TY - CHAP
T1 - Quantitative Fluorescence Microscopy for Detecting Mammalian Rab Vesicles within the Parasitophorous Vacuole of the Human Pathogen Toxoplasma gondii
AU - Romano, Julia D.
AU - Hartman, Eric J.
AU - Coppens, Isabelle
N1 - Publisher Copyright:
© 2021, The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.
PY - 2021
Y1 - 2021
N2 - Fluorescence microscopy and image analysis are powerful techniques to examine the distribution and interactions of different cellular compartments, including mammalian organelles with intravacuolar pathogens. Toxoplasma gondii is an obligate intracellular protozoan parasite that forms a membrane-bound compartment, the parasitophorous vacuole (PV), upon invasion of mammalian cells. From within the PV, the parasite interacts with many host organelles (without fusion), redirects host vesicles decorated with Rab GTPases to the PV, and internalizes many of these nutrient-filled Rab vesicles into the PV. Here, we report a method to distinguish the host Rab vesicles that are exclusively trapped in the Toxoplasma PV from those localized along the edge of the vacuole. Such a discrimination between the two Rab vesicle populations (inside versus outside of the PV) allows the selective characterization of the intra-PV Rab vesicles, for example, number per PV, volume, and distance from the PV centroid, as well as comparisons between wild-type and mutant Toxoplasma.
AB - Fluorescence microscopy and image analysis are powerful techniques to examine the distribution and interactions of different cellular compartments, including mammalian organelles with intravacuolar pathogens. Toxoplasma gondii is an obligate intracellular protozoan parasite that forms a membrane-bound compartment, the parasitophorous vacuole (PV), upon invasion of mammalian cells. From within the PV, the parasite interacts with many host organelles (without fusion), redirects host vesicles decorated with Rab GTPases to the PV, and internalizes many of these nutrient-filled Rab vesicles into the PV. Here, we report a method to distinguish the host Rab vesicles that are exclusively trapped in the Toxoplasma PV from those localized along the edge of the vacuole. Such a discrimination between the two Rab vesicle populations (inside versus outside of the PV) allows the selective characterization of the intra-PV Rab vesicles, for example, number per PV, volume, and distance from the PV centroid, as well as comparisons between wild-type and mutant Toxoplasma.
KW - Fluorescence imaging
KW - Host–pathogen interaction
KW - Parasitophorous vacuole
KW - Rab GTPase
KW - Toxoplasma gondii
KW - Vesicle trafficking
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U2 - 10.1007/978-1-0716-1346-7_21
DO - 10.1007/978-1-0716-1346-7_21
M3 - Chapter
C2 - 34453726
AN - SCOPUS:85114328881
T3 - Methods in Molecular Biology
SP - 295
EP - 305
BT - Methods in Molecular Biology
PB - Humana Press Inc.
ER -