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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

Macropinocytosis is a highly conserved endocytic process initiated by the formation of F-actin-rich sheet-like membrane projections, also known as membrane ruffles. Increased rate of macropinocytotic solute internalization has been implicated in various pathological conditions. This protocol presents a method to quantify membrane ruffle formation in vitro using scanning electron microscopy.

Abstract

Membrane ruffling is the formation of motile plasma membrane protrusions containing a meshwork of newly polymerized actin filaments. Membrane ruffles may form spontaneously or in response to growth factors, inflammatory cytokines, and phorbol esters. Some of the membrane protrusions may reorganize into circular membrane ruffles that fuse at their distal margins and form cups that close and separate into the cytoplasm as large, heterogeneous vacuoles called macropinosomes. During the process, ruffles trap extracellular fluid and solutes that internalize within macropinosomes. High-resolution scanning electron microscopy (SEM) is a commonly used imaging technique to visualize and quantify membrane ruffle formation, circular protrusions, and closed macropinocytic cups on the cell surface. The following protocol describes the cell culture conditions, stimulation of the membrane ruffle formation in vitro, and how to fix, dehydrate, and prepare cells for imaging using SEM. Quantification of membrane ruffling, data normalization, and stimulators and inhibitors of membrane ruffle formation are also described. This method can help answer key questions about the role of macropinocytosis in physiological and pathological processes, investigate new targets that regulate membrane ruffle formation, and identify yet uncharacterized physiological stimulators as well as novel pharmacological inhibitors of macropinocytosis.

Introduction

Macropinocytosis is an endocytic process responsible for internalizing a large amount of extracellular fluid and its content via the formation of dynamic and actin-driven plasma membrane protrusions called membrane ruffles1. Many of these membrane ruffles form cups that close and fuse back onto the cell and separate from the plasma membrane as large, heterogeneous intracellular endosomes also known as macropinosomes1. Although macropinocytosis is induced by growth factors such as macrophage colony-stimulating factor (M-CSF) and epidermal growth factor (EGF) in a wide range of cell types, an additional unique, calcium-dep....

Protocol

NOTE: The following is a general protocol used to quantify membrane ruffle formation in RAW 264.7 macrophages using SEM microscopy. Optimization may be required for different cell types.

1. Cell line and cell culture

  1. Grow RAW 264.7 macrophages in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% (vol/vol) heat-inactivated Fetal Bovine Serum (FBS), 100 IU/mL of penicillin and 100 µg/mL streptomycin in a humidified incubator at 37 °C and 5% CO2. R.......

Representative Results

Here, we describe the results from the presented technique. Representative SEM images shown in Figure 1 demonstrate membrane ruffle formation in RAW 264.7 macrophages following treatment with PMA and M-CSF. Images were first captured at a magnification of 3,500x for quantification purposes and then at higher levels of magnification (8,500x to 16,000x) to show the plasma membrane at greater details. Pretreatment of macrophages with the macropinocytosis inhibitor EIPA attenuated membrane ruffl.......

Discussion

The present SEM imaging protocol provides a tool to visualize and quantify membrane ruffle formation, circular protrusions, and macropinocytic cups on the cell surface in vitro. Although the current protocol focuses on macrophages, studies have shown that membrane ruffle formation also occurs on various other cell types including dendritic cells, fibroblasts, neurons, and cancer cells11,12,14,

Acknowledgements

The authors thank Libby Perry (Augusta University) for her help with the SEM sample preparation. This work was supported by the National Institutes of Health [R01HL139562 (G.C.) and K99HL146954 (B.S.)] and American Heart Association [17POST33661254 (B.S.)].

....

Materials

NameCompanyCatalog NumberComments
0.5% Trypsin-EDTAGibco15400-054
2% GlutaraldehydeElectron Microscopy Sciences16320
4% ParaformaldehydeSanta Cruz Biotechnology281692
5-(N-ethyl-N-isopropyl)-AmilorideSigma Life ScienceA3085
Accuri C6 Flow Cytometer
Carbon Adhesive TabsElectron Microscopy Sciences77825-09
Dimethyl SulfoxideCorning25-950-CQC
Dulbecco's Modified Eagle MediumCytiva Life SciencesSH30022.01
Falcon 24-well Clear Flat Bottom TC-treated Multiwell Cell Culture PlateFalcon353047
Fetal Bovine SerumGemini Bio900-108
FitC-dextranThermo Fisher ScientificD1823
FM 4-64Thermo Fisher ScientificT13320
HERAcell 150i CO2 incubatorThermo Fisher Scientific51026282
Hummer Model 6.2 Sputter CoaterAnatech USA58565
JSM-IT500HR scanning electron microscope
Microscope Cover GlassThermo Fisher Scientific12-545-82
Pen StrepGibco15140-122
phorbol 12-myristate 13-acetateMillipore Sigma524400
RAW 264.7 macrophageATCCATCC TIB-71
Recombinant Human M-CSFPeprotech300-25
Samdri-790 Critical Point DryerTousimis Research Corporation8778B
SEM Aluminum Specimen MountsElectron Microscopy Sciences75220
Sodium CacodylateElectron Microscopy Sciences12300
Texas red-dextraThermo Fisher ScientificD1864
Trypan Blue SolutionThermo Fisher Scientific15250061
Zeiss LSM 780 confocal microscope

References

  1. Bohdanowicz, M., Grinstein, S. Role of phospholipids in endocytosis, phagocytosis, and macropinocytosis. Physiological Reviews. 93 (1), 69-106 (2013).
  2. Canton, J. Macropinocytosis: New....

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