BackgroundThe translocation or shuttling of proteins between the nucleus and cytoplasm (nucleocytoplasmic transport [NCPT]) is often a rapid event following stimulation with growth factors or in response to stress or other experimental manipulations. Commonly used methods to separate nuclei from cytoplasm employ lengthy steps such as density gradient centrifugation which exposes cells to non-physiological hyperosmotic conditions for extended time periods resulting in varying degrees of leakage between the nucleus and cytoplasm. To help maintain and quantify nuclear:cytoplasmic ratios of proteins, agents such as leptomycin B have been employed to be able to better analyze NCPT by inhibiting nuclear export. To track NCPT in the absence of these experimental manipulations that could introduce unknown artefacts, we have developed a rapid method that appears to produce pure nuclear and cytoplasmic fractions, suitable for obtaining accurate estimates of the nuclear:cytoplasmic ratios of proteins known to undergo NCPT.FindingsWe have developed a Rapid, Efficient And Practical (REAP) method for subcellular fractionation of primary and transformed human cells in culture. The REAP method is a two minute non-ionic detergent-based purification technique requiring only a table top centrifuge, micro-pipette and micro-centrifuge tubes. This inexpensive method has proven to efficiently separate nuclear from cytoplasmic proteins as estimated by no detectible cross-contamination of the nucleoporin and lamin A nuclear markers or the pyruvate kinase and tubulin cytoplasmic markers. REAP fractions also mirrored TNFα induced NF-κB NCPT observed in parallel by indirect immunofluorescence.ConclusionsThis method drastically reduces the time needed for subcellular fractionation, eliminates detectable protein degradation and maintains protein interactions. The simplicity, brevity and efficiency of this procedure allows for tracking ephemeral changes in subcellular relocalization of proteins while maintaining protein integrity and protein complex interactions.

Cell fate choice and commitment of multipotent progenitor cells to a differentiated lineage requires broad changes of their gene expression profile. But how progenitor cells overcome the stability of their gene expression configuration (attractor) to exit the attractor in one direction remains elusive. Here we show that commitment of blood progenitor cells to the erythroid or myeloid lineage is preceded by the destabilization of their high-dimensional attractor state, such that differentiating cells undergo a critical state transition. Single-cell resolution analysis of gene expression in populations of differentiating cells affords a new quantitative index for predicting critical transitions in a high-dimensional state space based on decrease of correlation between cells and concomitant increase of correlation between genes as cells approach a tipping point. The detection of “rebellious cells” that enter the fate opposite to the one intended corroborates the model of preceding destabilization of a progenitor attractor. Thus, early warning signals associated with critical transitions can be detected in statistical ensembles of high-dimensional systems, offering a formal theory-based approach for analyzing single-cell molecular profiles that goes beyond current computational pattern recognition, does not require knowledge of specific pathways, and could be used to predict impending major shifts in development and disease.

Cell fate choice and commitment of multipotent progenitor cells to a differentiated lineage requires broad changes of their gene expression profile. But how progenitor cells overcome the stability of their gene expression configuration (attractor) to exit the attractor in one direction remains elusive. Here we show that commitment of blood progenitor cells to the erythroid or myeloid lineage is preceded by the destabilization of their high-dimensional attractor state, such that differentiating cells undergo a critical state transition. Single-cell resolution analysis of gene expression in populations of differentiating cells affords a new quantitative index for predicting critical transitions in a high-dimensional state space based on decrease of correlation between cells and concomitant increase of correlation between genes as cells approach a tipping point. The detection of "rebellious cells" that enter the fate opposite to the one intended corroborates the model of preceding destabilization of a progenitor attractor. Thus, early warning signals associated with critical transitions can be detected in statistical ensembles of high-dimensional systems, offering a formal theorybased approach for analyzing single-cell molecular profiles that goes beyond current computational pattern recognition, does not require knowledge of specific pathways, and could be used to predict impending major shifts in development and disease. Author SummaryA certain type of multipotent progenitor cell of the blood can commit to either the white (myeloid) or the red (erythroid) blood cell lineage, thus making a discrete binary cell fate decision. To test a theory on fundamental principles of cell fate dynamics (as opposed to the usually studied molecular mechanisms), we monitored such a fate decision in vitro using single-cell resolution gene expression analysis. We found that blood progenitor cells undergoing a fate decision to commit to either lineage after treatment with fate-determining cytokines, according to theory, first destabilized their original state. Cell states hereby PLOS Biology |

The INhibitor of Growth 1 (ING1) is stoichiometric member of histone deacetylase (HDAC) complexes and functions as an epigenetic regulator and a type II tumor suppressor. It impacts cell growth, aging, apoptosis, and DNA repair, by affecting chromatin conformation and gene expression. Down regulation and mislocalization of ING1 have been reported in diverse tumor types and Ser/Thr phosphorylation has been implicated in both of these processes. Here we demonstrate that both in vitro and in vivo, the tyrosine kinase Src is able to physically associate with, and phosphorylate ING1, which results in a nuclear to cytoplasmic relocalization of ING1 in cells and a decrease of ING1 stability. Functionally, Src antagonizes the ability of ING1 to induce apoptosis, most likely through relocalization of ING1 and down regulation of ING1 levels. These effects were due to both kinase-dependent and kinase-independent properties of Src, and were most apparent at elevated levels of Src expression. These findings suggest that Src may play a major role in regulating ING1 levels during tumorigenesis in those cancers in which high levels of Src expression or activity are present. These data represent the first report of tyrosine kinase-mediated regulation of ING1 levels and suggest that kinase activation can impact chromatin structure through the ING1 epigenetic regulator.