Structural organization of interphase 3T3 fibroblasts studied by total internal reflection fluorescence microscopy.

Abstract

We studied the laminar organization of 3T3 fibroblast cells growing on glass slides by use of total internal reflection illumination to excite fluorescence emission (TIRF) from labeled molecules and stained cellular compartments that are very close to the cell-substrate contact region. Mitochondria, distant from the contact regions and stained with the watersoluble cationic dye, diI-C3-(3), fluoresced only as the glass/cytoplasm critical angle was approached. A similar result was obtained when the nuclei were stained with Hoechst dye 33342. From this measured angle a cytoplasmic refractive index in the range 1.358-1.374 was computed. The plasma membrane of 3T3 cells was stained with diI-Ct~-(3), and the cytoplasmic compartment was stained with fluoresceinyl-dextran (FTC-dextran) or with carboxyfluorescein. We have demonstrated a high degree of correspondence between the low-reflectance zones in the reflection interference image of a live cell and the TIRF images of both the plasma membrane and cytoplasmic compartment. TIRF photometry of selected contact regions of cells provided data from which the absolute separation of cell and substrate was computed. From a population of 3T3 cells microinjected with fluorescein-labeled actin, motile and adherent interphase cells were selected for study. For adherent cells, which displayed fluorescent stress fibers, the TIRF image was composed of intense patches and less intense regions that corresponded, respectively, to the focal contact and close-contact zones of the reflection-interference image. The intense patches corresponded to the endpoints of the stress fibers. Cells of motile morphology, which formed some focal contacts and extensive close-contact zones, gave AF-actin TIRF images of relatively even intensity. Thin lamellar regions of the cytoplasm were found to contain concentrations of actin not significantly different from other close-contact regions of the cell. The major analytical problem of TIRF microscopy is separation of the effects of proximity to substrate, refractive index, and fluorescent probe concentration on the local brightness of the TIRF image. From our results, it appears possible to use TIRF microscopy to measure the proximity of different components of substrate contact regions of cells.Mammalian fibroblasts that are grown on a planar substrate exhibit a laminar morphology that is a result of spatial constraints and the forces of adhesion and motility. Cell-substrate contacts are classified by degree of apposition and by size. Reflection-interference microscopy (1-3) and electron microscopy (4) have been used to demonstrate the existence of close contacts (extended domains in which the distance between cell membrane and substrate is ~50 nm) and focal contacts (compact domains in which the separation may be as little as 10 nm).The distribution and interaction of the cytoskeletal and contractile proteins responsible for cell-substrate attachments and, by implication, the sites of application of forces to the substrate are now partly under...

Polymorphonuclear leukocyte histamine receptors: occurrence in cell surface clusters and their redistribution during locomotion.

“…Cell locomotion leads to redistribution of fluorescence to pseudopods or to the lamellipodium and uropod. These cellular sites are responsible for adherence of PMNs and fibroblasts to substrates (23)(24)(25)(26)(27). Therefore, accumulation of histamine ligand-receptor complexes at these sites may sterically exclude membrane components participating in adhesion or decrease adhesion through the action of a second messenger such as cAMP.…”

Quantification of the Cell-Substratum Contact and Cell Lift-off Under Different Intra/Extracellular Conditions

“…Nutrition is supplied during the experiment from the incubator through the chip system. In the literature (Lanni et al 1985;Bretschneider et al 2004;Weisswange et al 2005;Partridge and Marcantonio 2006;Manneville 2006), there are many examples of the use of EF in combination with TIRF for measuring global and membrane-specific events. However, in the present study, EF setup was used only for illuminating the whole field to locate the cells before and/or after the TIRF images were recorded.…”

“…Gingell et al (1987) developed a rigorous electromagnetic theory of total internal reflection and applied it to the establishment of a quantitative basis for mapping cell-substratum topography. The TIRF technique has been used in biological studies, such as cell-substrate contacts, vesicle fusion, and single-molecule observation (Lanni et al 1985;Thompson and Lagerholm 1987;Wang and Axelrod 1994;Burmeister et al 1998;Toomre and Manstein 2001;Li et al 2004;Bretschneider et al 2004;Manneville 2006;Young and Marcantonio 2007). Lanni et al (1985) observed fibroblast cells growing on glass slides and Fuhr et al (1998) reported the observation of cell adhesion and migration by using different microscopic techniques, including TIRF.…”

3D Coupling of Fibronectin Fibril Arrangement with Topology of Ventral Plasma Membrane

“…Using interference reflection microscopy (Izzard & Lochner 1976), electron microscopy (Chen & Singer 1982) and total internal reflection fluorescence (TIRF) microscopy (Lanni et al, 1985;Truskey et al, 1992), it has been revealed that distance between the VPM and the substratum is 10-20 nm at FAs and  100 nm in other regions. If one considers the size of an integrin molecule (8  12 nm for an extracellular headpiece and 18 nm for flexible legs (Weise et al, 1992)), the gap between the VPM and the substratum outside FAs is much larger than the reach of the molecule.…”