Visualizing Intracellular Transport of Cargo in Cultured Astrocytes

Abstract

Source: Creighton, B. A., et al. Visualizing and Analyzing Intracellular Transport of Organelles and Other Cargos in Astrocytes. J. Vis. Exp. (2019)

This video demonstrates the visualization of intracellular cargo transport in cultured astrocytes using a fluorescent acidotropic probe that selectively labels acidic endolysosomal vesicles. Time-lapse imaging and Z-stack acquisition with a confocal microscope allow tracking of fluorescently labeled cargo throughout the three-dimensional cell volume. The analysis differentiates stationary cargo from cargo moving towards the cell periphery or the nucleus, providing insights into intracellular transport dynamics.

Protocol

1. Transfection of fluorescently tagged plasmids

1. The day prior to transfection or labeling, seed astrocytes at desired density for imaging 24−48 h post-transfection. A recommended density for 24-well plates or 14 mm glass bottom wells is 2 x 104 cells/well.
   NOTE: This protocol is optimized for transfection of astrocytes plated on 12 mm glass coverslips on 24-well plates or 14 mm glass bottom wells using a lipofection-based method. Reagents should be scaled depending on the size of the culture dish in which the astrocytes are growing.
2. Dilute the lipofection reagent (Table of Materials) in reduced-serum media (Table of Materials). To optimize the ratio of lipofection reagent to DNA, test a range of adequate dilutions. For example, if using the reagent employed in this protocol (Table of Materials), dilute 2, 3, 4, and 5 μL of the lipofection reagent in 50 μL of reduced-serum media.
3. Dilute 5 μg of high purity DNA in 250 μL of reduced-serum media. Add 5 μL of lipofection enhancer reagent (Table of Materials). Combine the tube containing the lipofection reagent with an equal volume of the DNA-lipofection enhancer mix. Mix by pipetting and incubate at room temperature (RT) for 15 min.
4. Remove astrocyte culture media and add transfection mix (step 1.3) to cells dropwise. After a 6-h incubation at 37 °C and 5% CO2, replace the transfection complex with an appropriate volume of astrocyte culture media (2 mL for 14 mm glass bottom dishes). Incubate for an additional 24−72 h before proceeding to the image acquisition. Closely monitor the duration of the incubation step to achieve the best balance between protein expression and cell viability.

2. Labeling of late endosomes/lysosomes using fluorescent probes

NOTE: Some cargos can be labeled using fluorescent dyes with high affinity for cargo-specific proteins. The following example permits the labelling of late endosomes/lysosomes with a fluorescent acidotropic probe.

1. Dilute the lysosomal-labeling probe (Table of Materials) in 200 µL of astrocyte culture media to a working concentration of 1 μM and apply to astrocytes from step 1.1 at a density of 2 x 104 cells/well. Incubate for 30 min at 37 °C.
2. Wash the cells once with warm astrocyte culture media and replace with imaging media (Table of Materials). Proceed to live imaging immediately.

3. Image acquisition using a time-lapse imaging system

NOTE: Time-lapse live imaging should be done using a fluorescence microscope equipped with a high-speed camera, definite focus, incubation chamber, and a 40x oil objective with a high numeric aperture (e.g., Plan-Apochromat 1.4NA). A variety of acquisition software is available for time-lapse imaging. Selection of the microscopy system and acquisition software should be based on their availability and suitability for the goals of the particular study. Some general guidelines are provided below.

1. Place the culture chamber or dish in the proper adaptor on the microscope stage. Using epifluorescence light, select the cell(s) that express fluorescent proteins or probe to record. Adjust the fluorescent sample illumination to visualize the selected cell(s) using the digital camera. Avoid using high illumination that might cause photobleaching and phototoxicity. Adjust focus and zoom.
2. Acquire single Z-stack time-lapse series at a frequency of 1 frame every 2 s for time intervals ranging between 300 s and 500 s using the zoom and definite focus functions.
   NOTE: Trafficking dynamics (velocity, frequency of motion, etc.) will vary depending on the protein of interest, thus, the time course of acquisition may require adjustment. For instance, mitochondria are less motile than lysosomes and exhibit frequent pauses. In this instance, it is more appropriate to adjust the acquisition parameters to 1 frame every 5 s for 800 s.
3. Save time-lapse images and export them as AVI or TIFF stack files.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Dulbecco's Modified Eagle Medium	Gen Clone	25-500
Fetal Bovine Serum	Gemini	100-106	Heat-Inactivated
FIJI (Fiji is Just Image J)	NIH	Version 1.52i
Fine Tip Tweezers	F.S.T	11254-20	Style #5
Fluorescence light source	Excelitas	012-63000	X-Cite 120Q
Glass Bottom Dishes	Mattek corporation	P35G-1.5-14-C	35 mm Dish | No. 1.5 Coverslip | 14 mm Glass Diameter | Uncoated
Graefe Forceps	F.S.T	11054-10	Graefe Iris Forceps with curved tips
Green fluorescent dye that stains acidic compartments (late endosomes and lysosomes)	Life Technologies	L7526	LysoTracker Green DND 26. Pre-dissolved in DMSOS to a 1mM stock solution. Dilute to the final working concentration in the growth medium or buffer of choice.
Hank's Balanced Salt Solution (10x)	Gibco	14065-056	Magnesium and calcium free
Imaging Media	Life Technologies	A14291DJ	Live Cell Imaging Solution
Inverted Confocal Microscope	Zeiss		LSM 780
KymoToolBox			https://github.com/fabricecordelieres/IJ_KymoToolBox
Lipofection Enhancer Reagent	Life Technologies	11514015	Plus Reagent
Lipofection Reagent	Life Technologies	15338100	Lipofectamine LTX reagent
Orbital shaking incubator	New Brunswick Scientific	8261-30-1008	Innova 4230 , orbital shaking incubator with temperature and speed control
Penicillin/Strepomycin solution (100x)	Gen Clone	25-512
Phosphate Buffered Saline (10x)	Gen Clone	25-507x
Poly-D-Lysine Hydrobromide	Sigma	P7405	Dissolve in Tris buffer, pH 8.5, at 1mg/mL. Freeze for long term storage. Avoid cycles of freezing and thawing
Reduced serum medium	Gibco	31985-062	OPTI-MEM
Tissue Culture Flasks	Olympus Plastics	25-209	75 cm^2. 100% angled neck access, 0.22um hydrophobic vented filter cap
Tissue culture incubator	Thermo Scientific	51030285	HERAcell VIOS 160i, tissue culture incubator with temperature, humidity, and CO2 control
Tris-Base	Sigma	T1503	8.402 g dissolved to one liter in water with 4.829 g Tris HCl to make 0.1 M Tris buffer, pH 8.5
Tris-HCl	Sigma	T3253	4.829 g dissolved to one liter in water with 8.402 g Tris Base to make 0.1 M Tris buffer, pH 8.5