Rab-regulated interaction of early endosomes with lipid droplets.

Recent studies indicate that lipid droplets isolated from a variety of different cells are rich in proteins known to regulate membrane traffic. Among these proteins are multiple Rab GTPases. Rabs are GTP switches that regulate intracellular membrane traffic through an ability to control membrane-membrane docking as well as vesicle motility. Here we present evidence that the multiple Rabs associated with droplets have a function in regulating membrane traffic. Droplet Rabs are removed by Rab GDP-dissociation inhibitor (RabGDI) in a GDP-dependent reaction, and are recruited to Rab-depleted droplets from cytosol in a GTP-dependent reaction. Rabs also control the recruitment of the early endosome (EE) marker EEA1 from cytosol. We use an in vitro reconstitution assay to show that transferrin receptor positive EEs bind to the droplet in a GTP/Rab-dependent reaction that appears not to lead to membrane fusion. This docking reaction is insensitive to ATP(gamma s) but is blocked by ATP. Finally, we show that when GTP bound active or GDP bound inactive Rab5 is targeted to the droplet, the active form recruits EEA1. We conclude that the Rabs associated with droplets may be capable of regulating the transient interaction of specific membrane systems, probably to transport lipids between membrane compartments.

Dual control of caveolar membrane traffic by microtubules and the actin cytoskeleton.

Live cell, time-lapse microscopy was used to study trafficking of caveolin-1-GFP in stably expressing CHO cells. Multiple cytological and biochemical tests verified that caveolin-1-GFP was a reliable marker for endogenous caveolin-1. At steady state, most caveolin-1-GFP was either at the cell surface associated with invaginated caveolae or near the centrosome in caveosomes. Live cell fluorescence imaging indicated that while much of the caveolin-1-GFP in caveolae at the cell surface was relatively sessile, numerous, highly motile caveolin-1-GFP-positive vesicles were present within the cell interior. These vesicles moved at speeds ranging from 0.3-2 microm/second and movement was abolished when microtubules were depolymerized with nocodazole. In the absence of microtubules, cell surface invaginated caveolae increased more than twofold and they became organized into linear arrays. Complete depolymerization of the actin cytoskeleton with latrunculin A, by contrast, triggered rapid and massive movements of caveolin-positive structures towards the centrosomal region of the cell. The caveolar membrane system of CHO cells therefore appears to be comprised of three caveolin-1-containing compartments. These include caveolae that are confined to the cell surface by cortical actin filaments, the peri-centrosomal caveosomes and caveolar vesicles, which we call 'cavicles', that move constitutively and bi-directionally along microtubules between the cell surface and caveosomes. The behavior of cavicles suggests that they function as transport intermediates between caveolae and caveosomes.

A molecular sensor detects signal transduction from caveolae in living cells.

Biochemical and cell fractionation studies suggest caveolae contain functionally organized sets of signaling molecules that are capable of transmitting specific signals to the cell. It is not known, however, whether any signals actually originate from caveolae in living cells. To address this question, we have engineered the calcium sensor yellow cameleon so that it is targeted either to the plasma membrane, caveolae, or the cytoplasm of endothelial cells. Quantitative measurements of the three Ca2+ pools detected by these probes indicate that caveolae are preferred sites of Ca2+ entry when Ca2+ stores in the endoplasmic reticulum are depleted. These results suggest that the signaling machinery in control of Ca2+ entry is functionally organized in the caveolae of living cells.