We analyzed the assembly of caveolae in CV1 cells by following the fate of newly synthesized caveolin-1 (CAV1), caveolin-2 and polymerase I and transcript release factor (PTRF)/cavin-1 biochemically and using live-cell imaging. Immediately after synthesis in the endoplasmic reticulum (ER), CAV1 assembled into 8S complexes that concentrated in ER exit sites, due to a DXE sequence in the N-terminal domain. The coat protein II (COPII) machinery allowed rapid transport to the Golgi complex. Accumulating in the medial Golgi, the caveolins lost their diffusional mobility, underwent conformational changes, associated with cholesterol, and eventually assembled into 70S complexes. Together with green fluorescent protein-glycosyl-phosphatidylinositol (GFP-GPI), the newly assembled caveolin scaffolds underwent transport to the plasma membrane in vesicular carriers distinct from those containing vesicular stomatitis virus (VSV) G-protein. After arrival, PTRF/cavin-1 was recruited to the caveolar domains over a period of 25 min or longer. PTRF/cavin-1 itself was present in 60S complexes that also formed in the absence of CAV1. Our study showed the existence of two novel large complexes containing caveolar coat components, and identified a hierarchy of events required for caveolae assembly occurring stepwise in three distinct locations -the ER, the Golgi complex and the plasma membrane.

Identification of the pathway by which caveolin-1 is degraded when caveolae assembly is compromised suggests that “caveosomes” may be endosomal accumulations of the protein awaiting degradation.

Oligomers of the ATPase EHD2 confine caveolae to the plasma membrane through association with actinCaveolae are plasma membrane microdomains that play important roles in signalling and endocytosis. The ATPase EHD2 shuttles on and off the static population of caveolae in an ATPase cycledependent manner and links caveolae to actin filaments confining them to the plasma membrane.

Opioid receptors (ORs) precisely modulate behavior when activated by native peptide ligands but distort behaviors to produce pathology when activated by non-peptide drugs. A fundamental question is how drugs differ from peptides in their actions on target neurons. Here, we show that drugs differ in the subcellular location at which they activate ORs. We develop a genetically encoded biosensor that directly detects ligand-induced activation of ORs and uncover a real-time map of the spatiotemporal organization of OR activation in living neurons. Peptide agonists produce a characteristic activation pattern initiated in the plasma membrane and propagating to endosomes after receptor internalization. Drugs produce a different activation pattern by additionally driving OR activation in the somatic Golgi apparatus and Golgi elements extending throughout the dendritic arbor. These results establish an approach to probe the cellular basis of neuromodulation and reveal that drugs distort the spatiotemporal landscape of neuronal OR activation.

Diverse cargo molecules (i.e., receptors and ligand/receptor complexes) are taken into the cell by clathrin-mediated endocytosis (CME) utilizing a core machinery consisting of cargo-specific adaptors, clathrin and the GTPase dynamin. Numerous endocytic accessory proteins are also required, but their differential roles and functional hierarchy during CME are not yet understood. Here, we used a combination of quantitative live-cell imaging by total internal reflection fluorescence microscopy (TIR-FM), and decomposition of the lifetime distributions of clathrin-coated pits (CCPs) to measure independent aspects of CCP dynamics, including the turnover of abortive and productive CCP species and their relative contributions. Capitalizing on the sensitivity of this assay, we have examined the effects of specific siRNAmediated depletion of endocytic accessory proteins on CME progression. Of the 12 endocytic accessory proteins examined, we observed seven qualitatively different phenotypes upon protein depletion. From this data we derive a temporal hierarchy of protein function during early steps of CME. Our results support the idea that a subset of accessory proteins, which mediate coat assembly, membrane curvature, and cargo selection, can provide input into an endocytic restriction point/checkpoint mechanism that monitors CCP maturation. INTRODUCTIONClathrin-mediated endocytosis (CME) proceeds through the sequential stages of 1) clathrin nucleation at the membrane, 2) clathrin-coated pit (CCP) maturation and invagination, 3) CCP scission, and 4) vesicle uncoating (Conner and Schmid, 2003). The core components of the CME machinery, namely clathrin, the tetrameric adaptor protein AP-2, and the large GTPase dynamin, are assisted by numerous endocytic accessory proteins throughout all stages of CCP maturation (Schmid and McMahon, 2007). Although the function of the core components of the CME machinery is increasingly well understood, the exact function of most other endocytic accessory proteins remains unknown. Among these factors, some such as CALM, epsin1, and SNX9 recognize specific subclasses of cargo molecules (Howard et al., 1999;Harel et al., 2008;Kazazic et al., 2009). Others, such as intersectin, Eps15, SNX9, and endophilin have multiple protein-interaction domains (e.g., Eps15-homolgy [EH] or Src-homology 3 [SH3] domains) and are thought to have scaffolding functions (Miliaras and Wendland, 2004;Ungewickell and Hinrichsen, 2007). Endophilin, SNX9, and epsin have lipid curvature sensing and/or generating domains (e.g., BAR or ENTH domains) and are thought to regulate CME by introducing local biochemical and physical changes into the lipid bilayer, i.e., membrane curvature (Ford et al., 2002;Itoh and De Camilli, 2006). Finally, Hip1R, SNX9, and intersectin are thought to link CME to the actin cytoskeleton and actin dynamics (McPherson, 2002;Le Clainche et al., 2007;Yarar et al., 2007). How these diverse and partially redundant components of the endocytic machinery are integrated and function to create a robust mech...