Name: quantification of endosome and lysosome motilities in cultured neurons using fluorescent probes
Uploaded: 2017-05-22T13:00:00
Description: Watch this Scientific Journal Video about Quantification of Endosome and Lysosome Motilities in Cultured Neurons Using Fluorescent Probes at JoVE.com

We thank Dr. Ricardo Dolmetsch (Stanford University School of Medicine; present affiliation: Novartis Institutes for Biomedical Research) for helping to develop this analysis and Dr. Matthew Wood, Takuma Aihara, and Dongsook Kim for their critical reading of the manuscript.

All animal procedures were performed with the approval of the University of Tsukuba Animal Care and Use Committee (IACUC).
1. Dissection
<ol>
	<li>To prepare embryonic day 13-14 ICR or C57BL/6 fetuses, euthanize pregnant mice by cervical dislocation. Remove the uterus and put it into a petri dish with Hank&#39;s Balanced Salt Solution (HBSS). Rinse it well to remove the blood.</li>
	<li>Using forceps, transfer the uterus to a new petri dish with 70% ethanol to sterilize.</li>
	<li>Transfer t...

<ol>
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This protocol introduces the procedure for quantifying vesicle motility. In primary neurons, endosomes and lysosomes tend to show high motility in younger neurons (4-6 DIV). Given that neurons must deliver some components to the leading edges to elongate neural processes, membrane trafficking should occur dynamically during this stage. Thus, it is important to use younger neurons to observe dynamic motility in neuronal dendrites. In addition, larger vesicles do not tend to exhibit high motility, even in younger neurons. ...

Precise control of endosome-lysosome trafficking is indispensable for regulating neuronal function. Notably, the dynamic movements of these vesicles are a key factor underlying the regulation of neuronal morphology, development, and survival. Defects in this system cause severe neuronal disorders1,2. The molecular mechanisms that link vesicle trafficking to neuronal diseases are considered complicated, and several groups have sought to examine this relevance. For instance, it has been reported that perturbed late endosome motility is significantly associated with Niemann-Pick C disease3, an inherited neurodegenerative disorder caused by lysosome defects. Another example is a mutation in a lysosomal Ca2+ channel, trpml1, which impairs lysosomal motility, resulting in lysosomal storage diseases4,5,6. Our group has reported that dysregulation of PtdIns(3,5)P2 turnover suppresses endosome and lysosome motility in neurons, leading to an increase in vulnerability to the stress response7,8. The metabolic regulation of PtdIns(3,5)P2, which mostly localizes on late endosomes and lysosomes, plays an important role in a wide variety of cellular functions, including vesicle trafficking and fusion-fission processes9,10. Since impaired PtdIns(3,5)P2 turnover causes severe neurodegeneration11,12, the aberrant regulation of endosome-lysosome motility could be a key factor for understanding the pathogenesis of neurodegeneration. An investigation of the molecular mechanisms that underlie vesicle motility may thus provide promising clues that can deepen our understanding of several neuronal disorders.
In this paper, we introduce a valuable method to quantify vesicle motility in neurons using a free software package called Manual Tracking. The aim was to develop a fast quantification method to analyze vesicle motility. This quantification is directed through a standard approach of clicking on a reference point in each frame of a time-lapse movie. The use of the Manual Tracking software makes this approach quite simple and of broad utility, unlike other applications. Furthermore, this approach is also applicable to other cells, such as glial cells. Although this method is primitive, it can be applied to various analyses, including of cellular motility and morphological change. For instance, after defining a reference point across a sequence of images, information on the positions of the reference points and the time at each position can be extracted from sequential images using data analysis and image-processing software. Taken together, this method is simple but powerful and contributes to the development of improved efficiency in studies based on membrane trafficking, such as those examining endosome-lysosome function.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Neurobasal medium</td>
			<td>Thermo Fisher</td>
			<td>21103-049</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s modified eagle medium&#160;</td>
			<td>Wako</td>
			<td>044-29765</td>
			<td></td>
		</tr>
		<tr>
			<td>Opti-MEM</td>
			<td>Thermo Fisher</td>
			<td>31985070</td>
			<td>Serum free medium</td>
		</tr>
		<tr>
			<td>Hank&#39;s balanced salt solution</td>
			<td>Thermo Fisher</td>
			<td>14170112</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicilin Streptmycin</td>
			<td>Thermo Fisher</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Thermo Fisher</td>
			<td>25030081</td>
			<td></td>
		</tr>
		<tr>
			<td>B-27 Supplements</td>
			<td>Thermo Fisher</td>
			<td>17504044</td>
			<td></td>
		</tr>
		<tr>
			<td>2.5% Trypsine</td>
			<td>Thermo Fisher</td>
			<td>15090046</td>
			<td></td>
		</tr>
		<tr>
			<td>poly-D-lysine hydrobromide</td>
			<td>Sigma</td>
			<td>P7280</td>
			<td></td>
		</tr>
		<tr>
			<td>poly-L-ornithine&#160;</td>
			<td>Sigma</td>
			<td>P4957</td>
			<td></td>
		</tr>
		<tr>
			<td>Nylon cell strainer</td>
			<td>Corning</td>
			<td>431750</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipofectamine 2000</td>
			<td>Thermo Fisher</td>
			<td>11668027</td>
			<td>Transfection reagent</td>
		</tr>
		<tr>
			<td>Polyethyleneimine &#34;Max&#34;</td>
			<td>polysciences</td>
			<td>24765</td>
			<td>Transfection reagent. As an alternative to Lipofectamine2000, 0.1mg/ml Polyethyleneimine dissolved in sterilized water is available. But it is low efficiency and high toxicity.</td>
		</tr>
		<tr>
			<td>BIOREVO BZ-9000</td>
			<td>Keyence</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubation system INUG2-K13</td>
			<td>Tokay Hit</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>GraphPad Prism version 6.0</td>
			<td>GraphPad Software</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Excel version 15</td>
			<td>Microsoft</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ verion 1.47</td>
			<td>NA</td>
			<td>NA</td>
			<td></td>
		</tr>
	</tbody>
</table>

quantification of endosome and lysosome motilities in cultured neurons using fluorescent probes

In the brain, membrane trafficking systems play important roles in regulating neuronal functions, such as neuronal morphology, synaptic plasticity, survival, and glial communications. To date, numerous studies have reported that defects in these systems cause various neuronal diseases. Thus, understanding the mechanisms underlying vesicle dynamics may provide influential clues that could aid in the treatment of several neuronal disorders. Here, we describe a method for quantifying vesicle motilities, such as motility distance and rate of movement, using a software plug-in for the ImageJ platform. To obtain images for quantification, we labeled neuronal endosome-lysosome structures with EGFP-tagged vesicle marker proteins and observed the movement of vesicles using a time-lapse microscopy. This method is highly useful and simplify measuring vesicle motility in neurites, such as axons and dendrites, as well as in the soma of both neurons and glial cells. Furthermore, this method can be applied to other cell lines, such as fibroblasts and endothelial cells. This approach could provide a valuable advancement of our understanding of membrane trafficking.

This assay was designed to measure vesicle dynamics in in vitro culture. This assay was utilized to determine the vesicle motility associated with neuronal morphology and survival. Figure 1A and B display representative data showing lysosome motility in neurons. Cortical neurons were transfected with the lysosome marker, LAMP-EGFP, and observed using a standard fluorescence microscope. It has been previously reported that specific endosome-lysoso...

Watch this Scientific Journal Video about Quantification of Endosome and Lysosome Motilities in Cultured Neurons Using Fluorescent Probes at JoVE.com

Quantification of Endosome and Lysosome Motilities in Cultured Neurons Using Fluorescent Probes

The investigation of membrane trafficking is crucial for understanding neuronal functions. Here, we introduce a method for quantifying vesicle motility in neurons. This is a convenient method that can be adapted to the quantification of membrane trafficking in the nervous system.

In the brain, membrane trafficking systems play important roles in regulating neuronal functions, such as neuronal morphology, synaptic plasticity, ...

The overall goal of this imaging analysis is to observe vesicle motility in neurons and quantify it easily. This approach enables one to analyze membrane trafficking in neurons in a straightforward and time-effective manner. To begin this procedure, transfer the uterus to a new Petri dish with 70%ethanol.Next, transfer the uterus to another Petri dish with HBSS. Then remove the fetuses from the uterus using sterilized dissecting forceps and surgical scissors. After that place them in a new Petri dish with HBSS.Under a stereo microscope remove the skin and skulls and take out the brains using the sterilized dissecting forceps. Then, transfer them to another Petri dish with HBSS. Remove the meninges using sterilized dissecting forceps.Then trim off the extra regions. Next, transfer the cortices to a new Petri dish with HBSS. Collect all the cortices using a disposable dropper or a 25 milliliter pipette and place them into a 15 milliliter tube.Following that, remove the HBSS by pipetting. Then, add three milliliters of cerebral cortical enzyme solution to the tube containing the cortices. Place the tube in a water bath and incubate it at 37 degrees Celsius for five minutes.After that add another three milliliters of cerebral cortical enzyme solution into the tube containing the cortices and incubate them at 37 degrees Celsius in a water bath for an additional five minutes. After five minutes remove the cerebral cortical enzyme solution and add five milliliters of DMEM containing 10%fetal bovine serum. Dissociate the cortices by gently pipetting up and down using a five milliliter pipette with a sterile P1000 tip.Then pipette the cortices again using a five milliliter pipette with a P200 tip. Put a 40 micrometer nylon cell strainer on a 50 milliliter tube and filter the suspension to remove the debris. Subsequently, centrifuge the suspension at 420 times g for five minutes at room temperature, and then remove the DMEM medium.Afterward, add five milliliters of cerebral cortical culture medium and gently resuspend the cell pellet. Then count the cells using a hemocytometer and adjust the cell concentration using the cerebral cortical culture medium. Next, plate three times 10 to the six cortical neurons in two milliliters of cell suspension for each coded 35 milliliter glass bottom dish.In this procedure, mix four micrograms of plasmid with 200 microliters of serum-free medium. In a separate tube dilute eight microliters of the transfection reagent in 200 microliters of serum-free medium. Then, incubate the solution for five minutes at room temperature.After five minutes mix the plasmid solution and the transfection reagent solution and incubate the mixture for 20 minutes at room temperature. Following that, add the mixture to the coded glass bottom dishes. Incubate the neurons at 37 degrees Celsius for 30 minutes.Afterward, replace the medium with two milliliters of cerebral cortical culture medium and incubate the neurons at 37 degrees Celsius for one to two days. For imaging, use a fluorescence microscope equipped with a CCD camera with a 40X objective lenses. Keep the temperature at 37 degrees Celsius using an incubation system.Acquire neuron images at one frame every five seconds over a 100-second period controlled by the image acquisition software. To analyze the images, open all the sequential images in ImageJ software with Manual Tracking. To combine the sequential images choose Image, then Stacks, followed by Images to Stack and then click OK.After that, choose Plugins then Manual Tracking.A Tracking window will pop up. Click on the checkbox of Show Parameters and define the tracking parameters in the Parameters section. Then click Add track to start a new track.After determining the vesicles of interest click on the center of the signals in the sequential images to record the xy coordinates. Repeat this procedure to collect the xy coordinates of the vesicles from all sequential images, each image automatically advances to the successive image after the reference point is selected. This image shows the motility of LAMP-EGFP containing vesicles in the dendrites of the mouse cortical neurons after two days of transfection, started at four DIV.The inset shows the time-lapse image of the LAMP-EGFP positive vesicles. The red arrowhead indicates a moving vesicle and the blue arrowhead indicates a resting vesicle. The orange arrowheads indicate higher fluorescent signals.Here is the time-dependent X-axis position of the LAMP-EGFP positive vesicles. The vesicle in the red circle, labeled b, is moving and the vesicle in the blue circle, labeled a, is resting. This bar graph indicates the total distance of LAMP-EGFP movement.And this bar graph indicates the average motility distance of a LAMP-EGFP containing vesicles.

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