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

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 processes^1,2. 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 primary fibroblast culture obtained from the HD patients' 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 transport^1,2, 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' ends are in constant rearrangement, their assembly phases alternate with the disassembly phases, and this behavior is called "dynamic instability"^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's disease.

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 Modified Eagle Media (DMEM) medium supplemented with 50 µg/mL of penicillin and 50 U/mL of streptomycin.
   NOTE: A skin biopsy must be performed under sterile conditions by a physician after a patient signs an informed consent.
2. Place the biopsy tissue in a 6 cm Petri dish together with a small amount of the medium.
3. Using a sterile scalpel, cut the biopsy sample into pieces of about 0.5-1 mm in size. Place 1-2 obtained fragments into a 3.5 cm Petri dish and place a sterile coverslip over the biopsy pieces. Slowly add 1.5 mL of a growth medium to the following composition: DMEM, 50 U/mL penicillin-streptomycin, and 10% fetal bovine serum (FBS).
4. Culture fibroblasts in the growth medium inside a CO₂ incubator maintained at 5% CO₂, 37 °C, 80% humidity.
   ​NOTE: After 4-7 days, first keratinocytes, then fibroblasts, begin to migrate from the tissue to the bottom of the dish.

2. Storage, freezing, and unfreezing of primary culture

1. Remove cells from the culture dish (see points 3.2-3.4).
2. Transfer the cell suspension to a 15 mL conical tube and centrifuge for 5 min at 200 x g. Then discard the supernatant and resuspend the cell pellet in 900 µL of cooled FBS.
3. Transfer to a cryopreservation tube drop by drop and add 100 µL of dimethyl sulfoxide (DMSO).
4. Place the cryoprobe in a low-temperature freezer at -80 °C. Twenty-four hours later, transfer the cryovial to liquid nitrogen (−196 °C) for long-term storage.
5. To defrost the cryopreserved cells, remove the cryovial from the nitrogen storage and, within 1 min, transfer 1 mL of the content into a 15 mL conical tube containing 9 mL of the transport medium preheated to 37 °C.
6. Carefully resuspend and then centrifuge the tube for 5 min at 200 x g. Discard the supernatant, resuspend the cell pellet in the required volume of the growth medium and place them on a Petri dish of the required diameter.

3. Cell cultivation

1. Cover the dish bottom with autoclaved 0.1% gelatin solution prepared in distilled water. Incubate for 15 min.
   NOTE: For transfection visualization, glass-bottom plate dishes (confocal dishes with glass thickness 170 µm) should be used.
2. Prepare a culture medium having the following composition: DMEM supplemented with 10% FBS, 2 mM L-alanyl-L-glutamine, 50 U/mL penicillin-streptomycin. Mix thoroughly and store at 4 °C. Warm the medium to 37 °C before adding to the cells.
3. Assess the culture under the microscope. Remove the medium and wash the fibroblasts with Dulbecco's phosphate-salt solution (DPBS).
4. Add 1 mL 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 1 mL of the culture medium.
5. Transfer the cell suspension into a 15 mL conical tube. Centrifuge the tube at 200 x g for 5 min, remove the supernatant, and resuspend the cell pellet in 1 mL of the culture medium.
6. Count the number of cells. Calculate the required number of cells to seed them with a density of 8-15 x 10³/cm² and resuspend in 2 mL of the culture medium.
7. Remove the gelatin solution from the culture dish and immediately add 2 mL of the cell suspension. Cultivate fibroblasts at 37 °C in a CO₂ incubator.
8. Refresh the medium every 2-4 days.
   ​NOTE: For experiments, use the cells of 4-11 passages.

4. Transfection

1. Replace the culture medium with a fresh culture medium 24 h before transfection.
   NOTE: The cell confluence should be 70-80%.
2. Prepare a DNA-lipid complex based on the area and density of the cell seeding. Use liposome-based transfection agent.
   NOTE: Use the cell seeding density as 1 x 10⁴ cells/cm².
3. Add 3 µL of commercial transfection reagent to 125 µL of optimal minimal essential medium (Opti-MEM) not containing any antibiotics, without touching the walls of the tube. Gently resuspend.
4. Dilute plasmid DNA (GFP-EB3) by adding 1 µg of the plasmid DNA to 125 µL of Opti-MEM. Gently resuspend.
   NOTE: Expression vector encoding GFP-EB3¹¹ was received as a kind gift from Dr. I. Kaverina (Vanderbilt University, Nashville) with permission from Dr. A. Akhmanova (Erasmus University, Rotterdam)¹¹.
5. Add diluted plasmid DNA to each tube of diluted transfection reagent (1:1). Incubate for 30 min.
6. Add the DNA-lipid complex to the 6 cm Petri dish containing cells and mix with a cruciform swing for 30 s. Incubate cells with a transfection agent for 24 h and then change to fresh medium. Analyze the efficiency of transfection after 24 h and 48 h.
   ​NOTE: 24 h after transfection, the efficiency was 10-15%, and after 48 h up to 40%.

5. Preparing for imaging

1. Before live imaging of cells, change the culture medium to a medium without pH-indicator dye to reduce autofluorescence.
2. Carefully apply mineral oil to the medium surface to completely cover the medium, isolating it from the external environment to reduce the O₂ penetration and the medium evaporation.
3. Use a mercury lamp and oil immersion 60x or 100x objective lens with a high numeral aperture to take the images.
   NOTE: For in vivo observations, the microscope must be equipped with an incubator to maintain the necessary conditions for the cells, including heating the object table and the lens to +37 °C, a closed chamber with CO₂ supply, and humidity level support. Use double distilled water to create humidity. Check the level of double distilled water before filming.
4. Place the confocal dish with the cells in the microscope holder before imaging. Ensure that the dish and the camera are securely attached to the holder to avoid drifting while taking images.

6. Setting the imaging parameters

1. Choose the low exposure values since light induces cell-damaging reactive oxygen species (ROS).
   NOTE: To study the dynamics of microtubules in human skin fibroblasts, a 300 ms exposure was selected.
2. Focus on the object of interest.
   NOTE: For long-term time-lapse imaging, use the automatic focus stabilization system to compensate for a possible shift along the z-axis.
3. Choose the optimal imaging conditions depending on the cells' photosensitivity and the rate of the fluorochrome fading.
   NOTE: Since microtubules are highly dynamic structures, a reasonably short time interval can be selected, and the frame rate must be sufficiently high. To investigate the microtubule dynamics in skin fibroblasts, we used at a frequency of 1 frame/s for 3-5 min.
4. When selecting the next object to get the image, move away from the already imaged area. Since this area was under the influence of light, there will be noticeable photo-bleaching.
   NOTE: Since a relatively high imaging frequency was used, the shutter did not close between images, and the lamp was lit for the entire period of imaging, which is why fading increased.
5. Choose the optimal video for studying the microtubules' 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 in case of the analyzed cell (Figure 3).
6. Use the selected videos to study the microtubules' plus-ends dynamics by tracing them in the ImageJ or Fiji program (Figure 4).
   NOTE: For quantitative analysis instructions see Supplementary Figure 2 and Supplementary File 1.

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...

Materials

Name	Company	Catalog Number	Comments
Instrumentation
Camera iXon DU897 EMCCD	Andor Technology
Eppendorf Centrifuge 5804 R	Eppendorf Corporate
Fluorescence filter set HYQ FITC	Nikon		Alternative: Leica, Olympus, Zeiss
LUNA-II Automated Cell Counte	Logos Biosystems	L40002
Microscope incubator for lifetime filming	Okolab	Temperature 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)	Nikon		Alternative: Leica, Olympus, Zeiss
Widefield fluorescence light microscope Eclipse Ti-E	Nikon		Alternative: Leica, Olympus, Zeiss
Software
Fiji (Image J version 2.1.0/1.53c)	Open source image processing software
NIS Elements	Nikon		Alternative: Leica, Olympus, Zeiss
Additional reagents
Mineral oil (Light white oil)	MP	151694
Cell culture dish
Cell Culture Dish	SPL Lifesciences	20035
Confocal Dish (glass thickness 170 µm)	SPL Lifesciences	211350	Alternative: MatTek
Conical Centrifuge tube	SPL Lifesciences	50015
Cryogenic Vials	Corning-Costar	430659
Microcentrifuge Tube	Nest	615001
Cultivation
Lipofectamine 3000 Transfection Reagent	Thermo Fisher Scientific	L3000001
Dimethyl sulfoxide	PanEko	135
DMEM (Dulbecco's Modified Eagle Media)	PanEko	C420
DPBS (Dulbecco's phosphate-salt solution)	PanEko	P060
Fetal bovine serum (FBS)	Hyclone	K053/SH30071.03
Gelatin (bovine skin)	PanEko	070
GlutaMAX	Thermo Fisher Scientific	35050038
Opti-MEM (1x) + Glutamax	Gibco	519850026
Penicillin-streptomycin	PanEko	A063
Trypsin-EDTA (0.25%)	Thermo Fisher Scientific	25200072
Transfection
Plasmid DNA with EB3-GFP	Kind 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