A high-throughput small molecule screen identifies synergism between DNA methylation and Aurora kinase pathways for X reactivation.

X-chromosome inactivation is a mechanism of dosage compensation in which one of the two X chromosomes in female mammals is transcriptionally silenced. Once established, silencing of the inactive X (Xi) is robust and difficult to reverse pharmacologically. However, the Xi is a reservoir of >1,000 functional genes that could be potentially tapped to treat X-linked disease. To identify compounds that could reactivate the Xi, here we screened ∼367,000 small molecules in an automated high-content screen using an Xi-linked GFP reporter in mouse fibroblasts. Given the robust nature of silencing, we sensitized the screen by "priming" cells with the DNA methyltransferase inhibitor, 5-aza-2'-deoxycytidine (5azadC). Compounds that elicited GFP activity include VX680, MLN8237, and 5azadC, which are known to target the Aurora kinase and DNA methylation pathways. We demonstrate that the combinations of VX680 and 5azadC, as well as MLN8237 and 5azadC, synergistically up-regulate genes on the Xi. Thus, our work identifies a synergism between the DNA methylation and Aurora kinase pathways as being one of interest for possible pharmacological reactivation of the Xi.

Niche-based screening identifies small-molecule inhibitors of leukemia stem cells.

Efforts to develop more effective therapies for acute leukemia may benefit from high-throughput screening systems that reflect the complex physiology of the disease, including leukemia stem cells (LSCs) and supportive interactions with the bone marrow microenvironment. The therapeutic targeting of LSCs is challenging because LSCs are highly similar to normal hematopoietic stem and progenitor cells (HSPCs) and are protected by stromal cells in vivo. We screened 14,718 compounds in a leukemia-stroma co-culture system for inhibition of cobblestone formation, a cellular behavior associated with stem-cell function. Among those compounds that inhibited malignant cells but spared HSPCs was the cholesterol-lowering drug lovastatin. Lovastatin showed anti-LSC activity in vitro and in an in vivo bone marrow transplantation model. Mechanistic studies demonstrated that the effect was on target, via inhibition of HMG-CoA reductase. These results illustrate the power of merging physiologically relevant models with high-throughput screening.

A dataset of images and morphological profiles of 30 000 small-molecule treatments using the Cell Painting assay.

Background: Large-scale image sets acquired by automated microscopy of perturbed samples enable a detailed comparison of cell states induced by each perturbation, such as a small molecule from a diverse library. Highly multiplexed measurements of cellular morphology can be extracted from each image and subsequently mined for a number of applications. Findings: This microscopy dataset includes 919 265 five-channel fields of view, representing 30 616 tested compounds, available at "The Cell Image Library" (CIL) repository. It also includes data files containing morphological features derived from each cell in each image, both at the single-cell level and population-averaged (i.e., per-well) level; the image analysis workflows that generated the morphological features are also provided. Quality-control metrics are provided as metadata, indicating fields of view that are out-of-focus or containing highly fluorescent material or debris. Lastly, chemical annotations are supplied for the compound treatments applied. Conclusions: Because computational algorithms and methods for handling single-cell morphological measurements are not yet routine, the dataset serves as a useful resource for the wider scientific community applying morphological (image-based) profiling. The dataset can be mined for many purposes, including small-molecule library enrichment and chemical mechanism-of-action studies, such as target identification. Integration with genetically perturbed datasets could enable identification of small-molecule mimetics of particular disease- or gene-related phenotypes that could be useful as probes or potential starting points for development of future therapeutics.

Microtubule transport in the axon: Re-thinking a potential role for the actin cytoskeleton.

Microtubules are transported down the axon as short pieces by molecular motor proteins. One popular idea is that these microtubules are transported by forces generated against the actin cytoskeleton. The motor for such transport is thought to be cytoplasmic dynein. Here, the authors review this model and discuss recent studies that sought to test it. These studies suggest that the model is valid but incomplete. Microtubule transport is bidirectional and can utilize either actin filaments or longer microtubules as a substrate in the anterograde direction but only longer microtubules in the retrograde direction. Cytoplasmic dynein is one participating motor but not the only one. The authors speculate that the category of anterograde microtubule transport that involves actin filaments may have specialized functions. The relevant forces that transport short microtubules may also be crucial for the manner by which the longer immobile microtubules interact with actin filaments during events such as axonal retraction and growth cone turning.

Role of actin filaments in the axonal transport of microtubules.

Microtubules originate at the centrosome of the neuron and are then released for transport down the axon, in which they can move both anterogradely and retrogradely during axonal growth. It has been hypothesized that these movements occur by force generation against the actin cytoskeleton. To test this, we analyzed the movement, distribution, and orientation of microtubules in neurons pharmacologically depleted of actin filaments. Actin depletion reduced but did not eliminate the anterograde movements and had no effect on the frequency of retrograde movements. Consistent with the idea that microtubules might also move against neighboring microtubules, actin depletion completely inhibited the outward transport of microtubules under experimental conditions of low microtubule density. Interestingly, visualization of microtubule assembly shows that actin depletion actually enhances the tendency of microtubules to align with one another. Such microtubule-microtubule interactions are sufficient to orient microtubules in their characteristic polarity pattern in axons grown overnight in the absence of actin filaments. In fact, microtubule behaviors were only chaotic after actin depletion in peripheral regions of the neuron in which microtubules are normally sparse and hence lack neighboring microtubules with which they could interact. On the basis of these results, we conclude that microtubules are transported against either actin filaments or neighboring microtubules in the anterograde direction but only against other microtubules in the retrograde direction. Moreover, the transport of microtubules against one another provides a surprisingly effective option for the deployment and orientation of microtubules in the absence of actin filaments.

Workflow and metrics for image quality control in large-scale high-content screens.

Automated microscopes have enabled the unprecedented collection of images at a rate that precludes visual inspection. Automated image analysis is required to identify interesting samples and extract quantitative information for high-content screening (HCS). However, researchers are impeded by the lack of metrics and software tools to identify image-based aberrations that pollute data, limiting experiment quality. The authors have developed and validated approaches to identify those image acquisition artifacts that prevent optimal extraction of knowledge from high-content microscopy experiments. They have implemented these as a versatile, open-source toolbox of algorithms and metrics readily usable by biologists to improve data quality in a wide variety of biological experiments.

A chemical screen probing the relationship between mitochondrial content and cell size.

The cellular content of mitochondria changes dynamically during development and in response to external stimuli, but the underlying mechanisms remain obscure. To systematically identify molecular probes and pathways that control mitochondrial abundance, we developed a high-throughput imaging assay that tracks both the per cell mitochondrial content and the cell size in confluent human umbilical vein endothelial cells. We screened 28,786 small molecules and observed that hundreds of small molecules are capable of increasing or decreasing the cellular content of mitochondria in a manner proportionate to cell size, revealing stereotyped control of these parameters. However, only a handful of compounds dissociate this relationship. We focus on one such compound, BRD6897, and demonstrate through secondary assays that it increases the cellular content of mitochondria as evidenced by fluorescence microscopy, mitochondrial protein content, and respiration, even after rigorous correction for cell size, cell volume, or total protein content. BRD6897 increases uncoupled respiration 1.6-fold in two different, non-dividing cell types. Based on electron microscopy, BRD6897 does not alter the percent of cytoplasmic area occupied by mitochondria, but instead, induces a striking increase in the electron density of existing mitochondria. The mechanism is independent of known transcriptional programs and is likely to be related to a blockade in the turnover of mitochondrial proteins. At present the molecular target of BRD6897 remains to be elucidated, but if identified, could reveal an important additional mechanism that governs mitochondrial biogenesis and turnover.

A human islet cell culture system for high-throughput screening.

A small-molecule inducer of beta-cell proliferation in human islets represents a potential regeneration strategy for treating type 1 diabetes. However, the lack of suitable human beta cell lines makes such a discovery a challenge. Here, we adapted an islet cell culture system to high-throughput screening to identify such small molecules. We prepared microtiter plates containing extracellular matrix from a human bladder carcinoma cell line. Dissociated human islets were seeded onto these plates, cultured for up to 7 days, and assessed for proliferation by simultaneous Ki67 and C-peptide immunofluorescence. Importantly, this environment preserved beta-cell physiological function, as measured by glucose-stimulated insulin secretion. Adenoviral overexpression of cdk-6 and cyclin D(1), known inducers of human beta cell proliferation, was used as a positive control in our assay. This induction was inhibited by cotreatment with rapamycin, an immunosuppressant often used in islet transplantation. We then performed a pilot screen of 1280 compounds, observing some phenotypic effects on cells. This high-throughput human islet cell culture method can be used to assess various aspects of beta-cell biology on a relatively large number of compounds.

Effects of dynactin disruption and dynein depletion on axonal microtubules.

We investigated potential roles of cytoplasmic dynein in organizing axonal microtubules either by depleting dynein heavy chain from cultured neurons or by experimentally disrupting dynactin. The former was accomplished by siRNA while the latter was accomplished by overexpressing P50-dynamitin. Both methods resulted in a persistent reduction in the frequency of transport of short microtubules. To determine if the long microtubules in the axon also undergo dynein-dependent transport, we ascertained the rates of EGFP-EB3 "comets" observed at the tips of microtubules during assembly. The rates of the comets, in theory, should reflect a combination of the assembly rate and any potential transport of the microtubule. Comets were initially slowed during P50-dynamitin overexpression, but this effect did not persist beyond the first day and was never observed in dynein-depleted axons. In fact, the rates of the comets were slightly faster in dynein-depleted axons. We conclude that the transient effect of P50-dynamitin overexpression reflects a reduction in microtubule polymerization rates. Interestingly, after prolonged dynein depletion, the long microtubules were noticeably misaligned in the distal regions of axons and failed to enter the filopodia of growth cones. These results suggest that the forces generated by cytoplasmic dynein do not transport long microtubules, but may serve to align them with one another and also permit them to invade filopodia.

Monastrol, a prototype anti-cancer drug that inhibits a mitotic kinesin, induces rapid bursts of axonal outgrowth from cultured postmitotic neurons.

Terminally postmitotic neurons continue to express many of the kinesin-related proteins known to configure microtubules during mitosis. Drugs that inhibit these kinesins are being developed as anti-cancer agents with the hope that they will inhibit proliferation of tumor cells without having adverse effects on the nervous system. The prototype, termed monastrol, inhibits the kinesin known as Eg5, which is essential for maintaining separation of the half-spindles. Eg5 is also highly expressed in neurons, particularly during development. Exposure of cultured sympathetic neurons to monastrol for a few hours increased both the number and the growth rate of the axons. With additional time, the overall lengths of the axons were indistinguishable from controls. Sensory neurons showed a similar short-term increase in axonal growth rate. However, prolonged exposure resulted in shorter axons, suggesting that sensory neurons may be more sensitive to toxic effects of the drug. Nevertheless, the overall health of the cultures was still far more robust than cultures treated with taxol, a drug commonly used for anti-cancer therapy. On the basis of these results, we conclude that Eg5 normally generates forces that oppose axonal growth, presumably by partially suppressing the forward advance of microtubules. We speculate that local regulation of Eg5 could be a means by which neurons coordinate rapid bursts of axonal growth with appropriate environmental cues. The comparatively modest toxic effects on the neurons over time are a hopeful sign for clinicians interested in using anti-Eg5 drugs for cancer therapy.