Assesing Orthogonality of ER-HRP and cyt-APEX proximity labeling
Ontology highlight
ABSTRACT: This project was evaluating the orthogonality of proximity labeling peroxidases that are used to determine partitioning of proteins between the ER and the cytosol.
Project description:We sought to map RBM6 interactome using ascorbate peroxidase (APEX2)-based proximity labelling combined with mass spectrometry. Toward this end, we used CRISPR-cas9 methodology to establish MCF10A cell line expressing APEX2 fused to the N-terminal of RBM6, hereafter called MCF10AAPEX2-RBM6. The peroxidase activity of APEX2-RBM6 fusion was confirmed. Next, RBM6 proximal proteins that were biotinylated by APEX2 activation using hydrogen peroxidase (H2O2) in the presence of biotin-phenol were isolated and subjected to mass spectrometry. We identified 173 (P-value<0.05) RBM6 proximity-interaction partners.
Project description:Single cell RNA-seq study of induced pluripotent stem cell derived neural stem cells. Analysis of gene expression over cell clusters identified inherent presence of neurogenic progenitors and gliogenic progenitors in established nerual stem cells. This study aids to explain heterogeneity of neural stem cell identity and resolves gene expression enrichment in subpopulations of diverse progenitors. Processed and quality controlled data sets used for generating figure 3 in published article. Single cell raw data files for experiments are not available for public download.
Project description:Lipid droplet (LD) function is regulated by a complement of integral and peripheral proteins that associate with the LD phospholipid monolayer. Defining the composition of the LD proteome has remained a challenge due to the presence of contaminating proteins in LD-enriched buoyant fractions. To overcome this limitation, we developed a proximity labeling strategy that exploits LD-targeted APEX2 to biotinylate LD proteins in living cells. Application of this approach to U2OS and Huh7 cells identified the vast majority of previously validated LD proteins, excluded common contaminating proteins, and revealed new LD proteins.
Project description:Single cell RNA-seq study of induced pluripotent stem cell derived neural stem cells. Analysis of gene expression over cell clusters identified inherent presence of neurogenic progenitors and gliogenic progenitors in established neural stem cells. This study aids to explain heterogeneity of neural stem cell identity and resolves gene expression enrichment in subpopulations of diverse progenitors. Processed and quality controlled data sets used for generating figure 2 in published article. Single cell raw data files for experiments are not available for public download.
Project description:The Notch signaling pathway is a cell-cell communication system with fundamental roles in embryonic development and the nervous system, including neural stem cell proliferation and differentiation. To investigate the multivalency effect of ligands on the activation of the Notch receptor, we treated iPSc-derived neuroepithelial stem-like (lt-NES) cells with different Jag1 nanopatterns on DNA origami nanostructures.
Project description:Many intracellular bacteria, including the obligate intracellular pathogen Chlamydia trachomatis, grow within a membrane-bound “bacteria containing vacuole” (BCV). Secreted cytosolic effectors modulate host activity, but an understanding of the host-pathogen interactions that occur at the BCV membrane is limited by the difficulty in purifying membrane fractions from infected host cells. We used the ascorbate peroxidase proximity labeling system (APEX2), which labels proximal proteins with biotin in vivo, to study the protein-protein interactions that occur at the chlamydial vacuolar, or inclusion, membrane. An in vivo understanding of the secreted chlamydial inclusion membrane protein (Inc) interactions (e.g., Inc-Inc and Inc-eukaryotic protein) and how these contribute to overall host-chlamydial interactions at this unique membrane is lacking. We hypothesize some Incs organize the inclusion membrane whereas other Incs bind eukaryotic proteins to promote chlamydial-host interactions. To study this, Incs fused to APEX2 were expressed in C. trachomatis L2. Affinity purification-mass spectrometry (AP-MS) identified biotinylated proteins, which were analyzed for statistical significance using Significance Analysis of INTeractome (SAINT). Broadly supporting both Inc-Inc and Inc-host interactions, our Inc-APEX2 constructs labeled Incs as well as known and previously unreported eukaryotic proteins localizing to the inclusion. We demonstrate that endogenous LRRFIP1 (LRRF1) is recruited to the inclusion by the Inc, CT226, using bacterial two-hybrid and co-immunoprecipitation assays. We further demonstrate interactions between CT226 and the Incs used in our study to reveal a model for inclusion membrane organization. Combined, our data highlight the utility of APEX2 to capture the complex in vivo protein-protein interactions at the chlamydial inclusion.
Project description:Many intracellular bacteria, including the obligate intracellular pathogen Chlamydia trachomatis, grow within a membrane-bound “bacteria containing vacuole” (BCV). Secreted cytosolic effectors modulate host activity, but an understanding of the host-pathogen interactions that occur at the BCV membrane is limited by the difficulty in purifying membrane fractions from infected host cells. We used the ascorbate peroxidase proximity labeling system (APEX2), which labels proximal proteins with biotin in vivo, to study the protein-protein interactions that occur at the chlamydial vacuolar, or inclusion, membrane. An in vivo understanding of the secreted chlamydial inclusion membrane protein (Inc) interactions (e.g., Inc-Inc and Inc-eukaryotic protein) and how these contribute to overall host-chlamydial interactions at this unique membrane is lacking. We hypothesize some Incs organize the inclusion membrane whereas other Incs bind eukaryotic proteins to promote chlamydial-host interactions. To study this, Incs fused to APEX2 were expressed in C. trachomatis L2. Affinity purification-mass spectrometry (AP-MS) identified biotinylated proteins, which were analyzed for statistical significance using Significance Analysis of INTeractome (SAINT). Broadly supporting both Inc-Inc and Inc-host interactions, our Inc-APEX2 constructs labeled Incs as well as known and previously unreported eukaryotic proteins localizing to the inclusion. We demonstrate that endogenous LRRFIP1 (LRRF1) is recruited to the inclusion by the Inc, CT226, using bacterial two-hybrid and co-immunoprecipitation assays. We further demonstrate interactions between CT226 and the Incs used in our study to reveal a model for inclusion membrane organization. Combined, our data highlight the utility of APEX2 to capture the complex in vivo protein-protein interactions at the chlamydial inclusion.
Project description:Polyadenylation of mRNA precursors is mediated by a large multisubunit protein complex. Here we show that RBBP6 (retinoblastoma-binding protein 6), identified initially as an Rb- and p53-binding protein, is a component of this complex and functions in 3' processing in vitro and in vivo. RBBP6 associates with other core factors, and this interaction is mediated by an unusual ubiquitin-like domain, DWNN (domain with no name), that is required for 3' processing activity. The DWNN is also expressed, via alternative RNA processing, as a small single-domain protein (isoform 3 [iso3]). Importantly, we show that iso3, known to be down-regulated in several cancers, competes with RBBP6 for binding to the core machinery, thereby inhibiting 3' processing. Genome-wide analyses following RBBP6 knockdown revealed decreased transcript levels, especially of mRNAs with AU-rich 3' untranslated regions (UTRs) such as c-Fos and c-Jun, and increased usage of distal poly(A) sites. Our results implicate RBBP6 and iso3 as novel regulators of 3' processing, especially of RNAs with AU-rich 3' UTRs. RBBP6 siRNA or control siRNA was transfected in MCF7 cells in duplicates and RNA was hybridized to GeneChip Exon 1.0 ST arrays (Affymetrix)
Project description:Cell cycle quiescence is a critical feature contributing to haematopoietic stem cell (HSC) maintenance. Although various candidate stromal cells have been identified as potential HSC niches, the spatial localization of quiescent HSC in the bone marrow (BM) remains unclear. Here, using a novel approach that combines whole-mount confocal immunofluorescence imaging technique and computational modelling to analyse significant tridimensional associations among vascular structures, stromal cells and HSCs, we show that quiescent HSCs associate specifically with small arterioles that are preferentially found in endosteal BM. These arterioles are ensheathed exclusively by rare Nestin-GFP-peri/NG2+ pericytes, distinct from sinusoid-associated Nestin-GFP-retic/LepR+ cells. The present RNA-seq study sought to obtain a comprehensive understanding of the differences between the two distinct HSC cellular niches. mRNA profiles of sorted Nestin-GFP-peri and -GFP-retic bone marrow stromal cells were generated from pooled mice in triplicate by Illumina HiSeq 2000 sequencing.
Project description:A biosensor, consisting of the prokaryotic cis,cis-muconic acid-binding transcriptional activator BenM and an engineered reporter was successfully transplanted into bakers yeast Saccharomyces cerevisiae. RNAseq data used to assess the transcription orthogonality of BenM in yeast cells is presented here.