<em>In vivo</em> Assessment of Microtubule Dynamics and Orientation in <em>Caenorhabditis elegans</em> Neurons

Swagata Dey; Anindya Ghosh-Roy

doi:10.3791/62744

In vivo (in vivo) Ocena dynamiki i orientacji mikrotubul w neuronach Caenorhabditis ...

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JoVE Journal Neuroscience

In vivo Assessment of Microtubule Dynamics and Orientation in Caenorhabditis elegans Neurons

In vivo (in vivo) Ocena dynamiki i orientacji mikrotubul w neuronach Caenorhabditis elegans

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07:43 min

November 20, 2021

DOI: 10.3791/62744-v

Swagata Dey¹, Anindya Ghosh-Roy¹

¹DBT-National Brain Research Centre

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Overview

This study presents a protocol for in vivo imaging of dynamic microtubules using fluorescently labeled End binding protein EBP in the posterior lateral microtubule (PLM) neuron of C. elegans. The protocol aims to visualize microtubule dynamics, allowing researchers to assess the orientation and behavior of these cytoskeletal structures during neuronal development and regeneration.

Key Study Components

Area of Science

Neuroscience
Cytoskeletal Dynamics
Fluorescence Imaging

Background

Microtubules are integral to neuronal structure and function, influencing development and maturation.
They are composed of alpha and beta tubulin and exhibit dynamic behavior critical for neuronal processes.
End-binding proteins play a crucial role in promoting microtubule assembly and dynamics.

Purpose of Study

To develop a method for visualizing microtubule dynamics in vivo.
To analyze the behavior of microtubules during neuronal development and regeneration in C. elegans.
To adapt the technique for use in various cell types beyond neurons.

Methods Used

Fluorescence microscopy with a spinning disk unit was employed for imaging.
C. elegans served as the biological model, focusing on the PLM neurons.
Transgenic expression of EBP-2 fused to GFP was utilized for labeling.
Worms were mounted using polystyrene beads and viewed under a fluorescence microscope.
Time-lapse imaging and analysis were performed using ImageJ for quantitative measurements of microtubule dynamics.

Main Results

Comets observed represent dynamic microtubules, with movement classified as plus-end-out or minus-end-out.
The method enabled the assessment of growth direction, duration, and dynamics of microtubules, especially following axonal injury.
Observations indicated robust regeneration of axons with clear patterns of microtubule orientation.

Conclusions

This study facilitates the visualization and analysis of microtubule dynamics in neurons, advancing understanding of neuronal behavior during development and injury responses.
The findings have implications for exploring neuronal mechanisms and plasticity in various biological contexts.

Frequently Asked Questions

What are the advantages of using C. elegans for this study?

C. elegans is a well-established model organism for studying neuronal development and regeneration due to its simple nervous system and transparency, enabling direct observation.

How is the EBP reporter expressed in the neurons?

The EBP-2 transgene fused to GFP is expressed under specific promoters targeting PLM neurons to visualize microtubule dynamics.

What types of imaging outcomes are obtained?

The method produces time-lapse images of EBP comets, allowing for analysis of microtubule growth direction, duration, and structural dynamics.

How can this technique be adapted for other cell types?

The EBP reporter can be expressed in different cell types like muscles and skin by using appropriate promotors, enabling visualization of microtubule behavior in these cells.

What are potential limitations of this imaging technique?

Limitations may include phototoxicity, autofluorescence, and challenges with protein overexpression, which can affect data quality.

How does the technique measure microtubule dynamics quantitatively?

Quantitative measurement involves creating kymographs from time-lapse images to analyze the velocity and directionality of moving comets, which represent microtubule dynamics.

What potential cellular processes can this technique help elucidate?

The technique can enhance understanding of critical processes like cell division, migration, and neuronal regeneration through detailed analysis of microtubule dynamics.

Przedstawiono protokół obrazowania dynamicznych mikrotubul in vivo przy użyciu fluorescencyjnie znakowanego białka wiążącego End. Opisaliśmy metody znakowania, obrazowania i analizowania dynamicznych mikrotubul w neuronie tylnej bocznej mikrotubuli (PLM) C. elegans.

Celem tej procedury jest ocena organizacji i dynamiki mikrotubul in vivo. Neurony to spolaryzowane komórki z wyraźnymi przedziałami aksonalnymi, dendrytycznymi i synaptycznymi, utrzymywanymi przez leżący pod nimi cytoszkielet. Mikrotubule stanowią większość cytoszkieletu neuronalnego, a dynamika i orientacja determinują kluczowe zdarzenia podczas rozwoju i dojrzewania neuronów.

Mikrotubule składają się z heterodimerów alfa i beta tubuliny i są z natury spolaryzowane z wysoce dynamicznymi końcami dodatnimi i stabilnymi końcami ujemnymi. Podczas polimeryzacji na końcach dodatnich wielocząsteczkowy kompleks białek wiążących końce przejściowo łączy się z protofilamentami i sprzyja składaniu dimerów tubuliny. Ta technika pomoże nam uwidocznić mikrotubule neuronalne in vivo.

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