Biology subjects

Chapman, K. B.

Publications and source records attributed to Chapman, K. B..

5 recordsLinked to original sources

bioRxiv2026

Localization accuracy in single-molecule fluorescence microscopy

The two prevalent methods to determine the position of individual fluorophores in the focal plane of a microscope rely either on the centroid of the fluorescence pattern produced by the fluorophore on a camera or on targeting the fluorophore's coordinate with a spatially controlled illumination minimum, as realized in MINFLUX. In both methods, (sub)nanometer localization precisions have been reached, but little is known about their accuracy, i.e., the deviation of the obtained coordinate from its ground truth. Here we report a theoretical and experimental comparison of the accuracy provided by these two strategies. We quantify substantial systematic errors in camera-based localization due to the interplay of two difficult-to-control parameters: the orientation of the fluorophore in space and its axial deviation from the focal plane (defocus). For example, within an axial range of {+/-}120 nm around the focus, the median distance between two fluorophores on a nanobody measured by camera-based localization varies by 26.5 nm. Under the same conditions, a commercial MINFLUX system yields a median variation in distance of 1.4 nm, indicating an improvement in accuracy by an order of magnitude. The accuracy of MINFLUX localization can be further improved to (sub-)[a]ngstrom levels by reducing the average distance between the coordinates targeted by the illumination minimum and the fluorophore. As it delivers unrivaled precision and accuracy for mapping fluorophores and their distances, we consider coordinate-targeted localization essential for studying labeled cells at the sub-10 nm level and for exploring structural and conformational changes of labeled biomolecules.

biophysics↗

bioRxiv2025

Lamin B1 Safeguards the B Cell Genome and Shapes Lymphoma Outcome

Lamin B1 is a structural component of the nuclear lamina that participates in diverse cellular processes, including genome regulation and cellular senescence. During adaptive immune responses, B lymphocytes in germinal centres (GCs) undergo clonal expansion and programmed DNA damage at immunoglobulin loci, while simultaneously downregulating Lamin B1. Likewise, Lamin B1 downregulation has been observed in GC-derived lymphomas and myeloid malignancies, yet the functional consequences of Lamin B1 loss during B cell development remain poorly understood. Here, we used in vivo and in vitro B cell models of conditional hypomorphic expression of Lamin B1, which showed elevated DNA damage, altered chromatin accessibility, and disrupted transcriptional profiles. Using sBLISS (in situ labelling and sequencing of double-strand breaks), we identified non-random double-strand break hotspots in both mouse and human GC B cells, depleted of Lamin B1. These breaks are preferentially located near transcriptional start sites (TSSs) and regulatory elements that control translation and mRNA fate, implicating Lamin B1 in protecting fragile regulatory regions. Moreover, low LMNB1 expression correlated with poor clinical outcomes in patients with diffuse large B cell lymphoma (DLBCL). Together, this study reveals a crucial role for Lamin B1 in preserving genomic stability in B cells, underscoring its impact on the pathogenesis of B cell-derived malignancies.

cancer biology↗

bioRxiv2022

Multiplexed transcriptome discovery of RNA binding protein binding sites by antibody-barcode eCLIP

UV cross-linking and immunoprecipitation (CLIP) methodologies enable the identification of RNA binding sites of RNA-binding proteins (RBPs). Despite improvements in the library preparation of RNA fragments, the current enhanced CLIP (eCLIP) protocol requires 4 days of hands-on time and lacks the ability to process many RBPs in parallel. We present a new method termed antibody-barcode eCLIP (ABC) that utilizes DNA-barcoded antibodies and proximity ligation of the DNA oligonucleotides to RBP-protected RNA fragments to interrogate multiple RBPs simultaneously. We observe performance comparable to eCLIP with the advantage of a reduced hands-on time of 2 days and dramatically increased scaling while minimizing sample-to-sample variation and maintaining the same material requirement of a single eCLIP experiment.

genomics↗

bioRxiv2022

Scalable and deep profiling of mRNA targets for individual microRNAs with chimeric eCLIP

Our expanding knowledge of the roles small regulatory RNAs play across numerous areas of biology, coupled with the promise of RNA-targeted therapies and small RNA-based medicines, create an urgent need for tools that can accurately identify and quantify small RNA:target interactions at scale. MicroRNAs (miRNA) are a major class of small RNAs in plants and animals. The experimental capture of miRNA:mRNA interactions by ligation into chimeric RNA fragments in chimeric CrossLinking and ImmunoPrecipitation (CLIP) provides a direct readout of miRNA targets with high-throughput sequencing. Despite the power of this approach, widespread adoption of chimeric CLIP has been slow due to both methodological technical complexity as well as limited recovery of chimeric molecules (particularly beyond the most abundant miRNAs). Here we describe chimeric eCLIP, in which we integrate a chimeric ligation step into AGO2 eCLIP to enable chimeric read recovery. We show that removal of the cumbersome polyacrylamide gel and nitrocellulose membrane transfer step common to CLIP techniques can be omitted for chimeric AGO2 eCLIP to create a simplified high throughput version of the assay that maintains high signal- to-noise. With the increased yield of recovered miRNA:mRNA interactions in no-gel chimeric eCLIP, we show that simple enrichment steps using either PCR or on-bead probe capture can be added to chimeric eCLIP in order to target and enrich libraries for chimeric reads specific to one or more miRNAs of interest in both cell lines and tissue samples, resulting in 30- to 175-fold increases in recovery of chimeric reads for miRNAs of interest. We further demonstrate that the same probe-capture approach can be used to recover miRNA interactions for a targeted gene of interest, revealing both distinct miRNA targeting as well as co-targeting by several miRNAs from the same seed family. RNA-seq analysis of gene expression following miRNA overexpression confirmed miRNA-mediated repression of chimeric eCLIP-identified targets and indicated that probe-enriched chimeric eCLIP can provide additional sensitivity to detect regulated targets among genes that either contain or lack computationally predicted miRNA target sites. Thus, we believe that chimeric eCLIP will be a useful tool for quantitative profiling of miRNA targets in varied sample types at scale, and for revealing a deeper picture of regulatory networks for specific miRNAs of biological interest. HighlightsO_LINo-gel chimeric eCLIP improves recovery of miRNA:mRNA interactions by 70-fold C_LIO_LIProbe- and PCR-enrichment deeply profiles mRNA targets of miRNAs of interest C_LIO_LIChimeric eCLIP targets experimentally identify non-computationally predicted interactions C_LIO_LIIncreased depth recovers [~]6 million miRNA:target chimeras in HEK293T C_LI

genomics↗

bioRxiv2021

Differential Expression of α,β, and γ Protocadherin Isoforms During Differentiation, Aging, and Cancer

The cadherin family of cell surface glycoproteins plays a fundamental role in cell-cell recognition, thereby participating in diverse biological process such as embryonic morphogenesis and oncogenic transformation. The subset of clustered protocadherin (PCDH) genes generated from the , {beta}, and {gamma} loci, have been widely studied for their potential role in neuronal cell-cell recognition and neurogenesis, however their broader role in normal embryonic development and cancer has not been examined in detail. We utilized human embryonic stem (hES) cells to model early human development in vitro, comparing PCDH isoform transcription in diverse types of embryonic progenitors with normal adult-derived and cancer counterparts. Embryonic progenitors express genes from the and {beta} cluster at levels comparable to that seen in the CNS, while fetal and adult-derived cells express primarily from the {gamma} cluster. Replicative senescence left fibroblasts with markedly lower expression of all isoforms. We observe that an embryonic pattern of clustered protocadherin gene expression and associated CpG island methylation is commonly associated with cancer cell lines from diverse tissue types. The differential regulation of the , {beta}, and {gamma} loci coincide with alternate regions of DNA accessibility at CTCF binding sites and lamina-associated domains and CPL expression correlated with the expression of LMNA and LMNB1. These observations support a potential role for the differential regulation of genes within the clustered protocadherin locus in selective cell-cell adhesion during embryogenesis, regeneration, cancer and aging.

developmental biology↗