Caveolae are plasma membrane invaginations that may play an important role in numerous cellular processes including transport, signaling, and tumor suppression. By targeted disruption of caveolin-1, the main protein component of caveolae, we generated mice that lacked caveolae. The absence of this organelle impaired nitric oxide and calcium signaling in the cardiovascular system, causing aberrations in endothelium-dependent relaxation, contractility, and maintenance of myogenic tone. In addition, the lungs of knockout animals displayed thickening of alveolar septa caused by uncontrolled endothelial cell proliferation and fibrosis, resulting in severe physical limitations in caveolin-1-disrupted mice. Thus, caveolin-1 and caveolae play a fundamental role in organizing multiple signaling pathways in the cell.

No abstract

Although the exact etiology of Alzheimer's disease (AD) is a topic of debate, the consensus is that the accumulation of ␤-amyloid (A␤) peptides in the senile plaques is one of the hallmarks of the progression of the disease. The A␤ peptide is formed by the amyloidogenic cleavage of the amyloid precursor protein (APP) by ␤-and ␥-secretases. The endocytic system has been implicated in the cleavages leading to the formation of A␤. However, the identity of the intracellular compartment where the amyloidogenic secretases cleave and the mechanism by which the intracellularly generated A␤ is released into the extracellular milieu are not clear. Here, we show that ␤-cleavage occurs in early endosomes followed by routing of A␤ to multivesicular bodies (MVBs) in HeLa and N2a cells. Subsequently, a minute fraction of A␤ peptides can be secreted from the cells in association with exosomes, intraluminal vesicles of MVBs that are released into the extracellular space as a result of fusion of MVBs with the plasma membrane. Exosomal proteins were found to accumulate in the plaques of AD patient brains, suggesting a role in the pathogenesis of AD. multivesicular bodies ͉ rafts ͉ amyloid precursor protein ͉ ␤-secretase ͉ endocytosis A lzheimer's disease (AD) is a late-onset neurological disorder with progressive loss of memory and cognitive abilities as a result of excessive neurodegeneration (1). AD is characterized by extracellular aggregates of ␤-amyloid (A␤) peptides known as amyloid plaques (2). The A␤ peptide is derived from the sequential processing of the amyloid precursor protein (APP) by ␤-and ␥-secretases. ␤-secretase [(␤-APP cleaving enzyme (BACE)] is a type-1 transmembrane aspartyl protease and is mainly localized to endosomes, lysosomes and the transGolgi network (3). ␥-Secretase is a multicomponent complex that is composed of presenilin-1͞presenilin-2, nicastrin, Aph-1, and PEN-2 (4) and is localized to the early secretory (5, 6) and the endocytic compartments (7,8). Nonamyloidogenic processing of APP involves ␣-secretase that cleaves APP inside the A␤ region, giving rise to the ␣-cleaved ectodomain, thus precluding the formation of A␤ (9). Hence, the availability of APP to either ␣-or ␤-secretase determines whether A␤ peptide will be generated. Lateral organization of membranes (10) and subcellular localization (11, 12) of the substrate and the secretases have been documented to regulate A␤ generation. Recent work suggests that ␤-secretase associates with lipid rafts, liquid-ordered domains in the membrane (13,14), and that integrity of raft domains is required for ␤-cleavage of APP to occur (ref. 10; see, however, ref. 15). ␣-Cleavage, in contrast, occurs outside raft domains (10). The ␥-secretase complex is also raft-associated (16); hence, amyloidogenic processing of APP could occur in clustered raft domains to generate A␤ (10). Inhibition of endocytosis reduces ␤-cleavage but not ␣-cleavage, suggesting that ␤-cleavage mainly occurs in endosomes (10,11,(17)(18)(19). Accumulation of A␤ peptides in extracellular...

Lateral assemblies of glycolipids and cholesterol, “rafts,” have been implicated to play a role in cellular processes like membrane sorting, signal transduction, and cell adhesion. We studied the structure of raft domains in the plasma membrane of non-polarized cells. Overexpressed plasma membrane markers were evenly distributed in the plasma membrane. We compared the patching behavior of pairs of raft markers (defined by insolubility in Triton X-100) with pairs of raft/non-raft markers. For this purpose we cross-linked glycosyl-phosphatidylinositol (GPI)-anchored proteins placental alkaline phosphatase (PLAP), Thy-1, influenza virus hemagglutinin (HA), and the raft lipid ganglioside GM1 using antibodies and/or cholera toxin. The patches of these raft markers overlapped extensively in BHK cells as well as in Jurkat T–lymphoma cells. Importantly, patches of GPI-anchored PLAP accumulated src-like protein tyrosine kinase fyn, which is thought to be anchored in the cytoplasmic leaflet of raft domains. In contrast patched raft components and patches of transferrin receptor as a non-raft marker were sharply separated. Taken together, our data strongly suggest that coalescence of cross-linked raft elements is mediated by their common lipid environments, whereas separation of raft and non-raft patches is caused by the immiscibility of different lipid phases. This view is supported by the finding that cholesterol depletion abrogated segregation. Our results are consistent with the view that raft domains in the plasma membrane of non-polarized cells are normally small and highly dispersed but that raft size can be modulated by oligomerization of raft components.

An ability to mimic the boundaries of biological compartments would improve our understanding of self-assembly and provide routes to new materials for the delivery of drugs and biologicals and the development of protocells. We show that short designed peptides can be combined to form unilamellar spheres approximately 100 nanometers in diameter. The design comprises two, noncovalent, heterodimeric and homotrimeric coiled-coil bundles. These are joined back to back to render two complementary hubs, which when mixed form hexagonal networks that close to form cages. This design strategy offers control over chemistry, self-assembly, reversibility, and size of such particles.