Physiological polyspermy: Selection of a sperm nucleus for the development of diploid genomes in amphibians.

The union between a sperm and an egg nucleus in egg fertilization is necessary to mix genetic materials to create a new diploid genome for the next generation. In most animals, only one sperm is incorporated into the egg (monospermy), but several animals exhibit physiological polyspermy in which several sperms enter the egg during normal fertilization. However, only one sperm nucleus forms the zygote nucleus with the egg nucleus, even in a polyspermic egg. The cellular and molecular mechanisms involved in the selection of sperm nuclei in the egg cytoplasm have been well investigated in urodele amphibians. The principal sperm nucleus develops a larger sperm aster and contacts the egg nucleus to form a zygote nucleus, whereas other accessory sperm nuclei are unable to approach the egg nucleus. The diploid zygote nucleus induces cleavage and participates in embryonic development, whereas the accessory sperm nuclei undergo pyknosis and degenerate. We propose several models to account for the mechanisms of the selection of one sperm nucleus and the degeneration of accessory sperm nuclei. The roles of physiological polyspermy in animal reproduction are discussed by comparison with other polyspermic species.

Functional characterization of importin α8 as a classical nuclear localization signal receptor.

Importin α8 has recently been identified as an importin α family member based on its primary structure and binding ability to importin ß1 and to several karyophilic proteins. However, there has been no experimental evidence that importin α8 actually functions in the nuclear transport of classical nuclear localization signal (cNLS)-containing cargo. Here, using an in vitro transport assay, we demonstrate that purified recombinant importin α8 can transport SV40T antigen cNLS-containing cargo into the nucleus of HeLa cells, in conjunction with importin ß1. Pull-down assays, followed by mass spectrometry analysis, identified 179 putative importin α8-binding proteins, only 62 of which overlap with those of importin α1, the closest importin α family member. Among the importin α8-binding candidates, we showed that DNA damage-binding protein 2 (DDB2) was actually transported into the nucleus via the importin α8/ß1 pathway. Furthermore, we found that the other subtypes of importin α, which were also identified as importin α8-binding candidates, indeed form heterodimers with importin α8. Notably, we found that these importin α8-containing heterodimers were more stable in the presence of cNLS-substrates than heterodimers containing importin α1. From these findings, we propose that importin α8 functions as a cNLS receptor with distinct cargo specificity, and that heterodimerization by importin α8 is a novel regulatory mode of cNLS binding, in addition to the autoinhibitory regulation by the importin ß binding domain.

Importin alpha subtypes determine differential transcription factor localization in embryonic stem cells maintenance.

We recently demonstrated that the expression of the importin α subtype is switched from α2 to α1 during neural differentiation in mouse embryonic stem cells (ESCs) and that this switching has a major impact on cell differentiation. In this study, we report a cell-fate determination mechanism in which importin α2 negatively regulates the nuclear import of certain transcription factors to maintain ESC properties. The nuclear import of Oct6 and Brn2 was inhibited via the formation of a transport-incompetent complex of the cargo bound to a nuclear localization signal binding site in importin α2. Unless this dominant-negative effect was downregulated upon ESC differentiation, inappropriate cell death was induced. We propose that although certain transcription factors are necessary for differentiation in ESCs, these factors are retained in the cytoplasm by importin α2, thereby preventing transcription factor activity in the nucleus until the cells undergo differentiation.

Protein N-myristoylation is required for cellular morphological changes induced by two formin family proteins, FMNL2 and FMNL3.

The subcellular localization of 13 recently identified N-myristoylated proteins and the effects of overexpression of these proteins on cellular morphology were examined with the aim of understanding the physiological roles of the protein N-myristoylation that occurs on these proteins. Immunofluorescence staining of HEK293T cells transfected with cDNAs coding for the proteins revealed that most of them were associated with the plasma membrane or the membranes of intracellular compartments, and did not affect cellular morphology. However, two proteins, formin-like2 (FMNL2) and formin-like3 (FMNL3), both of them are members of the formin family of proteins, were associated mainly with the plasma membrane and induced significant cellular morphological changes. Inhibition of protein N-myristoylation by replacement of Gly2 with Ala or by the use of N-myristoylation inhibitor significantly inhibited membrane localization and the induction of cellular morphological changes, indicating that protein N-myristoylation plays critical roles in the cellular morphological changes induced by FMNL2 and FMNL3.