<meta charset="utf8"></head><body>GFP-Tagged RAB-3를 사용하여 살아있는 예쁜꼬마선충(C. elegans)의 축삭 수송 분석 향상

<ol>
	<li>Millecamps, S., Julien, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axonal+transport+deficits+and+neurodegenerative+diseases.">Axonal transport deficits and neurodegenerative diseases.</a> Nat Rev Neurosci. 14 (3), 161-176 (2013).</li><li>Brady, S. T., Morfini, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+motor+proteins,+axonal+transport+deficits+and+adult-onset+neurodegenerative+diseases.">Regulation of motor proteins, axonal transport deficits and adult-onset neurodegenerative diseases.</a> Neurobiol Dis. 105, 273-282 (2017).</li><li>Kevenaar, J. T., Hoogenraad, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+axonal+cytoskeleton:+from+organization+to+function.">The axonal cytoskeleton: from organization to function.</a> Front Mol Neurosci. 8, 44 (2015).</li><li>Roy, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Seeing+the+unseen:+the+hidden+world+of+slow+axonal+transport.">Seeing the unseen: the hidden world of slow axonal transport.</a> Neuroscientist. 20 (1), 71-81 (2014).</li><li>Watson, E. T., Pauers, M. M., Seibert, M. J., Vevea, J. D., Chapman, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+vesicle+proteins+are+selectively+delivered+to+axons+in+mammalian+neurons.">Synaptic vesicle proteins are selectively delivered to axons in mammalian neurons.</a> Elife. 12, e82568 (2023).</li><li>Schwartz, M. L., Jorgensen, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SapTrap,+a+toolkit+for+high-throughput+CRISPR/Cas9+gene+modification+in+Caenorhabditis+elegans.">SapTrap, a toolkit for high-throughput CRISPR/Cas9 gene modification in Caenorhabditis elegans.</a> Genetics. 202 (4), 1277-1288 (2016).</li><li>Niwa, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immobilization+of+Caenorhabditis+elegans+to+analyze+intracellular+transport+in+neurons.">Immobilization of Caenorhabditis elegans to analyze intracellular transport in neurons.</a> J Vis Exp. (128), e56690 (2017).</li><li>Caenorhabditis genetics center (CGC). University of Minnesota Available from: <a target="_blank" href="https://cgc.umn.edu/">https://cgc.umn.edu/</a> (2023)</li><li>Ghanta, K. S., Mello, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Melting+dsDNA+donor+molecules+greatly+improves+precision+genome+editing+in+Caenorhabditis+elegans.">Melting dsDNA donor molecules greatly improves precision genome editing in Caenorhabditis elegans.</a> Genetics. 216 (3), 643-650 (2020).</li><li>He, S., Cuentas-Condori, A., Miller, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NATF+(Native+and+Tissue-Specific+Fluorescence):+A+strategy+for+bright,+tissue-specific+GFP+labeling+of+native+proteins+in+Caenorhabditis+elegans.">NATF (Native and Tissue-Specific Fluorescence): A strategy for bright, tissue-specific GFP labeling of native proteins in Caenorhabditis elegans.</a> Genetics. 212 (2), 387-395 (2019).</li><li>Hammarlund, M., Hobert, O., Miller, D. M., Sestan, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+CeNGEN+Project:+The+complete+gene+expression+map+of+an+entire+nervous+system.">The CeNGEN Project: The complete gene expression map of an entire nervous system.</a> Neuron. 99 (3), 430-433 (2018).</li><li>Taylor, S. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+topography+of+an+entire+nervous+system.">Molecular topography of an entire nervous system.</a> Cell. 184 (16), 4329-4347 (2021).</li><li>Hammarlund, M., Hobert, O., Miller, D. M., Sestan, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+CeNGEN+project:+The+complete+gene+expression+map+of+an+entire+nervous+system.">The CeNGEN project: The complete gene expression map of an entire nervous system.</a> Neuron. 99 (3), 430-433 (2018).</li><li>Takihara, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+imaging+of+axonal+transport+of+mitochondria+in+the+diseased+and+aged+mammalian+CNS.">In vivo imaging of axonal transport of mitochondria in the diseased and aged mammalian CNS.</a> Proc Natl Acad Sci U S A. 112 (33), 10515-10520 (2015).</li><li>Li, L. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+neuronal+kinesin+UNC-104/KIF1A+is+a+key+regulator+of+synaptic+aging+and+insulin+signaling-regulated+memory.">The neuronal kinesin UNC-104/KIF1A is a key regulator of synaptic aging and insulin signaling-regulated memory.</a> Curr Biol. 26 (5), 605-615 (2016).</li><li>Viancour, T. A., Kreiter, N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vesicular+fast+axonal+transport+rates+in+young+and+old+rat+axons.">Vesicular fast axonal transport rates in young and old rat axons.</a> Brain Res. 628 (1-2), 209-217 (1993).</li><li>Cross, D. J., Flexman, J. A., Anzai, Y., Maravilla, K. R., Minoshima, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-related+decrease+in+axonal+transport+measured+by+MR+imaging+in+vivo.">Age-related decrease in axonal transport measured by MR imaging in vivo.</a> Neuroimage. 39 (3), 915-926 (2008).</li><li>Vagnoni, A., Hoffmann, P. C., Bullock, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reducing+Lissencephaly-1+levels+augments+mitochondrial+transport+and+has+a+protective+effect+in+adult+Drosophila+neurons.">Reducing Lissencephaly-1 levels augments mitochondrial transport and has a protective effect in adult Drosophila neurons.</a> J Cell Sci. 129 (1), 178-190 (2016).</li><li>Schindelin, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fiji:+an+open-source+platform+for+biological-image+analysis.">Fiji: an open-source platform for biological-image analysis.</a> Nat Methods. 9 (7), 676-682 (2012).</li><li>Thevenaz, P., Ruttimann, U. E., Unser, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+pyramid+approach+to+subpixel+registration+based+on+intensity.">A pyramid approach to subpixel registration based on intensity.</a> IEEE Trans Image Process. 7 (1), 27-41 (1998).</li><li>Neumann, S., Chassefeyre, R., Campbell, G. E., Encalada, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=KymoAnalyzer:+a+software+tool+for+the+quantitative+analysis+of+intracellular+transport+in+neurons.">KymoAnalyzer: a software tool for the quantitative analysis of intracellular transport in neurons.</a> Traffic. 18 (1), 71-88 (2017).</li><li>Hall, D. H., Russell, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+posterior+nervous+system+of+the+nematode+Caenorhabditis+elegans:+serial+reconstruction+of+identified+neurons+and+complete+pattern+of+synaptic+interactions.">The posterior nervous system of the nematode Caenorhabditis elegans: serial reconstruction of identified neurons and complete pattern of synaptic interactions.</a> J Neurosci. 11 (1), 1-22 (1991).</li><li>White, J. G., Southgate, E., Thomson, J. N., Brenner, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+structure+of+the+ventral+nerve+cord+of+Caenorhabditis+elegans.">The structure of the ventral nerve cord of Caenorhabditis elegans.</a> Philos Trans R Soc Lond B Biol Sci. 275 (938), 327-348 (1976).</li><li>White, J. G., Southgate, E., Thomson, J. N., Brenner, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+structure+of+the+nervous+system+of+the+nematode+Caenorhabditis+elegans.">The structure of the nervous system of the nematode Caenorhabditis elegans.</a> Philos Trans R Soc Lond B Biol Sci. 314 (1165), 1 (1986).</li><li>Park, J., Xie, Y., Miller, K. G., De Camilli, P., Yogev, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=End-binding+protein+1+promotes+specific+motor-cargo+association+in+the+cell+body+prior+to+axonal+delivery+of+dense+core+vesicles.">End-binding protein 1 promotes specific motor-cargo association in the cell body prior to axonal delivery of dense core vesicles.</a> Curr Biol. 33 (18), 3851-3864 (2023).</li><li>Dokshin, G. A., Ghanta, K. S., Piscopo, K. M., Mello, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Robust+genome+editing+with+short+single-stranded+and+long,+partially+single-stranded+DNA+donors+in+Caenorhabditis+elegans.">Robust genome editing with short single-stranded and long, partially single-stranded DNA donors in Caenorhabditis elegans.</a> Genetics. 210 (3), 781-787 (2018).</li><li>Paix, A., Folkmann, A., Rasoloson, D., Seydoux, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+efficiency,+homology-directed+genome+editing+in+Caenorhabditis+elegans+using+CRISPR-Cas9+ribonucleoprotein+complexes.">High efficiency, homology-directed genome editing in Caenorhabditis elegans using CRISPR-Cas9 ribonucleoprotein complexes.</a> Genetics. 201 (1), 47-54 (2015).</li><li>Goudeau, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Split-wrmScarlet+and+split-sfGFP:+tools+for+faster,+easier+fluorescent+labeling+of+endogenous+proteins+in+Caenorhabditis+elegans.">Split-wrmScarlet and split-sfGFP: tools for faster, easier fluorescent labeling of endogenous proteins in Caenorhabditis elegans.</a> Genetics. 217 (4), (2021).</li><li>Cranfill, P. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+assessment+of+fluorescent+proteins.">Quantitative assessment of fluorescent proteins.</a> Nat Methods. 13 (7), 557-562 (2016).</li><li>Fan, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SapTrap+assembly+of+Caenorhabditis+elegans+MosSCI+transgene+vectors.">SapTrap assembly of Caenorhabditis elegans MosSCI transgene vectors.</a> G3. 10 (2), 635-644 (2020).</li><li>Glomb, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+kinesin-1+adaptor+complex+controls+bimodal+slow+axonal+transport+of+spectrin+in+Caenorhabditis+elegans.">A kinesin-1 adaptor complex controls bimodal slow axonal transport of spectrin in Caenorhabditis elegans.</a> Dev Cell. 58 (19), 1847-1863 (2023).</li></ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Agarose</td>
			<td>Sigma-Aldrich</td>
			<td>A9539</td>
			<td></td>
		</tr>
		<tr>
			<td>Cover slips (22 mm x 22 mm, No1); Gold Seal Cover Glass</td>
			<td>Thomas Scientific</td>
			<td>6672A14</td>
			<td></td>
		</tr>
		<tr>
			<td>Levamisole</td>
			<td>ChemCruz</td>
			<td>sc-205730</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope: Nikon Ti2 inverted microscope, Yokogawa CSU-W1 SoRa Scanhead, Hamatsu Orca-Fusion BT sCMOS camera, Nikon CFI Plan Apo lambda 60x 1.4 NA oil immersion objective, Nikon photostimulation scanner at 488nm with an ET525/36 emission filter</td>
			<td>Nikon</td>
			<td></td>
			<td>Spinning Disc Confocal Microscope</td>
		</tr>
		<tr>
			<td>&#160;NIS-elements AR</td>
			<td>Nikon</td>
			<td></td>
			<td>Software for the Nikon Ti2&#160;</td>
		</tr>
		<tr>
			<td>Plain precleaned microscopy slides</td>
			<td>Thermo Scientific</td>
			<td>420-004T</td>
			<td></td>
		</tr>
		<tr>
			<td>Nematode strain</td>
			<td>Identifier</td>
			<td>Source</td>
			<td></td>
		</tr>
		<tr>
			<td>rab-3(ox699[GFP::flip-on::rab-3]) (II); shyIs43(glr-4p::FLP-NLSx2; odr-1p::RFP) (II)</td>
			<td>Park et al. (DOI: 10.1016/j.cub.2023.07.052)</td>
			<td>MTS1161&#160;</td>
			<td>Will be deposited at CGC (https://cgc.umn.edu/)</td>
		</tr>
	</tbody>
</table>

optimizing visualization of axonal transport of endogenous cargo by fluorescence microscopy in living caenorhabditis elegans

Throughout life, neurons rely on axonal transport to deliver proteins, lipids, and other molecules from the cell body to their final destination in the axon. Consequently, impairment of axonal transport is associated with a loss of neuronal function and is often involved in the pathology of neurodegenerative disorders1,2. Hence, understanding the mechanisms that underly axonal transport is of great interest. 
Several decades of research on axonal transport revealed many important insights into the molecular machinery that mediates this transport, their composition as well as regulatory mechanisms. Long-range axonal transport occurs on the microtubule cytoskeleton, which consists of partially overlapping microtubule polymers that are typically oriented with their plus end out in axons3. Consequently, anterograde transport is mediated by motor proteins that walk to the plus end of microtubules, kinesins, whereas retrograde transport depends on the minus end directed dynein motor. Although many aspects of transport have been revealed, for many axonal proteins it still remains unclear, how they are loaded into the transport machinery, how individual transport packages are organized, and how this transport is regulated3. 
Axonal transport was initially studied in radio-labeling experiments, in which radiolabeled amino acids were injected into the somatic compartment, where they were incorporated into nascent endogenous proteins and could be traced over time in the axonal compartment by autoradiography4. Although radiolabeling experiments allowed the study of axonal transport of endogenous proteins in vivo, it does not allow for the direct follow-up of the behavior of individual cargo to get mechanistic insights4. This limitation was overcome with the use of fluorescence microscopy. However, axonal transport is often not visualized on endogenous proteins but instead by expression of a fluorescent labeled copy. Especially for low expressed proteins, overexpression provides higher signal intensities which make visualization, preferably with single neuron resolution, possible. Moreover, ectopic expression of the fluorescent tagged protein circumvents the need and challenges of genome editing. Conversely, it has been argued that the behavior of ectopically expressed cargo may differ from the behavior of the endogenous cargo5. 
Recent improvements in genome editing made endogenous labeling strategies easier accessible. Hence, a lower signal intensity has become the major limitation to study axonal transport of a cargo by ectopic expression instead of endogenous labeling. Careful considerations in the endogenous labeling strategy paired with an optimization of the acquisition conditions can overcome this challenge. 
Nematodes provide an excellent research model to study axonal transport in vivo due to their transparency and ease in genetic manipulations. In this protocol, we describe a research strategy to visualize axonal transport of endogenous proteins with single neuron resolution in living Caenorhabditis elegans. We visualize the axonal transport of synaptic vesicle precursors by using a strain generated by the Jorgensen Lab6, in which the vesicle associated RAB GTPase, RAB3, is endogenously labeled with GFP, in the motor neuron DA9. By asking how small adaptations in different acquisition parameters and photobleaching can improve the visualization of individual transport events, the protocol provides ideas on how to optimize imaging conditions.

<meta charset="utf8"></head><body>저자 스포트라이트: Advanced Techniques for Visualizing Endogenous Axonal Transport Dynamics

Optimizing Visualization of Axonal Transport of Endogenous Cargo(내인성 화물의 축삭 수송 시각화 최적화)는 Living Caenorhabditis elegans에서 형광 현미경을 이용합니다.

Classical start is commonly used for axonal transport proteins for axonal transport visualization, but endogenous cargo behavior differs. Endogenous protein levels are low, making visualization challenging. We offer ideas to optimize the acquisition conditions for better visualization of endogenous GPR3, which labels synaptic vascular precursors.The abundance of cytosolic proteins in the axonal transport field, makes it difficult to visualize their dynamics, using conventional fluorescence microscopy. This protocol suggests using ablation step, along with temporally controlled labeling approaches to overcome these challenges. We have recently been able to visualize the axonal transport of the endogenous spectry.By optimizing the acquisition parameters described in this protocol, paired our temporal control labeling approach. This allowed us to identify the first components of the machinery that mediate the transport.

Watch this Scientific Journal Video about Optimizing Visualization of Axonal Transport of Endogenous Cargo by Fluorescence Microscopy in Living Caenorhabditis elegans at JoVE.com

Axonal transport is a prerequisite to deliver axonal proteins from their site of synthesis in the neuronal cell body to their destination in the axon. ...

optimizing-visualization-axonal-transport-endogenous-cargo

Research

JoVE Journal

Biology

Optimizing Visualization of Axonal Transport of Endogenous Cargo by Fluorescence Microscopy in Living Caenorhabditis elegans

480 조회수.  Yale School of Medicine. Classical start는 축삭 수송 시각화를 위해 축삭 수송 단백질에 일반적으로 사용되지만 내인성 화물 거동은 다릅니다. 내인성 단백질 수치가 낮아 시각화가 어렵습니다. 우리는 시냅스 혈관 전구체를 표지하는 내인성 GPR3의 더 나은 시각화를 위해 획득 조건을 최적화하기 위한 아이디어를 제공합니다.축삭 수송장에는 세포질 단백질이 풍부하기 때...