scholarly journals Effects of actin filament cross-linking and filament length on actin-myosin interaction.

1985 ◽  
Vol 101 (5) ◽  
pp. 1850-1857 ◽  
Author(s):  
T R Coleman ◽  
M S Mooseker
Keyword(s):  
Atpase Activity ◽  
Actin Filament ◽  
Actin Filaments ◽  
Actin Binding ◽  
Molar Ratio ◽  
Cross Linking ◽  
Filament Length ◽  
Maximum Enhancement ◽  
Actomyosin Atpase ◽  
Cross Linker

We have used two actin-binding proteins of the intestinal brush border, TW 260/240 and villin, to examine the effects of filament cross-linking and filament length on myosin-actin interactions. TW 260/240 is a nonerythroid spectrin that is a potent cross-linker of actin filaments. In the presence of this cross-linker we observed a concentration-dependent enhancement of skeletal muscle actomyosin ATPase activity (150-560% of control; maximum enhancement at a 1:70-80 TW 260/240:actin molar ratio). TW 260/240 did not cause a similar enhancement of either acto-heavy meromyosin (HMM) ATPase or acto-myosin subfragment-one (S1) ATPase. Villin, a Ca2+-dependent filament capping and severing protein of the intestinal microvillus, was used to generate populations of actin filaments of various lengths from less than 20 nm to 2.0 microns; (villin:actin ratios of 1:2 to 1:4,000). The effect of filament length on actomyosin ATPase was biphasic. At villin:actin molar ratios of 1:2-25 actin-activated myosin ATPase activity was inhibited to 20-80% of control values, with maximum inhibition observed at the highest villin:actin ratio. The ATPase activities of acto-HMM and acto-S1 were also inhibited at these short filament lengths. At intermediate filament lengths generated at villin:actin ratios of 1:40-400 (average lengths 0.26-1.1 micron) an enhancement of actomyosin ATPase was observed (130-260% of controls), with a maximum enhancement at average filament lengths of 0.5 micron. The levels of actomyosin ATPase fell off to control values at low concentrations of villin where filament length distributions were almost those of controls. Unlike intact myosin, the actin-activated ATPase of neither HMM nor S1 showed an enhancement at these intermediate actin filament lengths.

scholarly journals Bundling of actin filaments by alpha-actinin depends on its molecular length.

1990 ◽  
Vol 110 (6) ◽  
pp. 2013-2024 ◽  
Author(s):  
R K Meyer ◽  
U Aebi
Keyword(s):  
Smooth Muscle ◽  
Actin Filament ◽  
Actin Filaments ◽  
Actin Binding ◽  
Molar Ratio ◽  
Cross Linking ◽  
Actin Bundle ◽  
Actin Bundles ◽  
Chicken Gizzard ◽  
Molecular Length

Cross-linking of actin filaments (F-actin) into bundles and networks was investigated with three different isoforms of the dumbbell-shaped alpha-actinin homodimer under identical reaction conditions. These were isolated from chicken gizzard smooth muscle, Acanthamoeba, and Dictyostelium, respectively. Examination in the electron microscope revealed that each isoform was able to cross-link F-actin into networks. In addition, F-actin bundles were obtained with chicken gizzard and Acanthamoeba alpha-actinin, but not Dictyostelium alpha-actinin under conditions where actin by itself polymerized into disperse filaments. This F-actin bundle formation critically depended on the proper molar ratio of alpha-actinin to actin, and hence F-actin bundles immediately disappeared when free alpha-actinin was withdrawn from the surrounding medium. The apparent dissociation constants (Kds) at half-saturation of the actin binding sites were 0.4 microM at 22 degrees C and 1.2 microM at 37 degrees C for chicken gizzard, and 2.7 microM at 22 degrees C for both Acanthamoeba and Dictyostelium alpha-actinin. Chicken gizzard and Dictyostelium alpha-actinin predominantly cross-linked actin filaments in an antiparallel fashion, whereas Acanthamoeba alpha-actinin cross-linked actin filaments preferentially in a parallel fashion. The average molecular length of free alpha-actinin was 37 nm for glycerol-sprayed/rotary metal-shadowed and 35 nm for negatively stained chicken gizzard; 46 and 44 nm, respectively, for Acanthamoeba; and 34 and 31 nm, respectively, for Dictyostelium alpha-actinin. In negatively stained preparations we also evaluated the average molecular length of alpha-actinin when bound to actin filaments: 36 nm for chicken gizzard and 35 nm for Acanthamoeba alpha-actinin, a molecular length roughly coinciding with the crossover repeat of the two-stranded F-actin helix (i.e., 36 nm), but only 28 nm for Dictyostelium alpha-actinin. Furthermore, the minimal spacing between cross-linking alpha-actinin molecules along actin filaments was close to 36 nm for both smooth muscle and Acanthamoeba alpha-actinin, but only 31 nm for Dictyostelium alpha-actinin. This observation suggests that the molecular length of the alpha-actinin homodimer may determine its spacing along the actin filament, and hence F-actin bundle formation may require "tight" (i.e., one molecule after the other) and "untwisted" (i.e., the long axis of the molecule being parallel to the actin filament axis) packing of alpha-actinin molecules along the actin filaments.

scholarly journals Emergence and maintenance of different sized actin filaments in a common pool of building blocks

2021 ◽  
Author(s):  
Deb Sankar Banerjee ◽  
Shiladitya Banerjee
Keyword(s):  
Growth Inhibition ◽  
Actin Filament ◽  
Actin Filaments ◽  
Length Distribution ◽  
Building Blocks ◽  
Nucleation And Growth ◽  
Actin Binding ◽  
Filament Length ◽  
Growth Promoters ◽  
Spontaneous Nucleation

Actin is one of the key structural components of the eukaryotic cytoskeleton that regulates cellular architecture and mechanical properties. Dynamic regulation of actin filament length and organization is essential for the control of many physiological processes including cell adhesion, motility and division. While previous studies have mostly focused on the mechanisms controlling the mean length of individual actin filaments, it remains poorly understood how distinct actin filament populations in cells maintain different size using the same set of molecular building blocks. Here we develop a theoretical model for the length regulation of multiple actin filaments by nucleation and growth rate modulation by actin binding proteins in a limiting pool of monomers. We first show that spontaneous nucleation of actin filaments naturally leads to heterogeneities in filament length distribution. We then investigate the effects of filament growth inhibition by capping proteins and growth promotion by formin proteins on filament length distribution. We find that filament length heterogeneity can be increased by growth inhibition, whereas growth promoters do not significantly affect length heterogeneities. Interestingly, a competition between filament growth inhibitors and growth promoters can give rise to bimodal filament length distribution as well as a highly heterogeneous length distribution with large statistical dispersion. We quantitatively predict how heterogeneity in actin filament length can be modulated by tuning F-actin nucleation and growth rates in order to create distinct filament subpopulations with different lengths.

Coagulation factor Va is an actin filament binding and cross-linking protein

1995 ◽  
Vol 73 (1-2) ◽  
pp. 105-112 ◽  
Author(s):  
Emilia Furmaniak-Kazmierczak ◽  
Michael E. Nesheim ◽  
Graham P. Côté
Keyword(s):  
Actin Filament ◽  
Light Chain ◽  
Actin Filaments ◽  
Factor Xa ◽  
Coagulation Factor ◽  
Actin Binding ◽  
Cross Linking ◽  
Factor V ◽  
Proteolytic Activation ◽  
Factor Va

Bovine coagulation cofactor factor Va is shown to bind to filaments of skeletal muscle actin with a dissociation constant of 40–50 nM in the presence of 50 mM NaCl. At saturation, approximately one molecule of factor Va was bound for every two actin molecules. The binding of factor Va to F-actin was inhibited by increasing ionic strength, being approximately 20-fold weaker at 150 mM NaCl. Addition of factor Va dramatically increased both the low-speed sedimentation and the low-shear viscosity of actin filament solutions, indicating that factor Va cross-links actin filaments. Factor Va also bound to actin filaments saturated with myosin. The isolated 74-kilodalton light chain of factor Va displayed actin binding and cross-linking properties indistinguishable from those of intact factor Va. The procofactor factor V bound weakly to F-actin, indicating that proteolytic activation is required to uncover the actin binding sites within the light chain domain. Actin filaments had only a slight inhibitory effect on the prothombinase activity of the factor Va – factor Xa – phospholipid complex. Since high concentrations of actin filaments can be exposed to the circulation when cells are damaged, the interaction of factor Va with actin may be of physiological relevance.Key words: blood coagulation, factor V, actin.

scholarly journals Modulation of gelsolin-induced actin-filament severing by caldesmon and tropomyosin and the effect of these proteins on the actin activation of myosin Mg2+-ATPase activity

1996 ◽  
Vol 315 (3) ◽  
pp. 753-759 ◽  
Author(s):  
R Dąbrowska ◽  
H Hinssen ◽  
B Gałązkiewicz ◽  
E Nowak
Keyword(s):  
Atpase Activity ◽  
Actin Filaments ◽  
Smooth Muscle Actin ◽  
Direct Analysis ◽  
Inhibitory Effect ◽  
Microscopic Investigation ◽  
Electron Microscopic Investigation ◽  
Actin Binding ◽  
Molar Ratio ◽  
Electron Microscopic

We have investigated the cumulative effects of three smooth-muscle actin-binding proteins, gelsolin, caldesmon and tropomyosin, on actin activation of myosin Mg2+-ATPase activity under low-ionic-strength conditions. A combination of tropomyosin (at a stoicheiometric ratio to actin) and gelsolin (at a molar ratio to actin of up to 1:100) showed essentially additive stimulatory effects that were counteracted by caldesmon. Suppression of the gelsolin-induced activation of the ATPase by caldesmon was higher in the presence of tropomyosin although it was not complete even at stoicheiometric amounts of both proteins to actin. Since activation of actin-activated ATPase activity of myosin by gelsolin is related to its severing action, it is concluded that caldesmon and tropomyosin cannot fully protect actin filaments against the severing activity of gelsolin. Direct analysis of the actin-severing activity of gelsolin by a fluorimetric assay using pyrene-labelled actin confirmed this conclusion. Tropomyosin and caldesmon in saturating amounts relative to actin inhibited the activity of gelsolin by between 21 and 40% and 25 and 48% respectively, depending on the molar ratio of gelsolin to actin. The inhibitory effect was increased with a combination of both (up to 67%) although it was evident that even under these conditions the actin filaments were not fully protected from being severed by gelsolin. These findings were corroborated by electron-microscopic investigation of actin filaments with or without tropomyosin and caldesmon after the addition of gelsolin.

scholarly journals Arp2/3 Complex from Acanthamoeba Binds Profilin and Cross-links Actin Filaments

1998 ◽  
Vol 9 (4) ◽  
pp. 841-852 ◽  
Author(s):  
R. Dyche Mullins ◽  
Joseph F. Kelleher ◽  
James Xu ◽  
Thomas D. Pollard
Keyword(s):  
Actin Filament ◽  
Actin Filaments ◽  
Analytical Ultracentrifugation ◽  
Acanthamoeba Castellanii ◽  
Leading Edge ◽  
Actin Binding ◽  
Cross Linking ◽  
Motile Cells ◽  
Mechanism Of Interaction ◽  
Cross Links

The Arp2/3 complex was first purified from Acanthamoeba castellanii by profilin affinity chromatography. The mechanism of interaction with profilin was unknown but was hypothesized to be mediated by either Arp2 or Arp3. Here we show that the Arp2 subunit of the complex can be chemically cross-linked to the actin-binding site of profilin. By analytical ultracentrifugation, rhodamine-labeled profilin binds Arp2/3 complex with a Kd of 7 μM, an affinity intermediate between the low affinity of profilin for barbed ends of actin filaments and its high affinity for actin monomers. These data suggest the barbed end of Arp2 is exposed, but Arp2 and Arp3 are not packed together in the complex exactly like two actin monomers in a filament. Arp2/3 complex also cross-links actin filaments into small bundles and isotropic networks, which are mechanically stiffer than solutions of actin filaments alone. Arp2/3 complex is concentrated at the leading edge of motileAcanthamoeba, and its localization is distinct from that of α-actinin, another filament cross-linking protein. Based on localization and actin filament nucleation and cross-linking activities, we propose a role for Arp2/3 in determining the structure of the actin filament network at the leading edge of motile cells.

scholarly journals Labelling the Ca2+-ATPase of skeletal-muscle sarcoplasmic reticulum with the cross-linker o-phthalaldehyde

1996 ◽  
Vol 317 (2) ◽  
pp. 433-437 ◽  
Author(s):  
Yamin M. KHAN ◽  
Matthew WICTOME ◽  
Malcolm EAST ◽  
Anthony G. J. LEE
Keyword(s):  
Skeletal Muscle ◽  
Sarcoplasmic Reticulum ◽  
Atpase Activity ◽  
Fluorescence Emission ◽  
Molar Ratio ◽  
Cross Linking ◽  
Fluorescence Emission Spectrum ◽  
Cross Link ◽  
Subsequent Reaction ◽  
Cross Linker

The Ca2+-ATPase in the sarcoplasmic reticulum of skeletal muscle reacts with o-phthalaldehyde (OPA) to form a fluorescent isoindole product. The stoichiometry of labelling of the ATPase is 9 nmol of isoindole/mg of ATPase, corresponding to a 1:1 molar ratio of isoindole:ATPase. There is no evidence for any intermolecular cross-linking. Isoindole formation is faster in the presence of methylamine, but the stoichiometry of labelling is unchanged, whereas in the presence of 2-mercaptoethanol the level of labelling is much higher. It is concluded that OPA reacts with a single Cys residue (defining the specificity of the reaction) in a fast step, subsequent reaction with a Lys residue to form the isoindole being rate-controlling. Labelling the ATPase with OPA in the absence of methylamine leads to total loss of ATPase activity, whereas in the presence of methylamine, the decrease in ATPase activity on reaction is small. We conclude that the loss of ATPase activity probably follows from formation of the intramolecular cross-link rather than from the initial modification of the Cys residue. Reaction with OPA is not affected by the presence of ATP, ADP or Ca2+, so that the reactive Cys is not part of a ligand-binding site. The fluorescence emission spectrum of the labelled ATPase indicates a hydrophobic environment for the isoindole ring.

scholarly journals The stable actin core of mechanosensory stereocilia features continuous turnover of actin cross-linkers

2018 ◽  
Vol 29 (15) ◽  
pp. 1856-1865 ◽  
Author(s):  
Pallabi Roy ◽  
Benjamin J. Perrin
Keyword(s):  
Hair Cell ◽  
Hair Cells ◽  
Head Movement ◽  
Actin Filaments ◽  
Actin Dynamics ◽  
Actin Binding ◽  
Cross Linking ◽  
Sensory Hair Cells ◽  
Actin Turnover ◽  
Cross Linker

Stereocilia are mechanosensitive protrusions on the surfaces of sensory hair cells in the inner ear that detect sound, gravity, and head movement. Their cores are composed of parallel actin filaments that are cross-linked and stabilized by several actin-binding proteins, including fascin-2, plastin-1, espin, and XIRP2. The actin filaments are the most stable known, with actin turnover primarily occurring at the stereocilia tips. While stereocilia actin dynamics has been well studied, little is known about the behavior of the actin cross-linking proteins, which are the most abundant type of protein in stereocilia after actin and are critical for stereocilia morphogenesis and maintenance. Here, we developed a novel transgenic mouse to monitor EGFP-fascin-2 incorporation . In contrast to actin, EGFP-fascin-2 readily enters the stereocilia core. We also compared the effect of EGFP-fascin-2 expression on developing and mature stereocilia. When it was induced during hair cell development, we observed increases in both stereocilia length and width. Interestingly, stereocilia size was not affected when EGFP-fascin-2 was induced in adult stereocilia. Regardless of the time of induction, EGFP-fascin-2 displaced both espin and plastin-1 from stereocilia. Altering the actin cross-linker composition, even as the actin filaments exhibit little to no turnover, provides a mechanism for ongoing remodeling and repair important for stereocilia homeostasis.

scholarly journals Some perspectives on the viscosity of actin filaments.

1982 ◽  
Vol 93 (3) ◽  
pp. 987-991 ◽  
Author(s):  
K S Zaner ◽  
T P Stossel
Keyword(s):  
Shear Rate ◽  
Actin Filament ◽  
Actin Filaments ◽  
Cytochalasin B ◽  
Gel Filtration ◽  
Synthetic Polymers ◽  
Actin Binding ◽  
Filament Length ◽  
Meaningful Information ◽  
Shear Rate Dependence

Measurements of the dynamic viscosity of various actin filament preparations under conditions of low and controlled shear: (a) confirm the shear rate dependence of F-actin viscosities and show that this dependence obeys the power law relationship observed for entangled synthetic polymers; (b) permit estimation of the extent to which shear artifact amplifies changes in the apparent viscosity of F-actin measured in a falling ball viscometer; (c) show that gel-filtration chromatography of actin and the addition of cytochalasin B to F-actin bring about small (20-40%) changes in the viscosity of the F-actin solutions. These variations are consistent with alterations in the actin-binding protein concentrations required for incipient gelation, a parameter inversely related to average filament length. Therefore: (a) changes in the viscosity of F-actin can be magnified by use of the falling ball viscometer, and may exaggerate their biological importance; (b) chromatography of actin may not be required to obtain meaningful information about the rheology of actin filaments; (c) changes in actin filament length can satisfactorily explain alterations in F-actin viscosity exerted by cytochalasin B and by chromatography, obviating the need to postulate specific interfilament interactions.

scholarly journals αE-catenin actin-binding domain alters actin filament conformation and regulates binding of nucleation and disassembly factors

2013 ◽  
Vol 24 (23) ◽  
pp. 3710-3720 ◽  
Author(s):  
Scott D. Hansen ◽  
Adam V. Kwiatkowski ◽  
Chung-Yueh Ouyang ◽  
HongJun Liu ◽  
Sabine Pokutta ◽  
...  
Keyword(s):  
Actin Filament ◽  
Actin Filaments ◽  
Binding Domain ◽  
Actin Binding ◽  
Filamentous Actin ◽  
Filament Structure ◽  
Actin Binding Domain ◽  
Filament Bundles ◽  
Underlying Mechanisms ◽  
Cell Cell

The actin-binding protein αE-catenin may contribute to transitions between cell migration and cell–cell adhesion that depend on remodeling the actin cytoskeleton, but the underlying mechanisms are unknown. We show that the αE-catenin actin-binding domain (ABD) binds cooperatively to individual actin filaments and that binding is accompanied by a conformational change in the actin protomer that affects filament structure. αE-catenin ABD binding limits barbed-end growth, especially in actin filament bundles. αE-catenin ABD inhibits actin filament branching by the Arp2/3 complex and severing by cofilin, both of which contact regions of the actin protomer that are structurally altered by αE-catenin ABD binding. In epithelial cells, there is little correlation between the distribution of αE-catenin and the Arp2/3 complex at developing cell–cell contacts. Our results indicate that αE-catenin binding to filamentous actin favors assembly of unbranched filament bundles that are protected from severing over more dynamic, branched filament arrays.

Actin filaments regulate epithelial Na+ channel activity

1991 ◽  
Vol 261 (5) ◽  
pp. C882-C888 ◽  
Author(s):  
H. F. Cantiello ◽  
J. L. Stow ◽  
A. G. Prat ◽  
D. A. Ausiello
Keyword(s):  
Actin Filament ◽  
Actin Filaments ◽  
Functional Role ◽  
Cytochalasin D ◽  
Actin Binding ◽  
Na Channel ◽  
Na Channels ◽  
Channel Activity ◽  
Cortical Actin ◽  
Excised Patches

The functional role of the cytoskeleton in the control of ion channel activity is unknown. In the present study, immunocolocalization of Na+ channels with specific antibodies and fluorescein isothiocyanate-phalloidin to stain the cortical cytoskeleton indicates that actin is always present in close proximity to apical Na+ channels in A6 cells. The patch-clamp technique was used to assess the effect of cortical actin networks on apical Na+ channels in these A6 epithelial cells. The actin filament disrupter, cytochalasin D (5 micrograms/ml), induced Na+ channel activity in cell-attached patches within 5 min of addition. Cytochalasin D also induced and/or increased Na+ channel activity in 90% of excised patches tested within 2 min. Addition of short actin filaments (greater than 5 microM) to excised patches also induced channel activity. This effect was enhanced by addition of ATP and/or cytochalasin D. The effect of actin on Na+ channel activity was reversed by addition of the G actin-binding protein DNase I or completely prevented by treatment of the excised patches with this enzyme. Addition of the actin-binding protein, filamin, reversibly inhibited both spontaneous and actin-induced Na+ channels. Thus actin filament networks, achieved by either depolymerizing endogenous actin filaments by treatment with cytochalasin D, the addition of exogenous short actin filaments plus ATP, or actin plus cytochalasin D, regulate apical Na+ channel activity. This conclusion was supported by the observation that the addition of short actin filaments in the form of actin-gelsolin complexes in molar ratios less than 8:1 was also effective in activating Na+ channels. We have thus demonstrated a functional role for the cortical actin network in the regulation of epithelial Na+ channels that may complement a structural role for membrane protein targetting and assembly.

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