Dmitry Malkov

Actin and dynamin2 dynamics and interplay during clathrin-mediated endocytosis.

Actin assembly influences the precise temporal and quantitative recruitment of dynamin2 to sites of clathrin-mediated endocytosis.

Domain-Based Biosensor Assay to Screen for Epidermal Growth Factor Receptor Modulators in Live Cells

Traditional drug discovery efforts have resulted in the approval of a handful of receptor tyrosine kinase (RTK) inhibitors; however, their discovery relied solely on screening recombinant kinases, often with poor cellular activity outcome. The ability to screen RTKs in their natural environment is sought as an alternative approach. We have adapted a novel strategy utilizing a green fluorescent protein-labeled SRC homology 2 domain-based biosensor as a surrogate reporter of endogenous epidermal growth factor receptor (EGFR) activity in A549 cells. Upon activation of the receptor, EGFR function in live cells is measured by the number of green granules that form. Here we describe assay miniaturization and demonstrate specificity for EGFR through its chemical inhibition and RNAi-dependent knockdown resulting in complete abrogation of granule formation. Gefitinib and PD 153035 were identified as hits in a pilot screen. This approach allows for the identification of novel EGFR modulators in high-throughput formats for screening chemical and RNAi libraries.

Tagging of Genomic STAT3 and STAT1 with Fluorescent Proteins and Insertion of a Luciferase Reporter in the Cyclin D1 Gene Provides a Modified A549 Cell Line to Screen for Selective STAT3 Inhibitors

Signal transducer and activator of transcription 3 (STAT3) is an oncogenic protein that is constitutively activated in numerous cancer cell lines and human cancers. Another STAT family member, STAT1, possesses cancer-inhibitory properties and can promote apoptosis in tumor cells upon activation. To better characterize these important cancer related genes, we tagged STAT3 and STAT1 loci with fluorescent protein (FP) sequences (RFP and GFP respectively) by targeted integration via zinc finger nuclease (ZFN) - mediated homologous recombination in A549 cells that express aberrantly activated STAT3. We inserted the FP transgenes at the N-terminus of the STAT3 locus and at the C-terminus of the STAT1 locus. The integration resulted in endogenous expression of fluorescent STAT3 and STAT1 chimeric fusion proteins. When stimulated with IL-6 or IFN-γ, the cells showed robust nuclear translocation of RFP-STAT3 or STAT1-GFP, respectively. Pre-incubation of cells with a known specific STAT3 inhibitor showed that IFN-γ-induced translocation of STAT1-GFP was not impaired. STAT3 activates multiple downstream targets such as genes involved in cell cycle progression - e.g. cyclin D1. To detect changes in expression of endogenous cyclin D1, we used ZFN technology to insert a secreted luciferase reporter behind the cyclin D1 promoter and separated the luciferase and cyclin D1 coding regions by a 2A sequence to induce a translational skip. The luciferase insertion was made in the RFP-STAT3/STAT1-GFP cell line to have all three reporters in a single cell line. Addition of a STAT3 inhibitor led to suppression of cyclin D1 promoter activity and cell growth arrest. The triple-modified cell line provides a simple and convenient method for high-content screening and pre-clinical testing of potential STAT3 inhibitors in live cells while ensuring that the STAT1 pathway is not affected. This approach of reporting endogenous gene activities using ZFN technology could be applied to other cancer targets.

Endogenous gene tagging with fluorescent proteins.

Human genome manipulation has become a powerful tool for understanding the mechanisms of numerous diseases including cancer. Inserting reporter sequences in the desired locations in the genome of a cell can allow monitoring of endogenous activities of disease related genes. Native gene expression and regulation is preserved in these knock-in cells in contrast to cell lines with target overexpression under an exogenous promoter as in the case of transient transfection or stable cell lines with random integration. The fusion proteins created using the modern genome editing tools are expressed at their physiological level and thus are more likely to retain the characteristic expression profile of the endogenous proteins in the cell. Unlike biochemical assays or immunostaining, using a tagged protein under endogenous regulation avoids fixation artifacts and allows detection of the targets activity in live cells. Multiple gene targets could be tagged in a single cell line allowing for the creation of effective cell-based assays for compound screening to discover novel drugs.

Modulation of Genome Editing Outcomes by Cell Cycle Control of Cas9 Expression

Targeting specific chromosomal sequences for genome modification or regulation during particular phases of the cell cycle may prove useful in creating more precise, predictable genetic changes. Here, we present a system using a fusion protein comprised of a programmable DNA modification protein, Cas9, linked to a cell cycle regulated protein, geminin, as well as green fluorescent protein (GFP) for visualization. Despite the large size of Cas9 relative to geminin, cells were observed to express Cas9-GFP-geminin at levels which oscillate with the cell cycle. These fusion proteins are also shown to retain double-strand break (DSB) activity at specific chromosomal sequences to produce both indels and targeted integration of donor ssDNA. Most importantly, the ratio of ssDNA donor integration to non-homologous end joining (NHEJ) was observed to increase, suggesting that cell cycle control Cas9 expression may be an effective strategy to bias DNA repair outcomes.

Anomalous Irreversible “Conversion” of Tag-BFP Fluorescence Spectra in the Presence of Tag-RFP: Possible Model of Prion Interactions

Prions are misfolded protein molecules that can propagate by transmitting a misfolded protein state. If the infectious isoform of prion proteins (PrP), known as PrP scrapie (PrPSc), contacts the properly folded PrP called cellular PrP (PrPC), it can stimulate the refolding of normal PrPC into abnormal PrPSc. In the process, the abnormal PrPSc form acts as a template to guide the misfolding of normal PrPC to convert it into more abnormal protein. We encountered similar conversion of fluorescent proteins (FPs) after introducing different FP transgene constructs into several genomic target locations. We used monomeric FPs from Evrogen: TagBFP and RFPs (TagRFP and FusionRed); and Clontechs mCherry.In a variety of targets, across multiple cell lines generated, we noted TagBFP emitting light at RFP wavelengths under certain circumstances. This “conversion” phenomenon existed when the cell line contained both TagBFP and TagRFP expressed at high levels or when they were co-localized. We RFP-tagged α-tubulin (TUBA1B), a highly expressed cytoskeletal protein that polymerizes to form microtubules, using three RFP versions (TagRFP, FusionRed, and mCherry) in cells expressing a TagBFP Segmentation Marker driven off the highly expressed β-actin locus. The complex formed from TagBFP and TagRFP showed an increase in the red channel. In the case when only TagBFP-β-actin fusion was expressed in the absence of any other FP tags, a signal was also present in the red channel. We hypothesize that transient TagBFP-TagRFP heteromerization induces a red-emitting TagBFP conformation. The TagRFP mutations (94% a.a. homology TagBFP) make TagBFP emit in blue. Our results suggest that TagRFP may act as a template to guide the re-folding of TagBFP into a red-emitting conformation. It makes TagBFP-TagRFP pair a convenient tool to model protein-protein interactions during the prion conversion.

Abstract LB-506: Targeted integrations of fluorescent proteins into STAT3 and STAT1 Loci via ZFN mediated homologous recombination in human cancer cell lines provides a suitable and adequate model system for cell-based testing of new candidate drugs for human tumor treatment

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL STAT3 which is a member of the family of signal transducers and activators of transcription is an oncogene and it is constitutively activated in numerous cancer cell lines and human cancers including breast, lung, head and neck, liver, and pancreas. Another STAT family member STAT1 possesses cancer-inhibitory properties and once activated may promote apoptosis in tumor cells. Thus STAT3 has been identified as a promising drug discovery target for many cancers. Ideally the potential inhibitors of STAT3 should be very selective and should not inactivate STAT1. To better address these cancer-research important genes, we tagged STAT3 and STAT1 loci with fluorescent protein (FP) sequences (RFP and GFP respectively) by targeted integration via Zinc-Finger Nuclease (ZFN) mediated homologous recombination in cancer derived cell lines. ZFN technology is a fast and reliable way to manipulate the genome in a targeted fashion. ZFNs are synthetic proteins engineered to bind DNA at a sequence-specific location and create a double strand break (www.compozrzfn.com). The cells natural machinery repairs the break in one of two ways: non-homologous end joining or homologous recombination. Utilizing the homologous recombination pathway that ZFNs induce, we successfully inserted the FP transgenes into the desired target locations - at the N-terminus of the STAT3 locus and at the C-terminus of the STAT1 locus. Three cell lines were created, two with the single STAT tagged and a third with both STATs tagged. Integration resulted in endogenous expression of fluorescent STAT3 and STAT1 chimeric fusion proteins in the A549 cell lines. Functional analyses of knock-in cell lines indicated that both STAT3 and STAT1 native gene regulation is conserved resulting in normal levels of protein expression and preservation of protein function. When stimulated with IL-6 or INF-γ, A549 cells showed fast and robust nuclear translocation of RFP-STAT3 or STAT1-GFP respectively. Cell pre-incubation with a known specific STAT3 inhibitors showed that STAT1-GFP, INF-γ-induced translocation was not impaired. Thus, these cell lines provide a simple and convenient method for high-content screening and pre-clinical testing of potential STAT3 inhibitors while ensuring that the STAT1 pathway is not affected. This approach of zfn-tagging endogenous genes could be applied to other cancer targets. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-506. doi:1538-7445.AM2012-LB-506

Abstract 4895: Tagging endogenous genes with fluorescent reporters using CompoZr® zinc finger nuclease technology

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Zinc finger nucleases (ZFNs) are a class of engineered DNA-binding proteins that facilitate targeted editing of the genome by creating double-strand breaks (DSBs) in DNA at desired locations. DSBs are important for site-specific mutagenesis in that they stimulate the cells natural DNA-repair processes, namely homologous recombination (HR) and non-homologous end joining (NHEJ). The formation of a DSB increases the rate of HR by a thousand fold between a specific genomic target and a donor plasmid in somatic cells. Sigma-Aldrich CompoZr® ZFN technology is a useful tool to create site specific mutations, and to generate knock-in and knockout model lines for academic and clinical research. By this approach, we tagged an oncogene, several cytoskeletal genes and a chromatin gene by integrating a fluorescent protein sequence into the desired location (at the sequences encoding either N-terminus or C-terminus of the target protein) in the genome. The integration resulted in endogenous expression of the corresponding fusion proteins (green, red, or blue) that shows their native characteristic pattern. The following five loci were tagged: ERBB2/HER2 (human epidermal growth factor receptor 2, plasma membrane), TUBA1B (α-tubulin 1b, microtubule), ACTB (β-actin, actin stress fibers), LMNB1 (lamin B1, nuclear envelope) and HMGA1 (high mobility group AT-hook 1, nucleus). Single cell clones were isolated in SKOV3, U2OS, and MCF10 cells with one copy of a given gene tagged. Multiplexing was demonstrated by labeling different genes in the same cell line as well as different alleles of the same gene. We demonstrate that ZFN mediated gene tagging is an efficient way to generate knock-in cell lines to report endogenous gene expression. It provides the basis for development of various high content screening (HCS) assays for compound screening where target gene regulation and corresponding protein function are preserved. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4895. doi:10.1158/1538-7445.AM2011-4895