Biology subjects

Kelly, C. V.

Publications and source records attributed to Kelly, C. V..

8 recordsLinked to original sources

bioRxiv2026

Lipid composition-driven sorting of ABHD5 between monolayer and bilayer surfaces

Lipid droplets (LDs) are ubiquitous organelles that store neutral lipids and serve as central regulators of lipid homeostasis. Their structure includes a hydrophobic core of triacylglycerols and sterol esters surrounded by a phospholipid monolayer. This organization creates biophysical properties that guide selective protein recruitment. Among LD-associated proteins, /{beta}-hydrolase domain-containing protein 5 (ABHD5, also known as CGI-58) is a key regulator of lipolysis and broader lipid metabolism, yet the mechanisms guiding its distribution between endoplasmic reticulum (ER) bilayers and LD monolayers remain poorly understood. Because proper membrane association of ABHD5 is essential for activating PNPLA family lipases, identifying the determinants of its membrane selectivity is critical for understanding LD function in health and disease. In this study, we examined ABHD5 binding and sorting behavior using model membrane systems composed of giant unilamellar vesicles (GUVs) and droplet-embedded vesicles (DEVs) incorporating defined phospholipid and neutral lipid compositions. By integrating experimental assays with computational modeling, we quantified how ABHD5 partitions between bilayer membranes mimicking the ER and monolayer surfaces mimicking LDs. Systematic variation of membrane composition and physical properties allowed us to assess how packing defects and neutral lipid content shape ABHD5 localization. Our findings reveal the biophysical features that favor ABHD5 association with LD-like monolayers and provide new mechanistic insight into how cells target regulatory proteins to distinct membrane environments to control lipid metabolism.

biophysics↗

bioRxiv2025

Unraveling the Molecular Mechanisms of ABHD5 Membrane Targeting

ABHD5 is a master regulator of PNPLA family lipases, particularly PNPLA2 (ATGL), the rate-limiting triglyceride (TAG) hydrolase in metabolic tissues. Despite its central role in lipid metabolism, the molecular basis by which ABHD5 recognizes membranes and regulates enzymatic activity remains poorly understood. Here, we report an integrated computational-experimental study revealing how the /{beta}-hydrolase domain-containing protein 5 (ABHD5) dynamically engages lipid droplet (LD) and endoplasmic reticulum membranes to control lipolytic activation. Using multiscale molecular dynamics simulations, hydrogen-deuterium exchange mass spectrometry, and site-directed mutagenesis, we uncover a sequential dual-site membrane recognition mechanism, where the N-terminus provides initial anchoring and a lid helix within the insertion segment forms a crucial secondary contact. Membrane binding triggers a dramatic conformational switch in this lid, expanding the pseudosubstrate pocket and transforming ABHD5 into an active and membrane-localized regulator. This structural transition is coupled to membrane remodeling, inducing localized curvature and forming a triacylglycerol-enriched nanodomain beneath the ABHD5 pseudosubstrate pocket. This bidirectional interaction between ABHD5 and the membrane provides a persuasive mechanism for interfacial activation. Our findings establish new principles for how LD-binding proteins achieve functional specificity through membrane-dependent regulation, offering novel molecular targets for interventions in metabolic diseases. TeaserA key protein controls fat breakdown by changing shape when it binds membranes and reshapes local fat droplets.

biophysics↗

bioRxiv2024

Lipid Droplet Targeting of ABHD5 and PNPLA3 I148M is required to promote liver steatosis

The storage and release of triacylglycerol (TAG) in lipid droplets (LDs) is regulated by dynamic protein interactions. /{beta} hydrolase domain-containing protein 5 (ABHD5; also known as CGI-58) is a membrane/LD bound protein that functions as a co-activator of Patatin Like Phospholipase Domain Containing 2 (PNPLA2; also known as Adipose triglyceride lipase, ATGL) the rate-limiting enzyme for TAG hydrolysis. The dysregulation of TAG hydrolysis is involved in various metabolic diseases such as metabolic dysfunction-associated steatotic liver disease (MASLD). We previously demonstrated that ABHD5 interacted with PNPLA3, a closely related family member to PNPLA2. Importantly, a common missense variant in PNPLA3 (I148M) is the greatest genetic risk factor for MASLD. PNPLA3 148M functions to sequester ABHD5 and prevent co-activation of PNPLA2, which has implications for initiating MASLD; however, the exact mechanisms involved are not understood. Here we demonstrate that LD targeting of both ABHD5 and PNPLA3 I148M is required for the interaction. Molecular modeling demonstrates important resides in the C-terminus of PNPLA3 for LD binding and fluorescence cross-correlation spectroscopy demonstrates that PNPLA3 I148M greater associates with ABHD5 than WT PNPLA3. Moreover, the C-terminus of PNPLA3 is sufficient for functional targeting of PNPLAs to LD and the interaction with ABHD5. In addition, ABHD5 is a general binding partner of LD-bound PNPLAs. Finally, PNPLA3 I148M targeting to LD is required to promote steatosis in vitro and in the liver. Overall results suggest that PNPLA3 I148M is a gain of function mutation and that the interaction with ABHD5 on LD is required to promote liver steatosis.

cell biology↗

bioRxiv2023

Methods for making and observing model lipid droplets

The mechanisms by which the lipid droplet (LD) membrane is remodeled in concert with the activation of lipolysis incorporate a complex interplay of proteins, phospholipids, and neutral lipids. Model LDs (mLDs) provide an isolated, purified system for testing the mechanisms by which the droplet composition, size, shape, and tension affects triglyceride metabolism. Described here are methods of making and testing mLDs ranging from 0.1 to 40 {micro}m diameter with known composition. Methods are described for imaging mLDs with high-resolution microscopy during buffer exchanges for the measurement of membrane binding, diffusion, and tension via fluorescence correlation spectroscopy (FCS), fluorescence recovery after photobleaching (FRAP), fluorescence lifetime imaging microscopy (FLIM), atomic force microscopy (AFM), pendant droplet tensiometry, and imaging flow cytometry. These complementary, cross-validating methods of measuring LD membrane behavior reveal the interplay of biophysical processes in triglyceride metabolism.

biophysics↗

bioRxiv2023

Four-color fluorescence cross-correlation spectroscopy with one laser and one camera

The diffusion and reorganization of phospholipids and membrane-associated proteins are fundamental for cellular function. Fluorescence cross-correlation spectroscopy (FCCS) measures the diffusion and molecular interactions at nanomolar concentration in biological systems. We have developed a novel, economical method to simultaneously monitor diffusion and oligomerization with the use of super-continuum laser and spectral deconvolution from a single detector. Customizable excitation wavelengths were chosen from the wide-band source and spectral fitting of the emitted light revealed the interactions for up to four spectrally overlapping fluorophores simultaneously. This method was applied to perform four-color FCCS, as demonstrated with polystyrene nanoparticles, lipid vesicles, and membrane-bound molecules. Up to four individually customizable excitation channels were selected from the broad-spectrum fiber laser to excite the diffusers within a diffraction-limited spot. The fluorescence emission passed through a cleanup filter and a dispersive prism prior to being collected by a sCMOS or EMCCD camera with up to 10 kHz frame rates. The emission intensity versus time of each fluorophore was extracted through a linear least-square fitting of each camera frame and temporally correlated via custom software. Auto- and cross-correlation functions enabled the measurement of the diffusion rates and binding partners. We have measured the induced aggregation of nanobeads and lipid vesicles in solution upon increasing the buffer salinity. Because of the adaptability of investigating four fluorophores simultaneously with a cost-effective method, this technique will have wide application for examining complex homo- and heterooligomerization in model and living systems.

biophysics↗

bioRxiv2020

Coexisting lipid phases alter lipid dynamics and sort on nanoscale membrane curvature

The precise spatiotemporal control of nanoscale membrane shape and composition is the result of complex interplay of individual and collective molecular behaviors. Here, we employed single-molecule localization microscopy and computational simulations to observe single-lipid diffusion and sorting in model membranes with varying compositions, phase, temperature, and curvature. Supported lipid bilayers were created over 50-nm radius nanoparticles to mimic the size of naturally occurring membrane buds, such as endocytic pits and the formation of viral envelopes. The curved membranes recruited liquid-disordered lipid phases while altering the diffusion and sorting of tracer lipids. Disorder-preferring fluorescent lipids sorted to and experienced faster diffusion on the nanoscale curvature only when embedded in a membrane capable of sustaining lipid phase separation at low temperatures. The curvature-induced sorting and faster diffusion even occurred when the sample temperature was above the miscibility temperature of the planar membrane, implying that the nanoscale curvature could induce phase separation in otherwise homogeneous membranes. Further confirmation and understanding of these results are provided by continuum and coarse-grained molecular dynamics simulations with explicit and spontaneous curvature-phase coupling, respectively. The curvature-induced membrane compositional heterogeneity and altered dynamics were achieved only with a coupling of the curvature with a lipid phase separation. These cross-validating results demonstrate the complex interplay of lipid phases, molecular diffusion, and nanoscale membrane curvature that are critical for membrane functionality. SignificanceNanoscopic membrane organization and dynamics are critical for cellular function but challenging to experimentally measure. This work brings together super-resolution optical methods with multiscale computational approaches to reveal the interplay between curvature, composition, phase, and diffusion in model membranes. We report that curvature can induce phase separation in otherwise homogeneous membranes and that the phase-curvature coupling has a direct implication on lipid mobility. This discovery advances our understanding of the fundamental membrane biophysics that regulate membrane activities such as endocytosis and viral budding.

biophysics↗

bioRxiv2020

Single-lipid dynamics in phase-separated supported lipid bilayers

Phase separation is a fundamental organizing mechanism on cellular membranes. Lipid phases have complex dependencies on the membrane composition, curvature, tension, and temperature. Single-molecule diffusion measures a key characteristic of membrane behavior and relates to the effective membrane viscosity. Lipid diffusion rates vary by up to ten-fold between liquid-disordered (Ld) and liquid-ordered (Lo) phases depending on the membrane composition, measurement technique, and the surrounding environment. This manuscript reports the lipid diffusion on phase-separated supported lipid bilayers (SLBs) with varying temperature, composition, and lipid phase. Lipid diffusion is measured by single-particle tracking (SPT) and fluorescence correlation spectroscopy (FCS) via custom data acquisition and analysis protocols that apply to diverse membranes systems. We demonstrate agreement between FCS and SPT analyses with both the single-step length distribution and the mean squared displacement of lipids with significant immobile diffusers. Traditionally, SPT is sensitive to diffuser aggregation, whereas FCS largely excludes aggregates from the reported data. Protocols are reported for identifying and culling the aggregates prior to calculating diffusion rates via SPT. With aggregate culling, all diffusion measurement methods provide consistent results. With varying membrane composition and temperature, we demonstrate the importance of the tie-line length that separates the coexisting lipid phases in predicting the differences in diffusion between the Ld and Lo phases. HIGHLIGHTSO_LILipid diffusion varies with the lipid phases, temperature, and aggregation C_LIO_LIAggregate culling yields consistent measurements from single-particle tracking and fluorescence correlation spectroscopy C_LIO_LIMembrane with higher cholesterol content or at low temperature have more aggregates C_LIO_LIA more variation in the diffusion rates occurred between the coexisting lipid phases at low temperatures and low cholesterol content C_LI

biophysics↗

bioRxiv2020

Structured clustering of the glycosphingolipid GM1 is required for membrane curvature induced by cholera toxin

AB5 bacterial toxins and polyomaviruses induce membrane curvature as a mechanism to facilitate their entry into host cells. How membrane bending is accomplished is not yet fully understood but has been linked to the simultaneous binding of the pentameric B-subunit to multiple copies of their glycosphingolipid receptors. Here, we probe the toxin membrane binding and internalization mechanisms by using a combination of super-resolution and polarized localization microscopy. We show that cholera toxin subunit B (CTxB) can induce membrane curvature only when bound to multiple copies of its glycosphingolipid receptor, GM1, and the ceramide structure of GM1 is likely not a determinant of this activity as assessed in model membranes. A mutant CTxB capable of binding only a single GM1 fails to generate curvature either in model membranes or in cells and clustering the mutant CTxB-single-GM1 complexes by antibody cross-linking does not rescue the membrane curvature phenotype. We conclude that both the multiplicity and specific geometry of GM1 binding sites are necessary for the induction of membrane curvature. We expect this to be a general rule of membrane behavior for all AB5 toxins and polyomaviruses that bind glycosphingolipids to invade host cells. SIGNIFICANCE STATEMENTMembrane binding toxins demonstrate both a public health challenge and a bioengineering opportunity due to their efficient internalization into cells. These toxins multivalently bind to naturally occurring lipid receptors at the plasma membrane and initiate endocytosis. This manuscript reports the importance of structured lipid-receptor clustering for the induction of membrane bending. We also observed that the magnitude of membrane curvature was correlated to the stoichiometry of toxin-bound receptors. By identifying how these bacterial proteins initiate membrane curvature, these findings provide mechanistic insights into the early steps of pathogenic endocytosis.

biophysics↗