scholarly journals 14-3-3 Negatively Regulates Actin Filament Formation in the Deep Branching Eukaryote Giardia lamblia

2017 ◽  
Author(s):  
Jana Krtková ◽  
Jennifer Xu ◽  
Marco Lalle ◽  
Melissa Steele-Ogus ◽  
Germain C. M. Alas ◽  
...  
Keyword(s):  
Complex Formation ◽  
Actin Cytoskeleton ◽  
Actin Filament ◽  
Giardia Lamblia ◽  
Actin Filaments ◽  
Binding Proteins ◽  
Negative Regulator ◽  
Actin Binding ◽  
Filament Formation ◽  
Rho Family

AbstractThe phosphoserine/phosphothreonine-binding protein 14-3-3 is known to regulate actin, this function has been previously attributed to sequestration of phosphorylated cofilin. The deep branching eukaryote Giardia lamblia lacks cofilin and all other canonical actin-binding proteins (ABPs), and 14-3-3 was identified as an actin-associated protein in Giardia, yet its role in actin regulation was unknown. Gl14-3-3 depletion resulted in an overall disruption of actin organization characterized by ectopically distributed short actin filaments. Using phosphatase and kinase inhibitors, we demonstrated that actin phosphorylation correlated with destabilization of the actin network and increased complex formation with 14-3-3, while blocking actin phosphorylation stabilized actin filaments and attenuated complex formation. Giardia's sole Rho family GTPase, GlRac, modulates Gl14-3-3's association with actin, providing the first connection between GlRac and the actin cytoskeleton in Giardia. Giardia actin contains two putative 14-3-3 binding motifs, one of which (S330) is conserved in mammalian actin. Mutation of these sites reduced, but did not completely disrupt, the association with 14-3-3. Native gels and overlay assays indicate that intermediate proteins are required to support complex formation between 14-3-3 and actin. Overall, our results support a role for 14-3-3 as a negative regulator of actin filament formation.ImportanceGiardia lacks canonical actin binding proteins. 14-3-3 was identified as an actin interactor but the significance of this interaction was unknown. Loss of 14-3-3 results in ectopic short actin filaments, indicating that 14-3-3 is an important regulator of the actin cytoskeleton in Giardia. Drug studies indicate that 14-3-3 complex formation is in part phospho-regulated. We demonstrate that complex formation is downstream of Giardia’s sole Rho family GTPase, GlRac, this result provides the first mechanistic connection between GlRac and actin in Giardia. Native gels and overlay assays indicate intermediate proteins are required to support the interaction between 14-3-3 and actin suggesting that 14-3-3 is regulating multiple actin complexes. Overall, we find that 14-3-3 is a negative regulator of actin filament formation in Giardia.

scholarly journals 14-3-3 Regulates Actin Filament Formation in the Deep-Branching Eukaryote Giardia lamblia

mSphere ◽  
2017 ◽  
Vol 2 (5) ◽  
Author(s):  
Jana Krtková ◽  
Jennifer Xu ◽  
Marco Lalle ◽  
Melissa Steele-Ogus ◽  
Germain C. M. Alas ◽  
...  
Keyword(s):  
Complex Formation ◽  
Actin Cytoskeleton ◽  
Giardia Lamblia ◽  
Actin Filaments ◽  
Binding Proteins ◽  
Actin Binding ◽  
Actin Binding Proteins ◽  
Important Regulator ◽  
Rho Family ◽  
Drug Studies

ABSTRACT Giardia lacks canonical actin-binding proteins. Gl-14-3-3 was identified as an actin interactor, but the significance of this interaction was unknown. Loss of Gl-14-3-3 results in ectopic short actin filaments, indicating that Gl-14-3-3 is an important regulator of the actin cytoskeleton in Giardia. Drug studies indicate that Gl-14-3-3 complex formation is in part phospho-regulated. We demonstrate that complex formation is downstream of Giardia’s sole Rho family GTPase, Gl-Rac. This result provides the first mechanistic connection between Gl-Rac and Gl-actin in Giardia. Native gels and overlay assays indicate intermediate proteins are required to support the interaction between Gl-14-3-3 and Gl-actin, suggesting that Gl-14-3-3 is regulating multiple Gl-actin complexes. The phosphoserine/phosphothreonine-binding protein 14-3-3 is known to regulate actin; this function has been previously attributed to sequestration of phosphorylated cofilin. 14-3-3 was identified as an actin-associated protein in the deep-branching eukaryote Giardia lamblia; however, Giardia lacks cofilin and all other canonical actin-binding proteins (ABPs). Thus, the role of G. lamblia 14-3-3 (Gl-14-3-3) in actin regulation was unknown. Gl-14-3-3 depletion resulted in an overall disruption of actin organization characterized by ectopically distributed short actin filaments. Using phosphatase and kinase inhibitors, we demonstrated that actin phosphorylation correlated with destabilization of the actin network and increased complex formation with 14-3-3, while blocking actin phosphorylation stabilized actin filaments and attenuated complex formation. Giardia’s sole Rho family GTPase, Gl-Rac, modulates Gl-14-3-3’s association with actin, providing the first connection between Gl-Rac and the actin cytoskeleton in Giardia. Giardia actin (Gl-actin) contains two putative 14-3-3 binding motifs, one of which (S330) is conserved in mammalian actin. Mutation of these sites reduced, but did not completely disrupt, the association with 14-3-3. Native gels and overlay assays indicate that intermediate proteins are required to support complex formation between 14-3-3 and actin. Overall, our results support a role for 14-3-3 as a regulator of actin; however, the presence of multiple 14-3-3–actin complexes suggests a more complex regulatory relationship than might be expected for a minimalistic parasite. IMPORTANCE Giardia lacks canonical actin-binding proteins. Gl-14-3-3 was identified as an actin interactor, but the significance of this interaction was unknown. Loss of Gl-14-3-3 results in ectopic short actin filaments, indicating that Gl-14-3-3 is an important regulator of the actin cytoskeleton in Giardia. Drug studies indicate that Gl-14-3-3 complex formation is in part phospho-regulated. We demonstrate that complex formation is downstream of Giardia’s sole Rho family GTPase, Gl-Rac. This result provides the first mechanistic connection between Gl-Rac and Gl-actin in Giardia. Native gels and overlay assays indicate intermediate proteins are required to support the interaction between Gl-14-3-3 and Gl-actin, suggesting that Gl-14-3-3 is regulating multiple Gl-actin complexes.

scholarly journals Actin-binding proteins: the long road to understanding the dynamic landscape of cellular actin networks

2016 ◽  
Vol 27 (16) ◽  
pp. 2519-2522 ◽  
Author(s):  
Pekka Lappalainen
Keyword(s):  
Actin Cytoskeleton ◽  
Actin Filaments ◽  
Binding Proteins ◽  
Three Dimensional ◽  
Actin Dynamics ◽  
Actin Binding ◽  
Actin Binding Proteins ◽  
Vast Number ◽  
Actin Regulatory Proteins ◽  
The Individual

The actin cytoskeleton supports a vast number of cellular processes in nonmuscle cells. It is well established that the organization and dynamics of the actin cytoskeleton are controlled by a large array of actin-binding proteins. However, it was only 40 years ago that the first nonmuscle actin-binding protein, filamin, was identified and characterized. Filamin was shown to bind and cross-link actin filaments into higher-order structures and contribute to phagocytosis in macrophages. Subsequently many other nonmuscle actin-binding proteins were identified and characterized. These proteins regulate almost all steps of the actin filament assembly and disassembly cycles, as well as the arrangement of actin filaments into diverse three-dimensional structures. Although the individual biochemical activities of most actin-regulatory proteins are relatively well understood, knowledge of how these proteins function together in a common cytoplasm to control actin dynamics and architecture is only beginning to emerge. Furthermore, understanding how signaling pathways and mechanical cues control the activities of various actin-binding proteins in different cellular, developmental, and pathological processes will keep researchers busy for decades.

scholarly journals Diversity from similarity: cellular strategies for assigning particular identities to actin filaments and networks

Open Biology ◽  
2020 ◽  
Vol 10 (9) ◽  
pp. 200157
Author(s):  
Micaela Boiero Sanders ◽  
Adrien Antkowiak ◽  
Alphée Michelot
Keyword(s):  
Mechanical Properties ◽  
Actin Cytoskeleton ◽  
Actin Filaments ◽  
Binding Proteins ◽  
Molecular Mechanisms ◽  
Competitive Binding ◽  
Actin Binding ◽  
Actin Isoforms ◽  
Actin Networks ◽  
Covalent Modifications

The actin cytoskeleton has the particularity of being assembled into many functionally distinct filamentous networks from a common reservoir of monomeric actin. Each of these networks has its own geometrical, dynamical and mechanical properties, because they are capable of recruiting specific families of actin-binding proteins (ABPs), while excluding the others. This review discusses our current understanding of the underlying molecular mechanisms that cells have developed over the course of evolution to segregate ABPs to appropriate actin networks. Segregation of ABPs requires the ability to distinguish actin networks as different substrates for ABPs, which is regulated in three different ways: (1) by the geometrical organization of actin filaments within networks, which promotes or inhibits the accumulation of ABPs; (2) by the identity of the networks' filaments, which results from the decoration of actin filaments with additional proteins such as tropomyosin, from the use of different actin isoforms or from covalent modifications of actin; (3) by the existence of collaborative or competitive binding to actin filaments between two or multiple ABPs. This review highlights that all these effects need to be taken into account to understand the proper localization of ABPs in cells, and discusses what remains to be understood in this field of research.

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.

Biomechanical analysis of actin cytoskeleton function based on a spring network cell model

2016 ◽  
Vol 231 (7) ◽  
pp. 1308-1323 ◽  
Author(s):  
Hamed Ghaffari ◽  
Mohammad Said Saidi ◽  
Bahar Firoozabadi
Keyword(s):  
Actin Cytoskeleton ◽  
Actin Filaments ◽  
Cell Shape ◽  
Binding Proteins ◽  
Cell Model ◽  
Shape Change ◽  
Cell Deformation ◽  
Actin Binding ◽  
Actin Binding Proteins ◽  
Cell Shape Change

In this study, a new method for the simulation of the time-dependent behavior of actin cytoskeleton during cell shape change is proposed. For this purpose, a three-dimensional model of endothelial cell consisting of cell membrane, nucleus membrane, and main components of cytoskeleton, namely actin filaments, microtubules, and intermediate filaments is utilized. Actin binding proteins, which play a key role in regulating actin cytoskeleton behavior, are also simulated by using a novel technique. The actin cytoskeleton in this model is more dynamic and adoptable during cell deformation in comparison to previous models. The proposed model is subjected to compressive force between parallel micro plates in order to investigate actin cytoskeleton role in cell stiffening behavior, nucleus deformation, and cell shape change. The validity of the model is examined through the comparison of the obtained results with the data presented in previous literature. Not only does the model force deformation curve lie within a range of the experimental data, but also the elastic modulus of the cell model is in accordance with former studies. Our findings demonstrate that augmentation of actin filaments concentration within the cell reduces force transmission from cell membrane to the nucleus. Furthermore, actin binding proteins concentration increases by the enhancement of cell deformation and it is also indicated that cell stiffening with an increase in applied force is significantly affected by actin filaments reorientation, actin binding proteins reorganization and actin binding proteins augmentation.

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 Identification of Arabidopsis Cyclase-associated Protein 1 as the First Nucleotide Exchange Factor for Plant Actin

2007 ◽  
Vol 18 (8) ◽  
pp. 3002-3014 ◽  
Author(s):  
Faisal Chaudhry ◽  
Christophe Guérin ◽  
Matthias von Witsch ◽  
Laurent Blanchoin ◽  
Christopher J. Staiger
Keyword(s):  
Actin Cytoskeleton ◽  
Actin Filament ◽  
Binding Proteins ◽  
Actin Dynamics ◽  
Actin Binding ◽  
Nucleotide Exchange ◽  
Suspension Cells ◽  
Actin Binding Proteins ◽  
Cell Shapes

The actin cytoskeleton powers organelle movements, orchestrates responses to abiotic stresses, and generates an amazing array of cell shapes. Underpinning these diverse functions of the actin cytoskeleton are several dozen accessory proteins that coordinate actin filament dynamics and construct higher-order assemblies. Many actin-binding proteins from the plant kingdom have been characterized and their function is often surprisingly distinct from mammalian and fungal counterparts. The adenylyl cyclase-associated protein (CAP) has recently been shown to be an important regulator of actin dynamics in vivo and in vitro. The disruption of actin organization in cap mutant plants indicates defects in actin dynamics or the regulated assembly and disassembly of actin subunits into filaments. Current models for actin dynamics maintain that actin-depolymerizing factor (ADF)/cofilin removes ADP–actin subunits from filament ends and that profilin recharges these monomers with ATP by enhancing nucleotide exchange and delivery of subunits onto filament barbed ends. Plant profilins, however, lack the essential ability to stimulate nucleotide exchange on actin, suggesting that there might be a missing link yet to be discovered from plants. Here, we show that Arabidopsis thaliana CAP1 (AtCAP1) is an abundant cytoplasmic protein; it is present at a 1:3 M ratio with total actin in suspension cells. AtCAP1 has equivalent affinities for ADP– and ATP–monomeric actin (Kd ∼ 1.3 μM). Binding of AtCAP1 to ATP–actin monomers inhibits polymerization, consistent with AtCAP1 being an actin sequestering protein. However, we demonstrate that AtCAP1 is the first plant protein to increase the rate of nucleotide exchange on actin. Even in the presence of ADF/cofilin, AtCAP1 can recharge actin monomers and presumably provide a polymerizable pool of subunits to profilin for addition onto filament ends. In turnover assays, plant profilin, ADF, and CAP act cooperatively to promote flux of subunits through actin filament barbed ends. Collectively, these results and our understanding of other actin-binding proteins implicate CAP1 as a central player in regulating the pool of unpolymerized ATP–actin.

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.

Mechanotransduction by the Actin Cytoskeleton: Converting Mechanical Stimuli into Biochemical Signals

2018 ◽  
Vol 47 (1) ◽  
pp. 617-631 ◽  
Author(s):  
Andrew R. Harris ◽  
Pamela Jreij ◽  
Daniel A. Fletcher
Keyword(s):  
Actin Cytoskeleton ◽  
Actin Filaments ◽  
Binding Proteins ◽  
Shape Change ◽  
Cytoskeletal Proteins ◽  
Actin Binding ◽  
Mechanical Forces ◽  
Mechanical Stimuli ◽  
Force Transmission ◽  
Biochemical Signals

Force transmission through the actin cytoskeleton plays a central role in cell movements, shape change, and internal organization. Dynamic reorganization of actin filaments by an array of specialized binding proteins creates biochemically and architecturally distinct structures, many of which are finely tuned to exert or resist mechanical loads. The molecular complexity of the actin cytoskeleton continues to be revealed by detailed biochemical assays, and the architectural diversity and dynamics of actin structures are being uncovered by advances in super-resolution fluorescence microscopy and electron microscopy. However, our understanding of how mechanical forces feed back on cytoskeletal architecture and actin-binding protein organization is comparatively limited. In this review, we discuss recent work investigating how mechanical forces applied to cytoskeletal proteins are transduced into biochemical signals. We explore multiple mechanisms for mechanical signal transduction, including the mechanosensitive behavior of actin-binding proteins, the effect of mechanical force on actin filament dynamics, and the influence of mechanical forces on the structure of single actin filaments. The emerging picture is one in which the actin cytoskeleton is defined not only by the set of proteins that constitute a network but also by the constant interplay of mechanical forces and biochemistry.

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