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.
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.
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 behavior1,2,3,4,5,6.
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 cells7,8,9. 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.
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.
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.
3. Microscope set up for time-lapse imaging of RFP-H2B, α-tubulin-GFP expressing cells (Figure 1, Figure 2)
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.
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 progression10,11,12,13. Nevertheless, some perturbations only transiently delay spindle formation with cells ultimately proceeding through mitosis to complete anaphase chromosome segregation14. In the absence of cell synchronization approaches, which may themselves indirectly impact mitotic processes1,2, cells progress through mitosis asynchronously (Figure 2C). Using RFP-H2B to identify chromatin, mitotic cells with compact chromatin can be staged as being in prometaphase (those prior to complete metaphase alignment: Figure 2C, blue arrowheads), metaphase (complete chromosome alignment: Figure 2C, yellow arrowheads) and anaphase (chromosomes being segregated towards spindle poles: Figure 2C, white arrowheads). The capture of 5-8 coordinates over 4 h is sufficient to follow the progression of at least 50 cells through mitosis in a given condition. To investigate the dynamics of spindle assembly following alterations in centrosome number and/or inhibition of Aurora A kinase, an important regulator of spindle pole focusing14,15,16,17, we used the live cell time-lapse imaging of human cells with 2 centrosomes, or those containing supernumerary centrosomes. Cells were cultured in the presence or absence of the Aurora A kinase inhibitor prior to the start of imaging. Cells with 2 centrosomes experience the disruption in mitotic progression when treated with aurora A kinase inhibitor, but are ultimately able to achieve metaphase chromosome alignment (Figure 2C, yellow arrowhead). In contrast, cells with >2 centrosomes are exquisitely sensitive to Aurora A inhibition and exhibit an increase in prometaphase cells and an absence of metaphase cells, indicating a delay in spindle assembly and mitotic progression.
Figure 3 demonstrates that such time-lapse imaging approaches are sufficient to monitor both spindle assembly and mitotic fidelity. By visualizing α-tubulin-EGFP, it was observed that cells that experience spindle disruption (shown here due to centrosome overduplication) undergo dynamic changes as spindle pole focusing is achieved and a bipolar mitotic spindle is formed in preparation for cell division. Concurrent with spindle assembly, chromosome movement can be visualized with RFP-H2B to assess chromosome alignment and segregation fidelity. Mitotic cells that experience transient spindle multipolarity are susceptible to attachment errors that result in lagging chromosomes during anaphase and form micronuclei in the subsequent G1 phase of the cell cycle. Such defects are apparent with these live cell imaging approaches.
Changes in the dynamic progression of mitosis alter mitotic timing and impact mitotic cell fate
Following the nuclear envelope breakdown, spindle formation and chromosome movement can be tracked through the stages of mitosis to assess mitotic progression, duration, and the fate of cells that progress into anaphase. Centrosome amplification, a feature common in many types of cancer, is characterized by the presence of extra centrosomes and multipolar spindle formation18,19,20. As multipolar divisions result in highly aneuploid and likely unviable daughter cells, cancer cells actively cluster extra centrosomes to form a bipolar spindle and undergo a bipolar division5,20,21,22,23,24. Using live cell imaging approaches, our representative results show that cells with a normal centrosome content are able to proceed from nuclear envelope breakdown through metaphase alignment and anaphase onset to achieve a bipolar division in under 30 minutes (Figure 4A,D,E). In the presence of extra centrosomes, nearly 50% of cells are able to overcome a transient multipolar mitotic spindle and to form a bipolar spindle and complete cell division (Figure 4C,D). The remaining cells are unable to achieve a bipolar spindle and as a result exit mitosis through a multipolar division (Figure 4B,D). Regardless of whether spindle bipolarity is achieved, cells with extra centrosomes exhibit a significantly increased duration of mitosis compared to cells with 2 centrosomes, indicating that the dynamics of mitotic progression may be altered even when changes in mitotic outcome are not apparent (Figure 4E).
Figure 1: Selection of microscope and camera parameters. (A) Representation of the window to select the desired objective and appropriate filter cube using an image acquisition software. Shown is the selection of the 20x magnification objective and the brightfield filter cube. (B) Cells in the sample plate are found and brought into focus using brightfield or phase contrast microscopy. (C) Representation of the window to set exposure parameters for each image capture. Pixel binning can be used, if needed, to achieve reduce exposure time per capture and minimize photo-damage to cells. Scale bar is 10 μm. Please click here to view a larger version of this figure.
Figure 2: Image capture and analysis of time-lapse microscopy. (A and B) Representation of the window indicating how image acquisition parameters are set using NIS Elements HCA jobs image acquisition software. This software allows multi-coordinate, multi-well imaging over time. Each job can be modified to specify wells and coordinates to be captured. (C) Single time frames from four conditions of one experiment. RFP-H2B is used to track chromatin. Chromatin compaction and positioning are used to identify different stages of mitosis: Prometaphase (compaction in the absence of chromosome alignment, blue arrowhead), Metaphase (complete chromosome alignment at the cell equator, yellow arrowhead), and Anaphase (following initiation of synchronous chromosome segregation, white arrowhead). Scale bar is 10 µm. Please click here to view a larger version of this figure.
Figure 3: Time-lapse imaging visualizes defects in mitotic fidelity. Cells containing extra centrosomes form multipolar spindles and often cluster them into a bipolar spindle prior to completing cell division. Here, a cell enters mitosis with seven distinct spindle poles (white asterisks) which, over time, are clustered into two main spindle poles. At this point, chromosomes are able to align along the spindle equator, and the cell proceeds to anaphase. The transient multipolarity has permitted a mal attachment of a single chromosome. This unresolved error results in a lagging chromosome during anaphase which becomes incorporated into a micronucleus in one daughter cell (labeled with an arrow). Chromatin is visualized with RFP-Histone 2B and microtubules are visualized with α-tubulin-EGFP. Time stamps in each panel indicate minutes with respect to apparent nuclear envelope breakdown as judged by detection of tubulin within the nuclear boundary. Scale bar is 5 µm. Please click here to view a larger version of this figure.
Figure 4: Changes in the dynamic progression of mitosis alter mitotic timing and impact mitotic cell fate. RFP-H2B, shown in red, is used to track chromatin movement and α-tubulin-GFP, shown in green, is used to monitor centrosome/spindle pole number and organization. Loss of GFP-tubulin exclusion from the nucleus, concurrent with early chromatin compaction is an indication that the nuclear envelope has broken down (NEB) in preparation for mitotic cell division. Nuclear exclusion of GFP-tubulin is apparent upon mitotic exit and reformation of the nuclear envelope. (A) A cell with two centrosomes forms a bipolar spindle and progresses through anaphase to form two daughter cells. (B and C) Cells with extra centrosomes enter mitosis to form a multipolar spindle. (B) Some of these cells with extra centrosomes delay in mitosis without achieving a bipolar spindle and progress to complete multipolar anaphase. (C) Other cells with extra centrosomes exhibit a transient delay in mitotic progression while they cluster extra centrosomes to enable bipolar anaphase. (D) Cells with extra centrosomes are able to undergo a bipolar division approximately 50% of the time, while the remaining cells with extra centrosomes undergo a multipolar division. (E) Cells with extra centrosomes have increased mitotic timing regardless of the eventual mitotic fate (bipolar or multipolar anaphase). Scale bar is 5 µm. Please click here to view a larger version of this figure.
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 better equipped to follow and analyze both the dynamics of single cells, as well as the heterogeneity within a cellular population. In this way, time-lapse imaging is particularly useful in monitoring cell progression through the dynamic stages of mitosis and assessing how perturbations to this progression ultimately impact the fidelity of mitotic cell division and subsequent cell fate.
While there are a number of advantages to live cell imaging, significant challenges are associated that must be considered and mitigated when possible. One challenge is in maintaining appropriate environmental conditions to ensure cell viability and proliferation during imaging. To accomplish this, the imaging set-up must include an environmental chamber that regulates both temperature and CO2. Alternatively, cells may be imaged for a short-term with only temperature regulation if done so in a closed chamber. In both cases, use of an antivibration table and/or hardware-based approaches to mitigate axial focus fluctuations (e.g., Nikon's Perfect Focus) should be employed to maintain plate focus throughout the duration of the experiment. A second significant concern with live cell imaging is the potential for photodamage and photobleaching. Photobleaching is a concern where the fluorophore being imaged gradually decreases in fluorescence intensity as imaging progresses. However, the exposure to high intensity excitation light that results in photobleaching is also toxic to cells and care must be made to minimize cell exposure by decreasing exposure times, image acquisition intervals, and the total duration of the imaging sequence25. Consequences of not optimizing imaging conditions to minimize phototoxicity can include the generation of DNA damage and other cellular changes that can in turn compromise interpretation and understanding of the experiment. Should cell viability become a concern with long-term imaging, effort should be made to utilize pixel binning (to enable shorter exposures) and increase the duration between subsequent image captures. An additional concern is that the fluorescent tag may alter the behavior of the protein to which it is fused, or that the integration of the viral expression construct into the genome may itself impact cellular behavior. To account for the possibility that the addition of a fluorescent tag may impact protein function, an assessment of protein function following the addition of an N or C terminal fluorescent tag and comparison with non-tagged protein function is necessary. To determine if adverse effects on cell behavior arise due to the perturbation of the genomic locus in which the viral construct has been integrated, multiple single cell clones should be derived, compared to cells that lack the tagged protein, and tested to monitor that cellular fitness and mitotic progression are not perturbed. Alternatively, off target effects of random integration of the viral expression construct can be mitigated through targeted integration of the fusion protein into the endogenous locus, or other known regions of the genome, using CRISPR-based approaches.
Approaches to identify and characterize major regulators of mitotic progression have relied heavily on fixed cell imaging. Mitotic regulators identified in this way have subsequently been exploited in therapeutics targeting rapidly proliferating cancer cells7,8,9. However, antimitotic drugs do not always perform uniformly in different cancers and in many cases insight into the mitotic phenotypes that precede either successful or unsuccessful chemotherapeutic approaches remain unclear. Live cell time-lapse imaging to track mitotic cells in the presence and absence of spindle-perturbing drugs has the potential to provide the insight necessary to identify those modulators most likely to result in catastrophic mitoses and compromised viability of cancer cells with minimal impact on normal cells.
The authors have nothing to disclose.
DLM is supported by an NSF GRFP. ALM is supported by funding from the Smith Family Award for Excellence in Biomedical Research.
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 |