The midbody (MB) is a proteinaceous complex formed between the two daughter cells during cell division and is required for the final cell separation event in late cytokinesis. After cell division, the post-mitotic midbody, or midbody derivative (MBd), can be retained and accumulated in a subpopulation of cancer cells and stem cells, but not in normal diploid differentiated cells. However, the mechanisms by which MBds accumulate and function are unclear. Based on this, I hypothesize that the MBd is degraded by autophagy after cell division in normal diploid differentiated cells, whereas non-differentiated cells have low autophagic activity and would accumulate MBds. Indeed, I found this to be the case. MBd degradation occurred soon after cytokinesis in differentiated cells that possess high autophagic activity. Specifically, I found MBd degradation to be mediated by binding of the autophagy receptor, NBR1, to the MB protein Cep55. Moreover, by performing proteomic analysis of NBR1 interactions I found additional MB-localized proteins that are potential substrates for NBR1. In contrast to differentiated cells, stem and cancer cells have low autophagic activity thus MBds evade autophagosome encapsulation and accumulate. To examine whether MBds can define the differentiation status of a cell, we depleted NBR1 from differentiated fibroblasts causing an increase in MBd number. Strikingly, under these conditions, reprogramming of fibroblasts to pluripotent stem cells is increased. Equally interestingly, cancer cells with increased MBds have increased in vitro tumorigenicity. In conclusion, this study gives an insight into the fates of post-mitotic midbodies and also suggests a non-cytokinetic role of midbodies in enhancing pluripotency in stem cells and cancer stem cells.

Dissertation (Ph.D.)--University of South Florida, 2007. / Includes vita. Includes bibliographical references. Also available online.

Dissertation (Ph.D.)--University of South Florida, 2005. / Includes vita. Includes bibliographical references. Also available online in PDF format.

Dissertation (Ph.D.)--University of South Florida, 2007. / Title from PDF of title page. Document formatted into pages; contains 87 pages. Includes vita. Includes bibliographical references.

Thesis (Ph.D. in Molecular Biology) -- University of Colorado at Denver and Health Sciences Center, 2005. / Typescript. Includes bibliographical references (leaves 124-136).

Diss. (sammanfattning) Umeå : Umeå universitet, 2010.

Thesis (Ph. D.)--Case Western Reserve University, 2006. / [School of Medicine] Department of Pharmacology. Includes bibliographical references. Available online via OhioLINK's ETD Center.

A complex network of protein filaments collectively known as the cytoskeleton carries out several crucial cellular processes. These functions include, but are not limited to, motility, cell shape, mitosis and organelle trafficking. The cytoskeleton is also highly responsive, allowing the cell to alter its shape in response to its immediate needs and environment. One of the major components of the cytoskeleton is the microtubule network. To refer to the array of micro tubules in the cell as a skeleton is a misnomer. Microtubules, by virtue of their structure and nature, are highly dynamic, continuously growing and shrinking. They also bind a variety of accessory molecules that aid in regulating and directing their dynamic activity. In this way they provide a structural basis for integral cell functions that require rapid assembly and disassembly. In some cases, perturbations of the microtubule network results in structural anomalies that lead to undesirable outcomes for the cell, namely chromosomal missegregation events and instability. The accumulation of these events may induce aneuploidy, which has been a fundamental component of tumorigenesis. This dissertation examines the role of the microtubule cytoskeleton within three distinct contexts. The first chapter investigates the association of the anti-apoptotic protein survivin with the microtubule network and its potential impact upon the cell from interphase to cytokinesis. The second chapter of this dissertation explores a little-studied, microtubule-dense organelle, referred to as the midbody, and the highly orchestrated events that take place within it during cytokinesis. The third and final chapter describes a unique experimental condition that may further our understanding of the interaction between the tumor suppressor p53 and the centrosome in cell cycle regulation and tumorigenesis.

The main focus of my work has been the role of the MRN in the S-phase DNA damage checkpoint. The MRN plays many roles in cellular metabolism; some are checkpoint dependent and some are checkpoint independent. The multiple roles in cellular metabolism complicate study of the role of the MRN in the checkpoint. MRN mutations in budding yeast and mammals may display separation of function. Mechanistically, MRN, along with its cofactor Ctp1, is involved in 5’ resection to create single stranded DNA that is required for both signaling and homologous recombination. However, it is unclear if resection is essential for all of the cellular functions of MRN. Therefore I have made mutations to mimic those in budding yeast and mammals. I found that several alleles of rad32, as well as ctp1Δ, are defective in double-strand break repair and most other functions of the complex but maintain an intact S-phase DNA damage checkpoint. Thus, the MRN S-phase checkpoint role is separate from its Ctp1- and resection-dependent role in double-strand break repair. This observation leads me to conclude that other functions of MRN, possibly its role in replication fork metabolism, are required for S-phase DNA damage checkpoint function. One of the potential roles of Rad32 and the rest of the MRN complex is in sister chromatid exchange. The genetic requirements of sister chromatid exchange have been examined using unequal sister chromatid assays which only are able to assay exchanges that are illegitimate and produce changes in the genome. Most sister chromatid exchange must be equal to maintain genomic integrity and thus far there is no good assay for equal sister chromatid exchange. Yeast cells expressing the human equilibrative nucleoside transporter 1 (hENT1) and the herpes simplex virus thymidine kinase (tk) are able to incorporate exogenous thymidine into their DNA. This strain makes it possible for the fission yeast DNA to be labeled with halogenated thymidine analogs. This strain is being used to design an assay that will label one sister with BrdU and then DNA combing will be used to see equal sister chromatid exchange.

Proper regulation of cellular proliferation is critical for normal development and cancer prevention. Most, if not all, cancers contain mutations in the Rb/E2F pathway, which controls cellular proliferation. Inactivation of the retinoblastoma protein (Rb) can occur through Rb loss, mutation, or inactivation by cellular or viral oncoproteins leading to unrestrained proliferation. This occurs primarily by de-repression and activation of the E2F transcription factors, which promote the transition of cells from the G1to S phase of the cell cycle. In order to protect against loss of growth control, the p53 tumor suppressor is able to induce programmed cell death, or apoptosis, in response to loss of proper Rb cell cycle regulation. E2F1 serves as the primary link between the Rb growth control pathway and the p53 apoptosis pathway. While the pathway(s) linking E2F1 to p53 activation and apoptosis are unclear, it has been proposed that E2F1 activates p53-dependent apoptosis by transactivation of p19ARF leading to inhibition of Mdm2-promoted degradation of p53. We tested this hypothesis, and found that p19ARFis not required for E2F1-induced apoptosis. Instead, we find that expression of E2F1 leads to covalent modifications of p53 that correlate with p53 activation and are required for apoptosis. The observation that E2F1 induces covalent modification of p53 is consistent with the p53 modifications observed following DNA damage. We therefore hypothesized that E2F1 may be activating components of the DNA damage response to activate p53 and kill cells. Consistent with the DNA damage response, we find that E2F1-induced apoptosis is compromised in cells from patients with the related disorders ataxia telangiectasia and Nijmegen breakage syndrome, lacking functional Atm and Nbs1 gene products, respectively. E2F1-induced apoptosis and p53 modification also requires the human checkpoint kinase Chk2, another component of the DNA damage response. We find that the commitment step in E2F1-induced apoptosis is the induction of Chk2. Having found that E2F1 requires DNA damage kinases to induce apoptosis, we next examined events upstream of kinase activation. To this end, we observe relocalization of the DNA damage repair MRN complex (composed of Mre11, Nbs1, and Rad50) to nuclear foci specifically following expression of E2F1. Expression of E2F1 also induces relocalization of the DNA damage recognition proteins γH2AX and 53BP1 to nuclear foci, consistent with the location of these complexes observed following DNA double strand breaks. As a consequence of activating some or all of these DNA damage signaling proteins, expression of E2F1 blocks cell cycle progression in diploid human fibroblasts. The observed block in cell cycle progression is found to be, in part, due to activation of a p21-dependent cell cycle checkpoint. The E7 protein from the oncogenic human papillomavirus (HPV) is able to bind to and inactivate members of the Rb family. HPV infects quiescent, non-cycling cells that lack expression of DNA replication machinery that is essential for replication of the viral genome. By expression of the E7 protein, HPV is able to bypass normal Rb-mediated growth control and induce quiescent cells to enter S phase where the host cell DNA replication enzymes are present for viral replication. We find that expression of E7 can also result in apoptosis that is dependent specifically on E2F1. Additionally, E7-induced apoptosis, like E2F1-induced apoptosis, requires Atm, Nbs1, and Chk2. Expression of E7, like that of E2F1, induces E2F1-dependent covalent modification of p53 that correlates with apoptosis induction. These findings demonstrate that deregulation of the Rb/E2F growth control pathway leads to activation of an apoptosis program with some similarity to the pathways activated by DNA damage. Our observations suggest that E2F1 not only functions as a sensor for deregulation of Rb, but may also play an important role in regulating cellular growth control in response to other oncogenic stimuli.