Live Fluorescence, Inverse Imaging of Cell Ruffling, and Macropinocytosis

Summary

This protocol demonstrates dextran imaging in live cells using continuous uptake and inverse images to optimize visualization of ruffling, macropinosome maturation, and analysis of dextran and other cell labelings.

Abstract

Macropinocytosis is a highly conserved but still incompletely understood process that is essential for the uptake and ingestion of fluid, fluid-phase nutrients and other material in cells. The dramatic extension of cell surface ruffles, their closure to form macropinosomes, and the maturation of internalized macropinosomes are key events in this pathway that can be difficult to capture using conventional confocal imaging based on tracking a bolus of fluorescent cargo. Fluorescent dextrans are commonly used experimentally as fluid phase markers for macropinosomes and for other endocytic pathways. A method the lab has adopted to optimize the imaging of dextran uptake involves using live imaging of cells bathed in high concentrations of fluorescent dextran in the medium, with the unlabeled cells appearing in relief (as black). The cell ruffles are highlighted to visualize ruffle closure, and internalized macropinosomes appear as fluorescent vacuoles in the cell interior. This method is optimal for visualizing macropinosome features and allows for easy segmentation and quantification. This paper describes dual-labeling of pathways with different sized dextrans and the co-expression of lipid probes and fluorescent membrane proteins to demark macropinosomes and other endosomes. The detection of internalized dextran at an ultrastructural level using correlative light and electron microscopy (CLEM) is also demonstrated. These cell processes can be imaged using multiple live imaging modalities, including in 3D. Taken together, these approaches optimize macropinosome imaging for many different settings and experimental systems.

Introduction

Most vertebrate cell types share the innate capacity for the non-selective uptake of fluid with predecessors throughout evolution, dating back to single-cell amoeba¹. This highly conserved process of fluid-phase uptake by macropinocytosis (big drinking) is used by amoeba primarily for nutrient acquisition¹, while in vertebrate cells, it can similarly be a supply route for obtaining nutrients under stress, and it is used by immune cells for the sampling and surveillance of tissue environments². Macropinocytosis has features that distinguish it from other forms of endocytosis, including the distinct....

Protocol

1. Preparation of cells on 35 mm glass-bottom dishes (Day 0)

1. Maintain and passage cell lines in complete medium supplemented with 10% heat-inactivated (for RAW264.7) or regular fetal calf serum and 1% L-glutamine, at 37 °C in humidified 5% CO₂ incubator.
2. Plate the appropriate number of cells to achieve 60% confluency in 24 h, on 35 mm glass-bottom dishes.
   ​NOTE: The recommended cell density is between 2 mL of 0.15 x 10⁶ cells/mL to 0.25 x 10⁶ cells/mL for the RAW264.7, BV2 microglial cells, and MDA-MB 231 breast cancer cells described here.

2. Fluorescence DNA pla....

Results

The approach of imaging unlabeled live cells immersed in a high concentration of fluorescent dextran has several advantages over conventional imaging techniques for tracking macropinocytosis. By presenting the images in relief, the cell bodies appear as black and allow improved visualization of the dramatic cell surface ruffling against a bright, fluorescent background, followed by fluid-phase internalization of TMR dextran (70 kDa)-filled macropinosomes as depicted in Figure 1A. In this max.......

Discussion

This paper describes variations on more traditional ways of using dextran labeling to track macropinocytosis, based on live imaging of cells bathed continuously in fluorescent dextran(s) and visualizing the uptake on the black background of unlabeled cells. The optimized protocol provides the means to distinguish between different intracellular vesicles and allows a spatial-temporal tracking of multiple macropinocytotic and endocytic cargos and proteins. The method of immersing cells in a dextran medium to monitor the dy.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
2% Osmium Tetroxide	ProSciTech	EMS19192
647-Transferrin	Molecular Probes, Invitrogen	T23366
BV2 cells	Gift kindly given to us by Dr Liviu Bodea (Queensland Brain Institute)	-
CpG (Class B) B	Integrated DNA Technologies	Custom Order
Dextran Alexa Fluor 488  at 10 kDa MW	Life Technology Australia Pty Ltd	D22910
Dextran Alexa Fluor 647  at 10 kDa MW	Life Technology Australia Pty Ltd	D22914
Dextran Oregon Green 488 at 70 kDa MW	Life Technology Australia Pty Ltd	D1818
Dextran Tetramethylrhodamine at 70 kDa MW	Life Technology Australia Pty Ltd	D7173
DMEM medium with sodium pyruvate and L-glutamine	Gibco Invitrogen	#11995
Fetal Calf serum	Interpath Services Pty Ltd	SFBS-F
Glutaraldehyde aqueous solution, EM Grade 25%	ProSciTech	C002
Jeol 1011 electron microscope	JEOL	-
L-Glutamine	Gibco Invitrogen	25030081
Lipofectamine 2000	Gibco Invitrogen	11668019
MatTek  Glass Bottom Dish 35 mm, uncoated grid	MatTek Corporation	P35G-2-14-CGRD
MatTek glass bottom dishes 35 mm uncoated	MatTek Corporation	P35G-1.5-14C
MDA-MB 231 cells	ATCC	HTB-26
Opti-MEM reduced serum medium	Thermo Fisher Scientific	31985088
Plasmid mCherry-2XFYVE	Gift kindly given to us by Dr Frederic Meunier (University of Queensland)	-
Plasmid mCherry-PLCδ-PH	Gift kindly given to us by Dr Frederic Meunier (University of Queensland)	-
Raw264.7 cells	ATCC	TIB-71
RPMI medium 1640,without L-glutamine	Gibco Invitrogen	#21870
Ultrapure LPS	Jomar Life Research Pte Ltd	TLR-3PELPS
Uranyl Acetate	ProSciTech	C079
Zeiss inverted LSM880 confocal microscope	Zeiss	-