Cytoplasmic sampling

Figure 12.10. (a) Electrokinetic microinjector and (b) electropherogram generated from the removal and separation of cytoplasm samples from single nerve cells of Planorbis corneus, a pond snail.8 [Reprinted, by permission, from R. A. Wallingford and A. G. Ewing, Anal. Chem. 60 (No. 18), 1988, 1972-1975. Capillary Zone Electrophoresis with Electrochemical Detection in 12.7 pm Diameter Columns . 1988 by American Chemical Society.]... 

Single-cell CE experiments in neurochemistry were first performed in the late 1980s with initial cytoplasmic sampling from P. corneas completed in 1988 and whole cell injection of Helix aspersia in 1989 to separate fluorescently labeled amino acids. Since these pioneering studies, many different small molecule neurotransmitters and other related species have been detected in single cells by CE. 

A time course for the import of P-labeled RNAs microinjected into the cytoplasm is shown in Fig. 4. Each point represents a cohort of 15 oocytes microinjected within a few minutes of each other. For time management reasons it is best to inject the longer time point cohorts first. RNAs are extracted from the nuclear and cytoplasmic samples and analyzed as described above by acrylamide gel electrophoresis and autoradiography. The transport time course can be quantified by phosphorimaging, densitometry, or by cutting out the specific bands and scintillation counting. Phosphorimaging is now the method of choice. 

Mason, A. Z., and Storms, S. D. (1993). Applications of directly coupled SE-HPLC/ICP-MS in environmental toxicology studies A study of metal-ligand interactions in cytoplasmic samples. Mar. Environ. Res. 35(1-2), 19. 

The cytoplasmic domains reconstructed from negatively stained [90] and from frozen-hydrated samples [91,177] have similar shapes. Both include the protruding lobe and the bridge region that links the Ca " -ATPase molecules into dimers. The intramembranous peptide domains of the two ATPase molecules which make up a dimer spread apart as they pass through the bilayer toward the luminal side of the membrane, establishing contacts with the Ca -ATPase molecules in the neighboring dimer chains. The lateral association of dimer chains into extended crystal lattice is... 

Double-label ICC is another approach using a similar principle. It pairs a nuclear antibody with a cytoplasmic antibody on the same sample, such as keratin and estrogen receptor. At present, this method is mostly used in the research field. 

Ultrastructural examination of duckweed frond (Fig. 5) and root tissues treated with 18 (100 xM) revealed membrane damage to the tonoplast after 12 hours of exposure. The samples viewed through the transmission electron microscope showed ruptured tonoplasts, free-floating organelles and loss of cytoplasm relative to control tissues. The tonoplast may be the primary target for the phytotoxic effect of 18, which represents an unusual, if not unique, toxic mechanism among phytotoxic agents. 

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]. 

FIGURE 4.3 High magnification transmission electron micrographs of multilamellar membrane structures in the intercellular space of the cornified part of human epidermis. (A) cryo-electron micrograph of vitreous section. (B, C) conventional electron micrographs of resin embedded sections. The cell plasma membranes appear as 3.8 nm wide bilayers in (A) (open white arrow). A 16 nm broad zone of electron dense material, the cornified cell envelope (white asterix), is directly apposed to the cytoplasmic side of the bilayer plasma membranes in the native sample (A) (open white arrow). Scale bar 50 nm (A). Scale bars 25 nm (B, C) adapted from measures given in Swartzendruber et al. (1989). (A) reprinted from Norlen (2003). With permission from Blackwell Science Publications. (B, C) reprinted from Swartzendruber et al. (1989). With permission from Blackwell Science Publications. 

---