Biology subjects

Bag, N.

Publications and source records attributed to Bag, N..

7 recordsLinked to original sources

bioRxiv2026

Excited state Relaxation Activation Energy (ESRAct) of Di-4-ANEPPDHQ Maps Nanoscale Molecular Organization in Biomembranes

Live cell plasma membranes show spatially heterogeneous liquid-ordered (Lo)-like and liquid-disordered (Ld)-like regions similar to the co-existing Lo/Ld phases observed in lipid vesicles. The Lo-like regions are relatively less hydrated and less polar due to tight packing of the membrane components compared to the Ld-like regions. The steady-state fluorescence spectra of Di-4-ANEPPDHQ (Di-4), a widely used polarity-sensitive probe, is blue or red shifted when solvated in less polar (Ld- like) or more polar (Lo-like) regions respectively. However, quantification of Di-4 fluorescence in blue and red channels for the evaluation of membrane phase state suffers from the lack of specific wavelength choice for these two channels and Di-4s relatively higher concentration in Ld phase (red channel) due to its partitioning preference. To address these issues, we employed fluorescence lifetime of Di-4, a concentration independent photophysical parameter, to understand membrane biophysical properties. The fluorescence lifetime of Di-4 in lipid vesicles exhibits Arrhenius-like temperature dependence. Centred around this energetic feature of Di-4 photophysics, we developed a novel analytical module, namely excited state relaxation activation energy (ESRAct), that serves as an intrinsic descriptor of the membrane nano-environment sensed by this probe. We show that the ESRAact value scales with increasing disorder in nanoscale phase separation (i.e., ESRAct of pure Ld > mixed Ld/Lo > pure Lo phase). We then extended its applications to giant plasma membrane vesicles (GPMVs) isolated from MCF-7 cells and found that these vesicles exhibit nanoscale Lo/Ld co-existing phase within 16-37{degrees}C. We envisage wide applications of ESRAct to delineate plasma membrane phase behavior as well as general photophysical studies on other newly designed polarity-sensitive probes.

biophysics↗

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↗

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↗

bioRxiv2021

A palmitoylation code controls PI4KIIIα complex formation and PI(4,5)P2 homeostasis at the plasma membrane

PI4KIII is the major enzyme responsible for generating the phosphoinositide PI(4)P at the plasma membrane. This lipid kinase forms two multicomponent complexes, both including a palmitoylated anchor, EFR3. Whereas both PI4KIII complexes support production of PI(4)P, the distinct functions of each complex and mechanisms underlying the interplay between them remain unknown. Here, we present roles for differential palmitoylation patterns within a tri-Cys motif in EFR3B (Cys5/Cys7/Cys8) in controlling the distribution of PI4KIII between these two complexes at the plasma membrane and corresponding functions in phosphoinositide homeostasis. Spacing of palmitoyl groups within three doubly palmitoylated EFR3B "lipoforms" affects both its interactions with TMEM150A, a transmembrane protein governing formation of a PI4KIII complex functioning in rapid PI(4,5)P2 resynthesis following PLC signaling, and its partitioning within liquid-ordered and -disordered regions of the plasma membrane. This work identifies a palmitoylation code in controlling protein-protein and protein-lipid interactions affecting a plasma membrane-resident lipid biosynthetic pathway. SUMMARY STATEMENTDifferent palmitoylation patterns on a lipid kinase adaptor protein control partitioning of the kinase between two spatiotemporally and functionally distinct complexes within the plasma membrane.

cell biology↗

bioRxiv2020

Lipid-based, protein-based, and steric interactions synergize to facilitate transmembrane signaling stimulated by antigen-clustering of IgE receptors

Antigen (Ag) crosslinking of immunoglobulin E-receptor (IgE-Fc{varepsilon}RI) complexes in mast cells stimulates transmembrane (TM) signaling, requiring phosphorylation of the clustered Fc{varepsilon}RI by lipid-anchored Lyn tyrosine kinase. Previous studies showed that this stimulated coupling between Lyn and Fc{varepsilon}RI occurs in liquid ordered (Lo)-like nanodomains of the plasma membrane and that Lyn binds directly to cytosolic segments of Fc{varepsilon}RI that it initially phosphorylates for amplified activity. Net phosphorylation above a non-functional threshold is achieved in the stimulated state, but not in the resting state, and current evidence supports the hypothesis that this relies on disruption by Ag-crosslinking of a balance between Lyn and tyrosine phosphatase activities. However, the structural interactions that underlie the stimulation process remain poorly defined. This study evaluates the relative contributions and functional importance of different types of interactions leading to supra-threshold phosphorylation of Ag-crosslinked IgE-Fc{varepsilon}RI in live rat basophilic leukemia (RBL) mast cells. Our high-precision diffusion measurements by Imaging Fluorescence Correlation Spectroscopy (ImFCS) on multiple structural variants of Lyn and other lipid-anchored probes confirm subtle, stimulated stabilization of the Lo-like nanodomains and concomitant sharpening of segregation from liquid-disordered (Ld)-like regions. With other structural variants we determine that lipid-based interactions are essential for access by Lyn leading to phosphorylation of and protein-based binding to clustered Fc{varepsilon}RI. By contrast, TM tyrosine phosphatase, PTP, is excluded from these regions by steric repulsion of TM segments and preference for Ld-like regions. Overall, we establish a synergy of lipid-based, protein-based, and steric interactions underlying functional TM signaling in mast cells. SIGNIFICANCE STATEMENTLipid organization of the plasma membrane is known to be important for facilitating protein interactions in transmembrane signaling. However, the orchestration of these interactions in live cells has been elusive. We employed ImFCS to systemically investigate the interplay of lipids and proteins during signaling in mast cells, initiated as phosphorylation of Ag-crosslinked IgE-Fc{varepsilon}RI by lipid-anchored Lyn kinase. We find lipid-based interactions are first required for protein-based phosphorylation of the clustered Fc{varepsilon}RI within Lo-like nanodomains. Transmembrane phosphatases must be excluded from these regions, and we find this is mediated by their preference for Ld-like regions and by steric exclusion from the clustered Fc{varepsilon}RI proteins. ImFCS provides quantitative characterization of the functional link between features of plasma membrane organization and transmembrane signaling.

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↗