Long-Range and Directional Allostery of Actin Filaments Plays Important Roles in Various Cellular Activities

A wide variety of uniquely localized actin-binding proteins (ABPs) are involved in various cellular activities, such as cytokinesis, migration, adhesion, morphogenesis, and intracellular transport. In a micrometer-scale space such as the inside of cells, protein molecules diffuse throughout the cell interior within seconds. In this condition, how can ABPs selectively bind to particular actin filaments when there is an abundance of actin filaments in the cytoplasm? In recent years, several ABPs have been reported to induce cooperative conformational changes to actin filaments allowing structural changes to propagate along the filament cables uni- or bidirectionally, thereby regulating the subsequent binding of ABPs. Such propagation of ABP-induced cooperative conformational changes in actin filaments may be advantageous for the elaborate regulation of cellular activities driven by actin-based machineries in the intracellular space, which is dominated by diffusion. In this review, we focus on long-range allosteric regulation driven by cooperative conformational changes of actin filaments that are evoked by binding of ABPs, and discuss roles of allostery of actin filaments in narrow intracellular spaces.

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Evidence for a Conformational Change in Actin Induced by Fimbrin (N375) Binding

The Journal of Cell Biology ◽

10.1083/jcb.139.2.387 ◽

1997 ◽

Vol 139 (2) ◽

pp. 387-396 ◽

Cited By ~ 81

Author(s):

Dorit Hanein ◽

Paul Matsudaira ◽

David J. DeRosier

Keyword(s):

Actin Filaments ◽

Structural Changes ◽

Three Dimensional ◽

Binding Domain ◽

Actin Binding ◽

Dimensional Structure ◽

Binding Surface ◽

Actin Binding Domain ◽

Actin Structure ◽

Signal Transduction Proteins

Fimbrin belongs to a superfamily of actin cross-linking proteins that share a conserved 27-kD actin-binding domain. This domain contains a tandem duplication of a sequence that is homologous to calponin. Calponin homology (CH) domains not only cross-link actin filaments into bundles and networks, but they also bind intermediate filaments and some signal transduction proteins to the actin cytoskeleton. This fundamental role of CH domains as a widely used actin-binding domain underlines the necessity to understand their structural interaction with actin. Using electron cryomicroscopy, we have determined the three-dimensional structure of F-actin and F-actin decorated with the NH2-terminal CH domains of fimbrin (N375). In a difference map between actin filaments and N375-decorated actin, one end of N375 is bound to a concave surface formed between actin subdomains 1 and 2 on two neighboring actin monomers. In addition, a fit of the atomic model for the actin filament to the maps reveals the actin residues that line, the binding surface. The binding of N375 changes actin, which we interpret as a movement of subdomain 1 away from the bound N375. This change in actin structure may affect its affinity for other actin-binding proteins and may be part of the regulation of the cytoskeleton itself. Difference maps between actin and actin decorated with other proteins provides a way to look for novel structural changes in actin.

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Biased localization of actin binding proteins by actin filament conformation

10.1101/2020.02.21.959791 ◽

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.

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Salt-Mediated Stiffening, Destruction, and Resculpting of Actomyosin Network

Frontiers in Physics ◽

10.3389/fphy.2021.760340 ◽

2021 ◽

Vol 9 ◽

Author(s):

Bekele J. Gurmessa ◽

Michael J. Rust ◽

Moumita Das ◽

Jennifer L. Ross ◽

Rae M. Robertson-Anderson

Keyword(s):

Optical Tweezers ◽

Actin Filaments ◽

Viscoelastic Properties ◽

Structural Changes ◽

Myosin Ii ◽

Structural Evolution ◽

Confocal Imaging ◽

Actin Binding ◽

Time Varying ◽

High Concentrations

Cells dynamically change their viscoelastic properties by restructuring networks of actin filaments in the cytoskeleton, enabling diverse mechanical processes such as mobility and apoptosis. This restructuring is modulated, in part, by actin-binding proteins, such as myosin II, as well as counterions such as Mg2+ and K+. While high concentrations of Mg2+ can induce bundling and crosslinking of actin filaments, high concentrations of K+ destabilize myosin II minifilaments necessary to crosslink actin filaments. Here, we elucidate how the mechanics and structure of actomyosin networks evolve under competing effects of varying Mg2+ and K+ concentrations. Specifically, we couple microfluidics with optical tweezers microrheology to measure the time-varying linear viscoelastic moduli of actin networks crosslinked via myosin II as we cycle between low and high Mg2+ and K+ concentrations. Our complementary confocal imaging experiments correlate the time-varying viscoelastic properties with salt-mediated structural evolution. We find that the elastic modulus displays an intriguing non-monotonic time dependence in high-salt conditions, that correlates with structural changes, and that this process is irreversible, with the network evolving to a new steady-state as Mg2+ and K+ decrease back to their initial concentrations.

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Biased localization of actin binding proteins by actin filament conformation

Nature Communications ◽

10.1038/s41467-020-19768-9 ◽

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.

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Observation of long-range tertiary interactions during ligand binding by the TPP riboswitch aptamer

eLife ◽

10.7554/elife.12362 ◽

2015 ◽

Vol 4 ◽

Cited By ~ 21

Author(s):

Van K Duesterberg ◽

Irena T Fischer-Hwang ◽

Christian F Perez ◽

Daniel W Hogan ◽

Steven M Block

Keyword(s):

Ligand Binding ◽

Long Range ◽

Single Molecule ◽

Conformational Changes ◽

Structural Changes ◽

Regulatory Element ◽

Optical Trap ◽

Resonance Energy ◽

Thiamine Pyrophosphate ◽

Tertiary Interactions

The thiamine pyrophosphate (TPP) riboswitch is a cis-regulatory element in mRNA that modifies gene expression in response to TPP concentration. Its specificity is dependent upon conformational changes that take place within its aptamer domain. Here, the role of tertiary interactions in ligand binding was studied at the single-molecule level by combined force spectroscopy and Förster resonance energy transfer (smFRET), using an optical trap equipped for simultaneous smFRET. The ‘Force-FRET’ approach directly probes secondary and tertiary structural changes during folding, including events associated with binding. Concurrent transitions observed in smFRET signals and RNA extension revealed differences in helix-arm orientation between two previously-identified ligand-binding states that had been undetectable by spectroscopy alone. Our results show that the weaker binding state is able to bind to TPP, but is unable to form a tertiary docking interaction that completes the binding process. Long-range tertiary interactions stabilize global riboswitch structure and confer increased ligand specificity.

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Correlative cryo-imaging of the cellular universe with soft X-rays and laser light used to track F-actin structures in mammalian cells

Acta Crystallographica Section D Structural Biology ◽

10.1107/s2059798321010329 ◽

2021 ◽

Vol 77 (12) ◽

Author(s):

Mohamed Koronfel ◽

Ilias Kounatidis ◽

Dennis M. Mwangangi ◽

Nina Vyas ◽

Chidinma Okolo ◽

...

Keyword(s):

Actin Filaments ◽

Mammalian Cells ◽

Intracellular Transport ◽

Super Resolution ◽

X Rays ◽

Imaging Data ◽

Filamentous Actin ◽

Intracellular Space ◽

X Ray ◽

Integral Role

Imaging of actin filaments is crucial due to the integral role that they play in many cellular functions such as intracellular transport, membrane remodelling and cell motility. Visualizing actin filaments has so far relied on fluorescence microscopy and electron microscopy/tomography. The former lacks the capacity to capture the overall local ultrastructure, while the latter requires rigorous sample preparation that can lead to potential artefacts, and only delivers relatively small volumes of imaging data at the thinnest areas of a cell. In this work, a correlative approach utilizing in situ super-resolution fluorescence imaging and cryo X-ray tomography was used to image bundles of actin filaments deep inside cells under near-native conditions. In this case, fluorescence 3D imaging localized the actin bundles within the intracellular space, while X-ray tomograms of the same areas provided detailed views of the local ultrastructure. Using this new approach, actin trails connecting vesicles in the perinuclear area and hotspots of actin presence within and around multivesicular bodies were observed. The characteristic prevalence of filamentous actin in cytoplasmic extensions was also documented.

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Actin binding domain of Rng2 sparsely bound on F-actin strongly inhibits actin movement on myosin II

10.1101/2020.04.14.041046 ◽

2020 ◽

Author(s):

Yuuki Hayakawa ◽

Masak Takaine ◽

Taiga Imai ◽

Masafumi D. Yamada ◽

Keiko Hirose ◽

...

Keyword(s):

Fission Yeast ◽

Actin Filaments ◽

Structural Changes ◽

Myosin Ii ◽

Binding Domain ◽

Actin Binding ◽

Skeletal Muscle Myosin ◽

Actin Binding Domain ◽

Muscle Myosin

AbstractThe contraction of contractile rings (CRs) depends on interaction between actin filaments and myosin II filaments. The rate of contraction in the fission yeast Schizosaccharomyces pombe is less than 1/120 of the velocity of acto-myosin II movement in vitro, but the mechanism of inhibition has not been described. Here, we found that the calponin-homology actin binding domain of fission yeast IQGAP Rng2 (Rng2CHD) strongly inhibits the motility of actin filaments on skeletal muscle myosin II fragments in vitro, even at a low ratio of bound Rng2CHD to actin protomers, reducing the sliding velocity to half when the binding ratio was 1/75. Rng2CHD also induced structural changes of actin filaments and reduced the affinity between actin filaments and subfragment 1 (S1) of muscle myosin II carrying ADP. Intriguingly, actin-activated ATPase of S1 was only mildly inhibited, even by high concentrations of Rng2CHD. Moreover, the motility of actin filaments by myosin V was not inhibited by Rng2CHD. We propose a new regulatory mechanism for acto-myosin II movement that involves Rng2CHD-induced structural changes of actin filaments.

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Cofilin-induced unidirectional cooperative conformational changes in actin filaments revealed by high-speed atomic force microscopy

eLife ◽

10.7554/elife.04806 ◽

2015 ◽

Vol 4 ◽

Cited By ~ 66

Author(s):

Kien Xuan Ngo ◽

Noriyuki Kodera ◽

Eisaku Katayama ◽

Toshio Ando ◽

Taro QP Uyeda

Keyword(s):

Atomic Force Microscopy ◽

Conformational Changes ◽

Actin Filaments ◽

High Speed ◽

Structural Changes ◽

Force Microscopy ◽

Helical Pitch ◽

Atomic Force ◽

Microscopic Studies ◽

Pointed End

High-speed atomic force microscopy was employed to observe structural changes in actin filaments induced by cofilin binding. Consistent with previous electron and fluorescence microscopic studies, cofilin formed clusters along actin filaments, where the filaments were 2-nm thicker and the helical pitch was ∼25% shorter, compared to control filaments. Interestingly, the shortened helical pitch was propagated to the neighboring bare zone on the pointed-end side of the cluster, while the pitch on the barbed-end side was similar to the control. Thus, cofilin clusters induce distinctively asymmetric conformational changes in filaments. Consistent with the idea that cofilin favors actin structures with a shorter helical pitch, cofilin clusters grew unidirectionally toward the pointed-end of the filament. Severing was often observed near the boundaries between bare zones and clusters, but not necessarily at the boundaries.

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Structures of cofilin-induced structural changes reveal local and asymmetric perturbations of actin filaments

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1915987117 ◽

2020 ◽

Vol 117 (3) ◽

pp. 1478-1484 ◽

Cited By ~ 10

Author(s):

Andrew R. Huehn ◽

Jeffrey P. Bibeau ◽

Anthony C. Schramm ◽

Wenxiang Cao ◽

Enrique M. De La Cruz ◽

...

Keyword(s):

Conformational Changes ◽

Actin Filaments ◽

Structural Changes ◽

Structural Data ◽

Binding Mode ◽

Cryo Electron Microscopy ◽

Actin Structure ◽

Cluster A ◽

Pointed End ◽

Single Subunit

Members of the cofilin/ADF family of proteins sever actin filaments, increasing the number of filament ends available for polymerization or depolymerization. Cofilin binds actin filaments with positive cooperativity, forming clusters of contiguously bound cofilin along the filament lattice. Filament severing occurs preferentially at boundaries between bare and cofilin-decorated (cofilactin) segments and is biased at 1 side of a cluster. A molecular understanding of cooperative binding and filament severing has been impeded by a lack of structural data describing boundaries. Here, we apply methods for analyzing filament cryo-electron microscopy (cryo-EM) data at the single subunit level to directly investigate the structure of boundaries within partially decorated cofilactin filaments. Subnanometer resolution maps of isolated, bound cofilin molecules and an actin-cofilactin boundary indicate that cofilin-induced actin conformational changes are local and limited to subunits directly contacting bound cofilin. An isolated, bound cofilin compromises longitudinal filament contacts of 1 protofilament, consistent with a single cofilin having filament-severing activity. An individual, bound phosphomimetic (S3D) cofilin with weak severing activity adopts a unique binding mode that does not perturb actin structure. Cofilin clusters disrupt both protofilaments, consistent with a higher severing activity at boundaries compared to single cofilin. Comparison of these structures indicates that this disruption is substantially greater at pointed end sides of cofilactin clusters than at the barbed end. These structures, with the distribution of bound cofilin clusters, suggest that maximum binding cooperativity is achieved when 2 cofilins occupy adjacent sites. These results reveal the structural origins of cooperative cofilin binding and actin filament severing.

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Protein-induced conformational changes in 16S rRNA during assembly of the Escherichia Coli 30S ribosomal subunit: A STEM study

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100128833 ◽

1987 ◽

Vol 45 ◽

pp. 910-911

Author(s):

M. Boublik ◽

V. Mandiyan ◽

J.F. Hainfeld ◽

J.S. Wall

Keyword(s):

16S Rrna ◽

Conformational Changes ◽

Ribosomal Proteins ◽

Structural Changes ◽

Ribosomal Subunit ◽

Compact Structure ◽

E Coli ◽

Freeze Dried ◽

16S Rna ◽

30S Subunit

The aim of this study is to understand the mechanism of 16S rRNA folding into the compact structure of the small 30S subunit of E. coli ribosome. The assembly of the 30S E. coli ribosomal subunit is a sequence of specific interactions of 16S rRNA with 21 ribosomal proteins (S1-S21). Using dedicated high resolution STEM we have monitored structural changes induced in 16S rRNA by the proteins S4, S8, S15 and S20 which are involved in the initial steps of 30S subunit assembly. S4 is the first protein to bind directly and stoichiometrically to 16S rRNA. Direct binding also occurs individually between 16S RNA and S8 and S15. However, binding of S20 requires the presence of S4 and S8. The RNA-protein complexes are prepared by the standard reconstitution procedure, dialyzed against 60 mM KCl, 2 mM Mg(OAc)2, 10 mM-Hepes-KOH pH 7.5 (Buffer A), freeze-dried and observed unstained in dark field at -160°.

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