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PubMedDate not supplied

Tracking of individual cell cohorts in asynchronous Saccharomyces cerevisiae populations.

A novel flow cytometric procedure has been developed with the aim to obtain the growth properties of individual Saccharomyces cerevisiae cells in asynchronous culture. The method is based on labeling of the cell surface with FITC-conjugated concanavalin A and detection of the single-cell fluorescence with flow cytometry after cell exposure to growth conditions. Because the formation of new cell wall material in budded cells is restricted to the bud tip, exposure of the stained cells to growth conditions results in three cell types: (i) stained cells, (ii) partially stained cells, and (iii) unstained cells. Analysis of the staining pattern over time permits the determination of the specific growth rate of the cell population, the length of the budded cell cycle phase, and the growth pattern during the cell cycle of newly formed, partially stained daughter cells. The procedure has been tested with yeast cell populations growing at different rates. The data suggest an exponential increase in the size of individual cells during the cell cycle, as reflected by the forward angle light scattering (FALS) signals. It has been found that the apparent single-cell specific cell size growth rates, determined by FALS intensity, are significantly lower than the specific growth rates of the overall population. This could indicate that the tracking of a cohort of cells is significantly perturbed by a distribution of staining levels of daughter cells at cell division and that FALS may not be a good indicator of the cell size.

Cell Cycle↗
PubMed1989

Radiobiology of ultrasoft X rays. III. Normal human fibroblasts and the significance of terminal track structure in cell inactivation.

Ultrasoft characteristic X rays from carbon (0.28 keV) are severely attenuated as they pass through biological material, causing a nonuniform distribution of dose to cell nuclei. Complications of studying ultrasoft X rays can be minimized in this context by using cells with very thin cytoplasm and nuclei (e.g., less than the attenuation length of the X rays), and which exhibit a more nearly exponential dose response to cell killing, such as normal human fibroblasts compared with V79 cells. Using this cell system, we report the relative biological effectiveness (RBE) of A1-K and C-K X rays to be near unity. Previous studies of cell inactivation by characteristic carbon X rays gave RBEs of 3 to 4, supporting the idea that localized energy depositions from secondary electrons and primary track ends represent the principal mode of biological action for other low-LET radiations. In part, the reported high RBEs result from the use of mean dose to describe energy deposited within the cell nuclei by these poorly penetrating radiations. Implicit in the use of mean dose is that cellular damage varies linearly with dose within a critical target(s), an assumption that is of questionable validity for cells that exhibit pronounced curvilinear dose responses. The simplest interpretation of the present findings is that most energy depositions caused by track-end effects are not necessarily more damaging than the sparsely ionizing component.

Cell Survival↗
PubMed2000

Tat peptide-derivatized magnetic nanoparticles allow in vivo tracking and recovery of progenitor cells.

The ability to track the distribution and differentiation of progenitor and stem cells by high-resolution in vivo imaging techniques would have significant clinical and research implications. We have developed a cell labeling approach using short HIV-Tat peptides to derivatize superparamagnetic nanoparticles. The particles are efficiently internalized into hematopoietic and neural progenitor cells in quantities up to 10-30 pg of superparamagnetic iron per cell. Iron incorporation did not affect cell viability, differentiation, or proliferation of CD34+ cells. Following intravenous injection into immunodeficient mice, 4% of magnetically CD34+ cells homed to bone marrow per gram of tissue, and single cells could be detected by magnetic resonance (MR) imaging in tissue samples. In addition, magnetically labeled cells that had homed to bone marrow could be recovered by magnetic separation columns. Localization and retrieval of cell populations in vivo enable detailed analysis of specific stem cell and organ interactions critical for advancing the therapeutic use of stem cells.

Animals↗
PubMed1988

Association of thrombospondin of endothelial cells with other matrix proteins and cell attachment sites and migration tracks.

Different biochemical and cytochemical techniques were applied to characterize the sites of localization of thrombospondin in cultured endothelial cells. The results obtained by [35S]methionine labeling, immunoblotting, immunoprecipitation, fluorescence microscopy, ultracytochemistry, immunogold labeling, and silver enhancement experiments revealed that thrombospondin secreted by endothelial cells is structurally organized together with proteoheparan sulfate in spherical granules at the cell surface. These granules are about 100 to 300 nm in size. Heparin or enzymatic degradation with heparitinase, but not with ABC lyase, release thrombospondin from the cell surface. Fibronectin is expressed in the extracellular matrix of endothelial cells in a fibrillar organization, clearly distinct from the punctate pattern of thrombospondin on the cell surface. Furthermore, secreted thrombospondin is highly enriched together with fibronectin and proteoheparan sulfate in cell attachment sites and in cell migration tracks. In cell migration tracks proteoheparan sulfate more clearly resembles the fibrillar distribution pattern of fibronectin, whereas thrombospondin reveals a rather monodisperse pattern. The obtained data suggest preferential sites of interaction between thrombospondin and heparan sulfate proteoglycans on the cell surface and a participation of thrombospondin in cell adhesion and cell migration.

Animals↗
PubMed1973

Freeze-etching and x-ray diffraction of the isolated double-track layer from the cell wall of a gram-negative marine pseudomonad.

The isolated double-track layer of the cell wall of the gram-negative marine pseudomonad studied here contains a cleavage plane. This finding localizes the single cleavage plane of the cell wall and shows that the molecular architecture of this layer provides the lipid-enriched layer which cleaves preferentially in the frozen cell. The observation that the isolated double-track layer of the cell wall is sufficiently ordered at the molecular level to yield a well-defined X-ray diffraction pattern with a d-spacing of 0.44 nm shows that its molecular architecture is very similar to that of true membranes. This specific d-spacing is produced by the highly ordered packing of the hydrophobic portions of phospholipid molecules. Therefore, the double-track layer of the cell wall has been shown, by these two biophysical means, to have a molecular architecture which would allow it to function as the membrane-like "molecular sieve" layer, whose presence has been deduced from physiological data. This layer is important in the retention of cell wall-associated enzymes and in the control of the movement of large molecules through the cell wall.

Bacteria↗
PubMed2000

Tracking single proteins within cells.

We present experiments in which single proteins were imaged and tracked within mammalian cells. Single proteins of R-phycoerythrin (RPE) were imaged by epifluorescence microscopy in the nucleoplasm and cytoplasm at 71 frames/s. We acquired two-dimensional trajectories of proteins (corresponding to the projection of three-dimensional trajectories onto the plane of focus) for an average of 17 frames in the cytoplasm and 16 frames in the nucleus. Diffusion constants were determined from linear fits to the mean square displacement and from the mean displacement squared per frame. We find that the distribution of diffusion constants for RPE within cells is broader than the distributions obtained from RPE in a glycerol solution, from a Monte Carlo simulation, and from the theoretical distribution for simple diffusion. This suggests that on the time scales of our measurements, the motion of single RPE proteins in the cytoplasm and nucleoplasm cannot be modeled by simple diffusion with a unique diffusion constant. Our results demonstrate that it is possible to follow the motion of single proteins within cells and that the technique of single molecule tracking can be used to probe the dynamics of intracellular macromolecules.

Animals↗
PubMed1996

Single particle tracking of cell-surface HLA-DR molecules using R-phycoerythrin labeled monoclonal antibodies and fluorescence digital imaging.

The mobility of cell surface MHC molecules and their ability to form dynamic associations may be related to the physiological status of the cell and to the potential to bind effector T lymphocytes. To investigate these properties, we have prepared HLA DR specific monoclonal antibodies coupled in a 1:1 mole ratio to the fluorescent phycobiliprotein, R-phycoerythrin (PE). We show that these small particles can be sequentially imaged using a cooled slow-scan charge coupled device camera and hence can be used for single particle tracking experiments. We have applied this technique to investigate the movements of HLA DR molecules on fibroblasts transfected with human DR alpha and DR beta genes. PE-IgG was bound to the transfected fibroblasts and particle tracks were obtained by sequential imaging over a period of typically 30 minutes. Analysis of particle tracks revealed the presence of directed motion and domain-limited diffusion in addition to random diffusion. The contributions of these three types of motion showed cell to cell variability. Velocities of directed motion were of the order of 2 nm second-1 whilst domain diameters were in the range 200-800 nm. Diffusion coefficients for random diffusion were in the range 1 x 10(-13)-5 x 10(-12) cm2 second-1. The higher mobilities were observed for the lower intensity fluorescent spots, which possibly correspond to images of single particles. Much lower mobility was observed with a cell where the spot intensities were approximately double that of the lower intensity spots. These spots could be images of double particles implying the association of at least two HLA DR alpha beta dimers. These data are relevant to the study of MHC class II cell surface redistribution and antigen presentation in specific immunity.

Antibodies, Monoclonal↗
PubMed1999

Use of caged fluorochromes to track macromolecular movement in living cells.

One way to visualize and track the movement of macromolecules in the living cell is to follow their movement after tagging the molecule with a 'caged' or chemically masked fluorochrome. The fluorochrome does not fluoresce until the caging group is released by spot photoactivation, and the bright fluorescent signal can then be tracked as it moves into the dark surrounding area of the cell. When coupled with rapid imaging microscopy, it is possible to measure rates of movement as fast as macromolecular diffusion. This article describes the use of photoactivatable fluorochromes to track the intracellular movement of both proteins and nucleic acids and to track cell lineages.

Animals↗
PubMed1998

Identification of the adult resting progenitor cell by autoradiographic tracking of oligodendrocyte precursors in experimental CNS demyelination.

The new population of oligodendrocytes remyelinating an experimental demyelinating optic nerve lesion has been tracked backwards in time. Using autoradiography combined with electron microscopy and immunocytochemistry, serial sections of optic nerves from young adult cats were studied from 42 h (2 days) post-injection to 93 h (4 days) post-injection. The remyelinating oligodendrocyte lineage was found to commence with the single division of a resting progenitor cell residing in a central fascicular location outside the demyelinative lesion. The division of the founding progenitor cell occurs at 2 days post-lesion and results in two motile daughter cells. These cells, previously described by us as precursor cells, migrate to the lesion, closely appose demyelinated axons, produce axon wrapping processes and differentiate to become remyelinating oligodendrocytes. This study confirms that remyelinating oligodendrocytes originate from resting progenitor cells outside the lesion and not from mature oligodendrocytes, and implies that repeated demyelinative injury could exhaust the reparative capacity of such a region.

Animals↗
PubMed1995

In vivo characterization of two cell types in the rat globus pallidus which have opposite responses to dopamine receptor stimulation: comparison of electrophysiological properties and responses to apomorphine, dizocilpine, and ketamine anesthesia.

Extracellular single-unit recording techniques were used to examine the rat globus pallidus (GP). In both locally anesthetized, paralyzed rats and ketamine-anesthetized rats, we observed two distinct biphasic extracellular waveforms, which we have labeled Type I (negative/positive waveform) and Type II (positive/negative waveform). No significant differences were observed in the firing pattern or number of cells per track between these cell types, although the Type II neurons had a faster mean firing rate in the locally anesthetized animals. A portion of both cell types could be antidromically activated from the subthalamic nucleus, although Type II neurons had significantly slower conduction velocities. The most striking pharmacological difference between the two cell types was that Type I GP neurons were inhibited by systemic administration of the dopamine agonist apomorphine; previous studies have repeatedly shown that Type II GP cells are excited by this treatment. Pretreatment with a subthreshold dose of apomorphine reduced the responsiveness of Type I cells to a subsequent high dose of apomorphine, as has been shown for Type II cells. However, pretreatment with the NMDA antagonist dizocilpine (MK801) produced a significant change in the pattern of response to apomorphine for Type II GP neurons only. Relative to observations in locally anesthetized, paralyzed rats, ketamine anesthesia reduced the firing rate of both cell types, but did not significantly alter their direction of response to apomorphine. Thus, this study has confirmed the existence of two GP cell types with distinct extracellular waveforms and different responses to dopamine receptor stimulation. These data may necessitate a reevaluation of general theoretical models of basal ganglia function in order to account for these opposite effects of dopamine receptor stimulation on pallidal output.

Anesthesia↗
PubMed1982

Computer-based tracking of living cells.

A computer-based tracing technique has been developed to follow the movement of living cells and keep them centered in the field of view of an optical microscope. With the use of an image-processing system, the video image of a cell can be sufficiently processed to allow computer-recognition of the cell boundaries. Determination of the location of the center of the cell enables comparison of successive cell positions and correction for any cell movement. In order to illustrate the versatility of this technique, patterns of movement were obtained of cancerous and non-cancerous cells in an effort to determine the difference in motility between the two cell types. After examination of the data gathered, it was found that there is no difference in the motility between the two cell types over 1-h periods.

Animals↗
PubMed1989

Microcomputer-based interactive tracking of blood cells at biomaterial surfaces.

A microcomputer-based system for analyzing the motion of human platelets and leukocytes at synthetic surfaces from a sequence of video frames on tape is described. The software is designed to provide convenient interaction with an operator to reduce the burden of manual analysis. In addition, the system computes and stores the cell movement data on disk for subsequent statistical analysis. Measurement include the number and nature of cell-to-surface collisions, residence times, and distances traveled.

Biocompatible Materials↗