Caveolae are 50-60 nm flask-shaped invaginations of the plasma membrane that protect the plasma membrane from damage under stretch forces. They are highly abundant in cells that experience high levels of stress forces such as adipocytes, endothelial cells and muscle cells. Caveolae are generated by the oligomerisation and association of caveolin and cavin proteins, which form the caveolar coat complex at the caveolar bulb and are progressively well characterised. However, less is known about the proteins that localise to the caveolar neck. Using the CRIPSR/Cas9 system to generate gene knock-in and knockout cell lines, the role of EHD proteins at caveolae was investigated. It was found that, in addition to EHD2 being at the neck, both EHD1 and EHD4 were also present. The recruitment of other EHD proteins was markedly increased in the absence of EHD2. This functional redundancy was confirmed by the generation of EHD1, 2 and 4 triple knockout cell lines, which displayed two striking sets of phenotypes. Firstly, the characteristic higher-order clusters of caveolae are lost in the absence of EHD proteins. And secondly, caveolae are destabilised and the plasma membrane is more likely to rupture when the EHD1,2,4 knockout cells are subjected to cycles of stretch forces. The data identify the first molecular components that cluster caveolae into a membrane ultrastructure that potentially extends stretch buffering capacity. A second series of experiments tested different ideas about the function of caveolae in adipocytes. The insulin receptor and CD36 were found to at most partially colocalise with caveolae, and the role of caveolae in regulating signalling processes remains unclear. In contrast, the plasma membrane of adipocytes without caveolae is clearly more prone to rupture, confirming a mechanoprotective function.

Caveolin-1 is a marker protein for caveolae and can be a regulator of intracellular signaling pathways that contribute to the pathogenesis of human diseases. In the present study, the structural and functional changes of the lung in caveolin-1 null mice (Cav-1-/-) were assessed. Respiratory mechanics, measured using a small animal ventilator, revealed heightened central airway resistance (Rn), tissue resistance (G) and tissue elastance (H) in response to inhaled methacholine. The respiratory hyperreactivity is associated with increased collagen deposition around central and peripheral airways in Cav-1-/- mice; however, no difference was found in smooth muscle α-actin quantity between mouse strains. Similar to our in vivo findings, tracheal rings from Cav-1-/- mice mounted on an isometric wire myograph exhibited enhanced maximum active contractile force without a change in sensitivity (EC50) to methacholine. Rho kinase (ROCK1/2), protein kinase C (PKC) and extracellular signal regulated kinase 1/2 (ERK1/2) signaling were assessed as possible sources of the enhanced airway reactivity observed in Cav-1-/- mice. Inhibition of Rho kinase markedly blunted in vivo lung function responses (Rn) and (G) and ex vivo smooth muscle responses to methacholine. In fact, inhibition of Rho kinase completely eliminated any difference in response between mouse strains. Thus, our data indicate that Cav-1 may regulate mechanisms, such as Rho/Rho kinase signaling, that determine airway smooth muscle contraction and airway fibrosis; thus, it could be an important regulator of airway biology and physiology in health and disease.

Caveolin-1 is a marker protein for caveolae and can be a regulator of intracellular signaling pathways that contribute to the pathogenesis of human diseases. In the present study, the structural and functional changes of the lung in caveolin-1 null mice (Cav-1-/-) were assessed. Respiratory mechanics, measured using a small animal ventilator, revealed heightened central airway resistance (Rn), tissue resistance (G) and tissue elastance (H) in response to inhaled methacholine. The respiratory hyperreactivity is associated with increased collagen deposition around central and peripheral airways in Cav-1-/- mice; however, no difference was found in smooth muscle α-actin quantity between mouse strains. Similar to our in vivo findings, tracheal rings from Cav-1-/- mice mounted on an isometric wire myograph exhibited enhanced maximum active contractile force without a change in sensitivity (EC50) to methacholine. Rho kinase (ROCK1/2), protein kinase C (PKC) and extracellular signal regulated kinase 1/2 (ERK1/2) signaling were assessed as possible sources of the enhanced airway reactivity observed in Cav-1-/- mice. Inhibition of Rho kinase markedly blunted in vivo lung function responses (Rn) and (G) and ex vivo smooth muscle responses to methacholine. In fact, inhibition of Rho kinase completely eliminated any difference in response between mouse strains. Thus, our data indicate that Cav-1 may regulate mechanisms, such as Rho/Rho kinase signaling, that determine airway smooth muscle contraction and airway fibrosis; thus, it could be an important regulator of airway biology and physiology in health and disease.

Skeletal muscle function is important to the human body for daily activities. Mechanical signals are critical to the maintenance of that function. Muscle diseases, such as the muscular dystrophies, in which the force transmission apparatus is compromised, have devastating effects on muscle function and quality of life. Mechanical signals activate intracellular signaling to maintain function. ERK2 has been shown to be quickly and strongly upregulated following stretch, leading to cell proliferation. Stretch has been shown to cause deformation of caveolae, invaginations of the plasma membrane that inhibit ERK signaling. This leads to the hypothesis that stretch induced deformation of caveolae may initiate mechanotransduction by activating ERK2. Reducing caveolin-3 expression via siRNA knockdown eradicated the stretch-induced effect on ERK2 activation, indicating that caveolin is required for the stretch response. Stabilizing caveolae structure by temperature reduction or destabilizing caveolae by cholesterol depletion resulted in changes consistent with the hypothesis that proper caveolae structure plays an important role in inhibition of signaling molecules and that deformation mediates mechanotransduction, resulting in changes in activation of ERK2.

Rotavirus NSP4, the first described viral enterotoxin, localizes to the plasma membrane of infected cells, possibly through interaction with caveolin-1. A direct interaction between NSP4 and caveolin-1, the structural protein of caveolae, was shown by yeast two-hybrid, peptide binding, and FRET analyses. To dissect the precise NSP4 binding domain to caveolin-1, mutants were prepared by altering either the charged or hydrophobic face of the NSP4 C-terminal amphipathic alpha-helix and examined for binding to caveolin-1. Replacing six charged residues with alanine (FLNSP4Ala) disrupted the charged face, while the hydrophobic face was disrupted by replacing selected hydrophobic residues with charged amino acids (aa) (FLNSP4HydroMut). In yeast two-hybrid and peptide binding assays, FLNSP4Ala retained its binding capacity, whereas FLNSP4HydroMut failed to bind caveolin-1. Mutants were generated with an Nterminal truncated clone (NSP446-175), which removed the hydrophobic domains and aided in yeast-two hybrid assays. These mutants exhibited the same binding pattern as FLNSP4 confirming that the N-terminus of NSP4 lacks the caveolin-1 binding site and NSP446-175 is sufficient for binding. Seven additional mutants were prepared from NSP4HydroMut in which individually charged residues were reverted to the original hydrophobic aa or were replaced with alanine. Analyses of the interaction of these revertants with caveolin-1 localized the NSP4 binding domain to one critical hydrophobic aa (L116) and one or two additional aa (I113, L127, and/or L134) on the hydrophobic face. Those mutants that bound caveolin-1 bound both the N- and C-terminal caveolin-1 peptides, but lacked binding to a centrally located peptide. These data suggest conformational and hydrophobic constraints play a role in the NSP4-caveolin-1 association. The mutant NSP4 molecules also were evaluated for transport to the plasma membrane. Mammalian cells were transfected with FLNSP4, FLNSP41-175Ala, and NSP41-175HydroMut plasmid DNA, surface biotinylated, and examined by IFA or Western blot for NSP4 expression. Epifluorescence revealed FLNSP4 and FLNSP4Ala were exposed on the cell surface in the absence of other viral proteins, whereas NSP4HydroMut remained intracellular. Further, NSP4-transfected cells displayed an intracellular association of with caveolin-1 or the caveolin-1 chaperone complex proteins. These data indicate NSP4 interacts with caveolin-1 in the absence of other viral proteins.

No description available.

Rotavirus (RV) is an etiologic agent of viral gastroenteritis in children and infants worldwide, accounting for an estimated 500,000 deaths annually. NSP4, the first described viral enterotoxin, contributes to RV pathogenesis by mobilizing intracellular calcium through multiple mechanisms that promote abnormal ion transport and subsequent secretory diarrhea. NSP4 and the enterotoxic peptide 114-135 preferentially interact with model membranes mimicking caveolae in lipid composition and radius of curvature. Our laboratory has recently reported the colocalization and coimmunoprecipitation of NSP4 with caveolin-1, the structural protein of caveolae. Moreover, the caveolin-1 binding domain of NSP4 has been localized to the enterotoxic peptide. We now report that caveolin-1 binds NSP4 via the N- and C-termini and one terminus is sufficient for binding. A panel of caveolin-1 deletion mutants was expressed in a yeast two-hybrid assay against an NSP4 bait. Caveolin-1 mutants retaining at least one terminus were capable of binding the NSP4 bait. An in vitro binding assay confirmed the two-hybrid results and localized the NSP4 binding domains to caveolin-1 residues 2-22 and 161-178. These data support the hypothesis that caveolin-1 mediates NSP4 signaling and/or intracellular trafficking.

In adult cardiomyocytes, the established mechanism of excitation-contraction coupling is calcium-induced calcium release (CICR) mediated by L-type Ca2+ channels (Cav1.2). Briefly, membrane depolarization opens voltage-gated Cav1.2 to allow for the influx of extracellular Ca2+ into the cytosol. This small sarcolemmal (SL) Ca2+ influx is necessary for triggering a larger release of Ca2+ from the intracellular Ca2+ storage site, the sarcoplasmic reticulum (SR), through the SR Ca2+ release channel also known as the ryanodine receptor (RyR). RyR-mediated release of SR Ca2+ effectively raises the cytosolic free Ca2+ concentration, allowing for Ca2+ binding to troponin C on the troponin-tropomysin complex, leading to cross-bridge formation and cell contraction. However, previous functional data suggests an additional CICR modality involving reverse mode Na+-Ca2+ exchanger (NCX) activity also exists in neonate cardiomyocytes. To further our understanding of how CICR changes occur during development, we investigated the spatial arrangement of caveolin-3 (cav-3), the principle structural protein of small membrane invaginations named caveolae, and NCX in developing rabbit ventricular myocytes. Using traditional as well as novel image processing and analysis techniques, both qualitative and quantitative findings firmly establish the highly robust and organized nature of NCX and cav-3 distributions during development. Specifically, our results show that NCX and cav-3 are distributed on the peripheral membrane as discrete clusters and are not highly colocalized throughout development. 3D distance analysis revealed that NCX and cav-3 clusters are organized with a distinct longitudinal and transverse periodicity of 1-1.5 μm and that NCX and cav-3 cluster have a pronounced tendency to be mutually exclusive on the cell periphery. Although these findings do not support the original hypothesis that caveolae is the structuring element for a restricted microdomain facilitating NCX-CICR, our results cannot rule out the existence of such microdomain organized by other anchoring proteins. The developmentally stable distributions of NCX and cav-3 imply that the observed developmental CICR changes are achieved by the spatial re-organization of other protein partners of NCX or non-spatial modifications. In addition, the newly developed image processing and analysis techniques can have wide applicability to the investigations on the spatial distribution of other proteins and cellular structures.

Allergic asthma is a syndrome characterized by respiratory distress in response to environmental triggers. This atypical response to an allergen is an over reaction of the immune system causing an influx of inflammatory cells into the airway and concomitant airway smooth muscle constriction. Firstly, we demonstrate using whole house dust mite (HDM) extract as a sensitizing allergen produces an equivalent or more robust hyperresponsive and inflammatory reaction than can be achieved with the widely used ovalbumin (OVA) sensitization / challenge protocol. Secondly, we investigated the role of caveolin-1 in the pathophysiology of allergic asthma . Our data suggest an important role for cav-1 in down regulating allergic airway inflammation, leading to reduced airways hyperresponsiveness and mucus overproduction.

Background: Caveolin-1 (Cav-1), the major component of caveolae, is a multifunctional scaffolding protein that serves as a platform for the cell’s signal-transduction and plays a role in inflammation. However, its role in inflammatory bowel disease (IBD), a chronic inflammatory condition in the gastrointestinal tract, is not clear. A recent study shows that Cav-1 mediates angiogenesis in dextran sodium sulphate (DSS)-induced colitis. These results contradict our data, in which Cav-1 levels decreased significantly in 2,4,6-trinitrobenzene sulphonic acid (TNBS)–induced colitis. Methods: To test whether Cav-1 is involved in IBD pathogenesis, various models representing different dominant Th subtype responses and mimicking the immune pathologic mechanisms of different clinical IBD setting were employed: acute colitis was induced by intra-rectal administration of a single dose of TNBS in BALB/c and C57BL/6J mice, or by drinking 3% DSS water for 6 days in C57BL/6J mice. Chronic colitis was induced by administration of TNBS once a week for 7 weeks in BALB/c mice. To assess the effects of complete loss of Cav-1, Cav-1 knock-out (Cav-1-/-) and control wild-type C57BL/6J mice received a single TNBS administration. To further test the possible role of Cav-1, one of two peptides (that either mimicked (Caveolin scaffolding domain; CSD) or antagonized (Caveolin-1 binding domain; CBD1) Cav-1)) was administered intraperitoneally to mice receiving TNBS. Body weight and clinical scores were monitored. Colon Cav-1 and pro-inflammatory cytokine levels were quantified by ELISA. Inflammation was evaluated through histological analysis. Results: Cav-1 levels in mouse colon tissue were significantly decreased in TNBS-induced colitis mice when compared to normal mice and also inversely correlated with colon inflammation and cytokine levels. Furthermore, a loss of Cav-1 (Cav-1-/-) showed increased clinical and inflammatory scores and increased body weight loss. Mice receiving peptides to alter Cav-1 levels, showed surprising effects. The mimicking peptide (CSD) showed decreased Cav-1 levels, while the antagonizing peptide (CBD1) showed increased Cav-l levels. These changes in levels were associated with clinical and inflammatory scores and body weight loss that supported the TNBS-induced data. DSS-induced colitis mice showed increased disease activity index, however no significant difference in Cav-1 levels was found between colitis and normal mice. Conclusions: Cav-1 plays an important role in the protection of TNBS-induced colitis, but not in DSS-induced colitis, an entirely different result from a previous report, suggesting that enhancement of Cav-1 expression and functions may be beneficial to IBD treatment in some specific clinical settings. Further studies are warranted.