handling and preparation of caenorhabditis elegans for live imaging

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

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

Manuseio e preparação de Caenorhabditis elegans para imagens ao vivo

handling-and-preparation-of-caenorhabditis-elegans-for-live-imaging

Research

JoVE Journal

Biology

Handling and Preparation of Caenorhabditis elegans for Live Imaging

41 visualizações. Para começar, retire as placas de meio de crescimento do nematóide com Caenorhabditis elegans cultivadas em um gramado de bactérias. Usando um palito de fio de platina, transfira os vermes para uma gota de levamisol 0,5 milimolar e incube-os por 20 minutos. Pipeta de 12 microlitros M9 médio para um adesivo de 10% de agarose em uma lâmina de vidro.Usando um palito de cabelo, monte de 10 a 20 nematóides da gota de levamisol na gota M9 no adesivo de...

Vídeo: Manuseio e preparação de Caenorhabditis elegans para imagens ao vivo

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

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.

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. ...