High-throughput Measurement of <em>Dictyostelium discoideum</em> Macropinocytosis by Flow Cytometry

Abstract

Large-scale non-specific fluid uptake by macropinocytosis is important for the proliferation of certain cancer cells, antigen sampling, host cell invasion and the spread of neurodegenerative diseases. The commonly used laboratory strains of the amoeba Dictyostelium discoideum have extremely high fluid uptake rates when grown in nutrient medium, over 90% of which is due to macropinocytosis. In addition, many of the known core components of mammalian macropinocytosis are also present, making it an excellent model system for studying macropinocytosis. Here, the standard technique to measure internalized fluid using fluorescent dextran as a label is adapted to a 96-well plate format, with the samples analyzed by flow cytometry using a high-throughput sampling (HTS) attachment.Cells are fed non-quenchable fluorescent dextran for a pre-determined length of time, washed by immersion in ice-cold buffer and detached using 5 mM sodium azide, which also stops exocytosis. Cells in each well are then analyzed by flow cytometry. The method can also be adapted to measure membrane uptake and phagocytosis of fluorescent beads or bacteria.This method was designed to allow measurement of fluid uptake by Dictyostelium in a high-throughput, labor and resource efficient manner. It allows simultaneous comparison of multiple strains (e.g. knockout mutants of a gene) and conditions (e.g. cells in different media or treated with different concentrations of inhibitor) in parallel and simplifies time-courses.

Coordinated Ras and Rac Activity Shapes Macropinocytic Cups and Enables Phagocytosis of Geometrically Diverse Bacteria

“…Bulk macropinocytosis was measured by flow cytometry as in [ 64 ]. For Ax2 cells and their derivatives, 5 × 10 4 cells were seeded in 50 μl HL5 medium per well of a 96 well plate, with duplicate wells for 0, 5, 10, 30, 60, 90 and 120 min time points and left for 2 h to settle.…”

Akt kinases are required for efficient feeding by macropinocytosis inDictyostelium

“…All cell uptake measurements were performed as described (Williams and Kay, 2018a; Williams and Kay, 2018b). Briefly, cells were cultivated on SM agar with K. aerogenes bacteria, harvested, washed free of bacteria and inoculated in the indicated nutrient medium to 1×10 5 cells/ml, with triplicate 50 µl samples in wells of a 96-well plate.…”

“…Macropinocytosis in the model organism Dictyostelium discoideum is highly analogous to macropinocytosis in mammalian cells, however the situation is simplified as growth factor signalling and RTKs are not present. In Dictyostelium macropinocytosis is used for nutrient acquisition and accounts for the vast majority of fluid uptake, making accurate quantification simple (Hacker et al, 1997; Williams and Kay, 2018a). Additionally, both forward and reverse genetic approaches can be employed (Bloomfield et al, 2015; Paschke et al, 2018).…”

Living on soup: macropinocytic feeding in amoebae

“…This is not true in most mammalian cells, where only a small fraction of fluid uptake is by macropinocytosis, and microscopic assays have to be used instead (Commisso et al, 2014). The standard assay, in which fluorescent dextran uptake is measured by fluorimetry, has been adapted to high-throughput flow cytometry, allowing hundreds of samples to be assayed per day Kay, 2018b, Williams andKay, 2018a).…”