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Purification of infection-associated macropinosomes by magnetic isolation for proteomic characterization
Virginie Stévenin1,2,3, Quentin Giai Gianetto4,5, Magalie Duchateau4
1Institut Pasteur, Dynamics of Host-Pathogen Interactions Unit and CNRS UMR 3691, Paris, France. virginie.stevenin@ens-cachan.fr.
Abstract:
Macropinocytosis refers to the nonselective uptake of extracellular molecules into many different types of eukaryotic cells within large fluid-filled vesicles named macropinosomes. Macropinosomes are relevant for a wide variety of cellular processes, such as antigen sampling in immune cells, homeostasis in the kidney, cell migration or pathogen uptake. Understanding the molecular composition of the different macropinosomes formed during these processes has helped to differentiate their regulations from other endocytic events. Here, we present a magnetic purification protocol that segregates scarce macropinosomes from other endocytic vesicles at a high purity and in a low-cost and unbiased manner. Our protocol takes advantage of moderate-sized magnetic beads of 100 nm in diameter coupled to mass-spectrometry-based proteomic analysis. Passing the cell lysate through a table-top magnet allows the quick retention of the bead-containing macropinosomes. Unlike other cell-fractionation-based methodologies, our protocol minimizes sample loss and production cost without prerequisite knowledge of the macropinosomes and with minimal laboratory experience. We describe a detailed procedure for the isolation of infection-associated macropinosomes during bacterial invasion and the optimization steps to readily adapt it to various studies. The protocol can be performed in 3 d to provide highly purified and enriched macropinosomes for qualitative proteomic composition analysis.
Insights
We developed a low-cost magnetic bead protocol to isolate pure macropinosomes, essential vesicles for cellular processes like immune response and pathogen uptake. This method enables detailed proteomic analysis of macropinosome composition.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Macropinocytosis is a nonselective cellular uptake process involving large vesicles called macropinosomes.
- Macropinosomes play crucial roles in diverse cellular functions, including antigen sampling, kidney homeostasis, cell migration, and pathogen entry.
- Understanding macropinosome molecular composition is key to differentiating their regulation from other endocytic pathways.
Purpose of the Study:
- To present a novel, cost-effective, and unbiased magnetic purification protocol for isolating macropinosomes.
- To enable high-purity segregation of macropinosomes from other endocytic vesicles for subsequent analysis.
- To facilitate the study of macropinosome composition in various cellular contexts, such as during bacterial invasion.
Main Methods:
- Utilized 100 nm magnetic beads coupled with mass-spectrometry-based proteomic analysis.
- Employed a magnetic field to rapidly retain macropinosome-bound beads from cell lysates.
- Developed a protocol adaptable for isolating infection-associated macropinosomes during bacterial invasion.
Main Results:
- Achieved high purity and yield of macropinosomes using the magnetic bead-based protocol.
- Demonstrated a low-cost and unbiased method minimizing sample loss compared to traditional techniques.
- Successfully isolated infection-associated macropinosomes for proteomic analysis.
Conclusions:
- The presented magnetic purification protocol offers an efficient and accessible method for macropinosome isolation.
- This technique allows for detailed qualitative proteomic analysis of macropinosomes, advancing understanding of their function.
- The protocol is adaptable to various research needs, including the study of pathogen-host interactions.
