F-actin distribution in polymorphonuclear leucocytes

1987 ◽  
Vol 88 (4) ◽  
pp. 495-501
Author(s):  
W.S. Haston
Keyword(s):  
Three Dimensional ◽  
Time Lapse ◽  
Polymorphonuclear Leucocytes ◽  
Two Dimensional ◽  
Collagen Gels ◽  
Motile Cells ◽  
Low Concentrations ◽  
Chemotactic Factors ◽  
Basic Features ◽  
Cytoskeletal Elements

The polarization and amoeboid locomotion of neutrophil leucocytes is stimulated by chemotactic factors, which initiate waves of contraction in both adherent and non-adherent neutrophils. These cyclical contractile events have previously been analysed by time-lapse filming but the mechanisms involved in the coordination of the cytoskeleton during locomotion have not been elucidated, one reason being because of the problems involved in fixing motile cells. In this paper we show that improved fixation of motile neutrophils with low concentrations of glutaraldehyde followed by glycine quenching demonstrated significant differences in the pattern of staining with TRITC-phalloidin in neutrophils moving on different substrata. Previous film analysis had shown the basic features of locomotion to be similar on all substrata. A prominent feature of leucocyte locomotion on two-dimensional substrata (e.g. protein-coated glass), on three-dimensional collagen gels or in motile cells floating in suspension, is the wave of contraction that passes antero-posteriorly along the length of the cell. The organization of the cytoskeletal elements has not been demonstrated at contraction waves, but light fixation with glutaraldehyde followed by staining with TRITC-phalloidin demonstrated prominent bands of F-actin in neutrophils inside collagen gels. These bands were not present in neutrophils either in suspension or moving on a two-dimensional substratum. Although all motile neutrophils had brightly stained anterior lamellipodia, the cells moving on the two-dimensional substratum had very extensively ruffled leading lamellae stained very brightly with TRITC-phalloidin. The reasons for the absence of consistent bands of F-actin at contraction waves are discussed.

Contraction waves in lymphocyte locomotion

1984 ◽  
Vol 68 (1) ◽  
pp. 227-241
Author(s):  
W.S. Haston ◽  
J.M. Shields
Keyword(s):  
Three Dimensional ◽  
Collagen Gel ◽  
Time Lapse ◽  
Collagen Gels ◽  
Collagen Network ◽  
Longitudinal Contraction ◽  
Constriction Ring ◽  
Motile Cells ◽  
Transmission Electron ◽  
Contraction Wave

In this paper we propose that the constriction ring, a prominent feature of moving leucocytes, is a major source of locomotive force. Analysis of time-lapse films of lymphocytes in suspension and moving through three-dimensional collagen gels, demonstrated that the constriction ring was the morphological manifestation of a wave of circular contraction that moved antero-posteriorly. In lymphocytes in suspension the wave moved, although the cells could not. Analysis of lymphocytes moving through a collagen gel revealed that the waves remained stationary with respect to the external environment while the cell appeared to move forward through them. Passage of a single equatorial contraction wave resulted in cell lengthening: a shortening of the region posterior to the constriction was observed in cells moving through collagen gels, but not in lymphocytes held in suspension, suggesting that attachment of cells to the collagen network was necessary for longitudinal contraction. Lymphocyte attachment to collagen gels was mediated through the rapid extension of bleb-like structures into the collagen network. Transmission electron microscopy (TEM) failed to demonstrate any organized structure at the constriction ring. NBD-Phallacidin staining of lymphocytes together with TEM demonstrated that F-actin was distributed evenly throughout the length of the cell. Cell polarity was clearly recognizable by the distribution of coated vesicles, microvilli, and all organelles to the rear, and Thy 1–2 to the front, of motile cells, but polarity could be reversed by the passage of a single contraction wave starting at the rear of the cell, without prior redistribution of these structures.

scholarly journals Lymphocyte locomotion and attachment on two-dimensional surfaces and in three-dimensional matrices

1982 ◽  
Vol 92 (3) ◽  
pp. 747-752 ◽  
Author(s):  
WS Haston ◽  
JM Shields ◽  
PC Wilkinson
Keyword(s):  
Three Dimensional ◽  
Two Dimensional ◽  
Collagen Gels ◽  
Peripheral Lymph Node ◽  
Cellulose Esters ◽  
Peripheral Lymph ◽  
The Matrix ◽  
Identical Material ◽  
Variable Morphology ◽  
Collagen Lattices

The adhesion and locomotion of mouse peripheral lymph node lymphocytes on 2-D protein- coated substrata and in 3-D matrices were compared. Lymphocytes did not adhere to, or migrate on, 2-D substrata suck as serum- or fibronectin-coated glass. They did attach to and migrate in hydrated 3-D collagen lattices. When the collagen was dehydrated to form a 2-D surface, lymphocyte attachment to it was reduced. We propose that lymphocytes, which are poorly adhesive, are able to attach to and migrate in 3-D matrices by a nonadhesive mechanism such as the extension and expansion of pseudopodia through gaps in the matrix, which could provide purchase for movement in the absence of discrete intermolecular adhesions. This was supported by studies using serum-coated micropore filters, since lymphocytes attached to and migrated into filters with pore sizes large enough (3 or 8 mum) to allow pseudopod penetration but did not attach to filters made of an identical material (cellulose esters) but of narrow pore size (0.22 or 0.45 mum). Cinematographic studies of lymphocyte locomotion in collagen gels were also consistent with the above hypothesis, since lymphocytes showed a more variable morphology than is typically seen on plane surfaces, with formation of many small pseudopodia expanded to give a marked constriction between the cell and the pseudopod. These extensions often remained fixed with respect to the environment as the lymphocyte moved away from or past them. This suggests that the pseudopodia were inserted into gaps in the gel matrix and acted as anchorage points for locomotion.

scholarly journals The interaction of atoms and molecules with solid surfaces XII—Critical phenomena in a two-dimensional gas

1937 ◽  
Vol 163 (912) ◽  
pp. 132-138 ◽  
Keyword(s):  
Critical Temperature ◽  
Equation Of State ◽  
Critical Phenomena ◽  
Three Dimensional ◽  
Inert Gases ◽  
Two Dimensional ◽  
Lennard Jones ◽  
Low Concentrations ◽  
High Concentrations ◽  
The Right

In a paper recently published by Professor Lennard-Jones and the author (Lennard-Jones and Devonshire 1937) the equation of state of a gas at high concentrations has been calculated in terms of the interatomic fields. The equation found had the right kind of properties and, in particular, using the interatomic fields previously determined from the observed equation of state at low concentrations (Lennard-Jones 1931), the critical temperature was given correctly to within a few degrees for the inert gases. In this paper we shall apply the same method to determine the equation of state of a two-dimensional gas. Although such a gas cannot strictly be obtained in practice, an inert gas adsorbed on a surface (or in fact any gas held by van der Waals’ forces only) would probably behave very much like one, the fluctuations of the potential field over the surface not being of much importance. In confirmation of this it may be noted that the specific heat of argon adsorbed on charcoal was found by Simon (Simon 1935) to be equal to that of a perfect two-dimensional gas down to 60° K. A gas adsorbed on a liquid would be an even better representation of a two-dimensional one. Some measurements on the adsorption of krypton and xenon on liquid mercury have been made by Cassel and Neugebauer (Cassel and Neugebauer 1936), and they found no trace of any critical phenomena though they worked at temperatures considerably below the critical temperature of xenon. Our results are in agreement with this, for they show that the critical temperature of a two-dimensional gas should be about half that of the corresponding three-dimensional one.

scholarly journals Comparison of drug inhibitory effects (IC50) in monolayer and spheroid cultures

2020 ◽  
Author(s):  
Catherine Berrouet ◽  
Naika Dorilas ◽  
Katarzyna A. Rejniak ◽  
Necibe Tuncer
Keyword(s):  
Three Dimensional ◽  
Drug Dosage ◽  
Drug Action ◽  
Computational Studies ◽  
Two Dimensional ◽  
Inhibitory Effects ◽  
Action Mechanisms ◽  
Low Concentrations ◽  
Dimensional Cell

AbstractTraditionally, the monolayer (two-dimensional) cell cultures are used for initial evaluation of the ef-fectiveness of anticancer drugs. In particular, these experiments provide the IC50 curves that determine drug concentration that can inhibit growth of a tumor colony by half when compared to the cells grown with no exposure to the drug. Low IC50 value means that the drug is effective at low concentrations, and thus will show lower systemic toxicity when administered to the patient. However, in these experiments cells are grown in a monolayer, all well exposed to the drug, while in vivo tumors expand as three-dimensional multicellular masses, where inner cells have a limited access to the drug. Therefore, we performed computational studies to compare the IC50 curves for cells grown as a two-dimensional monolayer and a cross section through a three-dimensional spheroid. Our results identified conditions (drug diffusivity, drug action mechanisms and cell proliferation capabilities) under which these IC50 curves differ significantly. This will help experimentalists to better determine drug dosage for future in vivo experiments and clinical trials.

scholarly journals Glycosaminoglycans facilitate the movement of fibroblasts through three-dimensional collagen matrices

1989 ◽  
Vol 92 (2) ◽  
pp. 263-270 ◽  
Author(s):  
R. Docherty ◽  
J.V. Forrester ◽  
J.M. Lackie ◽  
D.W. Gregory
Keyword(s):  
Cell Invasion ◽  
Type I Collagen ◽  
Chondroitin Sulphate ◽  
Three Dimensional ◽  
Collagen Matrix ◽  
Type I ◽  
Collagen Gels ◽  
Collagen Concentration ◽  
Collagen Matrices ◽  
Low Concentrations

The effect of glycosaminoglycans on the invasion of choroid fibroblasts into type I collagen gels was studied. Both hyaluronate and chondroitin sulphate, when incorporated into the gel, facilitated invasion of the collagen matrix, although hyaluronate was considerably more effective. Hyaluronate-induced fibroblast invasion was markedly concentration-dependent, being reduced at both high and low concentrations. Increased cell invasion appeared to correlate with denser packing of collagen fibrils within the gel, since the same effect could be achieved by increasing the collagen concentration of native, i.e. glycosaminoglycan-free gels. Scanning electron microscopy of the interior of the collagen gels suggested that changes in packing arrangement of fibrils in gels that had polymerized in the presence of glycosaminoglycans might account in part for different rates of cell invasion.

High Resolution Microscopy of Multi-Layered Biological Crystals

1982 ◽  
Vol 40 ◽  
pp. 80-83
Author(s):  
H.A. Cohen ◽  
T.W. Jeng ◽  
W. Chiu
Keyword(s):  
High Resolution ◽  
Low Dose ◽  
Three Dimensional ◽  
Data Interpretation ◽  
Two Dimensional ◽  
Biological Objects ◽  
Density Maps ◽  
Density Map ◽  
Complex Crystal ◽  
High Resolution Microscopy

This tutorial will discuss the methodology of low dose electron diffraction and imaging of crystalline biological objects, the problems of data interpretation for two-dimensional projected density maps of glucose embedded protein crystals, the factors to be considered in combining tilt data from three-dimensional crystals, and finally, the prospects of achieving a high resolution three-dimensional density map of a biological crystal. This methodology will be illustrated using two proteins under investigation in our laboratory, the T4 DNA helix destabilizing protein gp32*I and the crotoxin complex crystal.

Instrumentation For Quantitative Microscopy

1977 ◽  
Vol 35 ◽  
pp. 206-209
Author(s):  
B. Ralph ◽  
A.R. Jones
Keyword(s):  
Volume Fraction ◽  
Three Dimensional ◽  
Two Dimensional ◽  
Automatic Data ◽  
Phase Volume Fraction ◽  
Statistical Confidence ◽  
Resultant Data ◽  
Automatic Data Collection ◽  
Third Dimension ◽  
Evolution Of Microstructure

In all fields of microscopy there is an increasing interest in the quantification of microstructure. This interest may stem from a desire to establish quality control parameters or may have a more fundamental requirement involving the derivation of parameters which partially or completely define the three dimensional nature of the microstructure. This latter categorey of study may arise from an interest in the evolution of microstructure or from a desire to generate detailed property/microstructure relationships. In the more fundamental studies some convolution of two-dimensional data into the third dimension (stereological analysis) will be necessary.In some cases the two-dimensional data may be acquired relatively easily without recourse to automatic data collection and further, it may prove possible to perform the data reduction and analysis relatively easily. In such cases the only recourse to machines may well be in establishing the statistical confidence of the resultant data. Such relatively straightforward studies tend to result from acquiring data on the whole assemblage of features making up the microstructure. In this field data mode, when parameters such as phase volume fraction, mean size etc. are sought, the main case for resorting to automation is in order to perform repetitive analyses since each analysis is relatively easily performed.

A linearity test for thick specimens imaged by HVEM over a 180° angular range

1991 ◽  
Vol 49 ◽  
pp. 552-553
Author(s):  
Yu Liu
Keyword(s):  
Phase Contrast ◽  
Three Dimensional ◽  
Angular Range ◽  
Two Dimensional ◽  
Back Projection ◽  
Line Integral ◽  
Exponential Law ◽  
Transmission Electron ◽  
Absorption Law ◽  
Linearity Test

The image obtained in a transmission electron microscope is the two-dimensional projection of a three-dimensional (3D) object. The 3D reconstruction of the object can be calculated from a series of projections by back-projection, but this algorithm assumes that the image is linearly related to a line integral of the object function. However, there are two kinds of contrast in electron microscopy, scattering and phase contrast, of which only the latter is linear with the optical density (OD) in the micrograph. Therefore the OD can be used as a measure of the projection only for thin specimens where phase contrast dominates the image. For thick specimens, where scattering contrast predominates, an exponential absorption law holds, and a logarithm of OD must be used. However, for large thicknesses, the simple exponential law might break down due to multiple and inelastic scattering.

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