Macropinocytosis refers to the nonselective uptake of extracellular molecules into many different types of eukaryotic cells within large fluid-filled vesicles named macropinosomes. Macropinosomes... Show moreMacropinocytosis 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. Show less
Schink, K.O.; Tan, K.W.; Spangenberg, H.; Martorana, D.; Sneeggen, M.; Stévenin, V.; ... ; Stenmark, H. 2021
Uptake of large volumes of extracellular fluid by actin-dependent macropinocytosis has an important role in infection, immunity and cancer development. A key question is how actin assembly and... Show moreUptake of large volumes of extracellular fluid by actin-dependent macropinocytosis has an important role in infection, immunity and cancer development. A key question is how actin assembly and disassembly are coordinated around macropinosomes to allow them to form and subsequently pass through the dense actin network underlying the plasma membrane to move towards the cell center for maturation. Here we show that the PH and FYVE domain protein Phafin2 is recruited transiently to newly-formed macropinosomes by a mechanism that involves coincidence detection of PtdIns3P and PtdIns4P. Phafin2 also interacts with actin via its PH domain, and recruitment of Phafin2 coincides with actin reorganization around nascent macropinosomes. Moreover, forced relocalization of Phafin2 to the plasma membrane causes rearrangement of the subcortical actin cytoskeleton. Depletion of Phafin2 inhibits macropinosome internalization and maturation and prevents KRAS-transformed cancer cells from utilizing extracellular protein as an amino acid source. We conclude that Phafin2 promotes macropinocytosis by controlling timely delamination of actin from nascent macropinosomes for their navigation through the dense subcortical actin network. Show less
