The endocytic itineraries of lipid raft markers, such as glycosyl phosphatidylinositol (GPI)-anchored proteins and glycosphingolipids, are incompletely understood. Here we show that different GPI-anchored have intracellular distributions; some (such the folate receptor) accumulate in transferrin-containing compartments, others CD59 GPI-linked green fluorescent protein [GFP]) Golgi apparatus. Selective photobleaching shows pool both GPI-GFP CD59-GFP constantly rapidly exchanges with these...

Lipid rafts are conceptualized as membrane microdomains enriched in cholesterol and glycosphingolipid that serve platforms for protein segregation signaling. The properties of these domains vivo unclear. Here, we use fluorescence recovery after photobleaching to test if raft association affects a protein's ability laterally diffuse large distances across the cell surface. diffusion coefficients (D) several types putative nonraft proteins were systematically measured under steady-state...

Flotillins are membrane proteins that form microdomains in the plasma of all mammalian cell types studied to date. They span evolutionary spectrum, with related flotillins present bacteria, fungi, plants and metazoans, which suggests they perform important, probably conserved, functions. have been implicated myriad processes include endocytosis, signal transduction regulation cortical cytoskeleton, yet molecular mechanisms underlie flotillin function these different cases still poorly...

Several different endocytic pathways have been proposed to function in mammalian cells. Clathrin-coated pits are well defined, but the identity, mechanism and of alternative controversial. Here we apply universal chemical labelling plasma membrane proteins define all primary vesicles, specific with a reducible SNAP-tag substrate. These approaches provide high temporal resolution stringent discrimination between surface-connected intracellular membranes. We find that at least 95% earliest...

Many endocytosed proteins in yeast travel to the vacuole, but some are recycled plasma membrane. We have investigated recycling of chimeras containing green fluorescent protein (GFP) and exocytic SNARE Snc1p. GFP-Snc1p moves from cell surface internal structures when Golgi function or exocytosis is blocked, suggesting continuous via Golgi. Internalization mediated by a conserved cytoplasmic signal, whereas diversion vacuolar pathway requires sequences within adjacent transmembrane domain....

Homotypic vacuole fusion in yeast requires Sec18p (N-ethylmaleimide–sensitive protein [NSF]), Sec17p (soluble NSF attachment [α-SNAP]), and typical vesicle (v) target membrane (t) SNAP receptors (SNAREs). We now report that vacuolar v- t-SNAREs are mainly found with as v–t-SNARE complexes vivo on purified vacuoles rather than only transiently forming such during docking, disrupting them upon fusion. In the priming reaction, ATP dissociate this complex, accompanied by release of Sec17p. SNARE...

Dishevelled is a crucial effector upstream in the Wnt signalling pathway, but molecular mechanism by which it transduces signal remains elusive. cytoplasmic protein with strong tendency to form puncta, correlates its potent activity stimulating transduction when overexpressed. These puncta are thought reflect vesicles. However, we show here that mammalian Dvl2 does not colocalise known vesicle markers for clathrin-mediated or clathrin-independent endocytic pathways. Furthermore, do stain...

Caveolae are an abundant feature of the plasma membrane many mammalian cell types, and have key roles in mechano-transduction, metabolic regulation, vascular permeability. Caveolin cavin proteins, as well EHD2 pacsin 2, all present caveolae. How these proteins assemble to form a protein interaction network for caveolar morphogenesis is not known. Using vivo crosslinking, velocity gradient centrifugation, immuno-isolation, tandem mass spectrometry, we determine that cavins caveolins into...

The pacsin (also termed syndapin) protein family is well characterised structurally. They contain F-BAR domains associated with the generation or maintenance of membrane curvature. cell biology these proteins remains less understood. Here, we initially confirm that EHD2, a previously shown biochemically to be present in caveolar fractions and bind pacsins, protein. We go on report GFP–pacsin 2 can recruited caveolae, endogenous partially colocalises caveolin 1 at plasma membrane. Analysis...

Caveolae are abundant in endothelial cells and thought to have important roles cell biology. The cavin proteins key components of caveolae, expressed at varied amounts different tissues. Here we use knockout mice determine the cavins 2 3 caveolar morphogenesis vivo. Deletion causes loss caveolae lung adipose tissue, but has no effect on abundance heart other Changes morphology endothelium null correlate with changes abundance. Cavin is not required for making tissues examined. determines...

Caveolae are strikingly abundant in endothelial cells, yet the physiological functions of caveolae endothelium and other tissues remain incompletely understood. Previous studies suggest a mechanoprotective role, but whether this is relevant under mechanical forces experienced by cells vivo unclear. In study we have sought to determine disassemble increased hemodynamic forces, help prevent acute rupture plasma membrane these conditions. Experiments cultured established biochemical assays for...

Abstract Caveolae have long been implicated in endocytosis. Recent data question this link, and the absence of specific cargoes potential cellular function caveolar endocytosis remains unclear. Here we develop new tools, including doubly genome-edited cell lines, to assay subcellular dynamics caveolae using tagged proteins expressed at endogenous levels. We find that around 5% pool is present on dynamic endosomes, delivered endosomes a clathrin-independent manner. Furthermore, show are...

Flotillin-1 and flotillin-2 co-assemble into plasma membrane microdomains that are involved in the endocytosis of molecules such as glycosyl phosphatidylinositol (GPI)-linked proteins. Previous studies suggest budding flotillin from is a tightly regulated process. Here, we demonstrate flotillins by Src family kinase Fyn. The inhibitor PP2 prevents EGF-induced internalisation, internalisation does not occur SYF cells lacking Src, Yes Expression Fyn, but or Yes, restores cells. an active form...