The quantification and regulation of microtubule dynamics in the mitotic spindle

Abstract

Microtubules play essential roles in cellular organization, cargo transport and chromosome segregation during cell division. During mitosis microtubules form a macromolecular structure known as the mitotic spindle that is responsible for the accurate segregation of chromosomes between the two daughter cells. This is accomplished thanks to finely tuned control of microtubule dynamics. Even small changes in microtubule dynamics during spindle formation and/or operation may lead to chromosome miss-segregation, chromosome instability and aneuploidy. These three events are directly correlated with human diseases like cancer and developmental defects. Precise measurements of microtubule dynamics in the spindle will allow us to discover new molecules involved in regulating microtubule dynamics and enable a deeper understanding of the the mechanisms that underlie mitosis and cancer emergence and development. Moreover, many chemotherapeutic agents for cancer treatment are targeted to microtubules, so continued investigation of their dynamics with utmost precision will facilitate the development of new drugs. Measuring microtubule dynamics in the spindle has been a difficult task until recently. With the development of new and gentler microscopic techniques, and new computer programs, we can perform better and more accurate measurements of microtubule dynamics during mitosis.

Introduction

Microtubules (MTs) are highly dynamic polymers composed of tubulin dimers that follow an assembly/disassembly cycle known as dynamic instability [1]. Dynamic instability can be described as the co-existence of growing and shrinking microtubules in the same solution conditions. A great deal has been learned about the structural and energetic properties of tubulin that drive the spontaneous assembly and disassembly of individual MTs [2]. Furthermore, a large number of accessory proteins have been identified that can alter the rate constants associated with dynamic instability and, accordingly, influence cellular MT turnover and dynamics. However, because MT turnover occurs rapidly and requires specialized, high resolution imaging equipment to measure, the details of this process in living cells are not fully fleshed out.

Microtubule dynamics in the spindle and Chromosome Instability

Turnover of MT polymer in the mitotic spindle is quite rapid, taking place with a half-life of just a few minutes [3]. The extent to which the behavior of microtubules is either individually stochastic, or coordinated within the spindle, is dependent upon a variety of factors including the proximity of chromatin, the association of MT end-binding complexes, the spatial distribution of regulators and kinetochore capture. For example, individual MTs may be captured or released from kinetochores presumably without significantly affecting others nearby. Alternatively, groups of MTs attached to the kinetochore can be coordinately assembled and disassembled to facilitate chromosome movement [4]. How MT assembly and disassembly is coordinated within the kinetochore bundle to facilitate chromosome movement is not fully understood. In addition to MT attachment and release, MTs in the spindle that are bound to kinetochores can assemble at the kinetochore-MT interface and disassemble at the centrosome leading to flux of polymer along the length of the kinetochore MT bundle [5]. Considerable lateral and longitudinal sliding of polymer has also been detected within spindles and kinetochore fiber bundles, complicating measurements of turnover. Previously identified dynamic MT behaviors that contribute to MT turnover in the spindle are summarized in Figure 1a.

Figure 1:

Overview of spindle microtubule dynamics. (a) Diagram depicting dynamic microtubule behaviors in the spindle that contribute to microtubule turnover. (b) Diagram depicting a selection of MT regulators and their proposed region of action within the spindle (c) Mitotic cell showing microtubules (green) and chromosomes (magenta). (d) Live mitotic spindle expressing EB3-GFP. Projected temporal color overlay in which blue, green and red are successive one second time points.

Drugs that bind tubulin have been useful as anti-cancer therapies due to the broad participation of MTs in diverse cellular processes. Use of these drugs in the lab and in the clinic has demonstrated that very small perturbations of dynamic MT turnover will adversely impact spindle function. Most dramatic are dynamic changes in MT turnover that trigger the spindle assembly checkpoint (SAC) leading to mitotic catastrophe or cell death. However, even subtle alterations in MT dynamics, those that do not immediately arrest cell division, have long-term consequences for the fidelity of chromosome segregation. For example, modest (10–20%) increases in microtubule assembly rates in spindle astral MTs are correlated with increased chromosome instability over the long-term without having an overt effect on cell division in the short-term [6, 7]. It is worth considering that these modest alterations in MT dynamics are highly relevant to human disease because they are correlated with cancer predisposition. Because there are many pathways that can modestly but significantly impact MT dynamics, it is critical to develop sensitive methods to measure MT turnover and dynamics within the densely populated mitotic spindle.

Microtubule dynamics change in phase with both the cell cycle and with cell size. A marvelous study from the Dumont lab uncovered a decreasing and surprisingly linear relationship between MT assembly rates in spindles and cell/spindle size in developing C. elegans embryo. They hypothesize that this observation underlies the mechanism for scaling spindle length to cell volume because MT growth rate is the only MT dynamic parameter that tracked with spindle length [8]. Such scaling can be tuned in Xenopus extracts by addition of the MT polymerase XMAP215/ch-TOG which correspondingly increases MT assembly rates [9]. Another study that addresses how cellular components influence MT self organization by changing growth parameters comes from the Gueroui group. While MTs and actin filaments have long been observed to exhibit antagonistic cytoplasmic space filling activities, their study adds a new facet to this crosstalk. They show convincingly in vitro that MT growth rates can be tuned higher with the addition of linear actin filaments and then down when the same actin filaments are branched [10] an observation likely to be relevant to spindle positioning among other things. Collectively, these studies underscore the importance of MT assembly rates in development and cellular patterning. Fortunately, MT growth rates are one of the more accessible parameters to measure in live cells.

New players regulating MT assembly in mitosis

There are hundreds of proteins that are able to regulate MT dynamics in the cell, either in interphase or during mitosis. Examples of some well-studied proteins that influence MT polymerization are the plus end binding proteins EB and ch-TOG [11]. Another example would be the microtubule nucleation factor TPX2. This protein is required for mitotic spindle formation and is able to suppress tubulin subunit off-rate during MT assembly and disassembly [12, 13]. However, there are also a growing number of proteins whose function is not obviously correlated with MT function that have been shown to alter MT dynamics. FKBP25 is a nuclear prolyl isomerase that interacts with nucleic acids and also with MTs. This protein affects MT dynamics promoting polymerization and stabilization of MTs. Cells lacking this protein show increased levels of chromosome instability [14]. In Drosophila the adaptor protein ALIX is able to control the orientation of the mitotic spindle by promoting the accumulation of gamma-tubulin at the centrosomes and facilitating the nucleation of astral MTs [15]. RITA (RBPJ interacting and tubulinassociated protein) is a negative regulator of the Notch signaling pathway that interacts with tubulin and localizes to MT structures. RITA also interacts with HDAC6 (tubulin/histone deacetylase 6). Loss of RITA decreases the interaction of HDAC6 with MTs and increases the levels of acetylated a-tubulin. The absence of RITA also increases MT stability and reduces MT dynamics, triggering chromosome congression and segregation errors. Moreover, the reexpression of a mutant form of RITA lacking the MT interaction domain is not able to rescue the wild type phenotypes, suggesting that RITA’s function is achieved through the interaction with MTs [16]. DRG1 (Developmentally regulated GTP binding protein 1) is a MT binding protein that promotes polymerization and stabilizes MTs without GTP hydrolysis despite its identity as a GTPase. Cells lacking DRG1 proceed slower through mitosis due to delayed spindle formation [17]. GTSE1 (G2 And S Phase-Expressed Protein 1) is a p53 binding protein that is able to repress apoptosis. Decreased GTSE1 promotes MT turnover and chromosome congressional problems via unrestricted MCAK/Kif2C activity whereas increased GTSE1 promotes chromosome missegregation and chromosome instability [18]. An alternate mechanism of action involving Aurora B and the kinesin Kif4A proposed by Tipton et al. [19] underscores the complexity of action of GTSE1 and the importance of tuning MT dynamics in different regions of the spindle. ASK1 (Apoptosis signal-regulating kinase 1, also known as MAPK/ERK Kinase Kinase 5) plays an important role in apoptosis and innate immune response. It is also overexpressed in pancreatic cancer. In their paper the authors show that ASK1 phosphorylates EB1 and the depletion of this protein decreases MT dynamics in spindles of pancreatic cancer cells [20]. Finally, there are two new papers describing the role of actin and profilin in MT dynamics. An intriguing study described above used Xenopus extracts with intact actin filaments to demonstrate how actin affects MT dynamics. They observed that branched actin filaments are, specifically able to reduce MT growth rates and trigger MT disassembly [10]. Profilin is an actin binding protein that regulates actin function, and that can indirectly interact with MTs through formins. In this paper the authors show that profilin is able to increase MT polymerization rates and that this MT activity depends on residues that are mutated in patients with amyotrophic lateral sclerosis (ALS). Interestingly these effects on MTs could be attenuated by increasing the concentration of actin, suggesting a competition between actin and tubulin to bind profilin [21].

A well-studied group of proteins regulating MT dynamics are the kinesin-related molecular motors. These motors are able to regulate MT dynamics promoting MT polymerization (e.g. CenpE), depolymerization (e.g. Kip3, MCAK/kif2C and Kar3) or pausing/suppressing the dynamics at the MT ends (e.g. Kif18A). One of the most intriguing groups of these motors is the Kinesin-8 family and new information regarding their mechanism of action is steadily accumulating. Some of kinesin-8 members depolymerize MTs (Kip3, Klp5/6 and Kif19) and others, like human Kif18A appear to suppress MT growth at the tip. The Kinesin-8 motor from Drosophila Klp67A regulates MT dynamics thanks to both plus-end stabilizing and destabilizing activity. During mitosis it stabilizes the microtubule-kinetochore (MT-KT) connections. Moreover, in this same work they show how in HeLa cells Kif18A is important to assure correct MT-KT connections. GFP-Mad2 signal depleted from metaphase KTs returns to the KT in the absence of Kif18A indicating a return to a less stable attachment state [22]. In yeast, Kip3 regulates both MT dynamics and spindle positioning in mitosis. Interestingly the region proximal to the motor domain regulates astral MT stability, while the distal tail regulates spindle disassembly [23]. KIf18B also is able to regulate MT dynamics and affect mitosis. Kif18B is able to promote catastrophe, regulate the length of astral MTs which, in turn, influences spindle positioning [24]. These proteins and their proposed principal site of action are diagrammed in Figure 1b.

Measurement of Microtubule Assembly in Mitotic Spindles

The metaphase mitotic spindle is a remarkable structure in static form (Figure 1c) but the complexity of the behavior of individual MTs in the spindle renders microtubule dynamics within the spindle difficult to parameterize. Even in interphase cells it can be difficult to impossible to distinguish one MT from its neighbor. Live fluorescently labeled MTs are below the level of resolution of most super-resolution microscopes making it difficult to characterize individual MT behavior in densely populated areas of the cell. This problem is compounded in the mitotic spindle as it is densely packed with MTs whose individual behavior is close to impossible to parse. It is possible to measure bulk turnover of MTs using techniques such as Fluorescence Recovery After Photobleaching microscopy (FRAP) or, alternatively, a photoactivatable form of tubulin.

Two well-established methods to investigate MT dynamics in the mitotic spindle are: 1) Speckle microscopy and 2) tracking of End-Binding (EB) proteins. Fluorescent EB proteins enable researchers to study the assembly dynamics of individual MTs in the spindle (Figure 1d). Speckle microscopy, in contrast, employs limited label and sensitive detection equipment to evaluate the movement of fiducial marks on individual MTs in the spindle. Speckle microscopy has proven useful to describe flux and lateral movements of MT polymer throughout the spindle [25]. These features cannot normally be discerned when MTs are uniformly labeled. However, speckle microscopy has limitations in that it does not reveal MT end dynamics except by inference during flux [26]. To achieve a more direct measure of MT end dynamics researchers have taken advantage of the transient association of EB proteins to assembling MT tips. The translocation of these visualizable comets of EB proteins across the cell can be used as a measure of the number and assembly rate of growing MTs in the spindle. Photo-inactivatable versions of EB1 and EB3 are available that enable the functional investigation of these proteins in cell behavior beyond their use as a marker for MT end assembly [27]. These constructs will prove to be very powerful tools to study MT dynamics in the spindle. The photodissociation with blue light of this EB variant attenuates microtubule growth. Would it be possible to use this EB variant to change MT dynamics at the KT-MT interface? Would it be possible to destabilize hyperstable attachments in cells with impaired error correction system? These are very interesting questions that could be addressed with this new tool. A disadvantage in using EB proteins as a readout for MT dynamics is that fluorescently labeled EB proteins dissociate from disassembling MTs rendering those MTs invisible. Additionally, the translocation of an EB comet cannot always reliably distinguish between the velocity of MT growth and the extent to which a MT may be translocating laterally as assembled polymer. Paused MTs can sometimes be discerned using EB but they are dimmer and are sometimes difficult to score above background. Despite these limitations, EB binding can be surprisingly sharp and bright and is amenable to automated [28] and semi-automated (see below) tracking in cells to investigate the spatial distribution of MT assembly rates.

Automated tracking of assembling MTs in the mitotic spindle is difficult because the density of EB comets confuses many automated trackers. Even so, automated tracking using custom MatLab software has been used by the Ellenberg lab in conjunction with RNAi screens to successfully identify genetic lesions that impact MT assembly rates in mitotic spindles [29]. An alternative to tracking software is the use of kymographs. A kymograph is a representation of a spatial position over time (Figure 2a–c) in which one of the axis represents time and the other one distance. A kymograph of the comets generated by EB proteins results in a straight line with a certain angle (Figure 2c–d) in the distance-time graph. The angle in this graph will allow determination of the speed of the comet and, thus, the MT polymerization rate. Measuring MT polymerization rates in spindles using kymographs is not new, but it can be grueling and tedious work. However, a semiautomatic method for evaluating kymographs makes use of the “Directionality” plugin from Fiji which is supported by Jean-Yves Tinevez. This plugin makes the task of measuring angles less biased when performing manual measurements (Figure 2). The plugin is able to identify all the lines in a kymograph and measure the angle of those lines. Now, using basic trigonometry to relate angles, distance and time it is possible calculate the speed of the EB comets to evaluate the MT polymerization rates in both astral and densely packed spindle MTs. As far as we know, we haven’t seen any paper using this plugin to measure MT polymerization rates in spindles. We encourage the mitosis community to download and try this wonderful plugin to standardize these measurements and make them more comparable among laboratories.

Figure 2:

Quantification of Microtubule polymerization rates in mitotic spindles. (a) Screenshot of a mitotic HeLa cell transfected with EB3-GFP. (b) To measure MT polymerization rates using a kymograph the cell is rotated until both centrosomes are in the same horizontal line. A rectangle around the centrosomes is drawn to obtain a kymograph. (c) Kymograph of the rectangle shown in (b). The horizontal line represents distance (in μm) and the vertical line time (in seconds). Once we apply the “Directionality” plugin to the region between the centrosomes (blue square), we get a new window with the same kymograph with different color lines. The different colors represent the different angles of the kymograph lines. (d) Histogram with the frequency distribution of the angles obtained from (c).

There are also some technical difficulties inherent in the measurement of MT assembly rates in spindles. The first problem often encountered is spindle movement. The mitotic spindle can rotate in the XY plane as well as in the Z axis. This movement of the spindle will affect the measurement of MT polymerization rates and it is an artifact that is very difficult to avoid. Spindles that move a great deal during imaging must be discarded. Another problem often encountered when measuring MT polymerization rates in the spindle is phototoxicity meaning that cells are damaged by exposure to light [30]. As the rates of MT assembly in human cells are between 0.1 and 0.5 um/sec [7, 27], it is advisable to image cells with frame rate of at least 1 exposure per second to track individual MT tips. In mitotic cells some aspects of phototoxicity are visible. The mitotic spindle begins to shorten when experiencing phototoxicity and also becomes less dynamic, which can impact MT assembly rates. Previously this problem could only be solved by reducing the number of Z focal planes, shortening exposures and limiting the movie length to avoid light damage. Lattice Light-Sheet Microscopy (LLSM) will enable researchers to capture more frames per second and more focal planes. This kind of microscopy is gentler on the cells than regular widefield or confocal microscopy and it has already been used to image a whole mitotic spindle and track and measure MT polymerization rates [31]. An adaptation of LLSM called LITE microscopy that is compatible with conventional microscope stands and coverslip-based objectives makes this technique more widely available to general cell biology labs [32].

Alternative Read-outs for Microtubule Assembly changes in Mitotic Spindles.

Subtle increases in MT assembly rates are correlated with increased levels of CIN [7]. The mechanism by which CIN is increased is presently unknown, however, the observation is interesting because there may be many unexpected pathways that subtly impact MTs in this manner [33]. As it turns out there exists another read-out that has been shown to correlate with increased MT assembly rates and that does not rely on live imaging or rate measurements. For reasons that are imperfectly understood, cells that exhibit increased MT assembly rates manifest a higher proportion of eccentric monasters in drugs that inhibit mitotic centrosome separation such as Monastrol (Figure 3). This curious occurrence was first described in the context of BRCA1 depletion [6] and further exploited as a potential method to screen for changes in MT modulation [33]. Depletion of known modulators of MT assembly also exhibit eccentric monasters, suggesting that they could serve as a rapid method to score cells with increased risk of CIN (Figure 3). Finally, alterations in MT assembly rates are also likely to perturb MT homeostasis and promote autoregulation of tubulin mRNA levels [34]. It is a formal possibility that changes in tubulin mRNA levels could serve as a general readout for cells expressing altered MT assembly rates.

Figure 3:

Symmetric and asymmetric monopolar spindles. HeLa cells treated with Monastrol for 5 hours, fixed and labeled for DNA (blue), Tubulin (green) and Pericentrin (red). Left: symmetric monopolar spindle with the centrosomes in the center of the cell. Right: asymmetric monopolar spindle with the centrosomes displaced from the center of the cell and closer to the cell membrane.

Conclusions

Recent studies suggest that MT dynamics is fine-tuned such that even small disruptions to MT assembly rates in mitotic spindles can impact cell division and the development of cancer. For this reason, it is important to develop methods to measure MT assembly rates in a rapid and unbiased manner and to further characterize what these changes in MT assembly rates mean in the cell. Often changes in MT dynamics will subtly change the distribution of MTs within the cell in accordance with a change in assembly dynamics. For example, depletion of the MT depolymerizer MCAK/Kif2C appears to promote longer MTs [35, 36] in addition to altering MT assembly rates [37]. This raises the possibility that MT length rather than assembly rates is a key feature that mechanistically impacts chromosome segregation and that the assembly rates represent a read-out for dynamic changes that influence MT length. Even steady state dynamic instability of purified microtubules in vitro will lead to longer MTs without appreciable changes in MT polymer [38]. Because MT polymer in living cells also exhibits dynamic instability [39] it is not unreasonable that MTs in cells would be subject to similar kinetic constraints. Presently, we do not have good methods to measure global MT length throughout the cell except in some interesting specialized systems such as mechanically elongated eggs [40]. We also lack tools to accurately and rapidly measure cellular MT polymer and dimer levels, MT end concentration, MT number and MT length within cells. Only by knowing these parameters can we understand how changes in MT dynamics impact details of spindle function. Fortunately, such tools are in development. Expansion microscopy, for example, has the potential to resolve dense MT arrays in fixed cells [41, 42]. Machine learning algorithms will enable bench scientists to teach microscopes to recognize and rapidly quantify cellular structures. This builds on the expertise of cell biologists without the need to become a proficient computer programmer [43]. Finally, advances in molecular modeling will enable cell biologists to kinetically test in silico changes in MT dynamics within a closed cellular system [8, 44, 45].