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 1.0 nm as measured by AFM. Streptavidin binding to biotinylated bR confirmed that the original 1.0 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

Isolation, Characterization, and Stability of Discretely-Sized Nanolipoprotein Particles Assembled with Apolipophorin-III

Background: Nanolipoprotein particles (NLPs) are discoidal, nanometer-sized particles comprised of self-assembled phospholipid membranes and apolipoproteins. NLPs assembled with human apolipoproteins have been used for myriad biotechnology applications, including membrane protein solubilization, drug delivery, and diagnostic imaging. To expand the repertoire of lipoproteins for these applications, insect apolipophorin-III (apoLp-III) was evaluated for the ability to form discretely-sized, homogeneous, and stable NLPs. Methodology: Four NLP populations distinct with regards to particle diameters (ranging in size from 10 nm to.25 nm) and lipid-to-apoLp-III ratios were readily isolated to high purity by size exclusion chromatography. Remodeling of the purified NLP species over time at 4uC was monitored by native gel electrophoresis, size exclusion chromatography, and atomic force microscopy. Purified 20 nm NLPs displayed no remodeling and remained stable for over 1 year. Purified NLPs with 10 nm and 15 nm diameters ultimately remodeled into 20 nm NLPs over a period of months. Intra-particle chemical cross-linking of apoLp-III stabilized NLPs of all sizes. Conclusions: ApoLp-III-based NLPs can be readily prepared, purified, characterized, and stabilized, suggesting their utilit

Yeast Screens Identify the RNA Polymerase II CTD and SPT5 as Relevant Targets of BRCA1 Interaction

BRCA1 has been implicated in numerous DNA repair pathways that maintain genome integrity, however the function responsible for its tumor suppressor activity in breast cancer remains obscure. To identify the most highly conserved of the many BRCA1 functions, we screened the evolutionarily distant eukaryote Saccharomyces cerevisiae for mutants that suppressed the G1 checkpoint arrest and lethality induced following heterologous BRCA1 expression. A genome-wide screen in the diploid deletion collection combined with a screen of ionizing radiation sensitive gene deletions identified mutants that permit growth in the presence of BRCA1. These genes delineate a metabolic mRNA pathway that temporally links transcription elongation (SPT4, SPT5, CTK1, DEF1) to nucleopore-mediated mRNA export (ASM4, MLP1, MLP2, NUP2, NUP53, NUP120, NUP133, NUP170, NUP188, POM34) and cytoplasmic mRNA decay at P-bodies (CCR4, DHH1). Strikingly, BRCA1 interacted with the phosphorylated RNA polymerase II (RNAPII) carboxy terminal domain (P-CTD), phosphorylated in the pattern specified by the CTDK-I kinase, to induce DEF1-dependent cleavage and accumulation of a RNAPII fragment containing the P-CTD. Significantly, breast cancer associated BRCT domain defects in BRCA1 that suppressed P-CTD cleavage and lethality in yeast also suppressed the physical interaction of BRCA1 with human SPT5 in breast epithelial cells, thus confirming SPT5 as a relevant target of BRCA1 interaction. Furthermore, enhanced P-CTD cleavage was observed in both yeast and human breast cells following UV-irradiation indicating a conserved eukaryotic damage response. Moreover, P-CTD cleavage in breast epithelial cells was BRCA1-dependent since damage-induced P-CTD cleavage was only observed in the mutant BRCA1 cell line HCC1937 following ectopic expression of wild type BRCA1. Finally, BRCA1, SPT5 and hyperphosphorylated RPB1 form a complex that was rapidly degraded following MMS treatment in wild type but not BRCA1 mutant breast cells. These results extend the mechanistic links between BRCA1 and transcriptional consequences in response to DNA damage and suggest an important role for RNAPII P-CTD cleavage in BRCA1-mediated cancer suppression

Stability of various types of nanolipoprotein particles (NLPs) upon lyophilization

Nanolipoprotein particles (NLPs) have many potential uses in modern medicine, from cancer therapeutics to vaccine alternatives. As with all pharmaceuticals, the ease of storage and adequate stability of a compound is always a question. It has been found that NLPs can be very stable upon lyophilization, a freeze-drying technique in which all of the water in a sample is removed, if the initial conditions are suitable. In these experiments the stability of NLPs prepared with different combinations of lipids were tested in order to determine the optimum NLP conditions. NLPs composed of different types of lipids were constructed and then lyophilized in a buffered solution containing the sugar trehalose as an excipient. Then the samples were rehydrated and analyzed using size-exclusion chromatography (SEC) to determine if the NLP remained intact. Results indicated that the lipid composition of the NLP plays and important role in the stability upon lyophilization. NLPs prepared with the saturated DMPC lipid are more stable upon lyophilization than those prepared with unsaturated lipids (DOPC and/or DOGS-NTA-Ni), and require less excipient (i.e. trehalose) upon lyophilization to retain structure. These studies will have implications on NLP and storage formulations for vaccine and therapeutic applications

Fluid-Phase Chain Unsaturation Controlling Domain Microstructure and Phase in Ternary Lipid Bilayers Containing GalCer and Cholesterol

We report the microstructure and phase behavior of three ternary mixtures each containing a long-chain saturated glycosphingolipid, galactosylceramide (GalCer), and cholesterol at room temperature. The unsaturation level of the fluid-phase component was varied by lipid choice, i.e., saturated 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), singly unsaturated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), or doubly unsaturated 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). GalCer was used because of its biological significance, for example, as a ligand in the sexual transmission of HIV and stimulator of natural killer T-cells. Supported lipid bilayers of the ternary mixtures were imaged by atomic force microscopy and GalCer-rich domains were characterized by area/perimeter ratios (A/P). GalCer domain phase transitions from solid (S) to liquid (L) phase were verified by domain behavior in giant unilamellar vesicles, which displayed two-dimensional microstructure similar to that of supported lipid bilayers. As cholesterol concentration was increased, we observed ∼2.5, ∼10, and ∼20-fold decreases in GalCer domain A/P for bilayers in L-S phase coexistence containing DOPC, POPC, and DLPC, respectively. The transition to L-L phase coexistence occurred at ∼10 mol % cholesterol for bilayers containing DOPC or POPC and was accompanied by maintenance of a constant A/P. L-L phase coexistence did not occur for bilayers containing DLPC. We systematically relate our results to the impact of chain unsaturation on the interaction of the fluid-phase lipid and cholesterol. Physiologically, these observations may give insight into the interplay of fatty acid chain unsaturation, sterol concentration, and lipid hydrophobic mismatch in membrane phenomena