Genetically engineered sensors of cell signaling

Measuring electrical activity in large numbers of cells with high spatial and temporal resolution is a fundamental problem for the study of neural development and information processing. To address this problem, we have constructed FlaSh: a novel, genetically-encoded probe that can be used to measure trans-membrane voltage in single cells. We fused a modified green fluorescent protein (GFP) into a voltage-sensitive potassium channel so that voltage dependent rearrangements in the potassium channel induce changes in the fluorescence of GFP. A voltage sensor encoded into DNA has the advantage that it may be introduced into an organism non-invasively and targeted to specific developmental stages, brain regions, cell types, and sub-cellular compartments.

We also describe modifications to FlaSh that shift its color, kinetics, and dynamic range. We used multiple green fluorescent proteins to produce variants of the FlaSh sensor that generate ratiometric signal output via fluorescence resonance energy transfer (FRET). Finally, we describe initial work toward FlaSh variants that are sensitive to G-protein coupled receptor (GPCR) activation. These sensors can be used to design functional assays for receptor activation in living cells.

Cell fate specification during Caenorhabditis elegans male tail development

The cells of the specialized mating structures of the nematode Caenorhabditis elegans adult male tail develop from sex-specific divisions of postembryonic blast cells. One male-specific blast cell, B, is the precursor to all the cells of the copulatory spicules. Both cell interactions and autonomous fate specification mechanisms are utilized in the B lineage to specify fate.

During development the anterior daughter of B, B.a, generates four distinct pairs of cells. Cell ablation experiments indicate that the cells of each pair respond to positional cues provided by other male-specific blast cells. F and U promote anterior fates, Y.p promotes some posterior fates, and the B.a progeny promote posterior fates. The cells within each pair may also interact.

The lin-3/let-23 signalling pathway, identified for its function in C. elegans hermaphrodite vulval induction, mediates the signal from F and U. Reduction-of-function mutations in lin-3 (EGF-like signal), let-23 (receptor), sem-5 (adaptor), let-60 (ras), or lin-45 (raf) disrupt the fates of the anterior cells, and mimic F and U ablation. In addition, ectopically expressed lin-3 disrupts the fates of the posterior cells, and can promote anterior fates even in the absence of F and U.

A genetic screen of over 9000 mutagenized gametes recovered 22 mutations in 20 loci that disrupt fate specification in male tail lineages. Seven of these mutations may represent new genes that play a role in male tail development.

The first division of the B cell is asymmetric. The gene vab-3 is required for specification of B.a fates, and it may represent a factor whose activity is localized to the B.a cell via the gene lin-17. lin-17 acts both at the first division of the B cell and at specific other cell divisions in the lineage.

Analysis of a transcriptional network involving PU.1, Notch, and Gata3 in the lymphomyeloid lineage decision during early T-cell development

Hematopoiesis is a well-established system used to study developmental choices amongst cells with multiple lineage potentials, as well as the transcription factor network interactions that drive these developmental paths. Multipotent progenitors travel from the bone marrow to the thymus where T-cell development is initiated and these early T-cell precursors retain lineage plasticity even after initiating a T-cell program. The development of these early cells is driven by Notch signaling and the combinatorial expression of many transcription factors, several of which are also involved in the development of other cell lineages. The ETS family transcription factor PU.1 is involved in the development of progenitor, myeloid, and lymphoid cells, and can divert progenitor T-cells from the T-lineage to a myeloid lineage. This diversion of early T-cells by PU.1 can be blocked by Notch signaling. The PU.1 and Notch interaction creates a switch wherein PU.1 in the presence of Notch promotes T-cell identity and PU.1 in the absence of Notch signaling promotes a myeloid identity. Here we characterized an early T-cell cell line, Scid.adh.2c2, as a good model system for studying the myeloid vs. lymphoid developmental choice dependent on PU.1 and Notch signaling. We then used the Scid.adh.2c2 system to identify mechanisms mediating PU.1 and Notch signaling interactions during early T-cell development. We show that the mechanism by which Notch signaling is protecting pro-T cells is neither degradation nor modification of the PU.1 protein. Instead we give evidence that Notch signaling is blocking the PU.1-driven inhibition of a key set of T-regulatory genes including Myb, Tcf7, and Gata3. We show that the protection of Gata3 from PU.1-mediated inhibition, by Notch signaling and Myb, is important for retaining a T-lineage identity. We also discuss a PU.1-driven mechanism involving E-protein inhibition that leads to the inhibition of Notch target genes. This is mechanism may be used as a lockdown mechanism in pro-T-cells that have made the decision to divert to the myeloid pathway.

Single-cell analysis of the physiology of mechanosensation in bacteria

Escherichia coli is one of the best studied living organisms and a model system for many biophysical investigations. Despite countless discoveries of the details of its physiology, we still lack a holistic understanding of how these bacteria react to changes in their environment. One of the most important examples is their response to osmotic shock. One of the mechanistic elements protecting cell integrity upon exposure to sudden changes of osmolarity is the presence of mechanosensitive channels in the cell membrane. These channels are believed to act as tension release valves protecting the inner membrane from rupturing. This thesis presents an experimental study of various aspects of mechanosensation in bacteria. We examine cell survival after osmotic shock and how the number of MscL (Mechanosensitive channel of Large conductance) channels expressed in a cell influences its physiology. We developed an assay that allows real-time monitoring of the rate of the osmotic challenge and direct observation of cell morphology during and after the exposure to osmolarity change. The work described in this thesis introduces tools that can be used to quantitatively determine at the single-cell level the number of expressed proteins (in this case MscL channels) as a function of, e.g., growth conditions. The improvement in our quantitative description of mechanosensation in bacteria allows us to address many, so far unsolved, problems, like the minimal number of channels needed for survival, and can begin to paint a clearer picture of why there are so many distinct types of mechanosensitive channels.

Polarization-dependent pulse compression in an argon-filled cell through filamentation

Pulse compression through filamentation in an argon-filled cell was experimentally demonstrated by using circularly and linearly polarized pulses. A 53 fs circularly polarized pulse was successfully compressed to 15 fs. By using circularly polarized pulse input, the broadened spectrum was much wider and the incident energy in the gas cell can be increased by more than 3/2 times. Much shorter pulse could be compressed by using circularly polarized pulse input. [GRAPHICS] The temporal profile of the compressed pulse (C) 2008 by Astro Ltd. Published exclusively by WILEY-VCH Verlag GmbH & Co. KGaA.

Preliminary investigation into implication of a single cell organism in fish feed bouyancy and flotation

Two Isocaloric Isoproteic 30% crude protein diets were formulated for Clariid catfish and Tilapia with wheat grain starch (WGS) and cassava tuber starch (CTS) incorporated at 10 percent as binding agents. Saccharomyces cerevisiae was included at 2% as floating additive. The water stability, nutrient retention and flotation of pelleted feeds were observed for 60 minutes. There were generally decreasing trends in stability and retention at increasing time of immersion in water. The lipid retention was higher (P>0.05) than proteins in both diets. WGS diet was better (P>0.05) than CTS diet in flotation, which has attributed to the presence of gluten protein in wheat products. It is envisaged that a break through in floating feed development in Nigeria aquaculture would save the Nigeria economy from extruded (floating) feed importation

Biotechnologies for cancer diagnostics : cell sorting, protein analysis and imaging of cellular metabolism

This thesis presents the development of chip-based technology for informative in vitro cancer diagnostics. In the first part of this thesis, I will present my contribution in the development of a technology called “Nucleic Acid Cell Sorting (NACS)”, based on microarrays composed of nucleic acid encoded peptide major histocompatibility complexes (p/MHC), and the experimental and theoretical methods to detect and analyze secreted proteins from single or few cells.

Secondly, a novel portable platform for imaging of cellular metabolism with radio probes is presented. A microfluidic chip, so called “Radiopharmaceutical Imaging Chip” (RIMChip), combined with a beta-particle imaging camera, is developed to visualize the uptake of radio probes in a small number of cells. Due to its sophisticated design, RIMChip allows robust and user-friendly execution of sensitive and quantitative radio assays. The performance of this platform is validated with adherent and suspension cancer cell lines. This platform is then applied to study the metabolic response of cancer cells under the treatment of drugs. Both cases of mouse lymphoma and human glioblastoma cell lines, the metabolic responses to the drug exposures are observed within a short time (~ 1 hour), and are correlated with the arrest of cell-cycle, or with changes in receptor tyrosine kinase signaling.

The last parts of this thesis present summaries of ongoing projects: development of a new agent as an in vivo imaging probe for c-MET, and quantitative monitoring of glycolytic metabolism of primary glioblastoma cells. To develop a new agent for c-MET imaging, the one-bead-one-compound combinatorial library method is used, coupled with iterative screening. The performance of the agent is quantitatively validated with cell-based fluorescent assays. In the case of monitoring the metabolism of primary glioblastoma cell, by RIMChip, cells were sorting according to their expression levels of oncoprotein, or were treated with different kinds of drugs to study the metabolic heterogeneity of cancer cells or metabolic response of glioblastoma cells to drug treatments, respectively.

Wnt and FGF signaling in C. elegans vulval cell lineage polarity

The interpretation of extracellular cues leading to the polarization of intracellular components and asymmetric cell divisions is a fundamental part of metazoan organogenesis. The C. elegans vulva, with its invariant cell lineage and interaction of multiple cell signaling pathways, provides an excellent model for the study of cell polarity within an organized epithelial tissue. Herein I discuss the interaction of Wnt and FGF signaling in controlling vulval cell lineage polarity with emphasis on the posterior-most cell that forms the vulva, P7.p.

The mirror symmetry of the C. elegans vulva is achieved by the opposite division orientation of the vulval precursor cells (VPCs) flanking the axis of symmetry. Opposing Wnt signals control the division patterns of the VPCs by controlling the localization of SYS-1/ β-catenin toward the direction of the Wnt gradient. Multiple Wnt signals, expressed at the axis of symmetry, promote the wild-type, anterior-facing, P7.p orientation, whereas Wnts EGL-20 and CWN-1 from the tail and posterior body wall muscle, respectively, promote the daughter cells of P7.p to face the posterior. EGL-20 acts through a member of the LDL receptor superfamily, LRP-2, along with Ror/CAM-1 and Van Gogh/VANG-1. All three transmembrane proteins control orientation through the localization of the SYS-1.

The Fibroblast Growth Factor (FGF) pathway acts in concert with LIN-17/Frizzled to regulate the localization of SYS-1. The source of the FGF ligand is the 1° VPC, P6.p, which controls the polarity of the neighboring 2° VPC, P7.p, by signaling through the sex myoblasts (SMs), activating the FGF pathway. The Wnt, cwn-1, is expressed in the posterior body wall muscle of the worm as well as the SMs, making it the only Wnt expressed on the posterior and anterior sides of P7.p at the time of the polarity decision. Both sources of cwn-1 act instructively to influence P7.p polarity in the direction of the Wnt gradient. The FGF pathway leads to the regulation of cwn-1 transcripts in the SMs. These results illustrate the first evidence of the interaction between FGF and Wnt in C. elegans development and vulval cell lineage polarity as well as highlight the promiscuous nature of Wnt signaling within C. elegans.

Phase-matched high-order harmonic generation in a gas cell with midinfrared femtosecond pulses

We experimentally investigate the generation of high-order harmonics in a 4-mm-long gas cell using midinfrared femtosecond pulses at various wavelengths of 1240 nm, 1500 nm, and 1800 nm. It is observed that the yield and cutoff energy of the generated high-order harmonics critically depend on focal position, gas pressure, and size of the input beam which can be controlled by an aperture placed in front of the focal lens. By optimizing the experimental parameters, we achieve a cutoff energy at similar to 190 eV with the 1500 nm driving pulses, which is the highest for the three wavelengths chosen in our experiment.

Compression of intense laser pulse through filamentation in an argon-filled cell

We demonstrate a pulse compression technique through filamentation in an argon-filled cell. By using a pair of chirped mirrors for dispersion compensation, we have successfully compressed the 53 fs pulse to 15 fs with good spatial qualities and good pulse stability. The total transmitted efficiency is more than 75%. The influence of the experiment parameters to the compressed pulses is also studied experimentally.