Biology subjects

Fowler, D.

Publications and source records attributed to Fowler, D..

13 recordsLinked to original sources

bioRxiv2026

High-resolution measurement of ERK activation state reveals determinants of BRAF regulation and drug sensitivity

High-resolution measurements of pathway activity make it possible to characterize how signaling responds to changing cellular conditions, to evaluate pathway-targeted inhibitors, and to dissect how sequence variation shapes signaling output. Here, we describe a sequencing-based assay that uses engineered, phospho-state-selective affinity reagents to quantify ERK activation state as a high-resolution, ratiometric readout of RAS/ERK MAPK pathway activity. Profiling ~1,600 BRAF variants at basal and elevated RAS-GTP levels allowed us to assign activity scores to somatic cancer and germline RASopathy variants and to distinguish residues that stabilize autoinhibition from those required for active dimer formation. We identified a large class of dominant negative variants that suppress signaling below endogenous levels through enhanced RAS engagement and an inability to form productive RAF dimers. Measuring the intracellular potency of the RAF inhibitor LXH254 against ~1,000 variants in parallel revealed hinge and activation-loop positions conferring resistance and showed that disrupting the BRAF-MEK interface is broadly sensitizing.

cell biology↗

bioRxiv2026

Embryo-scale Visual Cell Sorting reveals a conserved transcriptomic signature of nucleolar size linked to proteostasis

Nucleoli, nuclear speckles and other compartments regulate transcription, RNA processing, and chromatin organization within the nucleus, yet the relationship of their morphology to developmental gene expression programs in vivo is poorly understood. Here, we develop a high-throughput Visual Cell Sorting (VCS) workflow for fixed cells and nuclei that combines antibody-based photoconversion; GPU-accelerated, real-time image analysis; and three-level single-cell combinatorial indexing RNA-seq (sci-RNA-seq3) to link nuclear compartment morphology to single-nucleus transcriptomes at embryo scale. We use VCS to analyze and sort over 1 million mouse embryo-derived nuclei by nucleolar, nuclear speckle, or nuclear size and construct a transcriptional atlas annotated with nuclear compartment phenotypes. Nuclear compartment size varies both between and within lineages and is shaped by proliferation and differentiation. In extracellular matrix protein-producing cell types such as fibroblasts, chondrocytes, and osteoblasts, nucleolar enlargement is uncoupled from cell cycle, and in erythroid cells exhibit a sharp nucleolar contraction preceding cell-cycle exit. We identify a 41-gene transcriptional signature whose expression tracks nucleolar size, enriched for ribosome biogenesis, mitochondrial metabolism, unfolded protein response, stress granule, and ubiquitin-proteasome pathway components. We used this nucleolar transcriptional signature to annotate mouse, zebrafish and human developmental atlases with nucleolar size information, revealing a conserved coupling between nucleolar activity and proteostasis programs. Our work establishes Visual Cell Sorting as a scalable platform for mapping image-based phenotypes to molecular programs; details the relationship between nuclear compartment phenotypes and development; and provides a transcriptional signature to estimate nucleolar size from existing single-cell datasets.

genomics↗

bioRxiv2026

Postnatal Reprogramming Shapes Human Intestinal Epithelial Immune Competency

At birth, the intestine must rapidly adapt to enable nutritional function and immune microbial tolerance. Here, integrating single-cell multi-omics and spatial transcriptomics we define the circuits underpinning this process. We identify asynchronous developmental trajectories with postnatal epithelial reprogramming characterised by coordinated changes in metabolism, junctional structure and innate defence. At birth epithelial stem cells demonstrate dynamic enhancer remodelling, with accessibility often preceding transcription. Fetal stemness elements remain accessible despite reduced transcription across epithelial lineages, retaining plasticity potential. Post-natal epithelia experience sequential homing of myeloid cells followed by innate T cells with peri-epithelial B cells localising later in infancy. Using developmentally staged organoids, we show that epithelial responses to inflammatory stimuli are age-dependent and constrained in early life. We identify BHLHE40 as an early-life regulator that attenuates the impact of interferon- and NF-{kappa}B-driven signalling. Altogether we define the events driving epithelial licensing and barrier adaptation at birth and through infancy.

cell biology↗

bioRxiv2024

Multiplexed, multimodal profiling of the intracellular activity, interactions, and druggability of protein variants using LABEL-seq

Multiplexed assays of variant effect are powerful tools for assessing the impact of protein sequence variation, but are limited to measuring a single protein property and often rely on indirect readouts of intracellular protein function. Here, we developed LAbeling with Barcodes and Enrichment for biochemicaL analysis by sequencing (LABEL-seq), a platform for the multimodal profiling of thousands of protein variants in cultured human cells. Multimodal measurement of [~]20,000 variant effects for [~]1,600 BRaf variants using LABEL-seq revealed that variation at positions that are frequently mutated in cancer had minimal effects on folding and intracellular abundance but could dramatically alter activity, protein-protein interactions, and druggability. Integrative analysis of our multimodal measurements identified networks of positions with similar roles in regulating BRafs signaling properties and enabled predictive modeling of variant effects on complex processes such as cell proliferation and small molecule-promoted degradation. LABEL-seq provides a scalable approach for the direct measurement of multiple biochemical effects of protein variants in their native cellular context, yielding insight into protein function, disease mechanisms, and druggability.

molecular biology↗

bioRxiv2024

A missense variant effect map for the human tumour suppressor protein CHK2

The tumour suppressor CHEK2 encodes the serine/threonine protein kinase CHK2 which, upon DNA damage, is important for pausing the cell cycle, initiating DNA repair and inducing apoptosis. CHK2 phosphorylation of the tumour suppressor BRCA1 is also important for mitotic spindle assembly and chromosomal stability. Consistent with its cell cycle checkpoint role, both germline and somatic variants in CHEK2 have been linked to breast and multiple other cancer types. Over 90% of clinical germline CHEK2 missense variants are classified as variants of uncertain significance, complicating diagnosis of CHK2-dependent cancer. We therefore sought to test the functional impact of all possible missense variants in CHK2. Using a scalable multiplexed assay based on the ability of human CHK2 to complement DNA sensitivity of a S. cerevisiae lacking its ortholog RAD53, we generated a systematic missense variant effect map for CHEK2 missense variation. Map scores reflect known biochemical features of CHK2 and exhibit good performance in separating pathogenic from benign clinical missense variants. Thus, the missense variant effect map for CHK2 offers value in understanding both known and yet-to-be-observed CHK2 variants.

genetics↗

bioRxiv2023

Pseudotime analysis for time-series single-cell sequencing and imaging data

Many single-cell RNA-sequencing studies have collected time-series data to investigate transcriptional changes concerning various notions of biological time, such as cell differentiation, embryonic development, and response to stimulus. Accordingly, several unsupervised and supervised computational methods have been developed to construct single-cell pseudotime embeddings for extracting the temporal order of transcriptional cell states from these time-series scRNA-seq datasets. However, existing methods, such as psupertime, suffer from low predictive accuracy, and this problem becomes even worse when we try to generalize to other data types such as scATAC-seq or microscopy images. To address this problem, we propose Sceptic, a support vector machine model for supervised pseudotime analysis. Whereas psupertime employs a single joint regression model, Sceptic simultaneously trains multiple classifiers with separate score functions for each time point and also allows for non-linear kernel functions. Sceptic first generates a probability vector for each cell and then aims to predict chronological age via conditional expectation. We demonstrate that Sceptic achieves significantly improved prediction power (accuracy improved by 1.4 - 38.9%) for six publicly available scRNA-seq data sets over state-of-the-art methods, and that Sceptic also works well for single-nucleus image data. Moreover, we observe that the pseudotimes assigned by Sceptic show stronger correlations with nuclear morphology than the observed times, suggesting that these pseudotimes accurately capture the heterogeneity of nuclei derived from a single time point and thus provide more informative time labels than the observed times. Finally, we show that Sceptic accurately captures sex-specific differentiation timing from both scATAC-seq and scRNA-seq data.

genomics↗

bioRxiv2023

Antigen perception in T cells by long-term Erk and NFAT signaling dynamics.

Immune system threat detection hinges on T cells ability to perceive varying peptide major-histocompatibility complex (pMHC) antigens. As the Erk and NFAT pathways link T cell receptor engagement to gene regulation, their signaling dynamics may convey information about pMHC inputs. To test this idea, we developed a dual reporter mouse strain and a quantitative imaging assay that, together, enable simultaneous monitoring of Erk and NFAT dynamics in live T cells over day-long timescales as they respond to varying pMHC inputs. Both pathways initially activate uniformly across various pMHC inputs, but diverge only over longer (9+ hrs) timescales, enabling independent encoding of pMHC affinity and dose. These late signaling dynamics are decoded via multiple temporal and combinatorial mechanisms to generate pMHC-specific transcriptional responses. Our findings underscore the importance of long timescale signaling dynamics in antigen perception, and establish a framework for understanding T cell responses under diverse contexts. SIGNIFICANCE STATEMENTTo counter diverse pathogens, T cells mount distinct responses to varying peptide-major histocompatibility complex ligands (pMHCs). They perceive the affinity of pMHCs for the T cell receptor (TCR), which reflects its foreignness, as well as pMHC abundance. By tracking signaling responses in single living cells to different pMHCs, we find that T cells can independently perceive pMHC affinity vs dose, and encode this information through the dynamics of Erk and NFAT signaling pathways downstream of the TCR. These dynamics are jointly decoded by gene regulatory mechanisms to produce pMHC-specific activation responses. Our work reveals how T cells can elicit tailored functional responses to diverse threats and how dysregulation of these responses may lead to immune pathologies.

immunology↗

bioRxiv2022

OPS-γδ: allogeneic opsonin-secreting γδT cell immunotherapy for solid tumours mediates direct and bystander immunity

T cell-based cancer immunotherapy has typically relied on membrane-bound cytotoxicity enhancers such as chimeric antigen receptors expressed in autologous {beta}T cells. These approaches are limited by tonic signalling of synthetic constructs and costs associated with manufacture of bespoke patient products. {gamma}{delta}T cells are an emerging alternative chassis for cellular therapy, possessing innate anti-tumour activity, potent antibody-dependent cytotoxicity (ADCC) and minimal alloreactivity. We present an immunotherapeutic platform technology built around the V{gamma}9V{delta}2 {gamma}{delta}T cell chassis, harnessing specific characteristics of this cell type and offering an allo-compatible means of delivering cellular therapy that recruits bystander immunity. We engineered {gamma}{delta}T cells to secrete synthetic opsonins and stabilized IL15 (stIL15). Using GD2 as a model antigen we show how opsonin-secreting V{gamma}9V{delta}2 (OPS-{gamma}{delta}) have enhanced cytotoxicity and also confer this benefit on lymphoid and myeloid bystander cells. Reflecting the secreted nature of the engineered efficacy modules, the entire product rather than just the gene-modified fraction exhibited enhanced activation and cytotoxic profiles, superior persistence and proliferative capacity even upon repeated tumour challenge. Secretion of stIL15 abrogated the need for exogenous cytokine supplementation during expansion and further mediated functional licensing of bystander NK cells. Compared to unmodified {gamma}{delta}T cells, stIL15-OPS-{gamma}{delta} cells exhibited superior in-vivo control of subcutaneous tumour and persistence in the blood. stIL15-OPS-{gamma}{delta} cells were further efficacious in 3D patient-derived osteosarcoma models, where efficacy could be boosted with the addition of immunomodulatory aminobisphosphonate drug, zoledronic acid. Together the data identify stIL15-OPS-{gamma}{delta} cells as a novel allogeneic platform combining direct cytolysis with bystander activation to effect solid tumour control. One Sentence SummaryArmoured, opsonin-secreting OPS-{gamma}{delta} cell immunotherapy is built on the innate strengths of the V{gamma}9V{delta}2 cell chassis for allogeneic solid tumour targeting.

immunology↗

bioRxiv2022

A versatile, chemically-controlled DNA binding switch enables temporal modulation of Cas9-based effectors

CRISPR-Cas9s RNA-guided genome targeting ability has been leveraged to develop a plethora of effectors including targeted transcriptional activators, DNA base editors, and DNA prime editors. Although systems for inducibly modulating Cas9 activity have been developed, current approaches for conferring temporal control require extensive screening of functional protein components. A simpler and general strategy for conferring temporal control over diverse Cas9-based effector activities is needed. Here we describe a versatile chemically-controlled and rapidly-activated DNA binding Cas9 module (ciCas9) that is able to confer temporal control over a variety of Cas9-based effectors. Using the ciCas9 module, we engineer temporally-controlled cytidine and adenine DNA base editors. We employ the ciCas9 base editors to reveal that in vivo bystander editing kinetics occurs via a dependent process where editing at a preferred nucleotide position increases the frequency of edits at a second nucleotide within a target site. Finally, we demonstrate the versatility of the ciCas9 module by creating a temporally-controlled transcriptional activator, a dual cytidine and adenine base editor, and a prime editor.

molecular biology↗

bioRxiv2021

Profiling of the drug resistance of thousands of Src tyrosine kinase mutants uncovers a regulatory network that couples autoinhibition to the dynamics of the catalytic domain

Kinase inhibitors are effective cancer therapies but resistance often limits clinical efficacy. Despite the cataloguing of numerous resistance mutations, our understanding of kinase inhibitor resistance is still incomplete. Here, we comprehensively profiled the resistance of [~]3500 Src tyrosine kinase mutants to four different ATP-competitive inhibitors. We found that ATP-competitive inhibitor resistance mutations are distributed throughout Srcs catalytic domain. In addition to inhibitor contact residues, residues that participate in regulating Srcs phosphotransferase activity were prone to the development of resistance. Unexpectedly, we found that a resistance-prone cluster of residues located on the top face of the N-terminal lobe of Srcs catalytic domain contributes to autoinhibition by reducing catalytic domain dynamics, and mutations in this cluster led to resistance by lowering inhibitor affinity and promoting kinase hyperactivation. Together, our studies demonstrate how drug resistance profiling can be used to define potential resistance pathways and uncover new mechanisms of kinase regulation.

biochemistry↗

bioRxiv2021

The Forget-Me-Not dHCP study: 7 Tesla high resolution diffusion imaging in the unfixed post-mortem neonatal brain

Diffusion MRI of the neonatal brain allows investigation of the organisational structure of maturing fibres during brain development. Post-mortem imaging has the potential to achieve high resolution by using long scan times, enabling precise assessment of small structures. The Forget-Me-Not study, part of the Developing Human Connectome Project (dHCP), aims to acquire and publicly distribute high-resolution diffusion MRI data for unfixed post-mortem neonatal brain at 7T with a custom-built head coil. This paper describes how the study addressed logistical, technical and ethical challenges relating to recruitment pipeline, care pathway, tissue preservation, scan setup and protocol optimisation. Results from the first subject recruited to the study demonstrate high-quality diffusion MRI data. Preliminary voxel-wise and tractography-based analyses are presented for the cortical plate, subplate and white matter pathways, with comparison to age-matched in vivo dHCP data. These results demonstrate that high quality post-mortem data can be acquired and provide a sensitive means to explore the developing human brain, as well as altered diffusion properties consistent with post-mortem changes, at high resolution.

bioengineering↗

bioRxiv2020

High-throughput discovery of trafficking-deficient variants in the cardiac potassium channel KCNH2

BackgroundKCHN2 encodes the KV11.1 potassium channel responsible for IKr, a major repolarization current during the cardiomyocyte action potential. Variants in KCNH2 that decrease IKr can cause Type 2 Long QT syndrome, usually due to mistrafficking to the cell surface. Accurately discriminating between variants with normal and abnormal trafficking would help clinicians identify and treat individuals at risk of a major cardiac event. The volume of reported non-synonymous KCNH2 variants preclude the use of conventional electrophysiologic methods for functional study. ObjectiveTo report a high-throughput, multiplexed screening method for KCNH2 genetic variants capable of measuring the cell surface abundance of hundreds of missense variants in KCNH2. MethodsWe develop a method to quantitate KCNH2 variant trafficking on a pilot region of 11 residues in the S5 helix, and generate trafficking scores for 220/231 missense variants in this region. ResultsFor 5/5 variants, high-throughput trafficking scores validated when tested in single variant flow cytometry and confocal microscopy experiments. We additionally compare our results with planar patch electrophysiology and find that loss-of-trafficking variants do not produce IKr, but that some variants which traffic normally may still be functionally compromised. ConclusionsHere, we describe a new method for detecting trafficking-deficient variants in KCNH2 in a multiplexed assay. This new method accurately generates trafficking data for variants in KCNH2 and can be readily extended to all residues in Kv11.1 and to other cell surface proteins. CLINICAL IMPLICATIONSHundreds of KCNH2 variants have been observed to date, and thousands more will be found as clinical and population sequencing efforts become increasingly widespread. The major mechanism of KV11.1 loss of function is misfolding and failure to traffic to the cell surface. Deep mutational scanning of KCNH2 trafficking is a scalable, high-throughput method that can help identify new loss of function variants and decipher the large number of KCNH2 variants being found in the population.

genetics↗

bioRxiv2019

Visual Cell Sorting: A High-throughput, Microscope-based Method to Dissect Cellular Heterogeneity

Microscopy is a powerful tool for characterizing complex cellular phenotypes, but linking these phenotypes to genotype or RNA expression at scale remains challenging. Here, we present Visual Cell Sorting, a method that physically separates hundreds of thousands of live cells based on their visual phenotype. Visual Cell Sorting uses automated imaging and phenotypic analysis to direct selective illumination of Dendra2, a photoconvertible fluorescent protein expressed in live cells; these photoactivated cells are then isolated using fluorescence-activated cell sorting. First, we use Visual Cell Sorting to assess the effect of hundreds of nuclear localization sequence variants in a pooled format, identifying variants that improve nuclear localization and enabling annotation of nuclear localization sequences in thousands of human proteins. Second, we use Visual Cell Sorting to recover cells that retain normal nuclear morphologies after paclitaxel treatment, then derive their single cell transcriptomes to identify multiple pathways associated with paclitaxel resistance in human cancers. Unlike alternative methods, Visual Cell Sorting depends on inexpensive reagents and commercially available hardware. As such, it can be readily deployed to uncover the relationships between visual cellular phenotypes and internal states, including genotypes and gene expression programs.

molecular biology↗