scholarly journals Actin filament organization in the fish keratocyte lamellipodium.

1995 ◽  
Vol 129 (5) ◽  
pp. 1275-1286 ◽  
Author(s):  
J V Small ◽  
M Herzog ◽  
K Anderson
Keyword(s):  
Electron Microscope ◽  
Actin Filament ◽  
Actin Filaments ◽  
Lower Layer ◽  
Anterior Part ◽  
Length Distribution ◽  
Fixation Method ◽  
Release Mechanism ◽  
Filamentous Actin ◽  
Phalloidin Staining

From recent studies of locomoting fish keratocytes it was proposed that the dynamic turnover of actin filaments takes place by a nucleation-release mechanism, which predicts the existence of short (less than 0.5 microns) filaments throughout the lamellipodium (Theriot, J. A., and T. J. Mitchison. 1991. Nature (Lond.). 352:126-131). We have tested this model by investigating the structure of whole mount keratocyte cytoskeletons in the electron microscope and phalloidin-labeled cells, after various fixations, in the light microscope. Micrographs of negatively stained keratocyte cytoskeletons produced by Triton extraction showed that the actin filaments of the lamellipodium are organized to a first approximation in a two-dimensional orthogonal network with the filaments subtending an angle of around 45 degrees to the cell front. Actin filament fringes grown onto the front edge of keratocyte cytoskeletons by the addition of exogenous actin showed a uniform polarity when decorated with myosin subfragment-1, consistent with the fast growing ends of the actin filaments abutting the anterior edge. A steady drop in filament density was observed from the mid-region of the lamellipodium to the perinuclear zone and in images of the more posterior regions of lower filament density many of the actin filaments could be seen to be at least several microns in length. Quantitative analysis of the intensity distribution of fluorescent phalloidin staining across the lamellipodium revealed that the gradient of filament density as well as the absolute content of F-actin was dependent on the fixation method. In cells first fixed and then extracted with Triton, a steep gradient of phalloidin staining was observed from the front to the rear of the lamellipodium. With the protocol required to obtain the electron microscope images, namely Triton extraction followed by fixation, phalloidin staining was, significantly and preferentially reduced in the anterior part of the lamellipodium. This resulted in a lower gradient of filament density, consistent with that seen in the electron microscope, and indicated a loss of around 45% of the filamentous actin during Triton extraction. We conclude, first that the filament organization and length distribution does not support a nucleation release model, but is more consistent with a treadmilling-type mechanism of locomotion featuring actin filaments of graded length. Second, we suggest that two layers of filaments make up the lamellipodium; a lower, stabilized layer associated with the ventral membrane and an upper layer associated with the dorsal membrane that is composed of filaments of a shorter range of lengths than the lower layer and which is mainly lost in Triton.

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.

scholarly journals F-actin bundles in Drosophila bristles are assembled from modules composed of short filaments.

1996 ◽  
Vol 135 (5) ◽  
pp. 1291-1308 ◽  
Author(s):  
L G Tilney ◽  
P Connelly ◽  
S Smith ◽  
G M Guild
Keyword(s):  
Actin Filament ◽  
Actin Filaments ◽  
Modular Design ◽  
Thin Sections ◽  
Actin Bundle ◽  
Actin Bundles ◽  
Phalloidin Staining ◽  
Filament Bundles ◽  
End To End ◽  
As If

The actin bundles in Drosophila bristles run the length of the bristle cell and are accordingly 65 microns (microchaetes) or 400 microns (macrochaetes) in length, depending on the bristle type. Shortly after completion of bristle elongation in pupae, the actin bundles break down as the bristle surface becomes chitinized. The bundles break down in a bizarre way; it is as if each bundle is sawed transversely into pieces that average 3 microns in length. Disassembly of the actin filaments proceeds at the "sawed" surfaces. In all cases, the cuts in adjacent bundles appear in transverse register. From these images, we suspected that each actin bundle is made up of a series of shorter bundles or modules that are attached end-to-end. With fluorescent phalloidin staining and serial thin sections, we show that the modular design is present in nondegenerating bundles. Decoration of the actin filaments in adjacent bundles in the same bristle with subfragment 1 of myosin reveals that the actin filaments in every module have the same polarity. To study how modules form developmentally, we sectioned newly formed and elongating bristles. At the bristle tip are numerous tiny clusters of 6-10 filaments. These clusters become connected together more basally to form filament bundles that are poorly organized, initially, but with time become maximally cross-linked. Additional filaments are then added to the periphery of these organized bundle modules. All these observations make us aware of a new mechanism for the formation and elongation of actin filament bundles, one in which short bundles are assembled and attached end-to-end to other short bundles, as are the vertical girders between the floors of a skyscraper.

Characterization of the actin filament capping state in human erythrocyte ghost and cytoskeletal preparations

2000 ◽  
Vol 349 (1) ◽  
pp. 105-111 ◽  
Author(s):  
Philip A. KUHLMAN
Keyword(s):  
Actin Filament ◽  
Human Erythrocyte ◽  
Actin Filaments ◽  
Striated Muscle ◽  
Length Distribution ◽  
Shearing Stress ◽  
Bivalent Cation ◽  
Thin Filaments ◽  
Cation Concentration ◽  
Bivalent Cations

The narrow Gaussian-length-distribution of actin filaments forming the cytoskeleton of the human erythrocyte indicates the existence of strict mechanisms for length determination and maintenance. A similar regulation is achieved in striated muscle by the capping of both the ends of the thin filaments, which consequently prevents monomer exchange. However, the ability of erythroid cytoskeletal preparations to nucleate actin polymerization has led to the proliferation of the idea that at least the barbed ends of the actin filaments are uncapped. The mechanism by which the length of the filaments is thus maintained has been left open to debate. In an effort to resolve any doubt regarding length-maintenance in human erythrocytes we have characterized the capping state of the actin filaments in a number of different ghost and cytoskeletal preparations. Under conditions of sufficiently high bivalent-cation concentration the actin filaments retain functional caps at both the barbed and pointed ends. Hence filament capping at both ends prevents redistribution of the actin monomer in a similar manner to that proposed for the thin filaments of striated muscle. Actin filament uncapping is apparently caused by the centrifugal shearing stress imposed during ghost preparation. The uncapping is more pronounced when the bivalent-cation concentration is reduced or when the membrane is removed by detergents. The effects of bivalent cations seem to be mediated through the erythroid protein spectrin, consistent with the hypothesis of Wallis et al. [Wallis, Babitch and Wenegieme (1993) Biochemistry 32, 5045-5050] that the ability of spectrin to resist shearing stress is dependent on the degree of bound bivalent cations.

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.

scholarly journals Kinetic analysis of F-actin depolymerization in polymorphonuclear leukocyte lysates indicates that chemoattractant stimulation increases actin filament number without altering the filament length distribution.

1991 ◽  
Vol 115 (3) ◽  
pp. 677-687 ◽  
Author(s):  
M L Cano ◽  
D A Lauffenburger ◽  
S H Zigmond
Keyword(s):  
Actin Filaments ◽  
Time Course ◽  
Average Length ◽  
Length Distribution ◽  
Fold Increase ◽  
Dissociation Rate ◽  
Actin Binding ◽  
Filamentous Actin ◽  
Actin Depolymerization ◽  
Time Courses

The rate of filamentous actin (F-actin) depolymerization is proportional to the number of filaments depolarizing and changes in the rate are proportional to changes in filament number. To determine the number and length of actin filaments in polymorphonuclear leukocytes and the change in filament number and length that occurs during the increase in F-actin upon chemoattractant stimulation, the time course of cellular F-actin depolymerization in lysates of control and peptide-stimulated cells was examined. F-actin was quantified by the TRITC-labeled phalloidin staining of pelletable actin. Lysis in 1.2 M KCl and 10 microM DNase I minimized the effects of F-actin binding proteins and G-actin, respectively, on the kinetics of depolymerization. To determine filament number and length from a depolymerization time course, depolymerization kinetics must be limited by the actin monomer dissociation rate. Comparison of time courses of depolymerization in the presence (pointed ends free) or absence (barbed and pointed ends free) of cytochalasin suggested depolymerization occurred from both ends of the filament and that monomer dissociation was rate limiting. Control cells had 1.7 +/- 0.4 x 10(5) filaments with an average length of 0.29 +/- 0.09 microns. Chemo-attractant stimulation for 90 s at room temperature with 0.02 microM N-formylnorleucylleucylphenylalanine caused a twofold increase in F-actin and about a two-fold increase in the total number of actin filaments to 4.0 +/- 0.5 x 10(5) filaments with an average length of 0.27 +/- 0.07 microns. In both cases, most (approximately 80%) of the filaments were quite short (less than or equal to 0.18 micron). The length distributions of actin filaments in stimulated and control cells were similar.

scholarly journals Identification of actin filaments in the rhabdomeral microvilli of Drosophila photoreceptors.

1990 ◽  
Vol 110 (6) ◽  
pp. 1993-1998 ◽  
Author(s):  
K Arikawa ◽  
J L Hicks ◽  
D S Williams
Keyword(s):  
Actin Filament ◽  
Brush Border ◽  
Actin Filaments ◽  
Immunogold Labeling ◽  
Entire Length ◽  
Filamentous Actin ◽  
Fast Growing ◽  
Intestinal Brush Border ◽  
Myosin Subfragment ◽  
Myosin Subfragment 1

The phototransductive microvilli of arthropod photoreceptors each contain an axial cytoskeleton. The present study shows that actin filaments are a component of this cytoskeleton in Drosophila. Firstly, actin was detected in the rhabdomeral microvilli and in the subrhabdomeral cytoplasm by immunogold labeling with antiactin. Secondly, the rhabdomeres were labeled with phalloidin, indicating the presence of filamentous actin. Finally, the actin filaments were decorated with myosin subfragment-1. The characteristic arrowhead complex formed by subfragment-1 decoration points towards the base of the microvilli, so that the fast growing end of each filament is at the distal end of the microvillus, where it is embedded in a detergent-resistant cap. Each microvillus contains more than one actin filament. Decorated filaments extend the entire length of each microvillus and project into the subrhabdomeral cytoplasm. This organization is comparable to that of the actin filaments in intestinal brush border microvilli. Similar observations were made with the photoreceptor microvilli of the crayfish, Procambarus. Our results provide an indication as to how any myosin that is associated with the rhabdomeres might function.

scholarly journals Direct observation of actin filament severing by gelsolin and binding by gCap39 and CapZ.

1991 ◽  
Vol 115 (6) ◽  
pp. 1629-1638 ◽  
Author(s):  
E L Bearer
Keyword(s):  
Actin Filament ◽  
Direct Observation ◽  
Actin Filaments ◽  
Molecular Mechanisms ◽  
Actin Binding ◽  
Filamentous Actin ◽  
Rhodamine Phalloidin ◽  
Capping Protein ◽  
Calcium Dependent ◽  
Definition Of

Dynamic behavior of actin filaments in cells is the basis of many different cellular activities. Remodeling of the actin filament network involves polymerization and depolymerization of the filaments. Proteins that regulate these behaviors include proteins that sever and/or cap actin filaments. This report presents direct observation of severing of fluorescently-labeled actin filaments. Coverslips coated with gelsolin, a multi-domain, calcium-dependent capping and severing protein, bound rhodamine-phalloidin-saturated filaments along their length in the presence of EGTA. Upon addition of calcium, attached filaments bent as they broke. Actophorin, a low molecular weight, monomer sequestering, calcium-independent severing protein did not sever phalloidin-saturated filaments. Both gCap 39, a gelsolin-like, calcium-dependent capping protein that does not sever filaments, and CapZ, a heterodimeric, non-calcium-dependent capping protein, bound the filaments by one end to the coverslip. Visualization of individual filaments also revealed severing activity present in mixtures of actin-binding proteins isolated by filamentous actin affinity chromatography from early Drosophila embryos. This activity was different from either gelsolin or actophorin because it was not inhibited by phalloidin, but was calcium independent. The results of these studies provide new information about the molecular mechanisms of severing and capping by well-characterized proteins as well as definition of a novel type of severing activity.

Tropomodulin assembles early in myofibrillogenesis in chick skeletal muscle: evidence that thin filaments rearrange to form striated myofibrils

1999 ◽  
Vol 112 (8) ◽  
pp. 1111-1123 ◽  
Author(s):  
A. Almenar-Queralt ◽  
C.C. Gregorio ◽  
V.M. Fowler
Keyword(s):  
Skeletal Muscle ◽  
Actin Filament ◽  
Actin Filaments ◽  
Primary Cultures ◽  
General Mechanism ◽  
Thin Filaments ◽  
Phalloidin Staining ◽  
Capping Protein ◽  
Filament Bundles ◽  
End Capping

Actin filament lengths in muscle and nonmuscle cells are believed to depend on the regulated activity of capping proteins at both the fast growing (barbed) and slow growing (pointed) filament ends. In striated muscle, the pointed end capping protein, tropomodulin, has been shown to maintain the lengths of thin filaments in mature myofibrils. To determine whether tropomodulin might also be involved in thin filament assembly, we investigated the assembly of tropomodulin into myofibrils during differentiation of primary cultures of chick skeletal muscle cells. Our results show that tropomodulin is expressed early in differentiation and is associated with the earliest premyofibrils which contain overlapping and misaligned actin filaments. In addition, tropomodulin can be found in actin filament bundles at the distal tips of growing myotubes, where sarcomeric alpha-actinin is not always detected, suggesting that tropomodulin caps actin filament pointed ends even before the filaments are cross-linked into Z bodies by alpha-actinin. Tropomodulin staining exhibits an irregular punctate pattern along the length of premyofibrils that demonstrate a smooth phalloidin staining pattern for F-actin. Strikingly, the tropomodulin dots often appear to be located between the closely spaced, dot-like Z bodies that are stained for (α)-actinin. Thus, in the earliest premyofibrils, the pointed ends of the thin filaments are clustered and partially aligned with respect to the Z bodies (the location of the barbed filament ends). At later stages of differentiation, the tropomodulin dots become aligned into regular periodic striations concurrently with the appearance of striated phalloidin staining for F-actin and alignment of Z bodies into Z lines. Tropomodulin, together with the barbed end capping protein, CapZ, may function from the earliest stages of myofibrillogenesis to restrict the lengths of newly assembled thin filaments by capping their ends; thus, transitions from nonstriated to striated myofibrils in skeletal muscle are likely due principally to filament rearrangements rather than to filament polymerization or depolymerization. Rearrangements of actin filaments capped at their pointed and barbed ends may be a general mechanism by which cells restructure their actin cytoskeletal networks during cell growth and differentiation.

scholarly journals Structural effects and functional implications of phalloidin and jasplakinolide binding to actin filaments

2019 ◽  
Author(s):  
Sabrina Pospich ◽  
Felipe Merino ◽  
Stefan Raunser
Keyword(s):  
High Resolution ◽  
Actin Filament ◽  
Actin Filaments ◽  
Inorganic Phosphate ◽  
Binding Proteins ◽  
Conformational Space ◽  
Actin Binding ◽  
List Type ◽  
Filamentous Actin ◽  
Actin Binding Proteins

SummaryActin undergoes structural transitions during polymerization, ATP hydrolysis and subsequent release of inorganic phosphate. Several actin binding proteins sense specific states during this transition and can thus target different regions of the actin filament. Here we show in atomic detail that phalloidin, a mushroom toxin that is routinely used to stabilize and label actin filaments, suspends the structural changes in actin, likely influencing its interaction with actin binding proteins. Furthermore, high-resolution cryo-EM structures reveal structural rearrangements in F-actin upon inorganic phosphate release in phalloidin-stabilized filaments. We find that the effect of the sponge toxin jasplakinolide differs from the one of phalloidin, despite their overlapping binding site and similar interactions with the actin filament. Analysis of structural conformations of F-actin suggests that stabilizing agents trap states within the natural conformational space of actin.Abstract FigureHighlightsFive high-resolution cryo-EM structures of stabilized filamentous actinPhalloidin traps different structural states depending on when it is addedThe effect of phalloidin and jasplakinolide on filamentous actin is not identicalBoth toxins likely interfere with the binding of proteins sensing F-actin’s nucleotide state

Myofibrillar degeneration with diphtheria toxin

2020 ◽  
Vol 45 (4) ◽  
pp. 351-357
Author(s):  
Bilge Özerman Edis ◽  
Muhammet Bektaş ◽  
Rüstem Nurten
Keyword(s):  
Protein Synthesis ◽  
Actin Filaments ◽  
Diphtheria Toxin ◽  
Cardiac Tissue ◽  
Culture Conditions ◽  
Bacterial Toxin ◽  
Filamentous Actin ◽  
Cardiac Damage ◽  
Tissue Samples

AbstractObjectivesCardiac damage in patient with diphtheritic myocarditis is reported as the leading cause of mortality. Diphtheria toxin (DTx) is a well-known bacterial toxin inducing various cytotoxic effects. Mainly, catalytic fragment inhibits protein synthesis, induces cytotoxicity, and depolymerizes actin filaments. In this study, we aimed to demonstrate the extent of myofibrillar damage under DTx treatment to porcine cardiac tissue samples.MethodsTissue samples were incubated with DTx for 1–3 h in culture conditions. To analyze whole toxin (both fragments) distribution, conjugation of DTx with FITC was performed. Measurements were carried out with fluorescence spectrophotometer before and after dialysis. Immunofluorescence microscopy was used to show localization of DTx-FITC (15 nM) on cardiac tissue incubated for 2 h. Ultrastructural characterization of cardiac tissue samples treated with DTx (15 or 150 nM) was performed with transmission electron microscopy.ResultsDTx exerts myofibrillar disorganization. Myofilament degeneration, mitochondrial damage, vacuolization, and abundant lipid droplets were determined with 150 nM of DTx treatment.ConclusionsThis finding is an addition to depolymerization of actin filaments as a result of the DTx-actin interactions in in vitro conditions, indicating that myofilament damage can occur with DTx directly besides protein synthesis inhibition. Ultrastructural results support the importance of filamentous actin degeneration at diphtheritic myocarditis.

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