Biology subjects

Sanabria, H.

Publications and source records attributed to Sanabria, H..

6 recordsLinked to original sources

bioRxiv2026

Multidomain Coupling Governs FoxP1 Assembly and Nuclear Compartmentalization

FoxP1 is a multidomain transcription factor implicated in development, immunity, and cancer, widely proposed to function as a dimer. However, the molecular mechanisms governing its assembly and nuclear organization in living cells remain unclear. Here, we combine biochemical assays and live-cell fluorescence lifetime imaging to resolve FoxP1 homotypic interactions. We show that FoxP1 forms heterogeneous complexes whose stability is governed by antagonistic coupling between its leucine-zipper (ZIP) and Forkhead (FKH) domains. The FoxP1 ZIP domain promotes dimerization while suppressing FKH-mediated interactions, revealing a competing interdomain mechanism that tunes complex formation. Pathogenic and deletion variants disrupt this intricate balance, altering interaction stability and promoting the formation of dense nuclear condensates upon loss of DNA binding. Together, our results demonstrate that FoxP1 assembly is encoded by its multidomain architecture. Our findings show how competing interaction domains regulate transcription factor complex formation, nuclear organization, and DNA binding, with implications for disease-associated dysregulation. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=85 SRC="FIGDIR/small/728184v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@bb8cbcorg.highwire.dtl.DTLVardef@1149339org.highwire.dtl.DTLVardef@1cd87d7org.highwire.dtl.DTLVardef@9147a4_HPS_FORMAT_FIGEXP M_FIG C_FIG HIGHLIGHTSO_LIFoxP1 complexes assemble through two distinct mechanisms mediated by the FKH and ZIP domains. C_LIO_LIInterdomain coupling switches the configuration of the FoxP1 complex. C_LIO_LIFKH-mediated FoxP1 oligomers and other higher-order complexes accumulate at the nuclear periphery, whereas ZIP-mediated dimers remain more uniformly distributed. C_LIO_LIDisruption of FKH-DNA binding drives formation of protein-dense FoxP1 condensates. C_LI

biophysics↗

bioRxiv2023

Increasing Signal Intensity of Fluorescent Oligo-Labeled Antibodies

Full-spectrum flow cytometry has increased antibody-based multiplexing, yet further increases remain potentially impactful. We recently proposed how fluorescence Multiplexing using Spectral Imaging and Combinatorics (MuSIC) could do so using tandem dyes and an oligo-based antibody labeling method. In this work, we found that such labeled antibodies had significantly lower signal intensity than conventionally-labeled antibodies in human cell experiments. To improve signal intensity, we tested moving the fluorophores from the original external (ext.) 5 or 3 end-labeled orientation to internal (int.) fluorophore modifications. Cell-free spectrophotometer measurements showed a [~]6-fold signal intensity increase of the new int. configuration compared to the previous ext. configuration. Time-resolved fluorescence and fluorescence correlation spectroscopy showed that [~]3-fold brightness difference is due to static quenching most likely by the oligo or solution in the ext. configuration. Spectral flow cytometry experiments using peripheral blood mononuclear cells show int. MuSIC probe-labeled antibodies (i) retained increased signal intensity while having no significant difference in the estimated % of CD8+ lymphocytes and (ii) labeled with Atto488, Atto647, and Atto488/647 combinations can be demultiplexed in triple-stained samples. The antibody labeling approach is general and can be broadly applied to many biological and diagnostic applications where spectral detection is available.

molecular biology↗

bioRxiv2022

A Theory for High-Throughput Genetic Interaction Screening

Systematic, genome-scale genetic screens have been instrumental for elucidating genotype-phenotype relationships, but approaches for probing genetic interactions have been limited to at most [~]100 pre-selected gene combinations in mammalian cells. Here, we introduce a theory for high-throughput genetic interaction screens. The theory extends our recently developed Multiplexing using Spectral Imaging and Combinatorics (MuSIC) approach to propose [~]105 spectrally unique, genetically-encoded MuSIC barcodes from 18 currently available fluorescent proteins. Simulation studies based on constraints imposed by spectral flow cytometry equipment suggest that genetic interaction screens at the human genome-scale may be possible if MuSIC barcodes can be paired to guide RNAs. While experimental testing of this theory awaits, it offers transformative potential for genetic perturbation technology and knowledge of genetic function. More broadly, the availability of a genome-scale spectral barcode library for non-destructive identification of single-cells could find more widespread applications such as traditional genetic screening and high-dimensional lineage tracing.

biochemistry↗

bioRxiv2022

Fuzzy Supertertiary Interactions within PSD-95 Enable Ligand Binding

The scaffold protein PSD-95 links postsynaptic receptors to sites of presynaptic neurotransmitter release. Flexible linkers between folded domains in PSD-95 enable a dynamic supertertiary structure. Interdomain interactions within the PSG supramodule, formed by PDZ3, SH3 and GuK domains, regulate PSD-95 activity. Here we combined Discrete Molecular Dynamics and single molecule FRET to characterize the PSG supramodule, with time resolution spanning picoseconds to seconds. We used a FRET network to measure distances in full-length PSD-95 and model the conformational ensemble. We found that PDZ3 samples two conformational basins, which we confirmed with disulfide mapping. To understand effects on activity, we measured binding of the synaptic adhesion protein neuroligin. We found that PSD-95 bound neuroligin well at physiological pH while truncated PDZ3 bound poorly. Our hybrid structural models reveal how the supertertiary context of PDZ3 enables recognition of this critical synaptic ligand.

biophysics↗

bioRxiv2021

Inferring kinetic rate constants from single-molecule FRET trajectories - a blind benchmark of kinetic analysis tools

Single-molecule FRET (smFRET) is a versatile technique to study the dynamics and function of biomolecules since it makes nanoscale movements detectable as fluorescence signals. The powerful ability to infer quantitative kinetic information from smFRET data is, however, complicated by experimental limitations. Diverse analysis tools have been developed to overcome these hurdles but a systematic comparison is lacking. Here, we report the results of a blind benchmark study assessing eleven analysis tools used to infer kinetic rate constants from smFRET trajectories. We tested them against simulated and experimental data containing the most prominent difficulties encountered in analyzing smFRET experiments: different noise levels, varied model complexity, non-equilibrium dynamics, and kinetic heterogeneity. Our results highlight the current strengths and limitations in inferring kinetic information from smFRET trajectories. In addition, we formulate concrete recommendations and identify key targets for future developments, aimed to advance our understanding of biomolecular dynamics through quantitative experiment-derived models.

biophysics↗

bioRxiv2021

Ensemble Switching Unveils a Kinetic Rheostat Mechanism of the Eukaryotic Thiamine PyrophosphateRiboswitch

Thiamine pyrophosphate (TPP) riboswitches regulate thiamine metabolism by inhibiting the translation of enzymes essential to thiamine synthesis pathways upon binding to thiamine pyrophosphate in cells across all domains of life. Recent work on the Arabidopsis thaliana TPP riboswitch suggests a multi-step TPP binding process involving multiple riboswitch conformational ensembles and that Mg2+ dependence underlies the mechanism of TPP recognition and subsequent transition to the translation-inhibiting state of the switching sequence followed by changes in the expression platform. However, details of the relationship between TPP riboswitch conformational changes and interactions with TPP and Mg2+ in the aptamer domain constituting this mechanism are unknown. Therefore, we integrated single-molecule multiparameter fluorescence and force spectroscopy with atomistic molecular dynamics simulations and found that conformational transitions within the aptamer domain associated with TPP and Mg2+ ligand binding occurred between at least five different ensembles on timescales ranging from s to ms. These dynamics are at least an order of magnitude faster than folding and unfolding kinetics associated with translation-state switching in the switching sequence. Moreover, we propose that two pathways exist for ligand recognition. Together, our results suggest a dynamic ensemble switching of the aptamer domain that may lead to the translation-inhibiting state of the riboswitch. Additionally, our results suggest that multiple configurations could enable inhibitory tuning manifested through ligand-dependent changes via ensemble switching and kinetic rheostat-like behavior of the Arabidopsis thaliana TPP riboswitch.

biophysics↗