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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
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Integrin-based diffusion barrier separates membrane domains enabling the formation of microbiostatic frustrated
Michelle E Maxson1, Xenia Naj2, Teresa R O'Meara3
1Program in Cell Biology, Hospital for Sick Children, Toronto, Canada.
Elife
|March 20, 2018
Summary
Macrophages engulf long Candida albicans hyphae into tubular phagosomes. A diffusion barrier forms, separating phagosome and cell membranes, enabling antimicrobial defense against fungal growth.
Area of Science:
- Cell Biology
- Immunology
- Microbiology
Background:
- Candida albicans hyphae can evade phagocytosis due to their large size.
- Macrophages engulf portions of hyphae, forming incompletely sealed tubular phagosomes.
Purpose of the Study:
- To investigate the membrane dynamics and barrier formation in phagosomes engulfing Candida albicans hyphae.
- To understand the molecular mechanisms underlying the distinct lipid composition and separation of phagosomal and plasma membranes.
Main Methods:
- Fluorescence microscopy
- Lipid analysis
- Fluorescence recovery after photobleaching (FRAP)
- Investigating protein involvement (Syk, Pyk2/Fak, formin)
Main Results:
- Frustrated phagosomes are stabilized by F-actin cuffs via integrin activation.
- Distinct lipid compositions exist between the plasmalemma and phagosomal membrane (PtdIns(4,5)P2 absent in phagosome, PtdIns(3)P and PtdIns(3,4,5)P3 present).
- A diffusion barrier forms, maintaining phagosome identity, dependent on Syk, Pyk2/Fak, and formin-mediated actin assembly.
Conclusions:
- Macrophages generate specialized, distinct phagosomal compartments to contain Candida albicans hyphae.
- The formation of a diffusion barrier and distinct lipid domains is crucial for deploying antimicrobial mechanisms and limiting fungal growth.
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