INVESTIGATING THE CAUSES AND CONSEQUENCES OF PROTEIN NUCLEOCYTOPLASMIC PARTITIONING IN VERTEBRATE DEVELOPMENT

Abstract

Eukaryotic cells consist of two main compartments, the cytoplasm and the nucleus. The partition is well defined; the nucleus hosts the genome, and mRNA translation into proteins occurs exclusively in the cytoplasm. The distribution of proteins between these compartments regulates many eukaryotic cellular processes, and its defects can result in developmental defects or cancer. One standard estimation for nucleocytoplasmic protein partitioning is its corresponding volume ratio. The ratio is typically 1/10 and largely deviates from this canonical value in disease phenotypes. Despite an important role and a strong implication for diseases, the understanding of which proteins localize in the nucleus and how this varies in different cell types and conditions remain largely unknown. This thesis investigates nucleocytoplasmic protein partitioning using Xenopus laevis embryos. Here, the nuclear-to-cytoplasmic volume ratio naturally drops by ~four orders of magnitude after fertilization, followed by a rapid doubling rate as development progresses without transcriptions. Past studies have observed that after 12 rapid cell divisions, the embryo senses a critical nuclear-to-cytoplasmic volume ratio to trigger first genes transcription. How the nuclear proteome changes throughout this period are poorly understood. I hypothesized that the proteome partition changes while the nuclear volume exponentially increases, subsequently controlling protein access to the genome and altering transcription timing. Here, I followed the questions of how protein partitioning changes in early development and how this change impacts developmental progression. During my thesis, I developed an imaging method to quantify the changes in the nuclear volume and a quantitative proteomics workflow to quantify protein abundance in frog embryos and oocytes. Combining these techniques, I studied protein nucleocytoplasmic partitioning in frog embryos as a function of developmental progression. I find a sequential nuclear entry of proteins into embryonic nuclei, such as transcription factors and RNA polymerases. The measured entry times of proteins into the nucleus correlate with the onset of downstream nuclear functions, like gene transcription. Finally, proteins affinity to importin, a nuclear transport receptor, largely determined this timing mechanism. The study concludes with a model in which early embryos organize themselves via encoding the timing of gene expression in the biochemical affinities to importin.