Depolymerization of actin filaments by Cucurbitacin I through binding G‐actin
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ABSTRACT:
SUBMITTER: Haciosmanoglu Aldogan E
PROVIDER: S-EPMC10867458 | biostudies-literature | 2023 Nov
REPOSITORIES: biostudies-literature
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Project description:Although small molecule actin modulators have been widely used as research tools, only one cell-permeable small molecule inhibitor of actin depolymerization (jasplakinolide) is commercially available. We report that the natural product cucurbitacin E inhibits actin depolymerization and show that its mechanism of action is different from jasplakinolide. In assays using pure fluorescently labeled actin, cucurbitacin E specifically affects depolymerization without affecting polymerization. It inhibits actin depolymerization at substoichiometric concentrations up to 1:6 cucurbitacin E:actin. Cucurbitacin E specifically binds to filamentous actin (F-actin) forming a covalent bond at residue Cys257, but not to monomeric actin (G-actin). On the basis of its compatibility with phalloidin staining, we show that cucurbitacin E occupies a different binding site on actin filaments. Using loss of fluorescence after localized photoactivation, we found that cucurbitacin E inhibits actin depolymerization in live cells. Cucurbitacin E is a widely available plant-derived natural product, making it a useful tool to study actin dynamics in cells and actin-based processes such as cytokinesis.
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Project description: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.
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Project description:Calponin from chicken gizzard induced polymerization of actin in the presence of 10 mM KCl. Only 2 min after the addition of KCl in the presence of a 0.0625-0.25:1 molar ratio of calponin to actin, a Poisson-type length distribution (with an average length of approx. 0.7 micron) was observed with formed actin filaments. This result suggests that calponin-actin complexes served as nuclei for rapid elongation. Calponin caused a rapid polymerization of actin even in G-buffer (2 mM Tris/HCl, pH 8.0) which is usually used for depolymerization of actin filaments. Binding of calponin at a level of up to 1.25 mol per mol of actin was observed in the actin filaments formed in the presence of calponin at very low ionic strengths. When actin filaments were exposed to 3.3 mM KCl, by dilution with G-buffer, a rapid depolymerization occurred. Addition of calponin greatly retarded the depolymerization process and, in the presence of an equimolar ratio of calponin to actin, depolymerization hardly occurred. In the presence of calmodulin, this inhibitory effect on depolymerization was reversed by Ca2+, releasing calponin from actin filaments.
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Project description:The actin cytoskeleton orchestrates cell mechanics and facilitates the physical integration of cells into tissues, while tissue-scale forces and extracellular rigidity in turn govern cell behaviour. Here, we discuss recent evidence that actin filaments (F-actin), the core building blocks of the actin cytoskeleton, also serve as molecular force sensors. We delineate two classes of proteins, which interpret forces applied to F-actin through enhanced binding interactions: 'mechanically tuned' canonical actin-binding proteins, whose constitutive F-actin affinity is increased by force, and 'mechanically switched' proteins, which bind F-actin only in the presence of force. We speculate mechanically tuned and mechanically switched actin-binding proteins are biophysically suitable for coordinating cytoskeletal force-feedback and mechanical signalling processes, respectively. Finally, we discuss potential mechanisms mediating force-activated actin binding, which likely occurs both through the structural remodelling of F-actin itself and geometric rearrangements of higher-order actin networks. Understanding the interplay of these mechanisms will enable the dissection of force-activated actin binding's specific biological functions.
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Project description:Actin-depolymerizing factor (ADF)/cofilins contribute to cytoskeletal dynamics by promoting rapid actin filament disassembly. In the classical view, ADF/cofilin sever filaments, and capping proteins block filament barbed ends whereas pointed ends depolymerize, at a rate that is still debated. Here, by monitoring the activity of the three mammalian ADF/cofilin isoforms on individual skeletal muscle and cytoplasmic actin filaments, we directly quantify the reactions underpinning filament severing and depolymerization from both ends. We find that, in the absence of monomeric actin, soluble ADF/cofilin can associate with bare filament barbed ends to accelerate their depolymerization. Compared to bare filaments, ADF/cofilin-saturated filaments depolymerize faster from their pointed ends and slower from their barbed ends, resulting in similar depolymerization rates at both ends. This effect is isoform specific because depolymerization is faster for ADF- than for cofilin-saturated filaments. We also show that, unexpectedly, ADF/cofilin-saturated filaments qualitatively differ from bare filaments: their barbed ends are very difficult to cap or elongate, and consequently undergo depolymerization even in the presence of capping protein and actin monomers. Such depolymerizing ADF/cofilin-decorated barbed ends are produced during 17% of severing events. They are also the dominant fate of filament barbed ends in the presence of capping protein, because capping allows growing ADF/cofilin domains to reach the barbed ends, thereby promoting their uncapping and subsequent depolymerization. Our experiments thus reveal how ADF/cofilin, together with capping protein, control the dynamics of actin filament barbed and pointed ends. Strikingly, our results propose that significant barbed-end depolymerization may take place in cells.
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Project description:Tropomyosin is an elongated α-helical coiled coil that binds to seven consecutive actin subunits along the long-pitch helix of actin filaments. Once bound, tropomyosin polymerizes end-to-end and both stabilizes F-actin and regulates access of various actin-binding proteins including myosin in muscle and nonmuscle cells. Single tropomyosin molecules bind weakly to F-actin with millimolar Kd , whereas the end-to-end linked tropomyosin associates with about a 1000-fold greater affinity. Despite years of study, the assembly mechanism of tropomyosin onto actin filaments remains unclear. In this study, we used total internal reflection fluorescence microscopy to directly monitor the cooperative binding of fluorescently labeled tropomyosin molecules to phalloidin-stabilized actin filaments. We find that tropomyosin molecules assemble from multiple growth sites after random low-affinity binding of single molecules to actin. As the length of the tropomyosin chain increases, the probability of detachment decreases, which leads to further chain growth. Tropomyosin chain extension is linearly dependent on the concentration of tropomyosin, occurring at approximately 100 monomers/(μM*s). The random tropomyosin binding to F-actin leads to discontinuous end-to-end association where gaps in the chain continuity smaller than the required seven sequential actin monomers are available. Direct observation of tropomyosin detachment revealed the number of gaps in actin-bound tropomyosin, the time course of gap annealing, and the eventual filament saturation process.
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Project description:Actin-binding proteins (ABPs) regulate the assembly of actin filaments (F-actin) into networks and bundles that provide the structural integrity of the cell. Two of these ABPs, filamin and alpha-actinin, have been extensively used to model the mechanical properties of actin networks grown in vitro; however, there is a lack in the understanding of how the molecular interactions between ABPs and F-actin regulate the dynamic properties of the cytoskeleton. Here, we present a native-like assay geometry to test the rupture force of a complex formed by an ABP linking two quasiparallel actin filaments. We readily demonstrate the adaptability of this assay by testing it with two different ABPs: filamin and alpha-actinin. For filamin/actin and alpha-actinin/actin, we measured similar rupture forces of 40-80 pN for loading rates between 4 and 50 pN/s. Both ABP unfolding and conformational transition events were observed, demonstrating that both are important and may be a significant mechanism for the temporal regulation of the mechanical properties of the actin cytoskeleton. With this modular, single-molecule assay, a wide range of ABP/actin interactions can be studied to better understand cytoskeletal and cell dynamics.
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Project description:Toxoplasma gondii relies on its actin cytoskeleton to glide and enter its host cell. However, T. gondii tachyzoites are known to display a strikingly low amount of actin filaments, which suggests that sequestration of actin monomers could play a key role in parasite actin dynamics. We isolated a 27-kDa tachyzoite protein on the basis of its ability to bind muscle G-actin and demonstrated that it interacts with parasite G-actin. Cloning and sequence analysis of the gene coding for this protein, which we named Toxofilin, showed that it is a novel actin-binding protein. In in vitro assays, Toxofilin not only bound to G-actin and inhibited actin polymerization as an actin-sequestering protein but also slowed down F-actin disassembly through a filament end capping activity. In addition, when green fluorescent protein-tagged Toxofilin was overexpressed in mammalian nonmuscle cells, the dynamics of actin stress fibers was drastically impaired, whereas green fluorescent protein-Toxofilin copurified with G-actin. Finally, in motile parasites, during gliding or host cell entry, Toxofilin was localized in the entire cytoplasm, including the rear end of the parasite, whereas in intracellular tachyzoites, especially before they exit from the parasitophorous vacuole of their host cell, Toxofilin was found to be restricted to the apical end.
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Project description:The ATP-binding cassette sub-family G member 1 (ABCG1) exports cellular cholesterol to high-density lipoproteins (HDL). However, a number of recent studies have suggested ABCG1 is predominantly localised to intracellular membranes. In this study, we found that ABCG1 was organized into two distinct cellular pools: one at the plasma membrane and the other associated with the endoplasmic reticulum (ER). The plasma membrane fraction was organized into filamentous structures that were associated with cortical actin filaments. Inhibition of actin polymerization resulted in complete disruption of ABCG1 filaments. Cholesterol loading of the cells increased the formation of the filamentous ABCG1, the proximity of filamentous ABCG1 to actin filaments and the diffusion rate of membrane associated ABCG1. Our findings suggest that the actin cytoskeleton plays a critical role in the plasma membrane localization of ABCG1.
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