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Caveolae are specialized plasma membrane domains with a unique lipid composition. Lipid peroxidation has recently been implicated in triggering caveola disassembly, releasing cavin proteins to regulate oxidative-stress-associated cellular processes, particularly ferroptosis. Here, we investigated how specific lipids influence caveola formation and their response to oxidative stress. A targeted screening of pro-ferroptotic enzymes identified ACSL4, a key enzyme in synthesizing polyunsaturated fatty acid (PUFA)-linked phospholipids, and ether phospholipid biosynthesis enzymes as critical regulators of caveola formation. Membrane-incorporated omega-6 PUFAs promoted caveola formation, while their displacement by omega-3 PUFAs or monounsaturated fatty acids disrupted this process. Importantly, oxidation of omega-6 PUFA chains in phosphatidylethanolamine (PE) triggered caveola disassembly during lipid peroxidation, potentially by affecting cavin-membrane interactions. These findings unveil a new model for caveola formation and signaling, linking caveola dynamics to ferroptosis with pro-ferroptotic lipids as essential caveolar components and key control points for caveola disassembly under oxidative stress.

Caveolae are atypical plasma membrane invaginations that take part in lipid sorting and regulation of oxidative and mechanical plasma membrane stress. Caveola formation requires caveolin, cavin, and specific lipid types. The recent advances in understanding the structure and assembly of caveolin and cavin complexes within the membrane context have clarified the fundamental processes underlying caveola biogenesis. In addition, the curvature of the caveola membrane is controlled by the regulatory proteins EHD2, pacsin2, and dynamin2, which also function to restrain the scission of caveolae from the plasma membrane (PM). Here, this is integrated with novel insights on caveolae as lipid and mechanosensing complexes that can dynamically flatten or disassemble to counteract mechanical, and oxidative stress.

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 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 the bulb of a subset of caveolae, but was not required for formation or fission of caveolae. Dyn2-positive 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 is not a core component of the caveolae machinery, but rather functions as an accessory protein that restrains caveola internalization.

The plasma membrane of many cell types, in particular, endothelia, smooth muscle cells, and adipocytes, contains numerous small invaginations termed caveolae. In nonmuscle cells, caveolae are formed by lipid-driven assembly of the integral membrane protein caveolin 1 (Cav1) and the peripherally attached protein cavin1. Accessory proteins such as Eps15 homology domain-containing 2 (EHD2) control the cell surface association of caveolae, together providing a unique invaginated membrane structure with distinct dynamics and protein and lipid compositions. These features enable caveolae to survey the plasma membrane integrity and to adjust membrane tension, and sort lipids according to the cellular requirements. Currently, characteristics of the protein and lipid interface of caveola are being unraveled, and this chapter is focused on the present knowledge of caveolae biogenesis and dynamics and describes methods that are being used to study the role of caveolae in lipid flux and lipid composition at the cell surface.

Caveolae are small plasma membrane invaginations, important for control of membrane tension, signaling cascades, and lipid sorting. The caveola 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 modeling to show that Cavin1 inserts into membranes. We establish that initial phosphatidylinositol (4, 5) bisphosphate [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 HR1 is further enhanced by the presence of flanking negatively charged disordered regions, which was found important for the coassembly 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.

The dynamic assembly of proteins at the membrane interphase is key to many cell biological processes such as the generation and stabilization of caveolae at the cell surface via coat proteins. The liposome co-sedimentation assay has been widely used for studies of protein and lipid interactions and has provided important information about binding mechanisms, lipid-binding specificity, and curvature preference of proteins. Here, we describe this technique in detail and how it can be used as a tool to address the membrane-binding ability and lipid specificity of caveolae-associated proteins.

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. Here, we found that global genetic ablation of EHD2 in mice leads to increased lipid droplet size in fat tissue. This organismic phenotype was paralleled at the cellular level by increased fatty acid uptake via a caveolae- and CD36-dependent pathway that also involves dynamin. Concomitantly, elevated numbers of detached caveolae were found in brown and white adipose tissue lacking EHD2, and increased caveolar mobility in mouse embryonic fibroblasts. EHD2 expression itself was down-regulated in the visceral fat of two obese mouse models and obese patients. Our data suggest that EHD2 controls a cell-autonomous, caveolae-dependent fatty acid uptake pathway and imply that low EHD2 expression levels are linked to obesity.

Caveolae are small Omega-shaped invaginations of the plasma membrane that play important roles in mechanosensing, lipid homeostasis and signaling. Their typical morphology is characterized by a membrane funnel connecting a spherical bulb to the membrane. Membrane funnels (commonly known as necks and pores) are frequently observed as transient states during fusion and fission of membrane vesicles in cells. However, caveolae display atypical dynamics where the membrane funnel can be stabilized over an extended period of time, resulting in cell surface constrained caveolae. In addition, caveolae are also known to undergo flattening as well as short-range cycles of fission and fusion with the membrane, requiring that the membrane funnel closes or opens up, respectively. This mini-review considers the transition between these different states and highlights the role of the protein and lipid components that have been identified to control the balance between surface association and release of caveolae.

Caveolae are bulb-shaped invaginations of the plasma membrane (PM) that undergo scission and fusion at the cell surface and are enriched in specific lipids. However, the influence of lipid composition on caveolae surface stability is not well described or understood. Accordingly, we inserted specific lipids into the cell PM via membrane fusion and studied their acute effects on caveolae dynamics. We demonstrate that sphingomyelin stabilizes caveolae to the cell surface, whereas cholesterol and glycosphingolipids drive caveolae scission from the PM. Although all three lipids accumulated specifically in caveolae, cholesterol and sphingomyelin were actively sequestered, whereas glycosphingolipids diffused freely. The ATPase EHD2 restricts lipid diffusion and counteracts lipid-induced scission. We propose that specific lipid accumulation in caveolae generates an intrinsically unstable domain prone to scission if not restrained by EHD2 at the caveolae neck. This work provides a mechanistic link between caveolae and their ability to sense the PM lipid composition.

Despite increasing interests in non-lamellar liquid crystalline dispersions, such as hexosomes, for drug delivery, little is known about their interactions with cells and mechanism of cell entry. Here we examine the cellular uptake of hexosomes based on phytantriol and mannide monooleate by HeLa cells using live cell microscopy in comparison to conventional liposomes. To investigate the importance of specific endocytosis pathways upon particle internalization, we silenced regulatory proteins of major endocytosis pathways using short interfering RNA. While endocytosis plays a significant role in liposome internalization, hexosomes are not taken up via endocytosis but through a mechanism that is dependent on cell membrane tension. Biophysical studies using biomembrane models highlighted that hexosomes have a high affinity for membranes and an ability to disrupt lipid layers. Our data suggest that direct biomechanical interactions of hexosomes with membrane lipids play a crucial role and that the unique morphology of hexosomes is vital for their membrane activity. Based on these results, we propose a mechanism, where hexosomes destabilize the bilayer, allowing them to "phase through" the membrane. Understanding parameters that influence the uptake of hexosomes is critical to establish them as carrier systems that can potentially deliver therapeutics efficiently to intracellular sites of action.