1. Quantitative Mapping of Endosomal DNA Processing by Single Molecule Counting
- Author
-
Nils G. Walter, Konstantinos Tsekouras, Steve Pressé, Ved Prakash, Muthukumaran Venkatachalapathy, Yamuna Krishnan, and Laurie A. Heinicke
- Subjects
Fluorescence-lifetime imaging microscopy ,Endosome ,Endocytic cycle ,02 engineering and technology ,Endosomes ,010402 general chemistry ,01 natural sciences ,Catalysis ,Cell Line ,chemistry.chemical_compound ,03 medical and health sciences ,Mice ,Immune system ,Fluorescence microscope ,Animals ,030304 developmental biology ,Fluorescent Dyes ,0303 health sciences ,Innate immune system ,010405 organic chemistry ,Chemistry ,Macrophages ,General Chemistry ,General Medicine ,DNA ,Carbocyanines ,021001 nanoscience & nanotechnology ,Photobleaching ,Single Molecule Imaging ,0104 chemical sciences ,Cell biology ,Hydrazines ,Microscopy, Fluorescence ,0210 nano-technology - Abstract
Extracellular DNA is engulfed by innate immune cells and digested by endosomal DNase II to generate an immune response. Quantitative information on endosomal stage-specific cargo processing is a critical parameter to predict and model the innate immune response. Biochemical assays quantify endosomal processing but lack organelle-specific information, while fluorescence microscopy has provided the latter without the former. Herein, we report a single molecule counting method based on fluorescence imaging that quantitatively maps endosomal processing of cargo DNA in innate immune cells with organelle-specific resolution. Our studies reveal that endosomal DNA degradation occurs mainly in lysosomes and is negligible in late endosomes. This method can be used to study cargo processing in diverse endocytic pathways and measure stage-specific activity of processing factors in endosomes.
- Published
- 2018