Importin-β Proteins Expression Modulation in Embryonic Stem Cells and Embryonic Fibroblasts of Mouse
Percival P. Sangel*
Institute of Animal Science, College of Agriculture and Food Science
University of the Philippines Los Baños, College 4031 Laguna, Philippines
Importin-β proteins are transport proteins important in the shuttling of cargo proteins by binding to either nuclear localization signal (NLS) or nuclear export signal (NES). This study investigated the in vitro expression modulation of selected importin-βs. Specifically, this study characterized the culture behavior of mouse embryonic stem cells after knockdown of selected importin-β proteins like Cse1L, IPO7, KPNB1, RanBP16, RanBP17, or XPO4. Also, this study assessed the effects of overexpressing RanBP17 or IPO7 during cellular reprogramming of mouse embryonic fibroblasts (MEFs). Results showed that Cse1L and KPNB1 are essential for the viability of mouse embryonic stem cells since knockdown of either one of these proteins resulted in the death of mouse embryonic stem cells. Meanwhile, the growth characteristics of RanBP17, XPO4, IPO7, or RanBP16 knockdown mouse embryonic stem cells were comparable with the control. Aside from round colonies, the appearance of flat cells and spreading growth characteristics in some colonies were observed, which indicated early signs of differentiation. On the other hand, the number of colonies with overexpressed Oct4, Sox2, Klf4, cMYC (OSKM) + RanBP17, or OSKM+ IPO7 was comparable to OSKM+Flag or OSKM (controls). This suggests that RanBP17 or IPO7 has limited application in the generation of induced pluripotent stem cells.
Many intricate trafficking pathways involve the transport of various substances in a living cell. In the case of the eukaryotic cell, its biomembrane creates compartmentalization, giving rise to the different organelles and affecting the transport of all cargo molecules. Central to these transport processes is the nucleocytoplasmic transport system. This regulates the shuttle of the different cargo proteins in and out of the nucleus as they pass through the nuclear pore complex (NPC) of the nuclear membrane. The major key players mediating most cargo protein transport via this system are small proteins collectively called importins (alpha and beta). Between the two importins, there are more reports available for importin-α contributing to a better understanding of its roles as a transporter. The previous works of Sekimoto et al. (1997) lead to the identification of the different subtypes of importin-α. This opened a research platform to identify and analyze respective NLS in different cargo proteins and elucidate the specific mechanisms involved in their nuclear transport. The study of Yasuhara et al. (2007) showed that functional switching of the importin-α subtype may lead to neural lineage differentiation of embryonic stem cells. Their study also demonstrated significance in the regulation of the activity of importin-α subtypes, which influence mostly the transport of transcription factors as protein cargoes. Moreover, the same report indicated the role of importin-αs as indicators of cell lineage determination. . . . read more
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