Biology subjects

Carlson, R. J.

Publications and source records attributed to Carlson, R. J..

6 recordsLinked to original sources

bioRxiv2026

The accessible human surfaceome

Proteins associated with the extracellular face of the plasma membrane ("surface proteins" or the "surfaceome") are important therapeutic targets because they can direct therapeutics to specific cell types and deliver them into cells. However, experimental and computational databases disagree substantially on which proteins make up the human plasma membrane proteome, and none focus specifically on the set of accessible surface proteins that would be useful for therapeutic applications. Here, we used an agentic LLM pipeline to curate the accessible human surfaceome from literature. To assess performance, we built SurfaceBench, a manually curated benchmark of 147 proteins on which five reference databases frequently disagree. Our LLM triage agent classified surface status more accurately than existing databases and recovered 1,108 candidate surface proteins not present in any of them. On held-out clinical and viral-entry surface targets, the pipeline recovered 98-100% of established surface antigens. A literature-anchored deep-dive pipeline run on over 5,000 candidates then returned 1,783 canonical surface proteins and 1,252 with weaker or context-dependent surface evidence. Each queryable record contains 24 LLM-based annotation fields--including confidence, primary localization, and expression breadth--and seven deterministic structural and topological features. Unlike existing databases, our records trace every claim to the agent's reasoning and to exact quotes from the full-text literature it could access. All records can be browsed in an open-source viewer. These results show that LLMs reasoning over unstructured scientific text can build updatable, bespoke databases that outperform expert-curated resources, although performance remains limited by agent access to full-text literature.

bioinformatics↗

bioRxiv2024

Classification and functional characterization of regulators of intracellular STING trafficking identified by genome-wide optical pooled screening

STING is an innate immune sensor that traffics across many cellular compartments to carry out its function of detecting cyclic di-nucleotides and triggering defense processes. Mutations in factors that regulate this process are often linked to STING-dependent human inflammatory disorders. To systematically identify factors involved in STING trafficking, we performed a genome-wide optical pooled screen and examined the impact of genetic perturbations on intracellular STING localization. Based on subcellular imaging of STING protein and trafficking markers in 45 million cells perturbed with sgRNAs, we defined 464 clusters of gene perturbations with similar cellular phenotypes. A higher-dimensional focused optical pooled screen on 262 perturbed genes which assayed 11 imaging channels identified 73 finer phenotypic clusters. In a cluster containing USE1, a protein that mediates Golgi to ER transport, we found a gene of unknown function, C19orf25. Consistent with the known role of USE1, loss of C19orf25 enhanced STING signaling. Other clusters contained subunits of the HOPS, GARP and RIC1-RGP1 complexes. We show that HOPS deficiency delayed STING degradation and consequently increased signaling. Similarly, GARP/RIC1-RGP1 loss increased STING signaling by delaying STING exit from the Golgi. Our findings demonstrate that genome-wide genotype-phenotype maps based on high-content cell imaging outperform other screening approaches, and provide a community resource for mining for factors that impact STING trafficking as well as other cellular processes observable in our dataset.

systems biology↗

bioRxiv2024

Single-cell image-based genetic screens systematically identify regulators of Ebola virus subcellular infection dynamics

Ebola virus (EBOV) is a high-consequence filovirus that gives rise to frequent epidemics with high case fatality rates and few therapeutic options. Here, we applied image-based screening of a genome-wide CRISPR library to systematically identify host cell regulators of Ebola virus infection in 39,085,093 million single cells. Measuring viral RNA and protein levels together with their localization in cells identified over 998 related host factors and provided detailed information about the role of each gene across the virus replication cycle. We trained a deep learning model on single-cell images to associate each host factor with predicted replication steps, and confirmed the predicted relationship for select host factors. Among the findings, we showed that the mitochondrial complex III subunit UQCRB is a post-entry regulator of Ebola virus RNA replication, and demonstrated that UQCRB inhibition with a small molecule reduced overall Ebola virus infection with an IC50 of 5 M. Using a random forest model, we also identified perturbations that reduced infection by disrupting the equilibrium between viral RNA and protein. One such protein, STRAP, is a spliceosome-associated factor that was found to be closely associated with VP35, a viral protein required for RNA processing. Loss of STRAP expression resulted in a reduction in full-length viral genome production and subsequent production of non-infectious virus particles. Overall, the data produced in this genome-wide high-content single-cell screen and secondary screens in additional cell lines and related filoviruses (MARV and SUDV) revealed new insights about the role of host factors in virus replication and potential new targets for therapeutic intervention.

microbiology↗

bioRxiv2022

ESCRT-dependent STING degradation curtails steady-state and cGAMP-induced signaling

STING is an intracellular sensor of cyclic di-nucleotides involved in response to pathogen- or self-derived DNA that induces protective immunity, or if dysregulated, autoimmunity. STING trafficking is tightly linked to its activity. We aimed to systematically characterize genes regulating STING trafficking and to define their impact on STING responses. Based on proximity-ligation proteomics and genetic screens, an ESCRT complex containing HGS, VPS37A and UBAP1 was found to be required for STING degradation and signaling shutdown. Analogous to phosphorylated STING creating a platform for IRF3 recruitment, oligomerization-driven STING ubiquitination by UBE2N formed a platform for ESCRT recruitment at the endosome, responsible for STING signaling shutdown. A UBAP1 mutant that underlies human spastic paraplegia and disrupts ESCRT function led to STING-dependent type I IFN responses at the steady-state, defining ESCRT as a homeostatic regulator of STING signaling.

immunology↗

bioRxiv2019

IRAK1-mediated coincidence detection of microbial signals licenses inflammasome activation

The innate immune system signals through various higher order signaling complexes called supramolecular organizing centers (SMOCs), which typically organize components of a single pathway. While innate immune signaling pathways have been largely characterized using single receptor stimuli, responses to pathogens require the coordinated engagement of multiple pathways. Here, we report an IRAK1-containing SMOC formed specifically when multiple receptors are activated, which recruits select components of the TLR, MAPK and inflammasome pathways. This allows for signal flux redistribution from TLRs to inflammasomes and facilitates inflammasome licensing through an MKK7-JNK axis, which is defective in Irak1-/- mice. Furthermore, this defect in Irak1-/- mice manifests in increased susceptibility to inflammasome-sensitive pathogens and diminished IL1 production from inflammasomes after co-TLR priming. Thus, IRAK1 SMOCs form a multi-pathway coordinating hub for coincidence detection of microbial signals, which may be employed by innate immune cells as a threat assessment and thresholding mechanism for inflammasome activation.

immunology↗

bioRxiv2018

Pooled optical screens in human cells

Large-scale genetic screens play a key role in the systematic discovery of genes underlying cellular phenotypes. Pooling of genetic perturbations greatly increases screening throughput, but has so far been limited to screens of enrichments defined by cell fitness and flow cytometry, or to comparatively low-throughput single cell gene expression profiles. Although microscopy is a rich source of spatial and temporal information about mammalian cells, high-content imaging screens have been restricted to much less efficient arrayed formats. Here, we introduce an optical method to link perturbations and their phenotypic outcomes at the singlecell level in a pooled setting. Barcoded perturbations are read out by targeted in situ sequencing following image-based phenotyping. We apply this technology to screen a focused set of 952 genes across >3 million cells for involvement in NF-{kappa}B activation by imaging the translocation of RelA (p65) to the nucleus, recovering 20 known pathway components and 3 novel candidate positive regulators of IL-1{beta} and TNF-stimulated immune responses.

genomics↗