Q3DM Inc.
8030 La Messa Blvd. #189
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Website: http://www.q3dm.com
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Analysis of Nuclear-Cytoplasmic Translocation Using High Throughput Microscopy
Real-time investigation of cellular and sub-cellular processes.
by Paul Hoeprich, Ph.D.
Figure 1: Example 203 0.5 N.A. fluorescent micrographs of HeLa cells using the EIDAQ 100 High Throughput Microscopy (HTM) system. NF-kB is detected with Alexa 488 (green) and the nucleus is defined by Hoechst 33243 staining (blue, not shown), visualized before (top) and after (bottom) stimulation of NF-kB nuclear translocation. Note the clear but incomplete shift of green fluorescence from the cytoplasm to the nucleus.
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Abstract
It is increasingly recognized that gaining a detailed understanding of complex biological systems such as cellular pathways is fundamental to advances in basic research and drug discovery. Most importantly, there is a need for the direct visualization of complex cellular and sub-cellular processes that serve to improve early drug discovery and basic research. The EIDAQ™ 100 HTM cellular imaging and analysis system facilitates accurate investigation of cellular and sub-cellular processes in real time. Here we describe how image analysis using the system is applied to quantify intracellular translocation of the transcription factor NF-kB. Validated results of nuclear-cytoplasmic translocation are presented with representative fluorescent images and dose response curves.
Background and significance
Due to their central role in cellular life, signal transduction pathways and their component proteins are of great interest in drug discovery. Cell signal transduction pathways frequently activate the transcription of specific genes that co-ordinate programmed responses such as the initiation of cell division, exocytosis, differentiation, and apoptosis. Activation of certain transcription factors lead to the translocation of molecules from a cell's cytoplasm to its nucleus. Once in the nucleus, these molecules bind to regulatory sequences in nuclear DNA. These molecules may be fluorescently labeled using antibodies, or by expression with a GFP fusion protein. After labeling, activation can be measured by the relative movement of the fluorescent label within the cell, from the cytoplasm to the nucleus.
Current plate readers, which rely on overall measurements of fluorescent intensity, cannot quantify such intracellular translocation, since it is not accompanied by a change in overall fluorescence intensity but instead by shifts of fluorescence distribution between different intracellular compartments. We have developed a simple method of directly quantifying and analyzing such translocation in an accurate and rapid manner using the EIDAQ 100 HTM system for image analysis.
One specific family of nuclear signal transduction factors, known as kappa B (NF-kB), is activated by multiple signal transduction pathways. Proteins in the
NF-kB pathway are well-recognized targets for the development of anti-inflammatory drugs, and inhibitors of NF-kB translocation are believed to have therapeutic potential for the treatment of certain cancers. Following activation, the protein subunit NF-kB p65 translocates from the cytoplasm into the nucleus, where it binds to specific regulatory sequences that initiate transcription. We have analyzed and quantified this translocation using high-throughput microscopy.
Experimental methods
Human cervical carcinoma cells (HeLa) were grown in a monolayer and treated with tumor necrosis factor alpha (TNF-a). The movement of fluorescently labeled proteins between the cell cytoplasm and nucleus was monitored by measuring the distribution of fluorescence in defined sub-cellular compartments. The EIDAQ 100 automatically imaged populations of cells, defined nucleus and cytoplasm, measured the amount of fluorescent signal in each, and then quantified the proportion of labeled protein in each cell compartment as a measure of cellular response.
Specifically, NF-kB distribution in HeLa cells was tracked with Alexa 488 (green emission) conjugated to polyclonal p65 antibodies while nuclei were detected through Hoechst 33243 (blue emission). Q3DM's proprietary metric FLIN (fractional localized intensity in nucleus) was used to quantify the degree of translocation. In another experimental system, human synovial fibroblasts were treated with a combination of cytokines in a dose response experiment.
 
Figure 2: Distribution of the metric FLIN among cells in the well. FLIN is defined as the fractional localized intensity in the nucleus. As a ratio measurement, this metric is independent of cell to cell variations in signal intensity. Higher values of FLIN are expressed by a shift in the histogram peak to the right, and are indicative of nuclear translocation taking place. In the experiments described here, stimulation caused a shift to higher FLIN values.
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Experimental results
A clear shift in fluorescence from the cytoplasm to the nucleus was observed in response to stimulation (Figures 2 and 3). Figure 2 shows two representative histograms of FLIN measurements taken from distinct populations of cells before and after activation.
The mean FLIN response is generally reported on a well-by-well basis to measure dose response. Representative data are shown in Figure 3. In this experiment, the NF-kB induction by interleukin 1 beta (IL-1b) in synovial fibroblasts is determined using FLIN. The resulting EC50 value of 0.3 nM is consistent with values reported in the literature.
Figure 3: Concentration of IL-1b (nM). Four replicate wells are shown for each of an 8-point dose response curve demonstrating nuclear translocation of NF-kB induced by interleukin 1 beta in synovial fibroblasts. EC50 0.3 nM (mean std error 0.0002.)
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Conclusion
The EIDAQ 100 was used to generate data that measured protein translocation from cytoplasm to nucleus in several different cell types. Q3DM's proprietary image analysis technologies enabled the accurate monitoring of fluorescence signal variations across multiple sub-cellular compartments, and measurement protein translocation in response to pharmacological manipulation. Though the data shown here are specific to NF-kB response, the same techniques are used to observe many other translocations. These results are clear examples of the powerful possibilities of advanced image analysis to directly visualize complex intracellular events.
About the author
Paul Hoeprich is Chief Operating Officer of Q3DM. Q3DM's products, which can be used individually or in concert, collect and analyze data rapidly and accurately. The EIDAQ 100 HTM system enables the direct visualization of cellular processes, the accurate detection of rare events, and the rapid gathering of statistically reliable datasets.
More information about the EIDAQ 100 HTM and its applications is available from:
Q3DM, San Diego, CA. 866-336-0336; q3dm.com
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