Caveolae Are Highly Immobile Plasma Membrane Microdomains, Which Are not Involved in Constitutive Endocytic Trafficking (original) (raw)
Related papers
Journal of Cellular and Molecular Medicine, 2009
Endocytosis -the uptake of extracellular ligands, soluble molecules, protein and lipids from the extracellular surface -is a vital process, comprising multiple mechanisms, including phagocytosis, macropinocytosis, clathrin-dependent and clathrin-independent uptake such as caveolae-mediated and non-caveolar raft-dependent endocytosis. The best-studied endocytotic pathway for internalizing both bulk membrane and specific proteins is the clathrin-mediated endocytosis. Although many papers were published about the caveolar endocytosis, it is still not known whether it represents an alternative pathway with distinct cellular compartments to avoid lysosomal degradation or ligands taken up by caveolae can also be targeted to late endosomes/lysosomes. In this paper, we summarize data available about caveolar endocytosis. We are especially focussing on the intracellular route of caveolae and providing data supporting that caveolar endocytosis can join to the classical endocytotic pathway.
Phosphatidylserine dictates the assembly and dynamics of caveolae in the plasma membrane
Journal of Biological Chemistry
Caveolae are bulb-shaped nanodomains of the plasma membrane that are enriched in cholesterol and sphingolipids. They have many physiological functions, including endocytic transport, mechanosensing, and regulation of membrane and lipid transport. Caveola formation relies on integral membrane proteins termed caveolins (Cavs) and the cavin family of peripheral proteins. Both protein families bind anionic phospholipids, but the precise roles of these lipids are unknown. Here, we studied the effects of phosphatidylserine (PtdSer), phosphatidylinositol 4-phosphate (PtdIns4P), and phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P 2) on caveolar formation and dynamics. Using live-cell, single-particle tracking of GFP-labeled Cav1 and ultrastructural analyses, we compared the effect of PtdSer disruption or phosphoinositide depletion with caveola disassembly caused by cavin1 loss. We found that PtdSer plays a crucial role in both caveola formation and stability. Sequestration or depletion of PtdSer decreased the number of detectable Cav1-GFP puncta and the number of caveolae visualized by electron microscopy. Under PtdSer-limiting conditions, the co-localization of Cav1 and cavin1 was diminished, and cavin1 degradation was increased. Using rapamycin-recruitable phosphatases, we also found that the acute depletion of PtdIns4P and PtdIns (4,5)P 2 has minimal impact on caveola assembly but results in decreased lateral confinement. Finally, we show in a model of phospholipid scrambling, a feature of apoptotic cells, that caveola stability is acutely affected by the scrambling. We conclude that the predominant plasmalemmal anionic lipid PtdSer is essential for proper Cav clustering, caveola formation, and caveola dynamics and that membrane scrambling can perturb caveolar stability. Caveolae ("little-caves") are bulb-shaped invaginations of the plasma membrane (PM) 3 enriched in cholesterol and sphingolipids, with a diameter of 50-80 nm (1). A variety of physiological roles has been attributed to caveolae, including as endocytic carriers, mechanosensors, regulators of membrane stress, and as regulators of lipid transport (2, 3). The primary protein component of caveolae is the integral membrane protein caveolin encoded by three homologous genes in mammalian cells: Cav1, Cav2, and Cav3. Caveolin proteins are co-translationally inserted into the endoplasmic reticulum (ER) (4) and are delivered to the PM via the secretory pathway (5). Newly synthesized Cav1 molecules self-associate to form oligomers of 12 or 14 monomers (6, 7). These oligomers then traffic from the Golgi apparatus to the PM via a vesicular carrier containing syntaxin 6, the ganglioside GM1, and glycosylphosphatidylinositollinked proteins (8). Once the Cav1 oligomers reach the PM, they can assemble to form caveolae, higher order structures containing Ϸ144 molecules of Cav1 protein (9). This basic structure has been referred to as a quantal unit (9). However, sub-quantal oligomeric structures of Cav1 have been described in the PM (9), as well as caveolar clusters formed by multiple caveolae in close, diffraction-limited proximity (10) or as larger rosette structures (11). From a structural standpoint, Cav1 contains a hairpin loop structure, three palmitoylation sites, and a scaffolding domain
Journal of Cellular and Molecular Medicine, 2008
Here we addressed the further fate of internalized caveolae by inducing caveolae-mediated uptake of albumin by HepG2 cells. We followed the route of internalized caveolin-1 by immunogold labelling of ultrathin frozen sections and by Western blot analyses of purified membrane fractions. Longterm (1 and 3 hrs) albumin treatment resulted in the appearance of albumin-containing caveolae in special multi-caveolar complexes (consisting of multiple caveolae clustered together) connected to the plasma membrane and caveosome-like structures in the cytoplasm. In addition, numerous CD63 (LIMP-1) positive late endosomes/multi-vesicular bodies were found positive for caveolin-1, suggesting that upon albumin incubation, caveolin-1 is endocytosed and enters the degradative pathway. Surprisingly, the number of caveolae at the plasma membrane increased after addition of albumin. This increase was blocked by cycloheximide treatment, indicating that albumin internalization also stimulates de novo protein synthesis, which is necessary for new caveolae formation. Together, our results show that during long-term albumin uptake, caveolin-1 travels to late endosomes and is replaced by newly synthesized caveolin-1 at the plasma membrane.
Traffic, 2002
Caveolae are flask-shaped invaginations present in the plasma membrane of many cell types. They have long been implicated in endocytosis, transcytosis, and cell signaling. Recent work has confirmed that caveolae are directly involved in the internalization of membrane components (glycosphingolipids and glycosylphosphatidylinositol-anchored proteins), extracellular ligands (folic acid, albumin, autocrine motility factor), bacterial toxins (cholera toxin, tetanus toxin), and several nonenveloped viruses (Simian virus 40, Polyoma virus). Unlike clathrin-mediated endocytosis, internalization through caveolae is a triggered event that involves complex signaling. The mechanism of internalization and the subsequent intracellular pathways that the internalized substances take are starting to emerge.
American journal of physiology. Cell physiology, 2017
Caveolins (Cavs) are ~20 kDa scaffolding proteins that assemble as oligomeric complexes in lipid raft domains to form caveolae, flask-shaped plasma membrane (PM) invaginations. Caveolae ("little caves") require lipid-lipid, protein-lipid, and protein-protein interactions that can modulate the localization, conformational stability, ligand affinity, effector specificity, and other functions of proteins that are partners of Cavs. Cavs are assembled into small oligomers in the endoplasmic reticulum (ER), transported to the Golgi for assembly with cholesterol and other oligomers, and then exported to the PM as an intact coat complex. At the PM, cavins, ~50 kDa adapter proteins, oligomerize into an outer coat complex that remodels the membrane into caveolae. The structure of caveolae protects their contents (i.e., lipids and proteins) from degradation. Cellular changes, including signal transduction effects, can destabilize caveolae and produce cavin dissociation, restructuring...
Journal of Biological Chemistry, 2000
Caveolins form interlocking networks on the cytoplasmic face of caveolae. The cytoplasmically directed N and C termini of caveolins are separated by a central hydrophobic segment, which is believed to form a hairpin within the membrane. Here, we report that the caveolin scaffolding domain (CSD, residues 82-101), and the C terminus (residues 135-178) of caveolin-1 are each sufficient to anchor green fluorescent protein (GFP) to membranes in vivo. We also show that the first 16 residues of the C terminus (i.e. residues 135-150) are necessary and sufficient to attach GFP to membranes. When fused to the caveolin-1 C terminus, GFP co-localizes with two trans-Golgi markers and is excluded from caveolae. In contrast, the CSD targets GFP to caveolae, albeit less efficiently than full-length caveolin-1. Thus, caveolin-1 contains at least two membrane attachment signals: the CSD, dictating caveolar localization, and the C terminus, driving trans-Golgi localization. Additionally, we find that caveolin-1 oligomer/oligomer interactions require the distal third of the caveolin-1 C terminus. Thus, the caveolin-1 C-terminal domain has two separate functions: (i) membrane attachment (proximal third) and (ii) protein/protein interactions (distal third). Caveolae are flask-shaped invaginations of the plasma membrane that are found in most cell types. However, caveolae are most abundant in endothelial cells, adipocytes, epithelial cells, fibroblasts, and myocytes (1). These structures participate in three main areas of cell physiology: endocytosis (2), cholesterol traffic (3), and signal transduction (4). They are coated on their cytoplasmic face by a family of proteins, the caveolins. Three mammalian caveolin genes (caveolin-1,-2, and-3) have been identified and characterized (5). Whereas caveolin-1 and-2 have overlapping tissue distributions (6), caveolin-3 is limited to muscle and neuroglial cells (7-10). Although expression of caveolin-1 or-3 is sufficient to form caveolae in cells lacking these structures (11-14), caveolins are
Caveolin-1 is a negative regulator of caveolae-mediated endocytosis to the endoplasmic reticulum
The Journal of biological chemistry, 2002
Caveolae are flask-shaped invaginations at the plasma membrane that constitute a subclass of detergent-resistant membrane domains enriched in cholesterol and sphingolipids and that express caveolin, a caveolar coat protein. Autocrine motility factor receptor (AMF-R) is stably localized to caveolae, and the cholesterol extracting reagent, methyl-beta-cyclodextrin, inhibits its internalization to the endoplasmic reticulum implicating caveolae in this distinct receptor-mediated endocytic pathway. Curiously, the rate of methyl-beta-cyclodextrin-sensitive endocytosis of AMF-R to the endoplasmic reticulum is increased in ras- and abl-transformed NIH-3T3 cells that express significantly reduced levels of caveolin and few caveolae. Overexpression of the dynamin K44A dominant negative mutant via an adenovirus expression system induces caveolar invaginations sensitive to methyl-beta-cyclodextrin extraction in the transformed cells without increasing caveolin expression. Dynamin K44A expressio...
Src-dependent phosphorylation of caveolin-1 Tyr14 promotes swelling and release of caveolae
Molecular biology of the cell, 2016
Caveolin 1 (Cav1) is a required structural component of caveolae and its phosphorylation by Src is associated with an increase in caveolae-mediated endocytosis. Here, we demonstrate using quantitative live-cell 4D, TIRF, and FRET imaging that endocytosis and trafficking of caveolae is associated with a Cav1 Tyr14 phosphorylation-dependent conformational change which spatially separates, or loosens, Cav1 molecules within the oligomeric caveolar coat. When tracked by TIRF and spinning-disk microscopy, cells expressing phospho-mimicking Cav1 (Y14D) mutant formed vesicles which were greater in number and volume when compared with Y14F-Cav1-GFP. Furthermore, we observed in HEK cells cotransfected with wild-type, Y14D, or Y14F Cav1-CFP and -YFP constructs that FRET efficiency was greater with Y14F pairs than with Y14D, indicating pY14-Cav1 regulates the spatial organization of Cav1 molecules within the oligomer. In addition, albumin-induced Src activation or direct activation of Src using...
Oligomers of the ATPase EHD2 confine caveolae to the plasma membrane through association with actin
The EMBO Journal, 2012
Caveolae are specialized domains present in the plasma membrane (PM) of most mammalian cell types. They function in signalling, membrane regulation, and endocytosis. We found that the Eps-15 homology domain-containing protein 2 (EHD2, an ATPase) associated with the static population of PM caveolae. Recruitment to the PM involved ATP binding, interaction with anionic lipids, and oligomerization into large complexes (60-75S) via interaction of the EH domains with intrinsic NPF/KPF motifs. Hydrolysis of ATP was essential for binding of EHD2 complexes to caveolae. EHD2 was found to undergo dynamic exchange at caveolae, a process that depended on a functional ATPase cycle. Depletion of EHD2 by siRNA or expression of a dominant-negative mutant dramatically increased the fraction of mobile caveolar vesicles coming from the PM. Overexpression of EHD2, in turn, caused confinement of cholera toxin B in caveolae. The confining role of EHD2 relied on its capacity to link caveolae to actin filaments. Thus, EHD2 likely plays a key role in adjusting the balance between PM functions of stationary caveolae and the role of caveolae as vesicular carriers.