Browsing by Subject "Mitosis"

Now showing 1 - 4 of 4
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    APOBEC3 subcellular localization and genomic editing
    (2012-10) Lackey, Lela Lynn
    The APOBEC proteins are DNA cytosine deaminases with roles in immunity, including retroviral restriction and antibody maturation. Their activity theoretically makes them a danger to genomic DNA. The subfamily of APOBEC3 genes has expanded to included seven different genes in primates. Based on their subcellular localization, only a subset of these APOBEC3 proteins have access to genomic DNA, and may potentially deaminate genomic DNA. Although the nuclear envelope breaks down during mitosis, I demonstrate that none of the APOBEC3s gain access to genomic DNA during cell division. However, APOBEC3B and other APOBEC3 proteins have access to genomic DNA during interphase. I also show that APOBEC3B is actively imported into the nuclear compartment. In general, APOBEC3 nuclear localization and deaminase activity correlate with ability to affect cell cycle progression, implicating these APOBEC3s in deamination of genomic DNA. In support of these conclusions, I observed cell death, activation of the DNA damage response and DNA mutations after ectopic expression of APOBEC3A and APOBEC3B. Moreover, endogenous APOBEC3B is demonstrably nuclear and active in breast cancer cell lines where it causes genomic deamination and mutations. Endogenous APOBEC3B is highly expressed in more than half of human breast cancers compared to normal breast tissues. In addition, sequences from tumors with higher levels of APOBEC3B have more mutations, and these mutations match APOBEC3B's deamination signature. My thesis work further defines the subcellular localization of the APOBEC3 family and provides the first evidence that APOBEC3B is involved in a human cancer type.
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    Centromere Mechanical Maturation And Mitotic Fidelity During Mammalian Cell Mitosis
    (2018-05) Harasymiw, Lauren
    Mitotic fidelity, a cell’s ability to accurately and reliably replicate its genome during cell division, is frequently disrupted in cancer and may significantly impact disease course by selecting for tumor promoting mutations. Thus, the ability to target the mitotic fidelity of cancer cells could be an important therapeutic approach to reducing cancer-associated morbidity and mortality. The mechanical function of the centromere, a specialized region of the chromosome that interacts with the mitotic spindle, is likely to be intimately connected to mitotic fidelity. During mitosis, mechanical tension develops across the centromere as a result of spindle-based forces. This is important because tension-based force signaling may play a critical role in preventing chromosome segregation errors during mitosis that result from incorrect attachments between the mitotic spindle and the chromosomes. However, the role of the centromere in establishing the magnitude and cell-cycle specificity of tension signaling, and its effect on chromosome segregation outcomes, remains unknown. Further, centromere mechanics have not been quantitatively characterized in vivo for mitotic human cells, and so the effect of disease states such as cancer on their function is largely unknown. We combined quantitative, biophysical microscopy with computational analysis in order to elucidate the mechanics of the centromere in unperturbed, mitotic mammalian cells. Our approach revealed that the mechanics of the mammalian centromere mature with a signature pattern during mitotic progression. Importantly, this maturation leads to amplified centromere tension specifically at metaphase. Thus, centromere mechanical maturation provides a positive feedback mechanism to increase the centromere’s tension signal during mitotic progression. Further, we found that a disruption in centromere mechanical maturation led to diminished tension at metaphase in cancer cells, and that this disruption increased in severity with increasing chromosome number. Strikingly, in cells with disrupted centromere mechanical maturation, the frequency of tension-based chromosome attachment errors was elevated, and these errors were more likely to persist into segregation defects at telophase. Thus, we reveal a novel role for the centromere in regulating tension during mitosis, and demonstrate a direct link between aneuploidy, centromere mechanics, and chromosome mis-segregation.
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    Modeling and analysis of microtubule-mediated chromosome transport during mitosis.
    (2008-08) Gardner, Melissa Klein
    During mitosis, dynamic arrays of kinetochore-associated microtubules (kMTs) and molecular motors are organized into a mitotic spindle that serves to accurately segregate chromosomes into daughter cells. Understanding the dynamics and organization of mitotic spindle components could ultimately apply to clinical applications, such as in cancer treatment, because of the centrality of the mitotic spindle in mediating cell mitosis. Computer simulation can provide a bridge between mitotic spindle phenotypes and the individual dynamic spindle components that produce these phenotypes. I have found that by simulating the dynamics of kMTs mediating chromosome segregation during mitosis, it is possible to build a model for their regulation which results in specific predictions for molecular functions within the mitotic spindle. Specifically, by simulating the dynamics of molecular motors and chromosomes relative to kMT dynamics, and by comparing these simulations to experiments using fluorescent proteins and cryo-electron tomography, major mechanisms regulating proper chromosome congression in yeast have been uncovered. I have shown (1) that tension generated via the stretch of chromosomes between sister kinetochores is important in regulating the proper separation of sister kinetochores during metaphase, and (2) that a molecular motor, specifically the Kinesin-5 molecular motor Cin8p, is responsible for mediating a gradient in kMT catastrophe frequency that is required for proper chromosome congression. Dynamic microtubule plus-ends are responsible for the proper segregation of chromosomes during mitosis, as well as for other critical cellular functions. By performing molecular-level Monte Carlo simulations of microtubule assembly and comparing these simulations to in vitro measurements of microtubule assembly, I have found that microtubule assembly at the nanoscale is highly variable. This result supports a model for microtubule dynamic instability in which there is exists a substantial and dynamic GTP-cap during microtubule assembly that is critical for microtubule growth.
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    A Quantitative Exploration of Tension Sensing at Metaphase in Budding Yeast
    (2018-07) Mukherjee, Soumya
    During mitosis, motors associate with microtubules to exert forces that push spindle poles apart, thus establishing a mitotic spindle. These pushing forces in turn cause tension in the chromatin that connects oppositely attached sister chromosomes. This tension has been hypothesized to act as a mechanical signal that allows the cell to detect chromosome attachment errors during mitosis. However, the magnitude of changes in tension that could be detected by the cell to initiate an error correction response during metaphase has not been measured, and the underlying mechanics of tension based error detection and error correction remains unknown. In this study, we generated a gradient in tension over multiple isogenic budding yeast cell lines by genetically altering the magnitude of motor-based spindle forces. This allowed us, for the first time, to quantitatively elucidate the mechanics of tension based error detection pathway in mitosis. We found that a decreasing gradient in tension led to an increasing gradient in rates of kinetochore detachment and anaphase chromosome mis-segregration, with a corresponding gradient in metaphase times. Further, these tension-based cellular response gradients were abrogated in the absence of key error-correction pathway proteins. The underlying mechanism involves as increasing gradient in the degree of phosphorylation of proteins, comprising the load-bearing component of the kinetochore-microtubule interface, in response to a decreasing gradient in the magnitude of tension. We conclude that the cell is exquisitely tuned to the magnitude of tension as a signal to detect potential chromosome segregation errors during mitosis.