Caveolae are vesicular invaginations of the plasma membrane. The chief structural proteins of caveolae are the caveolins. Caveolins form a scaffold onto which many classes of signaling molecules can assemble to generate preassembled signaling complexes. In addition to concentrating these signal transducers within a distinct region of the plasma membrane, caveolin binding may functionally regulate the activation state of caveolae-associated signaling molecules. Because the responsibilities assigned to caveolae continue to increase, this review will focus on: (i) caveolin structure/function and (ii) caveolae-associated signal transduction. Studies that link caveolae to human diseases will also be considered.The Caveolin Gene Family: Caveolin-1, -2, and -3 Molecular cloning has identified three distinct caveolin genes (1-6), caveolin-1, caveolin-2, and caveolin-3. Two isoforms of caveolin-1 (Cav-1␣ and Cav-1␤) are derived from alternate initiation during translation. Caveolin-1 and -2 are most abundantly expressed in adipocytes, endothelial cells, and fibroblastic cell types, whereas the expression of caveolin-3 is muscle-specific.Caveolin proteins interact with themselves to form homo-and hetero-oligomers (7-9), which directly bind cholesterol (10) and require cholesterol for insertion into model lipid membranes (10,11). Caveolin oligomers may also interact with glycosphingolipids (12). These protein-protein and protein-lipid interactions are thought to be the driving force for caveolae formation (7). In addition, the caveolin gene family is structurally and functionally conserved from worms (Caenorhabditis elegans) to man (13), supporting the idea that caveolins play an essential role.Caveolin-1 assumes an unusual topology. A central hydrophobic domain (residues 102-134) is thought to form a hairpin-like structure within the membrane. As a consequence, both the N-terminal domain (residues 1-101) and the C-terminal domain (residues 135-178) face the cytoplasm. A 41-amino acid region of the N-terminal domain (residues 61-101) directs the formation of caveolin homooligomers (7), whereas the 44-amino acid C-terminal domain acts as a bridge to allow these homo-oligomers to interact with each other, thereby forming a caveolin-rich scaffold (14).Recent co-immunoprecipitation and dual labeling experiments directly show that caveolin-1 and -2 form a stable hetero-oligomeric complex and are strictly co-localized (9). Caveolin-2 localization corresponds to caveolae membranes as visualized by immunoelectron microscopy (9). Thus, caveolin-2 may function as an "accessory protein" in conjunction with caveolin-1. Caveolin-interacting ProteinsA number of studies support the hypothesis that caveolin proteins provide a direct means for resident caveolae proteins to be sequestered within caveolae microdomains. These caveolin-interacting proteins include G-protein ␣ subunits, Ha-Ras, Src family tyrosine kinases, endothelial NOS, 1 EGF-R and related receptor tyrosine kinases, and protein kinase C isoforms (11, 15-18, 20 -32).Heterotri...

Caveolae are cholesterol͞sphingolipid-rich microdomains of the plasma membrane that have been implicated in signal transduction and vesicular trafficking. Caveolins are a family of caveolae-associated integral membrane proteins. Caveolin-1 and -2 show the widest range of expression, whereas caveolin-3 expression is restricted to muscle cell types. It has been previously reported that little or no caveolin mRNA species are detectable in the brain by Northern blot analyses or in neuroblastoma cell lines. However, it remains unknown whether caveolins are expressed within neuronal cells. Here we demonstrate the expression of caveolin-1 and -2 in differentiating PC12 cells and dorsal root ganglion (DRG) neurons by using mono-specific antibody probes. In PC12 cells, caveolin-1 expression is up-regulated on day 4 of nerve growth factor (NGF) treatment, whereas caveolin-2 expression is transiently up-regulated early in the differentiation program and then rapidly down-regulated. Interestingly, caveolin-2 is up-regulated in response to the mechanical injury of differentiated PC12 cells; up-regulation of caveolin-2 under these conditions is strictly dependent on continued treatment with NGF. Robust expression of caveolin-1 and -2 is also observed along the entire cell surface of DRG neurons, including high levels on growth cones. These findings demonstrate that neuronal cells express caveolins.

More than 100 species of venomous cone snails (genus Conus) are highly effective predators of fish. The vast majority of venom components identified and functionally characterized to date are neurotoxins specifically targeted to receptors, ion channels, and transporters in the nervous system of prey, predators, or competitors. Here we describe a venom component targeting energy metabolism, a radically different mechanism. Two fish-hunting cone snails, Conus geographus and Conus tulipa, have evolved specialized insulins that are expressed as major components of their venoms. These insulins are distinctive in having much greater similarity to fish insulins than to the molluscan hormone and are unique in that posttranslational modifications characteristic of conotoxins (hydroxyproline, γ-carboxyglutamate) are present. When injected into fish, the venom insulin elicits hypoglycemic shock, a condition characterized by dangerously low blood glucose. Our evidence suggests that insulin is specifically used as a weapon for prey capture by a subset of fish-hunting cone snails that use a net strategy to capture prey. Insulin appears to be a component of the nirvana cabal, a toxin combination in these venoms that is released into the water to disorient schools of small fish, making them easier to engulf with the snail's distended false mouth, which functions as a net. If an entire school of fish simultaneously experiences hypoglycemic shock, this should directly facilitate capture by the predatory snail.insulin shock | cone snails | conotoxins | nirvana cabal | venom

Although the absorption, transport, and catabolism of dietary lipids have been studied extensively in great detail in mammals and other vertebrates, a tractable genetic system for identifying novel genes involved in these physiologic processes is not available. To establish such a model, we monitored neutral lipid by staining fixed zebrafish larvae with oil red o (ORO). The head structures, heart, vasculature, and swim bladder stained with ORO until the yolk was consumed 6 days after fertilization (6 dpf). Thereafter, the heart and vasculature no longer had stainable neutral lipids. Following a high-fat meal, ORO stained the intestine and vasculature of 6 dpf larvae, and whole-larval triacylglycerol (TAG) and apolipoprotein B levels increased. Levels of microsomal triglyceride transfer protein (Mtp), the protein responsible for packaging TAG and betalipoproteins into lipoprotein particles, were unchanged by feeding. Since the developing zebrafish embryo expresses mtp in the yolk cell layer, liver, and intestine, we determined the effect of targeted knockdown of Mtp expression using an antisense morpholino oligonucleotide approach (Mtp MO) on the transport of yolk and dietary lipids. Mtp MO injection led to loss of Mtp expression and of lipid staining in the vasculature, heart, and head structures. Mtp MO-injected larvae were smaller than age-matched, uninjected larvae, consumed very little yolk, and did not absorb dietary neutral lipids; however, they absorbed a short chain fatty acid that does not require Mtp for transport. Importantly, the vasculature appeared unaffected in Mtp MO-injected larvae. These studies indicate that zebrafish larvae are suitable for genetic studies of lipid transport and metabolism.

Developmental mechanisms regulating gene expression and the stable acquisition of cell fate direct cytodifferentiation during organogenesis. Moreover, it is likely that such mechanisms could be exploited to repair or regenerate damaged organs. DNA methyltransferases (Dnmts) are enzymes critical for epigenetic regulation, and are used in concert with histone methylation and acetylation to regulate gene expression and maintain genomic integrity and chromosome structure. We carried out two forward genetic screens for regulators of endodermal organ development. In the first, we screened for altered morphology of developing digestive organs, while in the second we screed for the lack of terminally differentiated cell types in the pancreas and liver. From these screens, we identified two mutant alleles of zebrafish dnmt1. Both lesions are predicted to eliminate dnmt1 function; one is a missense mutation in the catalytic domain and the other is a nonsense mutation that eliminates the catalytic domain. In zebrafish dnmt1 mutants, the pancreas and liver form normally, but begin to degenerate after 84 hours post fertilization (hpf). Acinar cells are nearly abolished through apoptosis by 100 hpf, though neither DNA replication, nor entry into mitosis are halted in the absence of detectable Dnmt1. However, endocrine cells and ducts are largely spared. Surprisingly, dnmt1 mutants and dnmt1 morpholino-injected larvae show increased capacity for pancreatic beta cell regeneration in an inducible model of pancreatic beta cell ablation. Thus, our data suggest that Dnmt1 is dispensable for pancreatic duct or endocrine cell formation, but not for acinar cell survival. In addition, Dnmt1 may influence the differentiation of pancreatic beta cell progenitors or the reprogramming of cells toward the pancreatic beta cell fate.

A pandemic of metabolic diseases (atherosclerosis, diabetes mellitus, and obesity), unleashed by multiple social and economic factors beyond the control of most individuals, threatens to diminish human life span for the first time in the modern era. Given the redundancy and inherent complexity of processes regulating the uptake, transport, catabolism, and synthesis of nutrients, magic bullets to target these diseases will be hard to find. Recent studies using the worm Caenorhabditis elegans, the fly Drosophila melanogaster, and the zebrafish Danio rerio indicate that these “lower” metazoans possess unique attributes that should help in identifying, investigating, and even validating new pharmaceutical targets for these diseases. We summarize findings in these organisms that shed light on highly conserved pathways of energy homeostasis.

Caveolae are plasma membrane invaginations where key signaling elements are concentrated. In this report, both biochemical and histochemical analyses demonstrate that the amyloid precursor protein (APP), a source of A␤ amyloid peptide, is enriched within caveolae. Caveolin-1, a principal component of caveolae, is physically associated with APP, and the cytoplasmic domain of APP directly participates in this binding. The characteristic C-terminal fragment that results from APP processing by ␣-secretase, an as yet unidentified enzyme that cleaves APP within the A␤ amyloid sequence, was also localized within these caveolae-enriched fractions. Further analysis by cell surface biotinylation revealed that this cleavage event occurs at the cell surface. Importantly, ␣-secretase processing was significantly promoted by recombinant overexpression of caveolin in intact cells, resulting in increased secretion of the soluble extracellular domain of APP. Conversely, caveolin depletion using antisense oligonucletotides prevented this cleavage event. Our current results indicate that caveolae and caveolins may play a pivotal role in the ␣-secretase-mediated proteolysis of APP in vivo.Senile plaques and paired helical filaments are the hallmarks of the brain pathology of Alzheimer's disease (1). The principal component of the senile plaque is the A␤ amyloid peptide, which is composed of 39 -43 amino acid residues. The A␤ amyloid peptide is derived from a full-length precursor protein, termed APP 1 (amyloid precursor protein) (2). Alternate splicing of the APP gene generates at least 10 distinct isoforms; APP 695 is the brain-specific isoform. The A␤ amyloid peptide is generated by the processing of APP with ␤-and ␥-secretases (3). Alternatively, APP is processed by ␣-secretase, which cleaves APP within the A␤ sequence, thereby precluding the formation of A␤ (4). The identities of these secretases remain unknown.In some inherited forms of Alzheimer's disease, point mutations have been identified within the coding sequence of the APP gene (5). These mutations co-segregate with the disease phenotype and cause Alzheimer's disease. Understanding the molecular function and processing of APP is therefore critical to unraveling the molecular basis of Alzheimer's disease.One approach to elucidate the function of APP is to identify APP-interacting proteins. At least five distinct classes of molecules have been identified as APP binding partners as follows: G o (6), Fe 65 (7), X11 protein (8), Fe 65 -like protein (9), and APP-BP1 (10). The APP domain that interacts with G o has been localized to residues His 657 -Lys 676 within the cytoplasmic domain of APP 695 . G o is a brain-specific member of heterotrimeric GTP-binding protein (G-protein) family. The in vivo interaction between APP 695 and G o results in apoptotic cell death (11) and inhibition of cAMP response element trans-activation (12). In contrast, functional consequences of interactions between APP and other binding partners have not yet been described. However, it is likely th...

To find new genes that influence liver lipid mass, we performed a genetic screen for zebrafish mutants with hepatic steatosis, a pathological accumulation of fat. The red moon (rmn) mutant develops hepatic steatosis as maternally deposited yolk is depleted. Conversely, hepatic steatosis is suppressed in rmn mutants by adequate nutrition. Adult rmn mutants show increased liver neutral lipids and induction of hepatic lipid biosynthetic genes when fasted. Positional cloning of the rmn locus reveals a loss-of-function mutation in slc16a6a (solute carrier family 16a, member 6a), a gene that we show encodes a transporter of the major ketone body b-hydroxybutyrate. Restoring wild-type zebrafish slc16a6a expression or introducing human SLC16A6 in rmn mutant livers rescues the mutant phenotype. Radiotracer analysis confirms that loss of Slc16a6a function causes diversion of livertrapped ketogenic precursors into triacylglycerol. Underscoring the importance of Slc16a6a to normal fasting physiology, previously fed rmn mutants are more sensitive to death by starvation than are wild-type larvae. Our unbiased, forward genetic approach has found a heretofore unrecognized critical step in fasting energy metabolism: hepatic ketone body transport. Since b-hydroxybutyrate is both a major fuel and a signaling molecule in fasting, the discovery of this transporter provides a new direction for modulating circulating levels of ketone bodies in metabolic diseases.