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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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 ...

The Huntington's disease, HD, is an incurable, neurodegenerative pathology caused by a mutation in gene encoding huntingtin protein. The huntingtin is primarily associated with vesicles and microtubules, and probably involved in microtubule-dependent transport processes. To study the influence of mutant huntingtin on the dynamism of microtubules, we used in vivo visualization of EB3 protein, which regulates the dynamic properties of microtubules by bending and stabilizing the growing plus-ends.To load fluorescently-labeled EB3 into human skin fibroblasts, we applied plasmid transfection. We used the primary fibroblast culture to obtain from the EB3 patient's skin biopsy for this study. Deliver the biopsy to laboratory within a few hours in DMEM medium, supplemented with 15 microgram per milliliter of penicillin, and 50 units per milliliter of streptomycin.Place biopsy tissue in a six centimeters Petri dish, together with a small amount of medium. Using a sterile scalpel, cut the biopsy sample into pieces about 0.5 to one millimeters in size. Place one, two, obtained fragments into a 3.5 centimeters Petri dish, and place a sterile cover slip over the biopsy pieces.Slowly add 1.5 milliliters of a growth medium. Culture fiberblasts in the growth medium in a CO2 incubator, in 5%CO2 that is seven Centigrade, 80%humidity. After four, seven days, first keratinocytes, then fibroblasts begin to migrate from the tissue to the bottom of the dish.Cell cultivation. For transfection visualization, glass confocal plate dishes, confocal dishes should be used. Cover the dish bottom with autoclaved 0.1%gelatin solution prepared in distillate water.Incubate for 15 minutes. Prepare the culture medium. Mix thoroughly, store tall at plus 4 Centigrade.Warm the medium to plus 37 Centigrade, before adding to the cells. Assess the culture under the microscope. Remove the medium, and wash the cells with a sterile DPBS.Add one milliliter of pre-warmed 0.25%trypsin solution to the cells. Check the cells under the microscope if they detach from the substrate completely. Deactivate trypsin with one milliliter of the culture medium.Transfer the cell suspension into a 15-milliliter conical tube. Centrifuge the tube at 200 G for five minutes. Remove the supernatant, resuspend the cell palette in one milliliter of the culture medium.Count the cell number. Calculate the required number of cells. De-seed them with a density of eight to 15, multiplied by 10 to the power of three, passed by a centimeter.This is panned into milliliters of the culture medium. Remove the gelatin solution from the culture dish prepared for inoculation, and immediately add two milliliters of the cell suspension. Cultivate fibroblasts at plus 37 Centigrade in a CO2 incubator.Refresh the medium every two, four days. For experiments, use the cells of four, 11 passages. By the time of transfection, the confluence should be no more than 70, 80%Replace the culture medium with a fresh one 24 hours before transfection.Prepare a DNA-lipid complex, based on the area and density of the cell seeding. Liposomal transfection agent, and the density of the cell seeding of one, multiplied by 10 to the power of four cells, passed by a centimeter was used. Add three microliters of commercial reagent to 125 microliters of Opti-MEM, containing no antibiotic.Without touching the tube walls, gently resuspend. Dilute plasmid DNA, GFP-EB3, adding one microgram of the plasmid DNA to 125 microliters of OptiMEM. Gently resuspend.Add diluted plasmid DNA to each tube of diluted transfection reagent, one to one ratio. Incubate for 30 minutes. Add the DNA lipid complex to the six centimeters Petri dish with cells.Mix with a cruciform swing for 30 seconds. Incubate cells with a transfection agent for 24 hours, then change the medium to fresh one. Analyze the efficiency after inspection in 24 and 48 hours.Preparing for imaging. Before live imaging of cells, change the culture medium to a medium without PH indicator dye to reduce autofluorescence. Carefully apply mineral oil to the medium cell phase to completely cover the medium, isolating it from the external environment to reduce the O2 penetration, and the medium evaporation.Use a macro lamp and all image in 60-fold, 100-fold objective lens with high numeral aperture to take image. For in viva observations, the microscope must be equipped with an incubator to maintain the necessary conditions for the cells, including hitting the optic table, and the lens to pass 37 Centigrade, a closed chamber was CO2 supply, and humidity-level support. Place the confocal dish with the cells in the holder of the microscope before imaging.Ensure that the dish and the camera are securely attached to the holder to avoid drifting while taking images. Setting the imaging parameters. Choose the low exposure values, since slide induces cell-damaging, reacts with oxygen spaces.To study the dynamics of microtubules in human skin fibroblasts, we selected a 300 millisecond exposure. Focus on the object of interest. For long-time, time-lapse imaging, the automatic focus stabilization system, perfect focus system is necessary, since there may be a shift along the set axis, and the object of interest will thus be out of focus.Choose the optimal imaging conditions, depending on the cell's photosensitivity, and the rate of the flare chrome fading. Since microtubules are highly dynamic structures, are reasonably short time in turn can be selected, and the frame rate must be sufficiently high. To investigate the microtubule dynamics in skin fibroblasts, we used a frequency of one frame per second, for three, five minutes.When selecting the next object to get image, move away from the already imaged area, since under the influence of light, there is an elapsable photo-bleeding. Since we used a relatively high imaging frequency, the shutter didn't close between images, and the lamp was late for the entire period of imaging, which is fading increased. Choose the optimal videos for studying the microtubule dynamics visually, taking into account the quality of transfection, the quality of the microtubules'images, optimal signal-to-noise ratio, and the absence of drifting of analyzed cell.Use the selected videos to study the microtubules plus-ends dynamics, by trussing them in the Fiji program. The most critical step in the protocol is transfection, ensuring sufficient protein expression. Good signal-to-noise ratio, no photobleaching of microtubules, and absence of cell drifting are critical for imaging.In vivo visualization of microtubule dynamics provides vital information on the properties of mutant huntingtin. Our protocol can be applied to studies of other diseases, whose pathology implicates dynamic properties of cytoskeleton.

Research

JoVE Journal

Bioengineering

The Three-Dimensional Human Skin Reconstruct Model: a Tool to Study Normal  Skin and Melanoma Progression

Most in vitro studies in experimental skin biology have been done in 2-dimensional (2D) monocultures, while accumulating evidence suggests that cells ...

Spatio-Temporal Manipulation of Small GTPase Activity at Subcellular Level and on Timescale of Seconds in Living Cells

Dynamic regulation of the Rho family of small guanosine triphosphatases (GTPases) with great spatiotemporal precision is essential for various cellular ...

Visualization of Cortex Organization and Dynamics in Microorganisms, using Total Internal Reflection Fluorescence Microscopy

TIRF microscopy has emerged as  a powerful imaging technology to study spatio-temporal dynamics of fluorescent molecules  in vitro and in living cells1. ...

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to ...

From Fast Fluorescence Imaging to Molecular Diffusion Law on Live Cell Membranes in a Commercial Microscope

It has become increasingly evident that the spatial distribution and the motion of membrane components like lipids and proteins are key factors in the ...

Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models ...

Cloning and Large-Scale Production of High-Capacity Adenoviral Vectors Based on the Human Adenovirus Type 5

High-capacity adenoviral vectors (HCAdV) devoid of all viral coding sequences represent one of the most advanced gene delivery vectors due to their high ...

Microfluidic Flow Chambers Using Reconstituted Blood to Model Hemostasis and Platelet Transfusion In Vitro

Microfluidic Flow Chambers Using Reconstituted Blood to Model Hemostasis and Platelet Transfusion In Vitro

Blood platelets prepared for transfusion gradually lose hemostatic function during storage. Platelet function can be investigated using a variety of ...

A Microfluidic Flow Chamber Model for Platelet Transfusion and Hemostasis Measures Platelet Deposition and Fibrin Formation in Real-time

Microfluidic models of hemostasis assess platelet function under conditions of hydrodynamic shear, but in the presence of anticoagulants, this analysis is ...

Isolation of Adult Human Dermal Fibroblasts from Abdominal Skin and Generation of Induced Pluripotent Stem Cells Using a Non-Integrating Method

Induced pluripotent stem cells (iPSCs) could be considered, to date, a promising source of pluripotent cells for the management of currently untreatable ...