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 distinctive, large (>0.2 mm diameter) vacuolar-like macropinosomes, the non-selective nature of fluid and solute uptake and the accompanying expanses of plasma membrane that are opportunistically internalized into macropinosomes³. The non-selective nature of this uptake means that sorting and recycling must occur rapidly to rescue essential soluble and plasma membrane proteins by recycling them back to the cell surface. Much of the material taken into macropinosomes is destined for degradation in lysosomes, and it is funneled into endo/lysosomal pathways through macropinosome maturation and fusion with other endosomes. Plasma membrane proteins can also be recycled back to the cell surface from macropinosomes. There is intense interest in the cancer field for understanding the process of macropinocytosis, which is activated in cancer cells and helps to sustain their nutrition and proliferation^2,4,5,6. Macropinocytosis occurs both constitutively and can be further induced upon receptor-mediated stimulation in immune cells, where macropinosomes host receptor signaling, contribute to endo/lysosomal processing for antigen presentation and provide an entry portal for a variety of viruses, bacteria, and other pathogens^3,7.

Macropinosomes have few distinctive or unique markers. They are most easily identified during their formation at the base of dramatic, actin-rich cell surface ruffles, which close to engulf fluid⁸. Immediately after closure, the F-actin around macropinosomes is depolymerized, and the macropinosomes themselves undergo dynamic changes associated with homo- and heterotypic fusion, tubulation, and shrinkage⁹. Macropinosomes are also difficult to identify at an ultrastructural level since they have no characteristic coats or other features and appear simply as vacuoles of varying sizes. Without definitive membrane markers, macropinosomes are most readily and traditionally defined by their fluid cargo, which experimentally is through the use of fluorescent dextran. Dextran is a glucose-derived complex polysaccharide that can be coupled to a large array of fluorescent or electron-dense markers and added to the medium for uptake by cells or perfused for uptake in tissues. Moreover, large molecular weight (MW) dextran (70 kDa) is now regarded as a size-selective cargo for macropinosomes since it does not enter other, smaller endocytic vesicles, and it has become the most common label for macropinocytosis^10,11. Viewing macropinocytosis typically involves incubating cells with a pulse of fluorescent dextran and using fixed or live cells for fluorescence imaging to view the bolus of dextran labeling inside cells in macropinosomes. Due to their large size, unlabeled macropinosomes can also be viewed in some cell types using phase contrast or bright-field microscopy, wherein macropinosomes appear as large white empty vacuoles. Some contextual information is gained from the phase-contrast images of the cells, and these images can then additionally be overlayed with fluorescence images to depict fluorescent dextran uptake^12,13. Dextrans of different sizes and with distinct labels can be used to monitor fluid uptake into multiple cellular and endocytic pathways^10,14,15 and dextran can be applied to cell cultures or perfused into mice for intra-vital imaging¹⁶ or even injected into fly embryos¹⁷ for live imaging.

Some of the difficulties incurred in using dextran to track macropinosomes include its leakage out of macropinosomes after fixation or permeabilization of cells, it's dilute or hard-to-detect levels in cells that do not perform aggressive macropinocytosis, and its dynamic deployment and dispersion inside cells during macropinosome maturation³. These issues are compounded in most experiments which are designed to track the uptake of a single pulse of dextran added to cells and then washed off.

Instead, this paper describes the advantages of applying fluorescent dextran to the cells and leaving it in the medium during live imaging to record continuous uptake into cells. This amplifies the dextran signal in macropinosomes and subsequent endosomes showing the whole pathway of maturation. Viewing labeled macropinosomes against the unlabeled (black) interior of the cells, as a high contrast inverse setting, reveals all the features of the macropinosomes and, conversely, outside the cells, the highly fluorescent medium highlights the dynamic ruffling of the cell surface. This method describes the live imaging of dextran for light microscopic, confocal imaging, and for its detection after cell fixation by correlative light and electron microscopy (CLEM). The imaging included here was performed on different cell types to demonstrate the broad applicability of these approaches, including activated macrophages and microglial cells, where macropinocytosis is very active and rapid, and in cancer cells where macropinocytosis is relatively less abundant.

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 plasmid transfection (Day 1) - Optional

NOTE: Different fluorescently-tagged proteins and probes can be expressed transiently or stably in cloned cell lines to specifically label actin ruffles and compartments in the endocytic pathway such as early endosomes, late endosomes, or lysosomes.

1. Transfect the cells using a lipid-based transfection kit according to the manufacturer's instruction, 1 day post plating in a tissue culture hood.
2. Dilute 2 µg of endotoxin-free purified DNA plasmid in 125 µL of minimum essential media (reduced serum). Mix gently and let it stand for 5 min at room temperature.
3. Dilute 5 µL of transfection agent in 125 µL of minimum essential media (reduced serum). Mix gently and let it stand for 5 min at room temperature.
4. Combine the diluted DNA plasmid and the diluted transfection reagent and incubate for another 10 min before adding dropwise to cells.
5. Incubate the cells with transfection complex at 37 °C in a humidified 5% CO2 incubator for 3-4 h, before replacing with fresh full culture medium.
6. Use the transfected cells the next day for experiments or subject to limited dilution cloning to obtain stably expressing cell lines.

3. Visualization of endocytic vesicles and macropinosomes (Day 2)

NOTE: Fluorescent dextrans (Dextran Alexa Fluor 488/647 at 10 kDa MW, and Dextran Oregon Green 488/Tetramethylrhodamine at 70 kDa MW, Anionic, Lysine fixable) of different molecular weights are used to monitor fluid-phase uptake into a range of endocytic pathways¹⁴. 70 kDa dextran for macropinosomes and 10 kDa for all pathways.

1. Prepare dextran(s) as a 2x concentrated suspension at 200-500 ng/mL in pre-warmed culture medium for addition to cells.
2. OPTIONAL: Experiments may require prior or simultaneous addition of supplements or drugs to influence endocytic activity. For instance, pre-treat macrophages and microglial cells with growth factors (e.g., 200 ng/mL CSF-1) or activating ligands (e.g., LPS 100 ng/mL or 300 ng/mL CpG) to enhance ruffling and macropinocytosis. Culture the cancer cells in an appropriate serum-free medium for 12-16 h before dextran uptake in the complete medium. Refer to step 1.2 for recommended cell density.
3. To prepare for live imaging, position the cells grown in glass-bottom dishes on the stage of an inverted confocal microscope fitted with 37 °C heating pad and a 5% CO₂ humidified incubator.
4. Aspirate the excess medium leaving 500 µL of the complete medium in the glass bottom dish.
5. Find a suitable region of cells and set focus. For time-lapse imaging, pre-select the appropriate fluorescence lasers, filters, and settings before acquisition. Use the following settings to resolve individual cells and macropinosomes.
   1. Image the region of interest on an inverted confocal microscope with a 63x 1.4 NA oil immersion Plan Apochromat objective.
   2. Select 512 x 512 for image size, bidirectional scanning for fast imaging.
   3. Focus on an individual focal plane of the cells. Set to capture as a single slice or set an optical slice thickness (Nyquist) of approximately 0.3 µm to combine as Z-stacks.
   4. Select time-lapse with 5 s interval, for a duration between 20-45 min depending on specific cell types.
   5. Select hardware auto-focusing/focus tracking to aid in image stabilization during capture for an appropriate duration according to the specific cell types.
   6. Add an equal volume of 2x dextran spiked medium and start live image capture immediately for macrophages.Wait 20 min before imaging the slower uptake in MDA-MB 231 cells.
      ​NOTE: With this method, the BV2 cells with two fluorophore channels were successfully imaged on a single plane with maximum speed tracking, continuously for 45 min upon addition of the dextran mixture. To detect early macropinosomes in RAW 264.7 cells, where macropinocytosis occurs very rapidly, acquire a single slice (for fast-imaging) or Z stack of 4-6 slices (for 3D capture) and time-lapse with 5 s interval for 20 min upon the addition of dextran. For MDA-MB 231 cells, acquire optimal Z-stacks between 10-15 slices, with 5 s intervals for 40 mins.

4. Correlative light and electron microscopy (CLEM) (Day 3) - Optional

1. Prepare cells according to steps 1, 2, and 3 of the protocol with adaptations. In brief, grow 0.2 x 10⁶ cells/mL RAW264.7 cells on gridded glass-bottom dishes (35 mm Dish, No. 1.5 Gridded coverslip, 14 mm glass diameter, uncoated), transfect with a fluorescent plasmid (e.g., mCherry-2xFYVE) and leave overnight before the addition of dextran-488 (70 kDa) for 20 mins at 37 °C, 5% CO₂ humidified incubator.
2. Wash the cells swiftly with 1 mL of ice-cold PBS and fix with 0.1% glutaraldehyde.
3. Acquire the fixed cell images of the dextran-488 and mCherry-2xFYVE on a confocal microscope.
4. Capture bright-field images to identify the grid positions before the dishes are processed for transmission electron microscopy (EM) as previously described¹⁸. In brief, further, fix cells in 2.5% glutaraldehyde, postfix in 1% reduced osmium tetroxide, en-bloc stain with 2% uranyl acetate, and then dehydrate through a series of ethanol before embedding in LX112 resin. Excise the gridded locations from the block and collect ultra-thin sections using an ultra-microtome. Capture images on an electron microscope at 80 kV using appropriate imaging system software.

5. Data processing and visualization (Day 4)

NOTE: FIJI-an open-source image analysis package-is used for visualization and analysis³³.

1. Duplicate the raw data for processing to comply with F.A.I.R. data principles, i.e., that research data is Findable, Accessible, Interoperable, and Reusable.
2. Adjust the brightness and contrast for image sets equally using the histogram data.
3. Display the images as maximum intensity projections from Z-stack images acquired.
4. Insert scale bar for image sets.
5. Crop/Rotate FOV data (optional). Create representative panels for the time series data.
6. For further analysis and quantification, quantify the fluorescence intensity of dextran taken up into cells as a measure of macropinocytosis activity. To confine the analysis to individual cells, use the imaging method described here.
   1. Invert the LUT to flip the color for cell areas devoid of fluorescence to perform thresholding.
   2. Segment the foreground object and create a cell mask to identify individual cells.
   3. Use the cell mask in conjunction with a method¹⁹ previously published that identifies and measures individual macropinosomes. Use these metrics to quantify macropinocytosis, including size, number, and total uptake per cell.

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

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	-