Tropomyosin Isoforms Specify Functionally Distinct Actin Filament Populations In Vitro

Actin filament assembly is critical for eukaryotic cell motility. Arp2/3 complex and capping protein (CP) regulate actin assembly in vitro. To understand how these proteins regulate the dynamics of actin filament assembly in a motile cell, we visualized their distribution in living fibroblasts using green flourescent protein (GFP) tagging. Both proteins were concentrated in motile regions at the cell periphery and at dynamic spots within the lamella. Actin assembly was required for the motility and dynamics of spots and for motility at the cell periphery. In permeabilized cells, rhodamine-actin assembled at the cell periphery and at spots, indicating that actin filament barbed ends were present at these locations. Inhibition of the Rho family GTPase rac1, and to a lesser extent cdc42 and RhoA, blocked motility at the cell periphery and the formation of spots. Increased expression of phosphatidylinositol 5-kinase promoted the movement of spots. Increased expression of LIM–kinase-1, which likely inactivates cofilin, decreased the frequency of moving spots and led to the formation of aggregates of GFP–CP. We conclude that spots, which appear as small projections on the surface by whole mount electron microscopy, represent sites of actin assembly where local and transient changes in the cortical actin cytoskeleton take place.

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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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Specification of Actin Filament Function and Molecular Composition by Tropomyosin Isoforms

Molecular Biology of the Cell ◽

10.1091/mbc.e02-04-0244 ◽

2003 ◽

Vol 14 (3) ◽

pp. 1002-1016 ◽

Cited By ~ 180

Author(s):

Nicole S. Bryce ◽

Galina Schevzov ◽

Vicki Ferguson ◽

Justin M. Percival ◽

Jim J.-C. Lin ◽

...

Keyword(s):

Cell Size ◽

Functional Properties ◽

Actin Filament ◽

Actin Filaments ◽

Myosin Ii ◽

Molecular Composition ◽

Cellular Migration ◽

Stress Fibers ◽

Human Homologue ◽

Tropomyosin Isoforms

The specific functions of greater than 40 vertebrate nonmuscle tropomyosins (Tms) are poorly understood. In this article we have tested the ability of two Tm isoforms, TmBr3 and the human homologue of Tm5 (hTM5NM1), to regulate actin filament function. We found that these Tms can differentially alter actin filament organization, cell size, and shape. hTm5NM1was able to recruit myosin II into stress fibers, which resulted in decreased lamellipodia and cellular migration. In contrast, TmBr3 transfection induced lamellipodial formation, increased cellular migration, and reduced stress fibers. Based on coimmunoprecipitation and colocalization studies, TmBr3 appeared to be associated with actin-depolymerizing factor/cofilin (ADF)-bound actin filaments. Additionally, the Tms can specifically regulate the incorporation of other Tms into actin filaments, suggesting that selective dimerization may also be involved in the control of actin filament organization. We conclude that Tm isoforms can be used to specify the functional properties and molecular composition of actin filaments and that spatial segregation of isoforms may lead to localized specialization of actin filament function.

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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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The F-actin bundler α-actinin Ain1 is tailored for ring assembly and constriction during cytokinesis in fission yeast

Molecular Biology of the Cell ◽

10.1091/mbc.e16-01-0010 ◽

2016 ◽

Vol 27 (11) ◽

pp. 1821-1833 ◽

Cited By ~ 35

Author(s):

Yujie Li ◽

Jenna R. Christensen ◽

Kaitlin E. Homa ◽

Glen M. Hocky ◽

Alice Fok ◽

...

Keyword(s):

Fission Yeast ◽

Actin Filament ◽

Actin Filaments ◽

Biochemical Properties ◽

Ring Formation ◽

Cross Linking ◽

Contractile Ring ◽

Mixed Polarity ◽

Dividing Cells

The actomyosin contractile ring is a network of cross-linked actin filaments that facilitates cytokinesis in dividing cells. Contractile ring formation has been well characterized in Schizosaccharomyces pombe, in which the cross-linking protein α-actinin SpAin1 bundles the actin filament network. However, the specific biochemical properties of SpAin1 and whether they are tailored for cytokinesis are not known. Therefore we purified SpAin1 and quantified its ability to dynamically bind and bundle actin filaments in vitro using a combination of bulk sedimentation assays and direct visualization by two-color total internal reflection fluorescence microscopy. We found that, while SpAin1 bundles actin filaments of mixed polarity like other α-actinins, SpAin1 has lower bundling activity and is more dynamic than human α-actinin HsACTN4. To determine whether dynamic bundling is important for cytokinesis in fission yeast, we created the less dynamic bundling mutant SpAin1(R216E). We found that dynamic bundling is critical for cytokinesis, as cells expressing SpAin1(R216E) display disorganized ring material and delays in both ring formation and constriction. Furthermore, computer simulations of initial actin filament elongation and alignment revealed that an intermediate level of cross-linking best facilitates filament alignment. Together our results demonstrate that dynamic bundling by SpAin1 is important for proper contractile ring formation and constriction.

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In vitro studies of actin filament and network dynamics

Current Opinion in Cell Biology ◽

10.1016/j.ceb.2012.11.007 ◽

2013 ◽

Vol 25 (1) ◽

pp. 6-13 ◽

Cited By ~ 33

Author(s):

R Dyche Mullins ◽

Scott D Hansen

Keyword(s):

Actin Filament ◽

Network Dynamics ◽

In Vitro Studies

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Myosin Va Vesicular Transport is Modulated by Actin Filament Density, Orientation, and Polarity in an In Vitro 3D Actin Network

Biophysical Journal ◽

10.1016/j.bpj.2017.11.1182 ◽

2018 ◽

Vol 114 (3) ◽

pp. 211a

Author(s):

Andrew T. Lombardo ◽

Shane R. Nelson ◽

Guy G. Kennedy ◽

Kathleen M. Trybus ◽

Sam Walcott ◽

...

Keyword(s):

Actin Filament ◽

Vesicular Transport ◽

Actin Network ◽

Myosin Va

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Spreading of perturbations in myosin group kinetics along actin filaments

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1904164116 ◽

2019 ◽

Vol 116 (35) ◽

pp. 17336-17344 ◽

Cited By ~ 1

Author(s):

Zsombor Balassy ◽

Anne-Marie Lauzon ◽

Lennart Hilbert

Keyword(s):

Actin Filament ◽

Actin Filaments ◽

Nearest Neighbor ◽

Global Changes ◽

Chain Model ◽

Mechanical Coupling ◽

In Vitro Motility ◽

Local Perturbations ◽

Nearest Neighbor Coupling

Global changes in the state of spatially distributed systems can often be traced back to perturbations that arise locally. Whether such local perturbations grow into global changes depends on the system geometry and the spatial spreading of these perturbations. Here, we investigate how different spreading behaviors of local perturbations determine their global impact in 1-dimensional systems of different size. Specifically, we assessed sliding arrest events in in vitro motility assays where myosins propel actin, and simulated the underlying mechanochemistry of myosins that bind along the actin filament. We observed spontaneous sliding arrest events that occurred more frequently for shorter actin filaments. This observation could be explained by spontaneous local arrest of myosin kinetics that stabilizes once it spreads throughout an entire actin filament. When we introduced intermediate concentrations of the actin cross-linker filamin, longer actin was arrested more frequently. This observation was reproduced by simulations where filamin binding induces persistent local arrest of myosin kinetics, which subsequently spreads throughout the actin filament. A spin chain model with nearest-neighbor coupling reproduced key features of our experiments and simulations, thus extending to other linear systems with nearest-neighbor coupling the following conclusions: 1) perturbations that are persistent only once they spread throughout the system are more effective in smaller systems, and 2) perturbations that are persistent upon their establishment are more effective in larger systems. Beyond these general conclusions, our work also provides a theoretical model of collective myosin kinetics with a finite range of mechanical coupling along the actin filament.

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Dehydroepiandrosterone Sulfate and Allopregnanolone Directly Stimulate Catecholamine Production via Induction of Tyrosine Hydroxylase and Secretion by Affecting Actin Polymerization

Endocrinology ◽

10.1210/en.2005-0263 ◽

2005 ◽

Vol 146 (8) ◽

pp. 3309-3318 ◽

Cited By ~ 56

Author(s):

I. Charalampopoulos ◽

Ε. Dermitzaki ◽

L. Vardouli ◽

C. Tsatsanis ◽

C. Stournaras ◽

...

Keyword(s):

Tyrosine Hydroxylase ◽

Actin Filament ◽

Chromaffin Cells ◽

Actin Polymerization ◽

Dehydroepiandrosterone Sulfate ◽

Neuroactive Steroids ◽

Zona Reticularis ◽

Catecholamine Synthesis ◽

Catecholamine Levels

Abstract Adrenal cortical cells of zona reticularis produce the neuroactive steroids dehydroepiandrosterone (DHEA), its sulfate ester dehydroepiandrosterone sulfate (DHEAS), and allopregnanolone (ALLO). An interaction between zona reticularis and adrenal medulla has been postulated based on their close proximity and their interwoven borders. The aim of this paper was to examine in vitro the possible paracrine effects of these steroids on catecholamine production from adrenomedullary chromaffin cells, using an established in vitro model of chromaffin cells, the PC12 rat pheochromocytoma cell line. We have found the following: 1) DHEA, DHEAS, and ALLO increased acutely (peak effect between 10–30 min) and dose-dependently (EC50 in the nanomolar range) catecholamine levels (norepinephrine and dopamine). 2) It appears that the acute effect of these steroids involved actin depolymerization/actin filament disassembly, a fast-response cellular system regulating trafficking of catecholamine vesicles. Specifically, 10−6m phallacidin, an actin filament stabilizer, completely prevented steroid-induced catecholamine secretion. 3) DHEAS and ALLO, but not DHEA, also affected catecholamine synthesis. Indeed, DHEAS and ALLO increased catecholamine levels at 24 h, an effect blocked by l-2-methyl-3-(-4hydroxyphenyl)alanine and 3-(hydrazinomethyl)phenol hydrochloride, inhibitors of tyrosine hydroxylase and l-aromatic amino acid decarboxylase, respectively, suggesting that this effect involved catecholamine synthesis. The latter hypothesis was confirmed by finding that DHEAS and ALLO increased both the mRNA and protein levels of tyrosine hydroxylase. In conclusion, our findings suggest that neuroactive steroids exert a direct tonic effect on adrenal catecholamine synthesis and secretion. These data associate the adrenomedullary malfunction observed in old age and neuroactive steroids.

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The scaffold-protein IQGAP1 enhances and spatially restricts the actin-nucleating activity of Diaphanous-related formin 1 (DIAPH1)

Journal of Biological Chemistry ◽

10.1074/jbc.ra119.010476 ◽

2020 ◽

Vol 295 (10) ◽

pp. 3134-3147 ◽

Cited By ~ 2

Author(s):

Anan Chen ◽

Pam D. Arora ◽

Christine C. Lai ◽

John W. Copeland ◽

Trevor F. Moraes ◽

...

Keyword(s):

Plasma Membrane ◽

Actin Filament ◽

Intramolecular Interaction ◽

Essential Feature ◽

Temporal Regulation ◽

Iq Motif ◽

Sequential Binding ◽

Spatio Temporal

The actin cytoskeleton is a dynamic array of filaments that undergoes rapid remodeling to drive many cellular processes. An essential feature of filament remodeling is the spatio-temporal regulation of actin filament nucleation. One family of actin filament nucleators, the Diaphanous-related formins, is activated by the binding of small G-proteins such as RhoA. However, RhoA only partially activates formins, suggesting that additional factors are required to fully activate the formin. Here we identify one such factor, IQ motif containing GTPase activating protein-1 (IQGAP1), which enhances RhoA-mediated activation of the Diaphanous-related formin (DIAPH1) and targets DIAPH1 to the plasma membrane. We find that the inhibitory intramolecular interaction within DIAPH1 is disrupted by the sequential binding of RhoA and IQGAP1. Binding of RhoA and IQGAP1 robustly stimulates DIAPH1-mediated actin filament nucleation in vitro. In contrast, the actin capping protein Flightless-I, in conjunction with RhoA, only weakly stimulates DIAPH1 activity. IQGAP1, but not Flightless-I, is required to recruit DIAPH1 to the plasma membrane where actin filaments are generated. These results indicate that IQGAP1 enhances RhoA-mediated activation of DIAPH1 in vivo. Collectively these data support a model where the combined action of RhoA and an enhancer ensures the spatio-temporal regulation of actin nucleation to stimulate robust and localized actin filament production in vivo.

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