Biology subjects

Floyd, B. M.

Publications and source records attributed to Floyd, B. M..

7 recordsLinked to original sources

bioRxiv2025

Color-neutral and reversible tissue transparency enables longitudinal deep-tissue imaging in live mice

Light scattering in biological tissue presents a significant challenge for deep in vivo imaging. Our previous work demonstrated the ability to achieve optical transparency in live mice using intensely absorbing dye molecules, which created transparency in the red spectrum while blocking shorter-wavelength photons. In this paper, we extend this capability to achieve optical transparency across the entire visible spectrum by employing molecules with strong absorption in the ultraviolet spectrum and sharp absorption edges that rapidly decline upon entering the visible spectrum. This new color-neutral and reversible tissue transparency method enables optical transparency for imaging commonly used fluorophores in the green and yellow spectra. Notably, this approach facilitates tissue transparency for structural and functional imaging of the live mouse brain labeled with yellow fluorescent protein and GCaMP through the scalp and skull. We show that this method enables longitudinal imaging of the same brain regions in awake mice over multiple days during development. Histological analyses of the skin and systemic toxicology studies indicate minimal acute or chronic damage to the skin or body using this approach. This color-neutral and reversible tissue transparency technique opens new opportunities for noninvasive deep-tissue optical imaging, enabling long-term visualization of cellular structures and dynamic activity with high spatiotemporal resolution and chronic tracking capabilities. Significance StatementTissue scattering represents a major barrier to deep tissue imaging in vivo. We recently showed that tissue can be rendered transparent in the red spectrum using intensely absorbing dye molecules. Here, we introduce a new, color-neutral and reversible tissue transparency approach. We demonstrate longitudinal structural and functional imaging in the deep tissue of awake mice.

bioengineering↗

bioRxiv2025

Mapping the nanoscale organization of the human cell surface proteome reveals new functional associations and surface antigen clusters

The cell surface is a dynamic interface that controls cell-cell communication and signal transduction relevant to organ development, homeostasis and repair, immune reactivity, and pathologies driven by aberrant cell surface phenotypes. The spatial organization of cell surface proteins is central to these processes. High-resolution fluorescence microscopy and proximity labeling have advanced studies of surface protein associations, but the spatial organization of the complete surface proteome remains uncharted. In this study, we systematically mapped the surface proteome of human T-lymphocytes and B-lymphoblasts using proximity labeling of 85 antigens, identified from over 100 antibodies tested for binding to surface-exposed proteins. These experiments were coupled with an optimized data-independent acquisition mass spectrometry workflow to generate a robust dataset. Unsupervised clustering of the resulting interactome revealed functional modules, including well-characterized complexes such as the T-cell receptor and HLA class I/II, alongside novel clusters. Notably, we identified mitochondrial proteins localized to the surface, including the transcription factor TFAM, suggesting previously unappreciated roles for mitochondrial proteins at the plasma membrane. A high-accuracy machine learning classifier predicted over 6,000 surface protein associations, highlighting functional associations such as IL10RBs role as a negative regulator of type I interferon signaling. Spatial modeling of the surface proteome provided insights into protein dispersion patterns, distinguishing widely distributed proteins, such as CD45, from localized antigens, such as CD226 pointing to active mechanisms of regulating surface organization. This work provides a comprehensive map of the human surfaceome and a resource for exploring the spatial and functional dynamics of the cell membrane proteome.

systems biology↗

bioRxiv2025

Combinatorial phosphorylation on CTD of RNA polymerase II selectively controls transcription and export of protein-coding mRNAs

C-terminal domain (CTD) of RNA polymerase II is crucial for recruiting transcription regulators via specific post-translational modifications (PTM), especially phosphorylation. The hypothesis of combination of PTMs, or CTD code, that can allow precise and dynamic recruitment of transcription machinery is highly attractive, yet the experimental evidence to support this hypothesis has been scarce. Here, despite lacking specific antibodies for combinatorial CTD phosphorylation, we developed an innovative approach that detects double phosphorylation patterns on the CTD in a whole-genomic fashion by leveraging the antibody masking effect with selectively removing the flanking interference. Using this method, we detected pT4pS5 double phosphosites occurring exclusively during the transcription of protein-coding genes. Furthermore, we showed that pT4pS5 marks recruit the Transcription and Export complex (TREX), which specifically facilitates mRNA processing and nucleocytoplasmic export of protein-coding mRNAs. The recruitment of TREX by pT4pS5 phosphosites is particularly important for the processing of lengthy neurogenesis-related genes. Our results provide experimental support for the notion that CTD coding system can function combinatorially and in a gene-specific manner, which encodes an exact information about the transcription of specific gene clusters. This method can be broadly applied to map all combinatorial PTM patterns on RNA polymerase II, paving the way for a deeper understanding of gene-specific transcription regulation at the molecular level.

molecular biology↗

bioRxiv2021

Photoredox-catalyzed decarboxylative C-terminal differentiation for bulk and single molecule proteomics

Methods for the selective labeling of biogenic functional groups on peptides are being developed and used in the workflow of both current and emerging proteomics technologies, such as single-molecule fluorosequencing. To achieve successful labeling with any one method requires that the peptide fragments contain the functional group for which the labeling chemistry is designed. In practice, only two functional groups are present on every peptide fragment regardless of the protein cleavage site, namely, an N-terminal amine and a C-terminal carboxylic acid. Developing a global-labeling technology, therefore, requires one to specifically target the N- and/or C-terminus of peptides. In this work, we showcase the first successful application of photocatalyzed C-terminal decarboxylative-alkylation for peptide mass-spectrometry and single molecule protein sequencing, that can be broadly applied to any proteome. We demonstrate that peptides in complex mixtures generated from enzymatic digests from bovine serum albumin, as well as protein mixtures from yeast and human cell extracts, can be site-specifically labeled at their C-terminal residue with a Michael acceptor. Using two distinct analytical approaches, we characterize C-terminal labeling efficiencies of greater than 50% across complete proteomes and document the proclivity of various C-terminal amino acid residues for decarboxylative-labeling, showing histidine and tryptophan to be the most disfavored. Finally, we combine C-terminal decarboxylative labeling with an orthogonal carboxylic acid labeling technology in tandem, to establish a new platform for fluorosequencing.

biochemistry↗

bioRxiv2021

Functional expression of opioid receptors and other human GPCRs in yeast engineered to produce human sterols

The yeast Saccharomyces cerevisiae is a powerful tool for studying G protein-coupled receptors (GPCRs) as they can be functionally coupled to its pheromone response pathway. However, some exogenous GPCRs, including the mu opioid receptor, are non-functional in yeast, which may be due to the presence of the fungal sterol ergosterol instead of the animal sterol cholesterol. We engineered yeast to produce cholesterol and introduced the human mu opioid receptor, creating an opioid biosensor capable of detecting the peptide DAMGO at an EC50 of 62 nM and the opiate morphine at an EC50 of 882 nM. Furthermore, introducing mu, delta, and kappa opioid receptors from diverse vertebrates consistently yielded active opioid biosensors that both recapitulated expected agonist binding profiles with EC50s as low as 2.5 nM and were inhibited by the antagonist naltrexone. Additionally, clinically relevant human mu opioid receptor alleles, or variants with terminal mutations, resulted in biosensors that largely displayed the expected changes in activity. We also tested mu opioid receptor-based biosensors with systematically adjusted biosynthetic intermediates of cholesterol, enabling us to relate sterol profiles with biosensor sensitivity. Finally, cholesterol-producing and sterol intermediate biosensor backgrounds were applied to other human GPCRs, resulting in SSTR5, 5-HTR4, FPR1 and NPY1R signaling with varying degrees of cholesterol dependence. Our sterol-optimized platform will be a valuable tool in generating human GPCR-based biosensors, aiding in ongoing receptor deorphanization efforts, and providing a framework for high-throughput screening of receptors and effectors.

synthetic biology↗

bioRxiv2020

Systematic Identification of Protein Phosphorylation-Mediated Interactions

Protein phosphorylation is a key regulatory mechanism involved in nearly every eukaryotic cellular process. Increasingly sensitive mass spectrometry approaches have identified hundreds of thousands of phosphorylation sites but the functions of a vast majority of these sites remain unknown, with fewer than 5% of sites currently assigned a function. To increase our understanding of functional protein phosphorylation we developed an approach for identifying the phosphorylation-dependence of protein assemblies in a systematic manner. A combination of non-specific protein phosphatase treatment, size-exclusion chromatography, and mass spectrometry allowed us to identify changes in protein interactions after the removal of phosphate modifications. With this approach we were able to identify 316 proteins involved in phosphorylation-sensitive interactions. We recovered known phosphorylation-dependent interactors such as the FACT complex and spliceosome, as well as identified novel interactions such as the tripeptidyl peptidase TPP2 and the supraspliceosome component ZRANB2. More generally, we find phosphorylation-dependent interactors to be strongly enriched for RNA-binding proteins, providing new insight into the role of phosphorylation in RNA binding. By searching directly for phosphorylated amino acid residues in mass spectrometry data, we identified the likely regulatory phosphosites on ZRANB2 and FACT complex subunit SSRP1. This study provides both a method and resource for obtaining a better understanding of the role of phosphorylation in native macromolecular assemblies.

systems biology↗

bioRxiv2020

Solid-Phase Peptide Capture and Release for Bulk and Single-Molecule Proteomics

The field of proteomics has expanded recently with more sensitive techniques for the bulk measurement of peptides as well as single-molecule techniques. One limiting factor for some of these methods is the need for multiple chemical derivatizations and highly pure proteins free of contaminants. We demonstrate a solid-phase capture strategy suitable for the proteolysis, purification, and subsequent chemical modification of peptides. We use this resin on an HEK293T cell lysate and perform one-pot proteolysis, capture, and derivatization to generate a cellular proteome that identified over 40,000 bead-bound peptides. We also show that this capture can be reversed in a traceless manner, such that it is amenable for single-molecule proteomics techniques. With this technique, we perform a fluorescent labeling and C-terminal derivatization on a peptide and subject it to fluorosequencing, demonstrating that washing the resin is sufficient to remove excess dyes and other reagents prior to single-molecule protein sequencing.

biochemistry↗