Name: visualizing and analyzing intracellular transport of organelles and other cargos in astrocytes
Uploaded: 2019-08-28T15:00:00
Description: Watch this Scientific Journal Video about Visualizing and Analyzing Intracellular Transport of Organelles and Other Cargos in Astrocytes at JoVE.com

DNL was supported by the University of North Carolina at Chapel Hill (UNC) School of Medicine as a Simmons Scholar. TWR was supported by UNC PREP Grant R25 GM089569. Work using the UNC Neuroscience Center Microscopy Core Facility was supported, in part, by funding from the NIH-NINDS Neuroscience Center Support Grant P30 NS045892 and the NIH-NICHD Intellectual and Developmental Disabilities Research Center Support Grant U54 HD079124.

All animal procedures were performed with the approval of the University of North Carolina at Chapel Hill Animal Care and Use Committee (IACUC).
1. Brain dissection and culture of primary mouse astrocytes
NOTE: The following protocol was adapted from published methods, which follows the original procedure developed by McCarthy and deVellis13,14,15. Mixed cell cultures of McCa...

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Here, we describe an experimental approach to express, visualize, and track fluorescently tagged organelles and membrane proteins of interest using time-lapse video microscopy in high-purity primary mouse cortical MD astrocytes. We also outline a methodology for measuring particle dynamics. Direct visualization of protein and organelle dynamics in primary astrocytes provides a powerful tool to study the regulation of intracellular transport in these cells in vitro.
The methodology for establis...

Astrocytes are the most abundant cells in the adult central nervous system, where they perform unique developmental and homeostatic functions1. Astrocytes modulate synaptic development through direct contact with pre- and postsynaptic terminals as part of the tri-partite synapse, which contains neurotransmitter receptors, transporters, and cell adhesion molecules that facilitate synapse formation and neuron-astrocyte communication2. In addition, astrocytes actively control synaptic transmission and prevent neuronal excitotoxicity by rapidly removing excitatory neurotransmitters from the synaptic cleft, recycling neurotransmitters, and participating in synaptic pruning3,4,5,6. To enable these highly interactive functions, astrocytes communicate among themselves, with other glial cells, and with neurons through specialized membrane proteins, including cell adhesion molecules, aquaporins, ion channels, neurotransmitter transporters, and gap junction molecules. Astrocytes actively change the surface levels of these proteins in response to fluctuations in their intra- and extracellular environment7. Furthermore, changes in the levels and distribution of mitochondria, lipid droplets, and degradative and recycling organelles modulate energy supply, metabolite availability, and cellular clearing processes that are essential for astrocyte function and survival.
The dynamic changes in membrane protein and organelle trafficking and positioning in astrocytes are facilitated by the concerted function of motor proteins and adaptors that promote cargo motility8,9. Similarly, surface levels of membrane proteins are modulated through internalization and recycling events10. These cargos are transported via an intricate network of actin, microtubules, and possibly intermediate filaments tracks8. Studies based on immunofluorescence staining of end-binding protein 1 (EB1), which accumulates at the growing microtubule plus ends, suggest that in astrocytes bundles of microtubules radiate out from the perinucleus and extend their plus end towards the periphery11. However, a comprehensive examination of the organization and polarity of microtubules and other cytoskeletal elements using live-cell imaging is still lacking. While many of the mechanisms underlying the dynamics of organelles and membrane proteins have been extensively studied in neurons and other cell types, cargo motility in astrocytes is less well understood. Most of our current knowledge about changes in protein and organelle distribution in astrocytes is based on traditional antibody-based labeling of fixed preparation, which precludes precise spatial and temporal examination of cargo dynamics7,12.
Here, we describe a method to label membrane proteins and organelles for live imaging in high purity primary mouse astrocyte cultures. Using this protocol, we provide examples in which we track the dynamic localization of green fluorescent protein (GFP)-tagged membrane proteins in transfected astrocytes, including the gap junction protein connexin 43 (Cx43-GFP) and the excitatory amino acid transporter 1 (EAAT1-GFP). We also describe the use of a fluorescent acidotropic probe to visualize acidic organelles and follow their trafficking dynamics in live astrocytes. Finally, we demonstrate how to analyze the time-lapse data to extract and evaluate transport parameters for individual cargos.

<table>
	<tbody>
		<tr>
			<td>2.5% Trypsin (10x)</td>
			<td>Gibco</td>
			<td>15090-046</td>
			<td></td>
		</tr>
		<tr>
			<td>Benchtop Centrifuge</td>
			<td>Thermo Scientific</td>
			<td>75-203-637</td>
			<td>Sorvall ST8 Centrifuge</td>
		</tr>
		<tr>
			<td>Cell Culture Grade Water</td>
			<td>Gen Clone</td>
			<td>25-511</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Culture Microscope</td>
			<td>Zeiss</td>
			<td>WSN-AXIOVERT A1</td>
			<td>Vert.A1 inverted scope, Inverted tissue culture microscope with fluorescent capabilities</td>
		</tr>
		<tr>
			<td>Cytosine Arabinoside</td>
			<td>Sigma</td>
			<td>C1768-100MG</td>
			<td>(AraC) Dissolve lyophilized powder in sterile cell culture grade water to make a 10mM stock, aliquot and freeze for long term storage</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Sigma</td>
			<td>D9542-5MG</td>
			<td>Dissolve lyophilized powder in deionized water to a maximum concentration of 20 mg/ml, heat or sonicate as necessary. Use at 300 nM for counterstaining.</td>
		</tr>
		<tr>
			<td>Dissecting Microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td>Stemi 305</td>
		</tr>
		<tr>
			<td>Dissecting Scissors</td>
			<td>F.S.T</td>
			<td>14558-09</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium</td>
			<td>Gen Clone</td>
			<td>25-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Gemini</td>
			<td>100-106</td>
			<td>Heat-Inactivated</td>
		</tr>
		<tr>
			<td>FIJI (Fiji is Just Image J)</td>
			<td>NIH</td>
			<td>Version 1.52i</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine Tip Tweezers</td>
			<td>F.S.T</td>
			<td>11254-20</td>
			<td>Style #5</td>
		</tr>
		<tr>
			<td>Fluorescence light source</td>
			<td>Excelitas</td>
			<td>012-63000</td>
			<td>X-Cite 120Q</td>
		</tr>
		<tr>
			<td>GFAP antibody</td>
			<td>Cell Signaling</td>
			<td>3670S</td>
			<td>GFAP (GA5) mouse monoclonal antibody</td>
		</tr>
		<tr>
			<td>Glass Bottom Dishes</td>
			<td>Mattek corporation</td>
			<td>P35G-1.5-14-C</td>
			<td>35 mm Dish | No. 1.5 Coverslip | 14 mm Glass Diameter | Uncoated</td>
		</tr>
		<tr>
			<td>Graefe Forceps</td>
			<td>F.S.T</td>
			<td>11054-10</td>
			<td>Graefe Iris Forceps with curved tips</td>
		</tr>
		<tr>
			<td>Green fluorescent dye that stains acidic compartments (late endosomes and lysosomes)</td>
			<td>Life Technologies</td>
			<td>L7526</td>
			<td>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.</td>
		</tr>
		<tr>
			<td>Hank&#39;s Balanced Salt Solution (10x)</td>
			<td>Gibco</td>
			<td>14065-056</td>
			<td>Magnesium and calcium free</td>
		</tr>
		<tr>
			<td>Imaging Media</td>
			<td>Life Technologies</td>
			<td>A14291DJ</td>
			<td>Live Cell Imaging Solution</td>
		</tr>
		<tr>
			<td>Inverted Confocal Microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td>LSM 780</td>
		</tr>
		<tr>
			<td>KymoToolBox</td>
			<td></td>
			<td></td>
			<td>https://github.com/fabricecordelieres/IJ_KymoToolBox</td>
		</tr>
		<tr>
			<td>Lipofection Enhancer Reagent</td>
			<td>Life Technologies</td>
			<td>11514015</td>
			<td>Plus Reagent</td>
		</tr>
		<tr>
			<td>Lipofection Reagent</td>
			<td>Life Technologies</td>
			<td>15338100</td>
			<td>Lipofectamine LTX reagent</td>
		</tr>
		<tr>
			<td>Orbital shaking incubator</td>
			<td>New Brunswick Scientific</td>
			<td>8261-30-1008</td>
			<td>Innova 4230 , orbital shaking incubator with temperature and speed control</td>
		</tr>
		<tr>
			<td>Penicillin/Strepomycin solution (100x)</td>
			<td>Gen Clone</td>
			<td>25-512</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (10x)</td>
			<td>Gen Clone</td>
			<td>25-507x</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-D-Lysine Hydrobromide</td>
			<td>Sigma</td>
			<td>P7405</td>
			<td>Dissolve in Tris buffer, pH 8.5, at 1mg/mL. Freeze for long term storage. Avoid cycles of freezing and thawing</td>
		</tr>
		<tr>
			<td>Reduced serum medium</td>
			<td>Gibco</td>
			<td>31985-062</td>
			<td>OPTI-MEM</td>
		</tr>
		<tr>
			<td>Tissue Culture Flasks</td>
			<td>Olympus Plastics</td>
			<td>25-209</td>
			<td>75 cm^2. 100% angled neck access, 0.22um hydrophobic vented filter cap</td>
		</tr>
		<tr>
			<td>Tissue culture incubator</td>
			<td>Thermo Scientific</td>
			<td>51030285</td>
			<td>HERAcell VIOS 160i, tissue culture incubator with temperature, humidity, and CO2 control</td>
		</tr>
		<tr>
			<td>Tris-Base</td>
			<td>Sigma</td>
			<td>T1503</td>
			<td>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</td>
		</tr>
		<tr>
			<td>Tris-HCl</td>
			<td>Sigma</td>
			<td>T3253</td>
			<td>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</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.25%), phenol red</td>
			<td>Gibco</td>
			<td>25200072</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum-Driven Filter Systems</td>
			<td>Olympus Plastics</td>
			<td>25-227</td>
			<td>500 ml, PES membrane, 0.22 &#181;m</td>
		</tr>
		<tr>
			<td>Vannas scissors straight</td>
			<td>Roboz</td>
			<td>RS-5620</td>
			<td></td>
		</tr>
	</tbody>
</table>

visualizing and analyzing intracellular transport of organelles and other cargos in astrocytes

Astrocytes are among the most abundant cell types in the adult brain, where they play key roles in a multiplicity of functions. As a central player in brain homeostasis, astrocytes supply neurons with vital metabolites and buffer extracellular water, ions, and glutamate. An integral component of the &#8220;tri-partite&#8221; synapse, astrocytes are also critical in the formation, pruning, maintenance, and modulation of synapses. To enable these highly interactive functions, astrocytes communicate among themselves and with other glial cells, neurons, the brain vasculature, and the extracellular environment through a multitude of specialized membrane proteins that include cell adhesion molecules, aquaporins, ion channels, neurotransmitter transporters, and gap junction molecules. To support this dynamic flux, astrocytes, like neurons, rely on tightly coordinated and efficient intracellular transport. Unlike neurons,&#160;where intracellular trafficking has been extensively delineated, microtubule-based transport in astrocytes has been less studied. Nonetheless, exo- and endocytic trafficking of cell membrane proteins and intracellular organelle transport orchestrates astrocytes&#8217; normal biology, and these processes are often affected in disease or in response to injury. Here we present a straightforward protocol to culture high quality murine astrocytes, to fluorescently label astrocytic proteins and organelles of interest, and to record their intracellular transport dynamics using time-lapse confocal microscopy. We also demonstrate how to extract and quantify relevant transport parameters from the acquired movies using available image analysis software (i.e., ImageJ/FIJI) plugins.

The protocol for establishing primary mouse MD astrocytes outlined above should yield reproducible, high quality cultures. Although cultures initially contain a mix of astrocytes, fibroblasts, and other glial cells, including microglia and oligodendrocytes (Figure 1Bi,Biv; red arrowheads), the addition of AraC to the mixed culture between DIV5-DIV7 minimizes the proliferation of these contaminant cells. The combined AraC treatment and shaking-based purification strategy enri...

Watch this Scientific Journal Video about Visualizing and Analyzing Intracellular Transport of Organelles and Other Cargos in Astrocytes at JoVE.com

Visualizing and Analyzing Intracellular Transport of Organelles and Other Cargos in Astrocytes

Here we describe an in vitro live-imaging method to visualize intracellular transport of organelles and trafficking of plasma membrane proteins in murine astrocytes. This protocol also presents an image analysis methodology to determine cargo transport itineraries and kinetics.

Astrocytes are among the most abundant cell types in the adult brain, where they play key roles in a multiplicity of functions. As a central player in ...

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