Image-based Screen Research Articles

Genome-Wide and Functional Annotation of Human E3 Ubiquitin Ligases Identifies MULAN, a Mitochondrial E3 that Regulates the Organelle's Dynamics and Signaling

Specificity of protein ubiquitylation is conferred by E3 ubiquitin (Ub) ligases. We have annotated ∼617 putative E3s and substrate-recognition subunits of E3 complexes encoded in the human genome. The limited knowledge of the function of members of the large E3 superfamily prompted us to generate genome-wide E3 cDNA and RNAi expression libraries designed for functional screening. An imaging-based screen using these libraries to identify E3s that regulate mitochondrial dynamics uncovered MULAN/FLJ12875, a RING finger protein whose ectopic expression and knockdown both interfered with mitochondrial trafficking and morphology. We found that MULAN is a mitochondrial protein – two transmembrane domains mediate its localization to the organelle's outer membrane. MULAN is oriented such that its E3-active, C-terminal RING finger is exposed to the cytosol, where it has access to other components of the Ub system. Both an intact RING finger and the correct subcellular localization were required for regulation of mitochondrial dynamics, suggesting that MULAN's downstream effectors are proteins that are either integral to, or associated with, mitochondria and that become modified with Ub. Interestingly, MULAN had previously been identified as an activator of NF-κB, thus providing a link between mitochondrial dynamics and mitochondria-to-nucleus signaling. These findings suggest the existence of a new, Ub-mediated mechanism responsible for integration of mitochondria into the cellular environment.

Small molecule regulators of autophagy identified by an image-based high-throughput screen

Autophagy is a lysosome-dependent cellular catabolic mechanism mediating the turnover of intracellular organelles and long-lived proteins. Reduction of autophagy activity has been shown to lead to the accumulation of misfolded proteins in neurons and may be involved in chronic neurodegenerative diseases such as Huntington's disease and Alzheimer's disease. To explore the mechanism of autophagy and identify small molecules that can activate it, we developed a series of high-throughput image-based screens for small-molecule regulators of autophagy. This series of screens allowed us to distinguish compounds that can truly induce autophagic degradation from those that induce the accumulation of autophagosomes as a result of causing cellular damage or blocking downstream lysosomal functions. Our analyses led to the identification of eight compounds that can induce autophagy and promote long-lived protein degradation. Interestingly, seven of eight compounds are FDA-approved drugs for treatment of human diseases. Furthermore, we show that these compounds can reduce the levels of expanded polyglutamine repeats in cultured cells. Our studies suggest the possibility that some of these drugs may be useful for the treatment of Huntington's and other human diseases associated with the accumulation of misfolded proteins.

Image-based chemical screening

Technological advances have made it feasible to conduct high-throughput small-molecule screens based on visual phenotypes of individual cells, using automated imaging and analysis. These screens are rapidly moving from being small, proof-of-principle tests to robust and widespread screens of hundreds of thousands of compounds. Automated imaging screens maximize the information obtained in an initial screen and improve the ability to select high-quality leads. In this Perspective, I highlight the key steps necessary for conducting a high-throughput image-based chemical compound screen.

C-Src protein kinase inhibitors block assembly and maturation of dengue virus

Dengue virus is a mosquito-borne flavivirus that represents an important emerging infectious disease and is an international health concern. Currently, there is no vaccine or effective antiviral therapy to prevent or to treat dengue virus infection. The slow progress in developing antiviral agents might be alleviated by the availability of efficient high-throughput anti-dengue virus screening assays. In this study, we report an immunofluorescence image-based assay suitable for identification of small molecule inhibitors of dengue virus infection and replication. Using this assay, we have discovered that inhibitors of the c-Src protein kinase exhibit a potent inhibitory effect on dengue virus (serotypes 1-4) and murine flavivirus Modoc. Mechanism of action studies demonstrated that the c-Src protein kinase inhibitor dasatinib prevents the assembly of dengue virions within the virus-induced membranous replication complex. These results demonstrate that this cell-based screen may provide a powerful means to identify new potential targets for anti-dengue drug development while simultaneously providing pharmacological probes to investigate dengue virus-host cell interactions at the biochemical level. Given the simplicity and excellent reproducibility of the assay, it should be useful in high-throughput screens of both small molecule and RNAi libraries when implemented on a robotic image-based high-throughput screen (HTS) platform. Given the reasonable clinical safety of inhibitors such as dasatinib and AZD0530, inhibitors of c-Src protein kinase may have the potential to become a new class of anti-dengue viral therapeutic agents.

A Synthetic Small Molecule That Induces Neuronal Differentiation of Adult Hippocampal Neural Progenitor Cells

Brain power: An image-based screen of a chemical library was carried out to identify molecules that regulate the differentiation of adult rat hippocampal neural stem cells. Neuropathiazol (1) induces selective neuronal differentiation and competitively suppresses astrocyte differentiation. This compound should be a useful tool to study the processes that control stem-cell fate both in vitro and in vivo.

Characterization of a novel, defense-related Arabidopsis mutant, cir1, isolated by luciferase imaging.

In order to identify components of the defense signaling network engaged following attempted pathogen invasion, we generated a novel PR-1::luciferase (LUC) transgenic line that was deployed in an imaging-based screen to uncover defense-related mutants. The recessive mutant designated cir1 exhibited constitutive expression of salicylic acid (SA), jasmonic acid (JA)/ethylene, and reactive oxygen intermediate-dependent genes. Moreover, this mutation conferred resistance against the bacterial pathogen Pseudomonas syringae pv. tomato DC3000 and a virulent oomycete pathogen Peronospora parasitica Noco2. Epistasis analyses were undertaken between cir1 and mutants that disrupt the SA (nprl, nahG), JA (jar1), and ethylene (ET) (ein2) signaling pathways. While resistance against both P. syringae pv. tomato DC3000 and Peronospora parasitica Noco2 was partially reduced by npr1, resistance against both of these pathogens was lost in an nahG genetic background. Hence, cirl-mediated resistance is established via NPR1-dependent and -independent signaling pathways and SA accumulation is essential for the function of both pathways. While jar1 and ein2 reduced resistance against P. syringae pv. tomato DC3000, these mutations appeared not to impact cir1-mediated resistance against Peronospora parasitica Noco2. Thus, JA and ET sensitivity are required for cir1-mediated resistance against P. syringae pv. tomato DC3000 but not Peronospora parasitica Noco2. Therefore, the cir1 mutation may define a negative regulator of disease resistance that operates upstream of SA, JA, and ET accumulation.

Three-dimensional imaging of the yeast actin cytoskeleton through the budding cell cycle.

The actin cytoskeleton of Saccharomyces cerevisiae is composed of a single, conventional actin isoform that is 86% identical to mammalian actins in addition to a battery of similarly conserved associated proteins. This conservation of components and their functions has led yeast geneticists and cell biologists to use the experimental power of yeast toward the study of the regulation of actin cytoskeleton assembly and the basic, housekeeping functions of the actin cytoskeleton (for a comprehensive review, see Botstein et al., 1997 ). Important to these studies has been the visualization of the actin cytoskeleton through the cell cycle and how mutations in regulators and components of the yeast cytoskeleton affect this organization. The first images of the yeast actin cytoskeleton showed that it consists of two filament-based structures: the actin cortical patch and the actin cables (Adams and Pringle, 1984 ; Kilmartin and Adams, 1984 ). The actin cortical patches show a polarized distribution that changes during the cell cycle: first they appear at the incipient bud site, suggesting a role in bud emergence; soon thereafter they are also found within the growing bud, indicating a role in bud growth; and late in the cell cycle they reorganize into two rings in the neck, where they are believed to be involved in septation and cytokinesis. By electron microscopy, the actin cortical patches have been shown to be invaginations of the plasma membrane around which actin filaments and actin-associated proteins are organized (Mulholland et al., 1994 ). Recently it has been shown that subsets of actin cortical patches can move at speeds of up to 1 μm/s (Doyle and Botstein, 1996 ; Waddle et al., 1996 ). The actin cables, which consist of bundled actin filaments, were observed to generally run along the long axis of budding cells. This organization fits well with the understanding that actin is involved in polarized cell growth, dynamic reorganization of the cell cortex, membrane trafficking at the cell cortex, and organelle segregation at cell division. Because certain aspects of the organization of the yeast actin cytoskeleton cannot be well addressed or documented by conventional two-dimensional microscopy, I have investigated the use of a DeltaVision deconvolution microscope for visualization of the yeast actin cytoskeleton in three dimensions. This instrument digitally captures focal sections in the Z plane and mathematically removes out-of-focus light by examining neighboring focal sections, from each section in a process called iterative deconvolution (Agard et al., 1989 ; Scalettar et al., 1996 ). The clarified focal sections can then be assembled to produce high-resolution three-dimensional images. I have found that this technology is extremely powerful for the study of yeast cell anatomy. With respect to the organization of the actin cytoskeleton, I have confirmed that the actin cables (like the patches) are cortical in their arrangement and can be observed to attach both at their ends and laterally to the cortical patches. In some cases, multiple cables can be seen to attach to single cortical patches. In addition, I have observed the earliest stages of bud emergence and have found that buds first emerge through a ring of cortical patches, and only after this stage do the cortical patches migrate into the bud. In my strain background (S288C), I do not observe a polarized distribution of cortical patches in the bud at any time during bud growth.