Intracellular Translocation/Compartmentalization Applications

Introduction

The ability to identify regions or domains in the cell and then quantify the presence of specific cellular constituents in those domains has wide-ranging applicability. Important areas of cell biology research include the study of signal transduction cascades, apoptosis, transcriptional regulation and the cell cycle, to name just a few. Two cases - the translocation of NFkB from cytoplasm to nucleus and the accumulation of signaling molecules as a result of GPCR ligation using the Norak Transfluor ® assay, are highlighted here.

Highlights of Analysis with iGeneration Imaging Cytometers

Nuclear–Cytoplasmic Translocation

Certain transcription factors and cell cycle regulatory molecules will primarily reside in either the cytoplasm or the nucleus. The iGeneration platform can distinguish nuclear regions by detecting a DNA-binding dye. The region surrounding each nucleus may be monitored by defining a ring around the nucleus using peripheral contours. A scattergram resolving cells based on their measured nuclear fluorescence from peripheral fluorescence allows identification of populations that have high levels of a specific marker, such as NFkB, in the nucleus vs. in the cytoplasm. In the population scattergram below, each dot represents a cell. Cells in the red region show high nuclear fluorescence while those in the green region have high cytoplasmic fluorescence.

Green Region
High Detection in the Cytoplasm

Red Region
High Detection in the Nucleus

iBrowser™ software may be used to compare all wells in a 96-well plate, allowing examination of both control and treated wells. In the example shown below, the number of cells falling into the scattergram region showing high nuclear fluorescence is plotted per well, providing a rapid quantitative assessment of the results from a full 96-well plate.

Similarly, a quick overview on a well-by-well basis can be obtained as shown below.

In this example, wells where cells have high nuclear fluorescence are identified by a temperature-like color scale, with red wells having the greatest number of cells with high nuclear fluorescence.

Norak Transfluor^® Assay

This assay detects the translocation of arrestin-GFP in response to agonist-stimulated GPCR intracellular signaling. The images below show the b-adrenergic receptor wild receptor that induces pit-like accumulation of arrestin-GFP, compared to a modified b-adrenergic receptor that results in vesicle-like accumulation of arrestin-GFP.

Data for this section provided by Kazuo Ozawa, Ph.D., Etsuo Shinohara, and Sachiko Karaki, Ph.D., Olympus Corporation, Genome Medical Business Division, Tokyo, Japan.

GFP fluorescence in:

Control cells

Stimulated cells with wild-type receptor (pit-type)

Stimulated cells with modified receptor (vesicle-type)

This translocation response was measured using MaxPixel, a morphometric feature acquired on the iGeneration platform which reports changes in brightness. As arrestin-GFP accumulates into pits or vesicle-like structures, the MaxPixel values rise. The plot below compares MaxPixel values measured in populations activated with varying concentrations of agonist (isoproterenol).

Benefits of Using iGeneration Technology

Using advanced data processing, subcellular compartmentalization and translocation can be monitored and quantified. Multiple probes can be analyzed in a single sample while obtaining widefield images, which may be correlated to the quantitative data in galleries or field images. The iGeneration platform combines the advantages of flow cytometry and image analysis in a single system. The iGeneration inverted format supports a diversity of carrier types, allowing flexibility in matching the needs of your application with the appropriate carrier type.

More Information

For more information, review the CompuCyte Published Materials and CompuCyte Bibliography.
Request the Transfluor Assay Application Note.