The mammalian Golgi apparatus exists as stacks of cisternae that are laterally linked to form a continuous membrane ribbon, but neither the molecular requirements for, nor the purpose of, Golgi ribbon formation are known. Here, we demonstrate that ribbon formation is mediated by specific membrane-fusion events that occur during Golgi assembly, and require the Golgi proteins GM130 and GRASP65. Furthermore, these GM130 and GRASP65-dependent lateral cisternal-fusion reactions are necessary to achieve uniform distribution of enzymes in the Golgi ribbon. The membrane continuity created by ribbon formation facilitates optimal processing conditions in the biosynthetic pathway.

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...

Oil-immersion microscope objective lenses have been designed and optimized for the study of thin, two-dimensional object sections that are mounted immediately below the coverslip in a medium that is index matched to the immersion oil. It has been demonstrated both experimentally and through geometrical- and physical-optics theory that, when the microscope is not used with the correct coverslip or immersion oil, when the detector is not located at the optimal plane in image space, or when the object does not satisfy specific conditions, aberration will degrade both the contrast and the resolution of the image. In biology the most severe aberration is introduced when an oil-immersion objective lens is used to study thick specimens, such as living cells and tissues, whose refractive indices are significantly different from that of the immersion oil. We present a model of the three-dimensional imaging properties of a fluorescence light microscope subject to such aberration and compare the imaging properties predicted by the model with those measured experimentally. The model can be used to understand and compensate for aberration introduced to a microscope system under nondesign optical conditions so that both confocal laser scanning microscopy and optical serial sectioning microscopy can be optimized.

Cell-substrate adherence is a fundamental property of microorganisms that enables them to exist in biofilms. Our study focuses on adherence of the fungal pathogen Candida albicans to one substrate, silicone, that is relevant to device-associated infection. We conducted a mutant screen with a quantitative flow-cell assay to identify thirty transcription factors that are required for adherence. We then combined nanoString gene expression profiling with functional analysis to elucidate relationships among these transcription factors, with two major goals: to extend our understanding of transcription factors previously known to govern adherence or biofilm formation, and to gain insight into the many transcription factors we identified that were relatively uncharacterized, particularly in the context of adherence or cell surface biogenesis. With regard to the first goal, we have discovered a role for biofilm regulator Bcr1 in adherence, and found that biofilm regulator Ace2 is a major functional target of chromatin remodeling factor Snf5. In addition, Bcr1 and Ace2 share several target genes, pointing to a new connection between them. With regard to the second goal, our findings reveal existence of a large regulatory network that connects eleven adherence regulators, the zinc-response regulator Zap1, and approximately one quarter of the predicted cell surface protein genes in this organism. This limited yet sensitive glimpse of mutant gene expression changes had thus defined one of the broadest cell surface regulatory networks in C. albicans.

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.

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.

Two fundamental parameters of the highly dynamic, ultrathin lamellipodia of migrating fibroblasts have been determined-its thickness in living cells (176 +/- 14 nm), by standing-wave fluorescence microscopy, and its F-actin density (1580 +/- 613 microm of F-actin/microm(3)), via image-based photometry. In combination with data from previous studies, we have computed the density of growing actin filament ends at the lamellipodium margin (241 +/- 100/microm) and the maximum force (1.86 +/- 0.83 nN/microm) and pressure (10.5 +/- 4.8 kPa) obtainable via actin assembly. We have used cell deformability measurements (. J. Cell Sci. 44:187-200;. Proc. Natl. Acad. Sci. USA. 79:5327-5331) and an estimate of the force required to stall the polymerization of a single filament (. Proc. Natl. Acad. Sci. USA. 78:5613-5617;. Biophys. J. 65:316-324) to argue that actin assembly alone could drive lamellipodial extension directly.

We describe a simple method for loading exogenous macromolecules into the cytoplasm of mammalian cells adherent to tissue culture dishes . Culture medium was replaced with a thin layer of fluorescently labeled macromolecules, the cells were harvested from the substrate by scraping with a rubber policeman, transferred immediately to ice cold media, washed, and then replated for culture . We refer to the method as "scrape-loading ." Viability of cells was 50-60% immediately after scrape-loading and was 90% for those cells remaining after 24 h of culture . About 40% of adherent, well-spread fibroblasts contained fluorescent molecules 18 h after scrape-loading of labeled dextrans, ovalbumin, or immunoglobulin-G . On average, 107 dextran molecules (70,000-mol wt) were incorporated into each fibroblast by scrape-loading in 10 mg/ml dextran . The extent of loading depended on the concentration and molecular weight of the dextrans used. A fluorescent analog of actin could also be loaded into fibroblasts where it labeled stress fibers. HeLa cells, a macrophage-like cell line, J774A.1, and human neutrophils were all successfully loaded with dextran by scraping. The method of scrape-loading should be applicable to a broad range of adherent cell types, and useful for loading of diverse kinds of macromolecules .Methods for loading macromolecules into cytoplasm are essential to studies in several recent and evolving fields of eucaryotic cell and molecular biology. Transformation ofcells with exogenous DNA, in vivo studies of the location and activity of cytoskeletal proteins and other macromolecules in living cells by fluorescent analog cytochemistry, and the analysis of cytoplasmic degradation of proteins all depend on the incorporation of large molecules into living cells .Certain characteristics are desirable of any method for loading macromolecules into cells. The method should be simple, applicable to diverse cell types, and capable of loading a wide range of macromolecule species. A significant proportion of cells in a population should be loaded with the macromolecule and to a measurable extent. The macromolecules should be introduced, initially, into the cell's cytoplasm only. Cell function, morphology, and viability should not be extensively compromised by the loading method.Several methods are now available for loading exogenous macromolecules into cell cytoplasm . Microneedles are used to inject diverse substances into individual cells (8; see 6 for a review) . Briefexposure to a hypotonic medium loads protein in that medium into the cytoplasm of a cell population (2) . 1556Hyposomotic shock ofpinosomes containing dextran or protein releases most of these substances into the cytoplasm of cells (14). Liposomes and red blood cells containing trapped substances can, after fusion with host cells, deliver such trapped substances into host cytoplasm (17,7,15). In addition, high voltage electric impulses enhance uptake of DNA into cells (13).No single one ofthese methods now available fulfills all the criteria li...