Biology subjects

Herr, A.

Publications and source records attributed to Herr, A..

8 recordsLinked to original sources

bioRxiv2026

Aiptasia larvae are phenotypically validated as a model of coral bleaching using high-throughput machine-learning image analysis

The sea anemone Aiptasia is a model system for understanding cnidarian loss of symbiotic algae under heat stress (bleaching). While Aiptasia polyps have been widely used to study this process, accurate symbiosis phenotyping grapples with discordant length scales: fine spatial resolution (~100 um) is needed across a whole organism (~5 mm). To address this, we consider small (~100 um), optically transparent Aiptasia larvae as a bleaching model suitable for whole-organism phenotyping by fluorescence microscopy with larvae classified as symbiotic when algae are localized within gastrodermal cells. To expedite phenotyping, we introduce a machine-learning (ML) image-analysis pipeline (SYMPHONY) designed for single-larva resolution analysis of intact larvae. SYMPHONY efficiently identifies the cellular location of internalized algae (accuracy: 79%, precision: 82%, recall: 79%, F1 score: 79%; training dataset composed of 1611 total objects). Additionally, SYMPHONY reports statistically significant larval bleaching under heat stress and corroborates manual phenotyping results, while significantly reducing operator labor from hours to minutes. The combination of the Aiptasia larvae model and the SYMPHONY pipeline aims to accelerate our understanding of symbiosis breakdown.

bioengineering↗

bioRxiv2025

Joint Mapping of Chromatin Accessibility and Targeted Proteomics in HER2-expressing Breast Cancer Systems

HER2 proteoforms promote therapeutic resistance and aggressiveness in HER2-positive breast cancer, yet their epigenetic consequences remain poorly defined. Here, we establish EpiBlot, a joint assay incorporating a customized plateATAC-seq workflow that minimizes sample inputs with single-cell western blotting to concurrently profile chromatin accessibility with protein and proteoform expression. We applied our method to engineered MCF7 cells expressing HER2 proteoforms - full-length p185HER2 or truncated 611-CTF -, where we evaluated the impact of such proteoforms on the epigenetic and protein profiles after lapatinib or doxorubicin exposure. Expression of 611-CTF elicits pervasive chromatin remodeling, whereas p185HER2 provokes only modest accessibility shifts under the same treatments. EpiBlot reveals that treatment with doxorubicin drives extensive genome-wide accessibility changes, while lapatinib treatment produces limited global effects but unmasks proteoform-specific responses. Concordance between chromatin accessibility and protein abundance is moderate, underscoring complex regulatory coupling. Extending this dual-modality approach to HER2-low patient-derived organoids uncovers distinct chromatin states and reveals a subpopulation of triple-negative breast-cancer cells expressing truncated HER2 proteoforms. We anticipate that EpiBlot will highlight the value of multimodal profiling with proteoform identification for dissecting tumor heterogeneity and therapeutic response in cancer. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=198 SRC="FIGDIR/small/678463v2_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@673263org.highwire.dtl.DTLVardef@11d28c5org.highwire.dtl.DTLVardef@15fa336org.highwire.dtl.DTLVardef@1d4426b_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2025

Paired Analyses of Nuclear Protein Targets and Genomic DNA by Single-Cell Western Blot and Single-Cell PCR

Single-cell multimodal assays measure multiple layers of molecular information. Existing single-cell tools have limited capability to analyze nuclear proteins and genomic DNA from the same originating single cell. To address this gap, we designed and developed a microfluidic single-cell assay (SplitBlot), that pairs measurements of genomic DNA (PCR-based) and nucleo-cytoplasmic proteins (nuclear histone H3 and cytoplasmic beta-actin). To accomplish this paired multiomic measurement, we utilize microfluidic precision to fractionate protein molecules (both nuclear and cytoplasmic) from genomic DNA (nuclear). We create a fractionation axis that prepends a comet-like encapsulation of genomic DNA in an agarose molded microwell to a downstream single-cell western blot in polyacrylamide gel (PAG). For single-cell genomic DNA analysis, the agarose-encapsulated DNA is physically extracted from the microfluidic device for in-tube PCR, after release of genomic DNA from a molten agarose pallet (86% of pallets resulted in amplification of TurboGFP). For protein analysis, nucleo-cytoplasmic proteins are photocaptured to the PAG (via benzophenone) and probed in-situ (15 kDa histone H3 resolved from 42 kDa beta-actin with a separation resolution Rs = 0.77, CV = 76%). The SplitBlot reported the amplification of TurboGFP DNA and the separation of nuclear histone H3 and cytoplasmic beta-actin from the same single U251 cells engineered to express TurboGFP. Demonstrated here, Split-Blot offers the capacity for precision genomic DNA vs. protein fractionation for subsequent split workflow consisting of in-tube PCR and on-chip single-cell western blotting, thus providing a tool for pairing genotype to nuclear and cytoplasmic protein expression at the single-cell level. TOC Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/646125v1_ufig1.gif" ALT="Figure 1"> View larger version (17K): org.highwire.dtl.DTLVardef@168541corg.highwire.dtl.DTLVardef@69ff17org.highwire.dtl.DTLVardef@ea4fa1org.highwire.dtl.DTLVardef@111daad_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2025

Single-cell differential detergent fractionation for detection of cytokeratin 8 proteoforms

Differential detergent fractionation (DDF) enables compartment-specific lysis, offering a strategy to analyze cytoplasmic proteins while preserving the nucleus for downstream assays. However, while this method facilitates sub-cellular resolution, current single-cell approaches using DDF remain limited in their ability to identify proteoforms without compromising nuclear integrity. This limitation is especially pronounced in proteins where their proteoforms present diverse biological functions such as cytokeratin 8 (CK8), a structural protein implicated in several disease states. Here, we present a single-cell western blot (scWB) integrated with DDF to selectively solubilize and separate CK8 proteoforms while preserving nuclear integrity. To evaluate assay applicability and nuclear stability, we profiled CK8 across breast cancer cell lines (MCF7, SKBR3, and MDA-MB-231), confirming proteoform detection only in MCF7 and preservation of nuclear content across all lines. We report on assay development, including screening a panel of lysis buffers based on nonionic detergents, and electrophoresis conditions to achieve a separation resolution between two proteoforms of up to 0.94, while preserving an intact nucleus. The cytoplasm-specific lysis (DDF buffer) yielded detectable proteoforms in 14.3% of solubilized cells, comparable to 10.3% with whole-cell lysis (RIPA buffer). Our approach allows for tailored solubilization, achieving reliable proteoform detection and nuclear retention across different cell types. Proteoform profiling at the single-cell level forms a basis for the exploration of the role of specific CK8 molecular forms in cellular processes.

bioengineering↗

bioRxiv2024

Electromigration of Charged Analytes Through Immiscible Fluids in Multiphasic Electrophoresis

Multiphasic buffer systems have been of greatest interest in electrophoresis and liquid-liquid electrotransfer; this study extends that foundation by exploring the interplay of the geometric and viscous properties of an interleaving oil layer on the electrotransfer of a charged analyte from an aqueous solution into a hydrogel. We utilized finite element analysis to examine two complementary configurations: one being electrotransfer of a charged analyte (protein) in an aqueous phase into a surrounding hydrogel layer and another being electrotransfer of the protein from that originating aqueous phase - through an interleaving oil layer of predetermined viscosity and thickness - and into a surrounding hydrogel layer. Results indicate that the presence of an oil layer leads to increased skew of the injected peak. To explain this difference in injection dispersion, we utilize Probsteins framework and compare the Peclet (Pe) number with the ratio between length scales characteristic to the axial and radial dispersion, respectively. The formulation assigns electrotransfer conditions into six different dispersion regimes. We show that the presence or absence of an interleaving oil layer moves the observed peak dispersion into distinct electrotransfer regimes; the presence of an oil layer augments the electrophoretic mobility mismatch between the different phases, resulting in a five-fold increase in Pe and a six-fold increase in the ratio between the axial to radial dispersion characteristic lengths. We further show that oil viscosity significantly influences resultant injection dispersion. A decrease in oil-layer viscosity from 0.08 Pa{middle dot}s to 0.02 Pa{middle dot}s results in a >100% decrease in injection dispersion. Our theoretical predictions were experimentally validated by comparing the electrotransfer regimes of three different mineral oil samples. We show that lowering the oil viscosity to 0.0039 Pa{middle dot}s results in an injection regime similar to that of the absence of an oil layer. Additionally, we measure the migration distance and show that average electromigration velocity over the transit duration is inversely proportional to the viscosity of an interleaving oil layer. Understanding of the impact of electrotransfer of charged species across multiple immiscible fluid layers on peak dispersion informs the design of multiphasic electrophoresis systems.

bioengineering↗

bioRxiv2023

Contact-Inhibited ERK Signaling is Determined by Cellular-Resolution Western Blotting

Extracellular signal-regulated kinase (ERK) signaling is essential to regulated cell behaviors, including cell proliferation, differentiation, and apoptosis. The influence of cell-cell contacts on ERK signaling is central to epithelial cells, yet few studies have sought to understand the same in cancer cells, particularly with single-cell resolution. To acquire same-cell measurements of both phenotypic (cell-contact state) and targeted-protein profile (ERK phosphorylation), we prepend high-content, whole-cell imaging prior to endpoint cellular-resolution western blot analyses for each of hundreds of individual HeLa cancer cells cultured on that same chip, which we call contactBlot. By indexing the phosphorylation level of ERK in each cell or cell-cluster to the imaged cell-contact state, we compare ERK signaling between isolated and in-contact cells. We observe attenuated ([~]2x) ERK signaling in HeLa cells which are in-contact versus isolated. Attenuation is sustained when the HeLa cells are challenged with hyperosmotic stress. Our findings show the impact of cell-cell contacts on ERK activation with isolated and in-contact cells, while introducing a multi omics tool for control and scrutiny of cell-cell interactions.

bioengineering↗

bioRxiv2023

Dissecting Mechanisms of Ligand Binding and Conformational Changes in the Glutamine-Binding Protein

The glutamin-binding protein GlnBP is part of an ATP-binding cassette transporter system in E. coli and uses two well-characterized conformational states, an open ligand-free and a closed-liganded state, to facilitate active amino-acid uptake. Existing literature on its ligand binding mechanism lacked sufficient evidence to univocally assign the kinetic type of binding mechanism for GlnBP: ligand binding prior to conformational change, i.e., an induced fit or the conformational selection, in which the ligand binds the matching conformation from a pre-existing ensemble. Since such mechanistic questions are relevant for our fundamental understanding of how this and other biomacromolecules regulate cellular processes, we here revisit the question for GlnBP. We present a biochemical and biophysical analysis using a combination of calorimetry, single-molecule and surface-plasmon resonance spectroscopy and molecular dynamics simulations. We found that both apo- and holo-GlnBP show no detectable exchange between open and (semi-)closed conformations on timescales between 100 ns and 10 ms and that ligand binding and conformational changes in GlnBP are correlated. A global analysis of our experimental results suggests that the conformational selection model is only compatible with GlnBP for the extreme scenario of very fast conformational exchange between the open and closed states on timescales <100 ns. In contrast all data remains compatible with an induced-fit mechanism, where the ligand binds GlnBP prior to conformational rearrangements. Importantly, our work demonstrates that it is an intricate task to identify the type of kinetic binding mechanism and that this requires not only a sufficient set of data, but also an integrative experimental and theoretical framework to address the question. Based on this concept, we propose that various protein systems, for which so far only insufficient kinetic data are available, should be revisited.

biophysics↗

bioRxiv2019

Separations-Encoded Microparticles For Single-Cell Western Blotting

Direct measurement of proteins from single cells has been realized at the microscale using microfluidic channels, capillaries, and semi-enclosed microwell arrays. Although powerful, these formats are constrained, with the enclosed geometries proving cumbersome for multistage assays, including electrophoresis followed by immunoprobing. We introduce a hybrid microfluidic format that toggles between a planar microwell array and a suspension of microparticles. The planar array is stippled in a thin sheet of polyacrylamide gel, for efficient single-cell isolation and protein electrophoresis of hundreds-to-thousands of cells. Upon mechanical release, array elements become a suspension of separations-encoded microparticles for more efficient immunoprobing due to enhanced mass transfer. Dehydrating microparticles offer improved analytical sensitivity owing to in-gel concentration of fluorescence signal for high-throughput single-cell targeted proteomics.

bioengineering↗