An intracellular traffic jam: Fc receptor-mediated transport of immunoglobulin G

Recent advances in imaging techniques along with more powerful in vitro and in vivo models of receptor-mediated ligand transport are facilitating advances in our understanding of how cells efficiently direct receptors and their cargo to target destinations within the cytoplasm and at the plasma membrane. Specifically, light and 3D electron microscopy studies examining the trafficking behavior of the neonatal Fc receptor (FcRn), a transport receptor for immunoglobulin G (IgG), have given us new insights into the dynamic interplay between the structural components of the cytosolic trafficking machinery, its protein regulators, and the receptors it directs to various locations within the cell. These studies build upon previous biochemical characterizations of FcRn transport and are allowing us to begin formulation of a more complete model for the intracellular trafficking of receptor–ligand complexes

Towards a Robuster Interpretive Parsing

The input data to grammar learning algorithms often consist of overt forms that do not contain full structural descriptions. This lack of information may contribute to the failure of learning. Past work on Optimality Theory introduced Robust Interpretive Parsing (RIP) as a partial solution to this problem. We generalize RIP and suggest replacing the winner candidate with a weighted mean violation of the potential winner candidates. A Boltzmann distribution is introduced on the winner set, and the distribution’s parameter $T$ is gradually decreased. Finally, we show that GRIP, the Generalized Robust Interpretive Parsing Algorithm significantly improves the learning success rate in a model with standard constraints for metrical stress assignment

Ligand Valency Affects Transcytosis, Recycling and Intracellular Trafficking Mediated by the Neonatal Fc Receptor

The neonatal Fc receptor (FcRn) transports IgG across epithelial cell barriers to provide maternal antibodies to offspring and serves as a protection receptor by rescuing endocytosed IgG and albumin from lysosomal degradation. Here we describe the generation of polarized Madin–Darby canine kidney (MDCK) cells expressing rat FcRn (rFcRn) to investigate the potential requirement for ligand bivalency in FcRn-mediated transport. The rFcRn-MDCK cells bind, internalize and bidirectionally transcytose the bivalent ligands IgG and Fc across polarized cell monolayers. However, they cannot be used to study FcRn-mediated transport of the monovalent ligand albumin, as we observe no specific binding, internalization or transcytosis of rat albumin. To address whether ligand bivalency is required for transport, the ability of rFcRn to transcytose and recycle wild-type Fc homodimers (wtFc; two FcRn-binding sites) and a heterodimeric Fc (hdFc; one FcRn-binding site) was compared. We show that ligand bivalency is not required for transcytosis or recycling, but that wtFc is transported more efficiently than hdFc, particularly at lower concentrations. We also demonstrate that hdFc and wtFc have different intracellular fates, with more hdFc than wtFc being trafficked to lysosomes and degraded, suggesting a role for avidity effects in FcRn-mediated IgG transport

Experimental investigation of helicity in turbulent swirling jet using dual-plane dye laser PIV technique

This paper reports a new method of generating two light sheets using a dye laser system and the use of this dual-plane dye laser system to analyse average helicity and energy dissipation in a turbulent swirling flow. The dual-plane PIV system that was used in this study consisted of three cameras and a single frequency Nd:YAG laser, which was used to generate two parallel light sheet planes with differing wavelengths(colour). The method of generating two different light sheet wavelengths using a single laser source is an innovative and new technique. Stereoscopic PIV measurements were obtained in one plane with the use of two CCD cameras, and standard PIV measurements were obtained in the other plane with the use of one CCD camera. The light scattered by the particles on two different light sheets were separated using appropriate optical filters. The measurements obtained were used to estimate the components of the velocity gradient tensor. The tensor components were then used to determine the average vorticity components and helicity quantities of the fluid that was investigated. To determine the average turbulent kinetic energy dissipation, the continuity equation was used to infer the out-of-plane gradient of the out-of-plane velocity. From the analysis of the results, it was found that regions with high helicity were correlated with regions of high turbulent kinetic energy dissipation. © 2008 Springer-Verlag

Comparison of FcRn- and pIgR-Mediated Transport in MDCK Cells by Fluorescence Confocal Microscopy

Protein delivery across polarized epithelia is controlled by receptor-mediated transcytosis. Many studies have examined basolateral-to-apical trafficking of polymeric IgA (pIgA) by the polymeric immunoglobulin receptor (pIgR). Less is known about apical-to-basolateral transcytosis, the direction the neonatal Fc receptor (FcRn) transports maternal IgGs across intestinal epithelia. To compare apical-to-basolateral and basolateral-to-apical transcytosis, we co-expressed FcRn and pIgR in Madin-Darby canine kidney (MDCK) cells and used pulse-chase experiments with confocal microscopy to examine transport of apically applied IgG Fcγ and basolaterally applied pIgA. Fcγ and pIgA trafficking routes were initially separate but intermixed at later chase times. Fcγ was first localized near the apical surface, but became more equally distributed across the cell, consistent with concomitant transcytosis and recycling. By contrast, pIgA transport was strongly unidirectional: pIgA shifted from near the basolateral surface to an apical location with increasing time. Some Fcγ and pIgA fluorescence colocalized in early (EEA1-positive), recycling (Rab11a-positive), and transferrin (Tf)-positive common/basolateral recycling endosomes. Fcγ became more enriched in Tf-positive endosomes with time, whereas pIgA was sorted from these compartments. Live-cell imaging revealed that vesicles containing Fcγ or pIgA shared similar mobility characteristics and were equivalently affected by depolymerizing microtubules, indicating that both trafficking routes depended to roughly the same extent on intact microtubules

The Chicken Yolk Sac IgY Receptor, a Mammalian Mannose Receptor Family Member, Transcytoses IgY across Polarized Epithelial Cells

In mammals the transfer of passive immunity from mother to young is mediated by the MHC-related receptor FcRn, which transports maternal IgG across epithelial cell barriers. In birds, maternal IgY in egg yolk is transferred across the yolk sac to passively immunize chicks during gestation and early independent life. The chicken yolk sac IgY receptor (FcRY) is the ortholog of the mammalian phospholipase A2 receptor, a mannose receptor family member, rather than an FcRn or MHC homolog. FcRn and FcRY both exhibit ligand binding at the acidic pH of endosomes and ligand release at the slightly basic pH of blood. Here we show that FcRY expressed in polarized mammalian epithelial cells functioned in endocytosis, bidirectional transcytosis, and recycling of chicken FcY/IgY. Confocal immunofluorescence studies demonstrated that IgY binding and endocytosis occurred at acidic but not basic pH, mimicking pH-dependent uptake of IgG by FcRn. Colocalization studies showed FcRY-mediated internalization via clathrin-coated pits and transport involving early and recycling endosomes. Disruption of microtubules partially inhibited apical-to-basolateral and basolateral-to-apical transcytosis, but not recycling, suggesting the use of different trafficking machinery. Our results represent the first cell biological evidence of functional equivalence between FcRY and FcRn and provide an intriguing example of how evolution can give rise to systems in which similar biological requirements in different species are satisfied utilizing distinct protein folds

Design of an airlift loop bioreactor and pilot scales studies with fluidic oscillator induced microbubbles for growth of a microalgae Dunaliella salina

This study was conducted to test the feasibility of growing microalgae on steel plant exhaust gas, generated from the combustion of offgases from steel processing, which has a high CO content. Two field trials of batch algal biomass growth, mediated by microbubble transfer processes in an airlift loop bioreactor showed only steady growth of biomass with 100% survival rate. The gas analysis of CO uptake in the 2200L bioreactor showed a specific uptake rate of 0.1g/L/h, an average 14% of the CO available in the exhaust gas with a 23% composition of CO. This uptake led to a steady production of chlorophyll and total lipid constituency in the bioreactor, and an accelerating exponential growth rate of biomass, with a top doubling time of 1.8days. The gas analysis also showed anti-correlation of CO uptake and O production, which along with the apparent stripping of the O to the equilibrium level by the microbubbles, strongly suggests that the bioreactor is not mass transfer limited, nor O inhibited. Removing O inhibition results in high growth rates and high density of biomass. © 2011 Elsevier Ltd

A freeze substitution fixation-based gold enlarging technique for EM studies of endocytosed nanogold-labeled molecules

We have developed methods to locate individual ligands that can be used for electron microscopy studies of dynamic events during endocytosis and subsequent intracellular trafficking. The methods are based on enlargement of 1.4 nm Nanogold attached to an endocytosed ligand. Nanogold, a small label that does not induce misdirection of ligand–receptor complexes, is ideal for labeling ligands endocytosed by live cells, but is too small to be routinely located in cells by electron microscopy. Traditional pre-embedding enhancement protocols to enlarge Nanogold are not compatible with high pressure freezing/freeze substitution fixation (HPF/FSF), the most accurate method to preserve ultrastructure and dynamic events during trafficking. We have developed an improved enhancement procedure for chemically fixed samples that reduced auto-nucleation, and a new pre-embedding gold enlarging technique for HPF/FSF samples that preserved contrast and ultrastructure and can be used for high-resolution tomography. We evaluated our methods using labeled Fc as a ligand for the neonatal Fc receptor. Attachment of Nanogold to Fc did not interfere with receptor binding or uptake, and gold-labeled Fc could be specifically enlarged to allow identification in 2D projections and in tomograms. These methods should be broadly applicable to many endocytosis and transcytosis studies