Name: quantitative approaches for scoring in vivo neuronal aggregate and organelle extrusion in large exopher vesicles in c. elegans
Uploaded: 2020-09-18T14:00:00
Duration: 9 min 6 s
Description: Watch this Scientific Journal Video about Quantitative Approaches for Scoring in vivo Neuronal Aggregate and Organelle Extrusion in Large Exopher Vesicles in C. elegans at JoVE.com

We acknowledge the following NIH grants: R01AG047101 and R37AG56510.&#160;Members of the Driscoll and Grant labs have contributed extensively to the development and fine tuning of protocols described, with rigorous experiments and strong communication.

<ol>
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The characterization of the in vivo molecular mechanisms of aggregate and organelle elimination in the form of large exophers is in its infancy. Questions as to the designation of cargoes for expulsion, the polarized collection of these cargoes within the cell, the regulation of the decision to generate exophers, the machinery that mediates extrusions, and the interaction of exophers with the degradative machinery in a neighboring cell all remain to be addressed. Furthermore, the in vivo visualization of tubular connections that can pass biological materials that include calcium, aggregates, and mitochondria is interesting and understudied biology in its own right. Questions of why certain cells are more prone to exopher production than others also are unresolved, but can begin to be genetically dissected with the approaches outlined in this protocol.
Described in detail in this protocol are the approaches to achieving reproducible scoring of exopher production, with attention to distinguishing exophers from nearby cell somas, timing of analyses to capture peak of exopher production, and strict control of growth conditions to eliminate unintended stresses that can modulate exopher levels. Both distinction of the large early exopher, or the &#8220;starry night&#8217; dispersion in the surrounding hypodermis can be quantitated as evidence of exopher production. That being said, neurons expressing mCherry under basal conditions are most often associated with 5-25% of neurons of a specific type producing an exopher. Controlled introduction of stress conditions could be applied to increase exopher production to detection as high as 90% of neurons producing extrusions, particularly useful for genetic or pharmacological screens for modifiers.
In human neurodegenerative disease, large aggregates can transfer from diseased neurons into neighboring cells to promote pathology spread. The exopher mechanism might transpire via a conserved mechanism used for aggregate extrusion across phyla. Defining the in vivo molecules that either enhance the efficiency of this process (considered more effective proteostasis control) or block it might be harnessed to influence design of novel strategies for combating multiple neurodegenerative diseases. As such, the protocol described here could be used for classical genetic mutagenesis screens, genome-wide RNAi screens that systematically knock down genes to identify enhancers and suppressors, or for drug intervention studies that identify candidate pharmacological modifiers of this process. The approach is straightforward, although somewhat laborious. Exophers are so large they can be viewed with a high-magnification dissecting microscope. Still, C. elegans neurons are relatively small and looking at their organelles or their membranes require higher power confocal images and is a slow process. Options for higher throughput could involve high content imaging approaches in multi-well plate format.
The application of a standardized approach to exopher scoring should underlie a concerted genetic dissection of the process by which neurons can organize and eliminate cellular debris.

The neurotoxic challenges of aggregates and dysfunctional mitochondria have long been considered to be cell-intrinsic, but more recently it has become clear that misfolded human disease proteins originating in one neuron can also spread to neighboring cells, promoting pathology1. Likewise, mammalian mitochondria can be sent out of the cell of their original production for transcellular degradation2 or for rescue of mitochondrial populations in challenged neighboring cells3. Vesicles of various sizes have generally been observed to transfer cellular materials to neighboring cells or to fluid surroundings4. Some extruded vesicles approach the size of the average neuronal soma (average touch neuron soma ~ 6 &#181;m) and can accommodate large aggregates and organelles.
A striking example of large vesicle extrusion that can carry protein aggregates and organelles occurs in C. elegans touch receptor neurons that express a high copy number reporter construct encoding a noxious aggregation-prone, degradation-resistant mCherry5. Extrusions from the touch neurons, called exophers, are ~4 &#181;m average diameter, selectively include mCherry or other aggregates, and are delivered directly into the neighboring hypodermis, which normally surrounds the touch receptor neurons. The hypodermis attempts lysosome-based degradation, but some non-digestible contents such as mCherry aggregates can be re-extruded by the hypodermis into the fluid-filled pseudocoelom of the animal, from which the mCherry can be taken up by remote scavenger cells called coelomocytes for long term storage (Figure 1, Figure 2)5.
The large extruded exopher vesicles leave the cell surrounded by touch receptor plasma membrane and can contain aggregated human disease proteins, mitochondria, and lysosomes. The process of exopher production appears to involve sorting of potentially toxic species (for example an aggregation-prone expressed mCherry is segregated from soluble, inoffensive proteins like GFP that remains mostly in the neuronal soma). In this way, directed expulsion of the threatening entities is accomplished by the neuron5. A proteostasis challenge, such as stress induced by&#160;autophagy knockdown, MG132-mediated proteasome inhibition, or transgenic expression of human disease proteins such as Huntington&#8217;s disease-associated expanded polyglutamine Q128 or Alzheimer&#8217;s disease-implicated fragment A&#946;1-42, can increase the numbers of neurons that produce exophers5.
As exophers have only recently been documented, what is known of their biology merits description. Exophers were discovered in, and are the most well studied in, C. elegans touch receptor neurons. There are six C. elegans mechanosensory touch neurons that have cell bodies distributed around the body (Figure 3A) and are called microtubule cells because their ultrastructure features distinctive 15 protofilament microtubules. The touch receptor neurons are the anterior AVM (anterior ventral microtubule neuron), ALMR, and ALML (anterior lateral microtubule neurons right and left), the more central PVM (posterior ventral microtubule neuron), and the posterior PLMR and PLML (posterior lateral microtubule neurons right and left) in the tail. Interestingly, the six touch receptor neurons produce exophers at different rates, despite expressing the same offensive transgene (Figure 3C). Of the six mechanosensory touch receptor neurons, the ALMR neuron undergoes exophergenesis more often than the other touch neurons. Quantitation of exopher numbers from touch neurons is thus usually established by focusing upon the ALMR.
Exophergenesis is a dynamic process that typically begins with swelling of the neuronal cytoplasm (Figure 1A-B). Cellular contents, organelles, or protein aggregates are collected to one side of the neuronal soma, most commonly toward the posterior end of the ALMR neuron (away from the projecting neurite), forming a pre-exopher domain (PED) (Figure 1B). The early protrusion is observed as the PED begins to project outwards, forming a recognizable protruded bud. The late bud is defined when the widest diameter of the pre-exopher domain is approximately &#8531; larger than the diameter of the constriction of the soma-exopher neck (Figure 1C).&#160;Exophers can be ejected in nearly any direction from the soma, but most exophers exit posteriorly from the cell body and remain in approximately the same focal plane as the originating soma.
The exopher can move away from the originating soma as the neck of the bud narrows into a thin filament. Exophers can remain attached to the soma via this filament (Figure 1D, arrow) and can later become detached. Cellular contents such as calcium, aggregates, and mitochondria can be transferred via this filament into the attached exopher5, although the bulk of extruded material is put into the exopher compartment by the massive budding event. Exophers are considered mature when there is no visible connecting tube or thin filament and the exopher is fully separated from the sending soma (Figure 1E).
Exophers produced by C. elegans touch neurons immediately encounter the hypodermis, the tissue that surrounds the touch neuron. Most commonly, the exopher vesicle appears to travel within the hypodermis posteriorly towards the tail, and can be fairly distant from the soma before exopher contents appear targeted for degradation (for example, the distance can be ~100 &#181;m away from the soma (Figure 1F)). The fluorescent exopher vesicle breaks up into many smaller vesicles within the hypodermis, taking on an appearance referred to as &#8220;starry night&#8221; (Figure 1G and Figure 2). In the &#8220;starry night&#8221; stage, punctate fluorescent material can be observed scattered across the hypodermal syncytium into many smaller points of fluorescence as compared to the original solitary exopher. Starry night can look punctate under low magnification and with higher magnification, can look punctate and/or networked within the hypodermis. The fluorescent signal of the starry night is typically dimmer than the exopher and the neuronally expressed fluorescence (Figure 2B-C). The dispersal of mCherry into many punctate vesicles is thought to involve phagosome maturation and fusion with the endosomal/lysosomal network of the hypodermal cell. Some exopher materials are likely degraded in the hypodermal lysosomal network, but residual species that are resistant to degradation (such as mCherry aggregates) are thrown out of the hypodermis into the pseudocoelom, a fluid compartment that can contain cellular debris. The fluorescent material is later taken in by remote scavenger cells called coelomocytes (Figure 2C), which can concentrate, store, and again attempt degradation of mCherry.
The phenomenon of aggregate extrusion and transfer appears conserved across phyla, having been reported in genetic models such as C. elegans5,6,7 and D. melanogaster8,9 as well as in multiple mammalian models. Exopher-like extrusions have been reported for mammalian cells10, an observation suggesting that conserved mechanisms might underlie aggregate and organelle expulsion. Exopher production may thus be a conserved mechanism of cellular debris management that constitutes a fundamental, but formerly unrecognized, branch of neuronal proteostasis and mitochondrial quality control, which, when imbalanced, might actively contribute to neurodegenerative disease. Identification of the molecules involved in debris discrimination and sorting, transport to a distinct subcellular locale, extrusion, formation/scission of the tubular connection linking the soma and late exopher, and recognition of the large extruded vesicle for remote degradation by a neighboring cell remain for future work. Studies in nematode and fly models will be critically important to defining mechanisms of aggregate and organelle collection and transfer, utilizing unbiased genetic approaches and powerful cell biological tools offered by these models to identify participating molecules in physiological context.
Critical first steps in deciphering mechanisms operative in exopher biology involve defining protocols for reproducible in vivo exopher quantification. The C. elegans model offers a particular advantage for such efforts since the body is transparent and exophers can be readily observed when they contain fluorescently tagged proteins or organelles. Exophers have been reported to be generated by C. elegans dopaminergic neurons PDE and CEP, ASE and ASER sensory neurons, and dye-filling amphid neurons5. Because exophers produced by touch receptor neurons are best characterized, the focus here is on the use of touch neurons for exopher analysis. However the basic approach can be applied to measure exopher production from any cell. Protocols to detect and quantitate exophers produced by C. elegans touch receptor neurons that transgenically express mCherry protein are outlined, with an emphasis on cargoes that can be monitored and temporal constraints in scoring. This article defines approaches toward in vivo exopher identification, and the quantitation of environmental and genetic conditions that modulate exopher production. Protocols emphasize critical attention to constant non-stress conditions for the determination of baseline exopher production and for comparisons across genotypes.

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			<td height="53" style="height:54px;width:191px;">95B Scientific CMOS camera</td>
			<td style="width:108px;">Photometrics Prime</td>
			<td style="width:128px;"></td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="53">
			<td height="53" style="height:54px;width:191px;">1,000 &#956;L low retention tips</td>
			<td style="width:108px;">Sarstedt</td>
			<td style="width:128px;"></td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="53">
			<td height="53" style="height:54px;width:191px;">10 mL serological pipette</td>
			<td style="width:108px;">Appleton Woods</td>
			<td style="width:128px;">CC214</td>
			<td style="width:177px;"></td>
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		<tr height="53">
			<td height="53" style="height:54px;width:191px;">10 &#956;L low retention tips</td>
			<td style="width:108px;">Sarstedt</td>
			<td style="width:128px;">70.1130.105</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="53">
			<td height="53" style="height:54px;width:191px;">13% sodium hypochlorite</td>
			<td style="width:108px;">Acros Organics</td>
			<td style="width:128px;">AC219255000</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="53">
			<td height="53" style="height:54px;width:191px;">15 mL centrifuge tubes</td>
			<td style="width:108px;">Fisher Scientific</td>
			<td style="width:128px;">05-539-12</td>
			<td style="width:177px;"></td>
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		<tr height="53">
			<td height="53" style="height:54px;width:191px;">2 L erlenmeyer flasks</td>
			<td style="width:108px;">Scientific Laboratory Supplies</td>
			<td style="width:128px;">FLA4036</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="53">
			<td height="53" style="height:54px;width:191px;">25 mL serological pipette</td>
			<td style="width:108px;">Appleton Woods</td>
			<td style="width:128px;">CC216</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="53">
			<td height="53" style="height:54px;width:191px;">300 &#956;L low retention tips</td>
			<td style="width:108px;">Sarstedt</td>
			<td style="width:128px;">70.765.105</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="53">
			<td height="53" style="height:54px;width:191px;">50 mL serological pipette</td>
			<td style="width:108px;">Appleton Woods</td>
			<td style="width:128px;">CC117</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="53">
			<td height="53" style="height:54px;width:191px;">5-Fluoro-2&#39;-deoxyuridine 98%</td>
			<td style="width:108px;">Alfa Aesar</td>
			<td style="width:128px;">L16497.ME</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="53">
			<td height="53" style="height:54px;width:191px;">9 cm sterile Petri dishes</td>
			<td style="width:108px;">Fisher Scientific</td>
			<td style="width:128px;">11309283</td>
			<td style="width:177px;"></td>
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			<td height="53" style="height:54px;width:191px;">absolute ethanol</td>
			<td style="width:108px;">Vwr</td>
			<td style="width:128px;">20821.33</td>
			<td style="width:177px;"></td>
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		<tr height="53">
			<td height="53" style="height:54px;width:191px;">Agar</td>
			<td style="width:108px;">Sigma Aldrich</td>
			<td style="width:128px;">A1296</td>
			<td style="width:177px;"></td>
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		<tr height="53">
			<td height="53" style="height:54px;width:191px;">C. elegans strain wild type</td>
			<td style="width:108px;">Supplied by CGC</td>
			<td style="width:128px;">N2</td>
			<td style="width:177px;">C. elegans strain</td>
		</tr>
		<tr height="53">
			<td height="53" style="height:54px;width:191px;">calcium chloride dihydrate</td>
			<td style="width:108px;">Sigma Aldrich</td>
			<td style="width:128px;">C3881</td>
			<td style="width:177px;"></td>
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			<td height="53" style="height:54px;width:191px;">cholesterol</td>
			<td style="width:108px;">Acros</td>
			<td style="width:128px;">110190250</td>
			<td style="width:177px;"></td>
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		<tr height="53">
			<td height="53" style="height:54px;width:191px;">dibasic sodium phosphate</td>
			<td style="width:108px;">Sigma Aldrich</td>
			<td style="width:128px;">S3264</td>
			<td style="width:177px;"></td>
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			<td height="53" style="height:54px;width:191px;">E. coli strain OP50</td>
			<td style="width:108px;">Supplied by CGC</td>
			<td style="width:128px;">Op50</td>
			<td style="width:177px;">E coli strain</td>
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			<td height="53" style="height:54px;width:191px;">FBS10 Standard microscope</td>
			<td style="width:108px;">Meyer Instruments</td>
			<td style="width:128px;">KSC 410-1-100-1</td>
			<td style="width:177px;">FBS10 Standard with Plate Base, 100/100 Trinocular Head and Flip zoom</td>
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			<td height="53" style="height:54px;width:191px;">glass pipette 270 mm</td>
			<td style="width:108px;">Fisherbrand</td>
			<td style="width:128px;">FB50255</td>
			<td style="width:177px;"></td>
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		<tr height="53">
			<td height="53" style="height:54px;width:191px;">Heraeus Multifuge X3R</td>
			<td style="width:108px;">Thermofisher scientific</td>
			<td style="width:128px;">75004515</td>
			<td style="width:177px;"></td>
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			<td style="width:108px;">Fisher Scientific</td>
			<td style="width:128px;">11821741</td>
			<td style="width:177px;"></td>
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		<tr height="53">
			<td height="53" style="height:54px;width:191px;">LB medium capsules</td>
			<td style="width:108px;">MP biomedicals</td>
			<td style="width:128px;">3002-031</td>
			<td style="width:177px;"></td>
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			<td height="53" style="height:54px;width:191px;">LDI &#8211; Laser Diode Illuminator</td>
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			<td style="width:128px;"></td>
			<td style="width:177px;"></td>
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		<tr height="53">
			<td height="53" style="height:54px;width:191px;">levamisole</td>
			<td style="width:108px;">Sigma Aldrich</td>
			<td style="width:128px;">16595-80-5</td>
			<td style="width:177px;"></td>
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		<tr height="53">
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			<td style="width:128px;">4982000012</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="53">
			<td height="53" style="height:54px;width:191px;">magnesium sulphate</td>
			<td style="width:108px;">Sigma Aldrich</td>
			<td style="width:128px;">M7506</td>
			<td style="width:177px;"></td>
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		<tr height="53">
			<td height="53" style="height:54px;width:191px;">monobasic potassium phosphate</td>
			<td style="width:108px;">Sigma Aldrich</td>
			<td style="width:128px;">P0662</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="53">
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		<tr height="53">
			<td height="53" style="height:54px;width:191px;">Nalgene 1 L Centrifuge pots</td>
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			<td style="width:128px;">3120-1000</td>
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			<td style="width:108px;">Sorvall</td>
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			<td style="width:128px;">22037242</td>
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		<tr height="53">
			<td height="53" style="height:54px;width:191px;">SK4005 zdIs5[Pmec-4GFP]</td>
			<td style="width:108px;">contract Driscoll lab</td>
			<td style="width:128px;"></td>
			<td style="width:177px;">GFP expressed in touch neurons</td>
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		<tr height="53">
			<td height="53" style="height:54px;width:191px;">sodium chloride</td>
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		</tr>
		<tr height="53">
			<td height="53" style="height:54px;width:191px;">Sodium hydroxide</td>
			<td style="width:108px;">Fisher Chemical</td>
			<td style="width:128px;">S/4880/53</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="53">
			<td height="53" style="height:54px;width:191px;">Tactrol 2 Autoclave</td>
			<td style="width:108px;">Priorclave</td>
			<td style="width:128px;"></td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="53">
			<td height="53" style="height:54px;width:191px;">Triton-X</td>
			<td style="width:108px;">Thermofisher scientific</td>
			<td style="width:128px;">28313</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="53">
			<td height="53" style="height:54px;width:191px;">Tween 20</td>
			<td style="width:108px;">Sigma Aldrich</td>
			<td style="width:128px;">9005-64-5</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="53">
			<td height="53" style="height:54px;width:191px;">X-Light V2 Spinning Disk Confocal Unit</td>
			<td style="width:108px;">CrestOptics</td>
			<td style="width:128px;"></td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="53">
			<td height="53" style="height:54px;width:191px;">ZB4065 bzIs166[Pmec-4mCherry]</td>
			<td style="width:108px;">contract Driscoll lab</td>
			<td style="width:128px;"></td>
			<td style="width:177px;">mCherry expressed in touch neurons</td>
		</tr>
		<tr height="53">
			<td height="53" style="height:54px;width:191px;">ZB4067 bzIs167[Pmec-4mitogfp Pmec-4mCherry4]; igIs1[Pmec-7YFP Pmec-3htt57Q128::cfp lin-15+]</td>
			<td style="width:108px;">contract Driscoll lab</td>
			<td style="width:128px;"></td>
			<td style="width:177px;">Q128 expressed in touch neurons</td>
		</tr>
		<tr height="53">
			<td height="53" style="height:54px;width:191px;">ZB4509 bzIs166[Pmec-4mCherry]; bzIs168[Pmec-7LMP-1::GFP]</td>
			<td style="width:108px;">contract Driscoll lab</td>
			<td style="width:128px;"></td>
			<td style="width:177px;">mitoROGFP expressed in touch neurons</td>
		</tr>
		<tr height="53">
			<td height="53" style="height:54px;width:191px;">ZB4528 bzIs166[Pmec-4mCherry]; zhsEx17 [Pmec-4mitoLS::ROGFP]</td>
			<td style="width:108px;">contract Driscoll lab</td>
			<td style="width:128px;"></td>
			<td style="width:177px;">autophagy marker expressed in touch neurons</td>
		</tr>
		<tr height="53">
			<td height="53" style="height:54px;width:191px;">ZEISS Axio Vert.A1</td>
			<td style="width:108px;">Zeiss</td>
			<td style="width:128px;"></td>
			<td style="width:177px;"></td>
		</tr>
	</tbody>
</table>

quantitative approaches for scoring in vivo neuronal aggregate and organelle extrusion in large exopher vesicles in c. elegans

Toxicity of misfolded proteins and mitochondrial dysfunction are pivotal factors that promote age-associated functional neuronal decline and neurodegenerative disease across species. Although these neurotoxic challenges have long been considered to be cell-intrinsic, considerable evidence now supports that misfolded human disease proteins originating in one neuron can appear in neighboring cells, a phenomenon proposed to promote pathology spread in human neurodegenerative disease.
C. elegans adult neurons that express aggregating proteins can extrude large (~4 &#181;m) membrane-surrounded vesicles that can include the aggregated protein, mitochondria, and lysosomes. These large vesicles are called &#8220;exophers&#8221; and are distinct from exosomes (which are about 100x smaller and have different biogenesis). Throwing out cellular debris in exophers may occur by a conserved mechanism that constitutes a fundamental, but formerly unrecognized, branch of neuronal proteostasis and mitochondrial quality control, relevant to processes by which aggregates spread in human neurodegenerative diseases.
While exophers have been mostly studied in animals that express high copy transgenic mCherry within touch neurons, these protocols are equally useful in the study of exophergenesis using fluorescently tagged organelles or other proteins of interest in various classes of neurons.
Described here are the physical features of C. elegans exophers, strategies for their detection, identification criteria, optimal timing for quantitation, and animal growth protocols that control for stresses that can modulate exopher production levels. Together, details of protocols outlined here should serve to establish a standard for quantitative analysis of exophers across laboratories. This document seeks to serve as a resource in the field for laboratories seeking to elaborate molecular mechanisms by which exophers are produced and by which exophers are reacted to by neighboring and distant cells.

Watch this Scientific Journal Video about Quantitative Approaches for Scoring in vivo Neuronal Aggregate and Organelle Extrusion in Large Exopher Vesicles in C. elegans at JoVE.com

Quantitative Approaches for Scoring in vivo Neuronal Aggregate and Organelle Extrusion in Large Exopher Vesicles in C. elegans

This protocol describes approaches for detection and quantitation of large aggregate and/or organelle extrusions (~4 &#181;m) produced by C. elegans cells in the form of membrane-bound exophers. We describe strains, growth conditions, scoring criteria, timing, and microscopy considerations needed to facilitate dissection of this debris expulsion mechanism.

Quantitative Approaches for Scoring in vivo Neuronal Aggregate and Organelle Extrusion in Large Exopher Vesicles in C. elegans

Toxicity of misfolded proteins and mitochondrial dysfunction are pivotal factors that promote age-associated functional neuronal decline and ...

Research

JoVE Journal

Neuroscience

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