Articles by Fabian E Ortega in JoVE
A Multi-well Format Polyacrylamide-based Assay for Studying the Effect of Extracellular Matrix Stiffness on the Bacterial Infection of Adherent Cells Effie E Bastounis1, Fabian E Ortega1, Ricardo Serrano2, Julie A Theriot3 1Department of Biochemistry, Stanford University School of Medicine, 2Department of Mechanical and Aerospace Engineering, University of California San Diego, 3Departments of Biochemistry, Microbiology and Immunology and Howard Hughes Medical Institute, Stanford University School of Medicine We have developed a multi-well format polyacrylamide-based assay for probing the effect of extracellular matrix stiffness on bacterial infection of adherent cells. This assay is compatible with flow cytometry, immunostaining, and traction force microscopy, allowing for quantitative measurements of the biomechanical interactions between cells, their extracellular matrix, and pathogenic bacteria.
Other articles by Fabian E Ortega on PubMed
Oxygen Regulation of Breathing Through an Olfactory Receptor Activated by Lactate Nature. | Pubmed ID: 26560302 Animals have evolved homeostatic responses to changes in oxygen availability that act on different timescales. Although the hypoxia-inducible factor (HIF) transcriptional pathway that controls long-term responses to low oxygen (hypoxia) has been established, the pathway that mediates acute responses to hypoxia in mammals is not well understood. Here we show that the olfactory receptor gene Olfr78 is highly and selectively expressed in oxygen-sensitive glomus cells of the carotid body, a chemosensory organ at the carotid artery bifurcation that monitors blood oxygen and stimulates breathing within seconds when oxygen declines. Olfr78 mutants fail to increase ventilation in hypoxia but respond normally to hypercapnia. Glomus cells are present in normal numbers and appear structurally intact, but hypoxia-induced carotid body activity is diminished. Lactate, a metabolite that rapidly accumulates in hypoxia and induces hyperventilation, activates Olfr78 in heterologous expression experiments, induces calcium transients in glomus cells, and stimulates carotid sinus nerve activity through Olfr78. We propose that, in addition to its role in olfaction, Olfr78 acts as a hypoxia sensor in the breathing circuit by sensing lactate produced when oxygen levels decline.
Trinucleotide Repeat Containing 6c (TNRC6c) is Essential for Microvascular Maturation During Distal Airspace Sacculation in the Developing Lung Developmental Biology. | Pubmed ID: 28811219 GW182 (also known asTNRC6) family members are critically involved in the final effector phase of miRNA-mediated mRNA repression. The three mammalian paralogs, TNRC6a, b and c, are thought to be redundant based on Argonaute (Ago) binding, tethering assays, and RNAi silencing of individual members in cell lines. To test this idea, we generated TNRC6a, b and c knockout mice. TNRC6a mutants die at mid-gestation, while b- and c- deleted mice are born at a Mendelian ratio. However, the majority of TNRC6b and all TNRC6c mutants die within 24h after birth, the latter with respiratory failure. Necropsy of TNRC6c mutants revealed normal-appearing airways that give rise to abnormally thick-walled distal gas exchange sacs. Immunohistological analysis of mutant lungs demonstrated a normal distribution of bronchiolar and alveolar cells, indicating that loss of TNRC6c did not abrogate epithelial cell differentiation. The cellular kinetics and relative proportions of endothelial, epithelial, and mesenchymal cells were also not altered. However, the underlying capillary network was simplified and endothelial cells had failed to become tightly apposed to the surface epithelium in TNRC6c mutants, presumably causing the observed respiratory failure. TGFβ family mutant mice exhibit a similar lung phenotype of thick-walled air sacs and neonatal lethality, and qRT-PCR confirmed dynamic downregulation of TGFβ1 and TGFβR2 in TNRC6c mutant lungs during sacculation. VEGFR, but not VEGF-A ligand, was also lower, likely reflecting the overall reduced capillary density in TNRC6c mutants. Together, these results demonstrate that GW182 paralogs are not functionally redundant in vivo. Surprisingly, despite regulating a general cellular process, TNRC6c is selectively required only in the distal lung and not until late in gestation for proper expression of the TGFβ family genes that drive sacculation. These results imply a complex and indirect mode of regulation of sacculation by TNRC6c, mediated in part by dynamic transcriptional repression of an inhibitor of TGFβ family gene expression.
Listeria Monocytogenes InlP Interacts with Afadin and Facilitates Basement Membrane Crossing PLoS Pathogens. | Pubmed ID: 29847585 During pregnancy, the placenta protects the fetus against the maternal immune response, as well as bacterial and viral pathogens. Bacterial pathogens that have evolved specific mechanisms of breaching this barrier, such as Listeria monocytogenes, present a unique opportunity for learning how the placenta carries out its protective function. We previously identified the L. monocytogenes protein Internalin P (InlP) as a secreted virulence factor critical for placental infection. Here, we show that InlP, but not the highly similar L. monocytogenes internalin Lmo2027, binds to human afadin (encoded by AF-6), a protein associated with cell-cell junctions. A crystal structure of InlP reveals several unique features, including an extended leucine-rich repeat (LRR) domain with a distinctive Ca2+-binding site. Despite afadin's involvement in the formation of cell-cell junctions, MDCK epithelial cells expressing InlP displayed a decrease in the magnitude of the traction stresses they could exert on deformable substrates, similar to the decrease in traction exhibited by AF-6 knock-out MDCK cells. L. monocytogenes ΔinlP mutants were deficient in their ability to form actin-rich protrusions from the basal face of polarized epithelial monolayers, a necessary step in the crossing of such monolayers (transcytosis). A similar phenotype was observed for bacteria expressing an internal in-frame deletion in inlP (inlP ΔLRR5) that specifically disrupts its interaction with afadin. However, afadin deletion in the host cells did not rescue the transcytosis defect. We conclude that secreted InlP targets cytosolic afadin to specifically promote L. monocytogenes transcytosis across the basal face of epithelial monolayers, which may contribute to the crossing of the basement membrane during placental infection.