In this paper, we report a protocol describing an in vivo method to measure microtubule dynamic instability in docetaxel-resistant breast cancer cells (MCF-7TXT). In this method, a deconvolution microscopy imaging system is used to detect the expression of GFP-tubulin in target cells.
Taxanes such as docetaxel belong to a group of microtubule-targeting agents (MTAs) that are commonly relied upon to treat cancer. However, taxane resistance in cancerous cells drastically reduces the effectiveness of the drugs’ long-term usage. Accumulated evidence suggests that the mechanisms underlying taxane resistance include both general mechanisms, such as the development of multidrug resistance due to the overexpression of drug-efflux proteins, and taxane-specific mechanisms, such as those that involve microtubule dynamics.
Because taxanes target cell microtubules, measuring microtubule dynamic instability is an important step in determining the mechanisms of taxane resistance and provides insight into how to overcome this resistance. In the experiment, an in vivo method was used to measure microtubule dynamic instability. GFP-tagged α-tubulin was expressed and incorporated into microtubules in MCF-7 cells, allowing for the recording of the microtubule dynamics by time lapse using a sensitive camera. The results showed that, as opposed to the non-resistant parental MCF-7CC cells, the microtubule dynamics of docetaxel-resistant MCF-7TXT cells are insensitive to docetaxel treatment, which causes the resistance to docetaxel-induced mitotic arrest and apoptosis. This paper will outline this in vivo method of measuring microtubule dynamic instability.
The leading cause of breast cancer mortality is through metastasis1,2. Taxanes, such as docetaxel and paclitaxel, are currently used as first-line regimens in the treatment of metastatic breast cancer2,3,4,5,6. They are part of a group of microtubule-targeting agents (MTAs) that disrupt microtubule dynamics. However, one of the greatest challenges to using taxanes in curative therapy is the development of taxane resistance in cancer cells, which leads to disease recurrence7. Drug resistance accounts for more than 90% of all deaths among patients with metastatic breast cancer7.
Microtubules are formed by the polymerization of α- and β-tubulin heterodimers8,9. The precise regulation of microtubule dynamics is important for many cellular functions, including cell polarization, cell cycle progression, intracellular transport, and cell signaling. Dysregulation of microtubules and their dynamics will disrupt cell function and result in cell death10,11. Depending on how they cause this dysregulation, MTA drugs can be classified as either microtubule stabilizing agents (i.e., taxanes) or microtubule-destabalizing agents (i.e., vinca alkaloids or colchicine-site binding agents)20. Despite their opposite effects on microtubule mass, at a sufficient dosage, both classes can kill cancer cells through their effects on microtubule dynamics21.
Taxanes function primarily by stabilizing the microtubule spindle12, leading to chromosomal misalignment. The subsequent perpetual activation of the spindle assembly checkpoint (SAC) arrests the cell in mitosis. Prolonged mitotic arrest then causes apoptosis13,14. Taxane interacts with microtubules through the taxane binding site on β-tubulin8,15, which is only present in assembled tubulin16.
Multiple mechanisms for taxane resistance have been proposed9,17. These mechanisms include both general multidrug resistance due to the overexpression of drug-efflux proteins and taxane-specific resistance5,9,18,19. For example, taxane-resistant cancer cells may have altered expression and function of certain β-tubulin isotypes5,9,19,20,21,22,23. By using an in vivo method to measure microtubule dynamic instability, we show that, when compared to non-resistant, parental MCF-7CC cells17, the microtubule dynamics of docetaxel-resistant MCF-7TXT cells are insensitive to docetaxel treatment.
To better understand the function of MTAs and the exact mechanism of taxane-resistance in cancerous cells, it is essential to measure microtubule dynamics. Here, we report an in vivo method of doing so. By using live imaging in combination with the expression of GFP-tagged tubulin in cells, we can measure the microtubule dynamics of MCF-7TXT and MCF-7CC cells with and without docetaxel treatment. The results can help us design more effective drugs that can overcome taxane resistance.
1. Preparing the Cells for Live Imaging
2. Live Imaging to Examine Microtubule Dynamic Instability
3. Image Processing and Data Analysis
Using the protocol presented here, we studied the effects of docetaxel on the microtubule dynamics of normal (MCF-7CC) and docetaxel-resistant (MCF-7TXT) breast cancer cells. Two sets of images show the effects of docetaxel (0.5 µM) on microtubule growth and shortening in MCF-7CC and MCF-7TXT cells (Figure 1A).
We also calculated the rate of microtubule growth or shortening under these conditions for the two cell lines and showed that at 0.5 µM, docetaxel strongly inhibits the tubulin dynamics of MCF-7CC cells but not MCF-7TXT cells (Figure 1B).
Figure 1: The Effects of Docetaxel on the Microtubule Dynamics of MCF-7TXT and MCF-7CC Cells. The live imaging was performed as described. (A) Selected images from the live imaging of microtubule dynamics in MCF-7CC and MCF-7TXT cells following treatment with 0.5 μM docetaxel for 1 h. The arrow indicates the extending microtubules. The arrowhead indicates the shortening microtubules. Size bar = 5 µm. (B) The extension rate and shortening rate of microtubules were measured from the recorded images. Each datum is the average of 20 measurements from at least 8 different cells. The error bar is the standard error. ** indicates that the difference is statistically significant, with p <0.01. Please click here to view a larger version of this figure.
There are two major methods to measure microtubule dynamic instability: in vitro and in vivo. In the in vitro method, purified tubulin is used to measure microtubule dynamic instability with computer-enhanced time-lapse differential interference-contrast microscopy. In the in vivo method, microinjected fluorescent tubulin, or expressed GFP-tubulin, is incorporated into microtubules. The dynamics (growth and shortening) of the microtubules is then recorded by time-lapse using a sensitive camera in the peripheral region of interphase cells10,20,25. While various types of microscopes have been used for this purpose, the protocol we reported here uses a deconvolution microscope.
In vivo, dynamic instability occurs only at microtubule plus ends (the ends with the β-tubulin facing outward), whereas the minus ends (the ends with the α-tubulin facing outward) remain the same length26. On the other hand, for microtubules assembled in vitro, dynamic instability occurs at both ends of the microtubules, with the plus ends being more robust than the minus ends27. The main advantage of analyzing dynamic instability in vitro is that it provides mechanistic information about the specific role of different agents on microtubule dynamics in the absence of cellular MAPs. Moreover, under in vitro conditions, dynamic instability can be studied at both ends. This provides insight into the mechanisms by which drugs or regulatory proteins affect microtubule stability at the opposite ends. Historically, the minus ends are tracked much less than the plus ends10. Much of our understanding of microtubule behavior comes from studies on microtubules assembled from purified tubulin in vitro.
However, while many of the basic principles of microtubule behavior gained from these studies can also be applied to microtubules in cells, there are certain differences in microtubule dynamics between in vitro and in vivo. In cells, microtubules often grow at a five- to ten-fold faster rate, and transitions between growth and/or shortening occur ~10 times more frequently than with microtubules assembled from purified tubulin in vitro. There are also dramatic changes in microtubule organization and dynamics throughout the cell cycle, which reflect a high degree of spatial and temporal regulation of cellular microtubule behavior. This behavior cannot be studied by in vitro research. Furthermore, the regulation of microtubule dynamics is achieved by tubulin posttranslational modification, tubulin isotype expression, and a large group of MAPs and other microtubule-interacting proteins that either stabilize or destabilize microtubules28,29. Therefore, studying microtubule instability in living cells is much more physiologically applicable.
The method described here is a simple and very reliable protocol to study microtubule dynamics in living cells. The steps that require more attention are the selection of the cell for observation and the determination of the exposure time. It is critical to select cells that are flat and have high fluorescence intensity of GFP-tubulin. It is also desirable to reduce the intensity of excitation and the exposure time to avoid the fading of the GFP fluorescence. Certainly, like all of the other in vivo methods, this protocol is not suitable to study microtubule dynamic instability under various manipulated conditions. For example, different isoforms of α- and β-tubulin may behave differently in terms of microtubule dynamics. Only in vitro methods can be used to study the effects of individual purified tubulin isoforms on microtubule dynamic instability. Moreover, compared with the microinjection of fluorescence-labelled tubulin, the expression of GFP-tagged tubulin by transfection or transduction of plasmids may exhibit a relatively lower signal-to-noise ratio. However, expression of GFP-tagged tubulin in cells by transfection or transduction of plasmids allows more freedom to experimentally alter other cellular characteristics.
The protocol reported here could be used for various studies. On one hand, it can provide novel and critical insights regarding the function of microtubules during various phases of the cell cycle, especially in mitosis. It can also be used to study the effects of various cellular processes, such as migration, on microtubule dynamics. On the other hand, it can be used to study the effects of various microtubule inhibitors (many of them are cancer drugs) on microtubule dynamics in normal and cancerous cells under more physiological conditions. We have used this method to study the effects of various anti-mitotic cancer drugs on microtubule dynamic instability in both docetaxel resistant and non-docetaxel resistant breast cancer cells.
The authors have nothing to disclose.
This research is supported by funding from CBCF (to ZW).
Dulbecco's Modified Eagle's Medium (DMEM) | Sigma-Aldrich | D5796 | |
Non-essential amino acids | Life Technologies, Invitrogen | 11140-050 | |
FBS | Gibco, Invitrogen | 12483 | |
Anti-Anti (100x) | Life Technologies, Invitrogen | 15240-062 | |
docetaxel | Sigma-Aldrich | 01885-5mg-F | |
DMEM phenol red-free | Gibco, Invitrogen | 21063 | |
CellLight Reagent *BacMam 2.0* GFP-tubulin | ThermoFisher Scientific | C10613 | Key reagent for expressing GFP tubulin in cells |
CellLight Reagent *BacMam 2.0* GFP | ThermoFisher Scientific | B10383 | Control |
Dimethyl Sulfoxide (DMSO) | Sigma-Aldrich+B9:AA9 | 472301 | for dissoving decetaxel |
22-mm glass coveslip | Fisher Scientifics | 12-545-101 | |
6-well culture plate | Greiner Bio-One International | 6 Well Celi Culture Plate | |
DeltaVision Microscopy Imaging Systems | GE Health | This system is equipped with weather station for controlling temperature and CO2. It also equipped with Worx Software for deconvolution and time lapse control. | |
Trypsin-EDTA (0.25%), phenol red | ThermoFisher Scientific | 25200056 | |
Bright-Line Hemacytometer Set, Hausser Scientific | Hausser Scientific, Distributed by VWR | Supplier No.: 1492 VWR No.:15170-172 |