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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

This protocol is dedicated to the microtubule plus-end visualization by EB3 protein transfection to study their dynamic properties in primary cell culture. The protocol was implemented on human primary skin fibroblasts obtained from Huntington's disease patients.

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.

Introduction

Huntington's disease (HD) is an incurable neurodegenerative pathology caused by a mutationin gene encodinghuntingtin protein (HTT). HTT is primarily associated with vesicles and microtubules and is probably involved in microtubule-dependent transport processes1,2. To study the influence of mutant HTT on the microtubule dynamics, we used in vitro visualization of the EB3 protein, that regulates the dynamic properties of microtubules by binding and stabilizing the growing plus-ends. To load fluorescently labeled EB3 into human skin fibroblasts, plasmid transfection was applied. We used the....

Protocol

This protocol follows the guidelines of the Federal Research and Clinical Center of Physical-Chemical Medicine of Federal Medical Biological Agency dated September 08, 2015.

NOTE: Figure 1 gives an overview of the protocol.

1. Obtaining a primary culture of human skin fibroblasts (Figure 2)

  1. Deliver the biopsy to a laboratory within a few hours in Dulbecco's .......

Representative Results

The resulting GFP-EB3 movies produced using the protocol (Figure 1) illustrate the microtubules' dynamic properties. Microtubules are involved in different cell processes, and their dynamic properties impact various life characteristics of the primary human cell culture from patients' biopsy material (Figure 2).

The following parameters determine the dynamic instability of microtubules: the rates of growth (polymerization) and.......

Discussion

Better quality results for microtubules' dynamics analysis can be obtained from high-quality microscopic images. It is important to observe all the necessary conditions for time-lapse imaging of living cells and to correctly adjust the imaging parameters. Using special cell culture dishes with a glass bottom (confocal dishes) is important, since glass has a different refractive index of light than plastic. The thickness of the glass and its uniformity over its entire area is also extremely important, since these paramete.......

Acknowledgements

This research was funded by the Ministry of Science and Higher Education of the Russian Federation, grant No. 075-15-2019-1669 (transfection of fibroblasts), by the Russian Science Foundation, grant No. 19-15-00425 (all other works on the cultivation of fibroblasts in vitro). It was partially supported by Lomonosov Moscow State University Development program PNR5.13 (imaging and analysis). The authors acknowledge the support of the Nikon Center of Excellence at A. N. Belozersky Institute of Physico-Chemical Biology. We want to offer our special thanks to Ekaterina Taran for her help assistance with voice acting. The authors also thank Pavel Belikov for his he....

Materials

NameCompanyCatalog NumberComments
Instrumentation
Camera iXon DU897 EMCCDAndor Technology
Eppendorf Centrifuge 5804 REppendorf Corporate
Fluorescence filter set HYQ FITCNikonAlternative: Leica, Olympus, Zeiss
LUNA-II Automated Cell CounteLogos BiosystemsL40002
Microscope incubator for lifetime filmingOkolabTemperature controller H301-T-UNIT-BL-PLUS
Gas controller CO2-O2-UNIT-BL
Objective lens CFI Plan Apo Lambda 60x Oil 1.4 (WD 0.13)NikonAlternative: Leica, Olympus, Zeiss
Widefield fluorescence light microscope Eclipse Ti-ENikonAlternative: Leica, Olympus, Zeiss
Software
Fiji (Image J version 2.1.0/1.53c)Open source image processing software
NIS ElementsNikonAlternative: Leica, Olympus, Zeiss
Additional reagents
Mineral oil (Light white oil)MP151694
Cell culture dish
Cell Culture DishSPL Lifesciences20035
Confocal Dish (glass thickness 170 µm)SPL Lifesciences211350Alternative: MatTek
Conical Centrifuge tubeSPL Lifesciences50015
Cryogenic VialsCorning-Costar430659
Microcentrifuge TubeNest615001
Cultivation
Lipofectamine 3000 Transfection ReagentThermo Fisher ScientificL3000001
Dimethyl sulfoxidePanEkofigure-materials-2766135
DMEM (Dulbecco's Modified Eagle Media)PanEkoC420figure-materials-2956
DPBS (Dulbecco's phosphate-salt solution)PanEkoP060figure-materials-3146
Fetal bovine serum (FBS)HycloneK053/SH30071.03
Gelatin (bovine skin)PanEkofigure-materials-3421070
GlutaMAXThermo Fisher Scientific35050038
Opti-MEM (1x) + GlutamaxGibco519850026
Penicillin-streptomycinPanEkoA063figure-materials-3804
Trypsin-EDTA (0.25%)Thermo Fisher Scientific25200072
Transfection
Plasmid DNA with EB3-GFPKind gift of Dr. I. Kaverina [Vanderbilt University, Nashville] with permission from Dr. A. Akhmanova
[Erasmus University, Rotterdam]
Stepanova et al., 2003 DOI: 10.1523/JNEUROSCI.23-07-02655.2003

References

  1. Engelender, S., et al. Huntingtin-associated Protein 1 (HAP1) Interacts with the p150 Glued Bubunit of Dynactin. Human Molecular Genetics. 6 (13), 2205-2212 (1997).
  2. Caviston, J. P., Ross, J. L., Antony, S. M., Tokito, M., Holzbaur, E. L.

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Microtubule DynamicsEB3 ProteinHuntington s DiseaseHuman Primary Skin FibroblastsCell CultureTransfectionFluorescent LabelingMicroscopy

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