Name: microtubule plus-end dynamics visualization in huntington's disease model based on human primary skin fibroblasts
Uploaded: 2022-01-08T13:00:00
Description: Watch this Scientific Journal Video about Microtubule Plus-End Dynamics Visualization in Huntington's Disease Model based on Human Primary Skin Fibroblasts at JoVE.com

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 help with the video editing. Figures in the manuscript were created with BioRender.com.

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)
<ol>
	<li>Deliver the biopsy to a laboratory within a few hours in Dulbecco&#39;s ...

<ol>
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C., Akhmanova, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microtubule+plus-end+tracking+proteins+in+neuronal+development.">Microtubule plus-end tracking proteins in neuronal development.</a> Cellular and Molecular Life Sciences. 73 (10), 2053-2077 (2016).</li><li>Applegate, K. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=plusTipTracker:+Quantitative+image+analysis+software+for+the+measurement+of+microtubule+dynamics.">plusTipTracker: Quantitative image analysis software for the measurement of microtubule dynamics.</a> Journal of Structural Biology. 176 (2), 168-184 (2011).</li><li>Komarova, Y. A., Vorobjev, I. A., Borisy, G. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Life+cycle+of+MTs:+persistent+growth+in+the+cell+interior,+asymmetric+transition+frequencies+and+effects+of+the+cell+boundary.">Life cycle of MTs: persistent growth in the cell interior, asymmetric transition frequencies and effects of the cell boundary.</a> Journal of Cell Science. 115 (17), 3527-3539 (2002).</li><li>Nahidiazar, L., Agronskaia, A. V., Broertjes, J., vanden Broek, B., Jalink, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimizing+imaging+conditions+for+demanding+multi-color+super+resolution+localization+microscopy.">Optimizing imaging conditions for demanding multi-color super resolution localization microscopy.</a> PLoS One. 11 (7), 0158884 (2016).</li><li>Dixit, R., Cyr, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+damage+and+reactive+oxygen+species+production+induced+by+fluorescence+microscopy:+effect+on+mitosis+and+guidelines+for+non-invasive+fluorescence+microscopy.">Cell damage and reactive oxygen species production induced by fluorescence microscopy: effect on mitosis and guidelines for non-invasive fluorescence microscopy.</a> The Plant Journal. 36 (2), 280-290 (2003).</li><li>Grzelak, A., Rychlik, B., Bartosz, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light-dependent+generation+of+reactive+oxygen+species+in+cell+culture+media.">Light-dependent generation of reactive oxygen species in cell culture media.</a> Free Radical Biology and Medicine. 30 (12), 1418-1425 (2001).</li></ol>

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...

Huntington&#39;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&#160;dynamics, we used in vitro&#160;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 primary fibroblast culture obtained from the HD patients&#39; skin biopsy for this study.
The mutation in the HTT protein gene leads to elongation of the polyglutamine tract3. HTT has a role in such cellular processes as endocytosis4, cell transport1,2, protein degradation5, etc. Substantial part of these processes involves various elements of the cell cytoskeleton, including the microtubules.
Human primary cells are the best model to reproduce events occurring in patient cells as closely as possible. To create such models, one needs to isolate cells from human biopsy material (e.g., from surgical samples). The resulting primary cell line is suitable to study pathogenesis using various genetic, biochemical, molecular, and cell biology methods. Also, human primary cell cultures serve as a precursor for creating various transdifferentiated and transgenic cultures6.
However, in contrast to immortalized cell cultures, the significant disadvantage of primary cells is their limited passage capacity. Therefore, we recommend using cells in the early passages stage (up to 15). Older cultures degenerate very quickly, losing their unique properties. Thus, the newly obtained primary cells should be kept frozen for long-term storage.
Primary cell cultures are susceptible to cultivation conditions. Therefore, they often require unique approaches and optimization of growing conditions. In particular, the human skin primary fibroblasts used in our experiments are demanding on the substrate. Hence, we used various additional coatings (e.g., gelatin or fibronectin) depending on the experiment type.
The cell cytoskeleton determines the cell shape, mobility, and locomotion. The dynamics of the cytoskeleton are crucial for many intracellular processes both in interphase and mitosis. In particular, the cytoskeleton polymerized from tubulin, are highly dynamic and polar structures, enabling motor protein-mediated directed intracellular transport. The microtubules&#39; ends are in constant rearrangement, their assembly phases alternate with the disassembly phases, and this behavior is called &#34;dynamic instability&#34;7,8,9. Various associated proteins shift the equilibrium of the polymerization reaction, leading either to the polymer formation or the protein monomer formation. The addition of tubulin subunits occurs mainly at the plus-end of microtubules10. The end-binding (EB) proteins family consists of three members: EB1, EB2, and EB3. They serve as plus-end-tracking proteins (+TIPs) and regulate the dynamic properties of microtubules by binding and stabilizing their growing plus-ends11.
Many studies use fluorescent molecule-labeled tubulin microinjection or transfection with time-lapse imaging and video analysis to visualize microtubules in vitro. These methods might be invasive and harmful to cells, especially primary human cells. The most challenging step is to find conditions for cell transfection. We tried to reach the highest possible level of transfection without affecting viability and native cell morphology. This study applies the classical method to study the differences in microtubule dynamics in skin fibroblasts of healthy donors and patients with Huntington&#39;s disease.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Instrumentation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Camera iXon DU897 EMCCD</td>
			<td>Andor Technology</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf Centrifuge 5804 R</td>
			<td>Eppendorf Corporate</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence filter set HYQ FITC</td>
			<td>Nikon</td>
			<td></td>
			<td>Alternative: Leica, Olympus, Zeiss</td>
		</tr>
		<tr>
			<td>LUNA-II Automated Cell Counte</td>
			<td>Logos Biosystems</td>
			<td>L40002</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope incubator for lifetime filming</td>
			<td>Okolab</td>
			<td>Temperature controller H301-T-UNIT-BL-PLUS</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>Gas controller CO2-O2-UNIT-BL</td>
			<td></td>
		</tr>
		<tr>
			<td>Objective lens CFI Plan Apo Lambda 60x Oil 1.4 (WD 0.13)</td>
			<td>Nikon</td>
			<td></td>
			<td>Alternative: Leica, Olympus, Zeiss</td>
		</tr>
		<tr>
			<td>Widefield fluorescence light microscope Eclipse Ti-E</td>
			<td>Nikon</td>
			<td></td>
			<td>Alternative: Leica, Olympus, Zeiss</td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fiji (Image J version 2.1.0/1.53c)</td>
			<td>Open source image processing software</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NIS Elements</td>
			<td>Nikon</td>
			<td></td>
			<td>Alternative: Leica, Olympus, Zeiss</td>
		</tr>
		<tr>
			<td>Additional reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mineral oil (Light white oil)</td>
			<td>MP</td>
			<td>151694</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture dish</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Culture Dish</td>
			<td>SPL Lifesciences</td>
			<td>20035</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal Dish (glass thickness 170 &#181;m)</td>
			<td>SPL Lifesciences</td>
			<td>211350</td>
			<td>Alternative: MatTek</td>
		</tr>
		<tr>
			<td>Conical Centrifuge tube</td>
			<td>SPL Lifesciences</td>
			<td>50015</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryogenic Vials</td>
			<td>Corning-Costar</td>
			<td>430659</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge Tube</td>
			<td>Nest</td>
			<td>615001</td>
			<td></td>
		</tr>
		<tr>
			<td>Cultivation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Lipofectamine 3000 Transfection Reagent</td>
			<td>Thermo Fisher Scientific</td>
			<td>L3000001</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide</td>
			<td>PanEko</td>
			<td><img alt="Equation 1" src="/files/ftp_upload/62963/62963eq01.jpg" />135</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM (Dulbecco&#39;s Modified Eagle Media)</td>
			<td>PanEko</td>
			<td>C420<img alt="Equation 2" src="/files/ftp_upload/62963/62963eq02.jpg" /></td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS (Dulbecco&#39;s phosphate-salt solution)</td>
			<td>PanEko</td>
			<td>P060<img alt="Equation 2" src="/files/ftp_upload/62963/62963eq02.jpg" /></td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Hyclone</td>
			<td>K053/SH30071.03</td>
			<td></td>
		</tr>
		<tr>
			<td>Gelatin (bovine skin)</td>
			<td>PanEko</td>
			<td><img alt="Equation 1" src="/files/ftp_upload/62963/62963eq01.jpg" />070</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMAX</td>
			<td>Thermo Fisher Scientific</td>
			<td>35050038</td>
			<td></td>
		</tr>
		<tr>
			<td>Opti-MEM (1x) + Glutamax</td>
			<td>Gibco</td>
			<td>519850026</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin</td>
			<td>PanEko</td>
			<td>A063<img alt="Equation 2" src="/files/ftp_upload/62963/62963eq02.jpg" /></td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.25%)</td>
			<td>Thermo Fisher Scientific</td>
			<td>25200072</td>
			<td></td>
		</tr>
		<tr>
			<td>Transfection</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Plasmid DNA with EB3-GFP</td>
			<td>Kind gift of Dr. I. Kaverina [Vanderbilt University, Nashville] with permission from Dr. A. Akhmanova 
			[Erasmus University, Rotterdam]</td>
			<td>Stepanova et al., 2003 DOI: 10.1523/JNEUROSCI.23-07-02655.2003</td>
			<td></td>
		</tr>
	</tbody>
</table>

microtubule plus-end dynamics visualization in huntington's disease model based on human primary skin fibroblasts

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.

The resulting GFP-EB3 movies produced using the protocol (Figure 1) illustrate the microtubules&#39; 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&#39; biopsy material (Figure 2).
The following parameters determine the dynamic instability of microtubules: the rates of growth (polymerization) and...

Watch this Scientific Journal Video about Microtubule Plus-End Dynamics Visualization in Huntington's Disease Model based on Human Primary Skin Fibroblasts at JoVE.com

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

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.

Transfection with a fluorescently labeled marker protein of interest in combination with time-lapse video microscopy is a classic method of studying the ...

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