Using fluorescence recovery after photobleaching, we have studied the diffusion of fluorescein-labeled, size fractionated Ficoll in the cytoplasmic space of living Swiss 3T3 cells as a probe of the physical chemical properties of cytoplasm. The results reported here corroborate and extend the results of earlier experiments with fluorescein-labeled, size-fractionated dextran: diffusion of nonbinding particles in cytoplasm is hindered in a size-dependent manner. Extrapolation of the data suggests that particles larger than 260 A in radius may be completely nondiffusible in the cytoplasmic space. In contrast, diffusion of Ficoll in protein solutions of concentration comparable to the range reported for cytoplasm is not hindered in a size-dependent manner. Although we cannot at present distinguish among several physical chemical models for the organization of cytoplasm, these results make it clear that cytoplasm possesses some sort of higher-order intermolecular interactions (structure) not found in simple aqueous protein solutions, even at high concentration. These results also suggest that, for native cytoplasmic particles whose smallest radial dimension approaches 260 A, size may be as important a determinant of cytoplasmic diffusibility as binding specificity. This would include most endosomes, polyribosomes, and the larger multienzyme complexes.The non-Newtonian properties of cytoplasm have been well documented during more than a century of study, but the physical chemical basis for the non-Newtonian properties of cytoplasm is not understood (for reviews, see refs. 1-12). While such macroscopic non-Newtonian phenomena as viscoelasticity and thixotropy imply that cytoplasm possesses some sort of submicroscopic intermolecular organization not found in a dilute, aqueous solution, the possible forms of this organization range from a liquid crystal structure due to the high concentration of protein in cytoplasm, to a meshwork of entangled filamentous proteins, to a crosslinked gel network. A fundamental problem in approaching this question has been the difficulty of studying living cells with high enough resolution. Until recently there has been no method of obtaining data on a molecular level without the necessity of first fixing the cells for electron microscopy or fractionating the cells for subsequent biochemical analysis. Each of these approaches contains the potential for artifacts that make it uncertain how far the results of such experiments can be extended to the structure and function of living cells. Two relatively new techniques have made it possible to study the behavior of specific molecules in living cells while keeping perturbation of the cells' normal structure and function to a minimum. Fluorescent analog cytochemistry (FAC) can be used to study the subcellular distribution of fluorescent derivatives (analogs) of specific molecules (13), and fluorescence recovery after photobleaching (FRAP) can be used to study quantitatively the mobility of these analogs within living cells (14-21). By com...
Abstract. We have used size-fractionated, fluorescent dextrans to probe the structure of the cytoplasmic ground substance of living Swiss 3T3 cells by fluorescence recovery after photobleaching and video image processing. The data indicate that the cytoplasm of living cells has a fluid phase viscosity four times greater than water and contains structural barriers that restrict free diffusion of dissolved macromolecules in a size-dependent manner. Assuming these structural barriers comprise a filamentous meshwork, the combined fluorescence recovery after photobleaching and imaging data suggest that the average pore size of the meshwork is in the range of 300 to 400 ~,, but may be as small as 200 A in some cytoplasmic domains.
Animal cells dividing in culture undergo a dramatic sequence of morphological changes, characterized by cytoskeletal disassembly as cells round up, redistribution of actin, myosins and other cytoplasmic and surface molecules into the cleavage furrow, and respreading, before daughter cells finally separate at the mid-body. Knowledge of forces governing these movements is critical to understanding their mechanisms, including whether formation of the cleavage furrow results from increased force generation at the equator or relaxation at the poles, and whether traction force subsequently mediates cytofission of the intercellular bridge. We have quantitatively mapped traction forces in dividing cells, by extending the silicone-rubber substratum method to detect forces of nanonewtons to micronewtons. We used a new silicone polymer to fabricate substrata whose compliance can be adjusted precisely by ultraviolet irradiation. We show that traction force appears locally at the furrow in the absence of relaxation at the poles during cleavage. Force also rises as connected daughter cells respread and attempt to separate, suggesting that tension contributes to the severing of the intercellular bridge when cytokinesis is completed.
The use of fluorescence microscopy for investigating the three-dimensional structure of cells and tissue is of growing importance in cell biology, biophysics and biomedicine. Three-dimensional data are obtained by recording a series of images of the specimen as it is stepped through the focal plane of the microscope. Whether by direct imaging or by confocal scanning, diffraction effects and noise generally limit axial resolution to about 0.5 microns. Here we describe a fluorescence microscope in which axial resolution is increased to better than 0.05 microns by using the principle of standing-wave excitation of fluorescence. Standing waves formed by interference in laser illumination create an excitation field with closely spaced nodes and antinodes, allowing optical sectioning of the specimen at very high resolution. We use this technique to obtain images of actin fibres and filaments in fixed cells, actin single filaments in vitro and myosin II in a living cell.
This paper describes the development and characterization of a microphysiology platform for drug safety and efficacy in liver models of disease that includes a human, 3D, microfluidic, four-cell, sequentially layered, self-assembly liver model (SQL-SAL); fluorescent protein biosensors for mechanistic readouts; as well as a microphysiology system database (MPS-Db) to manage, analyze, and model data. The goal of our approach is to create the simplest design in terms of cells, matrix materials, and microfluidic device parameters that will support a physiologically relevant liver model that is robust and reproducible for at least 28 days for stand-alone liver studies and microfluidic integration with other organs-on-chips. The current SQL-SAL uses primary human hepatocytes along with human endothelial (EA.hy926), immune (U937) and stellate (LX-2) cells in physiological ratios and is viable for at least 28 days under continuous flow. Approximately, 20% of primary hepatocytes and/or stellate cells contain fluorescent protein biosensors (called sentinel cells) to measure apoptosis, reactive oxygen species (ROS) and/or cell location by high content analysis (HCA). In addition, drugs, drug metabolites, albumin, urea and lactate dehydrogenase (LDH) are monitored in the efflux media. Exposure to 180μM troglitazone or 210μM nimesulide produced acute toxicity within 2–4 days, whereas 28μM troglitazone produced a gradual and much delayed toxic response over 21 days, concordant with known mechanisms of toxicity, while 600μM caffeine had no effect. Immune-mediated toxicity was demonstrated with trovafloxacin with lipopolysaccharide (LPS), but not levofloxacin with LPS. The SQL-SAL exhibited early fibrotic activation in response to 30nM methotrexate, indicated by increased stellate cell migration, expression of alpha-smooth muscle actin and collagen, type 1, alpha 2. Data collected from the in vitro model can be integrated into a database with access to related chemical, bioactivity, preclinical and clinical information uploaded from external databases for constructing predictive models.
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