Biology subjects

Granneman, J. G.

Publications and source records attributed to Granneman, J. G..

9 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↗

bioRxiv2026

Mitochondrial carrier SLC25A34 links clock, diet, and temperature control of interorganellar lipid cycling

Adipocyte lipid metabolism is coordinated by circadian rhythms, diet, and environmental temperature. Yet how these diverse signals are molecularly integrated remains unknown. Here we show that clock, diet, and temperature cues converge on the orphan mitochondrial transporter, SLC25A34, to orchestrate thermogenic cycling of lipid synthesis and oxidation. During sleep, the clock suppresses Slc25a34 transcription through REV-ERB. Waking, lipid-rich diets, or cold exposure abolish this repression, allowing lipolytic signals to stimulate Slc25a34 expression via PPAR. SLC25A34 then imports oxaloacetate into mitochondria to accelerate the export of substrates used for acetyl-CoA production in the cytosol. This feeds into cytosolic lipid synthesis and transcriptional induction of mitochondrial biogenesis, which collectively promote mitochondrial lipid oxidation. Thus, SLC25A34 confers circadian, dietary, and environmental control of thermogenic metabolism through interorganellar lipid cycling.

molecular biology↗

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↗

bioRxiv2025

PNPLA3 I148M Reduces Hepatic Triacylglycerol Secretion and Mitigates Left Ventricular Diastolic Dysfunction in MASH Diet Mice

Cardiovascular disease (CVD) is a leading cause of mortality among individuals with metabolic dysfunction-associated steatotic liver disease (MASLD). Paradoxically, the strongest genetic risk factor for MASLD, PNPLA3-I148M, is associated with a reduced risk of CVD. The mechanisms for how PNPLA3-I148M causes MASLD and preserves cardiac health are not well understood. To further investigate cardioprotective effects of PNPLA3-I148M we expressed human WT-PNPLA3, PNPLA3-I148M or a GFP control in the livers of PNPLA3-/- mice. In agreement with prior investigations, mice expressing PNPLA3-I148M displayed greater hepatic neutral lipid accumulation on chow and a Metabolic Dysfunction-Associated Steatohepatitis (MASH) promoting diet. Changes in hepatosteatosis between the groups was not due to alterations in whole body metabolic parameters, although WT-PNPLA3 promoted greater glucose intolerance on a MASH diet. Echocardiography revealed that hearts from PNPLA3-WT mice had changes in left ventricular mass and thickness following 16-weeks of MASH, but not chow diet, which was not seen in the GFP or PNPLA3-I148M groups. Moreover, PNPLA3-WT and GFP mice had reduced E/A ratios (diastolic function), post MASH diet, which was not detected in PNPLA3-I148M mice. In addition, PNPLA3-I148M reduced hepatic secretion of TAGs under a MASH, but not chow diet. The expression of PNPLA3-I148M modified the liver lipidome, while minimal effects were observed in the heart. No differences in atherosclerotic plaque formation were observed following 24 weeks of MASH diet. These findings indicate that hepatic PNPLA3-I148M promotes hepatosteatosis yet protects against diet induced cardiac remodeling and diastolic dysfunction primarily through reductions in hepatic TAG secretion.

physiology↗

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↗

bioRxiv2022

VPS13A and VPS13C influence lipid droplet abundance

Lipid transfer proteins mediate the exchange of lipids between closely apposed membranes at organelle contact sites and play key roles in lipid metabolism, membrane homeostasis, and cellular signaling. A recently discovered novel family of lipid transfer proteins, which includes the VPS13 proteins (VPS13A-D), adopt a rod-like bridge conformation with an extended hydrophobic groove that enables the bulk transfer of membrane lipids for membrane growth. Loss of function mutations in VPS13A and VPS13C cause chorea acanthocytosis and Parkinsons disease, respectively. VPS13A and VPS13C localize to multiple organelle contact sites, including endoplasmic reticulum (ER) - lipid droplet (LD) contact sites, but the functional roles of these proteins in LD regulation remains mostly unexplored. Here, we employ CRISPR-Cas9 genome editing to generate VPS13A and VPS13C knockout cell lines in U-2 OS cells via deletion of exon 2 and introduction of an early frameshift. Analysis of LD content in these cell lines revealed that loss of either VPS13A or VPS13C results in reduced LD abundance under oleate-stimulated conditions. These data implicate VPS13A and VPS13C in LD regulation and raise the intriguing possibility that VPS13A and VPS13C-mediated lipid transfer facilitates LD biogenesis.

cell biology↗

bioRxiv2022

Deconstructing cold-induced brown adipocyte neogenesis in mice

Cold-exposure triggers neogenesis in classic interscapular brown adipose tissue (BAT) that involves activation of b1-adrenergic receptors, proliferation of PDGFRA+ adipose tissue stromal cells (ASCs), and recruitment of immune cells whose phenotypes are presently unknown. Single- cell RNA-sequencing (scRNA-seq) identified three ASC subpopulations that occupied distinct tissue locations. Of these, interstitial ASC1 were found to be direct precursors of new brown adipocytes (BA). Surprisingly, knockout of b1-adrenergic receptors in ASCs did not prevent cold- induced neogenesis, whereas pharmacological activation of the b3-adrenergic receptor on BAs was sufficient, suggesting that signals derived from mature BAs indirectly trigger ASC proliferation and differentiation. In this regard, cold exposure induced the delayed appearance of multiple macrophage and dendritic cell populations whose recruitment strongly correlated with the onset and magnitude of neogenesis across diverse experimental conditions. High resolution immunofluorescence and single molecule fluorescence in situ hybridization demonstrated that cold-induced neogenesis involves dynamic interactions between ASC1 and recruited immune cells that occur on the micrometer scale in distinct tissue regions. Our results indicate that neogenesis is not a reflexive response of progenitors to b-adrenergic signaling, but rather is a complex adaptive response to elevated metabolic demands within brown adipocytes.

cell biology↗

bioRxiv2022

MEIG1/PACRG associated and non-associated functions of axonemal dynein light intermediate polypeptide 1 (DNALI1) in mammalian spermatogenesis

Axonemal dynein light intermediate polypeptide 1 (DNALI1) was originally cloned from Chlamydomonas reinhardtii in an effort to find motor proteins essential for flagellar motility. Here we report that DNALI1 is a binding partner of parkin co-regulated gene 1 (PACRG), which forms a complex with meiosis expressed gene 1 (MEIG1) in the manchette, a transient and unique structure only present in the elongating spermatids and required for normal spermiogenesis of the male germ cell differentiation process. DNALI1 recruits the PACRG protein in transfected CHO cells, and also stabilizes PACRG in bacteria and transfected mammalian cells. The untagged DNALI1 could also be co-purified with His-tagged PACRG in the gel filtration assay. Immunofluorescence staining on isolated male germ cells revealed that DNALI1 was present in the manchette of elongating spermatids, and colocalized with PACRG in this structure. In Pacrg mutant mice, localization of DNALI1 in the manchette was not changed, suggesting that DNALI1 and PACRG form a complex in the manchette, with DNALI1 being an upstream molecule. Mice deficiency in DNALI1 specifically in male germ cells showed dramatically reduced sperm numbers and were infertile. In addition, majority of the sperm exhibited abnormal morphology including misshapen heads, bent tails and enlarged midpiece, discontinuous accessory structure, and loss of sperm individualization, emphasizing the importance of DNALI1 in sperm development. Examination of testis histology revealed impaired spermiogenesis in the conditional Dnali1 knockout mice. Electron microscopy revealed disrupted ultrastructure in sperm of the Dnali1 mutant mice. Testicular levels of MEIG1, PACRG and SPAG16L proteins were not changed in the Dnali1 mutant mice. However, MEIG1 and SPAG16L were no longer present in the manchette in the absence of DNALI1. These findings demonstrate that DNALI1 is involved in the connection of the MEIG1/PACRG complex to carry cargo proteins along the manchette microtubules for sperm flagella formation. Given that Dnali1 mutant mice showed impaired sperm individualization that was not observed in the MEIG1 nor PACRG-deficient mice, DNALI1 might fulfill other functions beyond its role associated with the MEIG1/PACRG complex. Thus, DNALI1 plays multiple roles in sperm cell differentiation and function. Summary statementAxonemal dynein light intermediate polypeptide 1 (DNALI1) is required for sperm formation and male fertility. It associates with the MEIG1/PACRG complex in the manchette and is involved in a cargo transport system. In addition, it might be related to IFT and sperm individualization.

cell biology↗