Biology subjects

Klymchenko, A. S.

Publications and source records attributed to Klymchenko, A. S..

At least 19 recordsLinked to original sources

bioRxiv2026

Covalent Solvatochromic Membrane Probe for Sensing Lipid Surrounding of Proteins

Plasma membranes (PM) have been traditionally studied by fluorescent probes by either labelling lipid or protein parts. Based on our MemGraft concept, we report a fluorescent probe based on solvatochromic dye Nile Red (MemGraft-NR) for sensing lipid organization in the immediate surrounding of proteins, i.e. at the protein-lipid interface. Two sequentially attached MemGraft scaffolds are required to ensure robust grafting of Nile Red to PM. In contrast to classical lipid-bound membrane probes, MemGraft-NR exhibits high resistance to the presence of serum and fixation with permeabilization. Two-color confocal imaging of MemGraft-NR in live cells reveals higher polarity at the protein-lipid interface compared to lipid-bound probe NR12A. Surprisingly, MemGraft-NR displays higher sensitivity to variation of cholesterol content in PM compared to NR12A, suggesting that protein-lipid interface is particularly impacted by changes in the lipid composition. MemGraft-NR also enabled in situ monitoring of lipid exchange and permeabilization of PM, something impossible for classical solvatochromic PM probes. Unexpectedly, MemGraft-NR reveals a dramatic decrease in the local polarity in early endosomes, suggesting that endosomal negative curvature provides strong impact on organization of the protein-lipid interface. The developed probe opens a new way to study biological membranes by sensing lipid surrounding of membrane proteins.

biochemistry↗

bioRxiv2026

Cerebral microthrombi promote focal Cav-1-dependent blood-brain barrier impairment, triggering neuroinflammation and neuronal damage after traumatic brain injury

Traumatic brain injury (TBI) is frequently accompanied by blood-brain barrier (BBB) dysfunction, yet the microvascular events that initiate barrier failure and secondary neural injury remain poorly understood. Using highly sensitive fluorescent nanoscale tracers, correlative light and electron microscopy, single-cell transcriptomics, and genetic manipulation of Caveolin-1 (Cav-1), we show that BBB leakage after TBI in mice occurs focally at sites of cerebral microthrombus formation. Endothelial cells adjacent to microthrombi remain structurally intact, but exhibit a distinct transcriptional program enriched in both thrombosis- and transcytosis-related genes, including Cav-1. Microthrombi promote Cav-1-dependent transcytosis and size-selective extravasation of blood-borne proteins, exposing the surrounding parenchyma to circulating factors and inducing focal microglia activation and neuronal damage in vivo. In human induced pluripotent stem cell-derived neurons, these proteins directly impaired neuronal structure and network function. Genetic deletion of Cav-1 markedly reduces microthrombus-associated BBB leakage, immune-cell diapedesis, microglial activation and neuronal loss, whereas endothelial re-expression of Cav-1 using AAVs restores leakage. Human TBI data reveal early dynamic coagulation dysregulation associated with adverse clinical outcome, focal cerebral vascular injury and endothelial transcriptional signatures consistent with reduced barrier integrity and increased caveolae-associated transport. Together, these findings identify cerebral microthrombi as focal sites of Cav-1-dependent BBB dysfunction and establish pathological endothelial transcytosis as a mechanistic link between post-traumatic microvascular thrombosis, blood protein extravasation and secondary neuronal injury. Targeting Cav-1-dependent transcytosis may therefore provide a strategy to limit pathological BBB permeability and secondary injury after TBI.

neuroscience↗

bioRxiv2026

Rhodamine-derived ratiometric fluorescent molecular rotor for mitochondrial viscosity sensing

Fluorescent molecular rotors for sensing local microviscosity have become indispensable tools for better understanding the mechanisms of living systems. Current probes relay on intensity or lifetime measurements, while ratiometric molecular rotors are lacking. Here, we introduce a new design concept of molecular rotor using xanthene scaffold with freely rotating 9-aryl group. We studied three rhodamine derivatives, where 2-carboxyphenyl group was replaced with 4-methoxyphenyl, 2-thienyl and 2-benzofuryl. We found that five membered 9-aryl ring provides two major effects. First, free rotation of thienyl derivative, confirmed by theoretical calculations, makes it classical molecular rotor with intensity and lifetime-based response to viscosity. Second, benzofuryl derivative exhibits second emission band in near-infrared region and fluorescence ratiometric response to viscosity. Theoretical calculations suggest that the benzofuryl derivative can attain planar conformation in the excited state, which explains appearance of the NIR emitting band. Both new dyes efficiently target mitochondria, while the 9-benzofuryl derivative enable quantitative ratiometric measurement of viscosity of inner mitochondrial membrane. Ratiometric imaging revealed mitochondria heterogeneity with local viscosity values ranging from 250 to 460 cP. Oxidative stress induced significant rise in the local viscosity in mitochondria in addition to their morphological changes. Overall, we propose a concept that converts bright rhodamine dyes into molecules rotors with a valuable ratiometric response to viscosity, which opens a bunch of potential biological applications.

biochemistry↗

bioRxiv2026

Fluorescence based microviscosity mapping in membraneless organelles

Membraneless organelles (MLOs) are cellular biomolecular condensates formed by liquid-liquid phase separation. Their biological functions are intimately linked to their material properties including viscosity. Condensate viscosity is determined by the size, shape, concentration and molecular interactions between MLOs components. It impacts the diffusion of MLOs constituents and the selective permeability of the condensate, thereby regulating the rate of biochemical reactions. Viscosity modifications associated with liquid-to-gel transition of the condensates are related to pathologies. Current experimental approaches for characterizing the material properties of cellular condensates remain limited. In this study, we report the use of BODIPY-based molecular rotors, in combination with fluorescence lifetime imaging microscopy (FLIM), to monitor the microviscosity of cellular MLOs directly in living cells. The fluorescence lifetime of BODIPY derivatives increases with the viscosity of their microenvironment, enabling quantitative assessment of microviscosity within condensates. HaloTag technology was employed to specifically label MLO components. Our findings reveal that the nucleolus exhibits higher viscosity than the surrounding nucleoplasm and that microviscosity varies across nucleolar sub-compartments. Furthermore, nucleolar reorganization induced by inhibition of rRNA synthesis results in a measurable increase in microviscosity. Finally, we demonstrate that the microviscosity of stress granules is lower than that of the nucleolus. Overall, presented results demonstrate the strong potential of the BODIPY based molecular rotors as a versatile and powerful tools for probing the material properties of cellular MLOs.

biophysics↗

bioRxiv2025

Prodan-based solvatochromic probes for polarity imaging of organelles

Solvatochromic probes provide microscopic, structural and functional information on their targeted cellular compartments. In this field, the challenge lies in designing probes that are both sufficiently sensitive to environment, and specific in their subcellular localization. Here, we design Prodan-based polarity probes targeting the following organelles: mitochondria, endoplasmic reticulum, Golgi apparatus, lysosomes and lipid droplets. The new probes provide robust organelle targeting, except for the mitochondrial probe, whose targeting ability is cell type dependent. Due to operating range of Prodan fluorophore in the UV-Blue region, our probes can be easily combined with other fluorescent tags in the visible range. Therefore, polarity of sub-cellular compartments can be studied together with additional fluorescent reporters. We used these probes to show the polarity of organelle membranes in healthy cells and under starvation condition where we observed organelle-specific polarity remodeling. These probes are important addition to the repertoire of smart cellular probes and will find critical use in understanding spatiotemporal regulation of cellular physiology.

biophysics↗

bioRxiv2025

Functionalization of lipid nanoemulsions with humanized antibodies using plug-and-play cholesterol anchor for targeting cancer cells

Lipid NEs are promising green nanocarriers for diagnostic and therapeutic applications, but their functionalization with biomolecules, such as antibodies, remains a challenge due to liquid nature of their core. Here, we developed an original plug-and-play strategy to graft an antibody (trastuzumab) at the surface of NEs, using components generally recognized as safe (GRAS). We synthesized a reactive derivative of cholesterol and a Biotin-PEG3000-Lysine linker, which can react within one-pot formulation to form an amphiphilic carbamate Biotin-PEG3000-Cholesterol. The cholesterol ensures anchorage of the linker, which effectively exposes biotin moiety at the surface of NEs for further antibody grafting using a biotin-neutravidin coupling. The reaction between the Biotin-PEG3000-Lysine linker and NPC-Chol was confirmed by 1H-NMR and absorption spectroscopy. The obtained biotinylated 50-nm NEs loaded with a near-infrared dye were successfully targeted to neutravidin-coated glass surfaces and imaged at the single-droplet level. The biotinylated NEs bearing the trastuzumab antibody targeted specifically HER2-amplified breast cancer models HCC-1954 and SKBR3, in contrast to control MDA-MB-231 (HER2-low) cells. Altogether, our study proposes an efficient methodology for grafting antibodies to the surface of NEs, which offers new opportunities of application of these green nanocarriers in biomedicine.

pharmacology and toxicology↗

bioRxiv2025

Peptide-functionalized fluorescent polymeric nanoparticles: polysarcosine length defines stealth properties

Stealth properties of nanoparticles are essential for their proper functionalization in biological systems. To address limitations of polyethylene glycol (PEG), commonly used for this purpose, we explore the potential of polysarcosine (PSar) as stealth shell in peptide-functionalized dye-loaded polymeric NPs. To this end, polymeric NPs loaded with rhodamine dye with bulky hydrophobic counterion and bearing azide groups at their surface were grafted with PSar of different lengths ranging from 5 to 19 sarcosine units using strain-promoted cycloaddition. The obtained peptide-functionalized NPs showed remarkable colloidal stability in physiological media. The length of PSar showed a profound effect on stealth properties of NPs. The increase in the length of grafted PSar lead to decrease in the negative surface charge to nearly neutral values and decreased protein adsorption according to fluorescence correlation spectroscopy. The NPs with 19mer PSar showed minimal interactions with live cells and glass surfaces in a complex biological medium, in contrast to its shorter PSar analogues. These stealth NPs bearing HaloTag ligand enabled specific targeting of proteins at the cell surface. The obtained results show that a relatively short PSar peptide can be used for achieving stealth properties in polymeric NPs, allowing specific protein targeting with minimized non-specific interactions. The obtained PSar-functionalized polymeric NPs appear as a powerful platform for the fabrication of the next generation of nanomaterials for bioimaging and biosensing applications.

pharmacology and toxicology↗

bioRxiv2025

Cooperative effects in DNA-functionalized polymeric nanoparticles

DNA-functionalized nanoparticles (NPs), called as spherical nucleic acids (SNA), attract strong attention due to their unique properties and numerous applications. In particular, DNA-functionalized dye-loaded polymeric NPs (DNA-NPs), owing to their exceptional fluorescence brightness emerge as powerful nanomaterials for ultrasensitive detection and imaging of nucleic acids. Here, we addressed a fundamental question unexplored for polymeric DNA-NPs: how dense packing of oligonucleotides at the particle surface impacts their capacity to specifically hybridize with complementary sequences. Using Forster Resonance Energy Transfer (FRET) between DNA-NPs and labelled complementary strands, we found that the DNA at the NPs surface exhibit dramatic enhancement in the duplex stability compared to free DNA duplexes (>20 {degrees}C). This effect increases at higher densities of DNA on NPs surface, which suggests that DNA cooperativity is responsible for the duplex stability enhancement. For example, 8 nt DNA duplexes were perfectly stable at RT on the surface of DNA-NPs. Furthermore, these DNA-NPs preserve capacity to distinguish mutations, even at the single nucleotide level within 21 nt sequence, when appropriate hybridization temperature is used. The hybridization between DNA-NPs and the complementary sequences proceeds on the min time scale at probe and target concentrations [≥] 10 and [≥]100 pM, respectively. Below that, this diffusion-controlled processes becomes too slow, which points on the fundamental limitation in the DNA/RNA sensing assays that require sufficiently high nanoprobe concentration. The present study sheds light on the capacity of DNA-NPs to specifically hybridize with the target sequences and provides insights in the development of nucleic acid sensing assays.

bioengineering↗

bioRxiv2025

Order disorder phase transitions in the plasma membrane of HeLa cells measured by fluorescent analysis of solvatochromic probes

Using the solvatochromic membrane probes Laurdan and Pro12A we investigated order-disorder phase transitions in cellular lipid membranes of HeLa cells. Due to internalization of Laurdan its fluorescence signal yields information about inner and outer cellular membranes while Pro12A exclusively stains the plasma membrane. The two different membrane-embedded dyes show an emission redshift upon increasing disorder of the surrounding lipids that can be quantified by the Generalized Polarization (GP). First, we compare the sensitivity of both probes to lipid phase transitions by analyzing GP of synthetic lipid vesicles as function of temperature. Second, we investigate the temperature dependent lipid order of HeLa cell membranes and find that the plasma membrane shows a partially reversible order-disorder transition regime at temperatures between T = 20 {degrees}C and T = 70 {degrees}C. Third, we calorimetrically detect an irreversible transition at T = 55 {degrees}C and conclude that the optically detected restructuring of the plasma membrane can be partially attributed to protein denaturation. At last, it is shown that the reversible plasma membrane transition temperature Tm shifts from Tm = 25 {degrees}C to Tm =-16 {degrees}C upon cholesterol depletion and sharpens from a half width of {Delta}TFWHM = 60 K to {Delta}TFWHM = 8 K. The reversibility and the sensitivity to cholesterol of this transition indicate a temperature-induced lipid melting within the plasma membrane of HeLa cells.

biophysics↗

bioRxiv2024

Fluorescent anionic cyanine plasma membrane probes for live cell and in vivo imaging

Proper staining of cell plasma membrane is indispensable for fluorescence imaging. Herein, we present an array of five anionic cyanine-based turn-on plasma membrane probes with emission spanning from green to near infrared. They are analogous of commonly used MemBright probes family, where two zwitterionic anchor groups are replaced with anionic sulfonates with dodecyl chains. The developed probes provide selective wash-free staining of plasma membranes of live cells in vitro, featuring improved brightness and slower internalization inside the cells. In comparison to protein-based (wheat germ agglutinin) membrane markers, new membrane probes provide better staining in poorly assessable cell-cell contacts. A key challenge is to stain cell plasma membranes directly in vivo. During in vivo brain tissue imaging in living mice by two-photon microscopy, the anionic cyanine probes allowed us to visualize in detail the pyramidal neurons with high image quality, clearly resolving neuron soma, dendrites with dendritic spines and axons with axonal boutons. The developed anionic cyanine-based plasma membrane probes constitute an important extension of the toolbox of fluorescent probes for plasma membrane research.

biochemistry↗

bioRxiv2024

Lipid-directed covalent fluorescent labeling of plasma membranes for long-term imaging, barcoding and manipulation of cells

Fluorescent probes for cell plasma membrane (PM) are generally based on amphiphilic anchors that incorporate non-covalently into biomembranes. Therefore, they are not compatible with fixation and permeabilization, presence of serum, or cell co-culture because of their exchange with the medium. Here, we report a concept of lipid-directed covalent labeling of PM, which exploits transient binding to lipid membrane surface generating high local dye concentration, thus favoring covalent ligation to random proximal membrane proteins. This concept yielded a class of fluorescent probes for PM (MemGraft), where a cyanine dye (Cy3 and Cy5) bears at its two ends low-affinity membrane anchor and reactive group: an activated ester or a maleimide. We found that MemGraft probes with these reactive groups provide efficient PM labelling, in contrast to a series of control compounds, including commercial Cy3-based labels of amino and thiol groups, revealing the crucial role of the membrane anchor combined with high reactivity of activated ester and a maleimide groups. In contrast to conventional PM probes, based on non-covalent interactions, MemGraft labelling approach is compatible with cell fixation, permeabilization, trypsinization and presence of serum. The latter allows long-term cell tracking and video imaging of cell PM dynamics without signs of phototoxicity. The covalent strategy also enables staining and long-term tracking of co-cultured cells labelled in different colors without probes exchange. Moreover, combination of different ratios of MemGraft-Cy3 and MemGraft-Cy5 probes enabled long-term cell barcoding in at least 5 color codes, important for tracking and visualizing multiple cells populations. Ultimately, we found that MemGraft strategy enables efficient biotinylation of cell surface, opening the path to cell surface engineering and cell manipulation.

biochemistry↗

bioRxiv2024

A Cell-Permeable Fluorescent Probe Reveals Temporally Diverse PI(4,5)P2 Dynamics Evoked by Distinct GPCR Agonists in Neurons

Lipids, key constituents of cell-membranes, are the first responders to cell signals. At the crux of spatiotemporal dynamics of lipid-signaling response are phosphoinositides. Indeed, phosphoinositides like phosphatidylinositol-(4,5)-bisphosphate (PI(4,5)P2), present in the inner-leaflet of eukaryotic cell-membranes, form the link between signal reception and downstream signal-transmission. In this backdrop, reversible fluorescent probes that can track live PI(4,5)P2 dynamics on a seconds time-scale will afford key insights into lipid-mediated signaling. However, realizing cell-permeable PI(4,5)P2-selective sensors for imaging dynamics remains a challenge due to the presence of structurally similar lipids and low levels of PI(4,5)P2. We report a computationally-designed, rapid-response, reversible, photo-stable, fluorescent sensor that permeates living cells, neurons, and a multicellular organism within few min of direct incubation and distinctly visualizes PI(4,5)P2 pools. We used the sensor to interrogate the role of PI(4,5)P2 in driving heterogeneity of signaling responses and contrasting behavioral effects that ensue upon binding of distinct ligands to the same G protein-coupled receptor. Specifically, we asked whether probing PI(4,5)P2 dynamics using our novel sensor could uncover the earliest of signaling differences evoked by hallucinogenic versus non-hallucinogenic ligands at the serotonin2A (5-HT2A) receptor. Our results reveal that a hallucinogenic ligand at the 5-HT2A receptor leads to a slower rate of PI(4,5)P2-depletion when compared to a non-hallucinogenic ligand, within the initial seconds of ligand addition, but has a sustained effect. The ability of our designer chemical probe in timing early seconds-minute timescale lipid-dynamics in living cells opens avenues for tracking early time-point molecular events in neuronal response to chemical and physical stimuli.

biochemistry↗

bioRxiv2024

Using a Foerster-resonance energy transfer (FRET)-based detection system (FedEcs) to monitor nanoparticle cargo delivery to the brain

Nanotechnology holds great promise for improving the delivery of therapeutics to the brain. However, current approaches often operate at the organ or tissue level and are limited by the lack of tools to dynamically monitor cargo delivery in vivo. We have developed highly fluorescent lipid nanodroplets (LNDs) that enable tracking of nanocarrier behaviour at the subcellular level while also carrying a Forster resonance energy transfer (FRET)-based drug delivery detection system (FedEcs) capable of monitoring cargo release in vivo. Using two-photon microscopy, we demonstrate that circulating LNDs in naive mouse brain vasculature exhibit 3D real-time FRET changes, showing size-dependent stability over two hours in blood circulation. Further, in a novel nano-stroke model, dynamic intravital two-photon imaging revealed that LNDs accumulated within cerebral post-ischemic microthrombi, where they released their cargo significantly faster than in normal blood circulation. Furthermore, the blood-brain barrier (BBB) became permeable at the microclot sites thereby allowing accumulated FedEcs-LNDs to cross the BBB and deliver their cargo to the brain parenchyma. This microthrombi-associated translocation was confirmed at the ultrastructural level via volume correlative light-electron microscopy. Consequently, our FedEcs represents a novel tool to quantitatively study the biodistribution and cargo release of nanocarriers at high resolution in real time. By enabling us to resolve passive targeting mechanisms post-stroke, - specifically, accumulation, degradation and extravasation via post-stroke microthrombi - this system could significantly enhance the translational validation of nanocarriers for future treatments of brain diseases.

neuroscience↗

bioRxiv2024

High-throughput analysis of membrane fluidity unveils a hidden dimension in immune cell states

Cell membranes undergo biophysical remodelling as an adaptation to the surroundings and to perform specific biological functions. However, the extent and relevance of such changes in human immune cells remain unknown, largely due to the lack of single-cell and multidimensional methodologies. Here, we apply a cytometry-based method to fill this gap by combining biophysical profiling with simultaneous analysis of immune cell markers. This platform reveals notable cell type-dependent plasma membrane order heterogeneity in immune cells. By sorting immune cells according to their membrane order and performing transcriptome and spatial surface proteome analyses together with functional tests, we show that plasma membrane order can be used to identify subsets of immune cells with distinct phenotypes and functional behaviours. Our findings demonstrate a broad heterogeneity of plasma membrane order in immune cells that will provide a more precise definition of immune cell states based on their biophysical properties in health and disease.

biophysics↗

bioRxiv2023

ATUM-Tomo: A multi-scale approach to cellular ultrastructure by combined volume scanning electron microscopy and electron tomography

Like other volume electron microscopy approaches, Automated Tape Collecting Ultramicrotomy (ATUM) enables imaging of serial sections deposited on thick plastic tapes by scanning electron microscopy (SEM). ATUM is unique in enabling hierarchical imaging and thus efficient screening for target structures, as needed for correlative light and electron microscopy. However, SEM of sections on tape can only access the section surface, thereby limiting the axial resolution to the typical size of cellular vesicles with an order of magnitude lower than the acquired xy resolution. In contrast, serial-section electron tomography (ET), a transmission electron microscopy-based approach, yields isotropic voxels at full EM resolution, but requires deposition of sections on electron-stable thin and fragile films, thus making screening of large section libraries difficult and prone to section loss. To combine the strength of both approaches, we developed ATUM-Tomo, a hybrid method, where sections are first reversibly attached to plastic tape via a dissolvable coating, and after screening detached and transferred to the ET-compatible thin films. ATUM-SEM of serial semi-thick sections and consecutive ET of selected re-mounted sections combines SEMs fast target recognition and coarse rendering capability with high-resolution volume visualizations, respectively, thus enabling multi-scale interrogation of cellular ultrastructure. As a proof-of-principle, we applied correlative ATUM-Tomo to study ultrastructural features of blood brain barrier (BBB) leakiness around microthrombi in a mouse model of traumatic brain injury. Microthrombi and associated sites of BBB leakiness were identified by confocal imaging of injected fluorescent and electron-dense nanoparticles, then relocalized by ATUM-SEM, and finally interrogated by correlative ATUM-Tomo. This workflow is a seamless zoom-in on structural BBB pathology from the micro- to the nanometer scale. Overall, our new ATUM-Tomo approach will substantially advance ultrastructural analysis of biological phenomena that require cell- and tissue-level contextualization of the finest subcellular textures.

neuroscience↗

bioRxiv2023

POLCAM: Instant molecular orientation microscopy for the life sciences

Current methods for single-molecule orientation localization microscopy (SMOLM) require optical setups and algorithms that can be prohibitively slow and complex, limiting the widespread adoption for biological applications. We present POLCAM, a simplified SMOLM method based on polarized detection using a polarization camera, that can be easily implemented on any wide-field fluorescence microscope. To make polarization cameras compatible with single-molecule detection, we developed theory to minimize field of view errors, used simulations to optimize experimental design, and developed a fast algorithm based on Stokes parameter estimation which can operate over 1000 fold faster than the state of the art, enabling near instant determination of molecular anisotropy. To aid in the adoption of POLCAM, we developed open-source image analysis software, and a website detailing hardware installation and software use. To illustrate the potential of POLCAM in the life sciences, we applied our method to study alpha-synuclein fibrils, the actin cytoskeleton of mammalian cells, fibroblast-like cells and the plasma membrane of live human T cells.

biophysics↗

bioRxiv2022

Dissecting the mechanisms of environment sensitivity of smart probes for quantitative assessment of membrane properties

The plasma membrane serves as a crucial platform for a multitude of cellular processes. Its collective biophysical properties are largely determined by the structural diversity of the different lipid species it accommodates. Therefore, a detailed investigation of biophysical properties of the plasma membrane is of utmost importance for a comprehensive understanding of biological processes occurring therein. During the past two decades several environment-sensitive probes have been popular tools to investigate membrane properties. Although these probes are assumed to report on membrane order in similar ways, their individual mechanisms remain to be elucidated. In this study, using model membrane systems, we studied the probes Pro12A, NR12S and NR12A in depth and examined their sensitivity to parameters with potential biological implications, such as the degree of lipid saturation, double bond position and configuration, phospholipid headgroup and cholesterol content. Applying spectral imaging together with atomistic molecular dynamics simulations and time-dependent fluorescent shift analyses, we unravelled individual sensitivities of these probes to different biophysical properties, their distinct localizations and specific relaxation processes in membranes. Overall, Pro12A, NR12S and NR12A serve together as a toolbox with a wide range of applications allowing to select the most appropriate probe for each specific research question.

biophysics↗

bioRxiv2022

Pathogenic variants of sphingomyelin synthase SMS2 disrupt lipid landscapes in the secretory pathway

Sphingomyelin is a dominant sphingolipid in mammalian cells. Its production in the trans-Golgi traps cholesterol synthesized in the ER to promote formation of a sphingomyelin/sterol gradient along the secretory pathway. This gradient marks a fundamental transition in physical membrane properties that help specify organelle identify and function. We previously identified mutations in sphingomyelin synthase SMS2 that cause osteoporosis and skeletal dysplasia. Here we show that SMS2 variants linked to the most severe bone phenotypes retain full enzymatic activity but fail to leave the ER owing to a defective autonomous ER export signal. Cells harboring pathogenic SMS2 variants accumulate sphingomyelin in the ER and display a disrupted transbilayer sphingomyelin asymmetry. These aberrant sphingomyelin distributions also occur in patient-derived fibroblasts and are accompanied by imbalances in cholesterol organization, glycerophospholipid profiles and lipid order in the secretory pathway. We postulate that pathogenic SMS2 variants undermine the capacity of osteogenic cells to uphold nonrandom lipid distributions that are critical for their bone forming activity.

cell biology↗