Microtubule plus tips: A dynamic route to chromosomal instability.
Ontology highlight
ABSTRACT: Although chromosomal instability (CIN) is a recognized hallmark of cancer the underlying mechanisms and consequences are largely unknown. However, it is accepted that lagging chromosomes represent a major prerequisite for chromosome missegregation in cancer cells. Here, we discuss how lagging chromosomes are generated and our recent findings establishing increased microtubule assembly rates as a source of CIN.
Project description:Regulation of microtubule dynamics is essential for many cell biological processes and is likely to be variable between different subcellular regions. We describe a computational approach to analyze microtubule dynamics by detecting growing microtubule plus ends. Our algorithm tracked all EB1-EGFP comets visible in an image time-lapse sequence allowing the detection of spatial patterns of microtubule dynamics. We introduce spatiotemporal clustering of EB1-EGFP growth tracks to infer microtubule behaviors during phases of pause and shortening. We validated the algorithm by comparing the results to data for manually tracked, homogeneously labeled microtubules and by analyzing the effects of well-characterized inhibitors of microtubule polymerization dynamics. We used our method to analyze spatial variations of intracellular microtubule dynamics in migrating epithelial cells.
Project description:The persistent malattachment of microtubules to chromosomes at kinetochores is a major mechanism of chromosomal instability (CIN) [1, 2]. In normal diploid cells, malattachments arise spontaneously and are efficiently corrected to preserve genomic stability [3]. However, it is unknown whether cancer cells with CIN possess the ability to efficiently correct attachment errors. Here we show that kinetochore microtubule attachments in cancer cells with CIN are inherently more stable than those in normal diploid RPE-1 cells. The observed differences in attachment stability account for the persistence of malattachments into anaphase, where they cause chromosome missegregation. Furthermore, increasing the stability of kinetochore microtubule attachments in normal diploid RPE-1 cells, either by depleting the tumor suppressor protein APC or the kinesin-13 protein MCAK, is sufficient to promote chromosome segregation defects to levels comparable to those in cancer cells with CIN. Collectively, these data identify that cancer cells have a diminished capacity to correct erroneous kinetochore microtubule attachments and account for the widespread occurrence of CIN in tumors [4].
Project description:Kinesin-14 molecular motors represent an essential class of proteins that bind microtubules and walk toward their minus-ends. Previous studies have described important roles for Kinesin-14 motors at microtubule minus-ends, but their role in regulating plus-end dynamics remains controversial. Kinesin-14 motors have been shown to bind the EB family of microtubule plus-end binding proteins, suggesting that these minus-end-directed motors could interact with growing microtubule plus-ends. In this work, we explored the role of minus-end-directed Kinesin-14 motor forces in controlling plus-end microtubule dynamics. In cells, a Kinesin-14 mutant with reduced affinity to EB proteins led to increased microtubule lengths. Cell-free biophysical microscopy assays were performed using Kinesin-14 motors and an EB family marker of growing microtubule plus-ends, Mal3, which revealed that when Kinesin-14 motors bound to Mal3 at growing microtubule plus-ends, the motors subsequently walked toward the minus-end, and Mal3 was pulled away from the growing microtubule tip. Strikingly, these interactions resulted in an approximately twofold decrease in the expected postinteraction microtubule lifetime. Furthermore, generic minus-end-directed tension forces, generated by tethering growing plus-ends to the coverslip using λ-DNA, led to an approximately sevenfold decrease in the expected postinteraction microtubule growth length. In contrast, the inhibition of Kinesin-14 minus-end-directed motility led to extended tip interactions and to an increase in the expected postinteraction microtubule lifetime, indicating that plus-ends were stabilized by nonmotile Kinesin-14 motors. Together, we find that Kinesin-14 motors participate in a force balance at microtubule plus-ends to regulate microtubule lengths in cells.
Project description:Individual dynamic microtubules can generate pushing or pulling forces when their growing or shrinking ends are in contact with cellular objects such as the cortex or chromosomes. These microtubules can operate in parallel bundles, for example when interacting with mitotic chromosomes. Here, we investigate the force-generating capabilities of a bundle of growing microtubules and study the effect that force has on the cooperative dynamics of such a bundle. We used an optical tweezers setup to study microtubule bundles growing against a microfabricated rigid barrier in vitro. We show that multiple microtubules can generate a pushing force that increases linearly with the number of microtubules present. In addition, the bundle can cooperatively switch to a shrinking state, due to a force-induced coupling of the dynamic instability of single microtubules. In the presence of GMPCPP, bundle catastrophes no longer occur, and high bundle forces are reached more effectively. We reproduce the observed behavior with a simple simulation of microtubule bundle dynamics that takes into account previously measured force effects on single microtubules. Using this simulation, we also show that a constant compressive force on a growing bundle leads to oscillations in bundle length that are of potential relevance for chromosome oscillations observed in living cells.
Project description:Chromosomal instability (CIN) is defined as the perpetual missegregation of whole chromosomes during mitosis and represents a hallmark of human cancer. However, the mechanisms influencing CIN and its consequences on tumour growth are largely unknown. We identified an increase in microtubule plus-end assembly rates as a mechanism influencing CIN in colorectal cancer cells. This phenotype is induced by overexpression of the oncogene AURKA or by loss of the tumour suppressor gene CHK2, a genetic constitution found in 73% of human colorectal cancers. Increased microtubule assembly rates are associated with transient abnormalities in mitotic spindle geometry promoting the generation of lagging chromosomes and influencing CIN. Reconstitution of proper microtubule assembly rates by chemical or genetic means suppresses CIN and thereby, unexpectedly, accelerates tumour growth in vitro and in vivo. Thus, we identify a fundamental mechanism influencing CIN in cancer cells and reveal its adverse consequence on tumour growth.
Project description:The assembly of microtubule-based cellular structures depends on regulated tubulin polymerization and directional transport. Here, we purify and characterize tubulin heterodimers that have human β-tubulin isotype III (TUBB3), as well as heterodimers with one of two β-tubulin mutations (D417H or R262H). Both point mutations are proximal to the kinesin-binding site and have been linked to an ocular motility disorder in humans. Compared to wild-type, microtubules with these mutations have decreased catastrophe frequencies and increased average lifetimes of plus- and minus-end-stabilizing caps. Importantly, the D417H mutation does not alter microtubule lattice structure or Mal3 binding to growing filaments. Instead, this mutation reduces the affinity of tubulin for TOG domains and colchicine, suggesting that the distribution of tubulin heterodimer conformations is changed. Together, our findings reveal how residues on the surface of microtubules, distal from the GTP-hydrolysis site and inter-subunit contacts, can alter polymerization dynamics at the plus- and minus-ends of microtubules.
Project description:Microtubules undergo alternating periods of growth and shortening, known as dynamic instability. These dynamics allow microtubule plus ends to explore cellular space. The "search and capture" model posits that selective anchoring of microtubule plus ends at the cell cortex may contribute to cell polarization, spindle orientation, or targeted trafficking to specific cellular domains. Whereas cytoplasmic dynein is primarily known as a minus-end-directed microtubule motor for organelle transport, cortically localized dynein has been shown to capture and tether microtubules at the cell periphery in both dividing and interphase cells. To explore the mechanism involved, we developed a minimal in vitro system, with dynein-bound beads positioned near microtubule plus ends using an optical trap. Dynein induced a significant reduction in the lateral diffusion of microtubule ends, distinct from the effects of other microtubule-associated proteins such as kinesin-1 and EB1. In assays with dynamic microtubules, dynein delayed barrier-induced catastrophe of microtubules. This effect was ATP dependent, indicating that dynein motor activity was required. Computational modeling suggests that dynein delays catastrophe by exerting tension on individual protofilaments, leading to microtubule stabilization. Thus, dynein-mediated capture and tethering of microtubules at the cortex can lead to enhanced stability of dynamic plus ends.
Project description:In fission yeast, the lengths of interphase microtubule (iMT) arrays are adapted to cell length to maintain cell polarity and to help centre the nucleus and cell division ring. Here, we show that length regulation of iMTs is dictated by spatially regulated competition between MT-stabilising Tea2/Tip1/Mal3 (Kinesin-7) and MT-destabilising Klp5/Klp6/Mcp1 (Kinesin-8) complexes at iMT plus ends. During MT growth, the Tea2/Tip1/Mal3 complex remains bound to the plus ends of iMT bundles, thereby restricting access to the plus ends by Klp5/Klp6/Mcp1, which accumulate behind it. At cell ends, Klp5/Klp6/Mcp1 invades the space occupied by the Tea2/Tip1/Tea1 kinesin complex triggering its displacement from iMT plus ends and MT catastrophe. These data show that in vivo, whilst an iMT length-dependent model for catastrophe factor accumulation has validity, length control of iMTs is an emergent property reflecting spatially regulated competition between distinct kinesin complexes at the MT plus tip.
Project description:The development of cryo-electron microscopy (cryo-EM) allowed microtubules to be captured in their solution-like state, enabling decades of insight into their dynamic mechanisms and interactions with binding partners. Cryo-EM micrographs provide 2D visualization of microtubules, and these 2D images can also be used to reconstruct the 3D structure of the polymer and any associated binding partners. In this way, the binding sites for numerous components of the microtubule cytoskeleton-including motor domains from many kinesin motors, and the microtubule-binding domains of dynein motors and an expanding collection of microtubule associated proteins-have been determined. The effects of various microtubule-binding drugs have also been studied. High-resolution cryo-EM structures have also been used to probe the molecular basis of microtubule dynamic instability, driven by the GTPase activity of β-tubulin. These studies have shown the conformational changes in lattice-confined tubulin dimers in response to steps in the tubulin GTPase cycle, most notably lattice compaction at the longitudinal inter-dimer interface. Although work is ongoing to define a complete structural model of dynamic instability, attention has focused on the role of gradual destabilization of lateral contacts between tubulin protofilaments, particularly at the microtubule seam. Furthermore, lower resolution cryo-electron tomography 3D structures are shedding light on the heterogeneity of microtubule ends and how their 3D organization contributes to dynamic instability. The snapshots of these polymers captured using cryo-EM will continue to provide critical insights into their dynamics, interactions with cellular components, and the way microtubules contribute to cellular functions in diverse physiological contexts.
Project description:The exchange of tubulin dimer into steady-state microtubules was studied over a range of solution conditions, in order to assess the effects of various common buffer components on the dynamic instability of microtubules. In comparison with standard buffer conditions (100 mM-Pipes buffer, pH 6.5, containing 0.1 mM-EGTA, 1.8 mM-MgC12 and 1 M-glycerol), the rate and extent of exchange, and thus of dynamic instability, are suppressed by increasing the concentration of glycerol above 2 M. Exchange is enhanced by the addition of further Mg2+ (up to 17 mM) or by the addition of Ca2+ (up to 0.4 mM). Phosphate ion (150 mM) has relatively little effect on the dynamic behaviour of microtubules, as judged by the exchange method. The findings are interpreted within the framework of the Lateral Cap model for microtubule dynamic instability, in terms of the effects of these changes on the intrinsic rate constants of the system. By contrast, the extent of tubulin exchange depends selectively on the value of the dissociation rate constant for tubulin-GDP. A decrease in the extent of exchange, and hence in dynamic activity, is associated with a decreased value for this rate constant, and vice versa. The results also show good agreement of predictions of the model in treating the observed variations in the dynamic properties of individual microtubules, induced by different solution conditions.