<meta charset="utf8"></head><body>Aprimorando a análise de transporte axonal em C. elegans vivos usando RAB-3 marcado com GFP

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<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>Autor em destaque: técnicas avançadas para visualizar a dinâmica do transporte axonal endógeno

Otimizando a visualização do transporte axonal de carga endógena por microscopia de fluorescência em Caenorhabditis elegans vivos

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

533 visualizações.  Yale School of Medicine. O início clássico é comumente usado para proteínas de transporte axonal para visualização do transporte axonal, mas o comportamento da carga endógena difere. Os níveis de proteína endógena são baixos, tornando a visualização desafiadora. Oferecemos ideias para otimizar as condições de aquisição para melhor visualização do GPR3 endógeno, que rotula precursores vasculares sinápticos.A abundância de proteínas citosólicas no campo d...

Vídeo: Autor em destaque: técnicas avançadas para visualizar a dinâmica do transporte axonal endógeno

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. Consequently, loss of axonal transport impairs neuronal growth and function. Studying axonal transport therefore improves our understanding of neuronal cell biology. With recent improvements in CRISPR Cas9 genome editing, endogenous labeling of axonal cargos has become accessible, enabling to move beyond ectopic expression-based visualization of transport. However, endogenous labeling often comes at the cost of low signal intensity and necessitates optimization strategies to obtain robust data. Here, we describe a protocol to optimize the visualization of axonal transport by discussing acquisition parameters and a bleaching approach to improve the signal of endogenous labeled cargo over diffuse cytoplasmic background. We apply our protocol to optimize the visualization of synaptic vesicle precursors (SVPs) labeled by green fluorescent protein (GFP)-tagged RAB-3 to highlight how fine-tuning acquisition parameters can improve the analysis of endogenously labeled axonal cargo in Caenorhabditis elegans (C. elegans).

The paper describes the optimization of fluorescence microscopy acquisition parameters to visualize the axonal transport of endogenous labeled cargos at single-neuron resolution in a living nematode.