Live Cell Imaging to Assess the Dynamics of Metaphase Timing and Cell Fate Following Mitotic Spindle Perturbations

Summary

Here we present a protocol to assess the dynamics of spindle formation and mitotic progression. Our application of time-lapse imaging enables the user to identify cells at various stages of mitosis, track and identify mitotic defects, and analyze spindle dynamics and mitotic cell fate upon exposure to anti-mitotic drugs.

Abstract

Live cell time-lapse imaging is an important tool in cell biology that provides insight into cellular processes that might otherwise be overlooked, misunderstood, or misinterpreted by the fixed-cell analysis. While the fixed cell imaging and analysis is robust and sufficient to observe cellular steady-state, it can be limited in defining a temporal order of events at the cellular level and is ill-equipped to assess the transient nature of dynamic processes including mitotic progression. In contrast, live cell imaging is an eloquent tool that can be used to observe cellular processes at the single-cell level over time and has the capacity to capture the dynamics of processes that would otherwise be poorly represented in fixed cell imaging. Here we describe an approach to generate cells carrying fluorescently labeled markers of chromatin and microtubules and their use in live cell imaging approaches to monitor metaphase chromosome alignment and mitotic exit. We describe imaging-based techniques to assess the dynamics of spindle formation and mitotic progression, including the identification of cells at various stages in mitosis, identification and tracking of mitotic defects, and analysis of spindle dynamics and mitotic cell fate following the treatment with mitotic inhibitors.

Introduction

Image-based analysis of fixed cells is commonly used to assess the cell population level changes in response to various perturbations. When combined with cell synchronization, followed by the collection and imaging of serial time points, such approaches can be used to suggest a cellular sequence of events. Nevertheless, fixed cell imaging is limited in that temporal relationships are implied for a population and not demonstrated at the level of individual cells. In this way, while fixed cell imaging and analysis is sufficient to observe robust phenotypes and steady-state changes, the ability to detect transient changes over time and changes that impact only a subpopulation of the cells is imperfect. In contrast, live cell imaging is an eloquent tool that can be used to observe cellular and subcellular processes within a single cell, or cellular population, over time and without the aid of synchronization approaches that may themselves impact cellular behavior^1,2,3,4,5,6.

The formation of a bipolar mitotic spindle is essential for the proper chromosome segregation during cell division, resulting in two genetically identical daughter cells. Defects in mitotic spindle structure that corrupt mitotic progression and compromise the fidelity of chromosome segregation can result in catastrophic cell divisions and reduced cell viability. For this reason, mitotic poisons that alter spindle formation are promising therapeutics to limit the rapid proliferation of cancer cells^7,8,9. Nevertheless, fixed cell analysis of spindle structure following the addition of mitotic poisons is limited in its ability to assess the dynamic process of spindle formation and may not indicate whether observed changes in spindle structure are permanent or are instead transient and may be overcome to permit successful cell division.

In this protocol, we describe an approach to assess the dynamics of mitosis following spindle perturbations by live cell imaging. Using the hTERT immortalized RPE-1 cell line engineered to express an RFP-tagged Histone 2B to visualize chromatin, together with an EGFP-tagged α-tubulin to visualize microtubules, the timing of metaphase chromosome alignment, anaphase onset, and ultimately mitotic cell fate are assessed using visual cues of chromosome movement, compaction, and nuclear morphology.

Protocol

1. Generation of hTERT-RPE-1 cells stably expressing RFP-Histone 2B (RFP-H2B) and α-tubulin-EGFP (tub-EGFP)

NOTE: All steps follow aseptic techniques and take place in a biosafety level II+ (BSL2+) safety cabinet.

1. Generate retrovirus carrying the genes of interest (α-tubulin-EGFP and RFP-H2B) by the transfection of 293T cells with the appropriate lentiviral plasmids according to the manufacturer's instructions of the lipid-based transfection delivery system.
   1. Day 1: Use a disposable glass Pasteur pipette to aspirate the cell culture medium from a plate of sub-confluent 293T cells and wash off the residual medium with phosphate buffered saline (PBS) by adding 5 mL of PBS. Swirl to distribute PBS over the plate bottom, then aspirate the PBS with a sterile disposable glass Pasteur pipette.
   2. Add 2 mL of 0.05% trypsin that has been pre-warmed to 37 °C and return the plate to a humidified incubator at 37 °C with 5% CO₂ for 2 to 5 min to allow adherent cells to be released from the cell culture dish surface.
   3. Add 8 mL of fresh Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin to the plate containing trypsin.
   4. Resuspend the cells by gently pipetting and transfer the suspension to a sterile 15 mL conical tube. Place the conical tube in a balanced centrifuge and spin at 161 x g for 5 min at room temperature to gently pellet the cells.
   5. Aspirate the medium/trypsin solution from pelleted cells and resuspend cells in 10 mL of DMEM supplemented with 10% FBS and 1% penicillin/streptomycin. Use a hemocytometer to count the 293T cell suspension and plate 2 x 10⁶ 293T cells per well of a 6 well plate.
   6. Culture cells in a total volume of 2 mL of Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin and maintain in a humidified incubator at 37 °C with 5% CO₂.
   7. Day 2: Pipette 7 µL of lipid-based transfection delivery reagent and 100 µL of reduced serum medium into a 1.5 mL microcentrifuge tube and allow it to incubate at room temperature for 5 min (tube #1).
   8. In a separate 1.5 mL microcentrifuge tube, pipette 1 µg of RFP-H2B expression vector (or 1 µg of α-tubulin-EGFP expression vector), together with 5 µL enhancer reagent (as required per manufacturer's guidelines), 0.5 µg pMD2.G, and 1 µg psPAX2 and 100 µL of reduced serum medium (tube #2).
   9. Combine the contents of step 1.1.7 and step 1.1.8 by carefully pipetting tube #2 into tube #1 and incubate for 20 min at room temperature.
      NOTE: The reaction can be scaled as needed for transfection of cells in additional wells.
   10. Pipette the transfection reaction dropwise to the desired well containing 293T cells in 2 mL of medium and return the dish to the humidified incubator at 37 °C with 5% CO₂.
       NOTE: To generate cells expressing both RFP-H2B and α-tubulin-EGFP, generate separate virus for each expression construct (steps 1.1.7- 1.1.9).
   11. Day 3: Aspirate and replace the medium with 2 mL of fresh DMEM supplemented with 10% FBS and 1% penicillin/streptomycin. Return the dish to the humidified incubator at 37 °C with 5% CO₂.
2. Day 4: Collect the medium containing expressed virus particles, being cautious not to disrupt or remove 293T cells. Filter the medium containing virus particles by passing it through a 0.45 µM filter attached to a 5 mL syringe. Aliquot virus for short term storage at 4 °C, or long term storage at -80 °C.
   NOTE: Viral particles obtained in this manner will be present in a range of ~1 x 10⁷ to 1 x 10⁸ Transducing Units/mL. As the viral-producing cells and cells to be infected are both cultured in the same medium, viral concentration to replace the medium is not required and filtered viral particles can be used directly to infect cells.
3. Seed 2 x 10⁵ hTERT-RPE-1 cells per well of a 6-well dish in preparation for the viral infection. Culture cells in 2 mL of DMEM supplemented with 10% FBS and 1% penicillin/streptomycin and maintain in a humidified incubator at 37 °C with 5% CO₂.
4. Day 5: Add hexadimethrine bromide (e.g., polybrene)to the hTERT-RPE-1 cells to a final concentration of 8 µg/mL. Infect cells with a mixture of 500 µL of virus diluted in 500 µL of cell culture medium.
   NOTE: Hexadimethrine bromide stock solution is prepared in sterile distilled water, then filtered through a 0.45 μm filter.
5. Day 6: Using a disposable glass Pasteur pipette, aspirate the medium from wells containing the virus infected cells. Replace the cell culture medium with 2 mL of DMEM containing 10% FBS, 1% penicillin/streptomycin, and appropriate concentrations of antibiotic to select for the plasmid integration and expression.
   NOTE: The α-tubulin-EGFP expression plasmid used in the described experiments carries a puromycin-resistance gene and the RFP-H2B expression plasmid carries a blasticidin resistance gene. Therefore, 10 µg/mL of puromycin and 2 µg/mL of blasticidin are used for the selection of α-tubulin-EGFP, RFP-H2B expressing hTERT RPE-1 cells. Concentrations of antibiotic used for the selection may differ for various cell lines used.
6. Maintain cells under antibiotic selection for 5-7 days, replacing the medium every 3 days with fresh medium containing appropriate selection reagents.
   NOTE: Cells should be maintained at sub-confluence during the selection and should be expanded as needed, as described in steps 1.1.1 to 1.1.4.
7. Use immunofluorescence imaging to confirm the expression of tagged constructs.
   NOTE: If desired, single cell clones can be derived to obtain uniform expression levels within the cell population. Once stable clones are confirmed, cells can be maintained under the standard culture conditions in the absence of antibiotic selection.

2. Preparation of cells for live cell imaging following mitotic spindle perturbations

NOTE: Use aseptic techniques and perform the steps in a BSL2 safety cabinet.

1. Using a sterile disposable glass Pasteur pipette, aspirate the medium from the culture plate containing the cell line that carries the expression construct(s) (from step 1.7). Briefly wash the cells with 10 mL of sterile PBS. Swirl to distribute PBS over the plate bottom, then aspirate PBS with a sterile disposable glass Pasteur pipette.
2. Add 2 mL of 0.05% trypsin to the 10 cm plate. Incubate the plate at 37 °C for 2-5 min or until cells have detached from the plate surface.
3. Add 8 mL of fresh medium to the plate containing trypsin. Resuspend the cells by gently pipetting and transfer the suspension to a sterile 15 mL conical tube. Place the conical tube in a balanced centrifuge and spin at 161 x g for 5 min at room temperature to gently pellet the cells.
4. Carefully aspirate the supernatant and resuspend in 10 mL of PBS by pipetting gently with a 10 mL serological pipette. Place the conical tube in a balanced centrifuge and spin at 161 x g for 5 min at room temperature to gently pellet the cells.
5. Carefully aspirate the supernatant and resuspend in 10 mL of the fresh medium by pipetting gently with a 10 mL serological pipette.
6. Count cells, then calculate the cell number using a hemacytometer and dilute to a concentration of 1-2 x 10⁵ cells/mL in the cell culture medium. Seed 500 µL of the cell suspension to each well of a sterile 12-well imaging bottom plate. Place the plate in the cell culture incubator and allow cells to adhere to the plate surface.
   NOTE: Live cell imaging requires cells to be well-adhered so that they remain associated with the imaging plane during image acquisition. The duration of the time needed for cells to adhere following plating can differ from one cell line to the next and should be optimized for the cell line under study.
7. Up to 30 min prior to initiating time-lapse imaging, add a relevant concentration of a mitotic drug to one or more of the wells seeded with cells. To account for the potential impact of the organic solvent on cellular behavior, add an equal volume of the inhibitor's diluent to cells as controls. For example, ensure that the addition of 100 nM of the specific inhibitor of the mitotic kinase Aurora A (e.g., alisertib) is paralleled with an equal volume of DMSO, the diluent for this drug, in a control well of cells.
   NOTE: Comparative analysis of dynamic changes in the mitotic progression require wells of cells to be prepared in the absence of perturbations to enable imaging of normal mitoses in parallel to experimental conditions.

3. Microscope set up for time-lapse imaging of RFP-H2B, α-tubulin-GFP expressing cells (Figure 1, Figure 2)

1. Place the cell culture plate containing RFP-H2B, α-tubulin-GFP expressing hTERT RPE-1 cells to be imaged into an appropriate stage insert on an inverted epifluorescence microscope that is equipped with a high resolution camera (pixel size of 0.67 μm at 20x), an environmental chamber preheated to 37 °C, and a delivery system for humidified 5% CO₂.
   NOTE: Either enclosed environmental chambers or temperature controlled stage inserts may be appropriate provided humidified 5% CO₂ can be delivered and stable temperature control obtained.
2. Use a 20x air objective with a numerical aperture of 0.5 and equipped for the high contrast fluorescence and phase contrast or brightfield imaging. View cells with the phase contrast or brightfield and adjust the course and fine focus on the microscope to bring cells into focus.
3. Identify and set the optimal exposure times for brightfield, GFP, and RFP image acquisition by selecting the respective filter cube with appropriate excitation and emission for the fluorophores that will be imaged. Alternatively, click the auto exposure button to input a predetermined exposure time. If the signal is not sufficiently intense, select pixel binning to enable shorter exposure times by clicking on the binning tab and selecting 2 x 2 pixel binning from the drop down menu.
   NOTE: Phototoxicity can impair mitotic progression and cell viability. Failure of mitotic cells in control populations to complete normal mitoses may be an indication that exposure times and/or imaging duration needs to be further optimized.
4. Use an acquisition and analysis software that enables multi-coordinate, multi-well imaging to be acquired concurrently and define the parameters for the image acquisition.
   1. Select and calibrate the microscope stage to the multi-well dish, according to the manufacturer's instructions. Use the image acquisition software to highlight or otherwise select the wells that will be imaged by clicking on the relevant wells in the diagram of the plate format that is being used.
   2. Under the GeneratedPoints control panel (Figure 2), define the coordinates within each well that will be imaged by clicking on the Point Placement tab and selecting predefined or random coordinate placement from the drop down menu. Select the Working Area tab and select restricted from the drop down menu to restrict the coordinate selection area to exclude the boundaries of the well. Click the Count and Distribution tabs to select the number and distribution of points to be captured per well, respectively.
      NOTE: The number of coordinates that can be imaged per well within the 5 min timepoint increment will be limited by the number of wells to be imaged, as well as the exposure time for each channel. Typically, 5-8 coordinates per well are sufficient to observe at least 50 cells in each condition progress through mitosis within 4 h.
   3. Select and input the time interval and duration to collect images by clicking on and inputting the values in the TimeSequence control panel.
      NOTE: Acquisition of images every 1 to 5 minutes is appropriate to monitor the dynamics of mitotic progression. The overall duration of imaging should reflect the desired endpoint and proliferation rate of the cell line. For example, 4 hours is sufficient to see many cells progress through mitosis, 16 hours is sufficient to see most RPE-1 cells in an asynchronous population progress through mitosis.

4. Time-lapse image analysis to determine metaphase timing and mitotic cell fate following mitotic spindle perturbations

NOTE: Perform the image analysis using an image acquisition software (Table of Materials), ImageJ, or comparable image analysis software.

1. Visualize RFP-H2B labeled chromatin by selecting the images captured with the RFP filter cube in place. Identify a cell entering mitosis as indicated by initial chromatin compaction (Figure 2C, blue arrowhead) and nuclear envelope break down (when α-tubulin-EGFP is no longer excluded by the nuclear boundary).
2. Determine the mitotic timing of metaphase alignment and anaphase onset in individual cells: Track the cell through consecutive timepoints in the acquired movie to determine the number of timepoints/minutes from mitotic entry until RFP-H2B-labelled chromatin completes alignment at the cell equator during metaphase (Figure 2C, yellow arrowhead).
3. To monitor mitotic timing, mitotic fidelity, and cell fate, continue to track the cell through consecutive timepoints to identify the time coordinate at which anaphase chromosome segregation is apparent (Figure 2C, white arrowhead) and/or where chromatin decompaction and nuclear envelope reformation (as indicated by tubulin exclusion from the nucleus) has occurred.
   NOTE: Perturbations in spindle assembly or mitotic progression can be assessed as a function of the time required to obtain a bipolar mitotic spindle and achieve complete chromosome alignment, or to complete anaphase chromosome segregation.
4. Visualize RFP-H2B to identify cells in each population that exhibit mitotic defects including lagging chromosomes and chromatin bridges during anaphase chromosome segregation.
   NOTE: Compromised mitotic fidelity may also result in multinucleated or micronucleated daughter cells and can be visualized in cells post mitotic exit, as in Figure 3.

Results

Assessment of mitotic progression in the presence of spindle perturbations
The regulation of spindle pole focusing is an essential step in proper bipolar spindle formation. Disruption in this process through protein depletions, drug inhibition, or alterations in centrosome number corrupt spindle structure and delay or halt mitotic progression^10,11,12,

Discussion

The temporal resolution provided by the time-lapse imaging allows for the visualization and assessment of sequential cellular events within single cells. Approaches that make use of cellular synchronization followed by the collection and fixation of cells at sequential time points are limited in that comparisons are ultimately made between populations of cells. In contexts where the cellular response to perturbations may be non-uniform, or where the process being visualized is dynamic, live cell time-lapse imaging is bet...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.05% Trypsin	Gibo-Life sciences	25-510	A serine protease used to release adherent cells from culture dishes
15ml centrifuge tubes	Olympus Plastics	28-101
20x CFI Plan Fluor objective	Nikon		For use in Live-cell imaging to visualize both bright field and fluorescence
293T Cells	ATCC	CRL-3216	For use in retroviral transfection; used in step 1.1
a-tubulin-EGFP	Addgene	various numbers	Expression vector for alpha tubulin fused to a green fluorescent protein tag; for use in the visualization of tubulin in live-cell imaging: commercially available through addgene and other vendors
Alisertib	Selleckchem	S1133	Small molecule inhibitor of the mitotic kinase Aurora A. Stock concentration is prepared at 10mM in DMSO, and used at a final concentration of 100nM.
Blasticidin	Invitrogen	A11139-03	Antibiotic selection agent; used to select for a-tub-EGFP expressing cells
C02	Airgas		For use in cell culture and live cell imaging
Chroma ET-DS Red (TRITC/Cy3)	Chroma	49005	Single band filter set; excitation wavelength 545nm with 25nm bandwidth and emission at 605nm wavelength with 70nm bandwidth; for visualization of H2B-RFP
Chroma ET-EGFP (FITC/Cy2)	Chroma	49002	Single band filter set; excitation wavelength 470nm with 40nm bandwidth and emission at 525nm wavelength with 50nm bandwidth; for visualization of GFP-tubulin
disposable glass Pastuer pipets, sterilized	Fisher Scientific	13-678-6A	For use in aspirating cells
Dulbecco’s Modified Eagle Medium (DMEM)	Gibo-Life sciences	11965-084	Cell culture medium for growth of RPE-1 and 293T cells
Fetal Bovine Serum (FBS)	Gibo-Life sciences	10438-026	Cell culture medium supplement
Lipofectamine 3000 and p3000	Invitrogen	L3000-015	Lipid based transfection reagent for transfection of plasmids; used in 1.1.4
Multi well Tissue Culture dishes	Corning	various	for use in cell culture, transfection/infection, and live cell imaging
Nikon Ti-E microscope	Nikon		Inverted epifluorescence microscope for use in live-cell imaging
NIS Elements HC	Nikon	Version 4.51	Image acquisition and analysis software; used in sections 3 & 4
OPTI-MEM	Gibo-Life sciences	31985-070	Reduced serum medium for cell transfection; used in step 1.1.3
Penicillin/Streptomycin	Gibo-Life sciences	15140-122	antibiotic used in cell culture medium
phosphate bufferred saline (PBS)	Caisson labs	PBP06-10X1LT	sterile saline solution for use with cell culture
pMD2.G	Addgene	12259	Lentiviral VSV-G envelope expression construct; used in step 1.1.4
Polybrene	Sigma-Aldrich	H9268	Cationic polymer used to enhane viral infection efficiency; used in step 1.1.10
psPAX	Addgene	12260	2nd generation lentiviral packaging plasmid; used in step 1.1.4
Puromycin	Invitrogen	ant-pr-1	Antibiotic selection agent; used to select for RFP-H2B expressing cells
RFP- Histone 2B (H2B)	Addgene	various numbers	Expression vector for red fluorescent protein-tagged histone 2B; for use in the visualization of chromatin in live-cell imaging: commercially available through Addgene and other vendors
RNAi Max	Invitrogen	13778-150	Lipid based transfection reagent for transfection of siRNA constructs
RPE-1 cells	ATCC	CRL-4000	Human retinal pigment epithelial cell line
Tissue culture dish 100x20mm	Corning	353003	for use in culturing adherent cells
Zyla sCMOS camera	Nikon		Camera attached to the micrscope, used for capturing images of cells