Person:

Mitchison, Timothy

Vascular disrupting agents (VDAs), anti-cancer drugs that target established tumor blood vessels, fall into two main classes: microtubule targeting drugs, exemplified by combretastatin A4 (CA4), and flavonoids, exemplified by 5,6-dimethylxanthenone-4-acetic acid (DMXAA). Both classes increase permeability of tumor vasculature in mouse models, and DMXAA in particular can cause massive tumor necrosis. The molecular target of CA4 is clearly microtubules. The molecular target(s) of DMXAA remains unclear. It is thought to promote inflammatory signaling in leukocytes, and has been assumed to not target microtubules, though it is not clear from the literature how carefully this assumption has been tested. An earlier flavone analog, flavone acetic acid, was reported to promote mitotic arrest suggesting flavones might possess anti-microtubule activity, and endothelial cells are sensitive to even mild disruption of microtubules. We carefully investigated whether DMXAA directly affects the microtubule or actin cytoskeletons of endothelial cells by comparing effects of CA4 and DMXAA on human umbilical vein endothelial cells (HUVEC) using time-lapse imaging and assays for cytoskeleton integrity. CA4 caused retraction of the cell margin, mitotic arrest and microtubule depolymerization, while DMXAA, up to 500 µM, showed none of these effects. DMXAA also had no effect on pure tubulin nucleation and polymerization, unlike CA4. We conclude that DMXAA exhibits no direct anti-microtubule action and thus cleanly differs from CA4 in its mechanism of action at the molecular level.

Background: A recent crystal structure of monastrol in a ternary complex with the kinesin Eg5 motor domain highlights a novel, induced-fit drug binding site at atomic resolution. Mutational obliteration of the monastrol binding site results in a monastrol-resistant, but otherwise catalytically active Eg5 motor domain. However, considering the conformational changes at this site, it is unclear what specific interactions stabilize the interaction between monastrol and the Eg5 motor domain. Results: To study the molecular complementarity of the monastrol-Eg5 interaction, we used a combination of synthetic chemistry and targeted mutations in Eg5 to measure the contribution of specific contacts to inhibition of Eg5 in vitro and in cultured cells. Structure-activity data on chemical derivatives, sequence analysis of Eg5 homologs from different species, and the effect of mutations near the drug binding site were consistent with the crystal structure. Conclusion: The mechanism of monastrol revealed by our data rationalizes its specificity for Eg5 over other kinesins and highlights a potential mechanism of drug resistance for anti-cancer therapy targeting this site in Eg5.

Background: Cell migration is a complex phenomenon that requires the coordination of numerous cellular processes. Investigation of cell migration and its underlying biology is of interest to basic scientists and those in search of therapeutics. Current migration assays for screening small molecules, siRNAs, or other perturbations are difficult to perform in parallel at the scale required to screen large libraries. Results: We have adapted the commonly used scratch wound healing assay of tissue-culture cell monolayers to a 384 well plate format. By mechanically scratching the cell substrate with a pin array, we are able to create characteristically sized wounds in all wells of a 384 well plate. Imaging of the healing wounds with an automated fluorescence microscope allows us to distinguish perturbations that affect cell migration, morphology, and division. Readout requires ~1 hr per plate but is high in information content i.e. high content. We compare readouts using different imaging technologies, automated microscopy, scanners and a fluorescence macroscope, and evaluate the trade-off between information content and data acquisition rate. Conclusions: The adaptation of a wound healing assay to a 384 well format facilitates the study of aspects of cell migration, tissue reorganization, cell division, and other processes that underlie wound healing. This assay allows greater than 10,000 perturbations to be screened per day with a quantitative, high-content readout, and can also be used to characterize small numbers of perturbations in detail.

In this work, we describe pin-hole array correlation imaging, a multipoint version of fluorescence correlation spectroscopy, based upon a stationary Nipkow disk and a high-speed electron multiplying charged coupled detector. We characterize the system and test its performance on a variety of samples, including 40 nm colloids, a fluorescent protein complex, a membrane dye, and a fluorescence fusion protein. Our results demonstrate that pin-hole array correlation imaging is capable of simultaneously performing tens or hundreds of fluorescence correlation spectroscopy-style measurements in cells, with sufficient sensitivity and temporal resolution to study the behaviors of membrane-bound and soluble molecules labeled with conventional chemical dyes or fluorescent proteins.

Poly-ADP-ribosylation is a post-translational modification that regulates processes involved in genome stability. Breakdown of the poly(ADP-ribose) (PAR) polymer is catalysed by poly(ADP-ribose) glycohydrolase (PARG), whose endo-glycohydrolase activity generates PAR fragments. Here we present the crystal structure of PARG incorporating the PAR substrate. The two terminal ADP-ribose units of the polymeric substrate are bound in exo-mode. Biochemical and modelling studies reveal that PARG acts predominantly as an exo-glycohydrolase. This preference is linked to Phe902 (human numbering), which is responsible for low-affinity binding of the substrate in endo-mode. Our data reveal the mechanism of poly-ADP-ribosylation reversal, with ADP-ribose as the dominant product, and suggest that the release of apoptotic PAR fragments occurs at unusual PAR/PARG ratios.

Despite marked advances in breast cancer therapy, basal-like breast cancer (BBC), an aggressive subtype of breast cancer usually lacking estrogen and progesterone receptors, remains difficult to treat. In this study, we report the identification of MELK as a novel oncogenic kinase from an in vivo tumorigenesis screen using a kinome-wide open reading frames (ORFs) library. Analysis of clinical data reveals a high level of MELK overexpression in BBC, a feature that is largely dependent on FoxM1, a master mitotic transcription factor that is also found to be highly overexpressed in BBC. Ablation of MELK selectively impairs proliferation of basal-like, but not luminal breast cancer cells both in vitro and in vivo. Mechanistically, depletion of MELK in BBC cells induces caspase-dependent cell death, preceded by defective mitosis. Finally, we find that Melk is not required for mouse development and physiology. Together, these data indicate that MELK is a normally non-essential kinase, but is critical for BBC and thus represents a promising selective therapeutic target for the most aggressive subtype of breast cancer. DOI: http://dx.doi.org/10.7554/eLife.01763.001

Microtubule asters - radial arrays of microtubules organized by centrosomes - play a fundamental role in the spatial coordination of animal cells. The standard model of aster growth assumes a fixed number of microtubules originating from the centrosomes. However, aster morphology in this model does not scale with cell size, and we recently found evidence for non-centrosomal microtubule nucleation. Here, we combine autocatalytic nucleation and polymerization dynamics to develop a biophysical model of aster growth. Our model predicts that asters expand as traveling waves and recapitulates all major aspects of aster growth. With increasing nucleation rate, the model predicts an explosive transition from stationary to growing asters with a discontinuous jump of the aster velocity to a nonzero value. Experiments in frog egg extract confirm the main theoretical predictions. Our results suggest that asters observed in large fish and amphibian eggs are a meshwork of short, unstable microtubules maintained by autocatalytic nucleation and provide a paradigm for the assembly of robust and evolvable polymer networks. DOI: http://dx.doi.org/10.7554/eLife.19145.001

Purified microtubules have been shown to align along the static magnetic field (SMF) in vitro because of their diamagnetic anisotropy. However, whether mitotic spindle in cells can be aligned by magnetic field has not been experimentally proved. In particular, the biological effects of SMF of above 20 T (Tesla) have never been reported. Here we found that in both CNE-2Z and RPE1 human cells spindle orients in 27 T SMF. The direction of spindle alignment depended on the extent to which chromosomes were aligned to form a planar metaphase plate. Our results show that the magnetic torque acts on both microtubules and chromosomes, and the preferred direction of spindle alignment relative to the field depends more on chromosome alignment than microtubules. In addition, spindle morphology was also perturbed by 27 T SMF. This is the first reported study that investigated the cellular responses to ultra-high magnetic field of above 20 T. Our study not only found that ultra-high magnetic field can change the orientation and morphology of mitotic spindles, but also provided a tool to probe the role of spindle orientation and perturbation in developmental and cancer biology. DOI: http://dx.doi.org/10.7554/eLife.22911.001

Agonists of mouse STING (TMEM173) shrink and even cure solid tumor by activating innate immunity; human STING agonists are needed to test this therapeutic hypothesis in man. The endogenous STING agonist is 2′3′-cGAMP, a 2nd messenger that signals the presence of cytosolic dsDNA. We report activity-guided partial purification and identification of ENPP1 as the dominant 2′3′-cGAMP hydrolyzing activity in cultured cells. The hydrolysis activity of ENPP1 was confirmed using recombinant protein and was depleted in tissue extracts and plasma from Enpp1-/- mice. We synthesized a hydrolysis-resistant bis-phosphothioate analog of 2′3′-cGAMP (2′3′-cGsAsMP) with similar affinity for human STING in vitro and 10 times more potent at inducing IFN-β secretion from human THP1 monocytes. Studies in mouse Enpp1-/- lung fibroblasts indicate that resistance to hydrolysis contributes significantly to its higher potency. 2′3′-cGsAsMP is therefore improved over natural 2′3′-cGAMP as a model agonist, and has potential as a vaccine adjuvant and cancer therapeutic.

Mitochondria, which are essential organelles in resting and replicating cells, can vary in number, mass and shape. Past research has primarily focused on short-term molecular mechanisms underlying fission/fusion. Less is known about longer-term mitochondrial behavior such as the overall makeup of cell populations’ morphological patterns and whether these patterns can be used as biomarkers of drug response in human cells. We developed an image-based analytical technique to phenotype mitochondrial morphology in different cancers, including cancer cell lines and patient-derived cancer cells. We demonstrate that (i) cancer cells of different origins, including patient-derived xenografts, express highly diverse mitochondrial phenotypes; (ii) a given phenotype is characteristic of a cell population and fairly constant over time; (iii) mitochondrial patterns correlate with cell metabolic measurements and (iv) therapeutic interventions can alter mitochondrial phenotypes in drug-sensitive cancers as measured in pre- versus post-treatment fine needle aspirates in mice. These observations shed light on the role of mitochondrial dynamics in the biology and drug response of cancer cells. On the basis of these findings, we propose that image-based mitochondrial phenotyping can provide biomarkers for assessing cancer phenotype and drug response.