SUMMARYIntegrin-mediated adhesion regulates Rac1 membrane binding sites within lipid rafts. Detachment of cells from the substratum triggers clearance of rafts from the plasma membrane through caveolindependent internalization. The small GTPase Arf6 and microtubules also regulate Rac-dependent cell spreading and migration but the mechanisms are poorly understood. We now show that endocytosis of rafts after detachment requires F-actin, followed by microtubule-dependent trafficking to recycling endosomes (RE). When cells are replated on fibronectin, rafts exit from RE in an Arf6-dependent manner and return to the plasma membrane along microtubules. Both of these steps are required for plasma membrane targeting and activation of Rac1. These data therefore define a novel membrane raft trafficking pathway that is crucial for anchorage-dependent signaling.

Cholesterol is a critical component of cell membranes, and cellular cholesterol levels and distribution are tightly regulated in mammals. Recent evidence has revealed a critical role for pancreatic β cell-specific cholesterol homeostasis in insulin secretion as well as in β cell dysfunction in diabetes and the metabolic response to thiazolidinediones (TZDs), which are antidiabetic drugs. The ATP-binding cassette transporter G1 (ABCG1) has been shown to play a role in cholesterol efflux, but its role in β cells is currently unknown. In other cell types, ABCG1 expression is downregulated in diabetes and upregulated by TZDs. Here we have demonstrated an intracellular role for ABCG1 in β cells. Loss of ABCG1 expression impaired insulin secretion both in vivo and in vitro, but it had no effect on cellular cholesterol content or efflux. Subcellular localization studies showed the bulk of ABCG1 protein to be present in insulin granules. Loss of ABCG1 led to altered granule morphology and reduced granule cholesterol levels. Administration of exogenous cholesterol restored granule morphology and cholesterol content and rescued insulin secretion in ABCG1-deficient islets. These findings suggest that ABCG1 acts primarily to regulate subcellular cholesterol distribution in mouse β cells. Furthermore, islet ABCG1 expression was reduced in diabetic mice and restored by TZDs, implicating a role for regulation of islet ABCG1 expression in diabetes pathogenesis and treatment.

Calcium control of exocytosis has been reconstituted in a hybrid system with purified DCVs and supported target membranes.

The distribution of a paramagnetic amphiphile, N,N-dimethyl-N-nonyl-N-tempoylammonium ion, between the membranes of charged phospholipid vesicles and the surrounding aqueous medium was studied by electron paramagnetic resonance spectroscopy. By systematically varying the surface charge of the vesicles and the aqueous electrolyte concentration, the distribution was shown to indicate vesicle surface potential. At each fixed phospholipid composition, the surface potential exhibited a dependence on aqueous NaCl concentration very similar to that predicted by the Gouy equation. The ability to sense and quantitate surface potentials makes this facile and sensitive technique of value in the study of cell and organelle surfaces.

Intracellular transport of secretory proteins has been studied in the parotid to examine this process in an exocrine gland other than the pancreas and to explore a possible source of less degraded membranes than obtainable from the latter gland . Rabbit parotids were chosen on the basis of size (2-2 .5 g per animal), ease of surgical removal, and amylase concentration . Sites of synthesis, rates of intracellular transport, and sites of packaging and storage of newly synthesized secretory proteins were determined radioautographically by using an in vitro system of dissected lobules capable of linear amino acid incorporation for 10 hr with satisfactory preservation of cellular fine structure . Adequate fixation of the tissue with minimal binding of unincorporated labeled amino acids was obtained by using 10% formaldehyde-0 .175 M phosphate buffer (pH 7 .2) as primary fixative . Pulse labeling with leucine 3H, followed by a chase incubation, showed that the label is initially located (chase : 1-6 min) over the rough endoplasmic reticulum (RER) and subsequently moves as a wave through the Golgi complex (chase : 16-36 min), condensing vacuoles (chase : 36-56 min), immature granules (chase : 56-116 min), and finally mature storage granules (chase : 116-356 min) . Distinguishing features of the parotid transport apparatus are : low frequency of RER-Golgi transitional elements, close association of condensing vacuoles with the exit side of Golgi stacks, and recognizable immature secretory granules . Intracelular processing of secretory proteins is similar to that already found in the pancreas, except that the rate is slower and the storage is more prolonged . 290