Direct evidence for ADP-inorganic phosphate-F-actin as the major intermediate in ATP-actin polymerization. Rate of dissociation of inorganic phosphate from actin filaments

Biochemistry ◽  
1986 ◽  
Vol 25 (24) ◽  
pp. 7789-7792 ◽  
Author(s):  
M. F. Carlier ◽  
D. Pantaloni
Keyword(s):  
Actin Filaments ◽  
Inorganic Phosphate ◽  
Actin Polymerization ◽  
Direct Evidence ◽  
Polymerization Rate

scholarly journals Processive capping by formin suggests a force-driven mechanism of actin polymerization

2004 ◽  
Vol 167 (6) ◽  
pp. 1011-1017 ◽  
Author(s):  
Michael M. Kozlov ◽  
Alexander D. Bershadsky
Keyword(s):  
Actin Filaments ◽  
Actin Polymerization ◽  
Polymerization Rate ◽  
Elastic Stresses ◽  
Capping Proteins ◽  
Order Of Magnitude ◽  
Cellular Mechanosensing ◽  
Pulling Force ◽  
Actin Concentration

Regulation of actin polymerization is essential for cell functioning. Here, we predict a novel phenomenon—the force-driven polymerization of actin filaments mediated by proteins of the formin family. Formins localize to the barbed ends of actin filaments, but, in contrast to the standard capping proteins, allow for actin polymerization in the barbed direction. First, we show that the mechanism of such “leaky capping” can be understood in terms of the elasticity of the formin molecules. Second, we demonstrate that if a pulling force acts on the filament end via the leaky cap, the elastic stresses can drive actin polymerization. We estimate that a moderate pulling force of ∼3.4 pN is sufficient to reduce the critical actin concentration required for barbed end polymerization by an order of magnitude. Furthermore, the pulling force increases the polymerization rate. The suggested mechanism of force-driven polymerization could be a key element in a variety of cellular mechanosensing devices.

scholarly journals Actin-binding protein promotes the bipolar and perpendicular branching of actin filaments.

1980 ◽  
Vol 87 (3) ◽  
pp. 841-848 ◽  
Author(s):  
J H Hartwig ◽  
J Tyler ◽  
T P Stossel
Keyword(s):  
Actin Filaments ◽  
Actin Polymerization ◽  
Average Length ◽  
Binding Protein ◽  
Actin Binding ◽  
Branch Points ◽  
Heavy Meromyosin ◽  
Actin Binding Protein ◽  
Protein Dimers ◽  
Protein Molecules

Branching filaments with striking perpendicularity form when actin polymerizes in the presence of macrophage actin-binding protein. Actin-binding protein molecules are visible at the branch points. Compared with actin polymerized in the absence of actin-binding proteins, not only do the filaments branch but the average length of the actin filaments decreases from 3.2 to 0.63 micrometer. Arrowhead complexes formed by addition of heavy meromyosin molecules to the branching actin filaments point toward the branch points. Actin-binding protein also accelerates the onset of actin polymerization. All of these findings show that actin filaments assemble from nucleating sites on actin-binding protein dimers. A branching polymerization of actin filaments from a preexisting lattice of actin filaments joined by actin-binding protein molecules could generate expansion of cortical cytoplasm in amoeboid cells.

scholarly journals Tropomyosin inhibits ADF/cofilin-dependent actin filament dynamics

2002 ◽  
Vol 156 (6) ◽  
pp. 1065-1076 ◽  
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.

scholarly journals Forces drive basement membrane invasion inCaenorhabditis elegans

2018 ◽  
Vol 115 (45) ◽  
pp. 11537-11542 ◽  
Author(s):  
Rodrigo Cáceres ◽  
Nagagireesh Bojanala ◽  
Laura C. Kelley ◽  
Jes Dreier ◽  
John Manzi ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
Basement Membrane ◽  
Actin Filaments ◽  
Actin Polymerization ◽  
Force Production ◽  
Developmental Event ◽  
C Elegans ◽  
Anchor Cell

During invasion, cells breach basement membrane (BM) barriers with actin-rich protrusions. It remains unclear, however, whether actin polymerization applies pushing forces to help break through BM, or whether actin filaments play a passive role as scaffolding for targeting invasive machinery. Here, using the developmental event of anchor cell (AC) invasion inCaenorhabditis elegans, we observe that the AC deforms the BM and underlying tissue just before invasion, exerting forces in the tens of nanonewtons range. Deformation is driven by actin polymerization nucleated by the Arp2/3 complex and its activators, whereas formins and cross-linkers are dispensable. Delays in invasion upon actin regulator loss are not caused by defects in AC polarity, trafficking, or secretion, as appropriate markers are correctly localized in the AC even when actin is reduced and invasion is disrupted. Overall force production emerges from this study as one of the main tools that invading cells use to promote BM disruption inC. elegans.

scholarly journals Arp2/3 and Unc45 maintain heterochromatin stability in Drosophila polytene chromosomes

2019 ◽  
Author(s):  
George Dialynas ◽  
Laetitia Delabaere ◽  
Irene Chiolo
Keyword(s):  
Actin Filaments ◽  
Genome Stability ◽  
Actin Polymerization ◽  
Cultured Cells ◽  
Nuclear Periphery ◽  
Polytene Chromosomes ◽  
Nuclear Actin ◽  
Nuclear Dynamics ◽  
Ectopic Recombination ◽  
Repair Pathways

AbstractRepairing DNA double-strand breaks (DSBs) is particularly challenging in pericentromeric heterochromatin, where the abundance of repeated sequences exacerbates the risk of ectopic recombination. InDrosophilaKc cells, accurate homologous recombination (HR) repair of heterochromatic DSBs relies on the relocalization of repair sites to the nuclear periphery before Rad51 recruitment and strand invasion. This movement is driven by Arp2/3-dependent nuclear actin filaments and myosins’ ability to walk along them. Conserved mechanisms enable the relocalization of heterochromatic DSBs in mouse cells, and their defects lead to massive ectopic recombination in heterochromatin and chromosome rearrangements. InDrosophilapolytene chromosomes, extensive DNA movement is blocked by a stiff structure of chromosome bundles. Repair pathways in this context are poorly characterized, and whether heterochromatic DSBs relocalize in these cells is unknown. Here, we show that damage in heterochromatin results in relaxation of the heterochromatic chromocenter, consistent with a dynamic response in this structure. Arp2/3, the Arp2/3 activator Scar, and the myosin activator Unc45, are required for heterochromatin stability in polytene cells, suggesting that relocalization enables heterochromatin repair in this tissue. Together, these studies reveal critical roles for actin polymerization and myosin motors in heterochromatin repair and genome stability across different organisms and tissue types.Impact StatementHeterochromatin relies on dedicated pathways for ‘safe’ recombinational repair. In mouse and fly cultured cells, DNA repair requires the movement of repair sites away from the heterochromatin ‘domain’vianuclear actin filaments and myosins. Here, we explore the importance of these pathways inDrosophilasalivary gland cells, which feature a stiff bundle of endoreduplicated polytene chromosomes. Repair pathways in polytene chromosomes are largely obscure and how nuclear dynamics operate in this context is unknown. We show that heterochromatin relaxes in response to damage, and relocalization pathways are necessary for repair and stability of heterochromatic sequences. This deepens our understanding of repair mechanisms in polytenes, revealing unexpected dynamics. It also provides a first understanding of nuclear dynamics responding to replication damage or rDNA breaks, providing a new understanding of the importance of the nucleoskeleton in genome stability. We expect these discoveries to shed light on tumorigenic processes, including therapy-induced cancer relapses.

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.

scholarly journals Tropomyosin Tpm3.1 is required to maintain the structure and function of the axon initial segment

2019 ◽  
Author(s):  
Amr Abouelezz ◽  
Holly Stefen ◽  
Mikael Segerstråle ◽  
David Micinski ◽  
Rimante Minkeviciene ◽  
...  
Keyword(s):  
Actin Filaments ◽  
Hippocampal Neurons ◽  
Initial Segment ◽  
Actin Polymerization ◽  
Firing Frequency ◽  
Axon Initial Segment ◽  
Action Potential Initiation ◽  
Sodium Ion Channels ◽  
And Function ◽  
Filament Network

ABSTRACTThe axon initial segment (AIS) is the site of action potential initiation and serves as a vesicular filter and diffusion barrier that help maintain neuronal polarity. Recent studies have revealed details about a specialized structural complex in the AIS. While an intact actin cytoskeleton is required for AIS formation, pharmacological disruption of actin polymerization compromises the AIS vesicle filter but does not affect overall AIS structure. In this study, we found that the tropomyosin isoform Tpm3.1 decorates a population of relatively stable actin filaments in the AIS. Inhibiting Tpm3.1 in cultured hippocampal neurons led to the loss of AIS structure, the AIS vesicle filter, the clustering of sodium ion channels, and reduced firing frequency. We propose that Tpm3.1-decorated actin filaments form a stable actin filament network under the AIS membrane which provides a scaffold for membrane organization and AIS proteins.

Direct evidence of changes in myofilament responsiveness to Ca2+ during hypoxia and reoxygenation in myocardium

1990 ◽  
Vol 259 (3) ◽  
pp. H784-H795 ◽  
Author(s):  
R. J. Hajjar ◽  
J. K. Gwathmey
Keyword(s):  
Inorganic Phosphate ◽  
Direct Evidence ◽  
Ion Concentration ◽  
Force Curve ◽  
Force Development ◽  
Maximal Force ◽  
Phosphate Ion ◽  
Calcium Indicator ◽  
The Right ◽  
The Relationship

In the presence of 1 microM ryanodine, muscles loaded with the calcium indicator aequorin were stimulated at 15-20 Hz to produce steady levels of force and intracellular Ca2+ concentrations [( Ca2+]i) at various extracellular Ca2+ concentration ([Ca2+]o). After 5, 10, and 15 min of hypoxia and 3 min of reoxygenation, tetani were initiated. Force vs. [Ca2+]i relation was shifted to the right 0.11, 0.18, and 0.24 pCa units, and maximal force was down 66, 48, and 37% after start of hypoxia. During reoxygenation, the relationship was shifted up by 26%. In skinned fiber preparations, an increase in inorganic phosphate ion concentration from 0 to 10 mM and 15 mM decreased maximal force development by 32 and 53%, respectively, and shifted the pCa-force curve to the right by 0.08 and 0.14 pCa. A decrease in pH from 7.1 to 6.8 shifted the pCa-force curve to the right by 0.20 pCa units without affecting maximal force. These changes indicate that during hypoxia, a decrease in the sensitivity of the myofilaments to Ca2+ and a depression of maximal Ca2(+)-activated force occur, whereas during reoxygenation, there is an increase in maximal Ca2(+)-activated force.

Effect of acute hypercapnia on PTH-stimulated phosphaturia in dietary Pi-deprived rat

1987 ◽  
Vol 253 (1) ◽  
pp. F34-F40 ◽  
Author(s):  
J. Guntupalli ◽  
B. Matthews ◽  
B. Carlin ◽  
E. Bourke
Keyword(s):  
Glomerular Filtration Rate ◽  
Parathyroid Hormone ◽  
Glomerular Filtration ◽  
Inorganic Phosphate ◽  
Filtration Rate ◽  
Direct Evidence ◽  
Respiratory Acidosis ◽  
Urinary Ph ◽  
Renal Handling ◽  
Per Se

The effects of respiratory acidosis on renal inorganic phosphate (Pi) handling are controversial. Clearance experiments, therefore, were performed in fasted, chronically parathyroidectomized (PTX), dietary Pi-deprived rats. The objectives were twofold: to study the effects of compensated and uncompensated hypercapnia per se on renal Pi excretion and to examine the interaction between acute hypercapnia, dietary Pi, and parathyroid hormone (PTH) on the renal handling of Pi. Acute hypercapnia increased the plasma Pi (delta 2.82 +/- 0.65 mg/dl, P less than 0.05) without altering the glomerular filtration rate (GFR). The FEPi increased (delta 7.26 +/- 0.48%, P less than 0.001) but the TRPi/GFR also increased. PTH (3 U X kg-1 X h-1) superimposed on hypercapnia resulted in a plasma Pi comparable to hypercapnia alone. The FEPi (7.56 +/- 0.78 vs. 24.43 +/- 2.20%; P less than 0.001) was higher and the TRPi/GFR (117 +/- 4 vs. 80 +/- 2 micrograms/min, P less than 0.01) lower, in the former group. PTH infusion during normocapnia resulted in a lower FEPi (0.20 +/- 0.10 vs. 24.43 +/- 2.20%, P less than 0.001) and a higher TRPi/GFR (106 +/- 2 vs. 80 +/- 2 micrograms/min, P less than 0.01) compared with PTH infusion during hypercapnia. Urinary adenosine 3',5'-cyclic monophosphate (cAMP) excretion was similar between the groups. During hypercapnia, when the extracellular acidemia was neutralized, the phosphaturic action of PTH persisted. These studies offer direct evidence that in chronically PTX, dietary Pi-deprived rats, the phosphaturic action of PTH is restored by hypercapnia per se. This effect appears to be independent of extracellular acidemia, changes in the plasma Pi and calcium, urinary pH and Na and cAMP excretion.

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