Date Published: April 22, 2019
Publisher: Public Library of Science
Author(s): Predrag Jevtić, Richik N. Mukherjee, Pan Chen, Daniel L. Levy, Michael Klymkowsky.
More than just a container for DNA, the nuclear envelope carries out a wide variety of critical and highly regulated cellular functions. One of these functions is nuclear import, and in this study we investigate how altering the levels of nuclear transport factors impacts developmental progression and organismal size. During early Xenopus laevis embryogenesis, the timing of a key developmental event, the midblastula transition (MBT), is sensitive to nuclear import factor levels. How might altering nuclear import factors and MBT timing in the early embryo affect downstream development of the organism? We microinjected X. laevis two-cell embryos with mRNA to increase levels of importin α or NTF2, resulting in differential amounts of nuclear import factors in the two halves of the embryo. Compared to controls, these embryos exhibited delayed gastrulation, curved neural plates, and bent tadpoles with different sized eyes. Furthermore, embryos microinjected with NTF2 developed into smaller froglets compared to control microinjected embryos. We propose that altering nuclear import factors and nuclear size affects MBT timing, cell size, and cell number, subsequently disrupting later development. Thus, altering nuclear import factors early in development can affect function and size at the organismal level.
The nucleus plays many important roles in the cell. The nuclear envelope (NE) is composed of a double lipid bilayer. The outer nuclear membrane is continuous with the endoplasmic reticulum while the inner nuclear membrane is lined and supported by the nuclear lamina, composed of a meshwork of lamin intermediate filaments and lamin-associated proteins [1, 2]. Nuclear pore complexes (NPC) that mediate nucleocytoplasmic transport are inserted into the NE at sites where the inner and outer nuclear membranes fuse [2–5]. After mitosis and nuclear reassembly, lamins are imported into the nucleus along with other proteins containing nuclear localization signals (NLS). Classical nuclear import is mediated by importin α/β karyopherins, which bind NLS-containing proteins and ferry them across the NPC and into the nucleus. Within the nucleus, Ran in its GTP-bound state binds to importin β thereby releasing NLS cargos. Another key player in this process is NTF2, a dedicated nuclear import factor for Ran [6–10]. Associated with the NPC, NTF2 has been shown to reduce import of large cargos [11–13]. While nuclear import is critical for a wide variety of cell functions [14, 15], in this study we investigate how altering the levels of nuclear import factors impacts developmental progression and organismal size.
We microinjected one blastomere of two-cell stage embryos with mRNA encoding nuclear import factors along with fluorescent dextran to trace cells that received the mRNA (Fig 1A). One important advantage of this approach is that the uninjected half of the embryo serves as an internal control, thus facilitating the observation of any developmental differences between the two halves of the embryo. In some cases we differentially microinjected the two blastomeres to maximize potential nuclear import differences in the two halves (i.e. importin α/lamin B3 in one half and NTF2 in the other half). For control experiments, embryos were microinjected with mRNA encoding either GFP or histone H2B-GFP. Embryos were microinjected with mRNA amounts previously shown to maximally alter nuclear size in vivo (S1 Fig) [12, 13, 24]. Because importin α and NTF2 are essential, we felt that overexpression of these factors would be more informative than knockdown, which might have had pleiotropic effects due to reduced cell and embryo viability.