Highly multiplexed and quantitative cell-surface protein profiling using genetically barcoded antibodies.

Human cells express thousands of different surface proteins that can be used for cell classification, or to distinguish healthy and disease conditions. A method capable of profiling a substantial fraction of the surface proteome simultaneously and inexpensively would enable more accurate and complete classification of cell states. We present a highly multiplexed and quantitative surface proteomic method using genetically barcoded antibodies called phage-antibody next-generation sequencing (PhaNGS). Using 144 preselected antibodies displayed on filamentous phage (Fab-phage) against 44 receptor targets, we assess changes in B cell surface proteins after the development of drug resistance in a patient with acute lymphoblastic leukemia (ALL) and in adaptation to oncogene expression in a Myc-inducible Burkitt lymphoma model. We further show PhaNGS can be applied at the single-cell level. Our results reveal that a common set of proteins including FLT3, NCR3LG1, and ROR1 dominate the response to similar oncogenic perturbations in B cells. Linking high-affinity, selective, genetically encoded binders to NGS enables direct and highly multiplexed protein detection, comparable to RNA-sequencing for mRNA. PhaNGS has the potential to profile a substantial fraction of the surface proteome simultaneously and inexpensively to enable more accurate and complete classification of cell states

Cone-Rod Dystrophy Due to Mutations in a Novel Photoreceptor-Specific Homeobox Gene (CRX) Essential for Maintenance of the Photoreceptor

Genes associated with inherited retinal degeneration have been found to encode proteins required for phototransduction, metabolism, or structural support of photoreceptors. Here we show that mutations in a novel photoreceptor-specific homeodomain transcription factor gene (CRX) cause an autosomal dominant form of cone-rod dystrophy (adCRD) at the CORD2 locus on chromosome 19q13. In affected members of a CORD2-linked family, the highly conserved glutamic acid at the first position of the recognition helix is replaced by alanine (E80A). In another CRD family, a 1 bp deletion (E168 [delta1 bp]) within a novel sequence, the WSP motif, predicts truncation of the C-terminal 132 residues of CRX. Mutations in the CRX gene cause adCRD either by haploinsufficiency or by a dominant negative effect and demonstrate that CRX is essential for the maintenance of mammalian photoreceptorsThis work was supported by the RP Foundation of Canada (R. R. M.), the Foundation Fighting Blindness (R. R. M. and S. G. J.), the Canadian Genetic Disease Network (R. R. M. and A. D.), the Medical Research Council of Canada (R. R. M.), The Wellcome Trust (043825/Z/95) and the Human Genome Mapping Resource Centre (C. Y. G.-E. and S. S. B.), the Howard Hughes Medical Institute and NIH R01 EY0 8064 (C. L. C.), the Canadian Genome Analysis and Technology Genome Resource Facility (S. W. S. and L.-C. T.), the NIH/NEI (EY05627) (S. G. J.), and the Greek National Scholarship Foundation (M. P.). R. R. M. and L.-C. T. are International Research Scholars of the Howard Hughes Medical Institute

Transcription factor PRDM8 is required for rod bipolar and type 2 OFF-cone bipolar cell survival and amacrine subtype identity

Retinal bipolar (BP) cells mediate the earliest steps in image processing in the visual system, but the genetic pathways that regulate their development and function are incompletely known. We identified PRDI-BF1 and RIZ homology domain containing 8 (PRDM8) as a highly conserved transcription factor that is abundantly expressed in mouse retina. During development and in maturity, PRDM8 is expressed strongly in BP cells and a fraction of amacrine and ganglion cells. To determine whether Prdm8 is essential to BP cell development or physiology, we targeted the gene in mice. Prdm8(EGFP/EGFP) mice showed nonprogressive b-wave deficits on electroretinograms, consistent with compromised BP cell function or circuitry resembling the incomplete form of human congenital stationary night blindness (CSNB). BP cell specification was normal in Prdm8(EGFP/EGFP) retina as determined by VSX2(+) cell numbers and retinal morphology at postnatal day 6. BP subtype differentiation was impaired, however, as indicated by absent or diminished expression of BP subtype-specific markers, including the putative PRDM8 regulatory target PKCα (Prkca) and its protein. By adulthood, rod bipolar (RB) and type 2 OFF-cone bipolar (CB) cells were nearly absent from Prdm8-null mice. Although no change was detected in total amacrine cell (AC) numbers, increased PRKCA(+) and cholinergic ACs and decreased GABAergic ACs were seen, suggesting an alteration in amacrine subtype identity. These findings establish that PRDM8 is required for RB and type 2 OFF-CB cell survival and amacrine subtype identity, and they present PRDM8 as a candidate gene for human CSNB

Rom-1 is required for rod photoreceptor viability and regulation of disk morphogenesis

The homologous membrane proteins Rom-1 and peripherin-2 are localized to the disk rims of photoreceptor outer segments (OSs), where they associate as tetramers and larger oligomers1, 2, 3. Disk rims are thought to be critical for disk morphogenesis, OS renewal4 and the maintenance of OS structure5, but the molecules which regulate these processes are unknown. Although peripherin-2 is known to be required for OS formation (because Prph2−/− mice do not form OSs; ref. 6), and mutations in RDS (the human homologue of Prph2) cause retinal degeneration7, the relationship of Rom-1 to these processes is uncertain. Here we show that Rom1−/− mice form OSs in which peripherin-2 homotetramers are localized to the disk rims, indicating that peripherin-2 alone is sufficient for both disk and OS morphogenesis. The disks produced in Rom1−/− mice were large, rod OSs were highly disorganized (a phenotype which largely normalized with age) and rod photoreceptors died slowly by apoptosis. Furthermore, the maximal photoresponse of Rom1−/− rod photoreceptors was lower than that of controls. We conclude that Rom-1 is required for the regulation of disk morphogenesis and the viability of mammalian rod photoreceptors, and that mutations in human ROM1 may cause recessive photoreceptor degeneration