Date of Award

Fall 10-1-2021

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Molecular, Cellular, and Developmental Biology

First Advisor

Bahmanyar, Shirin

Abstract

The nucleus is the defining organelle of eukaryotic cells and compartmentalizes the genome from the rest of the cytoplasm. Compartmentalization is achieved by the nuclear envelope (NE), which is comprised of the inner and outer nuclear membranes. At fusion points are nuclear pore complexes (NPCs), which facilitate the transport of macromolecules across the NE. In metazoan cells that undergo open mitosis, the NE and NPCs disassemble to allow for chromosome segregation. With the completion of mitosis, the process must be reversed, and a new nucleus is formed. How thousands of NPCs rapidly assemble to form a functional NE is not well understood. Post-mitotic NPC assembly is initiated by the chromatin binding protein Elys and is followed by the recruitment of fenestrated ER membranes that contain pre-assembled NPC subcomplexes. Transmembrane nucleoporins are present on these ER membranes, but their function specifically during post-mitotic NPC assembly is unknown.In my thesis work I focus on the role of the conserved transmembrane nucleoporin Ndc1. Using the highly stereotypical one- and two-cell stage C. elegans embryo I first characterize a novel deletion allele of ndc1. Embryos lacking ndc1 experience a high degree of embryonic lethality and smaller brood sizes. Fixed and live cell imaging reveals ndc1 nuclei contain lower levels of inner and outer ring nucleoporins at the nuclear rim, suggesting defects in NPC assembly. Furthermore, depletion of ndc1 also decreases nuclear import and in turn nuclear size. Additionally, the reduced nuclear size of embryos lacking ndc1 can be partially rescued by overproduction of ER membranes, suggesting that decreased nuclear import resulting from decreased post-mitotic NPC assembly can be compensated for by providing additional membranes. To specifically investigate Ndc1 during post-mitotic NPC assembly, I next examined NE reformation after the first mitosis of C. elegans embryos. By 3D analysis of electron tomograms of nuclei immediately following NE formation, I observe ndc1 nuclei contain approximately four times fewer holes approximately the diameter of nuclear pores when compared to control. Additionally, initiation of nuclear import is delayed, and total accumulation of a nuclear import reporter is decreased after depletion of ndc1. I go on to show Ndc1 is required for the stable incorporation of the outer ring (scaffold nucleoporins) during this process of assembly. This suggests that Ndc1 is required for post-mitotic NPC assembly. I also find that Ndc1 itself is highly mobile at the NE and serves as a dynamic adapter to drive the self-assembly of NPCs after mitosis. Reduction of the Ndc1 binding partner, Nup53, phenocopies loss of Ndc1, but is more severe. Reduction of both ndc1 and nup53, completely inhibits NE and NPC formation. Taken together this suggests that Ndc1 is required for post-mitotic NPC assembly and that Ndc1 and Nup53, at least in part, independently regulate NPC assembly to ensure the formation of a functional NE. Together, my thesis work has thoroughly characterized the role of Ndc1 in the early C. elegans embryo during post-mitotic NPC assembly and nuclear expansion.

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