Measurement of Microtubule Dynamics by Spinning Disk Microscopy in Monopolar Mitotic Spindles

Naira Movsisyan; Luis A. Pardo

doi:10.3791/60478

Measurement of Microtubule Dynamics by Spinning Disk Microscopy in Monopolar Mitotic Spindles

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Biology

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Measurement of Microtubule Dynamics by Spinning Disk Microscopy in Monopolar Mitotic Spindles

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08:31 min

November 15, 2019

DOI: 10.3791/60478-v

Naira Movsisyan^1,2, Luis A. Pardo¹

¹AG Oncophysiology,Max-Planck Institute of Experimental Medicine, ²Göttingen Graduate School for Neurosciences, Biophysics, and Molecular Biosciences (GGNB)

Overview

This study presents a robust method for analyzing microtubule dynamics in cells synchronized in prometaphase, utilizing live-cell spinning disk confocal microscopy and MATLAB-based image processing. The technique facilitates reduced phototoxicity, allowing for the imaging of a larger number of cells and contributing to understanding microtubule dynamics in both normal and pathological conditions.

Key Study Components

Research Area

Microtubule dynamics
Cell synchronization
Live-cell imaging

Background

Microtubule dynamics are crucial for cell division and are altered in many diseases.
Understanding their regulation can aid in disease mechanism elucidation.
The method can also be adapted for drug discovery efforts.

Methods Used

Live-cell spinning disk confocal microscopy
HeLa cells synchronized in prometaphase
Synchronized imaging with red-shifted fluorescent proteins

Main Results

A comprehensive analysis of microtubule plus-end dynamics.
Data indicated minimal impact of imaging settings on growth speed, but significant effects on dynamicity.
Achieved robust tracking and characterization of microtubule behavior during cell division.

Conclusions

The study demonstrates a detailed method for investigating microtubule dynamics, which may be applicable in clinical research and drug development.
This work enhances understanding of microtubule behavior in cells and potential therapeutic targets for related diseases.

Frequently Asked Questions

What is the significance of analyzing microtubule dynamics?

Microtubule dynamics play a crucial role in cell division, and understanding their regulation can help identify therapeutic targets for diseases.

How does the method reduce phototoxicity?

The use of red-shifted fluorescent proteins in conjunction with spinning disk microscopy minimizes phototoxic effects, allowing for the imaging of more cells.

What cell type was used in the study?

HeLa cells were used for the analysis of microtubule dynamics.

How can the method be adapted for drug discovery?

The method can be modified to screen drugs against microtubules by changing fluorescence desynchronization protocols and analyzing different cell cycle phases.

What is the main technical challenge in this study?

Optimizing transfection protocols and seeding density for various cell types is essential for achieving accurate results.

What tools are used for image analysis?

A MATLAB-based platform and the u-track analysis software are employed for processing the imaging data.

What are the implications of the findings?

The findings provide insights into microtubule dynamics, which could inform therapeutic strategies for conditions where microtubule function is disrupted.

Here we present a robust and detailed method of microtubule dynamics analysis in cells synchronized in prometaphase using live-cell spinning disk confocal microscopy and MATLAB-based image processing.

What we present here is a robust and simple method to analyze microtubule dynamics using live-cell spinning disk confocal imaging in cells synchronized in prometaphase, and images are afterwards analyzed on a MATLAB-based platform. The use of red-shifted fluorescent protein in combination with spinning disk microscopy reduces phototoxicity. Therefore, a larger number of cells can be imaged within the same preparation.

Microtubule dynamicity is altered in a large number of pathological conditions. Therefore, understanding the regulation and the behavior of microtubule dynamics in those processes can help us understanding the mechanism of disease, and eventually, developing therapies to compensate for them. And the method is also upscaleable, so it could potentially be applied in a drug discovery effort.

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