Biology subjects

Lundmark, R.

Publications and source records attributed to Lundmark, R..

10 recordsLinked to original sources

bioRxiv2025

Membrane contacts between caveolae and the endoplasmic reticulum regulate uptake and metabolic trapping of long-chain fatty acids

The vital process of cellular fatty acid metabolism involves uptake, metabolic trapping and storage of long-chain fatty acids (LCFAs) in different lipid species. Although several different enzymes like transporters and acyl-CoA synthetases are involved, the sequential mechanistic steps of the process are poorly understood. Here, we have addressed the role of the caveola coat protein caveolin1 and the acyl-CoA synthetase FATP1 in the process as both have major impacts in the uptake and storage of fatty acids in vivo and in vitro. Using live cell microscopy and mass spectrometry-based quantification of LCFA metabolism, we found that FATP1-mediated uptake and metabolic processing of LCFAs is coupled to, and dependent on caveolin1. Furthermore, both proteins stimulate the incorporation of LCFA into phospholipids and triacylglycerol, but not in sphingolipids. Using correlative light and electron microscopy we found that membrane contact sites are formed between FATP1-enriched endoplasmic reticulum (ER) and caveolae. Analysis of their temporal stability showed that they are dynamic but frequently persist over minutes. We propose that caveolae directly couple the plasma membrane (PM) to the ER for directed LCFA transport and metabolic processing.

cell biology↗

bioRxiv2025

Lipid packing frustration in the outer leaflet of the plasma membrane prevents scission of caveolae

Lipid packing is a fundamental characteristic of bilayer membranes. It affects all membrane-associated processes ranging from curvature generation to membrane fission. Yet, we lack detailed mechanistic understanding of how lipid packing directly affects these processes in cellular membranes. Here, we address this by focusing on caveolae, small 0-shaped invaginations of the plasma membrane which serve as key regulators of cellular lipid sorting and mechano-responses. In addition to caveolae coat proteins, the lipid membrane is a core component of caveolae that critically impacts both the biogenesis, morphology and stability of such membrane invaginations. We show that the small compound Dyngo-4a adsorbs and inserts into the membrane, resulting in a dramatic dynamin-independent inhibition of caveola dynamics. Analysis of model membranes in combination with molecular dynamics simulations revealed that a substantial amount of Dyngo-4a was inserted and positioned at the level of cholesterol in the bilayer affecting lipid order in a cholesterol dependent manner. Dyngo-4a-treatment resulted in decreased lipid packing of the plasma membrane. This prevented caveolae internalization and lateral diffusion without affecting their morphology, associated proteins, or the overall cell stiffness. Artificially increasing plasma membrane cholesterol levels was found to counteract the block in caveola dynamics caused by Dyngo-4a-mediated lipid packing frustration. Therefore, we propose that the outer leaflet lipid packing of cholesterol in the the plasma membrane critically contributes to the confinement of caveolae to the plasma membrane. Significance statementLarsson et al., demonstrate that lipid packing critically impacts the stability of small 0-shaped plasma membrane cavities termed caveolae. The plasma membrane (PM) release of caveolae is dynamin independent and halted by PM incorporation of the small compound Dyngo-4a, which leads to decreased lipid packing as characterized by the authors. The block in caveola internalization can be counteracted by increased PM levels of cholesterol which is enriched in caveolae and increases the lipid packing. This proof-of-principle study shows that lipid packing controls membrane budding generating membrane vesicles that remain stably associated with the membrane for an extended period of time.

cell biology↗

bioRxiv2024

Cryo-electron tomography reveals coupled flavivirus replication, budding and maturation

Flaviviruses replicate their genomes in replication organelles (ROs) formed as bud-like invaginations on the endoplasmic reticulum (ER) membrane, which also functions as the site for virion assembly. While this localization is well established, it is not known to what extent viral membrane remodeling, genome replication, virion assembly, and maturation are coordinated. Here, we imaged tick-borne flavivirus replication in human cells using cryo-electron tomography. We find that the RO membrane bud is shaped by a combination of a curvature-establishing coat and the pressure from intraluminal template RNA. A protein complex at the RO base extends to an adjacent membrane, where immature virions bud. Naturally occurring furin site variants determine whether virions mature in the immediate vicinity of ROs. We further visualize replication in mouse brain tissue by cryo-electron tomography. Taken together, these findings reveal a close spatial coupling of flavivirus genome replication, budding, and maturation.

microbiology↗

bioRxiv2024

Unsaturated lipids as key control points for caveola formation and disassembly

Caveolae are specialized plasma membrane domains with a unique lipid composition. Recent work has implicated lipid peroxidation in triggering caveola disassembly, releasing caveolar proteins to regulate the cellular response to oxidative stress. Here we investigated the role of specific lipid species in caveola formation and response to oxidative stress. Systematic screening of lipid enzymes identified ACSL4, a critical enzyme in the synthesis of polyunsaturated fatty acid-containing lipids, and enzymes involved in the biosynthesis of pro-ferroptotic ether lipids, as key regulators of caveola formation. Omega-6 fatty acids promoted efficient caveola formation, while their oxidation or displacement by omega-3 or monounsaturated fatty acids disrupted this process. Moreover, we found that omega-6 fatty acid-containing membrane lipids were required for oxidative stress-induced caveola disassembly. These findings unveil a new model for caveola formation, highlighting specific unsaturated lipids as essential caveolar components and key control points for caveola disassembly in response to an oxidizing environment.

cell biology↗

bioRxiv2024

The flavivirus protein NS4B recruits the cis-Golgi protein ACBD3 to modify ER-Golgi trafficking for virion release

Flavivirus infection involves extensive remodeling of the endoplasmic reticulum (ER), which is key to both the replication of the viral RNA genome as well as the assembly and release of new virions. Yet, little is known about how viral proteins and host factors cooperatively facilitate such a vast transformation of the ER, and how this influences the different steps of the viral life cycle. In this study, we screened for host proteins that interact with the tick-borne encephalitis virus (TBEV) protein NS4B and found that the top candidates were coupled to trafficking between ER exit sites (ERES) and the Golgi. We characterized the role of ACBD3, one of the identified proteins, in flavivirus infection and show that it interacts with NS4B to promote infection across multiple flavivirus species. Using ACBD3 knockout cells, we found that the depletion of ACBD3 inhibited TBEV replication by preventing the trafficking of virions from the cell. We found that ACBD3 promotes flavivirus infection via a different mechanism than its previously described role in picornavirus infection. ACBD3 was enriched at modified ERES-Golgi contact sites to support virus replication. Therefore, we propose that ACBD3 promotes flavivirus replication by modifying the trafficking between the ERES and the Golgi to enable the release of new virions. Author summaryFlaviviruses including dengue virus and tick-borne encephalitis virus are group of viruses that widely affecting the health of human. During infection, flavivirus particles enter host cells and transform the endoplasmic reticulum (ER), which is the main structure for protein synthesis in cells. New flaviviral particles are produced in the transformed ER and then released to the Golgi apparatus, which is the main structure for protein transport in cells. It is unclear how the particles are transported from the ER to Golgi. Here we screened the factors that interact with viral proteins and identified a Golgi protein called ACBD3 as an important factor supporting flavivirus particles release from ER to Golgi. We showed that ACBD3 is recruited by flavivirus to a modified connection between ER and Golgi for viral particles release from the ER. Our work provides new insights into the fine coordination of virus replication and viral particles transport between organelles inside host cells.

cell biology↗

bioRxiv2022

Simultaneous Membrane and RNA Binding by Tick-Borne Encephalitis Virus Capsid Protein

Tick-borne encephalitis virus is an enveloped, pathogenic, RNA virus in the family Flaviviridae, genus Flavivirus. Viral particles are formed when the nucleocapsid, consisting of an RNA genome and multiple copies of the capsid protein, buds through the endoplasmic reticulum membrane and acquires the viral envelope and the associated proteins. The coordination of the nucleocapsid components to the sites of assembly and budding are poorly understood. Here, we investigate nucleocapsid assembly by characterizing the interactions of the wild-type and truncated capsid proteins with membranes by using biophysical methods and model membrane systems. We show that capsid protein initially binds membranes via electrostatic interactions with negatively-charged lipids which is followed by membrane insertion. Additionally, we show that membrane-bound capsid protein can recruit viral genomic RNA. We confirm the biological relevance of the biophysical findings by using mass spectrometry to show that purified virions contain negatively-charged lipids. Our results suggest that nucleocapsid assembly is coordinated by negatively-charged membrane patches on the endoplasmic reticulum and that the capsid protein mediates direct contacts between the nucleocapsid and the membrane.

biochemistry↗

bioRxiv2022

Dynamin2 stabilizes plasma membrane-connected caveolae by restraining fission

Caveolae are small membrane invaginations that generally are stably attached to the plasma membrane. Their release is believed to depend on the GTPase dynamin 2 (Dyn2), in analogy with its role in fission of clathrin-coated vesicles. The mechanistic understanding of caveola fission, and in particular the role of Dyn2, is however sparse. Here, we used microscopy-based tracking of individual caveolae in living cells to determine the role of Dyn2 in caveola dynamics. We report that Dyn2 stably associated with a subset of caveolae, but was not required for formation or fission of caveolae. The Dyn2-positive subset of caveolae displayed longer plasma membrane duration times, whereas depletion of Dyn2 resulted in shorter duration times and increased caveola fission. The stabilizing role of Dyn2 was independent of its GTPase activity and the caveola stabilizing protein EHD2. Thus, we propose that, in contrast to the current view, Dyn2 restrains caveolae at the plasma membrane and prevents fission.

cell biology↗

bioRxiv2021

Membrane insertion of Cavin1 facilitates caveolae assembly

Caveolae are small plasma membrane invaginations, important for control of membrane tension, signaling cascades and lipid sorting. The caveolae coat protein Cavin1 is essential for shaping such high curvature membrane structures. Yet, a mechanistic understanding of how Cavin1 assembles at the membrane interface is lacking. Here, we used model membranes combined with biophysical dissection and computational modelling to show that Cavin1 inserts into membranes. We establish that initial PI(4,5)P2-dependent membrane adsorption of the trimeric helical region 1 (HR1) of Cavin1 mediates the subsequent partial separation and membrane insertion of the individual helices. Insertion kinetics of the HR1 is further enhanced by the presence of flanking negatively charged disordered regions, which was found important for the co-assembly of Cavin1 with Caveolin1 in living cells. We propose that this intricate mechanism potentiates membrane curvature generation and facilitates dynamic rounds of assembly and disassembly of Cavin1 at the membrane. Significance statementCaveolae are cholesterol enriched membrane invaginations coupled to severe muscle and lipid disorders. Their formation is dependent on assembly of the protein Cavin1 at the lipid membrane interface driving membrane curvature. In this work, we dissect the mechanism for how Cavin1 binds and inserts into membranes using a combination of biochemical and biophysical characterization as well as computational modelling. The proposed model for membrane assembly potentiates dynamic switching between shielded and exposed hydrophobic helices used for membrane insertion and clarifies how Cavin1 can drive membrane curvature and the formation of caveolae.

cell biology↗

bioRxiv2020

Lipid accumulation promotes scission of caveolae

Caveolae, bulb-shaped invaginations of the plasma membrane (PM), show distinct behaviors of scission and fusion at the cell surface. Although it is known that caveolae are enriched in cholesterol and sphingolipids, exactly how lipid composition influences caveolae surface stability has not yet been elucidated. Accordingly, we inserted specific lipids into the PM of cells via membrane fusion and studied acute effects on caveolae dynamics. We demonstrate that cholesterol and glycosphingolipids specifically accumulate in caveolae, which decreases their neck diameter and drives their scission from the cell surface. The lipid-induced scission was counteracted by the ATPase EHD2. We propose that lipid accumulation in caveolae generates an intrinsically unstable domain prone to scission if not balanced by the restraining force of EHD2 at the neck. Our work advances the understanding of how lipids contribute to caveolae dynamics, providing a mechanistic link between caveolae and their ability to sense the PM lipid composition. SUMMARYCaveolae serve as mechanoprotectors and membrane buffers but their specific role in sensing plasma membrane lipid composition remains unclear. Hubert et al. show that cholesterol and glycosphingolipids accumulate in caveolae and drive subsequent scission from the cell surface. These results provide new insight into how lipids contribute to budding and scission of membrane domains in cells.

biochemistry↗

bioRxiv2019

EHD2-mediated restriction of caveolar dynamics regulates cellular lipid uptake

Eps15-homology domain containing protein 2 (EHD2) is a dynamin-related ATPase located at the neck of caveolae, but its physiological function has remained unclear. We found that global genetic ablation of EHD2 in mice led to increased fat deposits in several organs. This organismic phenotype was paralleled at the cellular level by an increased lipid uptake via a CD36-dependent pathway, an elevated number of detached caveolae and higher caveolar mobility. Following a high fat diet, various enzymes involved in de novo fatty acid synthesis were down-regulated in EHD2 KO mice. Furthermore, EHD2 expression itself was down-regulated in the visceral fat of two obese mouse models and in diet-induced obesity. Thus, EHD2 controls a cell-autonomous, caveolae-dependent lipid uptake pathway, implicating a role of EHD2 in fat accumulation and obesity.

cell biology↗