scholarly journals Polymerization of actin. VI. The polarity of the actin filaments in the acrosomal process and how it might be determined.

1979 ◽  
Vol 81 (3) ◽  
pp. 608-623 ◽  
Author(s):  
L G Tilney ◽  
N Kallenbach
Keyword(s):  
Low Temperature ◽  
Actin Filaments ◽  
Critical Concentration ◽  
Actin Monomer ◽  
Spontaneous Nucleation ◽  
Myosin Subfragment ◽  
Acrosomal Reaction ◽  
Myosin Subfragment 1

The polarity of the actin filaments which assemble from the nucleating body or actomere of Thyone and Pisaster sperm was determined using myosin subfragment 1 decoration. The polarity was found to be unidirectional with the arrowheads pointing towards the cell center. When polymerization is induced at low temperature with concentrations of actin near the critical concentration for polymerization, elongation of filaments occurs preferentially off the apical end. If the sperm are induced to undergo the acrosomal reaction with an ionophore, the polarity of the actin filaments attached to the actomere is the same as that already described, but the filaments which polymerize parallel to, but peripheral to, those extending from the actomere are randomly polarized. These randomly polarized filaments appear to result from spontaneous nucleation. When sperm are induced to undergo the acrosomal reaction with eggs, the polarity of the actin filaments is also unidirectional with the arrowheads pointing towards the cell center. From these results we conclude: (a) that the actomere, by nucleating the polymerization of actin filaments, controls the polarity of the actin filaments in the acrosomal process, (b) that the actomere recognizes a surface of the actin monomer that is different from that surface recognized by the dense material attached to membranes, and (c) that egg myosin could not act to pull the sperm into the egg. Included is a discussion of how the observation that monomers add largely to one end of a decorated filament in vitro relates to these in vivo observations.

scholarly journals How Listeria exploits host cell actin to form its own cytoskeleton. II. Nucleation, actin filament polarity, filament assembly, and evidence for a pointed end capper.

1992 ◽  
Vol 118 (1) ◽  
pp. 83-93 ◽  
Author(s):  
L G Tilney ◽  
D J DeRosier ◽  
A Weber ◽  
M S Tilney
Keyword(s):  
Host Cell ◽  
Actin Filaments ◽  
Critical Concentration ◽  
Filament Assembly ◽  
Tightly Coupled ◽  
Pointed End ◽  
Phagosomal Membrane ◽  
Simple Picture

After Listeria, a bacterium, is phagocytosed by a macrophage, it dissolves the phagosomal membrane and enters the cytoplasm. The Listeria than nucleates actin filaments from its surface. These newly assembled actin filaments show unidirectional polarity with their barbed ends associated with the surface of the Listeria. Using actin concentrations below the pointed end critical concentration we find that filament elongation must be occurring by monomers adding to the barbed ends, the ends associated with the Listerial surface. If Listeria with tails are incubated in G actin under polymerizing conditions, the Listeria is translocated away from its preformed tail by the elongation of filaments attached to the Listeria. This experiment and others tell us that in vivo filament assembly must be tightly coupled to filament capping and cross-bridging so that if one process outstrips another, chaos ensues. We also show that the actin filaments in the tail are capped on their pointed ends which inhibits further elongation and/or disassembly in vitro. From these results we suggest a simple picture of how Listeria competes effectively for host cell actin. When Listeria secretes a nucleator, the host's actin subunits polymerize into a filament. Host cell machinery terminate the assembly leaving a short filament. Listeria overcomes the host control by nucleating new filaments and thus many short filaments assemble. The newest filaments push existing ones into a growing tail. Thus the competition is between nucleation of filaments caused by Listeria and the filament terminators produced by the host.

scholarly journals Polymerization of actin. V. A new organelle, the actomere, that initates the assembly of actin filaments in Thyone sperm

1978 ◽  
Vol 77 (2) ◽  
pp. 551-564 ◽  
Author(s):  
LG Tilney
Keyword(s):  
Cell Surface ◽  
Actin Filaments ◽  
Critical Concentration ◽  
Amorphous Material ◽  
Dense Material ◽  
Dense Matrix ◽  
Spontaneous Nucleation ◽  
Acrosomal Reaction ◽  
Triton X

Between the acrosomal vacuole and the nucleus is a cup of amorphous material (profilactin) which is transformed into filaments during the acrosomal reaction. In the center of this cup in untreated Thyone sperm is a dense material which I refer to as the actomere; it is composed of 20-25 filaments embedded in a dense matrix. To visualize the substructure of the actomere, the profilactin around it must be removed. This is achieved either by demembranating the sperm with Triton X-100 and then raising the pH to 8.0, or by adding inophores to intact sperm at pH 8.0. Under these conditions, the actomere remains as a unit while the rest of the profilactin is solubilized or polymerized. When demembranated sperm are incubated under conditions in which the actin should polymerize, filaments grow from the end of the actomere: the actomere thus appears to behave as a nucleating body. This observation is strengthened by experiments in which untreated sperm are incubated in seawater or isotonic NaCl at pH 7.0 and the ionophore X537A is added; in this case, only a partial polymerization of the actin occurs and the acrosomal vacuole does not fuse with the cell surface. The actin filaments that do form, however, are attached to the apical end of the actomere. In fact, the elongating filaments push their way into and frequently through the acrosomal vacuole. Thus, it appears that the sperm organizes the actin filaments by controlling their nucleation. My model is that the cell controls the ammount of unbound actin such that it is slightly above the critical concentration for polymerization. Then, spontaneous nucleation is unfavored and polymerization would proceed from existing nuclei such as the actomer.

Myosin subfragment-1 is sufficient to move actin filaments in vitro

Nature ◽  
1987 ◽  
Vol 328 (6130) ◽  
pp. 536-539 ◽  
Author(s):  
Yoko Yano Toyoshima ◽  
Stephen J. Kron ◽  
Elizabeth M. McNally ◽  
Kenneth R. Niebling ◽  
Chikashi Toyoshima ◽  
...  
Keyword(s):  
Actin Filaments ◽  
Myosin Subfragment ◽  
Myosin Subfragment 1

scholarly journals Actin from Thyone sperm assembles on only one end of an actin filament: a behavior regulated by profilin.

1983 ◽  
Vol 97 (1) ◽  
pp. 112-124 ◽  
Author(s):  
L G Tilney ◽  
E M Bonder ◽  
L M Coluccio ◽  
M S Mooseker
Keyword(s):  
Isoelectric Point ◽  
Actin Filaments ◽  
Critical Concentration ◽  
Molar Ratio ◽  
Actin Assembly ◽  
Actin Monomer ◽  
Filament Bundles ◽  
Sperm Extract ◽  
Triton X

Thyone sperm were demembranated with Triton X-100 and, after washing, extracted with 30 mM Tris at pH 8.0 and 1 mM MgCl2. After the insoluble contaminants were removed by centrifugation, the sperm extract was warmed to 22 degrees C. Actin filaments rapidly assembled and aggregated into bundles when KCl was added to the extract. When we added preformed actin filaments, i.e., the acrosomal filament bundles of Limulus sperm, to the extract, the actin monomers rapidly assembled on these filaments. What was unexpected was that assembly took place on only one end of the bundle--the end corresponding to the preferred end for monomer addition. We showed that the absence of growth on the nonpreferred end was not due to the presence of a capper because exogenously added actin readily assembled on both ends. We also analyzed the sperm extract by SDS gel electrophoresis. Two major proteins were present in a 1:1 molar ratio: actin and a 12,500-dalton protein whose apparent isoelectric point was 8.4. The 12,500-dalton protein was purified by DEAE chromatography. We concluded that it is profilin because of its size, isoelectric point, molar ratio to actin, inability to bind to DEAE, and its effect on actin assembly. When profilin was added to actin in the presence of Limulus bundles, addition of monomers on the nonpreferred end of the bundle was inhibited, even though actin by itself assembled on both ends. Using the Limulus bundles as nuclei, we determined the critical concentration for assembly off each end of the filament and estimated the Kd for the profilin-actin complex (approximately 10 microM). We present a model to explain how profilin may regulate the extension of the Thyone acrosomal process in vivo: The profilin-actin complex can add to only the preferred end of the filament bundle. Once the actin monomer is bound to the filament, the profilin is released, and is available to bind to additional actin monomers. This mechanism accounts for the rapid rate of filament elongation in the acrosomal process in vivo.

scholarly journals Inhibition of actin filament depolymerization by the Dictyostelium 30,000-D actin-bundling protein.

1992 ◽  
Vol 119 (3) ◽  
pp. 559-567 ◽  
Author(s):  
S H Zigmond ◽  
R Furukawa ◽  
M Fechheimer
Keyword(s):  
Actin Filaments ◽  
Actin Polymerization ◽  
Initial Rate ◽  
Potent Inhibitor ◽  
Critical Concentration ◽  
Cross Linking ◽  
The Novel ◽  
Spontaneous Nucleation ◽  
Dose Dependent

We have studied the effect of the Dictyostelium discoideum 30,000-D actin-bundling protein on the assembly and disassembly of pyrenyl-labeled actin in vitro. The results indicate that the protein is a potent inhibitor of the rate of actin depolymerization. The inhibition is rapid, dose dependent, and is observed at both ends of the filament. There is little effect of 30-kD protein on the initial rate of elongation from F-actin seeds or on the spontaneous nucleation of actin polymerization. We could detect little or no effect on the critical concentration. The novel feature of these results is that the filament ends are free for assembly but are significantly impaired in disassembly with little change in the critical concentration at steady state. The effects appear to be largely independent of the cross-linking of actin filaments by the 30-kD protein. Actin cross-linking proteins may not only cross-link actin filaments, but may also differentially protect filaments in cells from disassembly and promote the formation of localized filament arrays with enhanced stability.

scholarly journals Myosin VI Stabilizes an Actin Network during Drosophila Spermatid Individualization

2006 ◽  
Vol 17 (6) ◽  
pp. 2559-2571 ◽  
Author(s):  
Tatsuhiko Noguchi ◽  
Marta Lenartowska ◽  
Kathryn G. Miller
Keyword(s):  
Green Fluorescent Protein ◽  
Actin Filaments ◽  
Fluorescent Protein ◽  
Cross Linking ◽  
Myosin Vi ◽  
Actin Network ◽  
Myosin Subfragment ◽  
Myosin Subfragment 1 ◽  
Green Fluorescent

Here, we demonstrate a new function of myosin VI using observations of Drosophila spermatid individualization in vivo. We find that myosin VI stabilizes a branched actin network in actin structures (cones) that mediate the separation of the syncytial spermatids. In a myosin VI mutant, the cones do not accumulate F-actin during cone movement, whereas overexpression of myosin VI leads to bigger cones with more F-actin. Myosin subfragment 1-fragment decoration demonstrated that the actin cone is made up of two regions: a dense meshwork at the front and parallel bundles at the rear. The majority of the actin filaments were oriented with their pointed ends facing in the direction of cone movement. Our data also demonstrate that myosin VI binds to the cone front using its motor domain. Fluorescence recovery after photobleach experiments using green fluorescent protein-myosin VI revealed that myosin VI remains bound to F-actin for minutes, suggesting its role is tethering, rather than transporting cargo. We hypothesize that myosin VI protects the actin cone structure either by cross-linking actin filaments or anchoring regulatory molecules at the cone front. These observations uncover a novel mechanism mediated by myosin VI for stabilizing long-lived actin structures in cells.

Structure of Myosin Subfragment-1 from Electron Microscopy and Crystallography

1988 ◽  
Vol 46 ◽  
pp. 156-157
Author(s):  
Donald A. Winkelmann
Keyword(s):  
Electron Microscopy ◽  
Molecular Weight ◽  
Actin Filaments ◽  
Light Chains ◽  
Myosin Filament ◽  
Primary Role ◽  
Heavy Chains ◽  
Myosin Subfragment ◽  
Myosin Subfragment 1 ◽  
Globular Head

The primary role of the interaction of actin and myosin is the generation of force and motion as a direct consequence of the cyclic interaction of myosin crossbridges with actin filaments. Myosin is composed of six polypeptides: two heavy chains of molecular weight 220,000 daltons and two pairs of light chains of molecular weight 17,000-23,000. The C-terminal portions of the myosin heavy chains associate to form an α-helical coiled-coil rod which is responsible for myosin filament formation. The N-terminal portion of each heavy chain associates with two different light chains to form a globular head that binds actin and hydrolyses ATP. Myosin can be fragmented by limited proteolysis into several structural and functional domains. It has recently been demonstrated using an in vitro movement assay that the globular head domain, subfragment-1, is sufficient to cause sliding movement of actin filaments.The discovery of conditions for crystallization of the myosin subfragment-1 (S1) has led to a systematic analysis of S1 structure by x-ray crystallography and electron microscopy. Image analysis of electron micrographs of thin sections of small S1 crystals has been used to determine the structure of S1 in the crystal lattice.

Parthenogenesis and cytoskeletal organization in ageing mouse eggs

Development ◽  
1986 ◽  
Vol 95 (1) ◽  
pp. 131-145
Author(s):  
Michelle Webb ◽  
Sarah K. Howlett ◽  
Bernard Maro
Keyword(s):  
Critical Concentration ◽  
Metaphase Plate ◽  
Meiotic Spindle ◽  
Cytoskeletal Organization ◽  
Spindle Poles ◽  
Different Types ◽  
Mouse Egg ◽  
Parthenogenetic Embryos

The cytoskeletal organization of the mouse egg changes during ageing in vivo and in vitro. The earliest change observed is the disappearance of the microfilament-rich area overlying the meiotic spindle. This is followed by the migration of the spindle towards the centre of the egg. Finally the spindle breaks down and the chromosomes are no longer organized on a metaphase plate. This spindle disruption may result from changes in the microtubule nucleating material found at the spindle poles and from an increase in the critical concentration for tubulin polymerization. It is possible to correlate the changes in the cytoskeletal organization of the egg occurring during ageing with the different types of parthenogenetic embryos obtained after ethanol activation. These observations strengthen the hypothesis that the actin-rich cortical area that overlies the meiotic spindle forms a domain to which the meiotic cleavage furrow is restricted and provides some insights into the mechanisms by which different types of parthenogenetic embryos are generated.

scholarly journals Capacity of human serum to depolymerize actin filaments

Blood ◽  
1987 ◽  
Vol 70 (2) ◽  
pp. 524-530
Author(s):  
PA Janmey ◽  
SE Lind
Keyword(s):  
Human Serum ◽  
Actin Filaments ◽  
Serum Proteins ◽  
Normal Serum ◽  
Slow Phase ◽  
Rapid Phase ◽  
Plasma Gelsolin ◽  
Preferential Formation

Human blood depolymerizes filamentous (F-)actin. The interaction of actin filaments and monomers with human serum was studied by following the kinetics and extent of the depolymerization of pyrene-labeled F- actin and by analysis of serum proteins adhering to immobilized actin monomers. In physiologic Ca2+ concentrations, the depolymerization of F- actin proceeds in two stages: a rapid phase, attributed to direct severing of filaments by plasma gelsolin, and a slow phase attributed to the binding of actin monomers to vitamin D-binding protein (DBP). Without Ca2+, only the slow phase is observed. Human serum can completely depolymerize 10 to 18 mumol/L of actin, of which approximately 5 mumol/L occurs rapidly. Depolymerization can be accounted for by the normal serum concentrations of gelsolin and DBP. Fibrin(ogen) and fibronectin, which bind actin in vitro, do not contribute to the kinetics or extent of its depolymerization. Affinity chromatography and functional assays for the presence of gelsolin-actin complexes show that addition of G-actin to serum results in preferential formation of actin-DBP complexes, but that addition of F- actin to serum produces both gelsolin-actin complexes and DBP-actin complexes. The distinctive binding of actin monomers and polymers to these two serum proteins suggests a means by which their coordinated actions are maximized in vivo, from the standpoint of depolymerizing filaments and clearing monomers from the circulation.

scholarly journals Arabidopsis ADF5 Acts as a Downstream Target Gene of CBFs in Response to Low-Temperature Stress

2021 ◽  
Vol 9 ◽  
Author(s):  
Pan Zhang ◽  
Dong Qian ◽  
Changxin Luo ◽  
Yingzhi Niu ◽  
Tian Li ◽  
...  
Keyword(s):  
Low Temperature ◽  
Actin Cytoskeleton ◽  
Temperature Stress ◽  
Regulatory Element ◽  
Low Temperature Stress ◽  
Knockout Mutant ◽  
Triple Mutant ◽  
Freezing Resistance

Low temperature is a major adverse environment that affects normal plant growth. Previous reports showed that the actin cytoskeleton plays an important role in the plant response to low-temperature stress, but the regulatory mechanism of the actin cytoskeleton in this process is not clear. C-repeat binding factors (CBFs) are the key molecular switches for plants to adapt to cold stress. However, whether CBFs are involved in the regulation of the actin cytoskeleton has not been reported. We found that Arabidopsis actin depolymerizing factor 5 (ADF5), an ADF that evolved F-actin bundling function, was up-regulated at low temperatures. We also demonstrated that CBFs bound to the ADF5 promoter directly in vivo and in vitro. The cold-induced expression of ADF5 was significantly inhibited in the cbfs triple mutant. The freezing resistance of the adf5 knockout mutant was weaker than that of wild type (WT) with or without cold acclimation. After low-temperature treatment, the actin cytoskeleton of WT was relatively stable, but the actin cytoskeletons of adf5, cbfs, and adf5 cbfs were disturbed to varying degrees. Compared to WT, the endocytosis rate of the amphiphilic styryl dye FM4-64 in adf5, cbfs, and adf5 cbfs at low temperature was significantly reduced. In conclusion, CBFs directly combine with the CRT/DRE DNA regulatory element of the ADF5 promoter after low-temperature stress to transcriptionally activate the expression of ADF5; ADF5 further regulates the actin cytoskeleton dynamics to participate in the regulation of plant adaptation to a low-temperature environment.

Sign in / Sign up

Export Citation Format

Share Document