Single-molecule imaging of IQGAP1 regulating actin filament dynamics

Molecular Biology of the Cell ◽

10.1091/mbc.e21-04-0211 ◽

2021 ◽

Author(s):

Gregory J. Hoeprich ◽

Amy N. Sinclair ◽

Shashank Shekhar ◽

Bruce L. Goode

Keyword(s):

Cell Adhesion ◽

Actin Filament ◽

Single Molecule ◽

Actin Dynamics ◽

Actin Binding ◽

Functional Effect ◽

Single Filament ◽

Filament Dynamics ◽

Do So

IQGAP is a conserved family of actin-binding proteins with essential roles in cell motility, cytokinesis, and cell adhesion, yet there remains a limited understanding of how IQGAP proteins directly influence actin filament dynamics. To close this gap, we used single-molecule and single-filament TIRF microscopy to observe IQGAP regulating actin dynamics in real time. To our knowledge, this is the first study to do so. Our results demonstrate that full-length human IQGAP1 forms dimers that stably bind to actin filament sides and transiently cap barbed ends. These interactions organize filaments into thin bundles, suppress barbed end growth, and inhibit filament disassembly. Surprisingly, each activity depends on distinct combinations of IQGAP1 domains and/or dimerization, suggesting that different mechanisms underlie each functional effect on actin. These observations have important implications for how IQGAP functions as an actin regulator in vivo, and how it may be regulated in different biological settings. [Media: see text] [Media: see text] [Media: see text]

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Single-molecule imaging of IQGAP1 regulating actin filament dynamics in real time

10.1101/2021.04.18.440338 ◽

2021 ◽

Author(s):

Gregory J Hoeprich ◽

Shashank Shekhar ◽

Bruce L Goode

Keyword(s):

Real Time ◽

Actin Filament ◽

Single Molecule ◽

Actin Dynamics ◽

Actin Binding ◽

Functional Effect ◽

Single Filament ◽

Filament Dynamics ◽

Do So

IQGAP is a conserved family of actin-binding proteins with essential roles in cell motility, cytokinesis, and cell adhesion, yet it has remained poorly understood how IQGAP proteins directly regulate actin filament dynamics. To close this gap, we used single-molecule and single-filament TIRF microscopy to directly visualize IQGAP regulating actin dynamics in real time. To our knowledge, this is the first study to do so. Our results show that full-length human IQGAP1 forms dimers that stably bind to filament sides and transiently cap barbed ends. These interactions organize actin filaments into thin bundles, suppress barbed end growth, and inhibit filament disassembly. Surprisingly, each activity depends on distinct combinations of IQGAP1 domains and/or dimerization, suggesting that different mechanisms underlie each functional effect on actin. These observations have important implications for how IQGAP functions as a direct actin regulator in vivo, and how it is deployed and regulated in different biological settings.

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Actin Depolymerizing Factor (ADF/Cofilin) Enhances the Rate of Filament Turnover: Implication in Actin-based Motility

The Journal of Cell Biology ◽

10.1083/jcb.136.6.1307 ◽

1997 ◽

Vol 136 (6) ◽

pp. 1307-1322 ◽

Cited By ~ 703

Author(s):

Marie-France Carlier ◽

Valérie Laurent ◽

Jérôme Santolini ◽

Ronald Melki ◽

Dominique Didry ◽

...

Keyword(s):

Actin Filament ◽

Atp Hydrolysis ◽

Fold Increase ◽

Actin Dynamics ◽

Actin Binding ◽

Actin Depolymerizing Factor ◽

Filament Dynamics ◽

Relevant Effect

Actin-binding proteins of the actin depolymerizing factor (ADF)/cofilin family are thought to control actin-based motile processes. ADF1 from Arabidopsis thaliana appears to be a good model that is functionally similar to other members of the family. The function of ADF in actin dynamics has been examined using a combination of physical–chemical methods and actin-based motility assays, under physiological ionic conditions and at pH 7.8. ADF binds the ADPbound forms of G- or F-actin with an affinity two orders of magnitude higher than the ATP- or ADP-Pi– bound forms. A major property of ADF is its ability to enhance the in vitro turnover rate (treadmilling) of actin filaments to a value comparable to that observed in vivo in motile lamellipodia. ADF increases the rate of propulsion of Listeria monocytogenes in highly diluted, ADF-limited platelet extracts and shortens the actin tails. These effects are mediated by the participation of ADF in actin filament assembly, which results in a change in the kinetic parameters at the two ends of the actin filament. The kinetic effects of ADF are end specific and cannot be accounted for by filament severing. The main functionally relevant effect is a 25-fold increase in the rate of actin dissociation from the pointed ends, while the rate of dissociation from the barbed ends is unchanged. This large increase in the rate-limiting step of the monomer-polymer cycle at steady state is responsible for the increase in the rate of actin-based motile processes. In conclusion, the function of ADF is not to sequester G-actin. ADF uses ATP hydrolysis in actin assembly to enhance filament dynamics.

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Myosin and gelsolin cooperate in actin filament severing and actomyosin motor activity

Journal of Biological Chemistry ◽

10.1074/jbc.ra120.015863 ◽

2020 ◽

pp. jbc.RA120.015863

Author(s):

Venukumar Vemula ◽

Tamás Huber ◽

Marko Ušaj ◽

Beáta Bugyi ◽

Alf Mansson

Keyword(s):

Motor Activity ◽

Actin Filament ◽

Single Molecule ◽

Actin Filaments ◽

Actin Binding ◽

In Vitro Motility Assay ◽

Cooperative Effects ◽

Myosin Motor ◽

Filament Dynamics

Actin is a major intracellular protein with key functions in cellular motility, signaling and structural rearrangements. Its dynamic behavior, such as polymerisation and depolymerisation of actin filaments in response to intra- and extracellular cues, is regulated by an abundance of actin binding proteins. Out of these, gelsolin is one of the most potent for filament severing. However, myosin motor activity also fragments actin filaments through motor induced forces, suggesting that these two proteins could cooperate to regulate filament dynamics and motility. To test this idea, we used an in vitro motility assay, where actin filaments are propelled by surface-adsorbed heavy meromyosin (HMM) motor fragments. This allows studies of both motility and filament dynamics using isolated proteins. Gelsolin, at both nanomolar and micromolar Ca2+ concentration, appreciably enhanced actin filament severing caused by HMM-induced forces at 1 mM MgATP, an effect that was increased at higher HMM motor density. This finding is consistent with cooperativity between actin filament severing by myosin-induced forces and by gelsolin. We also observed reduced sliding velocity of the HMM-propelled filaments in the presence of gelsolin, providing further support of myosin-gelsolin cooperativity. Total internal reflection fluorescence microscopy based single molecule studies corroborated that the velocity reduction was a direct effect of gelsolin-binding to the filament and revealed different filament severing pattern of stationary and HMM propelled filaments. Overall, the results corroborate cooperative effects between gelsolin-induced alterations in the actin filaments and changes due to myosin motor activity leading to enhanced F-actin severing of possible physiological relevance.

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Dynamics of Tpm1.8 domains on actin filaments with single molecule resolution

10.1101/2020.06.15.152033 ◽

2020 ◽

Cited By ~ 1

Author(s):

Ilina Bareja ◽

Hugo Wioland ◽

Miro Janco ◽

Philip R. Nicovich ◽

Antoine Jégou ◽

...

Keyword(s):

Actin Filament ◽

Single Molecule ◽

Actin Filaments ◽

High Probability ◽

Actin Binding ◽

Single Molecule Level ◽

Filament Dynamics ◽

Kinetics Of ◽

Insight Into

ABSTRACTTropomyosins regulate dynamics and functions of the actin cytoskeleton by forming long chains along the two strands of actin filaments that act as gatekeepers for the binding of other actin-binding proteins. The fundamental molecular interactions underlying the binding of tropomyosin to actin are still poorly understood. Using microfluidics and fluorescence microscopy, we observed the binding of fluorescently labelled tropomyosin isoform Tpm1.8 to unlabelled actin filaments in real time. This approach in conjunction with mathematical modeling enabled us to quantify the nucleation, assembly and disassembly kinetics of Tpm1.8 on single filaments and at the single molecule level. Our analysis suggests that Tpm1.8 decorates the two strands of the actin filament independently. Nucleation of a growing tropomyosin domain proceeds with high probability as soon as the first Tpm1.8 molecule is stabilised by the addition of a second molecule, ultimately leading to full decoration of the actin filament. In addition, Tpm1.8 domains are asymmetrical, with enhanced dynamics at the edge oriented towards the barbed end of the actin filament. The complete description of Tpm1.8 kinetics on actin filaments presented here provides molecular insight into actin-tropomyosin filament formation and the role of tropomyosins in regulating actin filament dynamics.

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A Balance of Capping Protein and Profilin Functions Is Required to Regulate Actin Polymerization in Drosophila Bristle

Molecular Biology of the Cell ◽

10.1091/mbc.e02-05-0300 ◽

2003 ◽

Vol 14 (1) ◽

pp. 118-128 ◽

Cited By ~ 34

Author(s):

Roberta Hopmann ◽

Kathryn G. Miller

Keyword(s):

Actin Cytoskeleton ◽

Actin Filament ◽

Actin Polymerization ◽

Genetic Interaction ◽

Actin Binding ◽

Actin Assembly ◽

Loss Of Function ◽

Capping Protein ◽

Filament Dynamics

Profilin is a well-characterized protein known to be important for regulating actin filament assembly. Relatively few studies have addressed how profilin interacts with other actin-binding proteins in vivo to regulate assembly of complex actin structures. To investigate the function of profilin in the context of a differentiating cell, we have studied an instructive genetic interaction between mutations in profilin (chickadee) and capping protein (cpb). Capping protein is the principal protein in cells that caps actin filament barbed ends. When its function is reduced in the Drosophila bristle, F-actin levels increase and the actin cytoskeleton becomes disorganized, causing abnormal bristle morphology. chickadee mutations suppress the abnormal bristle phenotype and associated abnormalities of the actin cytoskeleton seen in cpb mutants. Furthermore, overexpression of profilin in the bristle mimics many features of thecpb loss-of-function phenotype. The interaction betweencpb and chickadee suggests that profilin promotes actin assembly in the bristle and that a balance between capping protein and profilin activities is important for the proper regulation of F-actin levels. Furthermore, this balance of activities affects the association of actin structures with the membrane, suggesting a link between actin filament dynamics and localization of actin structures within the cell.

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Villin Severing Activity Enhances Actin-based Motility In Vivo

Molecular Biology of the Cell ◽

10.1091/mbc.e06-05-0423 ◽

2007 ◽

Vol 18 (3) ◽

pp. 827-838 ◽

Cited By ~ 22

Author(s):

Céline Revenu ◽

Matthieu Courtois ◽

Alphée Michelot ◽

Cécile Sykes ◽

Daniel Louvard ◽

...

Keyword(s):

Shigella Flexneri ◽

Actin Dynamics ◽

Actin Binding ◽

Loss Of Function ◽

Mesenchymal Transition ◽

Capping Protein ◽

Function Strategy ◽

In Cells

Villin, an actin-binding protein associated with the actin bundles that support microvilli, bundles, caps, nucleates, and severs actin in a calcium-dependant manner in vitro. We hypothesized that the severing activity of villin is responsible for its reported role in enhancing cell plasticity and motility. To test this hypothesis, we chose a loss of function strategy and introduced mutations in villin based on sequence comparison with CapG. By pyrene-actin assays, we demonstrate that this mutant has a strongly reduced severing activity, whereas nucleation and capping remain unaffected. The bundling activity and the morphogenic effects of villin in cells are also preserved in this mutant. We thus succeeded in dissociating the severing from the three other activities of villin. The contribution of villin severing to actin dynamics is analyzed in vivo through the actin-based movement of the intracellular bacteria Shigella flexneri in cells expressing villin and its severing variant. The severing mutations abolish the gain of velocity induced by villin. To further analyze this effect, we reconstituted an in vitro actin-based bead movement in which the usual capping protein is replaced by either the wild type or the severing mutant of villin. Confirming the in vivo results, villin-severing activity enhances the velocity of beads by more than two-fold and reduces the density of actin in the comets. We propose a model in which, by severing actin filaments and capping their barbed ends, villin increases the concentration of actin monomers available for polymerization, a mechanism that might be paralleled in vivo when an enterocyte undergoes an epithelio-mesenchymal transition.

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Cofilin is a pH sensor for actin free barbed end formation: role of phosphoinositide binding

The Journal of Cell Biology ◽

10.1083/jcb.200804161 ◽

2008 ◽

Vol 183 (5) ◽

pp. 865-879 ◽

Cited By ~ 122

Author(s):

Christian Frantz ◽

Gabriela Barreiro ◽

Laura Dominguez ◽

Xiaoming Chen ◽

Robert Eddy ◽

...

Keyword(s):

Actin Filament ◽

Structural Changes ◽

Ph Sensor ◽

Ph Sensitive ◽

Actin Binding ◽

Filamentous Actin ◽

Filament Dynamics ◽

Ph Dependent ◽

Dynamics Simulations

Newly generated actin free barbed ends at the front of motile cells provide sites for actin filament assembly driving membrane protrusion. Growth factors induce a rapid biphasic increase in actin free barbed ends, and we found both phases absent in fibroblasts lacking H+ efflux by the Na-H exchanger NHE1. The first phase is restored by expression of mutant cofilin-H133A but not unphosphorylated cofilin-S3A. Constant pH molecular dynamics simulations and nuclear magnetic resonance (NMR) reveal pH-sensitive structural changes in the cofilin C-terminal filamentous actin binding site dependent on His133. However, cofilin-H133A retains pH-sensitive changes in NMR spectra and severing activity in vitro, which suggests that it has a more complex behavior in cells. Cofilin activity is inhibited by phosphoinositide binding, and we found that phosphoinositide binding is pH-dependent for wild-type cofilin, with decreased binding at a higher pH. In contrast, phosphoinositide binding by cofilin-H133A is attenuated and pH insensitive. These data suggest a molecular mechanism whereby cofilin acts as a pH sensor to mediate a pH-dependent actin filament dynamics.

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Tropomyosin inhibits ADF/cofilin-dependent actin filament dynamics

The Journal of Cell Biology ◽

10.1083/jcb.200110013 ◽

2002 ◽

Vol 156 (6) ◽

pp. 1065-1076 ◽

Cited By ~ 180

Author(s):

Shoichiro Ono ◽

Kanako Ono

Keyword(s):

Actin Filament ◽

Actin Filaments ◽

Actin Polymerization ◽

Biological Properties ◽

Actin Dynamics ◽

Background Suppression ◽

Thin Filaments ◽

Actin Organization ◽

C Elegans ◽

Filament Dynamics

Tropomyosin binds to actin filaments and is implicated in stabilization of actin cytoskeleton. We examined biochemical and cell biological properties of Caenorhabditis elegans tropomyosin (CeTM) and obtained evidence that CeTM is antagonistic to ADF/cofilin-dependent actin filament dynamics. We purified CeTM, actin, and UNC-60B (a muscle-specific ADF/cofilin isoform), all of which are derived from C. elegans, and showed that CeTM and UNC-60B bound to F-actin in a mutually exclusive manner. CeTM inhibited UNC-60B–induced actin depolymerization and enhancement of actin polymerization. Within isolated native thin filaments, actin and CeTM were detected as major components, whereas UNC-60B was present at a trace amount. Purified UNC-60B was unable to interact with the native thin filaments unless CeTM and other associated proteins were removed by high-salt extraction. Purified CeTM was sufficient to restore the resistance of the salt-extracted filaments from UNC-60B. In muscle cells, CeTM and UNC-60B were localized in different patterns. Suppression of CeTM by RNA interference resulted in disorganized actin filaments and paralyzed worms in wild-type background. However, in an ADF/cofilin mutant background, suppression of CeTM did not worsen actin organization and worm motility. These results suggest that tropomyosin is a physiological inhibitor of ADF/cofilin-dependent actin dynamics.

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Side-binding proteins modulate actin filament dynamics

10.1101/008128 ◽

2014 ◽

Author(s):

Alvaro H. Crevenna ◽

Marcelino Arciniega ◽

Aurelie Dupont ◽

Kaja Kowalska ◽

Oliver Lange ◽

...

Keyword(s):

Actin Filament ◽

Brownian Dynamics ◽

Actin Polymerization ◽

Actin Dynamics ◽

Central Feature ◽

Filament Dynamics ◽

Filament Structure ◽

The Rich ◽

Dynamics Simulations ◽

Pointed End

Actin filament dynamics govern many key physiological processes from cell motility to tissue morphogenesis. A central feature of actin dynamics is the capacity of the filament to polymerize and depolymerize at its ends in response to cellular conditions. It is currently thought that filament kinetics can be described by a single rate constant for each end. Here, using direct visualization of single actin filament elongation, we show that actin polymerization kinetics at both filament ends are strongly influenced by proteins that bind to the lateral filament surface. We also show that the less dynamic end, called the pointed-end, has a non-elongating state that dominates the observed filament kinetic asymmetry. Estimates of filament flexibility and Brownian dynamics simulations suggest that the observed kinetic diversity arises from structural alteration. Tuning filament kinetics by exploiting the natural malleability of the actin filament structure may be a ubiquitous mechanism to generate the rich variety of observed cellular actin dynamics.

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