Donnelly Centre for Cellular and Biomolecular Research

PubMed

Recent Publications

Implementation of an antibody characterization procedure and application to the major ALS/FTD disease gene C9ORF72.

Related Articles

Implementation of an antibody characterization procedure and application to the major ALS/FTD disease gene C9ORF72.

Elife. 2019 10 15;8:

Authors: Laflamme C, McKeever PM, Kumar R, Schwartz J, Kolahdouzan M, Chen CX, You Z, Benaliouad F, Gileadi O, McBride HM, Durcan TM, Edwards AM, Healy LM, Robertson J, McPherson PS

Abstract
Antibodies are a key resource in biomedical research yet there are no community-accepted standards to rigorously characterize their quality. Here we develop a procedure to validate pre-existing antibodies. Human cell lines with high expression of a target, determined through a proteomics database, are modified with CRISPR/Cas9 to knockout (KO) the corresponding gene. Commercial antibodies against the target are purchased and tested by immunoblot comparing parental and KO. Validated antibodies are used to definitively identify the most highly expressing cell lines, new KOs are generated if needed, and the lines are screened by immunoprecipitation and immunofluorescence. Selected antibodies are used for more intensive procedures such as immunohistochemistry. The pipeline is easy to implement and scalable. Application to the major ALS disease gene C9ORF72 identified high-quality antibodies revealing C9ORF72 localization to phagosomes/lysosomes. Antibodies that do not recognize C9ORF72 have been used in highly cited papers, raising concern over previously reported C9ORF72 properties.

PMID: 31612854 [PubMed - in process]



▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄

Protein-Protein Interaction Profiling in Candida albicans Revealed by Biochemical Purification-Mass Spectrometry (BP/MS).

Related Articles

Protein-Protein Interaction Profiling in Candida albicans Revealed by Biochemical Purification-Mass Spectrometry (BP/MS).

Methods Mol Biol. 2019;2049:203-211

Authors: Pourhaghighi R, O'Meara TR, Cowen LE, Emili A

Abstract
BP/MS is a new experimental proteomic platform for systematic study of native protein complexes and global protein-protein interaction networks based on deep biochemical purification of soluble protein extracts and mass spectrometry identification of coeluting proteins. Herein, we describe the application of this methodology to draft a high-confidence protein interaction network for Candida albicans.

PMID: 31602613 [PubMed - in process]



▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄

Forward genetic screen in human podocytes identifies diphthamide biosynthesis genes as regulators of adhesion.

Forward genetic screen in human podocytes identifies diphthamide biosynthesis genes as regulators of adhesion.

Am J Physiol Renal Physiol. 2019 Sep 30;:

Authors: Cina DP, Ketela T, Brown KR, Chandrashekhar M, Mero P, Li C, Onay T, Fu Y, Han Z, Saleem MA, Moffat J, Quaggin SE

Abstract
Background: Podocyte function is tightly linked to the complex organization of its cytoskeleton, and adhesion to the underlying glomerular basement membrane. Adhesion of cultured podocytes to a variety of substrates is reported to correlate with podocyte health. Methods: To identify novel genes that are important for podocyte function, we designed an in vitrogenetic screen based on podocyte adhesion to plates coated with either fibronectin or soluble FLT1/Fc. Results: A genome-scale pooled RNA interference screen on immortalized human podocytes identified 77 genes that increased adhesion to fibronectin, 101 genes that increased adhesion to sFLT1/Fc, and 44 genes that increased adhesion to both substrates when knocked down. Multiple shRNAs against each of DPH1, DPH2, DPH3, and DPH4were top hits for increased adhesion. Immortalized human podocyte cells stably expressing these hairpins displayed increased adhesion to both substrates. We then used CRISPR-Cas9 to generate podocyte knockout cells for DPH1, DPH2,or DPH3which also displayed increased adhesion to both fibronectin and sFLT1/Fc, as well as a spreading defect. Last, we showed that Drosophila nephrocyte-specific knock-down of Dph1, Dph2, and Dph4results in altered nephrocyte function. Conclusions: In summary, we report a novel high-throughput method to identify genes important for podocyte function. Given the central role of podocyte adhesion as a marker of podocyte health, these data are a rich source of candidate regulators of glomerular disease.

PMID: 31566424 [PubMed - as supplied by publisher]



▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄

Neuronal-specific microexon splicing of TAF1 mRNA is directly regulated by SRRM4/nSR100.

Related Articles

Neuronal-specific microexon splicing of TAF1 mRNA is directly regulated by SRRM4/nSR100.

RNA Biol. 2019 Sep 27;:1-13

Authors: Capponi S, Stöffler N, Irimia M, Van Schaik FMA, Ondik MM, Biniossek ML, Lehmann L, Mitschke J, Vermunt MW, Creyghton MP, Graybiel AM, Reinheckel T, Schilling O, Blencowe BJ, Crittenden JR, Timmers HTM

Abstract
Neuronal microexons represent the most highly conserved class of alternative splicing events and their timed expression shapes neuronal biology, including neuronal commitment and differentiation. The six-nt microexon 34' is included in the neuronal form of TAF1 mRNA, which encodes the largest subunit of the basal transcription factor TFIID. In this study, we investigate the tissue distribution of TAF1-34' mRNA and protein and the mechanism responsible for its neuronal-specific splicing. Using isoform-specific RNA probes and antibodies, we observe that canonical TAF1 and TAF1-34' have different distributions in the brain, which distinguish proliferating from post-mitotic neurons. Knockdown and ectopic expression experiments demonstrate that the neuronal-specific splicing factor SRRM4/nSR100 promotes the inclusion of microexon 34' into TAF1 mRNA, through the recognition of UGC sequences in the poly-pyrimidine tract upstream of the regulated microexon. These results show that SRRM4 regulates temporal and spatial expression of alternative TAF1 mRNAs to generate a neuronal-specific TFIID complex.

PMID: 31559909 [PubMed - as supplied by publisher]



▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄

Single-cell transcriptomic profiling of the aging mouse brain.

Related Articles

Single-cell transcriptomic profiling of the aging mouse brain.

Nat Neurosci. 2019 Oct;22(10):1696-1708

Authors: Ximerakis M, Lipnick SL, Innes BT, Simmons SK, Adiconis X, Dionne D, Mayweather BA, Nguyen L, Niziolek Z, Ozek C, Butty VL, Isserlin R, Buchanan SM, Levine SS, Regev A, Bader GD, Levin JZ, Rubin LL

Abstract
The mammalian brain is complex, with multiple cell types performing a variety of diverse functions, but exactly how each cell type is affected in aging remains largely unknown. Here we performed a single-cell transcriptomic analysis of young and old mouse brains. We provide comprehensive datasets of aging-related genes, pathways and ligand-receptor interactions in nearly all brain cell types. Our analysis identified gene signatures that vary in a coordinated manner across cell types and gene sets that are regulated in a cell-type specific manner, even at times in opposite directions. These data reveal that aging, rather than inducing a universal program, drives a distinct transcriptional course in each cell population, and they highlight key molecular processes, including ribosome biogenesis, underlying brain aging. Overall, these large-scale datasets (accessible online at https://portals.broadinstitute.org/single_cell/study/aging-mouse-brain ) provide a resource for the neuroscience community that will facilitate additional discoveries directed towards understanding and modifying the aging process.

PMID: 31551601 [PubMed - in process]



▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄

High-throughput genome-wide phenotypic screening via immunomagnetic cell sorting.

Related Articles

High-throughput genome-wide phenotypic screening via immunomagnetic cell sorting.

Nat Biomed Eng. 2019 Sep 23;:

Authors: Mair B, Aldridge PM, Atwal RS, Philpott D, Zhang M, Masud SN, Labib M, Tong AHY, Sargent EH, Angers S, Moffat J, Kelley SO

Abstract
Genome-scale functional genetic screens are used to identify key genetic regulators of a phenotype of interest. However, the identification of genetic modifications that lead to a phenotypic change requires sorting large numbers of cells, which increases operational times and costs and limits cell viability. Here, we introduce immunomagnetic cell sorting facilitated by a microfluidic chip as a rapid and scalable high-throughput method for loss-of-function phenotypic screening using CRISPR-Cas9. We used the method to process an entire genome-wide screen containing more than 108 cells in less than 1 h-considerably surpassing the throughput achieved by fluorescence-activated cell sorting, the gold-standard technique for phenotypic cell sorting-while maintaining high levels of cell viability. We identified modulators of the display of CD47, which is a negative regulator of phagocytosis and an important cell-surface target for immuno-oncology drugs. The top hit of the screen, the glutaminyl cyclase QPCTL, was validated and shown to modify the N-terminal glutamine of CD47. The method presented could bridge the gap between fluorescence-activated cell sorting and less flexible yet higher-throughput systems such as magnetic-activated cell sorting.

PMID: 31548591 [PubMed - as supplied by publisher]



▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄

Pooled CRISPR-Based Genetic Screens in Mammalian Cells.

Related Articles

Pooled CRISPR-Based Genetic Screens in Mammalian Cells.

J Vis Exp. 2019 Sep 04;(151):

Authors: Chan K, Tong AHY, Brown KR, Mero P, Moffat J

Abstract
Genome editing using the CRISPR-Cas system has vastly advanced the ability to precisely edit the genomes of various organisms. In the context of mammalian cells, this technology represents a novel means to perform genome-wide genetic screens for functional genomics studies. Libraries of guide RNAs (sgRNA) targeting all open reading frames permit the facile generation of thousands of genetic perturbations in a single pool of cells that can be screened for specific phenotypes to implicate gene function and cellular processes in an unbiased and systematic way. CRISPR-Cas screens provide researchers with a simple, efficient, and inexpensive method to uncover the genetic blueprints for cellular phenotypes. Furthermore, differential analysis of screens performed in various cell lines and from different cancer types can identify genes that are contextually essential in tumor cells, revealing potential targets for specific anticancer therapies. Performing genome-wide screens in human cells can be daunting, as this involves the handling of tens of millions of cells and requires analysis of large sets of data. The details of these screens, such as cell line characterization, CRISPR library considerations, and understanding the limitations and capabilities of CRISPR technology during analysis, are often overlooked. Provided here is a detailed protocol for the successful performance of pooled genome-wide CRISPR-Cas9 based screens.

PMID: 31545321 [PubMed - in process]



▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄

Discovering genetic interactions bridging pathways in genome-wide association studies.

Related Articles

Discovering genetic interactions bridging pathways in genome-wide association studies.

Nat Commun. 2019 Sep 19;10(1):4274

Authors: Fang G, Wang W, Paunic V, Heydari H, Costanzo M, Liu X, Liu X, VanderSluis B, Oately B, Steinbach M, Van Ness B, Schadt EE, Pankratz ND, Boone C, Kumar V, Myers CL

Abstract
Genetic interactions have been reported to underlie phenotypes in a variety of systems, but the extent to which they contribute to complex disease in humans remains unclear. In principle, genome-wide association studies (GWAS) provide a platform for detecting genetic interactions, but existing methods for identifying them from GWAS data tend to focus on testing individual locus pairs, which undermines statistical power. Importantly, a global genetic network mapped for a model eukaryotic organism revealed that genetic interactions often connect genes between compensatory functional modules in a highly coherent manner. Taking advantage of this expected structure, we developed a computational approach called BridGE that identifies pathways connected by genetic interactions from GWAS data. Applying BridGE broadly, we discover significant interactions in Parkinson's disease, schizophrenia, hypertension, prostate cancer, breast cancer, and type 2 diabetes. Our novel approach provides a general framework for mapping complex genetic networks underlying human disease from genome-wide genotype data.

PMID: 31537791 [PubMed - in process]



▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄

Rewiring of the Human Mitochondrial Interactome during Neuronal Reprogramming Reveals Regulators of the Respirasome and Neurogenesis.

Related Articles

Rewiring of the Human Mitochondrial Interactome during Neuronal Reprogramming Reveals Regulators of the Respirasome and Neurogenesis.

iScience. 2019 Sep 04;19:1114-1132

Authors: Moutaoufik MT, Malty R, Amin S, Zhang Q, Phanse S, Gagarinova A, Zilocchi M, Hoell L, Minic Z, Gagarinova M, Aoki H, Stockwell J, Jessulat M, Goebels F, Broderick K, Scott NE, Vlasblom J, Musso G, Prasad B, Lamantea E, Garavaglia B, Rajput A, Murayama K, Okazaki Y, Foster LJ, Bader GD, Cayabyab FS, Babu M

Abstract
Mitochondrial protein (MP) assemblies undergo alterations during neurogenesis, a complex process vital in brain homeostasis and disease. Yet which MP assemblies remodel during differentiation remains unclear. Here, using mass spectrometry-based co-fractionation profiles and phosphoproteomics, we generated mitochondrial interaction maps of human pluripotent embryonal carcinoma stem cells and differentiated neuronal-like cells, which presented as two discrete cell populations by single-cell RNA sequencing. The resulting networks, encompassing 6,442 high-quality associations among 600 MPs, revealed widespread changes in mitochondrial interactions and site-specific phosphorylation during neuronal differentiation. By leveraging the networks, we show the orphan C20orf24 as a respirasome assembly factor whose disruption markedly reduces respiratory chain activity in patients deficient in complex IV. We also find that a heme-containing neurotrophic factor, neuron-derived neurotrophic factor [NENF], couples with Parkinson disease-related proteins to promote neurotrophic activity. Our results provide insights into the dynamic reorganization of mitochondrial networks during neuronal differentiation and highlights mechanisms for MPs in respirasome, neuronal function, and mitochondrial diseases.

PMID: 31536960 [PubMed - as supplied by publisher]



▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄

Age- and sex-dependent effects of metformin on neural precursor cells and cognitive recovery in a model of neonatal stroke.

Related Articles

Age- and sex-dependent effects of metformin on neural precursor cells and cognitive recovery in a model of neonatal stroke.

Sci Adv. 2019 Sep;5(9):eaax1912

Authors: Ruddy RM, Adams KV, Morshead CM

Abstract
Resident neural stem and progenitor cells, collectively termed neural precursor cells (NPCs), reside in a well-defined neurogenic niche in the subventricular zone (SVZ) and contribute to ongoing postnatal neurogenesis. It is well established that the NPC niche can alter the behavior of NPCs. NPC activation is a promising therapeutic strategy for brain repair. The drug metformin has been shown to activate neural stem cells, promote differentiation, and lead to functional motor recovery in a neonatal stroke model. We demonstrate that metformin-induced NPC expansion and functional recovery is sex hormone dependent. Metformin increases the size of the NPC pool in adult females, but not males, and promotes cognitive recovery in a model of brain injury in females, but not males. Our data demonstrate that metformin has age- and sex-dependent effects on NPCs that correlate with functional recovery, which has important implications for neural repair.

PMID: 31535024 [PubMed - in process]



▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄