20 research outputs found
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
Atomic force microscopy differentiates discrete size distributions between membrane protein containing and empty nanolipoprotein particles
AbstractTo better understand the incorporation of membrane proteins into discoidal nanolipoprotein particles (NLPs) we have used atomic force microscopy (AFM) to image and analyze NLPs assembled in the presence of bacteriorhodopsin (bR), lipoprotein E4 n-terminal 22k fragment scaffold and DMPC lipid. The self-assembly process produced two distinct NLP populations: those containing inserted bR (bR-NLPs) and those that did not (empty-NLPs). The bR-NLPs were distinguishable from empty-NLPs by an average increase in height of 1.0Â nm as measured by AFM. Streptavidin binding to biotinylated bR confirmed that the original 1.0Â nm height increase corresponds to br-NLP incorporation. AFM and ion mobility spectrometry (IMS) measurements suggest that NLP size did not vary around a single mean but instead there were several subpopulations, which were separated by discrete diameters. Interestingly, when bR was present during assembly the diameter distribution was shifted to larger particles and the larger particles had a greater likelihood of containing bR than smaller particles, suggesting that membrane proteins alter the mechanism of NLP assembly
Reconstructing Multiwell Potentials with Steep Gradients Using Stochastically Excited Spring Probes
Measurements of free
energy landscapes are critical for understanding
the basis of many physical, chemical, and biological interactions.
Statistical mechanics provide exact equations to calculate free energies,
but are built on the assumption that all possible configurations of
the system are sampled. The most pronounced limit to accurate free
energy computations is therefore the imperfect sampling of a potential
field, particularly in the case of interactions with steep gradients
and short reaction coordinates. We show through simulations that increasing
the stochastic fluctuations of a harmonic probe by active excitation
results in increased sampling times of high gradient adhesive interactions
and leads to the reconstruction of a more accurate energy landscape.
We use Brownian dynamics simulations to test the impact of probe approach
velocity, stiffness, and thermal energy to sample complex energy landscapes
with multiple wells of various depths and slopes to understand the
accuracy of energy surface reconstruction. We then show experimentally
that through the application of optimal stochastic excitations, we
are able to obtain accurate energy landscape reconstructions for different
probe and landscape parameters due to improved sampling of previously
poorly probed interactions
Continuous Sorting of Cells Based on Differential P Selectin Glycoprotein Ligand Expression Using Molecular Adhesion
Cell surface molecular
adhesions govern many important physiological
processes and are used to identify cells for analysis and purifications.
But most effective cell adhesion separation technologies use labels
or long-term attachments in their application. While label-free separation
microsystems typically separate cells by size, stiffness, and shape,
they often do not provide sufficient specificity to cell type that
can be obtained from molecular expression. We demonstrate a label-free
microfluidic approach capable of high throughput separation of cells
based upon surface molecule adhesion. Cells are flowed through a microchannel
designed with angled ridges at the top of the channel and coated with
adhesive ligands specific to target cell receptors. The ridges slightly
compress passing cells such that adhesive contact can be made with
sufficient surface area without unduly affecting cell trajectories
because of cell stiffness. Thus, sorting is sensitive to cell adhesion
but not to stiffness or cell size. The enforced interactions between
the cells and the ridges ensure that a high flow rate can be used
without lift forces quenching adhesion. As a proof of principle of
the method, we separate both Jurkat and HL60 cell lines based on their
differential expression of PSGL-1 ligand by using a ridged channel
coated with P selectin. We demonstrate 26-fold and 3.8-fold enrichment
of PSGL-1 positive and 4.4-fold and 3.2-fold enrichment of PSGL-1
negative Jurkat and HL60 cells, respectively. Increasing the number
of outlets to five allows for greater resolution in PSGL-1 selection
resulting in fractionation of a single cell type into subpopulations
of cells with high, moderate, and low PSGL-1 expression. The cells
can flow at a rate of up to 0.2 m/s, which corresponds to 0.045 million
cells per minute at the designed geometry, which is over 2 orders
of magnitude higher than previous adhesive-based sorting approaches.
Because of the short interaction time of the cells with the adhesive
surfaces, the sorting method does not further activate the cells due
to molecular binding. Such an approach may find use in label-free
selection of cells for a highly expressed molecular phenotype