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Daniël Lam: A method to quantify nucleo-cytoplasmic shuttling of proto-oncogene SET/I2PP2A

Abstract

SET is a nuclear protein that was initially found as an oncogene in human undifferentiated acute myeloid leukemia, fused to the nuclear porin Nup-214. Previously, we showed that SET associates to the small Rho family GTPase Rac1. Moreover, we found that Rac1 activity in conjunction with SET phosporylation at Ser 9, promotes nuclear exit of SET, driving the protein to he plasma membrane and into the cytoplasm. Finally, we found that the Rac1-induced recruitment of SET from the nucleus to the plasma membrane promotes Rac1-dependent cell migration. This event represents an entirely novel concept in the field of cell migration.

We have now generated fluorescent versions of the SET protein to analyse its nucleo-cytoplasmic shuttling in live cells. Initial studies showed that during steady-state culture, the protein, which is primarily nuclear, can translocate seemingly spontaneous to the cytosol. This translocation is paralleled by the nucleo-cytoplasmic shuttling of activated Rac1, which is also partially residing in the nucleus. We have also seen SET shuttling in spreading as well as dividing cells, although the intrinsic trigger is unknown.

Here we present a method for quantification of exit events in the context of SET nuclear shuttling. In brief the method is based on single cell tracking in combination with quantification of the area occupied by the fluorescent signal of YFP-SET. We have validated the method by quantifying nucleo-cytoplasmic shuttling of wild-type SET and of phospho-mimetic and phospho-deficient mutants. This method will be applicable to other signalling proteins that undergo nucleo-cytoplasmic shuttling as well.

Keywords

Fluorescence microscopy, nucleo-cytoplasmatic shuttling, cell migration, image analysis

Administrative data

Presenting author: Daniël Lam
Organisation: Department of Molecular cell biology, Sanquin Research and Landsteiner Laboratory, AMC, Amsterdam

co-authors: Eloise C. Anthony, Peter L. Hordijk

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