A single method for cryofixation and correlative light, electron microscopy and tomography of zebrafish embryos.

The zebrafish is a powerful vertebrate system for cell and developmental studies. In this study, we have optimized methods for fast freezing and processing of zebrafish embryos for electron microscopy (EM). We show that in the absence of primary chemical fixation, excellent ultrastructure, preservation of green fluorescent protein (GFP) fluorescence, immunogold labelling and electron tomography can be obtained using a single technique involving high-pressure freezing and embedding in Lowicryl resins at low temperature. As well as being an important new tool for zebrafish research, the maintenance of GFP fluorescence after fast freezing, freeze substitution and resin embedding will be of general use for correlative light and EM of biological samples.

Cavin4 interacts with Bin1 to promote T-tubule formation and stability in developing skeletal muscle.

The cavin proteins are essential for caveola biogenesis and function. Here, we identify a role for the muscle-specific component, Cavin4, in skeletal muscle T-tubule development by analyzing two vertebrate systems, mouse and zebrafish. In both models, Cavin4 localized to T-tubules, and loss of Cavin4 resulted in aberrant T-tubule maturation. In zebrafish, which possess duplicated cavin4 paralogs, Cavin4b was shown to directly interact with the T-tubule-associated BAR domain protein Bin1. Loss of both Cavin4a and Cavin4b caused aberrant accumulation of interconnected caveolae within the T-tubules, a fragmented T-tubule network enriched in Caveolin-3, and an impaired Ca2+ response upon mechanical stimulation. We propose a role for Cavin4 in remodeling the T-tubule membrane early in development by recycling caveolar components from the T-tubule to the sarcolemma. This generates a stable T-tubule domain lacking caveolae that is essential for T-tubule function.

Abnormal nuclear pore formation triggers apoptosis in the intestinal epithelium of elys-deficient zebrafish.

BACKGROUND & AIMS: Zebrafish mutants generated by ethylnitrosourea-mutagenesis provide a powerful tool for dissecting the genetic regulation of developmental processes, including organogenesis. One zebrafish mutant, "flotte lotte" (flo), displays striking defects in intestinal, liver, pancreas, and eye formation at 78 hours postfertilization (hpf). In this study, we sought to identify the underlying mutated gene in flo and link the genetic lesion to its phenotype. METHODS: Positional cloning was employed to map the flo mutation. Subcellular characterization of flo embryos was achieved using histology, immunocytochemistry, bromodeoxyuridine incorporation analysis, and confocal and electron microscopy. RESULTS: The molecular lesion in flo is a nonsense mutation in the elys (embryonic large molecule derived from yolk sac) gene, which encodes a severely truncated protein lacking the Elys C-terminal AT-hook DNA binding domain. Recently, the human ELYS protein has been shown to play a critical, and hitherto unsuspected, role in nuclear pore assembly. Although elys messenger RNA (mRNA) is expressed broadly during early zebrafish development, widespread early defects in flo are circumvented by the persistence of maternally expressed elys mRNA until 24 hpf. From 72 hpf, elys mRNA expression is restricted to proliferating tissues, including the intestinal epithelium, pancreas, liver, and eye. Cells in these tissues display disrupted nuclear pore formation; ultimately, intestinal epithelial cells undergo apoptosis. CONCLUSIONS: Our results demonstrate that Elys regulates digestive organ formation.

Ultrastructural identification of uncoated caveolin-independent early endocytic vehicles.

Using quantitative light microscopy and a modified immunoelectron microscopic technique, we have characterized the entry pathway of the cholera toxin binding subunit (CTB) in primary embryonic fibroblasts. CTB trafficking to the Golgi complex was identical in caveolin-1null (Cav1-/-) mouse embryonic fibroblasts (MEFs) and wild-type (WT) MEFs. CTB entry in the Cav1-/- MEFs was predominantly clathrin and dynamin independent but relatively cholesterol dependent. Immunoelectron microscopy was used to quantify budded and surface-connected caveolae and to identify noncaveolar endocytic vehicles. In WT MEFs, a small fraction of the total Cav1-positive structures were shown to bud from the plasma membrane (2% per minute), and budding increased upon okadaic acid or lactosyl ceramide treatment. However, the major carriers involved in initial entry of CTB were identified as uncoated tubular or ring-shaped structures. These carriers contained GPI-anchored proteins and fluid phase markers and represented the major vehicles mediating CTB uptake in both WT and caveolae-null cells.

The caveolin-cavin system plays a conserved and critical role in mechanoprotection of skeletal muscle.

Dysfunction of caveolae is involved in human muscle disease, although the underlying molecular mechanisms remain unclear. In this paper, we have functionally characterized mouse and zebrafish models of caveolae-associated muscle disease. Using electron tomography, we quantitatively defined the unique three-dimensional membrane architecture of the mature muscle surface. Caveolae occupied around 50% of the sarcolemmal area predominantly assembled into multilobed rosettes. These rosettes were preferentially disassembled in response to increased membrane tension. Caveola-deficient cavin-1(-/-) muscle fibers showed a striking loss of sarcolemmal organization, aberrant T-tubule structures, and increased sensitivity to membrane tension, which was rescued by muscle-specific Cavin-1 reexpression. In vivo imaging of live zebrafish embryos revealed that loss of muscle-specific Cavin-1 or expression of a dystrophy-associated Caveolin-3 mutant both led to sarcolemmal damage but only in response to vigorous muscle activity. Our findings define a conserved and critical role in mechanoprotection for the unique membrane architecture generated by the caveolin-cavin system.

Caveolin-1 is necessary for hepatic oxidative lipid metabolism: evidence for crosstalk between caveolin-1 and bile acid signaling.

Caveolae and caveolin-1 (CAV1) have been linked to several cellular functions. However, a model explaining their roles in mammalian tissues in vivo is lacking. Unbiased expression profiling in several tissues and cell types identified lipid metabolism as the main target affected by CAV1 deficiency. CAV1-/- mice exhibited impaired hepatic peroxisome proliferator-activated receptor α (PPARα)-dependent oxidative fatty acid metabolism and ketogenesis. Similar results were recapitulated in CAV1-deficient AML12 hepatocytes, suggesting at least a partial cell-autonomous role of hepatocyte CAV1 in metabolic adaptation to fasting. Finally, our experiments suggest that the hepatic phenotypes observed in CAV1-/- mice involve impaired PPARα ligand signaling and attenuated bile acid and FXRα signaling. These results demonstrate the significance of CAV1 in (1) hepatic lipid homeostasis and (2) nuclear hormone receptor (PPARα, FXRα, and SHP) and bile acid signaling.

Caveolin-1 deficiency leads to increased susceptibility to cell death and fibrosis in white adipose tissue: characterization of a lipodystrophic model.

Caveolin-1 (CAV1) is an important regulator of adipose tissue homeostasis. In the present study we examined the impact of CAV1 deficiency on the properties of mouse adipose tissue both in vivo and in explant cultures during conditions of metabolic stress. In CAV1(-/-) mice fasting caused loss of adipose tissue mass despite a lack of hormone-sensitive lipase (HSL) phosphorylation. In addition, fasting resulted in increased macrophage infiltration, enhanced deposition of collagen, and a reduction in the level of the lipid droplet protein perilipin A (PLIN1a). Explant cultures of CAV1(-/-) adipose tissue also showed a loss of PLIN1a during culture, enhanced secretion of IL-6, increased release of lactate dehydrogenase, and demonstrated increased susceptibility to cell death upon collagenase treatment. Attenuated PKA-mediated signaling to HSL, loss of PLIN1a and increased secretion of IL-6 were also observed in adipose tissue explants of CAV1(+/+) mice with diet-induced obesity. Together these results suggest that while alterations in adipocyte lipid droplet biology support adipose tissue metabolism in the absence of PKA-mediated pro-lipolytic signaling in CAV1(-/-) mice, the tissue is intrinsically unstable resulting in increased susceptibility to cell death, which we suggest underlies the development of fibrosis and inflammation during periods of metabolic stress.

Modern approaches for ultrastructural analysis of the zebrafish embryo.

The zebrafish is a powerful vertebrate system with great advantages for both forward and reverse genetic screens and as a model for human disease conditions. Light microscopy has been used extensively to study zebrafish development but less frequently have these studies been combined with ultrastructural information. Zebrafish embryos are ideal for electron microscopy (EM) with a single transverse section containing many different cell types and tissues. However, conventional methods of EM do not provide optimal preservation of all tissues and are usually incompatible with immunolabelling and visualisation of expressed fluorescently tagged proteins. Here we examine methods that overcome these problems. We summarise a range of methods, applicable to the ultrastructural analysis of zebrafish embryos, including methods for fast freezing and processing of zebrafish embryos. These methods preserve antigenicity, ultrastructure and GFP fluorescence even after embedding in resin. In addition, they are compatible with electron tomography. These methods provide a new set of research tools that provide an additional level of information, complementing current methods for study of this widely used model system.

MURC/Cavin-4 and cavin family members form tissue-specific caveolar complexes.

Polymerase I and transcript release factor (PTRF)/Cavin is a cytoplasmic protein whose expression is obligatory for caveola formation. Using biochemistry and fluorescence resonance energy transfer-based approaches, we now show that a family of related proteins, PTRF/Cavin-1, serum deprivation response (SDR)/Cavin-2, SDR-related gene product that binds to C kinase (SRBC)/Cavin-3, and muscle-restricted coiled-coil protein (MURC)/Cavin-4, forms a multiprotein complex that associates with caveolae. This complex can constitutively assemble in the cytosol and associate with caveolin at plasma membrane caveolae. Cavin-1, but not other cavins, can induce caveola formation in a heterologous system and is required for the recruitment of the cavin complex to caveolae. The tissue-restricted expression of cavins suggests that caveolae may perform tissue-specific functions regulated by the composition of the cavin complex. Cavin-4 is expressed predominantly in muscle, and its distribution is perturbed in human muscle disease associated with Caveolin-3 dysfunction, identifying Cavin-4 as a novel muscle disease candidate caveolar protein.

PTRF-Cavin, a conserved cytoplasmic protein required for caveola formation and function.

Caveolae are abundant cell-surface organelles involved in lipid regulation and endocytosis. We used comparative proteomics to identify PTRF (also called Cav-p60, Cavin) as a putative caveolar coat protein. PTRF-Cavin selectively associates with mature caveolae at the plasma membrane but not Golgi-localized caveolin. In prostate cancer PC3 cells, and during development of zebrafish notochord, lack of PTRF-Cavin expression correlates with lack of caveolae, and caveolin resides on flat plasma membrane. Expression of PTRF-Cavin in PC3 cells is sufficient to cause formation of caveolae. Knockdown of PTRF-Cavin reduces caveolae density, both in mammalian cells and in the zebrafish. Caveolin remains on the plasma membrane in PTRF-Cavin knockdown cells but exhibits increased lateral mobility and accelerated lysosomal degradation. We conclude that PTRF-Cavin is required for caveola formation and sequestration of mobile caveolin into immobile caveolae.

Evolutionary analysis and molecular dissection of caveola biogenesis.

Caveolae are an abundant feature of mammalian cells. Integral membrane proteins called caveolins drive the formation of caveolae but the precise mechanisms underlying caveola formation, and the origin of caveolae and caveolins during evolution, are unknown. Systematic evolutionary analysis shows conservation of genes encoding caveolins in metazoans. We provide evidence for extensive and ancient, local and genomic gene duplication, and classify distinct caveolin gene families. Vertebrate caveolin-1 and caveolin-3 isoforms, as well as an invertebrate (Apis mellifera, honeybee) caveolin, all form morphologically identical caveolae in caveolin-1-null mouse cells, demonstrating that caveola formation is a conserved feature of evolutionarily distant caveolins. However, coexpression of flotillin-1 and flotillin-2 did not cause caveola biogenesis in this system. In contrast to the other tested caveolins, C. elegans caveolin is efficiently transported to the plasma membrane but does not generate caveolae, providing evidence of diversity of function in the caveolin gene family. Using C. elegans caveolin as a template to generate hybrid caveolin constructs we now define domains of caveolin required for caveolae biogenesis. These studies lead to a model for caveola formation and novel insights into the evolution of caveolin function.

Caveolin-1 is required for lateral line neuromast and notochord development.

Caveolae have been linked to diverse cellular functions and to many disease states. In this study we have used zebrafish to examine the role of caveolin-1 and caveolae during early embryonic development. During development, expression is apparent in a number of tissues including Kupffer's vesicle, tailbud, intersomite boundaries, heart, branchial arches, pronephric ducts and periderm. Particularly strong expression is observed in the sensory organs of the lateral line, the neuromasts and in the notochord where it overlaps with expression of caveolin-3. Morpholino-mediated downregulation of Cav1alpha caused a dramatic inhibition of neuromast formation. Detailed ultrastructural analysis, including electron tomography of the notochord, revealed that the central regions of the notochord has the highest density of caveolae of any embryonic tissue comparable to the highest density observed in any vertebrate tissue. In addition, Cav1alpha downregulation caused disruption of the notochord, an effect that was enhanced further by Cav3 knockdown. These results indicate an essential role for caveolin and caveolae in this vital structural and signalling component of the embryo.

Caveolin-1 is essential for liver regeneration.

Liver regeneration is an orchestrated cellular response that coordinates cell activation, lipid metabolism, and cell division. We found that caveolin-1 gene-disrupted mice (cav1-/- mice) exhibited impaired liver regeneration and low survival after a partial hepatectomy. Hepatocytes showed dramatically reduced lipid droplet accumulation and did not advance through the cell division cycle. Treatment of cav1-/- mice with glucose (which is a predominant energy substrate when compared to lipids) drastically increased survival and reestablished progression of the cell cycle. Thus, caveolin-1 plays a crucial role in the mechanisms that coordinate lipid metabolism with the proliferative response occurring in the liver after cellular injury.

Regulated localization of Rab18 to lipid droplets: effects of lipolytic stimulation and inhibition of lipid droplet catabolism.

Rab GTPases are crucial regulators of membrane traffic. Here we have examined a possible association of Rab proteins with lipid droplets (LDs), neutral lipid-containing organelles surrounded by a phospholipid monolayer, also known as lipid bodies, which have been traditionally considered relatively inert storage organelles. Although we found close apposition between LDs and endosomal compartments labeled by expressed Rab5, Rab7, or Rab11 constructs, there was no detectable labeling of the LD surface itself by these Rab proteins. In contrast, GFP-Rab18 localized to LDs and immunoelectron microscopy showed direct association with the monolayer surface. Green fluorescent protein (GFP)-Rab18-labeled LDs underwent oscillatory movements in a localized area as well as sporadic, rapid, saltatory movements both in the periphery of the cell and toward the perinuclear region. In both adipocytes and non-adipocyte cell lines Rab18 localized to a subset of LDs. To gain insights into this specific localization, Rab18 was co-expressed with Cav3DGV, a truncation mutant of caveolin-3 shown to inhibit the catabolism and motility of lipid droplets. GFP-Rab18 and mRFP-Cav3DGV labeled mutually exclusive subpopulations of LDs. Moreover, in 3T3-L1 adipocytes, stimulation of lipolysis increased the localization of Rab18 to LDs, an effect reversed by beta-adrenergic antagonists. These results show that a Rab protein localizes directly to the monolayer surface of LDs. In addition, association with the LD surface was increased following stimulation of lipolysis and inhibited by a caveolin mutant suggesting that recruitment of Rab18 is regulated by the metabolic state of individual LDs.

Zebrafish as a model for caveolin-associated muscle disease; caveolin-3 is required for myofibril organization and muscle cell patterning.

Caveolae are an abundant feature of many animal cells. However, the exact function of caveolae remains unclear. We have used the zebrafish, Danio rerio, as a system to understand caveolae function focusing on the muscle-specific caveolar protein, caveolin-3 (Cav3). We have identified caveolin-1 (alpha and beta), caveolin-2 and Cav3 in the zebrafish. Zebrafish Cav3 has 72% identity to human CAV3, and the amino acids altered in human muscle diseases are conserved in the zebrafish protein. During embryonic development, cav3 expression is apparent by early segmentation stages in the first differentiating muscle precursors, the adaxial cells and slightly later in the notochord. cav3 expression appears in the somites during mid-segmentation stages and then later in the pectoral fins and facial muscles. Cav3 and caveolae are located along the entire sarcolemma of late stage embryonic muscle fibers, whereas beta-dystroglycan is restricted to the muscle fiber ends. Down-regulation of Cav3 expression causes gross muscle abnormalities and uncoordinated movement. Ultrastructural analysis of isolated muscle fibers reveals defects in myoblast fusion and disorganized myofibril and membrane systems. Expression of the zebrafish equivalent to a human muscular dystrophy mutant, CAV3P104L, causes severe disruption of muscle differentiation. In addition, knockdown of Cav3 resulted in a dramatic up-regulation of eng1a expression resulting in an increase in the number of muscle pioneer-like cells adjacent to the notochord. These studies provide new insights into the role of Cav3 in muscle development and demonstrate its requirement for correct intracellular organization and myoblast fusion.

RORalpha regulates the expression of genes involved in lipid homeostasis in skeletal muscle cells: caveolin-3 and CPT-1 are direct targets of ROR.

The staggerer mice carry a deletion in the RORalpha gene and have a prolonged humoral response, overproduce inflammatory cytokines, and are immunodeficient. Furthermore, the staggerer mice display lowered plasma apoA-I/-II, decreased plasma high density lipoprotein cholesterol and triglycerides, and develop hypo-alpha-lipoproteinemia and atherosclerosis. However, relatively little is known about RORalpha in the context of target tissues, target genes, and lipid homeostasis. For example, RORalpha is abundantly expressed in skeletal muscle, a major mass peripheral tissue that accounts for approximately 40% of total body weight and 50% of energy expenditure. This lean tissue is a primary site of glucose disposal and fatty acid oxidation. Consequently, muscle has a significant role in insulin sensitivity, obesity, and the blood-lipid profile. In particular, the role of RORalpha in skeletal muscle metabolism has not been investigated, and the contribution of skeletal muscle to the ROR-/- phenotype has not been resolved. We utilize ectopic dominant negative RORalpha expression in skeletal muscle cells to understand the regulatory role of RORs in this major mass peripheral tissue. Exogenous dominant negative RORalpha expression in skeletal muscle cells represses the endogenous levels of RORalpha and -gamma mRNAs and ROR-dependent gene expression. Moreover, we observed attenuated expression of many genes involved in lipid homeostasis. Furthermore, we show that the muscle carnitine palmitoyltransferase-1 and caveolin-3 promoters are directly regulated by ROR and coactivated by p300 and PGC-1. This study implicates RORs in the control of lipid homeostasis in skeletal muscle. In conclusion, we speculate that ROR agonists would increase fatty acid catabolism in muscle and suggest selective activators of ROR may have therapeutic utility in the treatment of obesity and atherosclerosis.

Inhibition of lipid raft-dependent signaling by a dystrophy-associated mutant of caveolin-3.

Specific point mutations in caveolin-3, a predominantly muscle-specific member of the caveolin family, have been implicated in limb-girdle muscular dystrophy and in rippling muscle disease. We examined the effect of these mutations on caveolin-3 localization and function. Using two independent assay systems, Raf activation in fibroblasts and neurite extension in PC12 cells, we show that one of the caveolin-3 point mutants, caveolin-3-C71W, specifically inhibits signaling by activated H-Ras but not by K-Ras. To gain insights into the effect of the mutant protein on H-Ras signaling, we examined the localization of the mutant proteins in fibroblastic cells and in differentiating myotubes. Unlike the previously characterized caveolin-3-DGV mutant, the inhibitory caveolin-3-C71W mutant reached the plasma membrane and colocalized with wild type caveolins. In BHK cells, caveolin-3-C71W associated with caveolae and in differentiating muscle cells with the developing T-tubule system. In contrast, the caveolin-3-P104L mutant accumulated in the Golgi complex and had no effect on H-Ras-mediated Raf activation. Inhibition by caveolin-3-C71W was rescued by cholesterol addition, suggesting that the mutant protein perturbs cholesterol-rich raft domains. Thus, we have demonstrated that a naturally occurring caveolin-3 mutation can inhibit signaling involving cholesterol-sensitive raft domains.