Caveolae: Mechanosensing and mechanotransduction devices linking membrane trafficking to mechanoadaptation

Mechanical forces (extracellular matrix stiffness, vascular shear stress, and muscle stretching) reaching the plasma membrane (PM) determine cell behavior. Caveolae are PM-invaginated nanodomains with specific lipid and protein composition. Being highly abundant in mechanically challenged tissues (muscles, lungs, vessels, and adipose tissues), they protect cells from mechanical stress damage. Caveolae flatten upon increased PM tension, enabling both force sensing and accommodation, critical for cell mechanoprotection and homeostasis. Thus, caveolae are highly plastic, ranging in complexity from flattened membranes to vacuolar invaginations surrounded by caveolae—rosettes—which also contribute to mechanoprotection. Caveolar components crosstalk with mechanotransduction pathways and recent studies show that they translocate from the PM to the nucleus to convey stress information. Furthermore, caveolae components can regulate membrane traffic from/to the PM to adapt to environmental mechanical forces. The interdependence between lipids and caveolae starts to be understood, and the relevance of caveolae-dependent membrane trafficking linked to mechanoadaption to different physiopathological processes is emerging.     

Membrane traffic in skeletal muscle

Skeletal muscle tissue is made up of highly organized multinuclear cells. The internal organization of the muscle cell is dictated by the necessary regular arrangement of repeated units within the protein myofibrils that mediate muscle contraction. Skeletal muscle cells have the usual membrane traffic pathways for partitioning newly synthesized proteins, internalizing cell surface receptors for hormones and nutrients, and mediating membrane repair. However, in muscle, these pathways must be further specialized to deal with targeting to and organizing muscle-specific membrane structures, satisfying the unique metabolic requirements of muscle and meeting the high demand for membrane repair in a tissue that is constantly under mechanical stress. Specialized membrane traffic pathways in muscle also play a role in the formation of muscle through fusion of myoblast membranes and the development of internal muscle-specific membrane structures during myogenesis and regeneration. It has recently become apparent that muscle-specific isoforms of proteins that are known to mediate ubiquitous membrane traffic pathways, as well as novel muscle-specific proteins, are involved in tissue-specific aspects of muscle membrane traffic. Here we describe the specialized membrane structures of skeletal muscle, how these are developed, maintained and repaired by specialized and generic membrane traffic pathways, and how defects in these pathways result in muscle disease.     

“Make way”: Pathogen exploitation of membrane traffic

Intracellular pathogens have evolved numerous strategies to manipulate their host cells to survive and replicate in a hostile environment. They often exploit membrane trafficking pathways to enter the cell, establish a replicative niche, avoid degradation and immune response, acquire nutrients and lastly, egress. Recent studies on membrane trafficking exploitation by intracellular pathogens have led to the discovery of novel and fascinating cell biology, including a noncanonical mechanism of ubiquitination and a novel mitophagy receptor. Thus, studying how pathogens target host cell membrane trafficking pathways is not only important for the development of new therapeutics, but also helps understanding fundamental mechanisms of cell biology.     

Osmomechanical regulation of membrane trafficking in polarized cells

The regulation of membrane trafficking is thought to be predominantly under the control of agonist-receptor transduction pathways. In the present study, osmomechanical stress due to swelling, a condition often accompanying cell activation, was shown to induce multiple membrane trafficking pathways in polarized absorptive epithelial cells in the absence of agonists. Osmomechanical stress activated rapidly (seconds) pathways of calcium-dependent membrane insertion into the basolateral domain, pathways of calcium-independent membrane retrieval from the basolateral domain, and a novel pathway of transcytosis (transcellular) between basolateral and apical cell domains. These pathways appear to underlie the transfer and regulation of transport proteins amongst cell compartments. This broad affect of osmomechanical stress on trafficking pathways may reflect a global mechanism for redistribution of transport proteins and other membrane components amongst cell compartments during states of mechanical stress.     

Phosphoinositides: regulators of membrane traffic and protein function

Phosphoinositides serve as important spatio-temporal regulators of intracellular trafficking and cell signalling events. In addition to their recognition by specific phosphoinositide binding domains present within cytoplasmic adaptor proteins or membrane integral channels and transporters phosphoinositides may affect membrane transport by eliciting conformational changes within proteins or by regulating enzymatic activities. During adaptor-mediated membrane traffic phosphoinositides form part of coincidence detection systems that aid in targeting pools of specific phosphoinositides to select intracellular transport pathways. In this review, we discuss potential mechanisms for conferring selectivity onto the phosphoinositide code as well as possible avenues for future research.     

Endosomal and non-endosomal functions of ESCRT proteins

The three endosomal sorting complexes required for transport (ESCRTs) are integral to the degradation of endocytosed membrane proteins and multivesicular body (MVB) biogenesis. Here, we review evidence that ESCRTs have evolved as a specialized machinery for the degradative sorting of ubiquitinated membrane proteins and we highlight recent studies that have shed light on the mechanisms by which these complexes mediate protein sorting, MVB biogenesis, tumour suppression and viral budding. We also discuss evidence that some ESCRT subunits have evolved additional functions that are unrelated to membrane trafficking.     

Probing intracellular vesicle trafficking and membrane remodelling by cryo-EM

Protein transport between the membranous compartments of the eukaryotic cells is mediated by the constant fission and fusion of the membrane-bounded vesicles from a donor to an acceptor membrane. While there are many membrane remodelling complexes in eukaryotes, COPII, COPI, and clathrin-coated vesicles are the three principal classes of coat protein complexes that participate in vesicle trafficking in the endocytic and secretory pathways. These vesicle-coat proteins perform two key functions: deforming lipid bilayers into vesicles and encasing selective cargoes. The three trafficking complexes share some commonalities in their structural features but differ in their coat structures, mechanisms of cargo sorting, vesicle formation, and scission. While the structures of many of the proteins involved in vesicle formation have been determined in isolation by X-ray crystallography, elucidating the proteins' structures together with the membrane is better suited for cryogenic electron microscopy (cryo-EM). In recent years, advances in cryo-EM have led to solving the structures and mechanisms of several vesicle trafficking complexes and associated proteins.     

Intracellular trafficking of raft/caveolae domains: insights from integrin signaling

Cells have a complex system for delivering and compartmentalizing proteins and lipids in order to achieve spatio-temporal coordination of signaling. Rafts/caveolae are plasma membrane microdomains that regulate signaling pathways and processes such as cell migration, polarization and proliferation. Regulation of raft/caveolae trafficking involves multiple steps regulated by different proteins to ensure coordination of signaling cascades. The best studied raft-mediated endocytic route is controlled by caveolins. Recent data suggest integrin-mediated cell adhesion is a key regulator of caveolar endocytosis. In this review we examine the regulation of caveolar trafficking and the interplay between integrins, cell adhesion and caveolae internalization.     

Caveolae: Formation,dynamics, and function

Caveolae are abundant surface pits formed by the assembly of cytoplasmic proteins on a platform generated by caveolin integral membrane proteins and membrane lipids. This membranous assembly can bud off into the cell or can be disassembled releasing the cavin proteins into the cytosol. Disassembly can be triggered by increased membrane tension, or by stress stimuli, such as UV. Here, we discuss recent mechanistic studies showing how caveolae are formed and how their unique properties allow them to function as multifunctional protective and signaling structures.     

Trafficking and function of the tetraspanin CD63

Tetraspanins comprise a large superfamily of cell surface-associated membrane proteins characterized by four transmembrane domains. They participate in a variety of cellular processes, like cell activation, adhesion, differentiation and tumour invasion. At the cell surface, tetraspanins form networks with a wide diversity of proteins called tetraspanin-enriched microdomains (TEMs). CD63 was the first characterized tetraspanin. In addition to its presence in TEMs, CD63 is also abundantly present in late endosomes and lysosomes. CD63 at the cell surface is endocytosed via a clathrin-dependent pathway, although recent studies suggest the involvement of other pathways as well and we here present evidence for a role of caveolae in CD63 endocytosis. In late endosomes, CD63 is enriched on the intraluminal vesicles, which by specialized cells are secreted as exosomes through fusion of endosomes with the plasma membrane. The complex localization pattern of CD63 suggests that its intracellular trafficking and distribution must be tightly regulated. In this review we discuss the latest insights in CD63 trafficking and its emerging function as a transport regulator of its interaction partners. Finally, the involvement of CD63 in cancer will be discussed.     

Pay attention to membrane tension: Mechanobiology of the cell surface

The cell surface is a mechanobiological unit that encompasses the plasma membrane, its interacting proteins, and the complex underlying cytoskeleton. Recently, attention has been directed to the mechanics of the plasma membrane, and in particular membrane tension, which has been linked to diverse cellular processes such as cell migration and membrane trafficking. However, how tension across the plasma membrane is regulated and propagated is still not completely understood. Here, we review recent efforts to study the interplay between membrane tension and the cytoskeletal machinery and how they control cell form and function. We focus on factors that have been proposed to affect the propagation of membrane tension and as such could determine whether it can act as a global or local regulator of cell behavior. Finally, we discuss the limitations of the available tool kit as new approaches that reveal its dynamics in cells are needed to decipher how membrane tension regulates diverse cellular processes.     

Oxidative Stress Induces Caveolin 1 Degradation and Impairs Caveolae Functions in Skeletal Muscle Cells

Increased level of oxidative stress, a major actor of cellular aging, impairs the regenerative capacity of skeletal muscle and leads to the reduction in the number and size of muscle fibers causing sarcopenia. Caveolin 1 is the major component of caveolae, small membrane invaginations involved in signaling and endocytic trafficking. Their role has recently expanded to mechanosensing and to the regulation of oxidative stress-induced pathways. Here, we increased the amount of reactive oxidative species in myoblasts by addition of hydrogen peroxide (H₂O₂) at non-toxic concentrations. The expression level of caveolin 1 was significantly decreased as early as 10 min after 500 μM H₂O₂ treatment. This reduction was not observed in the presence of a proteasome inhibitor, suggesting that caveolin 1 was rapidly degraded by the proteasome. In spite of caveolin 1 decrease, caveolae were still able to assemble at the plasma membrane. Their functions however were significantly perturbed by oxidative stress. Endocytosis of a ceramide analog monitored by flow cytometry was significantly diminished after H₂O₂ treatment, indicating that oxidative stress impaired its selective internalization via caveolae. The contribution of caveolae to the plasma membrane reservoir has been monitored after osmotic cell swelling. H₂O₂ treatment increased membrane fragility revealing that treated cells were more sensitive to an acute mechanical stress. Altogether, our results indicate that H₂O₂ decreased caveolin 1 expression and impaired caveolae functions. These data give new insights on age-related deficiencies in skeletal muscle.     

Key phases in the formation of caveolae

Caveolae are abundant plasma membrane pits formed by the coordinated action of peripheral and integral membrane proteins and membrane lipids. Here, we discuss recent studies that are starting to provide a glimpse of how filamentous cavin proteins, membrane-embedded caveolin proteins, and specific plasma membrane lipids are brought together to make the unique caveola surface domain. Protein assembly involves multiple low-affinity interactions that are dependent on ‘fuzzy’ charge-dependent interactions mediated in part by disordered cavin and caveolin domains. We propose that cavins help generate a lipid domain conducive to full insertion of caveolin into the bilayer to promote caveola formation. The synergistic assembly of these dynamic protein complexes supports the formation of a metastable membrane domain that can be readily disassembled both in response to cellular stress and during endocytic trafficking. We present a mechanistic model for generation of caveolae based on these new insights.     

Self-assemblies of Rab- and Arf-family small GTPases on lipid bilayers in membrane tethering

Small GTPases of the Ras superfamily, which include Ras-, Rho-, Rab-, Arf-, and Ran-family isoforms, are generally known to function as a nucleotide-dependent molecular switch in eukaryotic cells. In the GTP-loaded forms, they selectively recruit their cognate interacting proteins or protein complexes, termed “effectors,” to the cytoplasmic face of subcellular membrane compartments, thereby switching on the downstream effector functions, which are vital for fundamental cellular events, such as cell proliferation, cytoskeletal organization, and intracellular membrane trafficking. Nevertheless, in addition to acting as the classic nucleotide-dependent switches for the effectors, recent studies have uncovered that small GTPases themselves can be self-assembled specifically into homo-dimers or higher-order oligomers on membranes, and these assembly processes are likely responsible for their physiological functions. This Review focuses particularly on the self-assembly processes of Rab- and Arf-family isoforms during membrane tethering, the most critical step to ensure the fidelity of membrane trafficking. A summary of the current experimental evidence for self-assemblies of Rab and Arf small GTPases on lipid bilayers in chemically defined reconstitution system is provided     

Integrin-linked kinase controls microtubule dynamics required for plasma membrane targeting of caveolae

Caveolae are specialized compartments of the plasma membrane that are involved in signaling, endocytosis, and cholesterol transport. Their formation requires the transport of caveolin-1 to the plasma membrane, but the molecular mechanisms regulating the transport are largely unknown. Here, we?identify a critical role for adhesion-mediated signaling through β1 integrins and integrin-linked kinase (ILK) in caveolae formation. Mice lacking β1 integrins or ILK in keratinocytes have dramatically reduced numbers of plasma membrane caveolae in?vivo, which is due to impaired transport of caveolin-1-containing vesicles along microtubules (MT) to the plasma membrane. Mechanistically, ILK promotes the recruitment of the F-actin binding protein IQGAP1 to the cell cortex, which, in turn, cooperates with its?effector mDia1 to locally stabilize MTs and to allow?stable insertion of caveolae into the plasma membrane. Our results assign an important role to the integrin/ILK complex for caveolar trafficking to the cell surface.     

Proteomic analysis of membrane microdomains derived from both failing and non-failing human hearts

Eukaryotic cells plasma membranes are organized into microdomains of specialized function such as lipid rafts and caveolae, with a specific lipid composition highly enriched in cholesterol and glycosphingolipids. In addition to their role in regulating signal transduction, multiple functions have been proposed, such as anchorage of receptors, trafficking of cholesterol, and regulation of permeability. However, an extensive understanding of their protein composition in human heart, both in failing and non-failing conditions, is not yet available. Membrane microdomains were isolated from left ventricular tissue of both failing (n = 15) and non-failing (n = 15) human hearts. Protein composition and differential protein expression was explored by comparing series of 2-D maps and subsequent identification by LC-MS/MS analysis. Data indicated that heart membrane microdomains are enriched in chaperones, cytoskeletal-associated proteins, enzymes and protein involved in signal transduction pathway. In addition, differential protein expression profile revealed that 30 proteins were specifically up- or down-regulated in human heart failure membrane microdomains. This study resulted in the identification of human heart membrane microdomain protein composition, which was not previously available. Moreover, it allowed the identification of multiple proteins whose expression is altered in heart failure, thus opening new perspectives to determine which role they may play in this disease.     

Depletion of plasma membrane cholesterol dampens hydrostatic pressure and shear stress-induced mechanotransduction pathways in osteoblast cultures

The preferential association of cholesterol and sphingolipids within plasma membranes forms organized compartments termed lipid rafts. Addition of caveolin proteins to this lipid milieu induces the formation of specialized invaginated plasma membrane structures called caveolae. Both lipid rafts and caveolae are purported to function in vesicular transport and cell signaling. We and others have shown that disassembly of rafts and caveolae through depletion of plasma membrane cholesterol mitigates mechanotransduction processes in endothelial cells. Because osteoblasts are subjected to fluid-mechanical forces, we hypothesize that cholesterol-rich plasma membrane microdomains also serve the mechanotransduction process in this cell type. Cultured human fetal osteoblasts were subjected to either sustained hydrostatic pressure or laminar shear stress using a pressure column or parallel-plate apparatus, respectively. We found that sustained hydrostatic pressure induced protein tyrosine phosphorylation, activation of extracellular signal-regulated kinase (ERK)1/2, and enhanced expression of c-fos in both time- and magnitude-dependent manners. Similar responses were observed in cells subjected to laminar shear stress. Both sustained hydrostatic pressure- and shear stress-induced signaling were significantly reduced in osteoblasts pre-exposed to either filipin or methyl--cyclodextrin. These mechanotransduction responses were restored on reconstitution of lipid rafts and caveolae, which suggests that cholesterol-rich plasma membrane microdomains participate in the mechanotransduction process in osteoblasts. In addition, mechanical force-induced phosphoproteins were localized within caveolin-containing membranes. These data support the concept that lipid rafts and caveolae serve a general function as cell surface mechanotransduction sites within the plasma membrane. lipid rafts; caveolae; extracellular signal-regulated kinase     

Endoplasmic reticulum–plasma membrane contacts: Principals of phosphoinositide and calcium signaling

The endoplasmic reticulum (ER) forms an extensive network of membrane contact sites with intra-cellular organelles and the plasma membrane (PM). Interorganelle contacts have vital roles in membrane lipid and ion dynamics. In particular, ER–PM contacts are integral to numerous inter-cellular and intra-cellular signaling pathways including phosphoinositide lipid and calcium signaling, mechanotransduction, metabolic regulation, and cell stress responses. Accordingly, ER–PM contacts serve important signaling functions in excitable cells including neurons and muscle and endocrine cells. This review highlights recent advances in our understanding of the vital roles for ER–PM contacts in phosphoinositide and calcium signaling and how signaling pathways in turn regulate proteins that form and function at ER–PM contacts.     

The functions of Rab GTPases in plant membrane traffic

Rab GTPases are important determinants of membrane identity and membrane targeting. Higher plants have evolved a unique set of Rab GTPases that presumably reflects the specific demands of plant cell trafficking. In recent years, significant progress has been made in identifying Rab GTPases involved in endosome organisation, cytokinesis and in post-Golgi traffic to the plasma membrane and vacuoles. These include members of the Rab-F1, Rab-F2, Rab-A1, Rab-A2 and Rab-A4 subclasses. Some important regulators or effectors have also been identified for Rab-F, Rab-A1 and Rab-A4 proteins. However, uncertainties remain about the trafficking pathways that connect the compartments in the trans-Golgi/prevacuolar/endosomal system and there is still little or no insight into the functions of several major subclasses within the Rab GTPase family.     

Satnav for cells: Destination membrane fusion

All eukaryotic cells, from budding yeast to plants and mammals, are elaborately subdivided into functionally distinct, membrane-enclosed compartments – or organelles. Each organelle contains its own characteristic set of enzymes and other specialized molecules, which allows for the segregation of distinct biochemical reactions. A complex distribution system transports specific products (or cargos) from one compartment to another, involving a cycle of trafficking vesicle formation from a precursor membrane, vesicle transport to its destination (which may involve use of the cytoskeleton and specific motor proteins) and finally vesicle fusion with its target membrane.In the central nervous system (CNS), rapid communication between neurons at synapses is achieved using such a specialized trafficking pathway. Small synaptic vesicles move to the presynaptic plasma membrane where they fuse in response to Ca²⁺ influx, releasing chemical messengers (neurotransmitters) into the synaptic cleft. Vesicles are then recovered, reformed and refilled with neurotransmitter, ready for subsequent rounds of release. This recycling process may involve fusion with, and reformation from, a specific endosomal recycling station.As correct recycling of synaptic vesicles is essential to maintain neuronal signaling, every aspect of the process has been intensively studied. Amazingly, the general principals elucidated in this system are shared across membrane trafficking pathways in eukaryotes, and are largely mediated by common protein-based machineries. Hence, in this article, I will use the example of neuronal exocytosis to illustrate concepts which currently dominate our thinking about membrane trafficking pathways. In particular, I intend to focus on the all-important issue of how specificity in vesicle transport and fusion is achieved.

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