scholarly journals SCP1 encodes an actin-bundling protein in yeast

2003 ◽  
Vol 375 (2) ◽  
pp. 287-295 ◽  
Author(s):  
Steven J. WINDER ◽  
Thomas JESS ◽  
Kathryn R. AYSCOUGH
Keyword(s):  
Actin Filaments ◽  
Binding Proteins ◽  
Actin Binding ◽  
Filamentous Actin ◽  
Actin Bundles ◽  
Latrunculin A ◽  
The Stability ◽  
Order Structures

The association of F-actin (filamentous actin) with a large number of binding proteins is essential for cellular function. Actin-binding proteins control the dynamics of actin filaments, nucleate new filaments and facilitate formation of higher-order structures such as actin bundles. The yeast gene SCP1 encodes a small protein with significant homology to mammalian SM22/transgelin. We have investigated the role of Scp1p in budding yeast to probe the fundamental role of this family of proteins. Here, we demonstrate that Scp1p binds to F-actin and induces the formation of tight F-actin bundles in vitro. Deletion of SCP1 in yeast lacking the actin-bundling protein, fimbrin (Sac6p), exacerbates the disrupted actin phenotype and enhances latrunculin-A sensitivity. Furthermore, Scp1p co-localizes with actin in cortical patches and its localization is lost in the presence of latrunculin-A. Our data support a role for Scp1p in bundling actin filaments and, in concert with Sac6p, acting as a second actin-bundling activity crucial to the stability of the yeast actin cytoskeleton.

scholarly journals Actin depolymerizing factor controls actin turnover and gliding motility in Toxoplasma gondii

2011 ◽  
Vol 22 (8) ◽  
pp. 1290-1299 ◽  
Author(s):  
Simren Mehta ◽  
L. David Sibley
Keyword(s):  
Toxoplasma Gondii ◽  
Actin Filaments ◽  
Gliding Motility ◽  
Conditional Knockout ◽  
Host Cells ◽  
Actin Binding ◽  
Actin Depolymerizing Factor

Apicomplexan parasites rely on actin-based gliding motility to move across the substratum, cross biological barriers, and invade their host cells. Gliding motility depends on polymerization of parasite actin filaments, yet ∼98% of actin is nonfilamentous in resting parasites. Previous studies suggest that the lack of actin filaments in the parasite is due to inherent instability, leaving uncertain the role of actin-binding proteins in controlling dynamics. We have previously shown that the single allele of Toxoplasma gondii actin depolymerizing factor (TgADF) has strong actin monomer–sequestering and weak filament-severing activities in vitro. Here we used a conditional knockout strategy to investigate the role of TgADF in vivo. Suppression of TgADF led to accumulation of actin-rich filaments that were detected by immunofluorescence and electron microscopy. Parasites deficient in TgADF showed reduced speed of motility, increased aberrant patterns of motion, and inhibition of sustained helical gliding. Lack of TgADF also led to severe defects in entry and egress from host cells, thus blocking infection in vitro. These studies establish that the absence of stable actin structures in the parasite are not simply the result of intrinsic instability, but that TgADF is required for the rapid turnover of parasite actin filaments, gliding motility, and cell invasion.

scholarly journals Overexpression of human fibroblast caldesmon fragment containing actin-, Ca++/calmodulin-, and tropomyosin-binding domains stabilizes endogenous tropomyosin and microfilaments.

1994 ◽  
Vol 125 (2) ◽  
pp. 359-368 ◽  
Author(s):  
K S Warren ◽  
J L Lin ◽  
D D Wamboldt ◽  
J J Lin
Keyword(s):  
Cho Cells ◽  
Actin Filaments ◽  
Actin Binding ◽  
Expression Vectors ◽  
Binding Domains ◽  
Microfilament Bundles ◽  
The Stability ◽  
Triton X

Fibroblast caldesmon is a protein postulated to participate in the modulation of the actin cytoskeleton and the regulation of actin-based motility. The cDNAs encoding the NH2-terminal (aa.1-243, CaD40) and COOH-terminal (aa.244-538, CaD39) fragments of human caldesmon were subcloned into expression vectors and we previously reported that bacterially produced CaD39 protein retains its actin-binding properties as well as its ability to enhance low M(r) tropomyosin (TM) binding to actin and to inhibit TM-actin-activated HMM ATPase activity in vitro (Novy, R. E., J. R. Sellers, L.-F. Liu, and J. J.-C. Lin. 1993. Cell Motil. Cytoskeleton. 26:248-261). Bacterially produced CaD40 does not bind actin. To study the in vivo effects of CaD39 expression on the stability of actin filaments in CHO cells, we isolated and characterized stable CHO transfectants which express varying amounts of CaD39. We found that expression of CaD39 in CHO cells stabilized microfilament bundles as well as endogenous TM. CaD39-expressing clones displayed an increased resistance to cytochalasin B and Triton X-100 treatments and yielded increased amounts of TM-containing actin filaments in microfilament isolation procedures. In addition, analysis of these clones with immunoblotting and indirect immunofluorescence microscopy with anti-TM antibody revealed that stabilized endogenous TM and enhanced TM-containing microfilament bundles parallel increased amounts of CaD39 expression. The increased TM observed corresponded to a decrease in TM turnover rate and did not appear to be due to increased synthesis of endogenous TM. Additionally, the phenomenon of stabilized TM did not occur in stable CHO clones expressing CaD40. Therefore, it is likely that CaD39 can enhance TM's binding to F-actin in vivo, thus reducing TM's rate of turnover and stabilizing actin microfilament bundles.

scholarly journals Phosphoregulation of tropomyosin is crucial for actin cable turnover and division site placement

2019 ◽  
Vol 218 (11) ◽  
pp. 3548-3559 ◽  
Author(s):  
Saravanan Palani ◽  
Darius V. Köster ◽  
Tomoyuki Hatano ◽  
Anton Kamnev ◽  
Taishi Kanamaru ◽  
...  
Keyword(s):  
Fission Yeast ◽  
Actin Filaments ◽  
Coiled Coil ◽  
Actin Binding ◽  
Division Site ◽  
Actin Cable ◽  
Nonmuscle Cells ◽  
The Stability ◽  
Actin Cables

Tropomyosin is a coiled-coil actin binding protein key to the stability of actin filaments. In muscle cells, tropomyosin is subject to calcium regulation, but its regulation in nonmuscle cells is not understood. Here, we provide evidence that the fission yeast tropomyosin, Cdc8, is regulated by phosphorylation of a serine residue. Failure of phosphorylation leads to an increased number and stability of actin cables and causes misplacement of the division site in certain genetic backgrounds. Phosphorylation of Cdc8 weakens its interaction with actin filaments. Furthermore, we show through in vitro reconstitution that phosphorylation-mediated release of Cdc8 from actin filaments facilitates access of the actin-severing protein Adf1 and subsequent filament disassembly. These studies establish that phosphorylation may be a key mode of regulation of nonmuscle tropomyosins, which in fission yeast controls actin filament stability and division site placement.

scholarly journals Biased localization of actin binding proteins by actin filament conformation

2020 ◽  
Author(s):  
Andrew R Harris ◽  
Pamela Jreij ◽  
Brian Belardi ◽  
Andreas Bausch ◽  
Daniel A Fletcher
Keyword(s):  
Actin Filament ◽  
Single Molecule ◽  
Conformational Changes ◽  
Actin Filaments ◽  
Binding Proteins ◽  
Critical Role ◽  
Actin Binding ◽  
Cell Physiology ◽  
Physical Constraints

ABSTRACTThe assembly of actin filaments into distinct cytoskeletal structures plays a critical role in cell physiology, but how proteins localize differentially to these structures within a shared cytoplasm remains unclear. Here, we show that the actin-binding domains of accessory proteins can be sensitive to filament conformational changes. Using a combination of live cell imaging and in vitro single molecule binding measurements, we show that tandem calponin homology domains (CH1-CH2) can be mutated to preferentially bind actin networks at the front or rear of motile cells, and we demonstrate that the affinity of CH1-CH2 domain mutants varies as actin filament conformation is altered by perturbations that include stabilizing drugs, physical constraints, and other binding proteins. These findings suggest that conformational heterogeneity of actin filaments in cells could help to direct accessory binding proteins to different actin cytoskeletal structures through a biophysical feedback loop.

scholarly journals Utrophin Binds Laterally along Actin Filaments and Can Couple Costameric Actin with Sarcolemma When Overexpressed in Dystrophin-deficient Muscle

2002 ◽  
Vol 13 (5) ◽  
pp. 1512-1521 ◽  
Author(s):  
Inna N. Rybakova ◽  
Jitandrakumar R. Patel ◽  
Kay E. Davies ◽  
Peter D. Yurchenco ◽  
James M. Ervasti
Keyword(s):  
Molecular Weight ◽  
Actin Filaments ◽  
Binding Proteins ◽  
Full Length ◽  
Actin Binding ◽  
Lateral Association ◽  
Cortical Cytoskeleton ◽  
Molecular Dimensions ◽  
Native Molecular Weight

Dystrophin is widely thought to mechanically link the cortical cytoskeleton with the muscle sarcolemma. Although the dystrophin homolog utrophin can functionally compensate for dystrophin in mice, recent studies question whether utrophin can bind laterally along actin filaments and anchor filaments to the sarcolemma. Herein, we have expressed full-length recombinant utrophin and show that the purified protein is fully soluble with a native molecular weight and molecular dimensions indicative of monomers. We demonstrate that like dystrophin, utrophin can form an extensive lateral association with actin filaments and protect actin filaments from depolymerization in vitro. However, utrophin binds laterally along actin filaments through contribution of acidic spectrin-like repeats rather than the cluster of basic repeats used by dystrophin. We also show that the defective linkage between costameric actin filaments and the sarcolemma in dystrophin-deficientmdx muscle is rescued by overexpression of utrophin. Our results demonstrate that utrophin and dystrophin are functionally interchangeable actin binding proteins, but that the molecular epitopes important for filament binding differ between the two proteins. More generally, our results raise the possibility that spectrin-like repeats may enable some members of the plakin family of cytolinkers to laterally bind and stabilize actin filaments.

scholarly journals Biased localization of actin binding proteins by actin filament conformation

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Andrew R. Harris ◽  
Pamela Jreij ◽  
Brian Belardi ◽  
Aaron M. Joffe ◽  
Andreas R. Bausch ◽  
...  
Keyword(s):  
Actin Filament ◽  
Single Molecule ◽  
Conformational Changes ◽  
Actin Filaments ◽  
Binding Proteins ◽  
Critical Role ◽  
Actin Binding ◽  
Cell Physiology ◽  
Binding Domains

AbstractThe assembly of actin filaments into distinct cytoskeletal structures plays a critical role in cell physiology, but how proteins localize differentially to these structures within a shared cytoplasm remains unclear. Here, we show that the actin-binding domains of accessory proteins can be sensitive to filament conformational changes. Using a combination of live cell imaging and in vitro single molecule binding measurements, we show that tandem calponin homology domains (CH1–CH2) can be mutated to preferentially bind actin networks at the front or rear of motile cells. We demonstrate that the binding kinetics of CH1–CH2 domain mutants varies as actin filament conformation is altered by perturbations that include stabilizing drugs and other binding proteins. These findings suggest that conformational changes of actin filaments in cells could help to direct accessory binding proteins to different actin cytoskeletal structures through a biophysical feedback loop.

scholarly journals Requirements for Hirano Body Formation

2014 ◽  
Vol 13 (5) ◽  
pp. 625-634 ◽  
Author(s):  
Paul Griffin ◽  
Ruth Furukawa ◽  
Cleveland Piggott ◽  
Andrew Maselli ◽  
Marcus Fechheimer
Keyword(s):  
Actin Filament ◽  
Actin Filaments ◽  
Myosin Ii ◽  
Actin Binding ◽  
Hirano Bodies ◽  
Latrunculin B ◽  
Content Type ◽  
Inhibitory Domain

ABSTRACT Hirano bodies are paracrystalline F-actin-rich structures associated with diverse conditions, including neurodegeneration and aging. Generation of model Hirano bodies using altered forms of Dictyostelium 34-kDa actin-bundling protein allows studies of their physiological function and mechanism of formation. We describe a novel 34-kDa protein mutant, E60K, with a point mutation within the inhibitory domain of the 34-kDa protein. Expression of E60K in Dictyostelium induces the formation of model Hirano bodies. The E60K protein has activated actin binding and is calcium regulated, unlike other forms of the 34-kDa protein that induce Hirano bodies and that have activated actin binding but lack calcium regulation. Actin filaments in the presence of E60K in vitro show enhanced resistance to disassembly induced by latrunculin B. Actin filaments in model Hirano bodies are also protected from latrunculin-induced depolymerization. We used nocodazole and blebbistatin to probe the role of the microtubules and myosin II, respectively, in the formation of model Hirano bodies. In the presence of these inhibitors, model Hirano bodies can form but are smaller than controls at early times of formation. The ultrastructure of model Hirano bodies did not reveal any major difference in structure and organization in the presence of inhibitors. In summary, these results support the conclusion that formation of model Hirano bodies is promoted by gain-of-function actin filament bundling, which enhances actin filament stabilization. Microtubules and myosin II contribute to but are not required for formation of model Hirano bodies.

scholarly journals Characterizing interactions between the microtubule-binding protein CLIP-170 and F-actin

2021 ◽  
Author(s):  
Yueh-Fu O. Wu ◽  
Rachel A. Miller ◽  
Emily O. Alberico ◽  
Nora T. Nelson ◽  
Erin M. Jonasson ◽  
...  
Keyword(s):  
Cell Migration ◽  
Actin Polymerization ◽  
Actin Binding ◽  
Full Understanding ◽  
Filamentous Actin ◽  
Binding Region ◽  
Cellular Processes ◽  
The Stability ◽  
Competition Assays

AbstractThe cooperation between the actin and microtubule (MT) cytoskeletons is important for cellular processes such as cell migration and muscle cell development. Full understanding of how this cooperation occurs has yet to be sufficiently developed. The MT plus-end tracking protein (+TIP) CLIP-170 has been implicated in this actin-MT coordination by associating with the actin-binding signaling protein IQGAP1, and by promoting actin polymerization through binding with formins. Thus far, CLIP-170’s interactions with actin were assumed to be indirect. Here, we demonstrate that CLIP-170 can bind to filamentous actin (F-actin) directly. The affinity is relatively weak, but is strong enough to be significant in the actin-rich cortex, where actin concentrations can be extremely high. Using CLIP-170 fragments and mutants, we show that the direct CLIP-170:actin interaction is independent of the FEED domain, the region that mediates formin-dependent actin polymerization, and that the CLIP-170 F-actin-binding region overlaps with the MT-binding region. Consistent with these observations, in vitro competition assays indicate that CLIP-170:F-actin and CLIP-170:MT interactions are mutually exclusive. Taken together, these observations lead us to speculate that direct CLIP-170:F-actin interactions may function to reduce the stability of MTs in actin-rich regions of the cell, as previously proposed for EB1.

scholarly journals Structural effects and functional implications of phalloidin and jasplakinolide binding to actin filaments

2019 ◽  
Author(s):  
Sabrina Pospich ◽  
Felipe Merino ◽  
Stefan Raunser
Keyword(s):  
High Resolution ◽  
Actin Filament ◽  
Actin Filaments ◽  
Inorganic Phosphate ◽  
Binding Proteins ◽  
Conformational Space ◽  
Actin Binding ◽  
List Type ◽  
Filamentous Actin ◽  
Actin Binding Proteins

SummaryActin undergoes structural transitions during polymerization, ATP hydrolysis and subsequent release of inorganic phosphate. Several actin binding proteins sense specific states during this transition and can thus target different regions of the actin filament. Here we show in atomic detail that phalloidin, a mushroom toxin that is routinely used to stabilize and label actin filaments, suspends the structural changes in actin, likely influencing its interaction with actin binding proteins. Furthermore, high-resolution cryo-EM structures reveal structural rearrangements in F-actin upon inorganic phosphate release in phalloidin-stabilized filaments. We find that the effect of the sponge toxin jasplakinolide differs from the one of phalloidin, despite their overlapping binding site and similar interactions with the actin filament. Analysis of structural conformations of F-actin suggests that stabilizing agents trap states within the natural conformational space of actin.Abstract FigureHighlightsFive high-resolution cryo-EM structures of stabilized filamentous actinPhalloidin traps different structural states depending on when it is addedThe effect of phalloidin and jasplakinolide on filamentous actin is not identicalBoth toxins likely interfere with the binding of proteins sensing F-actin’s nucleotide state

scholarly journals Actin filaments in yeast are unstable in the absence of capping protein or fimbrin.

1995 ◽  
Vol 131 (6) ◽  
pp. 1483-1493 ◽  
Author(s):  
T S Karpova ◽  
K Tatchell ◽  
J A Cooper
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Actin Filament ◽  
Actin Filaments ◽  
Binding Proteins ◽  
Actin Binding ◽  
Actin Binding Proteins ◽  
Capping Protein ◽  
Filament Assembly

Many actin-binding proteins affect filament assembly in vitro and localize with actin in vivo, but how their molecular actions contribute to filament assembly in vivo is not understood well. We report here that capping protein (CP) and fimbrin are both important for actin filament assembly in vivo in Saccharomyces cerevisiae, based on finding decreased actin filament assembly in CP and fimbrin mutants. We have also identified mutations in actin that enhance the CP phenotype and find that those mutants also have decreased actin filament assembly in vivo. In vitro, actin purified from some of these mutants is defective in polymerization or binding fimbrin. These findings support the conclusion that CP acts to stabilize actin filaments in vivo. This conclusion is particularly remarkable because it is the opposite of the conclusion drawn from recent studies in Dictyostelium (Hug, C., P.Y. Jay, I. Reddy, J.G. McNally, P.C. Bridgman, E.L. Elson, and J.A. Cooper. 1995. Cell. 81:591-600). In addition, we find that the unpolymerized pool of actin in yeast is very small relative to that found in higher cells, which suggests that actin filament assembly is less dynamic in yeast than higher cells.

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