Identification of phagocytosis regulators using magnetic genome-wide CRISPR screens

Phagocytosis is required for a broad range of physiological functions, from pathogen defense to tissue homeostasis, but the mechanisms required for phagocytosis of diverse substrates remain incompletely understood. Here, we developed a rapid magnet-based phenotypic screening strategy, and performed eight genome-wide CRISPR screens in human cells to identify genes regulating phagocytosis of distinct substrates. After validating select hits in focused miniscreens, orthogonal assays and primary human macrophages, we show that (1) the previously uncharacterized gene NHLRC2 is a central player in phagocytosis, regulating RhoA-Rac1 signaling cascades that control actin polymerization and filopodia formation, (2) very-long-chain fatty acids are essential for efficient phagocytosis of certain substrates and (3) the previously uncharacterized Alzheimer’s disease–associated gene TM2D3 can preferentially influence uptake of amyloid-β aggregates. These findings illuminate new regulators and core principles of phagocytosis, and more generally establish an efficient method for unbiased identification of cellular uptake mechanisms across diverse physiological and pathological contexts.Eight genome-wide CRISPR screens identify genes required for substrate-specific phagocytosis. The study highlights roles for NHLRC2 in filopodia formation, very-long-chain fatty acids in substrate-specific phagocytosis and TM2D3 in uptake of amyloid-β aggregates.

[1]  Jun S. Liu,et al.  MAGeCK enables robust identification of essential genes from genome-scale CRISPR/Cas9 knockout screens , 2014, Genome Biology.

[2]  Yilong Li,et al.  Genome-wide recessive genetic screening in mammalian cells with a lentiviral CRISPR-guide RNA library , 2013, Nature Biotechnology.

[3]  J. Danska,et al.  SLAMF7 is critical for phagocytosis of haematopoietic tumour cells via Mac-1 integrin , 2017, Nature.

[4]  Michael C. Bassik,et al.  CRISPR-Cas9 screens in human cells and primary neurons identify modifiers of C9orf72 dipeptide repeat protein toxicity , 2017, bioRxiv.

[5]  J. Sulston,et al.  Mutations affecting programmed cell deaths in the nematode Caenorhabditis elegans. , 1983, Science.

[6]  E. van Goethem,et al.  Requirement for a Drosophila E3-ubiquitin ligase in phagocytosis of apoptotic cells. , 2007, Immunity.

[7]  Mary A. Logan,et al.  Negative regulation of glial engulfment activity by Draper terminates glial responses to axon injury , 2012, Nature Neuroscience.

[8]  B. Barres,et al.  Why is Wallerian degeneration in the CNS so slow? , 2007, Annual review of neuroscience.

[9]  P. O’Farrell,et al.  Identification of Drosophila Gene Products Required for Phagocytosis of Candida albicans , 2005, PLoS biology.

[10]  K. Ravichandran,et al.  Phagocytosis of apoptotic cells in homeostasis , 2015, Nature Immunology.

[11]  Thomas M. Norman,et al.  A Multiplexed Single-Cell CRISPR Screening Platform Enables Systematic Dissection of the Unfolded Protein Response , 2016, Cell.

[12]  W. Talbot,et al.  The Rag-Ragulator Complex Regulates Lysosome Function and Phagocytic Flux in Microglia. , 2016, Cell reports.

[13]  R. Bernards,et al.  High-Throughput Functional Genetic and Compound Screens Identify Targets for Senescence Induction in Cancer. , 2017, Cell reports.

[14]  Martin Kampmann,et al.  Integrated platform for genome-wide screening and construction of high-density genetic interaction maps in mammalian cells , 2013, Proceedings of the National Academy of Sciences.

[15]  Tony Pawson,et al.  Phosphoinositide 3-kinase enables phagocytosis of large particles by terminating actin assembly through Rac/Cdc42 GTPase-activating proteins , 2015, Nature Communications.

[16]  N. Gauthier,et al.  Squeezing in a Meal: Myosin Functions in Phagocytosis. , 2019, Trends in cell biology.

[17]  D. Morgens,et al.  Systematic comparison of CRISPR-Cas9 and RNAi screens for essential genes , 2016, Nature Biotechnology.

[18]  M. Freeman,et al.  Distinct molecular pathways mediate glial activation and engulfment of axonal debris after axotomy , 2012, Nature Neuroscience.

[19]  K. Ravichandran,et al.  Evidence for a Conserved Role for CrkII and Rac in Engulfment of Apoptotic Cells* , 2001, The Journal of Biological Chemistry.

[20]  Shiyou Zhu,et al.  High-throughput screening of a CRISPR/Cas9 library for functional genomics in human cells , 2014, Nature.

[21]  S. Grinstein,et al.  How nascent phagosomes mature to become phagolysosomes. , 2012, Trends in immunology.

[22]  James E. DiCarlo,et al.  RNA-Guided Human Genome Engineering via Cas9 , 2013, Science.

[23]  S. Gordon.  Phagocytosis: An Immunobiologic Process. , 2016, Immunity.

[24]  I. Miinalainen,et al.  NHLRC2 variants identified in patients with fibrosis, neurodegeneration, and cerebral angiomatosis (FINCA): characterisation of a novel cerebropulmonary disease , 2018, Acta Neuropathologica.

[25]  Horvitz,et al.  Genes required for the engulfment of cell corpses during programmed cell death in Caenorhabditis elegans. , 1991, Genetics.

[26]  T. Swigut,et al.  Selective silencing of euchromatic L1s revealed by genome-wide screens for L1 regulators , 2017, Nature.

[27]  Ash A. Alizadeh,et al.  Anti-CD47 Antibody Synergizes with Rituximab to Promote Phagocytosis and Eradicate Non-Hodgkin Lymphoma , 2010, Cell.

[28]  Norbert Perrimon,et al.  Drosophila RNAi Screen Reveals CD36 Family Member Required for Mycobacterial Infection , 2005, Science.

[29]  E. Lander,et al.  Genetic Screens in Human Cells Using the CRISPR-Cas9 System , 2013, Science.

[30]  J. Ting,et al.  Arp2/3 Complex Is Required for Macrophage Integrin Functions but Is Dispensable for FcR Phagocytosis and In Vivo Motility. , 2017, Developmental cell.

[31]  Joel A Swanson,et al.  Dynamics of cytoskeletal proteins during Fcgamma receptor-mediated phagocytosis in macrophages. , 2002, Molecular biology of the cell.

[32]  T. Maniatis,et al.  An RNA-Sequencing Transcriptome and Splicing Database of Glia, Neurons, and Vascular Cells of the Cerebral Cortex , 2014, The Journal of Neuroscience.

[33]  L. Jungbauer,et al.  Preparing synthetic Aβ in different aggregation states. , 2011, Methods in molecular biology.

[34]  M. Sixt,et al.  Multiple roles of filopodial dynamics in particle capture and phagocytosis and phenotypes of Cdc42 and Myo10 deletion , 2017, The Journal of Biological Chemistry.

[35]  J. Jongstra,et al.  Leukocyte-specific protein 1 (LSP1) , 2006, Immunologic research.

[36]  C. Kocks,et al.  Eater, a Transmembrane Protein Mediating Phagocytosis of Bacterial Pathogens in Drosophila , 2005, Cell.

[37]  A. Kihara.  Very long-chain fatty acids: elongation, physiology and related disorders. , 2012, Journal of biochemistry.

[38]  Sean R. Collins,et al.  Using light to shape chemical gradients for parallel and automated analysis of chemotaxis , 2015, Molecular systems biology.

[39]  L. Garver,et al.  The peptidoglycan recognition protein PGRP-SC1a is essential for Toll signaling and phagocytosis of Staphylococcus aureus in Drosophila. , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[40]  B. Stevens,et al.  New insights on the role of microglia in synaptic pruning in health and disease , 2016, Current Opinion in Neurobiology.

[41]  Chun Jimmie Ye,et al.  A Genome-wide CRISPR Screen in Primary Immune Cells to Dissect Regulatory Networks , 2015, Cell.

[42]  Xia Zhang,et al.  Measuring Opsonic Phagocytosis via Fcγ Receptors and Complement Receptors on Macrophages , 2011, Current protocols in immunology.

[43]  M. Hengartner,et al.  CED-12/ELMO, a Novel Member of the CrkII/Dock180/Rac Pathway, Is Required for Phagocytosis and Cell Migration , 2001, Cell.

[44]  A. Hofman,et al.  Rare Functional Variant in TM2D3 is Associated with Late-Onset Alzheimer's Disease , 2016, PLoS genetics.

[45]  S. Grinstein,et al.  Phagocytosis: receptors, signal integration, and the cytoskeleton , 2014, Immunological reviews.

[46]  Erin E. Gill,et al.  A Genome-Wide Knockout Screen in Human Macrophages Identified Host Factors Modulating Salmonella Infection , 2019, mBio.

[47]  P. Robinson,et al.  A rapid Percoll gradient procedure for preparation of synaptosomes , 2008, Nature Protocols.

[48]  Jennifer Doudna,et al.  RNA-programmed genome editing in human cells , 2013, eLife.

[49]  Michael T. McManus,et al.  A Systematic Mammalian Genetic Interaction Map Reveals Pathways Underlying Ricin Susceptibility , 2013, Cell.

[50]  S. Grinstein,et al.  Diversity and Versatility of Phagocytosis: Roles in Innate Immunity, Tissue Remodeling, and Homeostasis , 2017, Front. Cell. Infect. Microbiol..

[51]  Joel A. Swanson,et al.  Shaping cups into phagosomes and macropinosomes , 2008, Nature Reviews Molecular Cell Biology.

[52]  S. Shirasawa,et al.  ROS-induced cleavage of NHLRC2 by caspase-8 leads to apoptotic cell death in the HCT116 human colon cancer cell line , 2017, Cell Death & Disease.

[53]  W. Norton,et al.  Isolation of Myelin , 2006, Current protocols in cell biology.

[54]  J. Larrick,et al.  Characterization of a human macrophage-like cell line stimulated in vitro: a model of macrophage functions. , 1980, Journal of immunology.

[55]  M. Rämet,et al.  Functional genomic analysis of phagocytosis and identification of a Drosophila receptor for E. coli , 2002, Nature.

[56]  Brian Burke,et al.  A promiscuous biotin ligase fusion protein identifies proximal and interacting proteins in mammalian cells , 2012, The Journal of cell biology.

[57]  L. Borish,et al.  Apoptotic cell clearance by bronchial epithelial cells critically influences airway inflammation , 2012, Nature.

[58]  J. Settleman,et al.  Integrin signaling through Arg activates p190RhoGAP by promoting its binding to p120RasGAP and recruitment to the membrane. , 2006, Molecular biology of the cell.

[59]  M. Tyers,et al.  Expression of the protein kinase C substrate pleckstrin in macrophages: association with phagosomal membranes. , 1999, Journal of immunology.

[60]  B. Ozenberger,et al.  β-Amyloid Peptide-induced Apoptosis Regulated by a Novel Protein Containing a G Protein Activation Module* , 2001, The Journal of Biological Chemistry.

[61]  Neville E. Sanjana,et al.  Genome-Scale CRISPR-Cas9 Knockout Screening in Human Cells , 2014, Science.

[62]  Stephen J. Smith,et al.  Astrocytes mediate synapse elimination through MEGF10 and MERTK pathways , 2013, Nature.

[63]  C. Abrams,et al.  Pleckstrin Induces Cytoskeletal Reorganization via a Rac-dependent Pathway* , 1999, The Journal of Biological Chemistry.

[64]  Gaelen T. Hess,et al.  Genome-scale measurement of off-target activity using Cas9 toxicity in high-throughput screens , 2017, Nature Communications.

[65]  Le Cong,et al.  Multiplex Genome Engineering Using CRISPR/Cas Systems , 2013, Science.