Biology subjects

Hughes, R. M.

Publications and source records attributed to Hughes, R. M..

8 recordsLinked to original sources

bioRxiv2026

Characterization of a cofilin mutant with high actin bundling activity in living cells

Cofilin is a key regulator of actin dynamics that, along with a myriad of other actin-binding proteins, controls the balance of F- and G-actin in numerous cell types. While prior structural studies of the cofilin-actin binding interface have delineated many critical interactions between cofilin and actin, the roles of some residues within the cofilin-actin binding interface remain poorly defined. In this study, we investigate the role of cofilin S119 in the cofilin-actin interaction. Despite its unique position within the cofilin-actin interface and its putative role as a phosphorylation site, relatively little direct evidence exists to define whether it plays an important role in cofilin-actin dynamics. Using site-directed mutagenesis, we demonstrate that mutation of S119 to aromatic amino acids (W, F, Y) results in cofilins with strong actin bundling activity in living cells. This activity can be countered by the incorporation of mutants that disfavor actin rod forming activity (R21Q). Mutation of S119 to phospho-mimic (E) and non-phosphorylated (A) residues either strongly inhibits (E) or modestly increases (A) actin bundling activity. Expression of the S119W mutant in neurons reveals its impacts on spine length and size, while FRAP studies show that its mobile fraction is intermediate between that of LifeAct and WT cofilin. Finally, it is shown that the strong actin bundling phenotype associated with S119W inhibits the progression of optogenetically induced apoptosis.

cell biology↗

bioRxiv2026

Immobilized enzyme-assisted production of recombinant P113 peptide

Recombinant peptide production was pioneered in the 1970s for the generation of therapeutic peptides, with notable examples including insulin and somatostatin. These early methods required the use of cyanogen bromide (BrCN) for cleavage of the native peptide sequence from a fusion protein. Since that time, while numerous BrCN-dependent peptide methods continue to be reported, the accessibility and cost of site-specific proteases have improved dramatically. These developments have enabled alternative approaches to recombinant peptide generation that obviate the need for BrCN, an environmentally destructive toxin. We recently created an immobilized SUMO protease that can replace BrCN usage in recombinant peptide production workflows by releasing native peptides expressed as part of a SUMO-peptide fusion protein. We have used this approach to generate P113 peptide, the minimal active fragment of the antifungal peptide Histatin 5. In this report, we describe the creation and characterization of this immobilized SUMO protease and its application in the production of experimentally viable quantities of active P113 peptide.

biochemistry↗

bioRxiv2024

Optogenetic Regulation of EphA1 RTK Activation and Signaling

Eph receptors are ubiquitous class of transmembrane receptors that mediate cell-cell communication, proliferation, differentiation, and migration. EphA1 receptors specifically play an important role in angiogenesis, fetal development, and cancer progression; however, studies of this receptor can be challenging as its ligand, ephrinA1, binds and activates several EphA receptors simultaneously. Optogenetic strategies could be applied to circumvent this requirement for ligand activation and enable selective activation of the EphA1 subtype. In this work, we designed and tested several iterations of an optogenetic EphA1 - Cryptochrome 2 (Cry2) fusion, investigating their capacity to mimic EphA1-dependent signaling in response to light activation. We then characterized the key cell signaling target of MAPK phosphorylation activated in response to light stimulation. The optogenetic regulation of Eph receptor RTK signaling without the need for external stimulus promises to be an effective means of controlling individual Eph receptor-mediated activities and creates a path forward for the identification of new Eph-dependent functions.

synthetic biology↗

bioRxiv2023

OptoProfilin: A Single Component Biosensor of Applied Cellular Stress

The actin cytoskeleton is a biosensor of cellular stress and a potential prognosticator of human disease. In particular, aberrant cytoskeletal structures such as cofilin-actin rods and stress granules formed in response to energetic and oxidative stress are closely linked to neurodegenerative diseases such as Alzheimers, Parkinsons, and ALS. Whether these cytoskeletal phenomena can be harnessed for the development of biosensors for cytoskeletal dysfunction and, by extension, neurodegenerative disease progression, remains an open question. In this work, we describe the design and development of an optogenetic iteration of profilin, an actin monomer binding protein with critical functions in cytoskeletal dynamics. We demonstrate that this optically activated profilin ( OptoProfilin) can act as an optically triggered biosensor of applied cellular stress in select immortalized cell lines. Notably, OptoProfilin is a single component biosensor, likely increasing its utility for experimentalists. While a large body of preexisting work closely links profilin activity with cellular stress and neurodegenerative disease, this, to our knowledge, is the first example of profilin as an optogenetic biosensor of stress-induced changes in the cytoskeleton.

synthetic biology↗

bioRxiv2022

CRY-BARs: Versatile light-gated molecular tools for the remodeling of membrane architectures

BAR (Bin, Amphiphysin and Rvs) protein domains are responsible for the generation of membrane curvature and represent a critical mechanical component of cellular functions. Thus, BAR domains have great potential as components of membrane-remodeling tools for cell biologists. In this work, we describe the design and implementation of a family of versatile light-gated I-BAR domain containing tools ( CRY-BARs) with applications in the remodeling of membrane architectures and the control of cellular dynamics. By taking advantage of the intrinsic membrane binding propensity of the I-BAR domain, CRY-BARs can be used for spatial and temporal control of cellular processes that require induction of membrane protrusions. Using cell lines and primary neuron cultures, we demonstrate that the CRY-BAR optogenetic tool reports membrane dynamic changes associated with cellular activity. Moreover, we provide evidence that Ezrin acts as a relay between the plasma membrane and the actin cytoskeleton and therefore is an important mediator of switch function. Overall, CRY-BARs hold promise as a useful addition to the optogenetic toolkit to study membrane remodeling in live cells.

cell biology↗

bioRxiv2020

Collective ERK/Akt activity waves orchestrate epithelial homeostasis by driving apoptosis-induced survival

Cell death events continuously challenge epithelial barrier function, yet are crucial to eliminate old or critically damaged cells. How such apoptotic events are spatio-temporally organized to maintain epithelial homeostasis remains unclear. We observe waves of Extracellular Signal-Regulated Kinase (ERK) and AKT serine/threonine kinase (Akt) activity pulses that originate from apoptotic cells and propagate radially to healthy surrounding cells. This requires Epidermal Growth Factor Receptor (EGFR) and matrix metalloproteinase (MMP) signaling. At the single-cell level, ERK/Akt waves act as spatial survival signals that locally protect cells in the vicinity of the epithelial injury from apoptosis for a period of 3-4h. At the cell population level, ERK/Akt waves maintain epithelial homeostasis (EH) in response to mild or intense environmental insults. Disruption of this spatial signaling system results in the inability of a model epithelial tissue to ensure barrier function in response to environmental insults.

cell biology↗

bioRxiv2019

CofActor: A light and redox-gated optogenetic clustering tool to study abnormal cytoskeletal dynamics in stressed cells

The biochemical hallmarks of neurodegenerative diseases (neural fibrils, clumps, and tangles; heightened reactive oxygen species (ROS); cofilin-actin rods) have presented numerous challenges for development of in vivo diagnostic tools (1-7). Biomarkers such as amyloid beta (A{beta}) fibrils and Tau tangles in Alzheimers Disease (AD) are only accessible via invasive cerebrospinal fluid assay for peptide mass fingerprinting or post-mortem diagnosis (8-11), while ROS can be fleeting and thus challenging to monitor in vivo (12-15). While remaining a challenge for in vivo detection, the unique protein-protein interactions underlying these disease-specific biomarkers also present rich opportunities for the engineering of in vitro pathology-sensitive biosensors and bioactuators. These tools can be useful for the investigation of critical, early stage events in neurodegenerative diseases in both cellular and animal models (16, 17), while potentially leading to advanced detection reagents with clinical applications. In this report, we describe a light and redox-gated protein switch inspired by the phenomenon of cofilin-actin rod formation, which occurs in stressed neurons in the AD brain and following brain ischemia (18). By coupling the redox-sensitive interaction of cofilin and actin with the light responsive Cry2-CIB blue light switch, we accomplish both light- and ROS-gated control of this interaction. The resulting switch is referred to as the "CofActor" system. Site-directed mutagenesis of both cofilin and actin partners demonstrate which residues are critical for sustaining or abrogating the light and redox gated response. Furthermore, switch response varies depending on whether oxidative stress is generated via glycolytic inhibition or a combination of glycolytic inhibition and azide-induced oxidative stress. Finally, light and redox gated switch function was demonstrated in cultured hippocampal neurons. As a dual input biosensor, CofActor holds promise for the tracking of early stage events in neurodegeneration and the investigation of actin-binding protein interactions under oxidative stress.

neuroscience↗

bioRxiv2019

Imaging of Morphological and Biochemical Hallmarks of Apoptosis with Optimized Optogenetic Actuators

The creation of pathway-specific optogenetic switches for the activation of cell death pathways can provide insight into the mechanisms of apoptosis and also form a basis for non-invasive, next generation therapeutic strategies. Previous work has employed the Cryptochrome 2 (Cry2)/CIB, a blue light activated protein - protein dimerization module from A. thaliana in conjunction with BAX, an OMM targeting pro-apoptotic protein, for light-mediated initiation of mitochondrial outer membrane permeabilization (MOMP) and downstream apoptosis. In this work, we have further developed our original light activated Cry2-BAX system (henceforth referred to as "OptoBAX") by improving the photophysical properties and light-independent interaction of our optogenetic switch. The resulting optogenetic constructs have significantly reduced the frequency of light exposure required for the activation of membrane permeabilization, in addition to reducing dark state cytotoxicity. Furthermore, we have utilized OptoBAX in a series of experiments designed to measure the timing of the dramatic morphological and biochemical changes that occur in apoptotic cells following light-induced permeabilization of the outer mitochondrial membrane. Utilizing this data, we construct a timeline of biochemical and morphological events in early apoptosis, in addition to tracking the MOMP-induced redistribution of actin, a protein critical to apoptotic progression.

biochemistry↗