Zachary E. Perlman

Parallel Chemical Genetic and Genome-Wide RNAi Screens Identify Cytokinesis Inhibitors and Targets

Cytokinesis involves temporally and spatially coordinated action of the cell cycle and cytoskeletal and membrane systems to achieve separation of daughter cells. To dissect cytokinesis mechanisms it would be useful to have a complete catalog of the proteins involved, and small molecule tools for specifically inhibiting them with tight temporal control. Finding active small molecules by cell-based screening entails the difficult step of identifying their targets. We performed parallel chemical genetic and genome-wide RNA interference screens in Drosophila cells, identifying 50 small molecule inhibitors of cytokinesis and 214 genes important for cytokinesis, including a new protein in the Aurora B pathway (Borr). By comparing small molecule and RNAi phenotypes, we identified a small molecule that inhibits the Aurora B kinase pathway. Our protein list provides a starting point for systematic dissection of cytokinesis, a direction that will be greatly facilitated by also having diverse small molecule inhibitors, which we have identified. Dissection of the Aurora B pathway, where we found a new gene and a specific small molecule inhibitor, should benefit particularly. Our study shows that parallel RNA interference and small molecule screening is a generally useful approach to identifying active small molecules and their target pathways.

A high-throughput cell migration assay using scratch wound healing, a comparison of image-based readout methods

BackgroundCell 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.ResultsWe 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.ConclusionsThe 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.

The kinesin Eg5 drives poleward microtubule flux in Xenopus laevis egg extract spindles

Although mitotic and meiotic spindles maintain a steady-state length during metaphase, their antiparallel microtubules slide toward spindle poles at a constant rate. This “poleward flux” of microtubules occurs in many organisms and may provide part of the force for chromosome segregation. We use quantitative image analysis to examine the role of the kinesin Eg5 in poleward flux in metaphase Xenopus laevis egg extract spindles. Pharmacological inhibition of Eg5 results in a dose–responsive slowing of flux, and biochemical depletion of Eg5 significantly decreases the flux rate. Our results suggest that ensembles of nonprocessive Eg5 motors drive flux in metaphase Xenopus extract spindles.

Phenotypic Screening of Small Molecule Libraries by High Throughput Cell Imaging

We have developed high throughput fluorescence cell imaging methods to screen chemical libraries for compounds with effects on diverse aspects of cell physiology. We describe screens for compounds that arrest cells in mitosis, that block cell migration, and that block the secretory pathway. Each of these screens yielded specific inhibitors for research use, and the mitosis screen identified Eg5 as a potential target protein for cancer chemotherapy. Cell imaging provides a large amount of information from primary screening data that can be used to distinguish compounds with different effects on cells, and together with automated analysis, to quantitate compound effects.

A new method reveals microtubule minus ends throughout the meiotic spindle

Anastral meiotic spindles are thought to be organized differently from astral mitotic spindles, but the field lacks the basic structural information required to describe and model them, including the location of microtubule-nucleating sites and minus ends. We measured the distributions of oriented microtubules in metaphase anastral spindles in Xenopus laevis extracts by fluorescence speckle microscopy and cross-correlation analysis. We localized plus ends by tubulin incorporation and combined this with the orientation data to infer the localization of minus ends. We found that minus ends are localized throughout the spindle, sparsely at the equator and at higher concentrations near the poles. Based on these data, we propose a model for maintenance of the metaphase steady-state that depends on continuous nucleation of microtubules near chromatin, followed by sorting and outward transport of stabilized minus ends, and, eventually, their loss near poles.

High-content screening and profiling of drug activity in an automated centrosome-duplication assay

Maintenance of centrosome number is essential for cell‐cycle progression and genomic stability, but investigation of this regulation has been limited by assay difficulty. We present a fully automated image‐based centrosome‐duplication assay that is accurate and robust enough for both careful cell‐biology studies and high‐throughput screening, and employ this assay in a series of chemical‐genetic studies. We observe that a simple cytometric profiling strategy, which is based on organelle size, groups compounds with similar mechanisms of action; this suggests a simple strategy for excluding compounds that undesirably target such activities as protein synthesis and microtubule dynamics. Screening a library of compounds of known activity, we found unexpected effects on centrosome duplication by a number of drugs, most notably isoform‐specific protein kinase C inhibitors and retinoic acid receptor agonists. From a 16 320‐member library of uncharacterized small molecules, we identified five potent centrosome‐duplication inhibitors that do not target microtubule dynamics or protein synthesis. The analysis methodology reported here is directly relevant to studies of centrosome regulation in a variety of systems and is adaptable to a wide range of other biological problems.

Eg5 Causes Elongation of Meiotic Spindles When Flux-Associated Microtubule Depolymerization Is Blocked

In higher eukaryotes, microtubules (MT) in both halves of the mitotic spindle translocate continuously away from the midzone in a phenomenon called poleward microtubule flux. Because the spindle maintains constant length and microtubule density, this microtubule translocation must somehow be coupled to net MT depolymerization at spindle poles. The molecular mechanisms underlying both flux-associated translocation and flux-associated depolymerization are not well understood, but it can be predicted that blocking pole-based destabilization will increase spindle length, an idea that has not been tested in meiotic spindles. Here, we show that simultaneous addition of two pole-disrupting reagents p50/dynamitin and a truncated version of Xklp2 results in continuous spindle elongation in Xenopus egg extracts, and we quantitatively correlate this elongation rate with the poleward translocation of stabilized microtubules. We further use this system to demonstrate that this poleward translocation requires the activity of the kinesin-related protein Eg5. These results suggest that Eg5 is responsible for flux-associated MT translocation and that dynein and Xklp2 regulate flux-associated microtubule depolymerization at spindle poles.