TY - JOUR
T1 - Visualizing Membrane Ruffle Formation using Scanning Electron Microscopy
AU - Ahn, Wonmo
AU - Singla, Bhupesh
AU - Marshall, Brendan
AU - Csányi, Gábor
N1 - Funding Information:
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.)].
Publisher Copyright:
© 2021 JoVE Journal of Visualized Experiments.
PY - 2021/5
Y1 - 2021/5
N2 - 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.
AB - 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.
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U2 - 10.3791/62658
DO - 10.3791/62658
M3 - Article
C2 - 34125102
AN - SCOPUS:85108196823
VL - 2021
JO - Journal of visualized experiments : JoVE
JF - Journal of visualized experiments : JoVE
SN - 1940-087X
IS - 171
M1 - e62658
ER -