Differential Dynamic Behavior of Actin Filaments Containing Tightly-Bound Ca2+or Mg2+in the Presence of Myosin Heads Actively Hydrolyzing ATP†

Biochemistry ◽  
1999 ◽  
Vol 38 (40) ◽  
pp. 13288-13295 ◽  
Author(s):  
Conrad A. Rebello ◽  
Richard D. Ludescher
Keyword(s):  
Dynamic Behavior ◽  
Actin Filaments

scholarly journals Guard Cell Microfilament Analyzer Facilitates the Analysis of the Organization and Dynamics of Actin Filaments in Arabidopsis Guard Cells

2019 ◽  
Vol 20 (11) ◽  
pp. 2753
Author(s):  
Xin Li ◽  
Min Diao ◽  
Yanan Zhang ◽  
Guanlin Chen ◽  
Shanjin Huang ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Dynamic Behavior ◽  
Guard Cell ◽  
Actin Filaments ◽  
De Novo ◽  
Guard Cells ◽  
Stomatal Movement ◽  
Selection Of

The actin cytoskeleton is involved in regulating stomatal movement, which forms distinct actin arrays within guard cells of stomata with different apertures. How those actin arrays are formed and maintained remains largely unexplored. Elucidation of the dynamic behavior of differently oriented actin filaments in guard cells will enhance our understanding in this regard. Here, we initially developed a program called ‘guard cell microfilament analyzer’ (GCMA) that enables the selection of individual actin filaments and analysis of their orientations semiautomatically in guard cells. We next traced the dynamics of individual actin filaments and performed careful quantification in open and closed stomata. We found that de novo nucleation of actin filaments occurs at both dorsal and ventral sides of guard cells from open and closed stomata. Interestingly, most of the nucleated actin filaments elongate radially and longitudinally in open and closed stomata, respectively. Strikingly, radial filaments tend to form bundles whereas longitudinal filaments tend to be removed by severing and depolymerization in open stomata. By contrast, longitudinal filaments tend to form bundles that are severed less frequently in closed stomata. These observations provide insights into the formation and maintenance of distinct actin arrays in guard cells in stomata of different apertures.

scholarly journals Comparison of actin and cell surface dynamics in motile fibroblasts.

1992 ◽  
Vol 119 (2) ◽  
pp. 367-377 ◽  
Author(s):  
J A Theriot ◽  
T J Mitchison
Keyword(s):  
Actin Cytoskeleton ◽  
Dynamic Behavior ◽  
Actin Filaments ◽  
Half Life ◽  
Dorsal Surface ◽  
Cell Types ◽  
3T3 Cells ◽  
3T3 Fibroblasts ◽  
Continuous Filament ◽  
Time Required

We have investigated the dynamic behavior of actin in fibroblast lamellipodia using photoactivation of fluorescence. Activated regions of caged resorufin (CR)-labeled actin in lamellipodia of IMR 90 and MC7 3T3 fibroblasts were observed to move centripetally over time. Thus in these cells, actin filaments move centripetally relative to the substrate. Rates were characteristic for each cell type; 0.66 +/- 0.27 microns/min in IMR 90 and 0.36 +/- 0.16 microns/min in MC7 3T3 cells. In neither case was there any correlation between the rate of actin movement and the rate of lamellipodial protrusion. The half-life of the activated CR-actin filaments was approximately 1 min in IMR 90 lamellipodia, and approximately 3 min in MC7 3T3 lamellipodia. Thus continuous filament turnover accompanies centripetal movement. In both cell types, the length of time required for a section of the actin meshwork to traverse the lamellipodium was several times longer than the filament half-life. The dynamic behavior of the dorsal surface of the cell was also observed by tracking lectin-coated beads on the surface and phase-dense features within lamellipodia of MC7 3T3 cells. The movement of these dorsal features occurred at rates approximately three times faster than the rate of movement of the underlying bulk actin cytoskeleton, even when measured in the same individual cells. Thus the transport of these dorsal features must occur by some mechanism other than simple attachment to the moving bulk actin cytoskeleton.

Dynamic behavior of focal adhesions and associated actin filaments of sheared endothelial cells

2003 ◽  
Vol 2003.5 (0) ◽  
pp. 33-34
Author(s):  
Toshiro OHASHI ◽  
Tsugumasa YAMAMOTO ◽  
Naoki MOCHIZUKI ◽  
Masaaki SATO
Keyword(s):  
Endothelial Cells ◽  
Dynamic Behavior ◽  
Actin Filaments ◽  
Focal Adhesions

scholarly journals Role of fascin in filopodial protrusion

2006 ◽  
Vol 174 (6) ◽  
pp. 863-875 ◽  
Author(s):  
Danijela Vignjevic ◽  
Shin-ichiro Kojima ◽  
Yvonne Aratyn ◽  
Oana Danciu ◽  
Tatyana Svitkina ◽  
...  
Keyword(s):  
Rna Interference ◽  
Dynamic Behavior ◽  
Actin Filaments ◽  
Cellular Localization ◽  
Fluorescence Recovery After Photobleaching ◽  
Cross Linking ◽  
Wild Type ◽  
Actin Organization ◽  
Cross Linker

In this study, the mechanisms of actin-bundling in filopodia were examined. Analysis of cellular localization of known actin cross-linking proteins in mouse melanoma B16F1 cells revealed that fascin was specifically localized along the entire length of all filopodia, whereas other actin cross-linkers were not. RNA interference of fascin reduced the number of filopodia, and remaining filopodia had abnormal morphology with wavy and loosely bundled actin organization. Dephosphorylation of serine 39 likely determined cellular filopodia frequency. The constitutively active fascin mutant S39A increased the number and length of filopodia, whereas the inactive fascin mutant S39E reduced filopodia frequency. Fluorescence recovery after photobleaching of GFP-tagged wild-type and S39A fascin showed that dephosphorylated fascin underwent rapid cycles of association to and dissociation from actin filaments in filopodia, with t1/2 < 10 s. We propose that fascin is a key specific actin cross-linker, providing stiffness for filopodial bundles, and that its dynamic behavior allows for efficient coordination between elongation and bundling of filopodial actin filaments.

scholarly journals Arabidopsis Actin Depolymerizing Factor4 Modulates the Stochastic Dynamic Behavior of Actin Filaments in the Cortical Array of Epidermal Cells

2011 ◽  
Vol 23 (10) ◽  
pp. 3711-3726 ◽  
Author(s):  
Jessica L. Henty ◽  
Samuel W. Bledsoe ◽  
Parul Khurana ◽  
Richard B. Meagher ◽  
Brad Day ◽  
...  
Keyword(s):  
Dynamic Behavior ◽  
Actin Filaments ◽  
Epidermal Cells ◽  
Stochastic Dynamic ◽  
Cortical Array

scholarly journals Capping Protein Modulates the Dynamic Behavior of Actin Filaments in Response to Phosphatidic Acid in Arabidopsis

2012 ◽  
Vol 24 (9) ◽  
pp. 3742-3754 ◽  
Author(s):  
Jiejie Li ◽  
Jessica L. Henty-Ridilla ◽  
Shanjin Huang ◽  
Xia Wang ◽  
Laurent Blanchoin ◽  
...  
Keyword(s):  
Phosphatidic Acid ◽  
Dynamic Behavior ◽  
Actin Filaments ◽  
Capping Protein

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.

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.

Orientation of actin filaments during motion in motility assay

1995 ◽  
Vol 53 ◽  
pp. 52-53
Author(s):  
J. Borejdo ◽  
S. Burlacu
Keyword(s):  
Actin Filaments ◽  
Thin Filament ◽  
Striated Muscle ◽  
Myosin Head ◽  
Classical Method ◽  
Polarization Of Fluorescence ◽  
Single Protein ◽  
Intracellular Organelles ◽  
Change Orientation ◽  
Active Entity

Polarization of fluorescence is a classical method to assess orientation or mobility of macromolecules. It has been a common practice to measure polarization of fluorescence through a microscope to characterize orientation or mobility of intracellular organelles, for example anisotropic bands in striated muscle. Recently, we have extended this technique to characterize single protein molecules. The scientific question concerned the current problem in muscle motility: whether myosin heads or actin filaments change orientation during contraction. The classical view is that the force-generating step in muscle is caused by change in orientation of myosin head (subfragment-1 or SI) relative to the axis of thin filament. The molecular impeller which causes this change resides at the interface between actin and SI, but it is not clear whether only the myosin head or both SI and actin change orientation during contraction. Most studies assume that observed orientational change in myosin head is a reflection of the fact that myosin is an active entity and actin serves merely as a passive "rail" on which myosin moves.

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