Cell divisions can be visualized in real time using fluorescently tagged proteins and time-lapse microscopy. Using the protocol presented here, users can analyze cell division timing dynamics, mitotic spindle assembly, and chromosome congression and segregation. Defects in these events following RNA interference (RNAi)-mediated gene knockdown can be assessed and quantified.
Drosophila S2 cells are an important tool in studying mitosis in tissue culture, providing molecular insights into this fundamental cellular process in a rapid and high-throughput manner. S2 cells have proven amenable to both fixed- and live-cell imaging applications. Notably, live-cell imaging can yield valuable information about how loss or knockdown of a gene can affect the kinetics and dynamics of key events during cell division, including mitotic spindle assembly, chromosome congression, and segregation, as well as overall cell cycle timing. Here we utilize S2 cells stably transfected with fluorescently tagged mCherry:α-tubulin to mark the mitotic spindle and GFP:CENP-A (referred to as ‘CID’ gene in Drosophila) to mark the centromere to analyze the effects of key mitotic genes on the timing of cell divisions, from prophase (specifically at Nuclear Envelope Breakdown; NEBD) to the onset of anaphase. This imaging protocol also allows for the visualization of the spindle microtubule and chromosome dynamics throughout mitosis. Herein, we aim to provide a simple yet comprehensive protocol that will allow readers to easily adapt S2 cells for live imaging experiments. Results obtained from such experiments should expand our understanding of genes involved in the cell division by defining their role in several simultaneous and dynamic events. Observations made in this cell culture system can be validated and further investigated in vivo using the impressive toolkit of genetic approaches in flies.
Cell Division is a process critical to all multicellular organisms, both in their development and homeostasis1. Drosophila has long been used as a model for the study of cell division, with experiments in various tissue types and genetic conditions providing key insights into the process. While many of these insights come from fixed cell conditions, cell division is a dynamic procedure with many moving parts, making visualization of live cells integral to assessing RNAi or genetic knockout effects on numerous parts of cell division, including spindle formation, chromosome congressional and segregation, and cytokinesis.
Many protocols have been developed and utilized over the years to visualize Drosophila cell divisions in vivo. Various groups have cultivated techniques to image divisions in both larval imaginal discs and larval brains2,3,4,5,6,7,8. These techniques, while useful for imaging in specific tissues, are limited in throughput and often require generation and maintenance of genetic stocks to generate fluorescent components and alter the expression of genes of interest. Cultured S2 cells from Drosophila provide a higher throughput alternative to rapidly test the effects of various genes in cell division. Furthermore, with the ability to transfect various fluorescent proteins, S2 cells can be quickly modified to determine the effects of RNAi knockdown on numerous components of cell division. Fluorescently tagged genes of interest can also be observed in cell division, allowing for dynamic characterization of their function9.
Here we provide a detailed protocol for live imaging of mitotic S2 cells using methods we recently described10. Our method utilizes stably transfected cells with fluorescent markers for microtubules and centromeres whose expression are under control of a copper-inducible promotor (metallothionein; pMT). This methodology can be used to image spindle and chromosome dynamics in all phases of mitosis utilizing a relatively simple fluorescent microscope with basic imaging software. It can be further customized to fit individual research needs, with transient transfection and RNAi offering expanded possibilities for determining the role of candidate genes in mitosis. Because of the relative simplicity of the protocol, it can be used for small-scale loss-of-function screens to identify genes for which further study in vivo would be beneficial, allowing for more focused efforts and efficiency with subsequent genetic manipulation in flies.
1. Preparation of S2 Cells for Treatment with RNAi
2. Treatment of Seeded Cells with dsRNA Against Gene(s) of Interest
NOTE: The following example and Results section will use Shortstop (Shot), an actin-microtubule crosslinking agent as the gene of interest. Use a mock treatment with no dsRNA or with dsRNA directed against an irrelevant gene (e.g., beta-galactosidase, lacZ) as a negative control.
3. Induction of Fluorescent Protein Expression and Preparation of Cells for Imaging
4. Set Up a Live Cell Imaging Program
NOTE: Live Cell Imaging in this experiment was performed using an inverted imaging system and its associated software (e.g., Olympus IX83 with the cellSens Dimensions software package). Details will differ by microscope manufacturer and software package; thus, general guidelines and operations are listed below.
5. Image Dividing Cells Using Live Cell Imaging Program
NOTE: S2 cells do not require CO2 and grow optimally at 23-27 °C. All imaging was performed at ambient temperature in a well-controlled room.
Methods we describe above will result in the identification and imaging of Drosophila S2 cells undergoing cell division. Cells that are about to divide (i.e., just prior to entering M-phase) can be targeted by the presence of two centrosomes and an intact nucleus indicated by refracted light and a darker spot within the cell when viewed in the α-tubulin channel (Figure 1, left-most panels, red channel; arrowhead in Figure 1A). We estimate that approximately 2-5% of cells fall into this category, and between imaging experiments, we typically spend a maximum of 2-3 minutes scanning for another qualified cell. NEBD can be visualized through the disappearance of this dark spot, resulting in the uniform coloring of the cytoplasm (Figure 1, panels marked “00:00”, red). After NEBD, the time each cell takes to form the spindle, congress chromosomes, and segregate chromosomes can be measured simply by noting the time points of these events relative to NEBD.
We utilize these divisions to assess mitotic timing, specifically of NEBD to anaphase onset, noting the point at which chromosomes begin to separate. A majority (~90%) of copper-induced cells, expressed both mCherry:α-tubulin and GFP:CID. A small percentage of cells (~10%) expressed only one marker or neither. These cells were avoided during cell selection. Typically, S2 cells display NEBD to anaphase onset timing of between 20-30 minutes (Figure 1A, Control). We next treated cells with dsRNA targeted against Shortstop (Shot), an actin-microtubule crosslinking protein we suspected may affect cell cycle dynamics. Indeed, following Shot knockdown cells exhibited a significant mitotic delay (Figure 1B, ShotRNAi delay), with many cells arresting at metaphase and never transitioning to anaphase during the entire 2-3-hour imaging experiment (Figure 1D, ShotRNAi arrest). We reasoned this delay/arrest phenotype could likely be due to activation of the spindle assembly checkpoint (i.e. the M-phase checkpoint). To directly test this hypothesis, we co-treated cells with dsRNA against Shot and Rough deal (Rod), an important component of this checkpoint13. This led to a suppression of the arrest phenotype, resulting in NEBD to anaphase times similar to Control (Figure 1C, ShotRNAi+RodRNAi). Thus, this live imaging protocol allowed us to conclude that Shot is required for timely M-phase progression and that its loss leads to checkpoint activation that delays or arrests mitotic cells.
Figure 1: Live imaging reveals cell cycle defects due to mitotic checkpoint activation in S2 cells.
In all conditions, live-cell imaging was conducted on S2 cells stably co-transfected with inducible GFP:CID and mCherry:αTubulin as described herein. (A) Cartoons illustrate important landmarks during mitosis, and corresponding images are shown from representative movies of indicated genotypes with time points relative to NEBD (t=00:00) indicated. Control cells typically progress to anaphase within 20-30 min. ShotRNAi-treated cells display NEBD-anaphase delay (B) and frequently undergo metaphase arrest, never entering anaphase within the imaging experiment (D). Co-treatment of cells with ShotRNAi and RodRNAi, a component of the spindle assembly checkpoint, suppresses the Shot phenotype leading to anaphase onset kinetics similar to Control cells (C). The arrows in (A) indicate the ‘star-like’ centrosome structures, and the large arrowhead indicates the nucleus. Each scale bar represents 5 microns. This figure is adapted and republished with permission from10 (https://www.molbiolcell.org/info-for-authors). Please click here to view a larger version of this figure.
Video 1: Time-lapse movie of ‘Control’ S2 cell division. Video shows a representative movie of S2 cell division in ‘Control’ genotype. This movie corresponds to Figure 1A. This video is adapted and republished with permission from10 (https://www.molbiolcell.org/info-for-authors). Please click here to download this video.
Video 2: Time-lapse movie of delayed ‘ShotRNAi’ S2 cell division. Video shows a representative movie of S2 cell division in ‘ShotRNAi’ genotype that leads to a phenotypic mitotic delay. This movie corresponds to Figure 1B. Please click here to download this video.
Video 3: Time-lapse movie of ‘ShotRNAi+RodRNAi’ S2 cell division. Video shows a representative movie of S2 cell division in ‘ShotRNAi+RodRNAi’ genotype. This movie corresponds to Figure 1C. This video is adapted and republished with permission from10 (https://www.molbiolcell.org/info-for-authors). Please click here to download this video.
Video 4: Time-lapse movie of arrested ‘ShotRNAi’ S2 cell division. Video shows a representative movie of S2 cell division in ‘ShotRNAi’ genotype that leads to a phenotypic mitotic arrest. This movie corresponds to Figure 1D. Please click here to download this video.
Identifying an Appropriate Cell
Key to imaging dividing S2 cells is first locating the proper cell. Time can be wasted imaging cells that are mistakenly thought to be ready to divide but fail to do so in a reasonable timeframe. Cells must be identified that have two distinct and visible centrosomes and an intact nucleus. Centrosomes must have microtubule fibers emanating from them, giving them a “star-like” appearance. An intact nucleus refracts light when adjusting focus, and also makes the approximate center of the cell darker in appearance. The nucleus will also contain the GFP:CID “dots”, another distinguishing feature to help in its identification. Cells with >2 centrosomes, tubulin puncta without tubulin fibers emanating from them, or cells with only one centrosome should be avoided. Additionally, if two centrosomes are visible, but a nucleus is not, NEBD has already occurred and the cell is too far advanced in its division to be imaged if a complete M-phase analysis is desired. Once an appropriate cell is found, speed in starting imaging is crucial as NEBD occurs quickly (typically within 3-5 min) and delaying the start of imaging can lead to missed opportunities. Focus the cell in the imaging software quickly such that both centrosomes are visible, or if centrosomes are out of plane with each other, set the focal point between the two, such that each can be captured when Z stacks are collected above and below the defined center point. Once focused, immediately set the offset between the stage and the live cell chamber surface using the infrared focus check (if available) and begin the imaging program. Although the protocol presented here is specifically tailored for experiments assessing NEBD-to-anaphase dynamics, simple modifications could suit readers studying alternative mitotic events. For NEBD-to-metaphase and anaphase-to-telophase imaging, we suggest 10 s image capture intervals as these processes are highly dynamic and occur quickly in S2 cells (5-10 min). Metaphase-to-anaphase transition (our typical experimental focus) is a longer process (20-30 min) in S2 cells, and we collect images at 30 s intervals. Lastly, telophase-through-cytokinesis occurs quite slowly in S2 cells, and we suggest 60 s intervals providing enough resolution for this approximate hour-long process.
Maintaining Focus Throughout the Imaging Program
If an infrared focus check device is not available, be sure to avoid bumping the microscope or table it sits upon, as this can result in the cell moving out of focus during the imaging program, leading to ambiguous, un-interpretable results. Pre-coating the live cell chamber wells with Poly-L-lysine can help in cell adherence, which can help avoid cell movement and is especially useful for setups without infrared focus checks. Additionally, setups including shock-absorbing platforms or air tables can further help avoid cells moving out of focus. Lastly, some software programs have pause functions allowing the user to refocus and resume the program.
Imaging Multiple Cells
Helpful to accumulation of data is that often multiple cells ready to divide can be placed within the same field of view for imaging. This can be especially useful for experiments in which cells have long M-phase durations or prolonged arrest (e.g., conditions that induce activation of the SAC). For these cells, we typically image until the cells are photobleached (approximately 2-3 h), but the full timing of arrested cells should be at the discretion of the researcher. Sometimes the multiple pre-dividing cells can be in different focal planes, in which case it may be useful to add z-stacks to accommodate all cells. Adding more z stacks can also help improve resolution. Caution should be taken in doing this, however, as it will expose cells to more radiation and lead to quicker photobleaching, as well as lead to large file sizes on hard disk storage devices.
Avoiding Photobleaching and Phototoxicity
Our method describes specific settings for exposure times, imaging intervals, z stacks, and percent transmittance for our LED light source that generally work for our experimental setup and desired outcomes. As microscopes and experimental goals may vary, what might work well for our system may lead to premature photobleaching or phototoxicity in others. Photodamage may present a lack of spindle movement, microtubule fragmentation, defective microtubule dynamics, and prolonged spindle checkpoint activation. One potential method to avoid such pitfalls is to limit the exposure time. Most modern cameras now possess wide dynamic ranges, and histograms can be adjusted to visualize structures even at very low exposures. Another technique is to increase the imaging interval (e.g., to several minutes between images) such that the number of times a cell is exposed to light over a total collection interval is reduced. This is advantageous particularly in imaging for longer periods of time (many hours), and can additionally help in decreasing the file size of such experiments. Limiting the number of z stacks taken can also help reduce light exposure, using 2 or even just 1 instead of 3. Further, adjusting the percent transmittance of light (for LED light sources), or utilizing neutral density (ND) filters (for both Halogen Lamp and LED light sources) will decrease the light intensity and coupled with longer exposure times can preserve visibility. By choosing cells with centrosomes already separated, overall exposure can be limited in that these cells usually enter mitosis quickly (within a minute or two) after beginning imaging. One can also limit the time that cells are allowed to be imaged before NEBD. Our lab typically sets this time at 10 min, but a more conservative limit can easily be imposed by the user. Additionally, a more ‘invasive’ measure we recommend is treatment with dsRNA targeted against a component of the SAC (such as Rod or Mad2). If an arrest phenotype is due to non-specific cell damage (e.g., defective microtubule dynamics), such a treatment is less likely to suppress the arrest compared to a bona fide effect of the gene of interest in the original experiment.
Future Directions
The method described here can be utilized on a relatively simple epifluorescent microscope to rapidly image live cell divisions and can be easily adapted to suit a researcher’s particular experimental design and goals. Several excellent methods for culturing, RNAi knockdown approaches, transient transfection, and fluorescence microscopy have been published for S2 and other Drosophila cells9,14,15,16,17. Our protocol offers a couple of advantages. (1) The use of a double stable cell line (GFP:CID, mCherry:α-Tubulin) simultaneously marks two key mitotic structures, avoids the hassle and potential complications of transient transfection, and ensures that nearly all cells will express fluorescent markers as potential candidates for imaging. (2) The adaptation to mitosis adds to published protocols examining cytoskeletal dynamics in motile, non-dividing cells and extends to examining mitotic events in real time. While we believe ours is a powerful technique that adds to the repertoire of others, there can always be improvements made. One particular area of cell division that we have found hard to image is centrosome division. This occurs prior to NEBD and it is difficult to determine when a single centrosome is ready to separate into two. Using fluorescent cell cycle markers (Cyclin A and Cyclin B) could help resolve this problem, but comes at the expense of channels that could be used to visualize cell division components. Our best strategy for attempting to visualize centrosome division is to add multipoint acquisition to the imaging program (defining different points in the XY plane to image), but this can result in large files, and can also require (depending on the number of points) increasing the interval between image collection, decreasing time resolution of the resulting movie. Another solution could be synchronization of cells at a desired cell cycle stage using drugs that target cell cycle regulators followed by subsequent washout prior to imaging, although these methods may not be reliable in S2 cells specifically.
The protocol presented here provides a foundation for future applications in live cell imaging as well. With improved imaging and software technology, truly high throughput adaptations of this protocol using higher capacity, multi-chambered plates could be possible. Such innovations would make large-scale RNAi screens, such as those already achieved in fixed preparations12,18, more tractable in a live cell format. Small molecule drug screens could also be envisioned as a means of identifying novel compounds targeting cell division processes. Improvement of fluorophores and optical filters could also lead to imaging of multiple mitotic components (not just the two we describe), enabling imaging of specific mitotic regulators and their interactions with DNA and/or the spindle. For example, generating cells with fluorescent reporters that express following the DNA damage or during apoptosis would be useful tools to identify novel genes involved in these processes. Similar approaches could be used to examine cell cycle dynamics using the expression of fluorescently tagged cyclins19.
The authors have nothing to disclose.
This work was funded by the National Institutes of Health (R01 GM108756). We are grateful to Gary Karpen (University of California, Berkeley) for generously providing us with a GFP:CID S2 cell line stock from which our GFP:CID/mCherry:αTubulin line was generated10.
Bright-Line Hemacytometer | Sigma-Aldrich | Z359629-1EA | for cell counting |
cellSens imaging software | Olympus | ||
CELLSTAR Cell Culture Flask, 50ML, 25 CM2, PS, Red Filter Screw Cap, Clear, Sterile, 10PCS/BAG | Greiner Bio-One | 690175 | |
CELLSTAR Cell Culture Multiwell Plate, 6 well, PS, Clear, TC, Lid with condensation rings, sterile, single packed | Greiner Bio-One | 657160 | |
Centrifuge 5804 R | eppendorf | Cat. 022623508 | |
Copper(II) sulfate pentahydrate, minimum 98% | Sigma-Aldrich | C3036-250G | |
Corning Fetal Bovine Serum | Fisher Scientific | MT35015CV | |
Effectene Transfection Reagent 1 mL | Qiagen | 301425 | for transient transfection |
IX-83 Inverted Epifluorescent Microscope | Olympus | ||
LabTek Chambered Slide Insert | Applied Scientific Instruments | I-3016 | |
MEGAscript T7 Transcription Kit | Thermo Fisher Scientific | AM1334 | For dsRNA production |
MOXI Z Mini Automated Cell Counter Kit | ORFLO | MXZ001 | for cell counting |
MS-2000 XY Flat-Top Automated Stage and Controller | Applied Scientific Instruments | ||
Nunc Lab-Tek II Chambered Coverglass (no 1.5 borosilicate glass) 8-well | Thermo Fisher Scientific | 155409 | |
Orca-Flash 4.0 LT Camera | Hammamatsu Photonics K.K. | C11440-42U | |
pMT/V5-His A Drosophila Expression Vector | Thermo Fisher Scientific | V412020 | |
Poly-L-Lysine | Cultrex | 3438-100-01 | |
Purifier Logic+ Class II, Type A2 Biosafety Cabinets | Labconco | 302310000 | |
Schneider's insect medium | Sigma-Aldrich | S0146-100ML | |
Spectra Tub Centrifuge Tubes | VWR | 470224-998 | |
Uner Counter BOD Incubator | Sheldon manufacturing (VWR) | 89409-346 | |
X-CITE 120 LED | ExcelitasTechnologies | Led light source | |
Z -Drift Compensator (ZDC) | Olympus | infrared focus check |