Differential RhoA Dynamics in Migratory and Stationary Cells Measured by FRET and Automated Image Analysis

Genetically-encoded biosensors based on fluorescence resonance energy transfer (FRET) have been widely applied to study the spatiotemporal regulation of molecular activity in live cells with high resolution. The efficient and accurate quantification of the large amount of imaging data from these single-cell FRET measurements demands robust and automated data analysis. However, the nonlinear movement of live cells presents tremendous challenge for this task. Based on image registration of the single-cell movement, we have developed automated image analysis methods to track and quantify the FRET signals within user-defined subcellular regions. In addition, the subcellular pixels were classified according to their associated FRET signals and the dynamics of the clusters analyzed. The results revealed that the EGF-induced reduction of RhoA activity in migratory HeLa cells is significantly less than that in stationary cells. Furthermore, the RhoA activity is polarized in the migratory cells, with the gradient of polarity oriented toward the opposite direction of cell migration. In contrast, there is a lack of consistent preference in RhoA polarity among stationary cells. Therefore, our image analysis methods can provide powerful tools for high-throughput and systematic investigation of the spatiotemporal molecular activities in regulating functions of live cells with their shapes and positions continuously changing in time

Optimization of fluorescence lifetime imaging microscopy (FLIM) for studying the activity of enzymes in live cancer cells

This dissertation describes the process of optimizing a Fluorescence Lifetime Imaging Microscopy (FLIM) system in order to observe the dynamics of enzymes in live cancer cells. The enzyme studied throughout this research is Membrane Type 1 Matrix Metalloproteinase (MT1-MMP) which is a membrane-bound protein principally responsible for degrading extra-cellular matrix (ECM) proteins in the local environment of a migrating cell. However, MT1-MMP has an intricate role in the regulation of the cell’s migration separate from its simple proteolytic functions. In addition, the increased expression of MT1-MMP has been positively correlated with the invasive potential of tumor cells. In spite of the importance of MT1-MMP in understanding a cancer cell’s decision making as it leaves a tumor, very few reports have quantitatively studied the activity of this enzyme in live cells. Even fewer reports have examined the spatiotemporal activity of MT1-MMP in live cells cultured in 3-dimensional settings such as matrices of ECM proteins. These 3-dimensional settings can parallel the environment encountered by metastasizing cells in tissues. Studying live cells in 3-dimensional matrices is crucial for biologically relevant investigations. A cell’s morphology and migratory behavior can vary significantly when comparisons are made between cells cultured on two dimensional substrates and those cultured in 3-dimensional matrices. The purpose of this project was to understand the coordinated functions of MT1-MMP as live cancer cells interact with and move through a 3-dimensional matrix of ECM proteins. Specifically, we are ultimately interested in the spatiotemporal activation patterns of MT1-MMP in live cancer cells in order to build a quantitative (systems-level) model describing MT1-MMP’s role in the cell’s decision making as it is leaves a tumor site

Evolution of molecular structure during the sol-gel processing of lithium niobate and the development of microstructure in alkoxide-derived thin layers

Lithium niobate powders and thin-layers were prepared by sol-gel processing. Lithium niobium ethoxide and lithium niobium methoxyethoxide bimetallic alkoxides were used for both gel and thin-layer formation. Advantages of sol-gel processing were demonstrated by synthesis of LiNb(OCH\sb2CH\sb3)\sb6crystals having the proper cation ratio for stoichiometric LiNbO\sb3. The alkoxide deposition method also allowed for the formation of layers of uniform quality at 650\sp\circC, thereby, avoiding lithium oxide volatility.Lithium and niobium alkoxides reacted immediately to form bimetallic alkoxides. The structure of LiNb(OCH\sb2 CH \sb3)\sb6 consisted of niobium ethoxide octahedra linked by lithium. Studies of hydrolysis indicated a possible gelation mechanisms of cross-linking of individual lithium niobium ethoxide "polymers". Raman studies indicated the precursor alkoxide may be maintained during formation of an amorphous oxide, and considerable long range order developed prior to crystallization. Crystallization was followed by x-ray diffraction, infrared and Raman spectroscopies. At approximately 500\sp\circC, the material crystallized directly into the R3c phase of lithium niobate. Heat-treatment above 650\sp\circC gave changes associated with lithia volatility.Lithium and niobium methoxyethoxides also reacted to produce a bimetallic alkoxide. However, lithium niobium methoxyethoxide could not be crystallized from the parent alcohol. Raman studies indicated significantly less long-range order in the methoxyethoxide specimens after similar heat-treatments. The difference in long-range order was attributed to the similarity in structure of lithium niobium ethoxide with lithium niobate. During heat-treatment, structural relaxation may provide for long range order and hence crystal nuclei. Lithium niobium methoxyethoxide, on the other hand, may require greater structural rearrangement prior to crystallization.Ceramic microstructures depended on solution formulation in addition to heat-treatment conditions. Generally, rapid heating gave dense layers and a larger grain size, than by heating at 10\sp\circC/min. Water and ammonium hydroxide additions aided densification at lower heating rates. The results demonstrate the important relationships between solution chemistry, oligomeric structures, and final microstructures.U of I OnlyETDs are only available to UIUC Users without author permissio

Distinct Pathway of Human T-Cell Leukemia Virus Type 1 Gag Punctum Biogenesis Provides New Insights into Enveloped Virus Assembly

This report describes the results of experiments examining the pathway by which the human retroviral Gag protein is recruited to sites along the inner leaflet of the plasma membrane where Gag punctum biogenesis occurs. In particular, clever and sensitive experimental methods were devised to image in living cells fluorescently labeled Gag protein derivatives from human T-cell leukemia virus type 1 (HTLV-1) and human immunodeficiency virus type 1 (HIV-1) at the plasma membrane. The photoconvertible fluorescent protein mEos2 was strategically utilized, as the fluorescence emission of Gag at the plasma membrane could be differentiated from that of cytosolic Gag. This experimental strategy allowed for the determination of the Gag recruitment pathway into Gag puncta. For HTLV-1 Gag, puncta recruited Gag primarily from the plasma membrane, while HIV-1 Gag was recruited from the cytoplasm. These observations represent the first report of HTLV-1 particle biogenesis and its contrast to that of HIV-1. The observed differences in the Gag recruitment pathways used by HTLV-1 and HIV-1 Gag provide key information that is useful for informing the discovery of novel targets for antiretroviral therapies directed at eliminating virus infectivity and spread.The assembly of virus particles is a crucial aspect of virus spread. For retroviruses, the Gag polyprotein is the key driver for virus particle assembly. In order to produce progeny virus, once Gag is translated, it must translocate from the location in the cytoplasm where it is synthesized to the plasma membrane and form an oligomeric lattice that results in Gag puncta. The biogenesis of mature Gag puncta can trigger the budding process, resulting in virus particle production. While some aspects of the dynamics of Gag oligomerization and particle biogenesis have been observed with human immunodeficiency virus type 1 (HIV-1), the process of Gag punctum biogenesis remains poorly understood, particularly for other retroviruses. Here, we have conducted the most detailed studies thus far on Gag punctum biogenesis for human T-cell leukemia virus type 1 (HTLV-1). Using mEos2 photoconvertible fluorescent proteins and total internal reflection fluorescence microscopy (TIRF), we have found that HTLV-1 Gag was recruited to Gag puncta primarily from the plasma membrane. This was in stark contrast to HIV-1 Gag, which was recruited from the cytoplasm. These observations imply fundamental differences among retroviruses regarding the orchestration of Gag punctum biogenesis, which has important general implications for enveloped virus particle assembly

T cells use distinct topographical and membrane receptor scanning strategies that individually coalesce during receptor recognition

During immune surveillance, CD8 T cells scan the surface of antigen-presenting cells using dynamic microvillar palpation and movements as well as by having their receptors preconcentrated into patches. Here, we use real-time lattice light-sheet microscopy to demonstrate the independence of microvillar and membrane receptor patch scanning. While T cell receptor (TCR) patches can distribute to microvilli, they do so stochastically and not preferentially as for other receptors such as CD62L. The distinctness of TCR patch movement from microvillar movement extends to many other receptors that form patches that also scan independent of the TCR. An exception to this is the CD8 coreceptor which largely comigrates in patches that overlap with or are closely adjacent to those containing TCRs. Microvilli that assemble into a synapse contain various arrays of the engaged patches, notably of TCRs and the inhibitory receptor PD-1, creating a pastiche of occupancies that vary from microvillar contact to contact. In summary, this work demonstrates that localization of receptor patches within the membrane and on microvillar projections is random prior to antigen detection and that such random variation may play into the generation of many individually composed receptor patch compositions at a single synapse

Substrate perturbation alters the glycoside hydrolase activities and community composition of switchgrass-adapted bacterial consortia

Bacteria modulate glycoside hydrolase expression in response to the changes in the composition of lignocellulosic biomass. The response of switchgrass-adapted thermophilic bacterial consortia to perturbation with a variety of biomass substrates was characterized to determine if bacterial consortia also responded to changes in biomass composition. Incubation of the switchgrass-adapted consortia with these alternative substrates produced shifts in glycoside hydrolase activities and bacterial community composition. Substantially increased endoglucanase activity was observed upon incubation with microcrystalline cellulose and trifluororacetic acid-pretreated switchgrass. In contrast, culturing the microbial consortia with ionic liquid-pretreated switchgrass increased xylanase activity dramatically. Microbial community analyses of these cultures indicated that the increased endoglucanase activity correlated with an increase in bacteria related to Rhodothermus marinus. Inclusion of simple organic substrates in the culture medium abrogated glycoside hydrolase activity and enriched for bacteria related to Thermus thermophilus. These results demonstrate that the composition of biomass substrates influences the glycoside hydrolase activities and community composition of biomass-deconstructing bacterial consortia

Genomic analysis of xylose metabolism in members of the deinoccocus-thermus phylum from thermophilic biomass-deconstructing bacterial consortia

Members of the phylum Deinoccocus-Thermus are adapted to grow under extremes of temperature and radiation. Some of these members have broad applications in biotechnology. However, the specific role of members of Deinoccocus-Thermus in plant biomass deconstruction remains largely unknown. Adaptations of thermophilic communities to grow on plant biomass substrates as the sole carbon source have consistently produced consortia with abundant populations affiliated with the Deinoccocus-Thermus. One of these populations was closely related to cultured isolates of Thermus thermophilus, while the second population, termed NIC-1, was distantly related to Truepera radiovictrix. NIC-1 was abundant in adapted cultures grown on xylan-rich substrates, while the T. thermophilus was virtually absent. To begin to understand the origin of this selection, genomic comparisons of xylan and xylose metabolism were undertaken between NIC-1, recovered from the metagenome obtained from an ammonia fiber expansion (AFEX)-pretreated switchgrass-adapted consortium and a T. thermophilus isolate from a related high temperature switchgrass adaptation. While both genomes indicated relatively limited capabilities to hydrolyze xylan, the NIC-1 genome had a putative operon for xylose utilization, while xylose metabolism genes were absent from the T. thermophilus genome. Comparison of multiple T. thermophilus genomes indicated that the genes for xylose metabolism were present on a plasmid in only one strain. Inspection of metagenomic dataset for adapted communities that contain T. thermophilus indicated that the plasmid is present in the T. thermophilus populations but may be lost upon isolation