Name: quantitative cell biology of neurodegeneration in drosophila through unbiased analysis of fluorescently tagged proteins using imagej
Uploaded: 2018-08-03T14:00:00
Description: Watch this Scientific Journal Video about Quantitative Cell Biology of Neurodegeneration in Drosophila Through Unbiased Analysis of Fluorescently Tagged Proteins Using ImageJ at JoVE.com

This work is supported by the Sheila and David Fuente Neuropathic Pain Research Program Graduate Fellowship (to J.M.B.), the Lois Pope LIFE Fellows Program (to C.L., Y.Z., and J.M.B.), the Snyder-Robinson Foundation Predoctoral Fellowship (to C.L.), the Dr. John T. Macdonald Foundation (to C.L.), contracts, grants from National Institutes of Health (NIH) HHSN268201300038C, R21GM119018, and R56NS095893 (to R.G.Z.), and by Taishan Scholar Project (Shandong Province, People&#39;s Republic of China) (to R.G.Z.).

1. Considerations and Preparations for Designing the Image Analysis Experiment
<ol>
	<li>Predetermine suitable anatomical, cellular, or subcellular markers to serve as landmarks for standardizing a region of interest (ROI) across different samples, for example 4&#39;,6-diamidino-2-phenylindole (DAPI), membrane markers, localized fluorescent protein, etc.</li>
	<li>Select a fluorescent marker that can delimit discrete puncta beyond background levels for the structure of interest. 
	NOTE: This protocol is o...

<ol>
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The protocol outlined here can be used to robustly and reproducibly quantitate cell biological processes visualized by fluorescence-based imaging. Biological context and technical limitations need to be carefully considered to guide the experimental design. Fluorescent markers of subcellular structures of interest, whether immunohistochemical, dye-based, or genetically expressed, need to be distinguishable above background by morphology and intensity. The UAS/GAL4 system is widely used to drive targeted gene exp...

Neurodegenerative diseases affect millions of people each year and the incidence is increasing with an aging population1. While each neurodegenerative disease has a unique etiology, aggregation of misfolded proteins and breakdown of the proteostasis network are common pathological hallmarks of many of these diseases. Elucidating how disruption of these fundamental and interrelated processes goes awry to contribute to neuronal dysfunction and cell death is critical for understanding neurodegenerative diseases as well as guiding therapeutic interventions. Fluorescence-based imaging allows for investigation of these complex and dynamic processes in neurons and has greatly contributed to our understanding of neuronal cell biology. Analysis of fluorescently tagged proteins is challenging, particularly when experiments are carried out in vivo, due to highly compact tissues, diverse cell types, and morphological heterogeneity. Manually assisted quantification is affordable and straightforward, but is often time-consuming and subject to human bias. Therefore, there is a need for unbiased, reproducible, and accessible approaches for extracting quantifiable data from imaging studies.
We have outlined a simple and adaptable workflow using Fiji/ImageJ, a powerful and freely accessible image processing software2,3, to extract quantitative data from fluorescence imaging studies in experimental models of neurodegeneration using Drosophila. By following this protocol to quantify protein aggregation and autophagic flux &#8212; two cell biological features that are highly relevant to neurodegenerative disease pathology &#8212; we demonstrated the sensitivity and reproducibility of this approach. Analysis of fluorescently tagged mutant huntingtin (Htt) proteins in the Drosophila optic lobe revealed the number, size, and intensity of protein aggregates. We visualized a tandem fluorescent reporter of autophagic flux within the Drosophila visual system, which displays different emission signals depending on the compartmental environment4. Ratiometric-based analysis of the tandem reporter allowed for a quantitative and comprehensive view of autophagy-lysosome flux from autophagosome formation, maturation, and transport to degradation in the lysosome, and additionally highlighted vulnerable compartments disrupted in neurodegenerative conditions. Importantly, in both analyses we implemented semi-automated thresholding and segmentation steps in our protocol to minimize unconscious bias, increase sampling power, and provide a standard to facilitate comparisons between similar studies. The straightforward workflow is intended to make powerful Fiji/ImageJ plugins (developed by computer scientists based on mathematic algorithms) more accessible to neurobiologists and the life sciences community at large.

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	<tbody>
		<tr height="38">
			<td height="38" style="height:38px;width:347px;">SYLGARD(R) 184 Silicone Elastomer Kit</td>
			<td style="width:164px;">Dow Corning Corporation</td>
			<td style="width:243px;">PF184250</td>
			<td style="width:349px;">Dissection dish</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:347px;">Falcon 35 mm Not TC-Treated Easy-Grip Style Bacteriological Petri Dish</td>
			<td style="width:164px;">Corning</td>
			<td style="width:243px;">351008</td>
			<td style="width:349px;">Dissection dish</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Dumont #5 Forceps</td>
			<td style="width:164px;">Fine Science Tools</td>
			<td style="width:243px;">11251-20</td>
			<td style="width:349px;">Dissection tool</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Sodium Chloride</td>
			<td style="width:164px;">Sigma</td>
			<td style="width:243px;">S3014</td>
			<td style="width:349px;">PBS solution</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Sodium Phosphate Dibasic&#160;</td>
			<td style="width:164px;">Sigma</td>
			<td style="width:243px;">S5136</td>
			<td style="width:349px;">PBS solution</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Potassium Phosphate Monobasic</td>
			<td style="width:164px;">Sigma</td>
			<td style="width:243px;">P5655</td>
			<td style="width:349px;">PBS solution</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Triton&#160;X-100</td>
			<td style="width:164px;">Sigma</td>
			<td style="width:243px;">T9284</td>
			<td style="width:349px;">Washing and antibody incubaton solution.&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">37% Formaldehyde</td>
			<td style="width:164px;">VWR</td>
			<td style="width:243px;">10790-710</td>
			<td style="width:349px;">Fixation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Disposable Microcentrifuge Tubes (0.5mL, blue)</td>
			<td style="width:164px;">VWR</td>
			<td style="width:243px;">89000-022</td>
			<td style="width:349px;">Fixation, washing, and antibody incubaton.&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Plain and Frosted Micro Slides (25&#215;75mm)</td>
			<td style="width:164px;">VWR</td>
			<td style="width:243px;">48312-004</td>
			<td style="width:349px;">Slides for confocal imaging</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Micro Cover Glasses, rectangular (22&#215;40mm)</td>
			<td style="width:164px;">VWR</td>
			<td style="width:243px;">48393-172</td>
			<td style="width:349px;">Slides for confocal imaging</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Rubber Cement</td>
			<td style="width:164px;">Slime</td>
			<td style="width:243px;">1051-A</td>
			<td style="width:349px;">Mounting</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:347px;">VECTASHIELD Antifade Mounting Medium</td>
			<td style="width:164px;">Vector Laboratories, Inc.</td>
			<td style="width:243px;">H-1000</td>
			<td style="width:349px;">Mounting</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Scotch Magic 810 Invisible Tape (19mm&#215;25.4m)</td>
			<td style="width:164px;">3M Company</td>
			<td style="width:243px;">810</td>
			<td style="width:349px;">Mounting</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:347px;">Normal Goat Serum</td>
			<td style="width:164px;">Thermo Fisher Scientific</td>
			<td style="width:243px;">PCN5000</td>
			<td style="width:349px;">Antibody incubaton</td>
		</tr>
		<tr height="76">
			<td height="76" style="height:76px;width:347px;">DAPI (4&#39;,6-Diamidino-2-Phenylindole, Dihydrochloride)&#160;</td>
			<td style="width:164px;">Thermo Fisher Scientific</td>
			<td style="width:243px;">D1306</td>
			<td style="width:349px;">Nucleic acid staining. Dissolve in deionized water to make a 5 mg/mL stock solution and store at -80&#176;C. Dilute to a working concentration of 10-20 &#956;g/mL in PBTx.</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:347px;">3.5X-90X Stereo Zoom Inspection Industrial Microscope</td>
			<td style="width:164px;">AmScope</td>
			<td style="width:243px;">SM-1BNZ</td>
			<td style="width:349px;">Dissection scope. Equipped with 6W LED Dual Gooseneck Illuminator</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">ImageJ/Fiji</td>
			<td style="width:164px;">NIH</td>
			<td style="width:243px;">v1.51u</td>
			<td style="width:349px;">With SCF_MPI_CBG plugins (version 1.1.2)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">FV1000-IX81 Confocal-laser Scanning Microscope</td>
			<td style="width:164px;">Olympus</td>
			<td style="width:243px;"></td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Recombinant Construct</td>
			<td>Bloomington Drosophila Stock Center</td>
			<td style="width:243px;">BL37749</td>
			<td style="width:349px;"></td>
		</tr>
	</tbody>
</table>

quantitative cell biology of neurodegeneration in drosophila through unbiased analysis of fluorescently tagged proteins using imagej

With the rising prevalence of neurodegenerative diseases, it is increasingly important to understand the underlying pathophysiology that leads to neuronal dysfunction and loss. Fluorescence-based imaging tools and technologies enable unprecedented analysis of subcellular neurobiological processes, yet there is still a need for unbiased, reproducible, and accessible approaches for extracting quantifiable data from imaging studies. We have developed a simple and adaptable workflow to extract quantitative data from fluorescence-based imaging studies using Drosophila models of neurodegeneration. Specifically, we describe an easy-to-follow, semi-automated approach using Fiji/ImageJ to analyze two cellular processes: first, we quantify protein aggregate content and profile in the Drosophila optic lobe using fluorescent-tagged mutant huntingtin proteins; and second, we assess autophagy-lysosome flux in the Drosophila visual system with ratiometric-based quantification of a tandem fluorescent reporter of autophagy. Importantly, the protocol outlined here includes a semi-automated segmentation step to ensure all fluorescent structures are analyzed to minimize selection bias and to increase resolution of subtle comparisons. This approach can be extended for the analysis of other cell biological structures and processes implicated in neurodegeneration, such as proteinaceous puncta (stress granules and synaptic complexes), as well as membrane-bound compartments (mitochondria and membrane trafficking vesicles). This method provides a standardized, yet adaptable reference point for image analysis and quantification, and could facilitate reliability and reproducibility across the field, and ultimately enhance mechanistic understanding of neurodegeneration.

Quantification of the number, area, and intensity of fluorescently tagged mutant Htt aggregates in the Drosophila optic lobe
To investigate misfolded protein aggregation in the central nervous system of a Drosophila model of Huntington&#39;s disease, RFP-tagged mutant human Htt with a non-pathological (UAS-RFP-hHttQ15) or pathological expansion (UAS-RFP-hHttQ138) and membrane GFP (UAS-mCD8::G...

Watch this Scientific Journal Video about Quantitative Cell Biology of Neurodegeneration in Drosophila Through Unbiased Analysis of Fluorescently Tagged Proteins Using ImageJ at JoVE.com

Quantitative Cell Biology of Neurodegeneration in Drosophila Through Unbiased Analysis of Fluorescently Tagged Proteins Using ImageJ

We have developed a simple and adaptable workflow to extract quantitative data from fluorescence-imaging-based cell biological studies of protein aggregation and autophagic flux in the central nervous system of Drosophila models of neurodegeneration.

Quantitative Cell Biology of Neurodegeneration in Drosophila Through Unbiased Analysis of Fluorescently Tagged Proteins Using ImageJ

With the rising prevalence of neurodegenerative diseases, it is increasingly important to understand the underlying pathophysiology that leads to neuronal ...

Research

JoVE Journal

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