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"Phagosome Closure Assay" to Visualize Phagosome Formation in Three Dimensions Using Total Internal Reflection Fluorescent Microscopy (TIRFM)
Published on: August 26, 2016
ELECTRON MICROSCOPY OF IN VITRO ENDOCYTOSIS OF T2 PHAGE BY CELLS FROM RABBIT PERITONEAL EXUDATE
Abstract:
Macrophages from rabbit peritoneal exudate cells incubated in vitro with T(2) bacteriophage from 10 up to 120 minutes show phage particles adsorbed to cell membranes, in the process of being engulfed by means of rhopheocytosis, micropinocytosis, and phagocytosis, and localized within dense vacuoles, semi-dense vacuoles, and clear vacuoles of the cytoplasm. The electronmicrographs suggest that newly formed endocytic vacuoles containing phage particles fuse with one another and also fuse with dense bodies of the cytoplasm as they migrate towards the cell interior, thus yielding larger vacuoles of varying densities containing higher concentrations of phage. The polymorphonuclear cells present in a small proportion in the peritoneal exudate cells also endocytosed phage particles. The T(2) particles are found in large cytoplasmic vacuoles surrounded by an electron-opaque material presumably derived from cytoplasmic granules. No disintegration of T(2) phage within the macrophage following incubations up to 120 minutes could be demonstrated; however, disrupted phage particles were noted within cytoplasmic vacuoles of polymorphonuclear leucocytes after 15 minutes' incubation.
Insights
Rabbit macrophages engulf T(2) bacteriophage via multiple endocytic pathways, forming larger vacuoles. Polymorphonuclear cells also internalize phage, showing early disruption, unlike macrophages.
Area of Science:
- Cell Biology
- Microbiology
- Immunology
Background:
- Macrophages are key immune cells involved in pathogen clearance.
- Bacteriophages are viruses that infect bacteria and are studied for their biological mechanisms.
Purpose of the Study:
- To investigate the cellular mechanisms of T(2) bacteriophage uptake by macrophages.
- To characterize the intracellular fate of bacteriophages within macrophages and polymorphonuclear cells.
Main Methods:
- In vitro incubation of rabbit peritoneal exudate cells with T(2) bacteriophage.
- Electron microscopy to visualize phage-host cell interactions and intracellular localization.
- Time-course analysis of phage uptake and intracellular processing.
Main Results:
- Macrophages demonstrated phage adsorption to cell membranes and uptake via rhopheocytosis, micropinocytosis, and phagocytosis.
- Engulfed phages were localized in cytoplasmic vacuoles, which appeared to fuse and increase in size and phage concentration.
- Polymorphonuclear cells also endocytosed phages, with disrupted particles observed within 15 minutes, unlike macrophages.
Conclusions:
- Macrophages efficiently internalize T(2) bacteriophage through various endocytic routes.
- Intracellular phage processing in macrophages involves vacuole fusion, but no significant phage disintegration was observed within 120 minutes.
- Polymorphonuclear cells exhibit a more rapid degradation of T(2) bacteriophage compared to macrophages.
