scholarly journals Arginylation Regulates Intracellular Actin Polymer Level by Modulating Actin Properties and Binding of Capping and Severing Proteins

2010 ◽  
Vol 21 (8) ◽  
pp. 1350-1361 ◽  
Author(s):  
Sougata Saha ◽  
Maureen M. Mundia ◽  
Fangliang Zhang ◽  
Ryan W. Demers ◽  
Farida Korobova ◽  
...  
Keyword(s):  
Actin Filaments ◽  
Actin Polymerization ◽  
Molecular Mechanisms ◽  
Leading Edge ◽  
Biochemical Properties ◽  
Cytoskeletal Organization ◽  
Actin Networks ◽  
Severe Impairment ◽  
Associated Proteins

Actin arginylation regulates lamella formation in motile fibroblasts, but the underlying molecular mechanisms are unknown. To understand how arginylation affects the actin cytoskeleton, we investigated the biochemical properties and the structural organization of actin filaments in wild-type and arginyltransferase (Ate1) knockout cells. We found that Ate1 knockout results in a dramatic reduction of the actin polymer levels in vivo accompanied by a corresponding increase in the monomer level. Purified nonarginylated actin has altered polymerization properties, and actin filaments from Ate1 knockout cells show altered interactions with several associated proteins. Ate1 knockout cells have severe impairment of cytoskeletal organization throughout the cell. Thus, arginylation regulates the ability of actin to form filaments in the whole cell rather than preventing the collapse of preformed actin networks at the cell leading edge as proposed in our previous model. This regulation is achieved through interconnected mechanisms that involve actin polymerization per se and through binding of actin-associated proteins.

scholarly journals Direct visualization of how Actin Depolymerizing Factor’s filament severing and depolymerization synergizes with Capping Protein's "monomer funneling" to promote rapid polarized growth of actin filaments

2017 ◽  
Author(s):  
Shashank Shekhar ◽  
Marie-France Carlier
Keyword(s):  
Actin Filaments ◽  
Actin Polymerization ◽  
Molecular Mechanisms ◽  
Leading Edge ◽  
Bulk Solution ◽  
Direct Visualization ◽  
Actin Networks ◽  
Capping Protein ◽  
Visual Evidence ◽  
Pointed End

AbstractA living cell’s ability to assemble actin filaments in intracellular motile processes is directly dependent on the availability of polymerizable actin monomers which feed polarized filament growth. Continued generation of the monomer pool by filament disassembly is therefore crucial. Disassemblers like ADF/cofilin and filament cappers like Capping Protein (CP) are essential agonists of motility, but the exact molecular mechanisms by which they accelerate actin polymerization at the leading edge and filament turnover has been debated for over two decades. While filament fragmentation by ADF/cofilin has long been demonstrated by TIRF, filament depolymerization was only inferred from bulk solution assays. Using microfluidics-assisted TIRF microscopy, we provide the first direct visual evidence of ADF's simultaneous severing and rapid depolymerization of individual filaments. We have also built a conceptually novel assay to directly visualize ADF’s effect on a filament population. We demonstrate that ADF’s enhanced pointed-end depolymerization leads to an increase in polymerizable actin monomers co-existing with filaments, thus promoting faster barbed-end growth. We further reveal how ADF-enhanced filament depolymerization synergizes with CP’s long-predicted “monomer funneling” and leads to skyrocketing of filament growth rates, close to estimated rates in the lamellipodia. The “Funneling model” hypothesized, on thermodynamic grounds, that at high enough extent of capping, the few noncapped filaments transiently grow much faster, an effect proposed to be very important for motility. We provide the first direct microscopic evidence of monomer funneling by CP at the scale of individual filaments. We believe that these results enlighten our understanding of the turnover of cellular actin networks.

scholarly journals Contribution of Filopodia to Cell Migration: A Mechanical Link between Protrusion and Contraction

2010 ◽  
Vol 2010 ◽  
pp. 1-13 ◽  
Author(s):  
Fei Xue ◽  
Deanna M. Janzen ◽  
David A. Knecht
Keyword(s):  
Cell Migration ◽  
Actin Polymerization ◽  
Myosin Ii ◽  
Temporal Distribution ◽  
Leading Edge ◽  
Distal Portion ◽  
Actin Binding ◽  
Actin Networks ◽  
Motile Cells ◽  
A Cell

Numerous F-actin containing structures are involved in regulating protrusion of membrane at the leading edge of motile cells. We have investigated the structure and dynamics of filopodia as they relate to events at the leading edge and the function of the trailing actin networks. We have found that although filopodia contain parallel bundles of actin, they contain a surprisingly nonuniform spatial and temporal distribution of actin binding proteins. Along the length of the actin filaments in a single filopodium, the most distal portion contains primarily T-plastin, while the proximal portion is primarily bound byα-actinin and coronin. Some filopodia are stationary, but lateral filopodia move with respect to the leading edge. They appear to form a mechanical link between the actin polymerization network at the front of the cell and the myosin motor activity in the cell body. The direction of lateral filopodial movement is associated with the direction of cell migration. When lateral filopodia initiate from and move toward only one side of a cell, the cell will turn opposite to the direction of filopodial flow. Therefore, this filopodia-myosin II system allows actin polymerization driven protrusion forces and myosin II mediated contractile force to be mechanically coordinated.

scholarly journals Elasticity of dense actin networks produces nanonewton protrusive forces

2021 ◽  
Author(s):  
Marion Jasnin ◽  
Jordan Hervy ◽  
Stéphanie Balor ◽  
Anais Bouissou ◽  
Amsha Proag ◽  
...  
Keyword(s):  
Actin Filaments ◽  
Actin Polymerization ◽  
Electron Tomography ◽  
Force Production ◽  
Basal Membrane ◽  
Theoretical Modelling ◽  
Elastic Behavior ◽  
Integrative Approach ◽  
Cellular Functions ◽  
Actin Networks

AbstractActin filaments assemble into force-generating systems involved in diverse cellular functions, including cell motility, adhesion, contractility and division. It remains unclear how networks of actin filaments, which individually generate piconewton forces, can produce forces reaching tens of nanonewtons. Here we use in situ cryo-electron tomography to unveil how the nanoscale architecture of macrophage podosomes enables basal membrane protrusion. We show that the sum of the actin polymerization forces at the membrane is not sufficient to explain podosome protrusive forces. Quantitative analysis of podosome organization demonstrates that the core is composed of a dense network of bent actin filaments storing elastic energy. Theoretical modelling of the network as a spring-loaded elastic material reveals that it exerts forces of up to tens of nanonewtons, similar to those evaluated experimentally. Thus, taking into account not only the interface with the membrane but also the bulk of the network, is crucial to understand force generation by actin machineries. Our integrative approach sheds light on the elastic behavior of dense actin networks and opens new avenues to understand force production inside cells.

Small-Molecule Screen Identifies ROS as Key Regulators of Actin Dynamics in Neutrophils

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4641-4641
Author(s):  
Hidenori Hattori ◽  
Kulandayan K Subramanian ◽  
Hongbo R. Luo
Keyword(s):  
Small Molecule ◽  
Actin Polymerization ◽  
Neutrophil Migration ◽  
Physiological Role ◽  
Leading Edge ◽  
Actin Dynamics ◽  
Functional Screening ◽  
Cellular Processes

Abstract Precise spatial and temporal control of actin polymerization and depolymerization is essential for mediating various cellular processes such as migration, phagocytosis, vesicle trafficking and adhesion. In this study, we used a small-molecule functional screening approach to identify novel regulators of actin dynamics during neutrophil migration. Here we show that NADPH-oxidase dependent Reactive Oxygen Species act as negative regulators of actin polymerization. Neutrophils with pharmacologically inhibited oxidase or isolated from Chronic Granulomatous Disease (CGD) patient and mice displayed enhanced F-actin polymerization, multiple pseudopods formation and impaired chemotaxis. ROS localized to pseudopodia and inhibited actin polymerization by driving actin glutathionylation at the leading edge of migrating cells. Consistent with these in vitro results, adoptively transferred CGD murine neutrophils also showed impaired in vivo recruitment to sites of inflammation. Together, these results present a novel physiological role for ROS in regulation of action polymerization and shed new light on the pathogenesis of CGD.

scholarly journals Chloroquine enhances temozolomide cytotoxicity in malignant gliomas by blocking autophagy

2014 ◽  
Vol 37 (6) ◽  
pp. E12 ◽  
Author(s):  
Encouse B. Golden ◽  
Hee-Yeon Cho ◽  
Ardeshir Jahanian ◽  
Florence M. Hofman ◽  
Stan G. Louie ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Malignant Gliomas ◽  
Glioma Cells ◽  
In Vivo Model ◽  
Autophagosome Formation ◽  
Individual Drug ◽  
In Vivo Experiments ◽  
Associated Proteins

Object In a recent clinical trial, patients with newly diagnosed glioblastoma multiforme benefited from chloroquine (CQ) in combination with conventional therapy (resection, temozolomide [TMZ], and radiation therapy). In the present study, the authors report the mechanism by which CQ enhances the therapeutic efficacy of TMZ to aid future studies aimed at improving this therapeutic regimen. Methods Using in vitro and in vivo experiments, the authors determined the mechanism by which CQ enhances TMZ cytotoxicity. They focused on the inhibition-of-autophagy mechanism of CQ by knockdown of the autophagy-associated proteins or treatment with autophagy inhibitors. This mechanism was tested using an in vivo model with subcutaneously implanted U87MG tumors from mice treated with CQ in combination with TMZ. Results Knockdown of the autophagy-associated proteins (GRP78 and Beclin) or treatment with the autophagy inhibitor, 3-methyl adenine (3-MA), blocked autophagosome formation and reduced CQ cytotoxicity, suggesting that autophagosome accumulation precedes CQ-induced cell death. In contrast, blocking autophagosome formation with knockdown of GRP78 or treatment with 3-MA enhanced TMZ cytotoxicity, suggesting that the autophagy pathway protects from TMZ-induced cytotoxicity. CQ in combination with TMZ significantly increased the amounts of LC3B-II (a marker for autophagosome levels), CHOP/GADD-153, and cleaved PARP (a marker for apoptosis) over those with untreated or individual drug-treated glioma cells. These molecular mechanisms seemed to take place in vivo as well. Subcutaneously implanted U87MG tumors from mice treated with CQ in combination with TMZ displayed higher levels of CHOP/GADD-153 than did untreated or individual drug-treated tumors. Conclusions Taken together, these results demonstrate that CQ blocks autophagy and triggers endoplasmic reticulum stress, thereby increasing the chemosensitivity of glioma cells to TMZ.

Side-chain amino acid based cationic polymer induced actin polymerization

2017 ◽  
Vol 5 (6) ◽  
pp. 1218-1226 ◽  
Author(s):  
Binoy Maiti ◽  
Priyanka Dutta ◽  
Soma Seal ◽  
Sunirmal Pal ◽  
Priyadarsi De ◽  
...  
Keyword(s):  
Amino Acid ◽  
Actin Filaments ◽  
Actin Polymerization ◽  
Cationic Polymer ◽  
Side Chain

A side-chain amino acid (alanine) based cationic polymer is able to nucleate, polymerize and stabilize actin filaments in vitro and in vivo.

scholarly journals Actin-independent association of vinculin with the cytoplasmic aspect of the plasma membrane in cell-contact areas.

1983 ◽  
Vol 96 (6) ◽  
pp. 1622-1630 ◽  
Author(s):  
Z Avnur ◽  
J V Small ◽  
B Geiger
Keyword(s):  
Actin Filaments ◽  
Molecular Mechanisms ◽  
Cell Contact ◽  
Membrane Association ◽  
Focal Contact ◽  
Permeabilized Cells ◽  
Focal Contacts ◽  
Microfilament Bundles ◽  
Associated Proteins ◽  
Independent Association

We investigated the mode of association of vinculin with areas of contact between the termini of microfilament bundles and the cell membrane in sites of focal contact with the substrate by selective removal of actin from these areas. Opened-up substrate-attached membranes of chick fibroblasts as well as detergent-permeabilized cells were treated with fragmin from Physarum in the presence of Ca+2. This treatment removed actin filaments from the cytoplasmic faces of the membranes, along with several actin-associated proteins (alpha-actinin, tropomyosin, myosin, and filamin). Vinculin distribution was not affected by treatment. Moreover, rhodamine- or fluorescein-conjugated vinculin, when added to these preparations, became specifically associated with the focal contacts regardless of whether the latter were pretreated with fragmin or not. We conclude that the association of vinculin with focal contacts is largely actin-independent. We discuss the implications of these findings in the molecular mechanisms of microfilament membrane association in areas of cell contact.

scholarly journals Analysis of the Actin–Myosin II System in Fish Epidermal Keratocytes: Mechanism of Cell Body Translocation

1997 ◽  
Vol 139 (2) ◽  
pp. 397-415 ◽  
Author(s):  
Tatyana M. Svitkina ◽  
Alexander B. Verkhovsky ◽  
Kyle M. McQuade ◽  
Gary G. Borisy
Keyword(s):  
Cell Body ◽  
Actin Filaments ◽  
Actin Polymerization ◽  
Myosin Ii ◽  
Leading Edge ◽  
Time Lapse ◽  
Supramolecular Organization ◽  
Retrograde Flow ◽  
Body Boundary ◽  
Filament Bundles

While the protrusive event of cell locomotion is thought to be driven by actin polymerization, the mechanism of forward translocation of the cell body is unclear. To elucidate the mechanism of cell body translocation, we analyzed the supramolecular organization of the actin–myosin II system and the dynamics of myosin II in fish epidermal keratocytes. In lamellipodia, long actin filaments formed dense networks with numerous free ends in a brushlike manner near the leading edge. Shorter actin filaments often formed T junctions with longer filaments in the brushlike area, suggesting that new filaments could be nucleated at sides of preexisting filaments or linked to them immediately after nucleation. The polarity of actin filaments was almost uniform, with barbed ends forward throughout most of the lamellipodia but mixed in arc-shaped filament bundles at the lamellipodial/cell body boundary. Myosin II formed discrete clusters of bipolar minifilaments in lamellipodia that increased in size and density towards the cell body boundary and colocalized with actin in boundary bundles. Time-lapse observation demonstrated that myosin clusters appeared in the lamellipodia and remained stationary with respect to the substratum in locomoting cells, but they exhibited retrograde flow in cells tethered in epithelioid colonies. Consequently, both in locomoting and stationary cells, myosin clusters approached the cell body boundary, where they became compressed and aligned, resulting in the formation of boundary bundles. In locomoting cells, the compression was associated with forward displacement of myosin features. These data are not consistent with either sarcomeric or polarized transport mechanisms of cell body translocation. We propose that the forward translocation of the cell body and retrograde flow in the lamellipodia are both driven by contraction of an actin–myosin network in the lamellipodial/cell body transition zone.

scholarly journals Crosslinking actin networks produces compressive force

2019 ◽  
Author(s):  
Rui Ma ◽  
Julien Berro
Keyword(s):  
Actin Filaments ◽  
Brownian Dynamics ◽  
Actin Polymerization ◽  
Turgor Pressure ◽  
Compressive Force ◽  
Yeast Cells ◽  
Actin Networks ◽  
Dynamics Simulations ◽  
Brownian Dynamics Simulations ◽  
Over Time

AbstractActin has been shown to be essential for clathrin-mediated endocytosis in yeast. However, actin polymerization alone is likely insufficient to produce enough force to deform the membrane against the huge turgor pressure of yeast cells. In this paper, we used Brownian dynamics simulations to demonstrate that crosslinking of a meshwork of non-polymerizing actin filaments is able to produce compressive forces. We show that the force can be up to thousands of piconewtons if the crosslinker has a high stiffness. The force decays over time as a result of crosslinker turnover, and is a result of converting chemical binding energy into elastic energy.

scholarly journals Lamellipodin promotes actin assembly by clustering Ena/VASP proteins and tethering them to actin filaments

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Scott D Hansen ◽  
R Dyche Mullins
Keyword(s):  
Axon Guidance ◽  
Actin Filaments ◽  
Network Formation ◽  
Focal Adhesions ◽  
Leading Edge ◽  
Polymerase Activity ◽  
Actin Assembly ◽  
Filament Assembly

Enabled/Vasodilator (Ena/VASP) proteins promote actin filament assembly at multiple locations, including: leading edge membranes, focal adhesions, and the surface of intracellular pathogens. One important Ena/VASP regulator is the mig-10/Lamellipodin/RIAM family of adaptors that promote lamellipod formation in fibroblasts and drive neurite outgrowth and axon guidance in neurons. To better understand how MRL proteins promote actin network formation we studied the interactions between Lamellipodin (Lpd), actin, and VASP, both in vivo and in vitro. We find that Lpd binds directly to actin filaments and that this interaction regulates its subcellular localization and enhances its effect on VASP polymerase activity. We propose that Lpd delivers Ena/VASP proteins to growing barbed ends and increases their polymerase activity by tethering them to filaments. This interaction represents one more pathway by which growing actin filaments produce positive feedback to control localization and activity of proteins that regulate their assembly.

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