Investigation of the mechanisms and functional significance of nuclear size regulation in Xenopus laevis extracts and early X. laevis embryos

Size varies widely between all eukaryotic species. A little is known about how organelle size is regulated and how the sizes and/or numbers of organelles and other subcellular structures are regulated relative to cell size, a phenomenon referred to as scaling. Early Xenopus laevis embryogenesis is a robust system for investigating nuclear scaling and the functional significance of nuclear size during development and the mechanisms of developmental timing. The first major developmental transition is the midblastula transition (MBT), when zygotic transcription begins and cell cycles elongate. Fertilization is followed by a series of rapid cell divisions with concomitant reductions in cell size and it has long been recognized that the N/C volume ratio changes dramatically during early Xenopus development. Rather than examining the role of DNA amount in MBT timing, we investigated if MBT timing is regulated by nuclear size and developmental changes in the N/C volume ratio. We found that altering N/C volume ratio in early embryos alters the onset of zygotic transcription end lengthening of cell cycle at the MBT. It has been previously shown in Xenopus that rates of nuclear import influence nuclear size, with the levels of importin ? and nuclear transport factor 2 (NTF2) being important and both factors modulating lamin B3 import. Here we further investigated how lamins and NTF2 affect nuclear size by studying nuclei in Xenopus laevis egg and embryo extracts and in early stage X. laevis embryos. We found that nuclear growth and size are sensitive to the levels of any type of nuclear lamins, with low and high concentrations increasing and decreasing nuclear size, respectively. Next we investigated how NTF2 concentrations and mutants affect nuclear size. Using a mutant version of NTF2 that is defective for Ran binding, we found that the ability of NTF2 to inhibit nuclear growth and import of large cargos in X. laevis egg extract largely depends on its binding to Ran at the nuclear pore complex. This new functional and mechanistic information about nuclear size control will facilitate future studies into the functional significance of nuclear size during development, differentiation, and carcinogenesis.