SINGLE-CELL LEVEL INVESTIGATION OF CYTOSKELETAL/CELLULAR RESPONSE TO EXTERNAL STIMULI

A detailed understanding of the molecular mechanisms by which chemical signals control cell behavior is needed if the complex biological processes of embryogenesis, development, health and disease are to be completely understood. Yet, if we are to fully understand the molecular mechanisms controlling cell behavior, measurements at the single cell level are needed to supplement information gained from population level studies. One of the major challenges to accomplishing studies at the single cell level has been a lack of physical tools to complement the powerful molecular biological assays which have provided much of what we currently know about cell behavior. The goal of this exploratory project is the development of an experimental platform that facilitates integrated observation, tracking and analysis of the responses of many individual cells to controlled environmental factors (e.g. extracellular signals). Toward this goal, we developed chemically-patterned microarrays of both adherent and suspension mammalian cell types. A novel chemical patterning methodology, based on photocatalytic lithography, was developed to construct biomolecule and cell arrays that facilitate analysis of biological function. Our patterning techniques rely on inexpensive stamp materials and visible light, and do not necessitate mass transport or specified substrates. Patterned silicon and glass substrates are modified such that there is a non-biofouling polymer matrix surrounding the adhesive regions that target biomolecules and cells. Fluorescence and reflectance microscopy reveal successful patterning of proteins and single to small clusters of mammalian cells. In vitro assays conducted upon cells on the patterned arrays demonstrate the viability of cells interfacing with this synthetic system. Hence, we have successfully established a versatile cell measurement platform which can be used to characterize the molecular regulators of cellular behavior in a variety of important biological processes. The achievements realized in this project have enabled presentations and publication within the international scientific community, new collaborations with researchers at the University of California, and successful competition for three additional, separate research grants on studies of stem cell fate commitment and pathogen-host cell interactions

Decoupling Internalization, Acidification and Phagosomal-Endosomal/lysosomal Fusion during Phagocytosis of InlA Coated Beads in Epithelial Cells

BACKGROUND: Phagocytosis has been extensively examined in 'professional' phagocytic cells using pH sensitive dyes. However, in many of the previous studies, a separation between the end of internalization, beginning of acidification and completion of phagosomal-endosomal/lysosomal fusion was not clearly established. In addition, very little work has been done to systematically examine phagosomal maturation in 'non-professional' phagocytic cells. Therefore, in this study, we developed a simple method to measure and decouple particle internalization, phagosomal acidification and phagosomal-endosomal/lysosomal fusion in Madin-Darby Canine Kidney (MDCK) and Caco-2 epithelial cells. METHODOLOGY/PRINCIPAL FINDINGS: Our method was developed using a pathogen mimetic system consisting of polystyrene beads coated with Internalin A (InlA), a membrane surface protein from Listeria monocytogenes known to trigger receptor-mediated phagocytosis. We were able to independently measure the rates of internalization, phagosomal acidification and phagosomal-endosomal/lysosomal fusion in epithelial cells by combining the InlA-coated beads (InlA-beads) with antibody quenching, a pH sensitive dye and an endosomal/lysosomal dye. By performing these independent measurements under identical experimental conditions, we were able to decouple the three processes and establish time scales for each. In a separate set of experiments, we exploited the phagosomal acidification process to demonstrate an additional, real-time method for tracking bead binding, internalization and phagosomal acidification. CONCLUSIONS/SIGNIFICANCE: Using this method, we found that the time scales for internalization, phagosomal acidification and phagosomal-endosomal/lysosomal fusion ranged from 23-32 min, 3-4 min and 74-120 min, respectively, for MDCK and Caco-2 epithelial cells. Both the static and real-time methods developed here are expected to be readily and broadly applicable, as they simply require fluorophore conjugation to a particle of interest, such as a pathogen or mimetic, in combination with common cell labeling dyes. As such, these methods hold promise for future measurements of receptor-mediated internalization in other cell systems, e.g. pathogen-host systems

Porphyrin-Based Photocatalytic Lithography

Photocatalytic lithography is an emerging technique that couples light with coated mask materials in order to pattern surface chemistry. We excite porphyrins to create radical species that photocatalytically oxidize, and thereby pattern, chemistries in the local vicinity. The technique advantageously does not necessitate mass transport or specified substrates, it is fast and robust and the wavelength of light does not limit the resolution of patterned features. We have patterned proteins and cells in order to demonstrate the utility of photocatalytic lithography in life science applications

Influence of Biomolecular Site Density on Particle Interaction Lifetimes in Receptor-mediated Colloidal Assembly

Bio-inspired assembly of colloidal building blocks into functional nano- to microscale structures is expected to deliver profound developments in materials science and biotechnology. Previously, high surface densities of E-selectin and its ligand, sialyl Lewis ), were used to drive the assembly of bidisperse suspensions into various kinetically-trapped microstructures by varying relative particle concentrations (Hiddessen, A.L.; Rodgers, S.D.; Weitz, D.A., Hammer, D.A. Langmuir 2000, 16, 9744-9753). Here, the low affinity binding between E-selectin and sLe is exploited to create reversible particle interactions at low molecular surface densities, without the use of imposed stimuli (e.g., changes in temperature, solution chemistry, or external fields). Using videomicroscopy, hundreds of binding lifetimes between E-selectin-coated nano- (R = 0.47 m) and sLe -coated micro- (R = 2.75 m) particles are measured for a series of low surface densities (7 -- 51 sites/m ). The measurements reveal that average particle interaction times can be reduced from minutes down to single selectin-carbohydrate bond lifetimes ( 1 s) by decreasing sLe density. Distributions of selectin-mediated binding lifetimes are analyzed with a probabilistic receptor-ligand binding model (CozensRoberts, C.; Lauffenburger, D.A.; Quinn, J.A. Biophys. J. 1990, 58, 841-856), yielding estimates for molecular parameters, such as the on rate, 10 , and unstressed off rate, 0.25 s , which characterize the observed particle detachment behavior. The unstressed off-rate, r k , compares remarkably well with published values for E-selectin/ligand interactions. This new class of reversible, biophysically controlled colloidal interactions, driven by low affinity receptor-ligand interactions, h..

Biomolecular patterning vika photocatalytic lithography

We have developed a novel method for patterning surface chem.: Photocatalytic Lithog. This technique relies on inexpensive stamp materials and light; it does not necessitate mass transport or specified substrates, and the wavelength of light should not limit feature resoln. We have demonstrated the utility of this technique through the patterning of proteins, single cells and bacteria. [on SciFinder (R)