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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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&#039;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.

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 ...

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.

microtubule-plus-end-dynamics-visualization-huntington-s-disease

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 behave differently when they are grown within a 3D extra-cellular matrix and also interact with other cells (1-5). Mouse models have been broadly utilized to study tissue morphogenesis in vivo. However mouse and human skin have significant differences in cellular architecture and physiology, which makes it difficult to extrapolate mouse studies to humans. Since melanocytes in mouse skin are mostly localized in hair follicles, they have distinct biological properties from those of humans, which locate primarily at the basal layer of the epidermis. The recent development of 3D human skin reconstruct models has enabled the field to investigate cell-matrix and cell-cell interactions between different cell types. The reconstructs consist of a "dermis" with fibroblasts embedded in a collagen I matrix, an "epidermis", which is comprised of stratified, differentiated keratinocytes and a functional basement membrane, which separates epidermis from dermis. Collagen provides scaffolding, nutrient delivery, and potential for cell-to-cell interaction. The 3D skin models incorporating melanocytic cells recapitulate natural features of melanocyte homeostasis and melanoma progression in human skin. As in vivo, melanocytes in reconstructed skin are localized at the basement membrane interspersed with basal layer keratinocytes. Melanoma cells exhibit the same characteristics reflecting the original tumor stage (RGP, VGP and metastatic melanoma cells) in vivo. Recently, dermal stem cells have been identified in the human dermis (6). These multi-potent stem cells can migrate to the epidermis and differentiate to melanocytes.

In this report, we describe the three-dimensional skin reconstruct model which mimics human skin in architecture and composition. Melanocyte physiology, melanoma progression and the fate of dermal stem cells have been investigated using the skin reconstruct model. The model is also useful as a preclinical tool for drug assessment.

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 functions and events1, 2. Their spatiotemporally dynamic nature has been revealed by visualization of their activity and localization in real time3. In order to gain deeper understanding of their roles in diverse cellular functions at the molecular level, the next step should be perturbation of protein activities at a precise subcellular location and timing. 
To achieve this goal, we have developed a method for light-induced, spatio-temporally controlled activation of small GTPases by combining two techniques: (1) rapamycin-induced FKBP-FRB heterodimerization and (2) a photo-caging method of rapamycin. With the use of rapamycin-mediated FKBP-FRB heterodimerization, we have developed a method for rapidly inducible activation or inactivation of small GTPases including Rac4, Cdc424, RhoA4 and Ras5, in which rapamycin induces translocation of FKBP-fused GTPases, or their activators, to the plasma membrane where FRB is anchored. For coupling with this heterodimerization system, we have also developed a photo-caging system of rapamycin analogs. A photo-caged compound is a small molecule whose activity is suppressed with a photocleavable protecting group known as a caging group. To suppress heterodimerization activity completely, we designed a caged rapamycin that is tethered to a macromolecule such that the resulting large complex cannot cross the plasma membrane, leading to virtually no background activity as a chemical dimerizer inside cells6. Figure 1 illustrates a scheme of our system. With the combination of these two systems, we locally recruited a Rac activator to the plasma membrane on a timescale of seconds and achieved light-induced Rac activation at the subcellular level6.

A method for spatio-temporal control of small GTPase activity by light is described. This method is based on rapamycin-induced FKBP-FRB heterodimerization and photo-caging systems. Optimization of light-irradiation enables the spatio-temporally controlled activation of small GTPases at the subcellular level.

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. The optical phenomenon of total internal reflection occurs when light passes from a medium with high refractive index into a medium with low refractive index at an angle larger than a characteristic critical angle (i.e. closer to being parallel with the boundary). Although all light is reflected back under such conditions, an evanescent wave is created that propagates across and along the boundary, which decays exponentially with distance, and only penetrates sample areas that are 100-200 nm near the interface2. In addition to providing superior axial resolution, the reduced excitation of out of focus fluorophores creates a very high signal to noise ratios and minimizes damaging effects of photobleaching2,3. Being a widefield technique, TIRF also allows faster image acquisition than most scanning based confocal setups.
At first glance, the low penetration depth of TIRF seems to be incompatible with imaging of bacterial and fungal cells, which are often surrounded by thick cell walls. On the contrary, we have found that the cell walls of yeast and bacterial cells actually improve the usability of TIRF and increase the range of observable structures4-8. Many cellular processes can therefore be directly accessed by TIRF in small, single-cell microorganisms, which often offer powerful genetic manipulation techniques. This allows us to perform in vivo biochemistry experiments, where kinetics of protein interactions and activities can be directly assessed in living cells.
We describe here the individual steps required to obtain high quality TIRF images for Saccharomyces cerevisiae or Bacillus subtilis cells. We point out various problems that can affect TIRF visualization of fluorescent probes in cells and illustrate the procedure with several application examples. Finally, we demonstrate how TIRF images can be further improved using established image restoration techniques.

Total Internal Reflection Fluorescence (TIRF) microscopy is a powerful approach to observe structures close to the cell surface at high contrast and temporal resolution. We demonstrate how TIRF can be employed to study protein dynamics at the cortex of cell wall-enclosed bacterial and fungal cells.

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

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to evaluate new strategies that aim at improving vascularization, reliable methods are required to make the in-growth of new blood vessels into bio-artificial scaffolds visible and quantify the results. Over the past couple of years, our group has introduced a full skin defect model that enables the direct visualization of blood vessels by transillumination and provides the possibility of quantification through digital segmentation. In this model, one surgically creates full skin defects in the back of mice and replaces them with the material tested. Molecules or cells of interest can also be incorporated in such materials to study their potential effect. After an observation time of one&#8217;s own choice, materials are explanted for evaluation. Bilateral wounds provide the possibility of making internal comparisons that minimize artifacts among individuals as well as of decreasing the number of animals needed for the study. In comparison to other approaches, our method offers a simple, reliable and cost effective analysis. We have implemented this model as a routine tool to perform high-resolution screening when testing vascularization of different biomaterials and bio-activation approaches.

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

Vascularization is key to approaches in successful tissue engineering. Therefore, reliable technologies are required to evaluate the development of vascular networks in tissue-constructs. Here we present a simple and cost-effective method to visualize and quantify vascularization 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 regulation of many cellular functions. However, due to the fast dynamics and the tiny structures involved, a very high spatio-temporal resolution is required to catch the real behavior of molecules. Here we present the experimental protocol for studying the dynamics of fluorescently-labeled plasma-membrane proteins and lipids in live cells with high spatiotemporal resolution. Notably, this approach doesn&#8217;t need to track each molecule, but it calculates population behavior using all molecules in a given region of the membrane. The starting point is a fast imaging of a given region on the membrane. Afterwards, a complete spatio-temporal autocorrelation function is calculated correlating acquired images at increasing time delays, for example each 2, 3, n repetitions. It is possible to demonstrate that the width of the peak of the spatial autocorrelation function increases at increasing time delay as a function of particle movement due to diffusion. Therefore, fitting of the series of autocorrelation functions enables to extract the actual protein mean square displacement from imaging (iMSD), here presented in the form of apparent diffusivity vs average displacement. This yields a quantitative view of the average dynamics of single molecules with nanometer accuracy. By using a GFP-tagged variant of the Transferrin Receptor (TfR) and an ATTO488 labeled 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine (PPE) it is possible to observe the spatiotemporal regulation of protein and lipid diffusion on &#181;m-sized membrane regions in the micro-to-milli-second time range.

Spatial distribution and temporal dynamics of plasma membrane proteins and lipids is a hot topic in biology. Here this issue is addressed by a spatio-temporal image fluctuation analysis that provides conceptually the same physical quantities of single particle tracking, but it uses small molecular labels and standard microscopy setups.

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 reflect the human wound situation better than animal models. Although two-dimensional models are widely used to investigate processes such as cellular migration and proliferation, models that are more complex are required to gain a deeper knowledge about wound healing. Besides a suitable model system, the generation of precise and reproducible wounds is crucial to ensure comparable results between different test runs. In this study, the generation of a three-dimensional full thickness skin equivalent to study wound healing is shown. The dermal part of the models is comprised of human dermal fibroblast embedded in a rat-tail collagen type I hydrogel. Following the inoculation with human epidermal keratinocytes and consequent culture at the air-liquid interface, a multilayered epidermis is formed on top of the models. To study the wound healing process, we additionally developed an automated wounding device, which generates standardized wounds in a sterile atmosphere.

The goal of this protocol is to build up a three-dimensional full thickness skin equivalent, which resembles natural skin. With a specifically constructed automated wounding device, precise and reproducible wounds can be generated under maintenance of sterility.

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 packaging capacity (up to 35 kb), low immunogenicity, and low toxicity. However, for many laboratories the use of HCAdV is hampered by the complicated procedure for vector genome construction and virus production. Here, a detailed protocol for efficient cloning and production of HCAdV based on the plasmid pAdFTC containing the HCAdV genome is described. The construction of HCAdV genomes is based on a cloning vector system utilizing homing endonucleases (I-CeuI and PI-SceI). Any gene of interest of up to 14 kb can be subcloned into the shuttle vector pHM5, which contains a multiple cloning site flanked by I-CeuI and PI-SceI. After I-CeuI and PI-SceI-mediated release of the transgene from the shuttle vector the transgene can be inserted into the HCAdV cloning vector pAdFTC. Because of the large size of the pAdFTC plasmid and the long recognition sites of the used enzymes associated with strong DNA binding, careful handling of the cloning fragments is needed. For virus production, the HCAdV genome is released by NotI digest and transfected into a HEK293 based producer cell line stably expressing Cre recombinase. To provide all adenoviral genes for adenovirus amplification, co-infection with a helper virus containing a packing signal flanked by loxP sites is required. Pre-amplification of the vector is performed in producer cells grown on surfaces and large-scale amplification of the vector is conducted in spinner flasks with producer cells grown in suspension. For virus purification, two ultracentrifugation steps based on cesium chloride gradients are performed followed by dialysis. Here tips, tricks, and shortcuts developed over the past years working with this HCAdV vector system are presented.

A protocol for generation of high-capacity adenoviral vectors lacking all viral coding sequences is presented. Cloning of transgenes contained in the vector genome is based on homing endonucleases. Virus amplification in producer cells grown as adherent cells and in suspension relies on a helper virus providing viral genes in trans.

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

Blood platelets prepared for transfusion gradually lose hemostatic function during storage. Platelet function can be investigated using a variety of (indirect) in vitro experiments, but none of these is as comprehensive as microfluidic flow chambers. In this protocol, the reconstitution of thrombocytopenic fresh blood with stored blood bank platelets is used to simulate platelet transfusion. Next, the reconstituted sample is perfused in microfluidic flow chambers which mimic hemostasis on exposed subendothelial matrix proteins. Effects of blood donation, transport, component separation, storage and pathogen inactivation can be measured in paired experimental designs. This allows reliable comparison of the impact every manipulation in blood component preparation has on hemostasis. Our results demonstrate the impact of temperature cycling, shear rates, platelet concentration and storage duration on platelet function. In conclusion, this protocol analyzes the function of blood bank platelets and this ultimately aids in optimization of the processing chain including phlebotomy, transport, component preparation, storage and transfusion.

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

Platelet transfusion and hemostasis was modeled using blood reconstitution and microfluidic flow chambers to investigate the function of blood banking platelets. The data demonstrate the consequences of platelet storage lesion on hemostasis, 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 restricted to platelet deposition only. The intricate relationship between Ca2+-dependent coagulation and platelet function requires careful and controlled recalcification of blood prior to analysis. Our setup uses a Y-shaped mixing channel, which supplies concentrated Ca2+/Mg2+ buffer to flowing blood just prior to perfusion, enabling rapid recalcification without sample stasis. A ten-fold difference in flow velocity between both reservoirs minimizes dilution. The recalcified blood is then perfused in a collagen-coated analysis chamber, and differential labeling permits real-time imaging of both platelet and fibrin deposition using fluorescence video microscopy. The system uses only commercially available tools, increasing the chances of standardization. Reconstitution of thrombocytopenic blood with platelets from banked concentrates furthermore models platelet transfusion, proving its use in this research domain. Exemplary data demonstrated that coagulation onset and fibrin deposition were linearly dependent on the platelet concentration, confirming the relationship between primary and secondary hemostasis in our model. In a timeframe of 16 perfusion min, contact activation did not take place, despite recalcification to normal Ca2+ and Mg2+ levels. When coagulation factor XIIa was inhibited by corn trypsin inhibitor, this time frame was even longer, indicating a considerable dynamic range in which the changes in the procoagulant nature of the platelets can be assessed. Co-immobilization of tissue factor with collagen significantly reduced the time to onset of coagulation, but not its rate. The option to study the tissue factor and/or the contact pathway increases the versatility and utility of the assay.

This paper describes an experimental model of hemostasis that simultaneously measures platelet function and coagulation. Platelet and fibrin fluorescence is measured in real-time, and platelet adhesion rate, coagulation rate, and onset of coagulation are determined. The model is used to determine platelet procoagulant properties under flow in concentrates for transfusion.

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 diseases, for the reconstitution and regeneration of injured tissues and for the development of new drugs. Despite all the advantages related to the use of iPSCs, such as the low risk of rejection, the lessened ethical issues, and the possibility to obtain them from both young and old patients without any difference in their reprogramming potential, problems to overcome are still numerous. In fact, cell reprogramming conducted with viral and integrating viruses can cause infections and the introduction of required genes can induce a genomic instability of the recipient cell, impairing their use in clinic. In particular, there are many concerns about the use of c-Myc gene, well-known from several studies for its mutation-inducing activity. Fibroblasts have emerged as the suitable cell population for cellular reprogramming as they are easy to isolate and culture and are harvested by a minimally invasive skin punch biopsy. The protocol described here provides a detailed step-by-step description of the whole procedure, from sample processing to obtain cell cultures, choice of reagents and supplies, cleaning and preparation, to cell reprogramming by the means of a commercial non-modified RNAs (NM-RNAs)-based reprogramming kit. The chosen reprogramming kit allows an effective reprogramming of human dermal fibroblast to iPSCs and small colonies can be seen as early as 24 h after the first transfection, even with modifications with the respect to the standard datasheet. The reprogramming procedure used in this protocol offers the advantage of a safe reprogramming, without the risk of infections caused by viral vector-based methods, reduces the cellular defense mechanisms, and allows the generation of xeno-free iPSCs, all critical features that are mandatory for further clinical applications.

Cell reprogramming requires the introduction of key genes, which regulate and maintain the pluripotent cell state. The protocol described enables the formation of induced pluripotent stem cells (iPSCs) colonies from human dermal fibroblasts without viral/integrating methods but using non-modified RNAs (NM-RNAs) combined with immune evasion factors reducing cellular defense mechanisms.

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