431. Importin proteins and their role in maintenance of the stem cell state

Abstract

The importin (IMP) family of proteins mediates transport into the nucleus for many proteins larger than 40 kD. Through differential cargo recognition, IMPs regulate cellular events by controlling nuclear access of transcription factors and chromatin remodelling agents. During spermatogenesis, many IMPs change expression and localisation in a manner concordant with specific stages of spermatogenic development. To assess the potential role of IMPs in the transition between the stem cell and subsequent differentiation, we undertook analysis of the expression and subcellular localisation of several key murine IMPs in both pluripotent embryonic stem cells (mESCs) and embryoid bodies (EBs). All of the IMPs analysed (IMPα2, 3, 4, IMPβ1 and IMP5) were detected in undifferentiated mESCs by immunofluorescence, and each exhibited distinctive nucleocytoplasmic distribution patterns. Subcellular localisation of most IMPs altered after 10 days of mESC differentiation as EBs. This was paralleled by changes in the mRNA levels of IMPα1–4, IMPα6, IMPβ1 and IMP5, concomitant with alterations in the expression level of the pluripotency marker, Oct3/4. Reducing IMP-dependent nuclear import through overexpression of specific dominant-negative IMP constructs led to alterations in import or production of Oct3/4 protein, depending on the specific IMP. These findings indicate that IMPs may play very specific but distinct roles in cell fate choice between maintenance of pluri/multipotency and commitment to differentiation in ESCs and potentially in spermatogenesis or other organs that contain stem cells.