Cell Labeling with Magneto-Endosymbionts and the Dissection of the Subcellular Location, Fate, and Host Cell Interactions.

Autor: Lee KR; Bell Biosystems, San Francisco, CA, USA., Wakeel A; Bell Biosystems, San Francisco, CA, USA., Chakraborty P; Bell Biosystems, San Francisco, CA, USA., Foote CS; Bell Biosystems, San Francisco, CA, USA., Kajiura L; Bell Biosystems, San Francisco, CA, USA., Barrozo JC; Bell Biosystems, San Francisco, CA, USA., Chan AC; Bell Biosystems, San Francisco, CA, USA., Bazarov AV; Bell Biosystems, San Francisco, CA, USA., Spitler R; Pediatrics-Neonatology and Molecular Imaging Program (MIPS), Stanford University, Palo Alto, CA, USA., Kutny PM; Microinjection Service, Genetic Engineering Technologies, The Jackson Laboratory, Bar Harbor, ME, USA., Denegre JM; The Jackson Laboratory, Bar Harbor, ME, USA., Taft RA; Division of Reproductive Technologies, The Jackson Laboratory, Bar Harbor, ME, USA., Seemann J; Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, TX, USA., Rice BW; Bell Biosystems, San Francisco, CA, USA., Contag CH; Pediatrics-Neonatology and Molecular Imaging Program (MIPS), Stanford University, Palo Alto, CA, USA., Rutt BK; Radiology Department and Molecular Imaging Program (MIPS), Stanford University, Palo Alto, CA, USA., Bell CB 3rd; Bell Biosystems, San Francisco, CA, USA. caleb@bellbiosystems.com.
Jazyk: angličtina
Zdroj: Molecular imaging and biology [Mol Imaging Biol] 2018 Feb; Vol. 20 (1), pp. 55-64.
DOI: 10.1007/s11307-017-1094-6
Abstrakt: Purpose: The purposes of this study are to characterize magneto-endosymbiont (ME) labeling of mammalian cells and to discern the subcellular fate of these living contrast agents. MEs are novel magnetic resonance imaging (MRI) contrast agents that are being used for cell tracking studies. Understanding the fate of MEs in host cells is valuable for designing in vivo cell tracking experiments.
Procedures: The ME's surface epitopes, contrast-producing paramagnetic magnetosomal iron, and genome were studied using immunocytochemistry (ICC), Fe and MRI contrast measurements, and quantitative polymerase chain reaction (qPCR), respectively. These assays, coupled with other common assays, enabled validation of ME cell labeling and dissection of ME subcellular processing.
Results: The assays mentioned above provide qualitative and quantitative assessments of cell labeling, the subcellular localization and the fate of MEs. ICC results, with an ME-specific antibody, qualitatively shows homogenous labeling with MEs. The ferrozine assay shows that MEs have an average of 7 fg Fe/ME, ∼30 % of which contributes to MRI contrast and ME-labeled MDA-MB-231 (MDA-231) cells generally have 2.4 pg Fe/cell, implying ∼350 MEs/cell. Adjusting the concentration of Fe in the ME growth media reduces the concentration of non-MRI contrast-producing Fe. Results from the qPCR assay, which quantifies ME genomes in labeled cells, shows that processing of MEs begins within 24 h in MDA-231 cells. ICC results suggest this intracellular digestion of MEs occurs by the lysosomal degradation pathway. MEs coated with listeriolysin O (LLO) are able to escape the primary phagosome, but subsequently co-localize with LC3, an autophagy-associated molecule, and are processed for digestion. In embryos, where autophagy is transiently suppressed, MEs show an increased capacity for survival and even replication. Finally, transmission electron microscopy (TEM) of ME-labeled MDA-231 cells confirms that the magnetosomes (the MRI contrast-producing particles) remain intact and enable in vivo cell tracking.
Conclusions: MEs are used to label mammalian cells for the purpose of cell tracking in vivo, with MRI. Various assays described herein (ICC, ferrozine, and qPCR) allow qualitative and quantitative assessments of labeling efficiency and provide a detailed understanding of subcellular processing of MEs. In some cell types, MEs are digested, but the MRI-producing particles remain. Coating with LLO allows MEs to escape the primary phagosome, enhances retention slightly, and confirms that MEs are ultimately processed by autophagy. Numerous intracellular bacteria and all endosymbiotically derived organelles have evolved molecular mechanisms to avoid intracellular clearance, and identification of the specific processes involved in ME clearance provides a framework on which to develop MEs with enhanced retention in mammalian cells.
Databáze: MEDLINE
Externí odkaz: