization of actin filaments is not required to inhibit aggregation. Platelet activation by either G- or F- actin may thus be regulated by the local concentrations of the plasma actin-binding proteins gelsolin and DBP. Together, these proteins inhibit platelet aggregation in a manner that can be explained by their effects on actin's filament structure and the accessibility of its bound ADP. Depletion of DBP or gelsolin may allow actin released from injured tissues to stimulate purinergic receptors on platelets, and perhaps other cells, via its bound adenine nucleotides.     

Mini-thin filaments regulated by troponin-tropomyosin

Striated muscle thin filaments contain hundreds of actin monomers and scores of troponins and tropomyosins. To study the cooperative mechanism of thin filaments, "mini-thin filaments" were generated by isolating particles nearly matching the minimal structural repeat of thin filaments: a double helix of actin subunits with each strand approximately seven actins long and spanned by a troponin-tropomyosin complex. One end of the particles was capped by a gelsolin (segment 1-3)-TnT fusion protein (substituting for normal TnT), and the other end was capped by tropomodulin. EM showed that the particles were 46 +/- 9 nm long, with a knob-like mass attributable to gelsolin at one end. Average actin, tropomyosin, and gelsolin-troponin composition indicated one troponin-tropomyosin attached to each strand of the two-stranded actin filament. The minifilaments thus nearly represent single regulatory units of thin filaments. The myosin S1 MgATPase rate stimulated by the minifilaments was Ca2+-sensitive, indicating that single regulatory length particles are sufficient for regulation. Ca2+ bound cooperatively to cardiac TnC in conventional thin filaments but noncooperatively to cardiac TnC in minifilaments in the absence of myosin. This suggests that thin filament Ca2+-binding cooperativity reflects indirect troponin-troponin interactions along the long axis of conventional filaments, which do not occur in minifilaments. Despite noncooperative Ca2+ binding to minifilaments in the absence of myosin, Ca2+ cooperatively activated the myosin S1-particle ATPase rate. Two-stranded single regulatory units therefore may be sufficient for myosin-mediated Ca2+-binding cooperativity. Functional mini-thin filaments are well suited for biochemical and structural analysis of thin-filament regulation.     

Effects of actin filaments on fibrin clot structure and lysis.

The muscle and cytoskeletal protein actin is released from cells as a consequence of cell death and interacts with components of the hemostatic and fibrinolytic systems, including platelets, plasmin, and fibrin. We report here that incorporation of actin filaments into fibrin clots changes their viscoelastic properties by increasing their shear modulus at low deforming stresses and by nearly eliminating their tendency to become more rigid with increasing deformation (ie, exhibit strain-hardening). The viscoelastic effects depended on the length of the actin filaments as shown by the effects of the plasma filament-severing protein, gelsolin. Binding of actin to fibrin clots also varied with actin filament length. The plasma actin-binding proteins gelsolin and vitamin D-binding protein reduced, but did not eliminate, the incorporation of actin in the clot. Fluorescence microscopy showed a direct association of rhodamine-labeled actin filaments with the fibrin network. Incubation of clots containing long actin filaments in solutions containing physiologic concentrations of gelsolin (2 mumol/L) released 60% of the actin trapped in the clot. Reduction of the actin content of a fibrin clot by incubation in a gelsolin-containing solution resulted in an increased rate of clot lysis. The ability of plasma gelsolin to shorten actin filaments may therefore be of physiologic and potentially of therapeutic importance insofar as gelsolin-mediated diffusion of actin from the clot may restore the clot's rheologic properties and render it more sensitive to the lytic action of plasmin.     

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.     

Filament rigidity causes F-actin depletion from nonbinding surfaces

Proximity to membranes is required of actin networks for many key cell functions, including mechanics and motility. However, F-actin rigidity should hinder a filament''s approach to surfaces. Using confocal microscopy, we monitor the distribution of fluorescent actin near nonadherent glass surfaces. Initially uniform, monomers polymerize to create a depletion zone where F-actin is absent at the surface but increases monotonically with distance from the surface. At its largest, depletion effects can extend >35 μm, comparable with the average, mass-weighted filament length. Increasing the rigidity of actin filaments with phalloidin increases the extent of depletion, whereas shortening filaments by using capping protein reduces it proportionally. In addition, depletion kinetics are faster with higher actin concentrations, consistent with faster polymerization and faster Brownian-ratchet-driven motion. Conversely, the extent of depletion decreases with actin concentration, suggesting that entropy is the thermodynamic driving force. Quantitatively, depletion kinetics and extent match existing actin kinetics, rigidity, and lengths. However, explaining depletion profiles and concentration dependence (power-law of −1) requires modifying the rigid rod model. Within cells, surface depletion should slow membrane-associated F-actin reactions another ≈10-fold beyond hydrodynamically slowed diffusion of filaments (≈10-fold). In addition, surface depletion should cause membranes to bend spontaneously toward filaments. Such depletion principles underlie the thermodynamics of all surface-associated reactions with mechanical structures, ranging from DNA to filaments to networks. For various functions, cells must actively resist the thermodynamics of depletion.     

Single molecules of the bacterial actin MreB undergo directed treadmilling motion in Caulobacter crescentus

The actin cytoskeleton represents a key regulator of multiple essential cellular functions in both eukaryotes and prokaryotes. In eukaryotes, these functions depend on the orchestrated dynamics of actin filament assembly and disassembly. However, the dynamics of the bacterial actin homolog MreB have yet to be examined in vivo. In this study, we observed the motion of single fluorescent MreB-yellow fluorescent protein fusions in living Caulobacter cells in a background of unlabeled MreB. With time-lapse imaging, polymerized MreB [filamentous MreB (fMreB)] and unpolymerized MreB [globular MreB (gMreB)] monomers could be distinguished: gMreB showed fast motion that was characteristic of Brownian diffusion, whereas the labeled molecules in fMreB displayed slow, directed motion. This directional movement of labeled MreB in the growing polymer provides an indication that, like actin, MreB monomers treadmill through MreB filaments by preferential polymerization at one filament end and depolymerization at the other filament end. From these data, we extract several characteristics of single MreB filaments, including that they are, on average, much shorter than the cell length and that the direction of their polarized assembly seems to be independent of the overall cellular polarity. Thus, MreB, like actin, exhibits treadmilling behavior in vivo, and the long MreB structures that have been visualized in multiple bacterial species seem to represent bundles of short filaments that lack a uniform global polarity.     

Role of gelsolin in the formation and organization of triton-soluble F- actin during myeloid differentiation of HL-60 cells

Structurally and functionally distinct F-actin pools coexist with globular (G)-actin in a variety of eukaryotic cells, including polymorphonuclear leukocytes (PMNs). In PMNs, a Triton-soluble F-actin pool (TSF) exists as short cytoplasmic filaments capped with gelsolin, while Triton-insoluble F-actin (TIF) is a three-dimensional meshwork of F-actin associated with actin-binding protein 280 (ABP-280), alpha- actinin, and tropomyosin. The unique association of gelsolin with the TSF suggests a role for gelsolin in creation or regulation of TSF. To evaluate gelsolin's role in TSF formation, the quantities of actin and gelsolin were determined by quantitative sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblots in uninduced HL-60 cells (U-HL-60) and in HL-60 cells induced to myeloid differentiation with 1.25% dimethyl sulfoxide for 4 to 5 days (I-HL- 60). U-HL-60 cells contain 17.76 +/- 6.01 pmol actin per 10(6) cells (TIF, 5.3 +/- 1.5; TSF, 2.17 +/- 0.37; G, 10.3 +/- 5.7; n = 5) and 0.073 pmol gelsolin per 10(6) cells (TIF, 0; TSF, 0.002 +/- 0.005; G, 0.07 +/- 0.01; n = 3), representing molar actin to gelsolin (A:G) ratios of 1,085:1 for TSF and 147:1 for G. After myeloid differentiation, the actin content increases 1.80-fold (31.94 +/- 6.14 pmol/10(6) cells) equally in each actin pool (TIF, 9.36 +/- 2.35; TSF, 3.29 +/- 0.62; G, 19.29 +/- 4.83). Gelsolin increases 2.4-fold overall (0.178 +/- 0.02 pmol/10(6) cells) but 19-fold in TSF (0.038 +/- 0.009) and only 1.9-fold in G pool (0.139 +/- 0.006), resulting in A:G ratios of 87:1 in TSF and 139:1 in G. The findings of an increase in TSF gelsolin with decreased A:G ratios (1,085:1 v 87:1) with myeloid differentiation suggest shortening of TSF filaments, while the A:G ratios of unbound gelsolin are unchanged (147:1 v 139:1). Measurement of EGTA-resistant gelsolin/actin complexes in HL-60 cells shows that 95% to 100% of complexes exist in the TSF-actin pool only. These findings are consistent with a role for gelsolin in formation and organization of Triton-soluble F-actin. Furthermore, the apparent shortening of TSF-actin filaments with myeloid cellular differentiation and maturation may represent one mechanism of conversion of the nonmotile myeloblast to the motile PMN.     

Gelsolin secretion in interleukin-4-treated bronchial epithelia and in asthmatic airways

RATIONALE: The airway surface liquid, the thin layer of liquid covering the airways, is essential for mucociliary clearance and as a barrier against microbial and other noxious agents. Proteins secreted into the airway surface liquid by epithelial and nonepithelial cells may be important in innate immunity and to improve the fluidity of mucous secretions. OBJECTIVES: We aimed to identify proteins specifically secreted into the airway surface liquid by human bronchial epithelial cells, in resting conditions and after treatment with interleukin 4 (IL-4), a cytokine released in asthma. METHODS AND MAIN RESULTS: By using a proteomics approach, we found that one of the most abundant proteins was gelsolin, which breaks down actin filaments. Gelsolin mRNA and protein secretion were increased threefold in the airway surface liquid of epithelia treated with IL-4. These results were confirmed at the functional level by measuring actin depolymerization using a fluorescence assay. Gelsolin protein was also upregulated in the airways of subjects with asthma. CONCLUSIONS: Our findings indicate that gelsolin is released by epithelial cells into the airways and that its secretion is increased by IL-4 in vitro. In addition, we found that the concentration of both IL-4 and gelsolin were raised in the bronchoalveolar lavage of patients with asthma. These results suggest that gelsolin might improve the fluidity of airway surface liquid in asthma by breaking down filamentous actin that may be released in large amounts by dying cells during inflammation.     

Depression of plasma gelsolin level during acute liver injury.

Human plasma contains two actin-binding proteins, plasma gelsolin and vitamin D-binding protein. These proteins are considered to play an important role in the disposition of actin derived from injured tissue. To evaluate this actin-scavenger system, gelsolin concentrations were measured in serial plasma samples obtained from patients with acute liver injury using an enzyme-linked immunosorbent assay. Plasma gelsolin levels in 43 healthy persons were 226 +/- 52 micrograms/mL. They were markedly reduced to 80 +/- 40 micrograms/mL in 14 patients with an early stage of acute hepatitis and returned to normal levels of 232 +/- 38 micrograms/mL as the disease resolved. Moreover, they showed a significant negative correlation with serum aminotransferase and bilirubin levels. In 7 patients with hepatocellular carcinoma, plasma gelsolin levels rapidly decreased from 182 +/- 42 to 87 +/- 41 micrograms/mL after transcatheter arterial embolization therapy. Because plasma gelsolin is not a hepatic protein, the decreased levels are considered to depend exclusively on the extent of actin leakage from the injured liver.     

Failure of gelsolin overexpression to regulate lymphocyte apoptosis

The actin regulatory protein gelsolin cleaves actin filaments in a calcium- and polyphosphoinositide-dependent manner. Gelsolin has recently been described as a novel substrate of the cysteinyl protease caspase-3, an effector protease activated during apoptosis. Cleavage by caspase-3 generates an amino-terminal fragment of gelsolin that can sever actin filaments independently of calcium regulation. The disruption of the actin cytoskeleton by cleaved gelsolin is hypothesized to mediate many of the downstream morphological changes associated with apoptosis. In contrast, overexpression of full-length gelsolin has also been reported to inhibit apoptotic cell death upstream of the activation of caspase-3, suggesting that gelsolin may also act prior to commitment to cell death. The authors previously observed that actin stabilization by the cell permeant agent jasplakinolide enhanced cell death upon interleukin (IL)-2 or IL-3 withdrawal from growth-factor-dependent lymphocyte cell lines, and hypothesized that actin polymerization could alter the activity of gelsolin, thus enhancing apoptosis. Here the authors show that constitutive overexpression of gelsolin did not, however, inhibit or dramatically enhance apoptotic cell death upon growth-factor withdrawal, nor did it modify sensitivity to jasplakinolide. In contrast to previous reports, overexpression of gelsolin in Jurkat T cells did not prevent or delay apoptosis induced by Fas ligation or ceramide treatment. Overexpressed gelsolin protein was cleaved during apoptosis, as seen previously in this and other cell types. In these model systems, therefore, the level of gelsolin expression was not a rate-limiting determinant in commitment to or time to the morphological changes of apoptosis.     

Platelet-activating factor both stimulates and "primes" human polymorphonuclear leukocyte actin filament assembly

The phospholipid inflammatory mediator, platelet-activating factor (PAF), can stimulate polymorphonuclear leukocyte (PMN) chemotaxis. Conversion of cytoplasmic actin from monomers to filaments is associated with PMN motile functions. Using the fluorescent actin filament stain nitrobenzodiaxole phallicidin, we have investigated PAF's effects on human PMN actin polymerization. Concentrations of PAF between 1 x 10(-11) to 1 x 10(-6) mol/L induced actin filament (F- actin) assembly. An optimal concentration of PAF (1-5 x 10(-8) mol/L) induced a significantly lower rise in relative F-actin content (1.72 +/- 0.07 SEM) than an optimal concentration (5 x 10(-7) mol/L) of the chemotactic peptide FMLP (2.21 +/- 0.06). Unlike FMLP (F-actin content: 1.25 +/- 0.04 at five seconds), PAF stimulation was associated with a delay of more than five seconds (1.04 +/- 0.01 at five seconds) before an increase in F-actin could be detected. F-actin concentration reached maximum levels by 30 to 60 seconds. Prolonged stimulation (20 minutes) with PAF was associated with two phases of polymerization and depolymerization. Like FMLP, the initiation of actin filament assembly by PAF required receptor occupancy, this reaction being totally blocked by the PAF receptor inhibitor, SKI 63-441. As evidenced by the lack of inhibition by nordihydroguaiaretic acid (5 to 20 mumol/L), the production of leukotriene B4 was not required for the PAF-induced changes in F-actin. Like FMLP, PAF's ability to stimulate PMN actin polymerization was inhibited by pertussis toxin (.05 to 2.5 micrograms/mL) but not impaired by the addition of EGTA and/or the calcium ionophore A23187. Preincubation with 1 x 10(-11) to 1 x 10(-8) mol/L PAF for 2 to 60 minutes enhanced the rise in F-actin content induced by low concentrations of FMLP (5 x 10(-12) to 1 x 10(-10) mol/L) indicating that this phospholipid was capable of "priming" the PMN actin polymerization response.     

凝溶胶蛋白与脓毒症

凝溶胶蛋白是凝溶胶蛋白超家族的成员之一,是一种重要的肌动蛋白结合蛋白,其可通过切断、封端肌动蛋白丝,或使肌动蛋白聚集成核等方式来控制肌动蛋白的结构。凝溶胶蛋白除了在重组肌动蛋白丝中发挥作用以外,还在细胞运动、控制细胞程序性死亡等细胞活动中发挥着重要作用。本文重点回顾肌动蛋白的生物学特性、凝溶胶蛋白的基本结构和功能,以及在脓毒症患者中凝溶胶蛋白结合肌动蛋白的作用。临床上可通过监测血浆凝溶胶蛋白水平预测脓毒症患者的预后,评估脓毒症患者的病情严重程度;文章最后对外源性凝溶胶蛋白的治疗作用进行了简要介绍。     

Importance of free actin filament barbed ends for Arp2/3 complex function in platelets and fibroblasts

We investigated the effect of actin filament barbed end uncapping on Arp23 complex function both in vivo and in vitro. Arp23 complex redistributes rapidly and uniformly to the lamellar edge of activated wild-type platelets and fibroblasts but clusters in marginal actin filament clumps in gelsolin-null cells. Treatment of gelsolin-null platelets with the negative dominant N-WASp C-terminal CA domain has no effect on their residual actin nucleation activity, placing gelsolin actin filament severing, capping, and uncapping function upstream of Arp23 complex nucleation. Actin filaments capped by gelsolin or the gelsolin homolog CapG fail to enhance Arp23 complex nucleation in vitro, but uncapping of the barbed ends of these actin filaments restores their ability to potentiate Arp23 complex nucleation. We conclude that Arp23 complex contribution to actin filament nucleation in platelets and fibroblasts importantly requires free barbed ends generated by severing and uncapping.     

Actin polymerization induces a shape change in actin-containing vesicles.

We have encapsulated actin filaments in the presence and absence of various actin-binding proteins into lipid vesicles. These vesicles are approximately the same size as animal cells and can be characterized by the same optical microscopic and mechanical techniques used to study cells. We demonstrate that the initially spherical vesicles can be forced into asymmetric, irregular shapes by polymerization of the actin that they contain. Deformation of the vesicles requires that the actin filaments be on average at least approximately 0.5 micron long as shown by the effects of gelsolin, an actin filament-nucleating protein. Filamin, a filament-crosslinking protein, caused the surfaces of the vesicles to have a smoother appearance. Heterogeneous distribution of actin filaments within the vesicles is caused by interfilament interactions and modulated by gelsolin and filamin. The vesicles provide a model system to study control of cell shape and cytoskeletal organization, membrane-cytoskeleton interactions, and cytomechanics.     

Polymerization kinetics of ADP- and ADP-Pi-actin determined by fluorescence microscopy

We used fluorescence microscopy to determine how polymerization of Mg-ADP-actin depends on the concentration of phosphate. From the dependence of the elongation rate on the actin concentration and direct observations of depolymerizing filaments, we measured the polymerization rate constants of ADP-actin and ADP-P(i)-actin. Saturating phosphate reduces the critical concentration for polymerization of Mg-ADP-actin from 1.8 to 0.06 microM almost entirely by reducing the dissociation rate constants at both ends. Saturating phosphate increases the barbed end association rate constant of Mg-ADP-actin 15%, but this value is still threefold less than that of ATP-actin. Thus, ATP hydrolysis without phosphate dissociation must change the conformation of polymerized actin. Analysis of depolymerization experiments in the presence of phosphate suggests that phosphate dissociation near the terminal subunits is much faster than in the interior. Remarkably, 10 times more phosphate is required to slow the depolymerization of the pointed end than the barbed end, suggesting a weak affinity of phosphate near the pointed end. Our observations of single actin filaments provide clues about the origins of the difference in the critical concentration at the two ends of actin filaments in the presence of ATP.     

Ca2+ binding by domain 2 plays a critical role in the activation and stabilization of gelsolin

Gelsolin consists of six homologous domains (G1–G6), each containing a conserved Ca-binding site. Occupation of a subset of these sites enables gelsolin to sever and cap actin filaments in a Ca-dependent manner. Here, we present the structures of Ca-free human gelsolin and of Ca-bound human G1–G3 in a complex with actin. These structures closely resemble those determined previously for equine gelsolin. However, the G2 Ca-binding site is occupied in the human G1–G3/actin structure, whereas it is vacant in the equine version. In-depth comparison of the Ca-free and Ca-activated, actin-bound human gelsolin structures suggests G2 and G6 to be cooperative in binding Ca²⁺ and responsible for opening the G2–G6 latch to expose the F-actin-binding site on G2. Mutational analysis of the G2 and G6 Ca-binding sites demonstrates their interdependence in maintaining the compact structure in the absence of calcium. Examination of Ca binding by G2 in human G1–G3/actin reveals that the Ca²⁺ locks the G2–G3 interface. Thermal denaturation studies of G2–G3 indicate that Ca binding stabilizes this fragment, driving it into the active conformation. The G2 Ca-binding site is mutated in gelsolin from familial amyloidosis (Finnish-type) patients. This disease initially proceeds through protease cleavage of G2, ultimately to produce a fragment that forms amyloid fibrils. The data presented here support a mechanism whereby the loss of Ca binding by G2 prolongs the lifetime of partially activated, intermediate conformations in which the protease cleavage site is exposed.     

Regulation of the actin cycle in vivo by actin filament severing

Cycling of actin subunits between monomeric and filamentous phases is essential for cell crawling behavior. We investigated actin filament turnover rates, length, number, barbed end exposure, and binding of cofilin in bovine arterial endothelial cells moving at different speeds depending on their position in a confluent monolayer. Fast-translocating cells near the wound edge have short filament lifetimes compared with turnover values that proportionately increase in slower moving cells situated at increasing distances from the wound border. Contrasted with slow cells exhibiting slow actin filament turnover speeds, fast cells have less polymerized actin, shorter actin filaments, more free barbed ends, and less actin-associated cofilin. Cultured primary fibroblasts manifest identical relationships between speed and actin turnover as the endothelial cells, and fast fibroblasts expressing gelsolin have higher actin turnover rates than slow fibroblasts that lack this actin-severing protein. These results implicate actin filament severing as an important control mechanism for actin cycling in cells.     

Intermittent depolymerization of actin filaments is caused by photo-induced dimerization of actin protomers

Actin, one of the most abundant proteins within eukaryotic cells, assembles into long filaments that form intricate cytoskeletal networks and are continuously remodelled via cycles of actin polymerization and depolymerization. These cycles are driven by ATP hydrolysis, a process that also acts to destabilize the filaments as they grow older. Recently, abrupt dynamical changes during the depolymerization of single filaments have been observed and seemed to imply that old filaments are more stable than young ones [Kueh HY, et al. (2008) Proc Natl Acad Sci USA 105:16531-16536]. Using improved experimental setups and quantitative theoretical analysis, we show that these abrupt changes represent actual pauses in depolymerization, unexpectedly caused by the photo-induced formation of actin dimers within the filaments. The stochastic dimerization process is triggered by random transitions of single, fluorescently labeled protomers. Each pause represents the delayed dissociation of a single actin dimer, and the statistics of these single molecule events can be determined by optical microscopy. Unlabeled actin filaments do not exhibit pauses in depolymerization, which implies that, in vivo, older filaments become destabilized by ATP hydrolysis, unless this aging effect is overcompensated by actin-binding proteins. The latter antagonism can now be systematically studied for single filaments using our combined experimental and theoretical method. Furthermore, the dimerization process discovered here provides a molecular switch, by which one can control the length of actin filaments via changes in illumination. This process could also be used to locally "freeze" the dynamics within networks of filaments.     

Actin depolymerization under force is governed by lysine 113:glutamic acid 195-mediated catch-slip bonds

As a key element in the cytoskeleton, actin filaments are highly dynamic structures that constantly sustain forces. However, the fundamental question of how force regulates actin dynamics is unclear. Using atomic force microscopy force-clamp experiments, we show that tensile force regulates G-actin/G-actin and G-actin/F-actin dissociation kinetics by prolonging bond lifetimes (catch bonds) at a low force range and by shortening bond lifetimes (slip bonds) beyond a threshold. Steered molecular dynamics simulations reveal force-induced formation of new interactions that include a lysine 113(K113):glutamic acid 195 (E195) salt bridge between actin subunits, thus suggesting a molecular basis for actin catch-slip bonds. This structural mechanism is supported by the suppression of the catch bonds by the single-residue replacements K113 to serine (K113S) and E195 to serine (E195S) on yeast actin. These results demonstrate and provide a structural explanation for actin catch-slip bonds, which may provide a mechanoregulatory mechanism to control cell functions by regulating the depolymerization kinetics of force-bearing actin filaments throughout the cytoskeleton.