Biology subjects

Baird, B.

Publications and source records attributed to Baird, B..

9 recordsLinked to original sources

bioRxiv2025

Boxcar Imaging FCS Reveals Membrane Raft Stabilization Kinetics in Antigen-Stimulated Mast Cells

Antigen (Ag) crosslinking of immunoglobulin E-receptor (IgE-Fc{varepsilon}RI) complexes in mast cells and consequent coupling with Lyn tyrosine kinase in the plasma membrane inner leaflet stimulates transmembrane signaling to initiate allergic and inflammatory responses. As established previously, this coupling requires formation of liquid-ordered (Lo)-like regions (aka "rafts") around the nano-clustered receptors to facilitate lipid-based partitioning of Lyn via its membrane anchor, followed by receptor phosphorylation mediated by protein-protein interactions. Imaging fluorescence correlation spectroscopy (ImFCS) was previously used to measure diffusion of Lyn-EGFP and its lipid anchor PM-EGFP (both Lo-preferring) as well as EGFP-GG (inner leaflet lipid probe, liquid-disordered (Ld)-preferring) and showed that the membrane reorganized within 15 minutes after Ag addition. To quantify the transition kinetics between the resting and Ag-stimulated steady-states, we have now developed Boxcar ImFCS for time-resolved diffusion measurements on sub-minute scale. We found that Ag stimulation causes gradual diffusion decreases for Lyn-EGFP and PM-EGFP with distinctive half-times (t1/2) of 6.9 min and 12 min, respectively, showing that Lyns protein-based interactions accelerate its diffusional transition. Simultaneously, EGFP-GG gradually changes to faster diffusion with t1/2 = 9.4 min. In comparison, t1/2 = 5.0 min for recruitment of cytoplasmic Syk by phosphorylated Fc{varepsilon}RI, consistent with initiation of transmembrane signaling before global membrane reorganization and raft condensation is completed by large, stabilized Ag-IgE-Fc{varepsilon}RI clusters. Boxcar ImFCS extends the analytical power of ImFCS to reveal dynamic membrane processes that may accompany stimuli-receptor interactions and their sequalae. STATEMENT OF SIGNIFICANCEStimulated lipid reorganization and stabilization of liquid-ordered (Lo)- like regions ("rafts") in the plasma membrane inner leaflet are decisive for initiating IgE-receptor-mediated mast cell signaling. Here, we developed a new technique, termed Boxcar Imaging Fluorescence Correlation Spectroscopy, to determine the kinetics of raft stabilization after antigen binding and crosslinking IgE receptors. We provide one of the first characterizations of time-dependent raft condensation as stimulated in live cells. We envisage broad applications of this experimental strategy to quantitatively decipher intertwined processes of membrane phase-like separation and functional transmembrane signaling.

biophysics↗

bioRxiv2025

Wearable Focused Ultrasound Neuromodulation and Electrophysiological Recording Patch for REM Sleep Enhancement

The rise in sleep disease affecting the general population globally in the past decade has been detrimental to individually and socioeconomically. As of now, approaches often are temporary through medication, permanently using invasive implants with surgical complications or neuromodulation therapy. However, non-invasive, state-dependent neuromodulation during sleep is technically challenging, especially with the lack of flexibility, comfortability and robustness for sleep conditions. Here, we introduce a Non-invasive Electrophysiological Recording and Ultrasound Neuromodulation Sleep Patch (NEUSLeeP) in delivering focused ultrasound stimulation to the subthalamic nucleus (STN) overnight with simultaneous stable polysomnography recording. Our sleep patch integrates a custom eight-channel concentric ring transducer array with real-time electroencephalography (EEG), electrooculography (EOG), electromyography (EMG) recording, and individualized line-of-sight targeting to sonicate deep brain areas while preserving mobility. Our platform operated safely and comfortably across the two-nights sleep study. Stimulation of the left STN was delivered every 90 minutes throughout the night and was associated with a 25% increase in REM (Rapid Eye Movement) sleep duration and a 43 minutes reduction in REM sleep latency compared to a sham night in a study of 26 subjects. Blood Oxygen Level Dependent (BOLD) signal attenuation in functional Magnetic Resonance Imaging (fMRI) was localized primarily to a left ipsilateral basal-ganglia-midbrain-temporal circuit, consistent with selective network modulation rather than global arousal changes. Overall, NEUSLeeP demonstrates feasibility by (i) a light weight and wearable ultrasound neuromodulation and sleep recording during natural sleep; (ii) establishing a potential mechanism relating targeted ultrasound stimulation of STN/sleep networks to REM enhancement.

bioengineering↗

bioRxiv2025

Postprocessing-Enhanced Machine Learning for Reliable Real-Time Sleep Staging in Closed-Loop Neuromodulation

Real-time sleep stage classification is important for closed-loop neuromodulation at certain stages during sleep, yet current models often yield noisy and unstable outputs that risk false triggers. These fluctuations, especially near stage boundaries, can compromise the safety and reliability of stimulation. Existing methods frequently rely on model-specific architectures or require extensive tuning to maximize precision, which limits their generalizability and hinders deployment in real-time systems. To address this, we propose a lightweight, classifier-independent post-processing pipeline that stabilizes predictions without modifying the underlying classifier. Our method first applies temporal smoothing to predicted sleep-stage probabilities, followed by conservative control logic to enhance stimulation precision. We evaluate four smoothing techniques: Moving Average (MA), Exponential Smoothing (ES), Kalman Filtering (KF), and a novel Weighted Exponential Smoothing (WES), on a 29-subject open-source sleep dataset. To capture the trade-offs between stability and responsiveness, we introduce new real-time evaluation metrics. We identify an optimal smoothing intensity range and assess three control strategies: probability thresholding, naive waiting, and entropy constraint, as well as a hybrid method combining high confidence and low uncertainty. Smoothing improves generalization and robustness: under both low and high synthetic noise ({sigma} > 0.2), smoothed outputs retain 10-15% higher precision and recall across all stages. Control logic further enhances reliability: our hybrid method achieves 80% (Wake), 96% (N2), 98% (N3), and 86% (REM) precision. Finally, we introduce a stage-transition constraint matrix to suppress biologically implausible transitions (e.g., REM[->] N3), to further stabilize outputs during evidence accumulation, with potential applications in objective sleep quality assessment. To our knowledge, this is the first study to systematically characterize real-time trade-offs between smoothing, latency, and control logic in sleep staging. Overall, our generalizable framework is expected to improve safety, interpretability, and deployment feasibility of real-time sleep classification for both clinical and wearable closed-loop neuromodulation systems.

bioengineering↗

bioRxiv2024

The Synergy between Topography and Lipid Domains in the Plasma Membrane of Mast Cells Controls the Localization of Signaling Proteins and Facilitates their Coordinated Activation

Similar to T cells and B cells, mast cell surfaces are dominated by microvilli, and like these other immune cells we showed with microvillar cartography (MVC) that key signaling proteins for RBL mast cells localize to these topographical features. Although stabilization of ordered lipid nanodomains around antigen-crosslinked IgE-Fc{varepsilon}RI is known to facilitate necessary coupling with Lyn tyrosine kinase to initiate transmembrane signaling in these mast cells, the relationship of ordered-lipid nanodomains to membrane topography had not been determined. With nanoscale resolution provided by MVC, SEM and co-localization probability (CP) analysis, we found that Fc{varepsilon}RI and Lyn kinase are positioned primarily on the microvilli of resting mast cells in separate nano-assemblies. Upon antigen-activation, Fc{varepsilon}RI and Lyn merge into overlapping populations together with the LAT scaffold protein, accompanied by merger of microvilli into ridge-like ruffles. With selective lipid probes, we further found that ordered-lipid nanodomains preferentially occupy microvillar membranes, contrasting with localization of disordered lipids to flatter regions. With this proximity of signaling proteins and ordered lipid nanodomains in microvilli, the mast cells are poised to respond sensitively and efficiently to antigen but only in the presence of this stimulus. Use of a short chain ceramide to disrupt ordered-lipid regions of the plasma membrane and evaluation with MVC, CP, and flow cytometry provided strong evidence that the microvillar selective localization of signaling proteins and membrane environments is facilitated by the interplay between ordered-lipid nanodomains and actin attachment proteins, ERM (ezrin, radixin, moesin) and cofilin. Significance StatementParticipation of ordered-lipid nanodomains (aka "rafts") to target immune signaling in the plasma membrane has been established. Separately, membrane topography, specifically microvilli, has also emerged as a participant. Here, we show how these features are coordinated in mast cells that serve as gatekeepers for antigen-triggered, receptor-mediated immune responses, including allergies and inflammation. We found that these specific antigen receptors and a key kinase, together with ordered-lipid nanodomains, localize to microvilli in resting cells, forming separated nano-assemblies. Antigen causes the merger of microvilli into ruffles where receptors and kinases couple to initiate transmembrane signaling. Selective pre-organization of signaling proteins and targeting lipid domains in microvilli and their coordinated redistribution upon antigen stimulation facilitates sensitive and efficient immune responses.

immunology↗

bioRxiv2023

Alpha Synuclein Modulates Mitochondrial Ca2+ Uptake from ER During Cell Stimulation and Under Stress Conditions

Alpha synuclein (a-syn) is an intrinsically disordered protein prevalent in neurons, and aggregated forms are associated with synucleinopathies including Parkinson disease (PD). Despite the biomedical importance and extensive studies, the physiological role of a-syn and its participation in etiology of PD remain uncertain. We showed previously in model RBL cells that a-syn colocalizes with mitochondrial membranes, depending on formation of N-terminal helices and increasing with mitochondrial stress.1 We have now characterized this colocalization and functional correlates in RBL, HEK293, and N2a cells. We find that expression of a-syn enhances stimulated mitochondrial uptake of Ca2+ from the ER, depending on formation of its N-terminal helices but not on its disordered C-terminal tail. Our results are consistent with a-syn acting as a tether between mitochondria and ER, and we show increased contacts between these two organelles using structured illumination microscopy. We tested mitochondrial stress caused by toxins related to PD, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP/MPP+) and carbonyl cyanide m-chlorophenyl hydrazone (CCCP), and found that a-syn prevents recovery of stimulated mitochondrial Ca2+ uptake. The C-terminal tail, and not N-terminal helices, is involved in this inhibitory activity, which is abrogated when phosphorylation site serine-129 is mutated (S129A). Correspondingly, we find that MPTP/MPP+ and CCCP stress is accompanied by both phosphorylation (pS129) and aggregation of a-syn. Overall, our results indicate that a-syn can participate as a tethering protein to modulate Ca2+ flux between ER and mitochondria, with potential physiological significance. A-syn can also prevent cellular recovery from toxin-induced mitochondrial dysfunction, which may represent a pathological role of a-syn in the etiology of PD.

cell biology↗

bioRxiv2022

Lipid Driven Inter-leaflet Coupling of Plasma Membrane Order Regulates FcεRI Signaling in Mast Cells

Engagement of high affinity immunoglobulin E (IgE) receptor Fc{varepsilon}RI with extracellular, multivalent antigen (Ag) stabilizes co-existing ordered and disordered phases in the inner leaflet of the plasma membrane. This optimally controls biochemical interactions between signaling components required for transmembrane (TM) signaling in mast cells. The biophysical organization of the resting inner leaflet is poised to respond appropriately to this extracellular stimulation. The resting inner leaflet is generally less ordered than the outer leaflet, with a lipid composition that does not spontaneously phase separate in model membranes. We proposed that coupling between the two leaflets mediates separation into different phase-like domains in the inner leaflet. To test this hypothesis in live cells, we first established a straightforward approach to evaluate changes in membrane order due to inter-leaflet coupling by measuring inner leaflet diffusion of phase-specific lipid probes with Imaging Fluorescence Correlation Spectroscopy (ImFCS) before and after methyl--cyclodextrin (mCD)-catalyzed exchange of outer leaflet lipids (LEX) with exogenous order- or disorder-promoting phospholipids. We examined the functional impact of LEX by monitoring two Ag-stimulated cellular responses, namely early-stage recruitment of Syk kinase to the inner leaflet and late-stage exocytosis of secretory granules (degranulation). Based on changes in probe diffusion, we observed global increase or decrease of inner leaflet order when outer leaflet is exchanged with order or disorder promoting lipids, respectively, in unstimulated cells. Furthermore, the degree of stimulated Syk recruitment and degranulation correlates with the inner leaflet order of the resting cells, which was varied using LEX. Overall, combined LEX and ImFCS platform provides strong evidence of lipid-based control of stimulated TM signaling in live mast cells. In addition, our functional results imply that resting-state lipid composition and ordering of the outer leaflet sets the ordering of the inner leaflet, likely via interleaflet coupling, and correspondingly modulates TM signaling initiated by antigen-activated IgE-Fc{varepsilon}RI. STATEMENT OF SIGNIFICANCECoupling between plasma membrane leaflets, which are biochemically and biophysically asymmetric, results in a steady-state membrane organization that is thought to play fundamental roles in cellular functions. Here, we present a straightforward assay built around mCD-catalyzed lipid exchange (LEX) and Imaging Fluorescence Correlation Spectroscopy (ImFCS) to quantitatively characterize a novel, lipid-driven, interleaflet coupling mechanism and its functional impact in live mast cells. We showed that elevation of outer leaflet lipid order induces ordering throughout the inner leaflet in resting cells. This ordering enhances protein-based reactions during Ag-stimulated Fc{varepsilon}RI signaling and consequent cellular response. Overall, we provide a compelling evidence of functional relevance of plasma membrane organizational heterogeneity driven by lipid-based interleaflet coupling.

biophysics↗

bioRxiv2022

Transbilayer Coupling of Lipids in Cells Investigated by Imaging Fluorescence Correlation Spectroscopy

Plasma membrane hosts numerous receptors, sensors, and ion channels involved in cellular signaling. Phase separation of the plasma membrane is emerging as a key biophysical regulator of signaling reactions in multiple physiological and pathological contexts. There is much evidence that plasma membrane composition supports the co-existence liquid-ordered (Lo) and liquid-disordered (Ld) phases or domains at physiological conditions. However, this phase/domain separation is nanoscopic and transient in live cells. It is recently proposed that transbilayer coupling between the inner and outer leaflets of the plasma membrane is driven by their asymmetric lipid distribution and by dynamic cytoskeleton-lipid composites that contribute to the formation and transience of Lo/Ld phase separation in live cells. In this Perspective, we highlight new approaches to investigate how transbilayer coupling may influence phase separation. For quantitative evaluation of the impact of these interactions, we introduce an experimental strategy centered around Imaging Fluorescence Correlation Spectroscopy (ImFCS), which measures membrane diffusion with very high precision. To demonstrate this strategy we choose two well-established model systems for transbilayer interactions: crosslinking by multivalent antigen of immunoglobulin E bound to receptor Fc{varepsilon}RI, and crosslinking by cholera toxin B of GM1 gangliosides. We discuss emerging methods to systematically perturb membrane lipid composition, particularly exchange of outer leaflet lipids with exogenous lipids using methyl alpha cyclodextrin. These selective perturbations may be quantitatively evaluated with ImFCS and other high-resolution biophysical tools to discover novel principles of lipid-mediated phase separation in live cells in the context of their pathophysiological relevance.

biophysics↗

bioRxiv2019

Imaging FCS Delineates Subtle Heterogeneity in Plasma Membranes of Resting Mast Cells

A myriad of transient, nanoscopic lipid- and protein-based interactions confer a steady-state organization of plasma membrane in resting cells that is poised to orchestrate assembly of key signaling components upon reception of an extracellular stimulus. Although difficult to observe directly in live cells, these subtle interactions can be discerned by their impact on the diffusion of membrane constituents. Herein, we quantified the diffusion properties of a panel of structurally distinct lipid-anchored and transmembrane (TM) probes in RBL mast cells by multiplexed Imaging Fluorescence Correlation Spectroscopy. We developed a statistical analysis of data combined from many pixels over multiple cells to characterize differences as small as 10% in diffusion coefficients, which reflect differences in underlying interactions. We found that the distinctive diffusion properties of lipid-anchored probes can be explained by their dynamic partitioning into ordered proteo-lipid nanodomains, which encompass a major fraction of the membrane and whose physical properties are influenced by actin polymerization. Effects on diffusion by functional protein modules in both lipid-anchored and TM probes reflect additional complexity in steady-state membrane organization. The contrast we observe between different probes diffusing through the same membrane milieu represent the dynamic resting steady-state, which serves as a baseline for monitoring plasma membrane remodeling that occurs upon stimulation.

biophysics↗

bioRxiv2018

Regulation of Exocytosis and Mitochondrial Relocalization by Alpha-Synuclein in a Mammalian Cell Model

We characterized phenotypes in RBL-2H3 mast cells transfected with human alpha synuclein (a-syn) using stimulated exocytosis of recycling endosomes as a proxy for similar activities of synaptic vesicles in neurons. We found that low expression of a-syn inhibits stimulated exocytosis and that higher expression causes slight enhancement. NMR measurements of membrane interactions correlate with these functional effects: they are eliminated differentially by mutants that perturb helical structure in the helix 1 (A30P) or NAC/helix-2 (V70P) regions of membrane-bound a-syn, but not by other PD-associated mutants or C-terminal truncation. We further found that a-syn (but not A30P or V70P mutants) associates weakly with mitochondria, but this association increases markedly under conditions of cellular stress. These results highlight the importance of specific structural features of a-syn in regulating vesicle release, and point to a potential role for a-syn in perturbing mitochondrial function under pathological conditions.

cell biology↗