Global analysis of host-pathogen interactions that regulate early-stage HIV-1 replication.

Human Immunodeficiency Viruses (HIV-1 and HIV-2) rely upon host-encoded proteins to facilitate their replication. Here, we combined genome-wide siRNA analyses with interrogation of human interactome databases to assemble a host-pathogen biochemical network containing 213 confirmed host cellular factors and 11 HIV-1-encoded proteins. Protein complexes that regulate ubiquitin conjugation, proteolysis, DNA-damage response, and RNA splicing were identified as important modulators of early-stage HIV-1 infection. Additionally, over 40 new factors were shown to specifically influence the initiation and/or kinetics of HIV-1 DNA synthesis, including cytoskeletal regulatory proteins, modulators of posttranslational modification, and nucleic acid-binding proteins. Finally, 15 proteins with diverse functional roles, including nuclear transport, prostaglandin synthesis, ubiquitination, and transcription, were found to influence nuclear import or viral DNA integration. Taken together, the multiscale approach described here has uncovered multiprotein virus-host interactions that likely act in concert to facilitate the early steps of HIV-1 infection.

Label-free monitoring of T cell activation by the impedance-based xCELLigence system.

T cells play a critical role in maintaining the normal function of the adaptive immune response, with their dysfunction resulting in a variety of autoimmune and immunodeficiency diseases. Efficient and accurate detection of T cell function is therefore crucial to clinical diagnosis and development of immunomodulators. A variety of in vitro cellular systems are currently employed for analyzing T cell activation, yet all suffer from some combination of low throughput, unnatural conditions and long assay times. Label-free technologies are capable of detecting phenotypic responses to treatments under physiological conditions, thereby potentially accelerating drug discovery by facilitating the use of disease-relevant cell models for functional assessment and clinical diagnosis. The xCELLigence system is an impedance based label-free platform that allows for dynamic monitoring of subtle morphological and adhesive changes in cells, such as those induced during T cell activation. Here we describe the development and validation of a T cell activation assay based upon electrical impedance. Co-activation of Jurkat cells with anti-CD28 and anti-CD3 functional antibodies led to impedance changes that were rapidly and sensitively recorded (within 30 minutes). This phenomenon was also observed in human peripheral blood mononuclear cells. These changes reflect morphological and adhesive alterations correlated with cytoskeletal reorganization as verified by microscopy. They were functionally dependent on canonical T cell signaling pathways, including calcium-mediated signals and Src family kinases because relevant inhibitors impaired T cell activation. Our results provide a convenient approach to measure T cell activation in real-time and to elucidate the underlying mechanisms of action through probing with small molecules.

Rapid and quantitative assessment of cell quality, identity, and functionality for cell-based assays using real-time cellular analysis.

Strict quality control of cells is required for the standardization and interpretation of results in all areas of cell-based research, especially in drug discovery. Real-time cellular analysis using electrical impedance as a readout offers a rapid and highly reproducible method for quality control as it provides a quantitative measure of overall cell morphology and growth. In a case study, the authors demonstrate that samples of a single cell line obtained from several different labs show clear differences in their impedance profiles when compared with the corresponding standard cell line. A number of kinetic parameters were derived from the impedance profiles and used to quantify the differences among these cell lines. Our findings indicate that this methodology can detect cell line differences including mix-ups or contaminations, genetic alterations, and potential epigenetic changes occurring during passaging, all of which can occur in the time scale of a screening campaign. Finally, we provide evidence that these impedance profile differences can be predictive of different outcomes in cell-based functional assays for the effects of small molecules on otherwise seemingly identical cell lines.

A functional screen for regulators of CKDN2A reveals MEOX2 as a transcriptional activator of INK4a.

The CDKN2A locus encodes two important tumor suppressors, INK4a and ARF, which respond to oncogenic stresses by inducing cellular senescence. We conducted a genome-scale cDNA overexpression screen using a reporter containing INK4a regulatory sequences to identify novel transcriptional activators of this locus. This screen revealed 285 cDNAs that putatively regulate the transcriptional activation of INK4a. Of these, 56 are annotated as transcription factors, including two previously reported activators of the locus, ETS2 and JUNB. Fourteen genes were further validated for activity and specificity, including several homeodomain proteins. We found that the transcription of one of these, the homeodomain protein MEOX2 (GAX) is enhanced in primary cells during the induction of senescence, and forced expression of this protein results in the induction of premature senescence. We further demonstrate that MEOX2-induced senescence is dependent upon INK4a activity, and chromatin immunoprecipitation studies indicate that MEOX2 directly binds the INK4a promoter. These results support a role for this homeodomain protein as a direct regulator of INK4a transcription and senescence in human cells.

A role for IkappaB kinase 2 in bipolar spindle assembly.

IkappaB kinase 2 (IKK2 or IKKbeta) is a component of the IKK complex that coordinates the cellular response to a diverse set of extracellular stimuli, including cytokines, microbial infection, and stress. In response to an external stimulus, the complex is activated, resulting in the phosphorylation and subsequent proteasome-mediated degradation of IkappaB proteins. This event triggers the nuclear import of the NF-kappaB transcription factor, which activates the transcription of genes that regulate a variety of fundamental biological processes, including immune response, cell survival, and development. Here, we define an essential role for IKK2 in normal mitotic progression and the maintenance of spindle bipolarity. Chemical and genetic perturbation of IKK2 promotes the formation of multipolar spindles and chromosome missegregation. Depletion of IKK2 results in the deregulation of Aurora A protein stability and coincident hyperactivation of a putative Aurora A substrate, the mitotic motor KIF11. These data support a function for IKK2 as an antagonist of Aurora A signaling during mitosis. Additionally, our results indicate a direct role for IKK2 in the maintenance of genome stability and underscore the potential for oncogenic consequences in targeting this kinase for therapeutic intervention.