Date of Award

2014

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Graduate Group

Cell & Molecular Biology

First Advisor

Mitchell J. Weiss

Abstract

Red blood cells (erythrocytes) deliver oxygen to all tissues of the body. Defects in red blood cell production (erythropoiesis) can cause disease. Mammalian erythropoiesis culminates in enucleation, an incompletely understood process that entails the physical separation of the nucleus and cytoplasm. The work in this thesis investigated the role of a previously uncharacterized protein named Trim58 in erythropoiesis.

Human genetic studies suggested that TRIM58 played an important role in erythroid development. In humans and mice, Trim58 expression was found to be restricted to red blood cell precursors during late stage maturation. In fact, murine Trim58 was upregulated just prior to enucleation. Using short hairpin RNAs, Trim58 expression was inhibited in cultured murine erythroblasts. Through a variety of analyses, it was demonstrated that Trim58 is dispensable for early erythroid maturation. However, Trim58 knockdown impaired movement of the nucleus, thereby inhibiting enucleation.

Trim58 is a member of the tripartite motif-containing family of proteins, many of which function as E3 ubiquitin ligases that can facilitate protein degradation. Protein interaction studies demonstrated that Trim58 bound directly to the molecular motor protein complex dynein. Consistent with its putative role as an E3 ubiquitin ligase, ectopic Trim58 expression in HeLa cells caused dynein degradation in a proteasome-dependent fashion. Furthermore, dynein loss and efficient enucleation were coincident and dependent upon Trim58 induction during erythroid culture maturation.

Dynein mediates unidirectional nuclear movement toward the microtubule organizing center. Erythroid enucleation requires nuclear movement in the opposite direction. Hence, Trim58-mediated dynein degradation may be responsible for nuclear movement during enucleation. Our findings identify Trim58 as the first erythroid-specific protein that regulates this process. More broadly, regulated proteolysis represents a previously unappreciated mode of regulation for dynein, which is critical for many cellular processes.

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