Biology subjects

Klemm, S.

Publications and source records attributed to Klemm, S..

10 recordsLinked to original sources

bioRxiv2026

Repeated malaria exposure reprograms Vδ2+ γδT cell chromatin accessibility and immune function toward a reversible hyporesponsive state

V{delta}2+ {gamma}{delta} T cells contribute to antimalarial immunity via pro-inflammatory and cytotoxic mechanisms. Among children who have experienced repeated episodes of malaria, V{delta}2+ T cells develop reduced pro-inflammatory function through mechanisms that remain unknown. We hypothesized that recurrent malaria drives coordinated epigenetic and transcriptional remodeling of these semi-innate cells. Using PBMC from Ugandan children in cohorts spanning high and low transmission (10 children) and longitudinally across a marked decline in transmission (20 children, 3 timepoints), we profiled the epigenome and transcriptome of V{delta}2+ T cells by paired bulk ATAC-seq and RNA-seq and by CITE-seq. Higher malaria exposure was associated with reduced V{delta}2+ T cell TNF and IFN{gamma} production and with thousands of differentially accessible chromatin regions. Immunoregulatory genes including LYN, PLCG2, and HAVCR2 (Tim-3) showed increased chromatin accessibility and/or expression at high exposure, whereas pro-inflammatory gene (IFNG, GZMK, IL7R) and AP-1 (JUN/FOS) motif accessibility were increased at low exposure; linkage of regulatory elements with target genes corroborated these associations. Single-cell transcriptional analysis identified a cytotoxic CD8+CD16+CD57+ V{delta}2+ T cell subset. In our in vitro model, repeated stimulation with malaria-infected red blood cells or the phosphoantigen HMBPP was similarly associated with reduced V{delta}2+ T cell degranulation and cytokine production. Diminished responsiveness was predominantly TCR-dependent, accompanied by heightened p38 signaling, and was partially reversed by rest induced by the tyrosine kinase inhibitor dasatinib. Together these data reveal how repeated malaria may reshape cellular immunity by reprogramming V{delta}2+ T cells toward a reversible, hyporesponsive state and highlight targets for therapeutically restoring their function.

immunology↗

bioRxiv2025

Sensitive, direct detection of non-coding off-target base editor unwinding and editing in primary cells

Base editors create precise nucleotide changes in DNA, but their off-target activity remains challenging to quantify. Here, we develop and deploy a direct, in cellulo sequencing assay that simultaneously measures both Cas9-mediated unwinding and deaminase editing of genomic DNA (beCasKAS). Our strategy nominates >460-fold more potential off-target sites than other methods by enriching for Cas9-dependent R-loops immediately preceding editing. Using beCasKAS in primary human T-cells, we observe that mRNA-encoded ABE8e and PAMless ABE8e-SpRY base editors have distinct off-target profiles that can be mitigated by optimizing mRNA dose. Finally, we combine beCasKAS with base-resolution deep learning models to risk-stratify off-target edits by their likelihood of epigenetic dysregulation. Collectively, beCasKAS offers a sensitive and facile tool to optimize the balance between base editor on- and off-target activity.

genomics↗

bioRxiv2025

An emergent disease-associated motor neuron state precedes cell death in a mouse model of ALS

To uncover molecular determinants of motor neuron degeneration and selective vulnerability in amyotrophic lateral sclerosis (ALS), we generated longitudinal single-nucleus transcriptomes and chromatin accessibility profiles of spinal motor neurons from the SOD1-G93A ALS mouse model. Vulnerable alpha motor neurons showed thousands of molecular changes, marking a transition into a novel cell state we named disease-associated motor neurons (DAMNs). We identified transcription factor regulatory networks that govern how healthy cells transition into DAMNs as well as those linked to vulnerable and resistant motor neuron subtypes. Using spatial transcriptomics, we found reactive glia located near motor neurons early in disease, suggesting early signaling events between motor neurons and glia. Finally, we found that the human orthologs of genomic regions with differential accessibility in SOD1-G93A alpha motor neurons are enriched for single nucleotide polymorphisms associated with human ALS, providing evidence that the genetic underpinnings of motor neuron vulnerability are conserved.

neuroscience↗

bioRxiv2024

IQD2 recruits KLCR1 to the membrane-microtubule nexus to promote cytoskeletal mechano-responsiveness in leaf epidermis pavement cells

Plant cells experience a variety of mechanical stresses from both internal and external sources, including turgor pressure, mechanical strains arising from heterogeneous growth between neighboring cells, and environmental factors like touch from soil, rain, or wind [1,2]. These stresses serve as signals at the cell-, tissue- and organismal level to coordinate plant growth during development and stress responses [3]. In plants, the physical cell wall-plasma membrane-microtubule continuum is proposed to be integral in transducing mechanical signals from the exterior to intracellular components [4-6]. Cortical microtubules (CMTs) rapidly reorient in response to mechanical stress to align with the maximal tensile stress direction [7,8]. Several studies proposed that CMTs themselves may act as stress sensors; the precise mechanisms involved in the regulation of CMTs and the modes of sensing, however, are still not clearly understood. Here, we show that IQD2 and KLCR1 are enriched at CMTs in proximity to the plasma membrane. IQD2, which is a bona fide microtubule-associated protein, promotes microtubule localization of KLCR1. By combining cross-linking mass spectrometry (XL-MS) and computational modeling with structure-function studies, we present first experimental insights into the composition and structure of IQD2-KLCR1 complexes. Further, we demonstrate that the IQD2-KLCR1 module is a positive regulator of microtubule mechano-responses in pavement cells. Collectively, our work identifies the IQD2-KLCR1 module as novel regulator of mechanostress-mediated CMT reorientation and provides a framework for future mechanistic studies aimed at a functional dissection of mechanotransduction at the plasma membrane-CMT interface during growth and plant morphogenesis. HighlightsO_LIIQD2 and KLCR1 localize to the plasma membrane-microtubule nexus C_LIO_LIIQD2 is required for efficient microtubule targeting of KLCR1 in planta C_LIO_LIIQD2 physically interacts with KLCR1 and microtubules C_LIO_LIThe IQD2-KLCR1 module promotes mechano-stress induced microtubule reorganization C_LI

plant biology↗

bioRxiv2022

Alleviating cell lysate-induced inhibition to enable RT-PCR from single cells in picoliter-volume double emulsion droplets

Microfluidic droplet assays enable single-cell PCR and sequencing assays at unprecedented scale, with most methods encapsulating cells within nanoliter sized single emulsion droplets (water-in-oil). Encapsulating cells within picoliter double emulsion (DE) (water-in-oil-in-water) droplets allows sorting on commercially available FACS machines, making it possible to isolate single cells based on phenotypes of interest for downstream sequencing and PCR analyses. However, DE droplets must be within the range of large cells (<20 pl) for sorting on standard cytometers, posing challenges for molecular biology as prior reports suggest that reverse transcription (RT) and PCR amplification cannot proceed efficiently at volumes below 1 nL due to cell lysate-induced inhibition. To overcome this limitation, we used a plate-based RT-PCR assay designed to mimic reactions in picoliter droplets to systematically quantify and ameliorate the inhibition. We find that RT-PCR is blocked by lysate-induced cleavage of nucleic acid probes and primers, but that this cleavage can be efficiently alleviated through heat lysis that simultaneously inactivates inhibitory lysate components. We further show that the magnitude of RT-PCR inhibition depends strongly on cell type, but that RT-PCR can proceed in low-picoscale reaction volumes for most cell lines tested. Finally, we demonstrate one-step RT-PCR from single cells in 20 pL double emulsion droplets with fluorescence detectable via FACS. These results open up exciting new avenues for improving picoscale droplet RT-PCR reactions and expanding microfluidic droplet based single-cell analysis technologies.

bioengineering↗

bioRxiv2020

Single-cell transcriptomic analysis of the adult mouse spinal cord

The spinal cord is a fascinating structure responsible for coordinating all movement in vertebrates. Spinal motor neurons control the activity of virtually every organ and muscle throughout the body by transmitting signals that originate in the spinal cord. These neurons are remarkably heterogeneous in their activity and innervation targets. However, because motor neurons represent only a small fraction of cells within the spinal cord and are difficult to isolate, the full complement of motor neuron subtypes remains unknown. Here we comprehensively describe the molecular heterogeneity of motor neurons within the adult spinal cord. We profiled 43,890 single-nucleus transcriptomes using fluorescence-activated nuclei sorting to enrich for spinal motor neuron nuclei. These data reveal a transcriptional map of the adult mammalian spinal cord and the first unbiased characterization of all transcriptionally distinct autonomic and somatic spinal motor neuron subpopulations. We identify 16 sympathetic motor neuron subtypes that segregate spatially along the spinal cord. Many of these subtypes selectively express specific hormones and receptors, suggesting neuromodulatory signaling within the autonomic nervous system. We describe skeletal motor neuron heterogeneity in the adult spinal cord, revealing numerous novel markers that distinguish alpha and gamma motor neurons--cell populations that are specifically affected in neurodegenerative disease. We also provide evidence for a novel transcriptional subpopulation of skeletal motor neurons. Collectively, these data provide a single-cell transcriptional atlas for investigating motor neuron diversity as well as the cellular and molecular basis of motor neuron function in health and disease.

neuroscience↗

bioRxiv2019

Optimized double emulsion flow cytometry with high-throughput single droplet isolation

Droplet microfluidics has made large impacts in diverse areas such as enzyme evolution, chemical product screening, polymer engineering, and single-cell analysis. However, while droplet reactions have become increasingly sophisticated, phenotyping droplets by a fluorescent signal and sorting them to isolate variants-of-interest remains a field-wide bottleneck. Here, we present an optimized double emulsion workflow, sdDE-FACS, that enables high-throughput phenotyping, selection, and sorting of droplets using standard flow cytometers. Using a 130 m nozzle, we demonstrate robust post-sort recovery of intact droplets, with little to no shear-induced droplet breakage, at high sort frequency (12-14 kHz) across two industry-standard FACS instruments. We report the first quantitative plate statistics for double emulsion droplet isolation and demonstrate single droplet recovery with >70% efficiency. In addition, we establish complete downstream recovery of nucleic acids from single, sorted double emulsion droplets, an advance in droplet sorting comparable with the capabilities of single-cell FACS. This work resolves several hurdles in the field of high-throughput droplet analysis and paves the way for a variety of new droplet assays, including rare variant isolation and multiparameter single-cell analysis, marrying the full power of flow cytometry with droplet microfluidics.

bioengineering↗

bioRxiv2019

A single cell framework for multi-omic analysis of disease identifies malignant regulatory signatures in mixed phenotype acute leukemia

In order to identify the molecular determinants of human diseases, such as cancer, that arise from a diverse range of tissue, it is necessary to accurately distinguish normal and pathogenic cellular programs.1-3 Here we present a novel approach for single-cell multi-omic deconvolution of healthy and pathological molecular signatures within phenotypically heterogeneous malignant cells. By first creating immunophenotypic, transcriptomic and epigenetic single-cell maps of hematopoietic development from healthy peripheral blood and bone marrow mononuclear cells, we identify cancer-specific transcriptional and chromatin signatures from single cells in a cohort of mixed phenotype acute leukemia (MPAL) clinical samples. MPALs are a high-risk subtype of acute leukemia characterized by a heterogeneous malignant cell population expressing both myeloid and lymphoid lineage-specific markers.4, 5 Our results reveal widespread heterogeneity in the pathogenetic gene regulatory and expression programs across patients, yet relatively consistent changes within patients even across malignant cells occupying diverse portions of the hematopoietic lineage. An integrative analysis of transcriptomic and epigenetic maps identifies 91,601 putative gene-regulatory interactions and classifies a number of transcription factors that regulate leukemia specific genes, including RUNX1-linked regulatory elements proximal to CD69. This work provides a template for integrative, multi-omic analysis for the interpretation of pathogenic molecular signatures in the context of developmental origin.

genomics↗

bioRxiv2018

High-throughput chromatin accessibility profiling at single-cell resolution

We have developed a high-throughput single-cell ATAC-seq (assay for transposition of accessible chromatin) method to measure physical access to DNA in whole cells. Our approach integrates fluorescence imaging and addressable reagent deposition across a massively parallel (5184) nano-well array, yielding a nearly 20-fold improvement in throughput (up to ~1800 cells/chip, 4-5 hour on-chip processing time) and cost (~98{cents} per cell) compared to prior microfluidic implementations. We applied this method to measure regulatory variation in Peripheral Blood Mononuclear Cells (PBMCs) and show robust, de-novo clustering of single cells by hematopoietic cell type.

genomics↗

bioRxiv2018

Microtubule-associated protein IQ67 DOMAIN5 regulates interdigitation of leaf pavement cells in Arabidopsis thaliana

Plant microtubules form a highly dynamic intracellular network with important roles for regulating cell division, cell proliferation and cell morphology. Its organization and dynamics are coordinated by various microtubule-associated proteins (MAPs) that integrate environmental and developmental stimuli to fine-tune and adjust cytoskeletal arrays. IQ67 DOMAIN (IQD) proteins recently emerged as a class of plant-specific MAPs with largely unknown functions. Here, using a reverse genetics approach, we characterize Arabidopsis IQD5 in terms of its expression domains, subcellular localization and biological functions. We show that IQD5 is expressed mostly in vegetative tissues, where it localizes to cortical microtubule arrays. Our phenotypic analysis of iqd5 loss-of-function lines reveals functions of IQD5 in pavement cell (PC) shape morphogenesis, as indicated by reduced interdigitation of neighboring cells in the leaf epidermis of iqd5 mutants. Histochemical analysis of cell wall composition further suggests reduced rates of cellulose deposition in anticlinal cell walls, which correlate with reduced asymmetric expansion. Lastly, we provide evidence for IQD5-dependent recruitment of calmodulin calcium sensors to cortical microtubule arrays. Our work thus identifies IQD5 as a novel player in PC shape regulation, and, for the first time, links calcium signaling to developmental processes that regulate multi-polar growth in PCs.\n\nHighlightMicrotubule-localized Arabidopsis IQ67 DOMAIN5 regulates pavement cell morphogenesis in the leaf epidermis and links calcium-calmodulin signaling to lobe initiation and asymmetric expansion during early phases of interdigitated cell growth.

plant biology↗