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

Multiplex primer prediction software for divergent targets

We describe a Multiplex Primer Prediction (MPP) algorithm to build multiplex compatible primer sets to amplify all members of large, diverse and unalignable sets of target sequences. The MPP algorithm is scalable to larger target sets than other available software, and it does not require a multiple sequence alignment. We applied it to questions in viral detection, and demonstrated that there are no universally conserved priming sequences among viruses and that it could require an unfeasibly large number of primers (∼3700 18-mers or ∼2000 10-mers) to generate amplicons from all sequenced viruses. We then designed primer sets separately for each viral family, and for several diverse species such as foot-and-mouth disease virus (FMDV), hemagglutinin (HA) and neuraminidase (NA) segments of influenza A virus, Norwalk virus, and HIV-1. We empirically demonstrated the application of the software with a multiplex set of 16 short (10 nt) primers designed to amplify the Poxviridae family to produce a specific amplicon from vaccinia virus

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..

Porphyrin-based photocatalytic lithography

Photocatalytic lithography couples light with photoreactive coated mask materials 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 is suited for use with a wide variety of 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 to demonstrate the utility of photocatalytic lithography in life science applications

Phototocatalytic Lithography of Poly(propylene sulfide) Block Copolymers: Toward High-Throughput Nanolithography for Biomolecular Arraying Applications

Photocatalytic lithography (PCL) is an inexpensive, fast, and robust method of oxidizing surface chemical moieties to produce patterned substrates. This technique has utility in basic biological research as well as various biochip applications. We report on porphyrin-based PCL for patterning poly(propylene sulfide) block copolymer films on gold substrates on the micrometer and submicrometer scales. We confirm chemical patterning with imaging ToF-SIMS and low-voltage SEM. Biomolecular patterning on micrometer and submicrometer scales is demonstrated with proteins, protein-linked beads. and fluorescently labeled proteins