Biology subjects

Thoma, R.

Publications and source records attributed to Thoma, R..

3 recordsLinked to original sources

bioRxiv2026

Affinity-tag-based microfluidic protein isolation enables high-resolution Cryo-EM from minimal starting material

Cryo-EM is central to high-resolution structure determination, but conventional sample preparation consumes substantial amounts of protein and can destabilize sensitive complexes. Microfluidic approaches can miniaturize and accelerate these workflows while retaining the particle numbers required for single-particle analysis. Here, we present a generalized microfluidic isolation strategy that captures proteins directly from cell lysates or in vitro translation reactions using genetically encoded tags. The platform supports both affinitybased (ALFA-nanobody) and covalent (SpyTag3/SpyCatcher3) capture. Specificity derives from two sequential, orthogonal steps: (i) tag-mediated capture and concentration of the target protein on beads, and (ii) photoelution from the bead surface. By confining the sample to nanoliter volumes and minimizing transport, the approach suppresses non-specific carryover. Importantly, the isolation workflow is coupled to grid preparation, enabling preparation of three cryo-EM or negative-stain grids from a 25 nL eluate. Using this workflow, we isolated E. coli ferritin A, {beta}-galactosidase, and P. aeruginosa VgrG1 from cell lysate, obtaining reconstructions at 1.9-2.6 [A] resolution with B-factors comparable to optimized conventional workflows. We further isolated ferritin A from an in vitro translation reaction, yielding a 2.04 [A] reconstruction. A standardized 50 {micro}L starting volume enabled more than 16 independent microfluidic isolations. Tag-based microfluidic isolation thus provides a broadly applicable route to cryo-EM sample preparation, reducing sample volumes more than 1000-fold, reducing preparation times more than 3-fold for lysate-based workflows and up to 10-fold when combined with cell-free expression, and enabling high-throughput structural screening directly from in vitro translation reactions. Significance StatementCryo-electron microscopy (cryo-EM) enables protein structure determination at atomic resolution, but conventional sample preparation requires large amounts of purified protein and laborious workflows that can damage fragile complexes. We present a microfluidic platform that captures tagged proteins directly from minute amounts of crude lysates or cell-free synthesis reactions and deposits them onto cryo-EM grids in a single integrated workflow. Using this approach, we determine three protein structures at high resolution. By reducing sample volumes by roughly three orders of magnitude, the platform expands cryo-EM to proteins inaccessible to conventional workflows and provides a foundation for automated, high-throughput structural analysis.

molecular biology↗

bioRxiv2026

Tunable Tau Expression in C. elegans Neurons Reveals that Early-AD Tau Phosphorylation Selectively Impacts Behavior and Mitochondrial Quality Control

Tau protein accumulates myriad post-translational modifications as Alzheimers disease (AD) progresses, and early-disease tau modifications such as phosphorylation at threonine 231 (T231) likely play a key role in AD pathogenesis. Here, a series of "tunable tau" strains was developed in C. elegans to test the relative impact of tau pseudo-phosphorylation of T231 (T231E) compared to protein expression level as a driver of phenotypic penetrance and severity. Multiple copies of a cassette coding for pan-neuronal wildtype tau or T231E were inserted at a genomic safe harbor loci to create a repertoire of strains expressing tau from low to high levels. In stereotypical behavioral assays of locomotory activity, T231E selectively impacted phenotypic severity compared to wild-type human tau controls, which further tracked with age and tau expression level. However, deficits in associative memory were non-selective between tau and T231E. Moreover, genetic, pharmacologic, and molecular approaches indicated that mitophagy modulation could suppress T231E phenotypes. Additionally, a robust mitochondrial unfolded protein response (UPRmt) occurred in T231E, and loss of atfs-1, a transcription factor central to the UPRmt suppressed T231E toxicity. These results demonstrate that phenotypic severity is invariably associated with tau dosage, while early-AD relevant modifications can be causative drivers of selective deficits. Consistent with recent findings, enhancing mitophagy or suppressing potentially maladaptive consequences of persistent UPRmt induction can be beneficial. This provides a solid foundation for further interrogation into mitochondrial quality control disruption as a potential root cause for AD pathogenesis. HighlightsO_LIMatched sets of pan-neuronal, multi-copy tau strains enhance experimental control C_LIO_LIPhosphomimetic tau elicits selective behavioral and neuronal dysfunction C_LIO_LIPhosphomimetic tau triggers a unique mitochondrial unfolded response C_LIO_LITau depletion and mitochondrial interventions rescue observed deficits C_LI Visual Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=168 SRC="FIGDIR/small/701793v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@c5afb3org.highwire.dtl.DTLVardef@921b6corg.highwire.dtl.DTLVardef@466f8borg.highwire.dtl.DTLVardef@45452f_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2021

Trophic status and local conditions affect microbial potential for denitrification versus internal nitrogen cycling in lake sediments

The nitrogen (N) cycle is of global importance as N is an essential element and a limiting nutrient in terrestrial and aquatic ecosystems. Excessive anthropogenic N fertilizer usage threatens sensitive downstream aquatic ecosystems. Although freshwater lake sediments remove N through various microbial transformation processes, few studies have investigated the microbial communities involved. In an integrated biogeochemical and microbiological study on a eutrophic and oligotrophic lake, we estimated N removal rates in the sediments from porewater concentration gradients. Simultaneously, the abundance of different microbial N transformation genes was investigated using metagenomics on a seasonal and spatial scale. We observed that contrasting nutrient concentrations in the sediments were reflected in distinct microbial community compositions and significant differences in the abundance of various N transformation genes. Within each lake, we observed a more pronounced spatial than seasonal variability. The eutrophic Lake Baldegg showed a higher denitrification potential with higher nosZ gene (N2O reductase) abundance and higher nirS:nirK (nitrite reductase) ratio, indicating a greater capacity for complete denitrification. Correspondingly, this lake had a higher N removal efficiency. The oligotrophic Lake Sarnen, in contrast, had a higher potential for DNRA and nitrification, and specifically a high abundance of Nitrospirae, including some capable of comammox. In general, the oligotrophic lake ecosystems had a higher microbial diversity, thus acting as an important habitat for oligotrophic microbes. Our results demonstrate that knowledge of the genomic N transformation potential is important for interpreting N process rates and understanding the limitations of the N cycle response to environmental drivers. Importance{paragraph}Anthropogenic nitrogen (N) inputs can lead to eutrophication in aquatic systems, specifically in N limited coastal ecosystems. Lakes act as N sinks by transforming reactive N to N2 through denitrification or anammox. The N cycle in lake sediments is mediated by microbial processes and affected by environmental drivers such as the amount and quality of settling organic material or nitrate concentration. However, the microbial communities mediating the different N transformation processes and their impact on N removal in freshwater lake sediments remain largely unknown. We provide the first seasonally and spatially resolved metagenomic analysis of the N cycle in the sediments of two lakes with different trophic states. We show that the trophic state of lakes provokes other microbial communities with characteristic key players and functional potential for N transformation.

microbiology↗