Fluorescence depolymerization

Polyester composition can be determined by hydrolytic depolymerization followed by gas chromatography (28) to analyze for monomers, comonomers, oligomers, and other components including side-reaction products (ie, DEG, vinyl groups, aldehydes), plasticizers, and finishes. Mass spectroscopy and infrared spectroscopy can provide valuable composition information, including end group analysis (47,101,102). X-ray fluorescence is commonly used to determine metals content of polymers, from sources including catalysts, delusterants, or tracer materials added for fiber identification purposes (28,102,103). 

Molecules (chemoattractants) that stimulate neutrophil-directed migration (chemotaxis) bind to distinct receptors on neutrophil plasma membranes (discussed in Chapter 38 of this text). Within seconds after chemoattractant binding, neutrophils exhibit rapid oscillations in actin polymerization and depolymerization (12,13). The shape changes accompanying chemoattractant binding depend on the duration and extent of F-actin polymerization (3). These quantitative studies of F-actin content were performed utilizing a flow cytometric assay that detects the fluorescence intensity of individual, fixed, permeabihzed cells that have been stained with F-actin-specific, fluorescent phallotoxins (14,15). 

The data presented in Fig. 1 illustrate the changes in F-actin polymerization and depolymerization during 5 min of FMLP (UF M) stimulation at 37°C. Human neutrophils often exhibit a biphasic F-actin response under these conditions (18). All of the neutrophils exhibit changes in F-actin polymerization, as illustrated by the fluorescence histograms in Fig. 2, which... 

Fluorescein-labeled proteins are also used to measure the translational mobility of proteins and lipids by the Fluorescence Recovery After Photo-bleaching technique [54-59]. The uniformly labeled fluorescent sample is flashed with an intense light source to bleach a spot, thus producing a concentration gradient. The rate of recovery of fluorescence in that bleached area is measured and used to calculate the diffusion coefficient of the probe dye into the bleached zone. Such diffusion coefficient measurements have been used to determine the association constants of proteins in cells [60], to measure the exchange of tubulin between the cytoplasm and the microtubules [61,62], to study the polymerization-depolymerization process of actin [63-65] and to monitor the changes that occur upon cell maturation [66,67]. 

C2 toxin-induced depolymerization of actin filaments is visualized by fluorescence labeling of actin with FITC- or Rhodamine-phalloidin. 

A EXPERIMENTAL FIGURE 20-10 Fluorescence microscopy reveals in vivo growth and shrinkage of individual microtubules. Fluorescently-labeled tubulin was microinjected into cultured human fibroblasts. The cells were chilled to depolymerize preexisting microtubules into tubulin dimers and were then incubated at 37 °C to allow repolymerization. 

FIG. 13.6 Kinetoplasts of T. hrucei connected to basal bodies. Flagella were prepared by detergent lysis and cytoskeletal depolymerization. The flagellar are viewed by phase contrast and the kDNA by DAPI fluorescence. DAPI is a fluorescent dye which binds to DNA. (Reprinted with permission from ref. 64.)... 

Fig. 8 Confocal fluorescence microscopy of B50 neuroblastoma cells, (a, b) Immunolabeling of mtHSP70 green) in B50 cells in controls (a) and cisPt-treated cells (b). After cisPt, mitochondria are clustered around the nucleus and form dense masses in the cytoplasm (b). Nuclei are counterstained with Hoechst blue), (c, d) Double immunolabeling of filamentous actin red) and a-tubulin green) in B50 control cells (c) and in 48 h cisPt-treated cells (d). CisPt-induced cytoskeleton damage leads tubulin to reorganize into thick bundles (e) and to disruption of filamentous actin microfilaments and accumulation of depolymerized actin at cell periphery (f).

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