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Abstract

Bioengineering

Microtubule Plus-End Dynamics Visualization in Huntington's Disease Model based on Human Primary Skin Fibroblasts

Published: January 8th, 2022

DOI:

10.3791/62963

1A.N. Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, 2Faculty of Bioengineering and Bioinformatics, Lomonosov Moscow State University, 3Federal Research and Clinical Center of Physical-Chemical Medicine of Federal Medical Biological Agency, 4Center for Precision Genome Editing and Genetic Technologies for Biomedicine, Federal Research and Clinical Center of Physical-Chemical Medicine of Federal Medical Biological Agency

* These authors contributed equally

Abstract

Transfection with a fluorescently labeled marker protein of interest in combination with time-lapse video microscopy is a classic method of studying the dynamic properties of the cytoskeleton. This protocol offers a technique for human primary fibroblast transfection, which can be difficult because of the specifics of primary cell cultivation conditions. Additionally, cytoskeleton dynamic property maintenance requires a low level of transfection to obtain a good signal-to-noise ratio without causing microtubule stabilization. It is important to take measures to protect the cells from light-induced stress and fluorescent dye fading. In the course of our work, we tested different transfection methods and protocols as well as different vectors to select the best combination of conditions suitable for human primary fibroblast studies. We analyzed the resulting time-lapse videos and calculated microtubule dynamics using ImageJ. The dynamics of microtubules' plus-ends in the different cell parts are not similar, so we divided the analysis into subgroups - the centrosome region, the lamella, and the tail of fibroblasts. Notably, this protocol can be used for in vitro analysis of cytoskeleton dynamics in patient samples, enabling the next step towards understanding the dynamics of the various disease development.

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Keywords Microtubule Dynamics

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