<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

생체 내 Caenorhabditis elegans 뉴런의 미세 투덜역학 및 오리엔테이션 평가

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Neuroscience
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JoVE Journal Neuroscience

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

생체 내 Caenorhabditis elegans 뉴런의 미세 투덜역학 및 오리엔테이션 평가

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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.

형광 표지된 엔드 결합 단백질을 사용하여 생체 내의 동적 마이크로투부레를 이미징하기 위한 프로토콜이 제시되었다. 우리는 C. elegans의후방 측측 마이크로투룰 (PLM) 뉴런에서 동적 마이크로 튜브를 라벨, 이미지 및 분석하는 방법을 설명했다.

이 절차의 목적은 생체 내 의 마이크로 튜블러 조직 과 역학을 평가하는 것입니다. 뉴런은 뚜렷한 축축, 수지상 및 시냅스 구획을 가진 편광 세포이며, 그들의 근본적인 세포골격에 의해 유지됩니다. 마이크로 투부는 신경 세포 골격의 대부분을 형성하고, 역학및 방향은 신경 발달과 성숙 도중 중요한 사건을 결정합니다.

마이크로튜블러는 알파및 베타 튜룰린 이종수로 구성되어 있으며, 본질적으로 매우 역동적인 플러스 엔드와 안정적인 마이너스 엔드로 양극화됩니다. 플러스 엔드에서 중합 하는 동안, 최종 결합 단백질의 다중 분자 복합체, 일시적으로 프로토 필라멘트와 연결 하 고 tubulin dimers의 조립을 촉진. 이 기술은 우리가 생체 내에서 신경 마이크로 투튜를 시각화하는 데 도움이 될 것입니다.

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