Cytoplasm Of Single Cells Research Articles

Insights into In Vivo Environmental Effects on Quantitative Biochemistry in Single Cells.

Biomacromolecules exist and function in a crowded and spatially confined intracellular milieu. Single-cell analysis has been an essential tool for deciphering the molecular mechanisms of cell biology and cellular heterogeneity. However, a sound understanding of in vivo environmental effects on single-cell quantification has not been well established. In this study, via cell mimicking with giant unilamellar vesicles and single-cell analysis by an approach called plasmonic immunosandwich assay (PISA) that we developed previously, we investigated the effects of two in vivo environmental factors, i.e., molecular crowding and spatial confinement, on quantitative biochemistry in the cytoplasm of single cells. We find that molecular crowding greatly affects the biomolecular interactions and immunorecognition-based detection while the effect of spatial confinement in cell-sized space is negligible. Without considering the effect of molecular crowding, the results by PISA were found to be apparently under-quantitated, being only 29.5-50.0% of those by the calibration curve considering the effect of molecular crowding. We further demonstrated that the use of a calibration curve established with standard solutions containing 20% (wt) polyethylene glycol 6000 can well offset the effect of intracellular crowding and thereby provide a simple but accurate calibration for the PISA measurement. Thus, this study not only sheds light on how intracellular environmental factors influence biomolecular interactions and immunorecognition-based single-cell quantification but also provides a simple but effective strategy to make the single-cell analysis more accurate.

Quantitative measurement of transmitters in individual vesicles in the cytoplasm of single cells with nanotip electrodes.

The quantification of vesicular transmitter content is important for studying the mechanisms of neurotransmission and malfunction in disease, and yet it is incredibly difficult to measure the tiny amounts of neurotransmitters in the attoliter volume of a single vesicle, especially in the cell environment. We introduce a novel method, intracellular vesicle electrochemical cytometry. A nanotip conical carbon-fiber microelectrode was used to electrochemically measure the total content of electroactive neurotransmitters in individual nanoscale vesicles in single PC12 cells as these vesicles lysed on the electrode inside the living cell. The results demonstrate that only a fraction of the quantal neurotransmitter content is released during exocytosis. These data support the intriguing hypothesis that the vesicle does not open all the way during the normal exocytosis process, thus resulting in incomplete expulsion of the vesicular contents.

Quantitative Measurement of Transmitters in Individual Vesicles in the Cytoplasm of Single Cells with Nanotip Electrodes

AbstractThe quantification of vesicular transmitter content is important for studying the mechanisms of neurotransmission and malfunction in disease, and yet it is incredibly difficult to measure the tiny amounts of neurotransmitters in the attoliter volume of a single vesicle, especially in the cell environment. We introduce a novel method, intracellular vesicle electrochemical cytometry. A nanotip conical carbon‐fiber microelectrode was used to electrochemically measure the total content of electroactive neurotransmitters in individual nanoscale vesicles in single PC12 cells as these vesicles lysed on the electrode inside the living cell. The results demonstrate that only a fraction of the quantal neurotransmitter content is released during exocytosis. These data support the intriguing hypothesis that the vesicle does not open all the way during the normal exocytosis process, thus resulting in incomplete expulsion of the vesicular contents.

Microphotometry of Underwater Shadowing by a Moss from a Niagara Escarpment Waterfall

Microscope and fiber-optic spectrophotometry of transmittance and backscattering both showed moss leaves to be capable of casting strong shadows, with a single leaf blocking approximately 90% of incident light from a point source. In leaves with only one layer of cells, the transmittance through the cytoplasm of single cells was similar to that for whole leaves. Analysis of cell wall birefringence by polarized-light interferometry indicated that cell walls might normally scatter rather than transmit light. Spectra transmitted through, or backscattered from, the upper green layers of moss were dominated by selective absorbance from chlorophyll, but there was also evidence of wavelength-dependent scattering, as detected in the lower layers of brown, dead moss. Specular reflectance from moss leaves was detected by polarimetry and may have contributed to the relatively high macroscopic transmittance of stationary moss in water. Shadowing by moss leaves was confirmed by dynamic measurements of mosses in turbulent water without bubbles. Flicker patterns from leaves were superimposed on the underwater flicker pattern created at the air-water interface, thus flecks of light were reduced in intensity, increased in frequency, and decreased in duration. This was detected with both point source and diffuse illumination of samples.

Analysis of differential lipofection efficiency in primary and established myoblasts.

In this study we have compared the process of lipid-mediated transfection in primary and established myoblasts, in an attempt to elucidate the mechanisms responsible for the scarce transfectability of the former. We determined the metabolic stability of cytoplasmically injected and lipofected DNA in primary and established myoblasts and carried out a comparative time course analysis of luciferase reporter-gene expression and DNA stability. The efficiency of the transcription-translation machinery of the two cell types was compared by intranuclear injection of naked plasmid DNA encoding luciferase. Subcellular colocalization of fluorescein-labeled lipopolyplexes with specific endosomal and lysosomal markers was performed by confocal microscopy to monitor the intracellular trafficking of plasmid DNA during transfection. The metabolic stability of plasmid DNA was similar in primary and established myoblasts after both lipofection and cytoplasmic injection. In both cell types, lipofection had no detectable effect on the rate of cell proliferation. Confocal analysis showed that nuclear translocation of transfected DNA coincided with localization in a compartment devoid of endosome- or lysosome-specific marker proteins. The residency time of plasmid DNA in this compartment differed for primary and established myoblasts. Our findings suggest that the lower transfectability of primary myoblasts is mostly due to a difference in the intracellular delivery pathway that correlates with more rapid delivery of internalized complex to the lysosomal compartment.

A fluorescent assay of proteinases in cultured mammalian cells.

We have demonstrated proteinase activity in unfixed cells grown on tissue culture plates with a technique using 5-nitrosalicylaldehyde and peptide derivatives of 4-methoxy-2-naphthylamine. The 4-methoxy-2-naphthylamine liberated by proteinase activity reacts with 5-nitrosalicylaldehyde to form a fluorescent product. The substrates CBZ-alanyl-arginyl-arginyl-4-methoxy-2-naphthylamine and lysyl-alanyl-4-methoxy-2-naphthylamine, were used for the direct visual detection of two arylamidase activities in BALB/c 3T3 and C3H 10T 1/2 cells. With low magnification these enzyme activities can be detected in single clones; with higher magnification the fluorescent product can be seen within the cytoplasm of single cells.

Simultaneous localization of myosin and tubulin in human tissue culture cells by double antibody staining

We have studied the distribution of myosin and tubulin molecules inside the same tissue culture cells by using two antibodies labeled with contrasting fluorochromes. Antimyosin raised against human platelet myosin was labeled with rhodamine. Antitubulin raised against sea urchin vinblastine-induced tubulin crystals was labeled with fluorescein. The two antibodies stained entirely different structures inside the same flat interphase cell: antimyosin bound to stress fibers and antitubulin bound to thin, wavy fibers thought to be individual microtubules. Compact interphase cells stained diffusely with both antibodies. From prophase through early anaphase both antibodies stained the mitotic spindle, although the fluorescence contrast between the spindle and the cytoplasm was much higher with antitubulin than with antimyosin. From anaphase through telophase, strong antimyosin staining occurred in the cleavage furrow, while antitubulin stained the region between the separated chromosomes. This study established the feasibility of high-resolution fluorescent antibody localization of pairs of motility proteins in the cytoplasm of single cells, an approach which will make it possible to map out the sites of the various contractile protein interactions in situ.