Cell-substrate contacts

Axelrod, D. (1981). Cell-substrate contacts illuminated by total internal reflection fluorescence. J. Cell Biol. 89, 141-5. 

Quantitative determination of the absolute distance from the surface to a labeled cell membrane at a cell/substrate contact region can be based on the variation of F(d) with 0.(1O6) This effort is challenging because corrections have to be made for 0-dependent reflection and transmission through four stratified layers (glass, culture medium, membrane, and cytoplasm), all with different refractive indices. For 3T3 cells, Lanni et a//1065 derived a plasma membrane/substrate spacing of 49 nm for focal contacts and 69 nm for close contacts elsewhere. They were also able to calculate an approximate refractive index for the cytoplasm of 1.358 to 1.374. 

One TIRF study found that some membrane proteins behave just oppositely to AChR they avoid the cell/substrate contact regions/ 1 When endothelial cells are grown on a bare glass surface or are brought into suspension, a specific membrane protein marked with antibodies appears all over the cell surface, as evidenced by epi-illumination and TIRF. However, when the cells are grown on (or returned to) a surface coated with their own extracellular matrix material, the protein disappears from the basal (cell/substrate-contacting) side of the cells. 

Cell/substrate contacts can be located by a nonfluorescence technique completely distinct from TIRF, known as internal reflection microscopy (IRM).(1 9) Using conventional illumination sources, IRM visualizes cell/substrate contacts as dark regions. IRM has the advantage that it does not require the cells to be labeled, but the disadvantages that it contains no information about biochemical specificities in the contact regions and that it is less sensitive to changes in contact distance (relative to TIRF) within the critical first lOOnm from the surface. 

Localization of cell/substrate contact regions in cell culture. 

Reduction of cell autofluorescence relative to fluorescence excited at cell/substrate contacts. 

Fig. 8 (a) Nanopatteming by EBL A silcion substrate is functionalized with an amino-silane and coated with BSA. A focused electron beam is employed to write nanopattems into the BSA film. Proteins from solution can selectively adsorb into the nanopattems and guide the formation of cell-substrate contacts, (b) Fibroblast on fibronectin 10 x 10 nanodot matrix created by electron beam lithography. Cells spread, and fluorescent staining of intracellular proteins shows that focal contacts are located on the nanodots actin green), fibronectin red), and vinculin blue). Areas a, b, and c are shown magnified. (Figure in part reproduced with permission from [103])... 

Ex cell substrate contacts the electrolyte outside the cell... 

The most obvious application of the QCM technology in biomedical research with living cells is the online observation of cell-substrate contacts, either when they form de novo as in attachment and spreading, or when established cell-substrate contacts reorganize under the influence of biological, chemical, or physical stimuli. It is a unique advantage of the QCM technique that these kinds of measurements are still possible when the quartz resonator is first coated with a thin layer of any technical material of interest like, for instance. 

Another striking new direction of the QCM in the field of cell biology are motihty measurements based on noise analysis of the resonance frequency. When the cells move and crawl on the surface of the quartz plate the resonance frequency fluctuates as a direct consequence of the continuous assembly and disassembly of cell-substrate contacts during cell movement. Pax and coworkers have recently shown that the contraction of heart muscle cells can be easily recorded from the associated alterations of the resonance parameters [55]. We recently found that even in stationary cell layers without any open spaces that would allow for lateral migration, metaboUcally driven mi-cromotion can be recorded [56]. 

Adhesive instability may be related, in part, to proteolytic processing of cell-substrate contacts (Pollanen et al., 1987). Proteases are important in many developmental events and especially in cell... 

Evanescent wave microscopy or total internal reflection microscopy (TIRM) has been employed in the fields of biology and chemistry since the 1970s. The TIRM technique has long been used in cell biology studies and more recently cell-substrate contacts, vesicle fusion, and single-molecule observation. Here, cells on a microscope cover glass are illuminated by an... 

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