scholarly journals Direct visualization of flow-induced conformational transitions of single actin filaments in entangled solutions

2014 ◽  
Vol 5 (1) ◽  
Author(s):  
Inka Kirchenbuechler ◽  
Donald Guu ◽  
Nicholas A. Kurniawan ◽  
Gijsje H. Koenderink ◽  
M. Paul Lettinga
Keyword(s):  
Actin Filaments ◽  
Conformational Transitions ◽  
Direct Visualization ◽  
Entangled Solutions

scholarly journals Direct Visualization of Actin Filaments and Actin-Binding Proteins in Neuronal Cells

2020 ◽  
Vol 8 ◽  
Author(s):  
Minkyo Jung ◽  
Doory Kim ◽  
Ji Young Mun
Keyword(s):  
Electron Microscopy ◽  
Actin Filaments ◽  
Actin Polymerization ◽  
Binding Proteins ◽  
Light And Electron Microscopy ◽  
Super Resolution ◽  
Actin Binding ◽  
Direct Visualization ◽  
Actin Binding Proteins ◽  
Synapse Plasticity

Actin networks and actin-binding proteins (ABPs) are most abundant in the cytoskeleton of neurons. The function of ABPs in neurons is nucleation of actin polymerization, polymerization or depolymerization regulation, bundling of actin through crosslinking or stabilization, cargo movement along actin filaments, and anchoring of actin to other cellular components. In axons, ABP–actin interaction forms a dynamic, deep actin network, which regulates axon extension, guidance, axon branches, and synaptic structures. In dendrites, actin and ABPs are related to filopodia attenuation, spine formation, and synapse plasticity. ABP phosphorylation or mutation changes ABP–actin binding, which regulates axon or dendritic plasticity. In addition, hyperactive ABPs might also be expressed as aggregates of abnormal proteins in neurodegeneration. Those changes cause many neurological disorders. Here, we will review direct visualization of ABP and actin using various electron microscopy (EM) techniques, super resolution microscopy (SRM), and correlative light and electron microscopy (CLEM) with discussion of important ABPs in neuron.

scholarly journals Direct visualization of myosin-binding protein C bridging myosin and actin filaments in intact muscle

2011 ◽  
Vol 108 (28) ◽  
pp. 11423-11428 ◽  
Author(s):  
P. K. Luther ◽  
H. Winkler ◽  
K. Taylor ◽  
M. E. Zoghbi ◽  
R. Craig ◽  
...  
Keyword(s):  
Actin Filaments ◽  
Protein C ◽  
Binding Protein ◽  
Direct Visualization ◽  
Myosin Binding Protein ◽  
Myosin Binding Protein C ◽  
Intact Muscle ◽  
Myosin Binding

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 Cofilin Drives Rapid Turnover and Fluidization of Entangled F-actin

2017 ◽  
Author(s):  
Patrick M. McCall ◽  
Frederick C. MacKintosh ◽  
David R. Kovar ◽  
Margaret L. Gardel
Keyword(s):  
Steady State ◽  
Stress Relaxation ◽  
Actin Filaments ◽  
Atp Hydrolysis ◽  
Animal Cell ◽  
Animal Cells ◽  
Single Filament ◽  
Actin Cortex ◽  
Non Equilibrium ◽  
Entangled Solutions

AbstractThe shape of most animal cells is controlled by the actin cortex, a thin, isotropic network of dynamic actin filaments (F-actin) situated just beneath the plasma membrane. The cortex is held far from equilibrium by both active stresses and turnover: Myosin-II molecular motors drive deformations required for cell division, migration, and tissue morphogenesis, while turnover of the molecular components of the actin cortex relax stress and facilitate network reorganization. While many aspects of F-actin network viscoelasticity are well-characterized in the presence and absence of motor activity, a mechanistic understanding of how non-equilibrium actin turnover contributes to stress relaxation is still lacking. To address this, we developed a reconstituted in vitro system wherein the steady-state length and turnover rate of F-actin in entangled solutions are controlled by the actin regulatory proteins cofilin, profilin, and formin, which sever, recycle, and nucleate filaments, respectively. Cofilin-mediated severing accelerates the turnover and spatial reorganization of F-actin, without significant changes to filament length. Microrheology measurements demonstrate that cofilin-mediated severing is a single-timescale mode of stress relaxation that tunes the low-frequency viscosity over two orders of magnitude. These findings serve as the foundation for understanding the mechanics of more physiological F-actin networks with turnover, and inform an updated microscopic model of single-filament turnover. They also demonstrate that polymer activity, in the form of ATP hydrolysis on F-actin coupled to nucleotide-dependent cofilin binding, is sufficient to generate a form of active matter wherein asymmetric filament disassembly preserves filament number in spite of sustained severing.Significance StatementWhen an animal cell moves or divides, a disordered network of actin filaments (F-actin) plays a central role in controlling the resulting changes in cell shape. While it is known that continual turnover of F-actin by cofilin-mediated severing aids in reorganization of the cellular cytoskeleton, it is unclear how the turnover of structural elements alters the mechanical properties of the network. Here we show that severing of F-actin by cofilin results in a stress relaxation mechanism in entangled solutions characterized by a single-timescale set by the severing rate. Additionally, we identify ATP hydrolysis and nucleotide-dependent cofilin binding as sufficient ingredients to generate a non-equilibrium steady-state in which asymmetric F-actin disassembly preserves filament number in spite of sustained severing.

Direct visualization of organelle movement along actin filaments dissociated from characean algae

Science ◽  
1985 ◽  
Vol 227 (4692) ◽  
pp. 1355-1357 ◽  
Author(s):  
B Kachar
Keyword(s):  
Actin Filaments ◽  
Direct Visualization ◽  
Organelle Movement ◽  
Characean Algae

Electron Microscopy of Cytoplasmic Contractile Proteins

1978 ◽  
Vol 36 (3) ◽  
pp. 606-614 ◽  
Author(s):  
T.D. Pollard ◽  
P. Maupin
Keyword(s):  
Electron Microscopy ◽  
Actin Filaments ◽  
Three Dimensional ◽  
Uranyl Acetate ◽  
Contractile Proteins ◽  
Dimensional Structure ◽  
X Ray Diffraction ◽  
Cytoplasmic Matrix ◽  
Computer Image Processing ◽  
Nonmuscle Cells

In this paper we review some of the contributions that electron microscopy has made to the analysis of actin and myosin from nonmuscle cells. We place particular emphasis upon the limitations of the ultrastructural techniques used to study these cytoplasmic contractile proteins, because it is not widely recognized how difficult it is to preserve these elements of the cytoplasmic matrix for electron microscopy. The structure of actin filaments is well preserved for electron microscope observation by negative staining with uranyl acetate (Figure 1). In fact, to a resolution of about 3nm the three-dimensional structure of actin filaments determined by computer image processing of electron micrographs of negatively stained specimens (Moore et al., 1970) is indistinguishable from the structure revealed by X-ray diffraction of living muscle.

Destruction of Actin Filaments by Osmium Tetroxide

1977 ◽  
Vol 35 ◽  
pp. 466-467
Author(s):  
P. Maupin-Szamier ◽  
T. D. Pollard
Keyword(s):  
Actin Filaments ◽  
Time Course ◽  
Osmium Tetroxide ◽  
Rabbit Muscle ◽  
Muscle Actin ◽  
Sodium Phosphate Buffer ◽  
Transmission Electron ◽  
The Difference ◽  
Rates Of Decay ◽  
Short Time

We have studied the destruction of rabbit muscle actin filaments by osmium tetroxide (OSO4) to develop methods which will preserve the structure of actin filaments during preparation for transmission electron microscopy.Negatively stained F-actin, which appears as smooth, gently curved filaments in control samples (Fig. 1a), acquire an angular, distorted profile and break into progressively shorter pieces after exposure to OSO4 (Fig. 1b,c). We followed the time course of the reaction with viscometry since it is a simple, quantitative method to assess filament integrity. The difference in rates of decay in viscosity of polymerized actin solutions after the addition of four concentrations of OSO4 is illustrated in Fig. 2. Viscometry indicated that the rate of actin filament destruction is also dependent upon temperature, buffer type, buffer concentration, and pH, and requires the continued presence of OSO4. The conditions most favorable to filament preservation are fixation in a low concentration of OSO4 for a short time at 0°C in 100mM sodium phosphate buffer, pH 6.0.

Direct visualization of phase-separated domains in lipid membranes

1978 ◽  
Vol 36 (2) ◽  
pp. 290-291
Author(s):  
S. W. Hui ◽  
T. P. Stewart
Keyword(s):  
Lipid Membranes ◽  
Structural Information ◽  
Freeze Fracture ◽  
Biological Molecules ◽  
Vacuum Drying ◽  
Direct Visualization ◽  
X Ray Diffraction ◽  
Electron Microscopic ◽  
X Ray ◽  
Hydrocarbon Chains

Direct electron microscopic study of biological molecules has been hampered by such factors as radiation damage, lack of contrast and vacuum drying. In certain cases, however, the difficulties may be overcome by using redundent structural information from repeating units and by various specimen preservation methods. With bilayers of phospholipids in which both the solid and fluid phases co-exist, the ordering of the hydrocarbon chains may be utilized to form diffraction contrast images. Domains of different molecular packings may be recgnizable by placing properly chosen filters in the diffraction plane. These domains would correspond to those observed by freeze fracture, if certain distinctive undulating patterns are associated with certain molecular packing, as suggested by X-ray diffraction studies. By using an environmental stage, we were able to directly observe these domains in bilayers of mixed phospholipids at various temperatures at which their phases change from misible to inmissible states.

Conformational Transitions in Escherichia coli Ribosomal RNAs as Visualized by Dedicated STEM

1988 ◽  
Vol 46 ◽  
pp. 176-177
Author(s):  
J.S. Wall ◽  
V. Maridiyan ◽  
S. Tumminia ◽  
J. Hairifeld ◽  
M. Boublik
Keyword(s):  
Ionic Strength ◽  
Three Dimensional ◽  
Conformational Transitions ◽  
Dark Field ◽  
Dimensional Structure ◽  
Ribosomal Rnas ◽  
Field Mode ◽  
Freeze Dried ◽  
High Resolution Images ◽  
Low Ionic Strength

The high contrast in the dark-field mode of dedicated STEM, specimen deposition by the wet film technique and low radiation dose (1 e/Å2) at -160°C make it possible to obtain high resolution images of unstained freeze-dried macromolecules with minimal structural distortion. Since the image intensity is directly related to the local projected mass of the specimen it became feasible to determine the molecular mass and mass distribution within individual macromolecules and from these data to calculate the linear density (M/L) and the radii of gyration.2 This parameter (RQ), reflecting the three-dimensional structure of the macromolecular particles in solution, has been applied to monitor the conformational transitions in E. coli 16S and 23S ribosomal RNAs in solutions of various ionic strength.In spite of the differences in mass (550 kD and 1050 kD, respectively), both 16S and 23S RNA appear equally sensitive to changes in buffer conditions. In deionized water or conditions of extremely low ionic strength both appear as filamentous structures (Fig. la and 2a, respectively) possessing a major backbone with protruding branches which are more frequent and more complex in 23S RNA (Fig. 2a).

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.

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