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Caveolae, flask-shaped invaginations of the plasma membrane, are particularly abundant in muscle cells. We have recently cloned a muscle-specific caveolin, termed caveolin-3, which is expressed in differentiated muscle cells. Specific antibodies to caveolin-3 were generated and used to characterize the distribution of caveolin-3 in adult and differentiating muscle. In fully differentiated skeletal muscle, caveolin-3 was shown to be associated exclusively with sarcolemmal caveolae. Localization of caveolin-3 during differentiation of primary cultured muscle cells and development of mouse skeletal muscle in vivo suggested that caveolin-3 is transiently associated with an internal membrane system. These elements were identified as developing transverse-(T)-tubules by double-labeling with antibodies to the α1 subunit of the dihydropyridine receptor in C2C12 cells. Ultrastructural analysis of the caveolin-3– labeled elements showed an association of caveolin-3 with elaborate networks of interconnected caveolae, which penetrated the depths of the muscle fibers. These elements, which formed regular reticular structures, were shown to be surface-connected by labeling with cholera toxin conjugates. The results suggest that caveolin-3 transiently associates with T-tubules during development and may be involved in the early development of the T-tubule system in muscle.
The plasma membrane of mammalian cells is divided into a number of different structural and functional microdomains. Much recent interest has been focused on one such domain, the caveola, a surface invagination with unique morphology which is readily identifiable by electron microscopy (Parton, 1996). Caveolae are extremely abundant in endothelial cells, adipocytes, and smooth muscle cells. In endothelia, caveolae appear to play a major role in transport across the endothelial monolayer (Ghitescu et al., 1986; Schnitzer et al., 1994). Other work has suggested a role for caveolae in signal transduction (Lisanti et al., 1994), in specialized endocytic uptake pathways (Anderson, 1993), and in calcium homeostasis (Fujimoto, 1993; Fujimoto et al., 1992).
The diversity of the proposed functions of caveolae raises the question of whether they have a single function, as does a clathrin-coated pit, or whether they are structural units used for many different purposes. Caveolae are enriched in cholesterol (Montesano et al., 1982; Rothberg et al., 1990) and in glycosphingolipids (Parton, 1994), and increasing evidence suggests that caveolae are built up around sphingolipid–cholesterol rafts (Simons and Ikonen, 1996). The plasma membrane of all mammalian cells appears to contain such rafts which, upon detergent treatment, can be isolated as insoluble glycosphingolipid-enriched complexes (DIGs;1 Parton and Simons, 1995). DIGs and caveolae share many features, but caveolae appear to be more restricted in distribution, being undetectable in some cell types (Fra et al., 1994; Gorodinsky and Harris, 1995). Caveolin-1, the major protein of caveolae in mammalian cells (Kurzchalia et al., 1994; Parton, 1996), is a 21-kD integral membrane protein which has been shown to bind cholesterol and to interact with glycosphingolipids (Fra et al., 1995a; Murata et al., 1995). Heterologous expression of caveolin-1 in cells lacking caveolae causes formation of surface invaginations with many of the features of caveolae (Fra et al., 1995b). The de novo produced caveolae were the same size and shape as endogenous caveolae, and cross-linked glycosylphosphatidylinositol-anchored proteins were shown to be concentrated within these invaginations. These results suggest that caveolin-1 has the capacity to interact with DIGs and create the characteristic caveolar invagination and to generate a microdomain with a distinct lipid composition (Parton and Simons, 1995). Recent work has identified two additional caveolin family members which share many structural features with caveolin-1 (Way and Parton, 1995; Scherer et al., 1996; Tang et al., 1996). The role of caveolin-1 and other caveolins is not restricted to generating and maintaining caveolar structure. In vitro studies have shown specific functional interactions with trimeric G protein α subunits (Li et al., 1995). These interactions may hold the G protein in an inactive state on the cytoplasmic face of the membrane.
We have recently cloned and characterized a novel muscle-specific homologue of caveolin-1, termed M-caveolin or caveolin-3 (Way and Parton, 1995). Expression of caveolin-3 is induced upon muscle differentiation, and mRNA is undetectable in undifferentiated C2C12 cells (Way and Parton, 1995; Tang et al., 1996). Caveolin-3 is ∼60% homologous to caveolin-1 with the major differences occurring in the NH2-terminal portion of the protein. The identification of a muscle-specific caveolin protein raises the question of the specific role of this protein, and of caveolae generally, in muscle. Caveolae of muscle cells (also termed subsarcolemmal vesicles or microvesicles) have been studied using a host of morphological techniques (Gabella, 1978). Several different hypotheses were proposed for the role of caveolae in muscle function and development. The three main theories proposed roles for caveolae in (a) provision of extra membrane during muscle extension (Prescott and Brightman, 1976), (b) calcium entry (Popescu, 1974), and (c) in the formation of the transverse (T)-tubule system during muscle development (Ishikawa, 1968). The first “stretch” theory has largely been discounted by subsequent studies suggesting that caveolae do not contribute membrane during muscle extension in vivo (Dulhunty and Franzini-Armstrong, 1975; Gabella and Blundell, 1978). However, the second theory, proposing a role for muscle caveolae in calcium intake or homeostasis, remains relevant, as recent studies have shown a striking concentration of two putative calcium-regulating molecules within caveolae of several different cell types, including muscle cells (Fujimoto, 1993; Fujimoto et al., 1992). The third theory, invoking a role for caveolae in formation of the T-tubule system, was suggested from early studies of the development of the T-tubule system in cultured myotubes (Ezerman and Ishikawa, 1967; Ishikawa, 1968). The T-tubules are an extensive surface-connected system of membranes which develop and maintain a protein and lipid composition distinct from the sarcolemma (for review see Flucher, 1992). Elegant morphological studies suggested that T-tubules form from the repeated budding of caveolae. Initially the caveolae formed short, beaded, tubular structures, and then these developed into extensive, three-dimensional networks which appeared to be formed from interconnected arrays of caveolae (Ezerman and Ishikawa, 1967; Ishikawa, 1968). Later studies of mouse muscle development in vivo showed a similar association of caveolae-like structures with forming T-tubules, at an early developmental stage (Franzini-Armstrong, 1991). These results were consistent with studies showing that inhibition of T-tubule formation resulted in an accumulation of caveolae-like structures (Schiaffino et al., 1977). This model is still favored by many investigators as some caveolar components are shared with T-tubules (Yuan et al., 1991). However, other studies using membrane-impermeant lipid probes also provided evidence for an internal T-tubule compartment which subsequently fuses with the sarcolemma (Flucher et al., 1991). In the absence of specific markers for muscle caveolae, all of these models have been difficult to test.
We have examined the localization of caveolin-3 in C2C12 cells, primary mouse muscle cultures, and mouse skeletal and cardiac muscle during development in vivo. We show that caveolin-3 is associated with the T-tubule system of developing muscle. We postulate that caveolae and specifically, caveolin-3, may play a role in the formation of the T-tubule system of muscle. In addition, we speculate that the underlying principles of organization and formation of T-tubules and caveolae may be similar.
Media and reagents for cell culture were purchased from GIBCO BRL (Eggenheim, Germany). C2C12 cells were cultured as described previously (Way and Parton, 1995). Transfection was carried out using Lipofectin ( GIBCO BRL) according to the manufacturer's instructions, using caveolin-3-HA in the CB6 vector described by Way and Parton (1995). Cell lines were selected using G418 ( GIBCO BRL). Colonies were subcloned and analyzed for caveolin-3 expression by immunofluorescence, as described below.
Primary cultures of mouse muscle were prepared from 18-d embryos exactly as described by Chu et al. (1995). Briefly, muscle fibers from the limbs of 18-d post coitus embryos were incubated in trypsin and the cells dissociated by trituration. Debris was removed by filtering the cells through gauze, and the dissociated cells were plated on matrigel ( GIBCO BRL) or on calf skin collagen ( Sigma Chemical Co., New South Wales, Australia) according to manufacturer's recommendations.
A peptide corresponding to the 15 NH2-terminal amino acids of mouse caveolin-3 but, with the addition of a COOH-terminal cysteine residue and a bridging glycine residue, was synthesized (CGMTEEHTDLEARIIKD). The cysteine residue was used for coupling to activated keyhole limpet hemocyanin before injection into rabbits using the Imject Activated Immunogen Conjugation Kit ( Pierce, Rockford, IL). The conjugated peptides were separated from free peptide using a Presto Desalting column (Pierce) and the concentration of the pooled conjugates determined using the Bio-Rad Laboratories (Richmond, CA) assay with BSA as a standard. Antisera were affinity purified on a column prepared by coupling the peptide through the cysteine residue (Harlow and Lane, 1988). Affinity-purified antibodies were characterized by immunofluorescence and Western blotting; in each case a specific signal was only detected in differentiated C2C12 cells, and this signal was competed by preincubation of the antibody with the specific caveolin-3 peptide. Antibodies to the NH2 terminus of caveolin-1 (VIP21-caveolin) have been characterized previously (Dupree et al., 1993). Mab427 to the α1 subunit of the dihydropyridine receptor (DHPR) was purchased from Chemicon Intl., Inc. (Temecula, CA). Antibodies to cholera toxin were raised in rabbits using fixed cholera toxin binding subunit ( Sigma Chemical Co.) as immunogen.
C2C12 cells were grown on glass coverslips coated with laminin ( Sigma Chemical Co.) according to manufacturer's instructions. Cells were either fixed in cold methanol or in paraformaldehyde and then permeabilized with 0.1% saponin, as described previously (Parton et al., 1994). After immunolabeling cells were mounted in Mowiol (Hoechst, Frankfurt, Germany) and examined using fluorescence microscopes (Axiovert; Zeiss, Inc.). Confocal microscopy was performed using the EMBL confocal microscope or the confocal microscope (model MRC600 head and laser; BioRad Laboratories; Axioscope, Zeiss, Inc.; used at the Vision, Touch and Hearing Research Centre University of Queensland, Brisbane, Australia). In each case, optical sections were 0.5 μm in the z-plane.
Mice were killed by cervical dislocation, and small pieces of muscle from the leg or atrium were rapidly excised and fixed by immersion in either 8% paraformaldehyde in 100 mM phosphate buffer, pH 7.35, or the same fixative containing 0.1% glutaraldehyde. Muscle pieces were embedded in gelatin and were then infiltrated with polyvinylpyrrolidone/sucrose overnight and processed for ultrathin, frozen sectioning (Griffiths, 1993). Semithick (0.5–1 μm) and ultrathin sections (50–60 nm) were cut on a Leica Ultracut with FCS attachment. Thick sections were transferred to polylysine-coated coverslips and were then labeled immediately or stored until needed, at −20°C with identical results. Labeling of ultrathin sections for electron microscopy or thick sections for light microscopy was performed as described previously (Lütcke et al., 1994; Parton et al., 1989).
C2C12 cells were labeled with cholera toxin binding subunit (10 μg/ml; Sigma Chemical Co.) or with cholera toxin binding subunit/horseradish peroxidase (10 μg/ml; Sigma Chemical Co.) at 4°C as described previously (Parton, 1994), except that all incubations were increased to 2 h to allow time for diffusion into all surface-connected compartments. Labeling of frozen sections or embedding in Epon were performed as described previously (Parton, 1994). Grids were viewed using an electron microscope (model 1010; Jeol, Japan) in the Centre for Microscopy and Microanalysis, University of Queensland (Brisbane, Australia).
As a first step to understanding the function of caveolin-3, we examined its localization in muscle tissues. A peptide corresponding to the NH2-terminal portion of caveolin-3, a region of the protein which is not shared with other members of the caveolin family (Way and Parton, 1995), was used to immunize rabbits, and the resulting antiserum was affinity purified on a peptide column. The affinity- purified antibody (anti-cav3-N) recognized a band of ∼20 kD, which was present in differentiated but not undifferentiated C2C12 cells, and was competed by the specific peptide to which it was raised (results not shown). We investigated the distribution of caveolin-3 and caveolin-1 in fully differentiated skeletal and cardiac muscle cells. Skeletal muscle tissue from adult mice was fixed and processed for semi-thick or ultrathin sections. Sections were labeled with anti-cav3-N, followed by fluorescent second antibodies or protein A–gold. Transverse or longitudinal muscle sections of adult skeletal muscle tissue showed strong peripheral staining of the muscle fibers for caveolin-3 (Fig. 1, A and B). At the ends of muscle fibers, a clearly organized network of labeling was apparent (Fig. 1 A). Immunogold labeling of ultrathin sections showed that caveolin-3 was localized to sarcolemmal caveolae with low labeling of the intervening membrane or the interior of the muscle fiber (Fig. 2). Caveolin-1 was undetectable in the myofibers by immunofluorescence (Fig. 1, C and D) but showed a strong labeling of endothelial cells (Fig. 1 C). Similar results were obtained with cardiac tissue. Double labeling immunoelectron microscopy of thin, frozen sections from mouse atrium with antibodies to caveolin-1 and caveolin-3 confirmed the lack of colocalization of the two proteins; caveolin-3 was restricted to caveolae of cardiomyocytes, whereas caveolin-1 was only detected in capillary endothelia (Fig. 3). In contrast to skeletal muscle where caveolin-3 was only observed in association with sarcolemmal caveolae, caveolin-3 positive caveolae were observed to be associated with both the plasma membrane and the T-tubules of cardiac muscle (not shown).
Immunolocalization of caveolin-1 and caveolin-3 in adult mouse skeletal muscle. 0.5 μm frozen sections of mouse skeletal muscle (A– D) were labeled with antibodies to caveolin-3 (A) or caveolin-1 (C). B and D show the corresponding phase images for A and C, respectively. (A) Caveolin-3 labels the periphery of the fiber with negligible internal staining. Note the regular meshwork of labeled elements at the ends of the muscle fiber. (B) In contrast to caveolin-3, antibodies to caveolin-1 specifically label the endothelial cells of muscle capillaries rather than the muscle fibers. Bars, 5 μm.
Immunoelectron microscopic localization of caveolin-3 in adult mouse skeletal muscle. Ultrathin frozen sections of mouse skeletal muscle were labeled with antibodies to caveolin-3. Specific labeling is associated with sarcolemmal caveolae (small arrowheads indicate the sarcolemmal region; large arrowheads indicate labeled caveolae), as shown at higher magnification in the inset. An endothelial cell (e) in B is unlabeled. Double arrowheads indicate regions of the T-tubule system which generally show negligible labeling. m, mitochondria; sl, sarcolemma; n, nucleus; z, Z-line. Bars: (A and B) 200 nm; (inset) 100 nm.
Immunoelectron microscopic localization of caveolin-3 and caveolin-1 in adult cardiac tissue. Ultrathin frozen sections of mouse atrium were labeled with antibodies to caveolin-3 and caveolin-1. Small gold in A represents labeling for caveolin-3 and in B, indicates labeling for caveolin-1. Caveolin-1 labeling (large arrows) is only detectable on caveolae of endothelial cells (e), whereas labeling for caveolin-3 is only evident within the cardiac muscle cells, showing the specificity of the two antibodies. In each case, labeling is associated with uncoated plasma membrane invaginations with the characteristics of caveolae. The arrowhead in A indicates an unlabeled clathrin-coated pit. p, plasma membrane. Bars, 100 nm.
Earlier work has suggested a role for caveolae in the formation of the T-tubule system. We therefore examined the localization of caveolin-3 during muscle development in vivo and in vitro.
Electron microscopy studies have shown that the T-tubule system of mouse skeletal muscle starts to develop in the period before birth (Franzini-Armstrong, 1991). At this time T-tubules are orientated in the longitudinal direction along the length of the fiber. After birth the T-tubule system is reorganized to form the transverse arrangement characteristic of adult tissue (for review see Flucher, 1992). We therefore examined the distribution of caveolin-3 in mouse skeletal muscle between embryonic day 16 and 3 d after birth by immunofluorescence (Fig. 4). In embryonic mouse leg muscle, strong labeling for caveolin-3 was detectable around the periphery of muscle fibers. In addition, at early stages of development, labeling was apparent within punctate structures throughout the cell (Fig. 4 A). A characteristic feature of the labeling at this developmental stage was lines of regularly spaced puncta close to the surface membrane (Fig. 4 A). Internal staining was particularly striking in 18-d embryonic muscle. At this stage, clearly defined tubules, which apparently extended from the sarcolemmal region into the muscle fiber, were labeled by caveolin-3 antibodies (Fig. 4 B). Such structures are reminiscent of forming T-tubules. However, available antibodies to the DHPR gave low labeling of embryonic muscle tissue, consistent with a low expression level before birth (Morton and Froehner, 1989). Internal labeling for caveolin-3, although decreased, was also evident in newborn mouse muscle (Fig. 4 C). In 3-d-old mice, internal labeling was barely detectable (Fig. 4 D), consistent with the lack of internal labeling in mature muscle.
Immunolocalization of caveolin-3 during muscle development. 0.5 μm frozen sections of mouse skeletal muscle were fixed by immersion at various stages of embryonic development (embryonic day 16, A; embryonic day 18, B), immediately after birth (C) or 3 d after birth (D). All sections were labeled at the same time, and images were prepared with the same exposure and development times. Specific labeling is associated with the periphery of the muscle cells at all stages. In addition, internal labeling is evident from embryonic day 16 up to birth. Labeling is often evident as punctate dots of labeling aligned along the longitudinal axis of the muscle cells (A, arrows). In the embryonic day 18, muscle tubular structures, which apparently lead from the sarcolemma, are labeled (B, arrows). In the newborn muscle and especially 3 d after birth, the internal labeling is decreased. Bars, 5 μm.
The above results suggest that caveolin-3 associates with an internal compartment during muscle development. To investigate this process further, we varied the use of cell culture systems to study caveolin-3 during muscle development. We examined caveolin-3 distribution in two wellcharacterized culture systems: the C2C12 mouse myoblast/ myotube cell line and primary cultured mouse muscle cells. C2C12 cells are a well-characterized model system for studies of muscle differentiation which, in the differentiated state, express caveolin-3 (Way and Parton, 1995). The distribution of caveolin-3 in C2C12 cells was examined using the affinity-purified anti-cav3-N antibodies and immunofluorescence microscopy. After fusion of C2C12 cells to form myotubes, caveolin-3 antibodies labeled an extensive tubular network within the cytoplasm (Fig. 5). As shown by confocal microscopy, the tubules are present within the depth of the muscle fiber, extend over many micrometers, and form a complex branching network throughout the cytoplasm. Labeling was specific for caveolin-3, as it was inhibited by the specific peptide to which the antibody was raised (results not shown). In addition, identical labeling was observed in a C2C12 cell line expressing epitope-tagged caveolin-3 (see Fig. 12).
Immunolocalization of caveolin-3 in C2C12 cells. C2C12 cells were maintained in culture in differentiating medium for 6–8 d after reaching confluency. Cells were fixed with paraformaldehyde and processed for immunofluorescent localization of caveolin-3. Cells were viewed by confocal microscopy. A and B show two sections of two C2C12 myotubes at different planes through the cell: (A close to the base of the cell; B midway through the cell). Tubules and reticular structures (inset) run throughout the entire depth of the cell and are mainly orientated in the longitudinal direction. Specific labeling is associated with the differentiated C2C12 cells, whereas the underlying layer of undifferentiated cells (in the plane of the section shown in A) show low labeling. Bars, 5 μm.
We next examined the nature of the labeled organelles. In view of the morphology of the labeled elements we used antibodies to a well-characterized T-tubule marker, the α1 subunit of the DHPR (α1-DHPR). As shown in Fig. 6, the two markers showed a high degree of colocalization, as analyzed by confocal microscopy. Some peripheral structures were labeled by antibodies to caveolin-3 but not antiDHPR, but, most of the tubular internal structures were labeled with both markers. The labeled elements were predominantly orientated in the longitudinal direction, characteristic of T-tubules in incompletely differentiated muscle cells in vivo (Franzini-Armstrong, 1991). C2C12 cells maintained for longer periods in culture (>10 d), when some of the myotubes showed spontaneous contractile activity showed lower internal labeling (not shown).
Caveolin-3 colocalizes with a T-tubule marker in C2C12 cells. C2C12 cells were cultured as described in the legend to Fig. 5. Cells were fixed with paraformaldehyde and double labeled for caveolin-3 (rhodamine, A, C, E, and G) and for the T-tubule specific marker α1DHPR (FITC, B, D, F, and H). Caveolin-3 colocalizes with α1-DHPR in many tubular/reticular structures throughout the cell (arrows). Note that not all caveolin-3 positive structures are α1DHPR positive (arrowheads). Note the variation in the labeling for α1-DHPR (for example see the relatively low level of labeling of the cell in F) but the clear colocalization with caveolin-3. Bars: (A–D) 5 μm; (E and F) 2.5 μm.
To investigate the association of caveolin-3 with putative T-tubule elements in a more physiological culture situation, we examined the distribution of caveolin-3 in primary cultured mouse muscle cells. Primary cultured myotubes have been used extensively for studies of muscle differentiation and T-tubule formation and show a defined sequence of development in culture (Flucher et al., 1992). Muscle cells from the limbs of 18-d-old mouse embryos were dissociated by trypsin treatment and cultured for up to 24 d in vitro. Three days after plating, the culture medium was changed to differentiation medium, and two days later the cells started to show spontaneous contractile activity. Cells at different developmental stages were labeled for immunofluorescent detection of caveolin-3 and viewed by confocal microscopy. From the first day after plating, caveolin-3 was detectable in a small number of putative myoblasts (Fig. 7 A). Labeling first appeared in the perinuclear region of these cells. As the cells differentiated more, caveolin-3 labeling was observed in the cell periphery (Fig. 7, B and C). After fusion of myoblasts to form myotubes, caveolin-3 labeling greatly increased and labeling appeared within an extensive system of tubular/reticular elements which penetrated the entire cytoplasm (Fig. 7, D–F) and appeared identical to the T-tubule labeling of C2C12 cells. Consistent with the studies of developing skeletal muscle tissue with longer times in culture an increasing number of cells was observed to show predominantly peripheral staining with a reduction in the level of internal T-tubule labeling (Fig. 7 G). The results show that in both primary cultured muscle cells and C2C12 cells, caveolin-3 associates with developing T-tubules. In differentiated cells and in mature muscle, caveolin-3 is no longer detectable within the T-tubule system but is associated with sarcolemmal caveolae.
Immunolocalization of caveolin-3 during differentiation of primary muscle cells in culture. Mouse muscle cells from embryonic day 18 were cultured for various periods at 37°C before fixation and labeling for caveolin-3, as described in Materials and Methods. Cells were fixed 1 day after plating (A) or at various times after adding differentiation medium; 1 d (B and C), 5 d (D), 11 d (E and F), and 24 d (G). Cells were viewed by confocal (A–E and G) or conventional microscopy (F). Specific labeling is associated with the perinuclear and peripheral regions of day 0 myoblasts (A–C). From day 5 onwards, labeling is apparent within the putative T-tubule reticulum of fused myotubes (e.g., compare F with Fig. 12 F). At later stages, an increasing number of cells show surface labeling but low intracellular labeling (G). n, nuclei. Bars: (A–C) 5 μm; (D– G) 10 μm.
The distribution of caveolin-3 in differentiating C2C12 cells was further examined by immunoelectron microscopy. C2C12 cells were fixed with glutaraldehyde and then processed for frozen sectioning. Sections were labeled with affinity-purified anti-cav3-N antibodies. Negligible labeling was found in undifferentiated cells, but in fused cells, caveolin-3 labeling was associated with elaborate networks of interconnected membranes apparently deep within the cytoplasm of the myotubes (Fig. 8). The networks were composed of regular repeating units which, at low magnification, almost had a crystalline appearance. Labeling for caveolin-3, although low, was apparent over these entire networks and was not detectable on other intracellular membranes or the plasma membrane. Labeling appeared to be higher over the periphery of these networks and in the less tightly clustered regions. In these areas the individual units of the reticulum were apparent and, as shown in Fig. 8 B, strongly resembled caveolae. The dimensions of the individual units of the reticulum (Fig. 9 A, inset) suggest that these networks may be composed of fused caveolae or caveolae which have formed repeatedly but have not been released through a fission event.
Immunoelectron microscopic localization of caveolin-3 in C2C12 cells. C2C12 cells were cultured as described in the legend to Fig. 5 and then fixed with a glutaraldehyde-containing fixative. The cells were then processed and immunolabeled for caveolin-3 followed by protein A–gold. Note the specific labeling (gold particles indicated by arrowheads) of extensive regular interconnected arrays of labeled membranes (asterisks). These structures are made up of unit structures with similar dimensions to caveolae (asterisks mark units of a reticulum in the inset). In the less compact arrays, (shown at higher magnification in B), the labeling is stronger, apparently due to greater access to caveolin-3 epitopes, and the individual caveolae-like elements are clearly evident (arrows). Note the similarity of these structures to caveolae clusters of nonmuscle cells. Bars, 200 nm.
Immunoelectron microscopic localization of caveolin-3 in C2C12 cells. C2C12 cells were cultured and processed for frozen sectioning as described in the legend to Fig. 8, except that the cells were fixed in paraformaldehyde. Sections were immunolabeled with affinity-purified antibodies to caveolin-3. Specific immunolabeling for caveolin-3 is associated with 50–60 nm budding profiles with characteristic caveolar morphology (arrowheads). The caveolin-3 labeled elements form complex, extended arrays which penetrate the center of the muscle cell. The complex, clustered arrangement of these structures, as viewed in thin sections, suggests that in three dimensions, the caveolae form large clusters resembling “bunches of grapes.” In some regions, reticular elements are evident (Fig. 4 B, asterisk) which resemble forming T-tubules, as described in early morphological studies (Ishikawa, 1968), but are less compact and regular than those seen in glutaraldehyde-fixed cells. Note that the caveolin-3 labeling is typically associated with the bud-like profiles rather than the tubular interconnecting regions. The double arrowhead indicates a clathrin-coated pit. Bars, 200 nm.
We examined this further in paraformaldehyde-fixed cells. As shown in Fig. 9, the reticular structures appeared to be less well preserved under these fixation conditions, as they were less extensive and more irregular. However, labeling for caveolin-3 was clearly increased compared to glutaraldehyde-fixed cells and was associated with large clusters of caveolae-like elements. Specific labeling was invariably associated with caveolae-like profiles and not with any intervening flat membrane. The labeled structures extended over several micrometers into the center of the cells (Fig. 9). The labeled elements also had associated clathrin-coated pits, consistent with their identification as T-tubule elements.
The similarity of the caveolin-3–labeled elements to the previously described precursor T-tubule elements (Ishikawa, 1968) and the colocalization of caveolin-3 with a T-tubule marker by confocal microscopy, strongly support the idea that caveolin-3 associates with developing T-tubules. However, the available antibodies to the DHPR gave weak labeling by electron microscopy. To confirm that these elements were surface connected, we incubated differentiating C2C12 cells with peroxidase-labeled cholera toxin binding subunit (CT-B) at 4°C, a temperature at which endocytosis is blocked. The plasma membrane receptor for cholera toxin, GM1, has previously been shown to be enriched in caveolae (Montesano et al., 1982; Parton, 1994). Surface-labeled cells were fixed and embedded in Epon. Semi-thick sections were cut parallel to the culture substratum. As shown in Fig. 10, CT-B–peroxidase labeled the cell surface as well as long tubules and reticular structures throughout the cell. The labeled tubular elements were preferentially orientated in the longitudinal direction, characteristic of developing T-tubules. Both the tubular and reticular elements (Fig. 10, insets) showed the characteristic morphology of “fused” caveolar elements and appeared similar to those labeled with caveolin-3. We then repeated the above experiment using unlabeled CT-B and processed the cells for frozen sectioning. Thawed sections were labeled with antibodies to caveolin-3 and CT-B. As shown in Fig. 11, labeling for CT-B was associated with the cell surface and also with the caveolin-3 positive putative T-tubule elements. These results confirm that a large number of the labeled elements are indeed surface connected. Taken together, the results suggest that during muscle differentiation, caveolin-3 associates with developing T-tubules. The developing T-tubule system takes the form of a reticulum made up of individual units with morphological features and components characteristic of caveolae. These observations are consistent with the involvement of repeated caveolae formation in T-tubule development.
Cholera toxin peroxidase labeling of differentiating C2C12 cells. C2C12 cells cultured as described in the legend to Fig. 5 were incubated with CT-B-peroxidase for 2 h at 4°C and then fixed and processed for embedding in epon. Semi-thick sections (∼200 nm) were cut parallel to the substratum. Peroxidase-labeled elements are seen within the depths of the cell (arrows). The labeled structures are composed of individual bud-like elements of ∼60 nm diam, which form chains or reticula of interconnected structures (insets). Bars, 200 nm.
Immunoelectron microscopic localization of caveolin-3 in cholera toxin surface-labeled C2C12 cells. C2C12 cells cultured as described in the legend to Fig. 5 were incubated with CT-B for 2 h at 4°C before fixation with paraformaldehyde. Ultrathin frozen sections were immunolabeled with antibodies to cholera toxin, followed by 15 nm protein A–gold (arrowheads) and to caveolin-3 detected with 10 nm protein A–gold. Cholera toxin labeling is evident on the cell surface and in the extensive caveolin-3–positive tubulovesicular elements. pm, plasma membrane. Bars, 200 nm.
Finally we sought to examine the localization of caveolin-3 with respect to caveolin-1. We have previously shown that caveolin-3 and caveolin-1 colocalize when expressed in fibroblasts. To examine the distribution of the two proteins in myoblasts, which express caveolin-1 but not caveolin-3 (Way and Parton, 1995), a stable C2C12 cell line containing caveolin-3 with a COOH-terminal HA tag was generated. We examined the distribution of epitope-tagged caveolin-3 and of caveolin-1 during differentiation by double labeling with antibodies to the NH2 terminus of endogenous caveolin-1 (Dupree et al., 1993) and the 12CA5 antibody against the HA tag. In undifferentiated C2C12-CAV3HA cells 24 h after plating there was clear colocalization of caveolin-1 and epitope-tagged caveolin-3 at the cell periphery (Fig. 12, A and B). Therefore, in these undifferentiated muscle cells, as in fibroblasts (Way and Parton, 1995), caveolin-3 is apparently directed to caveolae. In contrast, 48 h after plating, but before fusion into myotubes, a number of cells started to show a different labeling pattern for caveolin-1 and caveolin-3 (Fig. 12, C and D), with caveolin-1 generally showing a more diffuse labeling pattern. In fused C2C12-CAV3HA cells epitope-tagged caveolin-3 was localized to the T-tubule system running throughout the cytoplasm (Fig. 12, E, F, and H). In contrast, caveolin-1 showed negligible labeling in the differentiated cells (Fig. 12 G), suggesting that the expression of caveolin-1 is reduced upon muscle differentiation. It therefore appears that caveolin-3 expressed in C2C12 cells colocalizes with caveolin-1 in the nondifferentiated state, but, as the cells differentiate, the two markers are separated. Caveolin-1 is sorted away from caveolin-3, which eventually associates with the T-tubule system.
Immunolocalization of epitope-tagged caveolin-3 and endogenous caveolin-1 in C2C12 cells. C2C12 cells expressing caveolin-3 with a COOH-terminal HA tag were fixed after 1 d (A and B), 2 d (C and D), or after 6–8 d, 4–6 d after the cells reached confluency and differentiation medium was added), (E–H). The cells were then labeled with antibodies to caveolin-1 (A, C, and G) or to the HA tag (B, D, E, F, and H). A–D and G and H show cells double labeled for caveolin-1 and the epitope-tagged caveolin-3. In day 1 myoblasts (A and B), the endogenous caveolin-1 and expressed caveolin-3 colocalize (arrows). At later times (C and D), some cells clearly show a different labeling pattern for the two caveolin proteins (arrows indicate comparable regions of the two cells which are labeled for caveolin-3 but not caveolin-1). After fusion of myoblasts to form myotubes, the epitope-tagged caveolin-3 is present within the T-tubule system which runs thoughout the cell (E, F, and inset, arrows). G and H show cells double labeled for caveolin-1 and epitope-tagged caveolin3. While caveolin-1 labeling is present in neighboring, undifferentiated myoblasts, labeling is very low in the multinucleate myotube (arrows). Weak staining represented background labeling as shown by peptide inhibition (not shown). n, nucleus. Bars: (A–D) 2 μm (same magnification); (E–H) 5 μm (E and F, G and H, same magnifications).
The T-tubule system of mammalian cells is an extensive membranous system which penetrates the entire muscle fiber but is continuous with the muscle plasma membrane. The protein and lipid composition of the T-tubule system is distinct from that of the sarcolemma. How this system develops and maintains its unique composition is a fundamental problem in cell biology. In the present study we have shown that caveolin-3, a member of a family of integral membrane proteins proposed to be involved in organizing membrane form and composition, is associated with precursor T-tubule elements in skeletal muscle. Our studies of caveolin-3 and caveolin-1 in C2C12 cells, primary cultured myotubes, and developing muscle in vivo suggest the following series of events. As myoblasts fuse to form myotubes, expression of caveolin-1 starts to decrease, and expression of the muscle-specific protein, caveolin-3, is dramatically increased. Caveolin-3 then appears in association with tubular elements which penetrate the entire cytoplasm of the myotubes but are predominantly orientated longitudinally along the length of the muscle fiber. These tubules are labeled by antibodies to the α1 subunit of the dihydropyridine receptor. By electron microscopy, the caveolin-3–labeled structures resemble elaborate, regular arrays of interconnected clusters of caveolae and are connected to the cell surface. These elements therefore have the characteristics of precursor T-tubules (Flucher et al., 1992). Around the time of birth, the level of caveolin-3 associated with the T-tubules starts to decrease, and in adult muscle, caveolin-3 is no longer detectable within the T-tubule system but is highly concentrated in sarcolemmal caveolae. The association of caveolin-3 with T-tubules is therefore restricted to a precursor T-tubule stage in which the forming T-tubules appear to consist of interconnected caveolae-like elements. These results raise the intriguing possibility that caveolae, and in particular caveolin-3, are involved in the biogenesis of the T-tubule system during muscle differentiation.
Early electron microscopic studies were the first to suggest a role for caveolae, identified purely on morphological grounds, in early T-tubule formation in cultured myotubes (Ezerman and Ishikawa, 1967; Ishikawa, 1968). These studies suggested that caveolae form repeatedly but keep their connectivity with the surface, leading to the generation of extensive, regular arrays of interconnected caveolae-like structures. The resulting reticulum was proposed to represent the precursor T-tubule system based on its connectivity with the cell surface and its junctions with the sarcoplasmic reticulum. Later studies showed that these structures contained markers characteristic of the T-tubule system but excluded sarcolemmal components (Flucher et al., 1993). In vivo studies also confirmed the association of caveolae-like structures with developing T-tubules during embryonic muscle development (Franzini-Armstrong, 1991). Moreover, in regenerating muscle fibers, the reforming T-tubule system was shown to be composed of caveolar elements often having a “honeycomb” appearance (Miike et al., 1984). Taken together, these studies strongly argue for a role for caveolae-like structures at an early stage in T-tubule formation, but their identification as caveolae was based on morphology alone. The findings of the present study clearly show that these elements are indeed caveolae, as demonstrated by labeling with the caveolin-3 antibody. The reticular precursor T-tubule elements described in the above studies are apparently identical to the structures labeled with antibodies to caveolin-3 in C2C12 cells in the present study (e.g., compare Figs. 8 and 9 with Figs. 2 or 10 of Ishikawa, 1968) and show for the first time that a caveolin protein is associated with the developing T-tubule system. We have also shown that at least some of these structures are accessible to and labeled by a cholera toxin conjugate administered in the extracellular medium. Our results are therefore consistent with the proposed endocytic model for T-tubule formation (Flucher, 1992). A second model for T-tubule formation invokes a role for exocytic, rather than endocytic transport. In this model, the precursor T-tubule system is formed as a result of exocytic transport and is not initially connected to the plasma membrane. Recent studies provided evidence for a combination of these models, as some T-tubule precursor elements were shown to be discontinuous with the cell surface at early stages of development (Flucher et al., 1991). Further work will be required to ascertain how this compartment relates to the caveolin-labeled elements described here. At present we cannot rule out that newly formed caveolae can fuse with each other to form clusters which subsequently fuse with the cell surface. In many respects, the caveolae clusters shown here resemble the caveolin-1 positive caveolae in many nonmuscle cells. Caveolae in many cell types form extensive arrays of interconnected structures which penetrate the cytoplasm (Parton, 1996). This is particularly evident after okadaic acid treatment, when caveolae form large clusters which appear to be pulled into the center of the cell in an actin- and microtubule-dependent manner (Parton et al., 1994). These structures show a remarkable resemblance to the caveolin3 positive caveolae clusters seen in the present study (Figs. 8 and 10). Taken together, these studies suggest that caveolae have a propensity to form such structures, and in muscle cells, these structures form the basis for the development of the T-tubule system.
In view of the known characteristics of caveolins, the postulated role of caveolae in T-tubule formation, and the transient detection of caveolin-3 within the developing T-tubule system, we speculate that caveolin-3 might be required to generate the unique protein and lipid composition of the T-tubule system. We have previously shown that caveolin-1 expression in caveolae-deficient cells causes de novo formation of caveolae (Fra et al., 1995b). The high density of caveolin-1 in the caveolar membrane as well as the need for a threshold level of caveolin-1 in the plasma membrane to produce caveolae (Parton, 1996), both argue for a structural role of caveolin in caveolae formation. Two recently described properties of caveolin-1 might be important in caveolar-domain formation. First, caveolin-1 self-associates to form oligomers (Monier et al., 1995; Sargiacomo et al., 1995). Second, caveolin is a cholesterolbinding protein (Murata et al., 1995). Cholesterol is essential for caveolae form and function (Rothberg et al., 1990, 1992), and it has been proposed that the interaction of caveolin with cholesterol in glycosphingolipid-enriched domains may be necessary for caveolae formation (Parton and Simons, 1995). Caveolin-3 appears to show a similar propensity to form oligomers (Tang et al., 1996) and shows high sequence homology in that region of the caveolin molecule postulated to be involved in oligomer formation (Sargiacomo et al., 1995). The region of the molecule involved in cholesterol binding is still unknown, but the three known caveolins have particularly high homology in the intramembrane and membrane proximal regions (Way and Parton, 1995; Tang et al., 1996). Thus, we speculate that caveolins may be general modulators of the plasma membrane, being able to generate the unique protein and lipid composition of caveolae, or, in the case of caveolin-3, the precursor T-tubule domain. Intriguingly, T-tubules, like caveolae, are known to be enriched in cholesterol (Hidalgo et al., 1983; Horgan and Kuypers, 1987), and this has even been used as a marker in fractionation studies (Knudson and Campbell, 1989). In addition, in the present study we have shown that the precursor T-tubule system is labeled by cholera toxin conjugates, which are concentrated in caveolae of other cells (Parton, 1994). While further work will be required to ascertain whether the receptor for cholera toxin, the ganglioside GM1, is actually concentrated within T-tubules, a logical extension of this model for T-tubule formation is that the protein and lipid composition of the T-tubule system may be maintained by principles similar to those of caveolae. Both caveolae and T-tubules represent membrane systems which are continuous with the plasma membrane but have a distinct composition. Caveolin-3 might be involved in the initial process of generating the T-tubule domain, and then cytoskeletal elements would assume the role of maintaining this structure in its precise alignment with each side of the Z-lines. Perhaps caveolae at the neck of the T-tubules in mature muscle (Franzini-Armstrong et al., 1975; Zampighi et al., 1975) act as barriers to prevent lipids and proteins of the sarcolemma and T-tubules from intermixing, as previously hypothesized (Flucher, 1992). In view of the importance of lipid-based sorting mechanisms in a number of different aspects of cellular organization (Simons and Ikonen, 1996), the hypothesis that the distinct sarcolemmal and T-tubule compositions may be generated using principles similar to those of caveolae, clearly warrants further attention.
In mature skeletal muscle, caveolin-3 is restricted to sarcolemmal caveolae and is no longer detectable in the T-tubule system. A decrease in internal labeling was already apparent around birth. From birth onwards, the T-tubules are gradually reorganized from longitudinally orientated tubules to the regularly spaced radially orientated T-tubules characteristic of mature muscle (Franzini-Armstrong, 1991). Our observations of developing mouse muscle, primary mouse muscle cultures, and C2C12 cells suggest that the association of caveolin-3 with the T-tubule system is restricted to the predominantly longitudinal precursor T-tubules. This longitudinal arrangement has been shown to persist for many days in cultured C2C12 cells and in primary cultures but appears to be short-lived in vivo (Flucher, 1992), fitting well with the results of the present study. As the T-tubules reorganize, caveolin-3 may be removed from the T-tubules, by some unknown recycling mechanism, or newly synthesized protein may be directed away from the T-tubule system to the sarcolemma. Alternatively, the epitope recognized by the antibody, at the NH2 terminus, may be masked. A precedent exists for the latter, as intracellular labeling for caveolin-1 is not detectable within the trans-Golgi network of fibroblasts with antibodies to the NH2 terminus of the protein, but only with antibodies to the COOH terminus (Dupree et al., 1993). However, as the morphological features characteristic of caveolae are not detectable within the T-tubules of skeletal muscle, we favor the view that caveolin-3 is not present in the mature T-tubule system but only during its development. The fact that caveolin-3 is present in T-tubules during development but not in the final differentiated state, when it is restricted to sarcolemmal caveolae, suggests that caveolin-3 might have two distinct functions: as a morphogenetic element involved in forming the T-tubule domain, and as a component of sarcolemmal caveolae. The role in sarcolemmal caveolae is presumably similar to that of caveolin-1 in other cells, that is, maintenance of caveolar form and signal transduction. The functional interaction of caveolin-3 with trimeric G protein α subunits is consistent with a role in signaling in muscle cells similar to that of caveolin-1 in nonmuscle cells (Tang et al., 1996). It should be noted in addition that caveolin-3 is also present in smooth muscle cells which do not have a T-tubule system (Song et al., 1996). This again indicates that the function of caveolin-3 is not restricted to T-tubule formation.
Caveolin-1 was not detectable in differentiated C2C12 cells nor in adult muscle tissue by immunofluorescence, although endothelial capillaries were heavily labeled (Fig. 12). However, undifferentiated myoblasts express caveolin-1, and we show here that expression of caveolin-3 in these cells results in initial colocalization and then segregation. Future studies should establish the signals involved in the sorting of the two proteins to different cellular compartments. While caveolin-3 associates with the T-tubules, the level of caveolin-1 decreases, suggesting that caveolin-3 replaces caveolin-1 as the major caveolin of differentiated muscle cells. The absence of caveolin-1 in differentiated cultured muscle cells is consistent with two previous studies (Munoz et al., 1996; Tang et al., 1996). However, despite the absence of caveolin in differentiated cultured cells, Munoz et al. (1996) detected caveolin-1 within muscle tissue by Western blotting. This apparent discrepancy may be explained by the abundance of caveolin-1 in endothelial cells in the muscle tissue.
In conclusion, we speculate that caveolin-3 plays a role in T-tubule formation analogous to that of caveolin-1 in caveolae formation, and that caveolae and T-tubules may represent different manifestations of lipid-based sorting phenomenon. In the final mature muscle, caveolin-3 has an additional role in sarcolemmal caveolae where, as in other cells, it is presumably involved in interactions with signaling molecules. These studies raise the intriguing possibility that T-tubules and caveolae may use similar principles to generate and maintain their form and composition.
The authors would like to thank Brigitte Joggerst (European Molecular Biology Laboratory, Heidelberg, Germany) and Colin MacQueen (VTHRC, University of Queensland) for excellent technical assistance. We are indebted to Drs. Melissa Little, Jenny Stow, and David James for providing reagents and to Dr. Peter Noakes for advice regarding primary cultures. We are particularly grateful to Drs. David James and Kai Simons for enlightening discussions and for their comments on the manuscript.
This work was supported by grants to R.G. Parton from the National Health and Medical Research Council of Australia and from the Human Frontiers Science Foundation.
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industry_corpus2: Biopharmaceuticals


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This law of cause and effect is not a punishment but is wholly for the sake of education or learning.
– BE yourself and surround yourself with what you want to have present in your Life.
– True joy follows doing what we’re suppose to be doing and waiting for the reward to come in on its own time.
These beautiful Handmade Knots Bracelets handmade by Buddhist Monks and their families are tied as they recite mantras to allow the bracelets to absorb the powerful energy such that the positive energy can flow into its bearer. It protects you from “bad karma” and ill-will. Our bracelets have been seen worn by thought leaders, activists, and celebrities for their powerful energy.
Previous article This Might Be the Most Beautiful Poem Ever Written.

industry_corpus2: Others


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At Woolverstone, on the River Orwell using a Hawk 20 sailing boat (up to 4 persons) and a Wheely power boat (up to 8 persons and 4 wheelchairs) we take sailors out on the water for approximately 2 hours either up river towards Ipswich or down river towards Felixstowe depending upon the wind and tide. These sessions are generally for those who will not learn to sail solo. Carers come sailing with the group. Who goes in which craft is at the discretion of the Duty Senior Instructor.
We can provide waterproof clothing and buoyancy aids however you should consider brining hats, scarves, gloves suitable footwear and on the odd occasions sun block.
Although these spaces are applied on a first come first served basis, priority will be given to sailors with no other bookings. To apply for these spaces please email your request to info@woolverstone project.org.uk with details of sailor, personal assistants and number of wheelchairs you would like to bring on board.

industry_corpus2: Travel & Geography


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As in Alice in Wonderland … Thus begins the journey in the historic Bottega del Meneghello in Milan. It seems incredible that this place is located just steps away from Corso Como, the heart of the city of fashion and Piazza Gae Aulenti, the hub of the recent “vertical” transformation, built-up endeavor.
The shop in Via Fara, number 5 seems to not know the noise of the city and its construction sites, almost giving the idea of being there since the dawn of time. Far from any reference of time or space, it is the home of Osvaldo Menegazzi, the “Maestro”, which here creates, paints, collects and shows his works alongside his Muse, Cristina Dorsini, niece and art director at the Il Meneghello publishing house. It is she who accompanies Alice during her journey through the hidden wonders within the atelier and it is she who introduces the theme which has so inspired all the works of Il Meneghello, Tarot cards.
Tarot cards appeared in Italy in the 15th Century, most likely just in Milan in the Visconti Court (Lords of the city from 1277 to 1447), one of the main sites for the spreading of the culture of that period. Generated here were fashions, styles and games which then taken and adapted in all the European courts. Playing with Tarot cards was a way to pass the time but not only. The cards, in fact, had a strong symbolic value, contained religious and philosophical teachings and had images representative of the protagonists of the history of time. Cristina underlines the artistic and historical value of the so-called “Visconti di Modrone” deck, the oldest in which it is possible to note the presence of particular allegorical cards, identified with the name of Triumphs.
Tarot cards are as follows: 56 Minor Arcana (in turn divided into Numbered and Court cards) and 22 Major Arcana (or Triumphs). The latter cards have names such as “The Emperor”, “The Hierophant”, “The Magician”, but also of virtue and other images (“Temperance”, “Judgment”, “The High Priestess”…) which over the centuries have been enhanced with alchemical references important enough to make the Triumphs – from the end of the 18th Century – fundamental tool of the art of divination and synthesis of the esoteric dimension.
But what has drIven Osvaldo Menegazzi to become “the painter of Tarot cards” from that which he was, an antiquarian and metaphysical painter? “My passion came alive during the 1970s, when I came across the first deck of 78 cards, the result of an engraving effort dating back to the 18th Century.” Il Meneghello studied and gave much appreciated worth to this precious deck. In fact, in the cards, art and craftsmanship speak a common language which is transposed by all the senses: touching the prints, observing the color renderings and interpreting the author’s stylistic characteristics. This is the point in which the Maestro decided to embark on a new path and began to devote himself personally to the study and design of the cards. He is fully absorbed by this activity and it has allowed him to express an artistic streak which knows how to wander between the most diverse semantic fields.
The commitment of the Maestro does not stop there. Osvaldo Menegazzi founded the Il Meneghello publishing house to bring life back to ancient decks, giving them new luster and personally takes care of the reprints. The publishing house is soon put in light for the valuable work of giving worth to something old where nothing is left to chance; embossing, paper and colors everything exactly as it should bringing back the historical and artistic value of the Tarot cards. So much so that the museums themselves which preserve the cards have allowed for their printing. One of the most important and certainly one of pride for the Maestro is the “Sola Busca” deck, dating back to the late 15th Century and destined for the Este Court was reprinted in a limited edition in 2013 in occasion of the exhibition dedicated to it at the Pinacoteca di Brera .
In the Il Meneghello bottega, it is possible to breathe in all these experiences, just take a look at the walls, browse among the shelves or chat with Osvaldo and discover the secrets and mysteries of this magical world.
And thus come alive the decks dedicated to insects in which the arcane XVIII “The Moon” is represented by a firefly while the Praying Mantis embodies “The Devil” (XV) and a spider turns into ”The Hanged Man” (XII). From the world of nature, which is traveled far and wide (shells, fish, trees…), we move on to that of art with bouquets inspired by currents and styles as in the case of “The amazing journey of a mannequin by painter in 22 Major Arcana” (return to metaphysical painting) or to the one dedicated to Futurism.
Many are also the references to Eastern symbologies that are found in his paintings and in his artistic compositions: large collages where Tarot cards, drawings and sometimes ideograms as well as images of Indian gods together with various objects chasing each other – just like the characters of Alice’s world – yet to returning each time to a universe of varied and highly evocative symbols .
The ease with which the Maestro has been able to deal with subjects so diverse in the creation of his tarot cards suggests a hidden talent. In the universe of images of the Maestro, we find a rare alchemy of artistic sensibility, artisan skill and love for life.
Each year, the Maestro comes up with a new deck, starting with a sketch, a rough draft and works on it until the idea which guided his hand has not been reached. There are still plenty of worlds to explore and the work of Il Meneghello, in this sense, could go on forever. A niche of collectors and enthusiasts from Italy, but especially from abroad (France, USA and Japan, the countries more receptive to this form of art), renew interest year to year for the work of the Maestro. The limited editions, the careful attention paid to the packaging and all made by hand, the use of sealing wax and other craft materials, add value and uniqueness to each deck, real “Made in Italy” treasures.
Seventy is the number of decks printed so far over the past forty years of business at the shop, and certainly over time the number will increase at the expense of the physical space of the atelier which is shrinking.
At The Trendscendent we believe that every discovery we bring to our readers is a rendezvous with a destiny of excellence, like picking a card from a precious tarot deck of our maestro. Discover your card here , meet your destiny and let the Triumphs guide your path to excellence.


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With Only around 40,000 seniors, Alaska has the smallest state population of seniors and also the smallest percentage population of seniors (6.4%) . The small population of seniors spread over a relatively large area makes it difficult for businesses to support the local senior population. Alaska also has the smallest number of assisted living facilities of any state.
Alaska also ranks extremely high with a cost-of-living factor of 126 compared to the national average of 100. These factors combined to make Alaska one of the most difficult areas for retirement including assisted living. Two other factors have a significant impact on discouraging seniors from living in the area. The often harsh weather and driving conditions can be very dangerous for many seniors. The other critical factor is that emergency senior health services and other state-of-the-art medical services are more rare in Alaska when compared to more metropolitan areas. With all these negative factors in mind, most seniors still prefer to live in close proximity to their friends and family. In many cases it may still be best to live in an Alaska assisted living facility if you have a family that will visit frequently and be available as an advocate on any senior health care issues.
Please reduce your list of potential Alaska assisted living facilities to a minimum before you contact these government agencies for assistance.
This directory includes 71 assisted living options for Alaska. Use the "Advance Search" to find the nearest 40 senior services based on your desired location or select a city and then choose a tab to see different types of senior care including nursing homes, Alzheimer's care centers, CCRCs, independent living, in-home and hospice care.
Here are the direct links to Fairbanks Nursing Homes, Juneau Nursing Homes, or use the advanced search and limit your results to Nursing Homes.

industry_corpus2: Health & Medicine


text: This new home was conceived as a historic telescoping house. The brick ‘original’ was modeled after local buildings in Middleburg, VA. The house is connected to a guest house by a pergola. This element creates the transition between the formal front garden and the less formal pool, hot tub and rear yard.

industry_corpus2: Real Estate & Construction


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Balance is the key to life. Work is a very important area of life. We invest our creativity, our hard work, our commitment in our careers. We start focusing on our work life at a very young age when in schools we are asked to write about “what would I want to become when I grow up” kind of essays. So at the work front most people seem to take commitment very naturally. But we are not trained to focus on subtle aspects of life. Focusing on that takes a whole deal of awareness, choice, and questions. When we are growing up we cherish our time spent at home as well. As kids, going out with parents for movies, shopping, watching games, all become a part of our cherished memories. At home front every small thing counts, holidays, weddings, festivals, funerals.
When we start our career we are genuinely committed. Then as time passes we seek a romantic partner, we seek kids, a home, a comfortable life. That’s when balance steps in. We are sometimes too focused in one area more than other. Sometimes one area demands a great deal of our attention and other area feels neglected.
For example, if a person is nearing a promotion and has to show a lot of commitment in training for the new added responsibility but at the same time is required to help their kids in study/sports, or required to take care of their parents as well.
Such situations can be challenging and stressful for anyone and requires a balance. We are sometimes giving a lot of us to everyone around us but to ourselves. Sad thing is we are not taught how to handle such situation is schools itself. I personally believe if we are taught a little bit more about the practical and realistic aspects of life in schools it will be easy for us to incorporate it in our mind. Nevertheless I believe balance is an attitude rather than a skill. We can cultivate this attitude within us.
Sit down every morning for 30 minutes with an intention that I am allowing a 30 minutes of nothingness to myself. In a goal driven system meditation should at least be goal free. You can put an alarm of 30 minutes while you sit straight cross legged and simply focus on your breath. 30 minutes of nothingness will train your mind to slow down and be less reactive, to watch and maybe not react at all if not required, be proactive instead, and to take a break in essence.
A friend of mine once went backpacking on his bike for a month solo. He lived in different places in different cities and came back a changed man. One of the things he noticed in this solo journey was that he wasn’t taking a full breath at all, all his life. In that journey he realized his breath was too shallow. We need to take 2 minutes break every 1 hour and close our eyes just to focus on our breath. I promise you it will be amazing.
We live our life a lot on auto pilot. We have fixed schedules of what we are doing every work day and every day off. It helps when we stop to ask a few questions about our routines. What would I do in this situation if I am truly being me?
We worry about home at work and we stress about work related issues at home. So we are never truly present in the moment thereby getting robbed of some very precious moments of our life. Living in present is probably the most important step in learning the balance of life. Like most things in life, the ability to be in present moment can come with setting an intention, practicing it over and over again and enjoying the feel of it. The whole ‘being in present moment’ thing will be then anchored in your reality.
Happiness is also an attitude rather than a goal. By choosing to be happy in this moment we can help achieve a balance and feel content. It also helps us to be more motivated in life to do more if we choose to be happy.
There are many ways to seek a balance in life. One way will work for you more than others. Happy exploring your way to seek balance!

industry_corpus2: Subject Education


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Scroll ahead to learn more about death and injuries reported on amusement park and carnival rides.
10-year-old Caleb Schwab died on Verruckt, a water raft ride, at a Schlitterbahn in Kansas City on Aug. 7, 2016.
Samantha Aguilar, 16, was on a ride called the "Sizzler" at a church carnival in El Paso, Friday April, 29, 2016, before she was ejected from her seat and killed, multiple media reports said.
Four people were slightly injured in 2014 when the Ninja coaster at the Six Flags Magic Mountain amusement park hit a tree branch dislodging the front car and keeping nearly two dozen summer fun-seekers hanging 20 to 30 feet in the air for hours as day turned to night Monday, July 7, 2014, in Valencia, Calif. Two of the four people hurt on the Ninja coaster were taken to the hospital as a precaution, but all the injuries were minor, fire and park officials said.
A woman reportedly died on a wooden roller coaster called the Raven at Holiday World in Santa Claus in Indiana in 2003. Tamar Fellner, 32, fell from the ride while it was in the midst of a 69-foot drop, according to reports.


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1. Serving to disperse morbid matter; discussive; as, a discutient application.
2. An agent (as a medicinal application) which serves to disperse morbid matter.
1. A strong disapproval or dislike of someone or something which is considered undeserving of respect: The mayor regarded the proposal for building a shopping center in the city park with disdain.
2. Etymology: from Middle English from Old French desdeign, desdeignier; based on Latin dedignari from de-, "reversal" + dignari, "to consider worthy" from dignus,"worthy, deserving recognition". © ALL rights are reserved.
Conveying a strong hatred for a person, or something, that is thought does not deserve respect: "When Janine was at the restaurant, she looked at the plate which the waiter brought with a disdainful glare because it was not what she had ordered."
A reference to a person who has a strong disapproval or dislike for something, or someone, who is regarded as not deserving respect: "The artist disdainfully reacted when someone criticized his work at the exhibition."
To undo the process of envenoming; to deprive of its venom.
To view or to treat with dislike or disapproval.
1. An appearance that has been spoiled or is misshapen.
2. The result of being disfigured; such as, a permanent scar.
1. Misspellings of dysfunction, dysfunctional.
2. Any disturbance in the function of an organ or body part.
3. Abnormal or impaired functioning, especially of a bodily system or social group.
Showing page 14 out of 35 pages of 513 main-word entries or main-word-entry groups.


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Depart from Johannesburg International airport for Tel Aviv on flight LY052 at 21:35. Dinner on board.
Arrive Tel Aviv at 0740 after breakfast on board.
Transfer up the Mediterranean coastline for a visit to the Carmelite Monastery at the Murakah, where Elijah smote the false prophets. Drive by Cana of Galilee where Jesus performed the first miracle of turning water into wine. Continue onto Nazareth Village to see a unique reconstruction of what first century Nazareth looked like on an actual ancient agricultural site.
Sing praise on a morning sail across the Sea of Galilee. See a boat from the time of Jesus. Visit Tabgha, where Jesus multiplied five loaves and two fish. Continue onto Capernaum, the center of Jesus’ Ministries. Ascend to the top of the Mt. of Beatitudes, where the group leader can relive the Sermon on the Mount. In the afternoon drive up to the Golan Heights for the magnificent view of the Sea of Galilee from above and learn about the geopolitical issues in the region.
Start the day with a spectacular view of Jerusalem from Mt. of Olives. Walk down the Palm Sunday Road and visit the Church of all Nations at the Garden of Gethsemane Visit the Pools of Bethesda and St. Annes Church. Pass through Ecce Homo Arch to the Via Dolorosa. Visit each Station of the Cross to the Church of the Holy Sepulcher. Experience a spiritual visit to the Western Wall.
Experience Yad Vashem Holocaust Memorial. Visit the Room of the Last Supper on Mt Zion. See the dungeon where Jesus was imprisoned at the Church of St. Peter in Gallicantu. End your Jerusalem experience with Holy Communion at the Garden Tomb.
Arrive early morning in Johannesburg with fantastic memories of a wonderful trip.
Hotels are subject to change until confirmation.
5. Meals – Breakfast and Dinner daily in hotels. Festive farewell restaurant dinner.


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muting is an acceptable dictionary word for games like scrabble, words with friends, crossword, etc.
The word 'muting' is made up of 6 letters.
Using the word 'muting' in Scrabble will fetch you 9 points while using it in Words with Friends will fetch you 13 points (without taking into consideration the effect of any multipliers).
See all the words ending with muting.
See all the words which start with muting.
See all the words which contain muting.

industry_corpus2: Gaming


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As Rupert Murdock once put it, “The world is changing very fast. Big will not beat small anymore. It will be the fast beating the slow.” But it goes beyond speed, with the unspoken combination of effectiveness and efficiency that must accompany that speed lest you crash and burn. We also have to add flexibility to the mix, because what really counts is agility: the ability not just to roll with the punches, but to avoid them and other obstacles altogether while travelling down the right road.
Sometimes, I think we’re living not through the Information Era, but the Hurry Up Epoch. Things are changing faster than we can keep up with in all areas of our lives — technologically and socially. We have to be like the Red Queen in Alice in Wonderland, running as fast as we can just to stay in one place. That’s fine as long as you take the initiative to maximize flexibility along with efficiency and effectiveness, and you don’t have to give up all your free time to do it. In fact, one of the great things about all this newfangled speed is that the onrushing technological change encourages it, yanking it up by its own bootstraps.
The fact that your new smartphone will no longer be “bleeding edge” in three months is part of the price of this progress. All the changes not only make it possible to go faster but also easier to do so. We’re scrambling ahead in an upward spiral, widening and advancing personal and team productivity as we go.
Unless you like the taste of dust, you’d best be leading the pack. That’s always been good advice, but it applies now more than ever. But speed won’t suffice: like Jack the famous candlestick jumper, you’ve got to be quick and nimble. Cycle and time-to-market speed has steadily decreased due to new technology and social innovations. One of the latter is that growing realization that executives are no longer limited to the C-Suite. Anyone in a position to execute strategy affecting the organization, from CEO right down to the low-level manager and foreman, is an executive. That’s part of the agility equation. While we may come up against human limitations that do slow us down eventually, right now out-of-the-box thinking rules, due to that most demanding mother of invention — necessity. If you can’t move with agility, you’ll lose the game.
Speed and flexibility make that possible. So how do you do it?
Get buy-in from your teammates and motivate them to move it, move it, move it (sorry — I’m channeling the Lemur King from the Madagascar movie)! Track their performance metrics, so you can congratulate them on how well they’re doing … or have a little chat in private about picking it up. Do everything possible to establish buy-in from all team personnel. Sincerely encourage initiative in a non-punitive environment. When you reward the fastest, most innovative workers, the others will want the reward too, so they’ll work harder at doing the right things right the first time.
Keep careful track of performance, giving fast, effective employees special incentives while providing benchmarks for everyone to shoot for. Make sure they understand, too, that they don’t have to ask permission for every little thing, especially the obvious decisions. You can’t afford “slowest common denominator” thinking, because it destroys productivity. If someone just can’t get it, get them help or transfer them elsewhere.
Your systems have to be flexible enough to change with unexpected situations and the times. That said, at least half the price of speed involves developing and constantly refining processes. Athletes and soldiers depend in large part on muscle memory to help them do their jobs crisply and efficiently. The same can be true for any learned action. Once you’ve studied the documentation and done the task often enough, you can go on autopilot while you brain works on other things.
Routines get things done, so document every little thing required of each task. Not only does this help new people get up to speed and allow others to do your job for you if you’re ill or vacationing, it helps you become so good at your work that it becomes almost effortless. Training just reinforces this level of muscle and mental memory required to bring you up to a heightened level of efficiency and productivity.
Each team member must understand the values, mission/vision, and strategic goals of the organization, and how to align the team’s goals with them. Simple, straightforward communication is key here; don’t hesitate or beat around the bush. Just tell them what you all have to do, and how to do it (it’s up to them if they want to find a better way). Urge them to ask questions if they don’t understand, and be patient as they do. If they don’t, ask them questions to ensure they understand.
Tech companies constantly release new applications, hardware, programs, and patches to make their products easier to use. Microsoft is famous for it. Though there have been occasional stumbles, techno-tools like these typically help you increase your efficiency, which translates to greater speed and productivity. I love using Office365.com and having instant access to all updates; I don’t like it when the updates override the custom settings I’ve painstakingly created!
Games and social media have their place, but that place generally isn’t work. Limit them to the team’s off time, unless they have a productive use. Depending on your industry, social media may be an effective way to keep up with customers, provide useful tips, and network. If so, appoint someone to maintain and track your social media contacts and load tips as part of their job. But their use of social media should apply only to work needs.
Processes and tasks may have to change or evolve over time to meet new conditions. Hanging onto the old way will only work for so long. Last year, I convinced a team member who was still using the old Windows 97-2003 .doc format to upgrade. Now that he’s graduated to the modern Windows 8.1 .docs format, our communications have become smoother.
Agility doesn’t just mean running around as fast as you can. Speed, flexibility, and efficiency are all productive ingredients to getting your work done right. If you’re in “run in circles, scream and shout” mode, take a deep breath. Consider the practices above—and proceed in a systematic fashion.
Laura Stack, aka The Productivity Pro®, gives speeches and seminars on sales and leadership productivity. For over 25 years, she’s worked with Fortune 1000 clients to reduce inefficiencies, execute more quickly, improve output, and increase profitability. Laura is the author of seven books, including Doing the Right Things Right: How the Effective Executive Spends Time.
Laura's The Productivity Pro® Follow @LauraStack on Twitter Get Laura's book — What to do when there is too much to do?

industry_corpus2: Technology & Research


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Only 31% of professionals who use mobile learning have claimed it has a high level of effectiveness, according to a recent study by ATD Research.
However, the results also showed that organisations who did achieve success were more likely to report higher market performance and higher effectiveness in their overall learning efforts.
The survey included 411 learning and business professionals from organisations all around the world. The companies included a range of industries which operated both nationally and globally.
It found that just one in three organisations have m-learning in place in their organisations.
Moreover, 21% of respondents said they were using m-learning informally (unstructured programs), and just 13% said there mobile program was formal (courses and content have specified objectives and a specified structure).
A further 37% said they plan to implement m-learning in the future.
The biggest barrier for m-learning programs was lack of funding, while others included insufficient information technology infrastructure to support mobile services, and a shortage of skills required to design learning content for mobile devices.
Only about half of the survey participants said they have metrics in place to measure how effective the m-learning programs are. This comprises of both formal and informal measures, as well as a mix of the two.
Moreover, most learning programs support sales or operations functions, while business units likely to benefit included executive-level management, marketing and human resources.
L&D Professional recently spoke to Leila Wearing, Director Academie Accor & Talent Development, of Accor Hotels about the importance of m-learning to their organisation.
“As a business we are trying as much as possible to become more mobile accessible,” she said.
“We run a lot of learning on a mobile device, whether it be a smart phone or an iPad, and any videos we put together have to be mobile accessible.


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"On the Same Page" book club meets each month on the second and fourth Tuesday at 7 p.m., in the library conference room. This is a small group of members who enjoy reading and have fun discussing various books.
So, if you like to read, stop by and check out the wonderful group of book club members. Who knows, you might be "On the Same Page" and want to join the group. Snacks are served, too!
If you have any questions, please feel free to call the library at 986-7814 and ask for Joyce or Kathy.
Second & Fourth Tuesday 7 p.m.


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As wristband products constantly evolve the variations are almost endless so for this reason, you won’t currently find a standard product list on this website. Instead, we ask that you let us know your requirements so that we can then help find you a solution.
Ask us about Tyvek, Laser, Inkjet, Thermal, Vinyl, Plastic, fabric etc. You may also require accessories like tickets, lanyards, pouches, cards, crimping tools, black lights, hand stamps etc.

industry_corpus2: Manufacturing


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We regulate the construction of new subdivisions in the county to ensure they have adequate water and sewer systems. This protects the public’s health and safety and prevents the spread of disease, pollution, and general nuisances.
Get a referral from the planning agency of the municipality where the subdivision will be located and submit it to the Salt Lake County Health Department.
The health department may perform inspections, reviews, and investigations to assure compliance. Failure to comply is a class B misdemeanor and may result in civil enforcement action or criminal prosecution.
Water to a new subdivision can be supplied by either a public culinary water system or an individual culinary water system (an approved well).
If the subdivision will utilize public drinking water, the system must be approved by the Utah Division of Drinking Water. A letter from the water district stating that the system is adequate for the development (often called a "will-serve letter") can serve as a precondition of approval.
If the subdivision will utilize an individual drinking water system, you must submit documentation that the system complies with Salt Lake County Health Department regulations.
Wastewater service to a new subdivision can be through an existing public sewer system or an individual wastewater disposal (septic) system.
If the public sewer system will service the development, the sewer district should supply you with a letter assuring SLCoHD that the system can serve your development and that adding the development to the system will not overload the treatment plant.
If the development will use individual wastewater disposal systems (such as septic tanks), you must submit documentation assuring that the system meets Utah Administrative Code R317 requirements, as well as additional requirements contained in Salt Lake County's Health Regulations.

industry_corpus2: Water Resources & Marine


text: All the cartridges on this page are guaranteed to work with your HP LaserJet Enterprise MFP M775z Toner printer. Remanufactured LaserJet Enterprise MFP M775z cartridges are ideal replacements for original HP LaserJet Enterprise MFP M775z Toner Cartridges as they are cheaper and come with a 100% satisfaction guarantee. OEM Inkjet cartridges are also available for your HP LaserJet Enterprise MFP M775z. Choose from colour and black HP LaserJet Enterprise MFP M775z cartridges.

industry_corpus2: Computing & Telecommunications


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Thread: Traktor Scratch Pro 2 With Hercules Deejay trim 4&6?
Use the soundcard you got when you puchased scratch pro. sorted.
TSP only "Officially" works with NI Audio cards and Certified Mixers.
DJTT ethics (not my ethics) are that we do not provide help to people using cracked (copied/shared/torrent/borrowed) software. That Is Not An Accusation. But being the Traktor expert i am i know that it is not possible to own TSP unless you have bought a Certified mixer or bought it with an NI card so... It looks to be evident that you are using an illegal version of Traktor , no matter what the source (copied/shared/torrent/borrowed) as you state "the first Traktor Scratch Pro" meaning a TSP version previous to a certain build were a loophole could be abused to enable timecode with any soundcard.
Look for an Audio 4. Not in production anymore but i couldn't recommend one more. Works with TSP if you dont own an 8,6 or 10.
but I also heard this about the frst traktor scratch pro which i currently run timecode on using my hercules deejay trim 4&6.
I trust that you will update your legit version of Traktor when you get an NI card then..? You must be on 1.2.X for the timecode hack to work. Thats a pretty old version.
The Audio 4 is a great card.


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The goal/outcome of this course is for the learner to provide an overview of specific cultural influences at the end of life for African Americans, Asian Americans, Hispanic Americans, American Indian/Alaskan Natives, and Muslim Americans, including the impact of religion and spirituality on funeral and burial practices and the challenges for health-care professionals to become culturally competent with end-of-life care.
Identify cultural competence challenges at end of life.
Discuss cultural differences in response to death and dying.
Describe specific cultural considerations at the end of life for African Americans, Asian Americans, Hispanic Americans, American Indian/Alaskan Natives, and Muslim Americans.
Differentiate between specific cultural issues for each cultural group.
Describe the influence of culture on funeral and burial practices.
Describe the influence of religion and spirituality on funeral and burial practices.


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WET WEATHER: Yarrawarrah Public School, Old Bush Rd, Yarrawarrah.
Boot Camp training combines running, interval training, and many other exercises using weights and/or body weight to help increase cardiovascular endurance, increase strength, and help people get into a routine of regular exercise. It is called “boot camp” because we train groups of people, typically outdoors,. We are not similar to the military Boot Camps you may hear going around. We focus more on functional training and a variety of other calisthenic exercises. We try to give exercises and Equipment you may or may not see and be able to perform at the local gym.
We use the term ‘boot camp’ to describe group fitness classes that promote fat loss, encouragement and team effort in a friendly and safe outdoor setting. Boot Camp is designed to push people a little bit further than they would normally push themselves in the gym alone. The idea is that everyone involved works at their own pace as they team up and work towards one goal, either in pairs, small teams of three or four, or even two teams head on. We instill a lot of timed activities to motivate members to better their time next round, we find the level of intensity from this goes through the roof. We all like to beat past scores and times.
Our Boot camps provide social support for those taking part. This provides a different environment for those exercisers who get bored in a gym or feel intimidated by the gym setting. Participants make friends and socialise as they exercise. Our Boot camps are usually tested for fitness on Day 1 and then retested at the start of the new term. Boot Camps run for 4 weeks and you can jump in and start your 4 weeks whenever you like.
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