CycleTrak: A novel system for the semi-automated analysis of cell cycle dynamics

AbstractCell proliferation is crucial to tissue growth and form during embryogenesis, yet dynamic tracking of cell cycle progression and cell position presents a challenging roadblock. We have developed a fluorescent cell cycle indicator and single cell analysis method, called CycleTrak, which allows for better spatiotemporal resolution and quantification of cell cycle phase and cell position than current methods. Our method was developed on the basis of the existing Fucci method. CycleTrak uses a single lentiviral vector that integrates mKO2-hCdt1 (30/120), and a nuclear-localized eGFP reporter. The single vector and nuclear localized fluorescence signals simplify delivery into cells and allow for rapid, automated cell tracking and cell cycle phase readout in single and subpopulations of cells. We validated CycleTrak performance in metastatic melanoma cells and identified novel cell cycle dynamics in vitro and in vivo after transplantation and 3D confocal time-lapse imaging in a living chick embryo

Single -Molecule and Ensemble Fluorescence Studies of Single -Stranded DNA and Replication Proteins

108 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2006.DNA replication is a complex process involving many proteins that must be performed for a cell to proliferate. In this work, we will study three members of the replication system: single-stranded DNA, single-stranded DNA binding proteins, and the sliding clamp and clamp loader proteins. Fluorescence studies have been widely used to study the properties of biomolecules and here we will use both Fluorescence Polarization Anisotropy (FPA) and Fluorescence Resonance Energy Transfer (FRET) to study these biomolecules. First, we present a method by which single molecule FRET measurements can be used to determine the persistence length of single-stranded DNA. Our results indicate that the persistence length decreases from 3.0nm to 1.5nm as the salt concentration increases from 50mM NaCl to 2M NaCl. Furthermore, we use fluoresce anisotropy and FRET measurements in ensemble studies to examine properties of single stranded DNA binding proteins from archaeal organisms. Our results will show that the Archaea have exploited the single stranded DNA binding fold motif unlike the other domains of life to create a wide variety of proteins that have different binding properties leading to the probable conclusion that many of the proteins area a result of gene duplication and recombination events. Finally, we will examine the properties of the clamp loader protein as it loads a fluorescently labeled clamp to DNA on both the ensemble and single molecule measurements. Our findings show that loading the clamp is a multi-step process with different conformational states that can be observed for the first time at a single molecule level. We also will examine mutations of the clamp loader protein to bring further insight into the not fully understood Methanosarcina acetivorans clamp loader.U of I OnlyRestricted to the U of I community idenfinitely during batch ingest of legacy ETD

Evidence for dynamic rearrangements but lack of fate or position

Neural crest cells emerge from the dorsal trunk neural tube and migrate ventrally to colonize neuronal derivatives, as well as dorsolaterally to form melanocytes. Here, we test whether different dorsoventral levels in the neural tube have similar or differential ability to contribute to neural crest cells and their derivatives. To this end, we precisely labeled neural tube precursors at specific dorsoventral levels of the chick neural tube using fluorescent dyes and a photoconvertible fluorescent protein. Neural tube and neural crest cell dynamics were then examined in vivo and in slice culture using 2-photon and confocal time-lapse imaging. The results show that neural crest precursors undergo dynamic rearrangements within the neuroepithelium, yielding an overall ventral to dorsal movement toward the midline of the neural tube, where they exit in a stochastic manner to populate multiple derivatives. No differences were noted in the ability of precursors from different dorsoventral levels of the neural tube to contribute to neural crest derivatives, with the exception of sympathetic ganglia, which appeared to be "filled" by the first population to emigrate. Rather than restricted developmental potential, however, this is likely due to a matter of timing

Development of multilineage adult hematopoiesis in the zebrafish with a runx1 truncation mutation

Runx1 is required for the emergence of hematopoietic stem cells (HSCs) from hemogenic endothelium during embryogenesis. However, its role in the generation and maintenance of HSCs during adult hematopoiesis remains uncertain. Here, we present analysis of a zebrafish mutant line carrying a truncation mutation, W84X, in runx1. The runx1W84X/W84X embryos showed blockage in the initiation of definitive hematopoiesis, but some embryos were able to recover from a larval “bloodless” phase and develop to fertile adults with multilineage hematopoiesis. Using cd41–green fluorescent protein transgenic zebrafish and lineage tracing, we demonstrated that the runx1W84X/W84X embryos developed cd41+ HSCs in the aorta-gonad-mesonephros region, which later migrated to the kidney, the site of adult hematopoiesis. Overall, our data suggest that in zebrafish adult HSCs can be formed without an intact runx1