Biology subjects

Garcia, F. J.

Publications and source records attributed to Garcia, F. J..

4 recordsLinked to original sources

bioRxiv2022

Sequencing by avidity enables high accuracy with low reagent consumption

We present avidity sequencing - a novel sequencing chemistry that separately optimizes the process of stepping along a DNA template and the process of identifying each nucleotide within the template. Nucleotide identification uses multivalent nucleotide ligands on dye-labeled cores to form polymerase-polymer nucleotide complexes bound to clonal copies of DNA targets. These polymer-nucleotide substrates, termed avidites, decrease the required concentration of reporting nucleotides from micromolar to nanomolar, and yield negligible dissociation rates. We demonstrate the use of avidites as a key component of a sequencing technology that surpasses Q40 accuracy and enables a diversity of applications that include single cell RNA-seq and whole human genome sequencing. We also show the advantages of this technology in sequencing through long homopolymers.

genomics↗

bioRxiv2022

A partnership of the lipid scramblase XK and of the lipid transfer protein VPS13A at the plasma membrane

Chorea-acanthocytosis and McLeod syndrome are diseases with shared clinical manifestations caused by mutations in VPS13A and XK, respectively. Key features of these conditions are the degeneration of caudate neurons and the presence of abnormally shaped erythrocytes. XK belongs to a family of plasma membrane (PM) lipid scramblases whose action results in exposure of PtdSer at the cell surface. VPS13A is an ER-anchored lipid transfer protein with a putative role in the transport of lipids at contacts of the ER with other membranes. Recently VPS13A and XK were reported to interact by still unknown mechanisms. So far, however, there is no evidence for a colocalization of the two proteins at contacts of the ER with the PM, where XK resides, as VPS13A was shown to be localized at contacts between the ER and either mitochondria or lipid droplets. Here we show that VPS13A can also localize at ER-PM contacts via the binding of its PH domain to a cytosolic loop of XK, that such interaction is regulated by an intramolecular interaction within XK and that both VPS13A and XK are highly expressed in the caudate neurons. Binding of the PH domain of VPS13A to XK is competitive with its binding to intracellular membranes that mediate other tethering functions of VPS13A. Our findings support a model according to which VPS13A-dependent lipid transfer between the ER and the PM is coupled to lipid scrambling within the PM. They raise the possibility that defective cell surface exposure of PtdSer may be responsible for neurodegeneration.

cell biology↗

bioRxiv2021

Single-cell dissection of the human cerebrovasculature in health and disease

Despite the importance of the blood-brain barrier in maintaining normal brain physiology and in understanding neurodegeneration and CNS drug delivery, human cerebrovascular cells remain poorly characterized due to their sparsity and dispersion. Here, we perform the first single-cell characterization of the human cerebrovasculature using both ex vivo fresh-tissue experimental enrichment and post mortem in silico sorting of human cortical tissue samples. We capture 31,812 cerebrovascular cells across 17 subtypes, including three distinct subtypes of perivascular fibroblasts as well as vasculature-coupled neurons and glia. We uncover human-specific expression patterns along the arteriovenous axis and determine previously uncharacterized cell type-specific markers. We use our newly discovered human-specific signatures to study changes in 3,945 cerebrovascular cells of Huntingtons disease patients, which reveal an activation of innate immune signaling in vascular and vasculature-coupled cell types and the concomitant reduction to proteins critical for maintenance of BBB integrity. Finally, our study provides a comprehensive resource molecular atlas of the human cerebrovasculature to guide future biological and therapeutic studies.

neuroscience↗

bioRxiv2021

Chemoproteomics Enabled Discovery of Selective Probes for NuA4 Factor BRD8

Bromodomain-containing proteins frequently reside in multisubunit chromatin complexes with tissue or cell state-specific compositions. Recent studies have revealed tumor-specific dependencies on the BAF complex bromodomain subunit BRD9 that are a result of recurrent mutations afflicting the structure and composition of associated complex members. To enable the study of ligand engaged complex assemblies, we established a chemoproteomics approach using a functionalized derivative of the BRD9 ligand BI-9564 as an affinity matrix. Unexpectedly, in addition to known interactions with BRD9 and associated BAF complex proteins, we identify a previously unreported interaction with members of the NuA4 complex through the bromodomain-containing subunit BRD8. We apply this finding, alongside homology model guided design, to develop chemical biology approaches for the study of BRD8 inhibition, and to arrive at first-in-class selective and cellularly active probes for BRD8. These tools will empower further pharmacological studies of BRD9 and BRD8 within respective BAF and NuA4 complexes.

biochemistry↗