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.

Caveolin-1 secreting LNCaP cells induce tumor growth of caveolin-1 negative LNCaP cells in vivo.

Caveolin-1 (Cav-1) was originally identified as a structural protein of caveolae, which is a plasma membrane domain that regulates a variety of signaling pathways involved in cell growth and migration. Here, we show that expression of Cav-1 in the Cav-1-deficient human prostate cancer cell line LNCaP both stimulates cell proliferation and promotes tumor growth in nude mice. Unexpectedly, Cav-1 expressing LNCaP (LNCaP(Cav-1)) cells injected into one side of a nude mouse promoted tumor growth of Cav-1 negative LNCaP cells injected on the contralateral side of the same animal. The LNCaP tumors were positive for Cav-1, however, this signal was not caused by migrated LNCaP(Cav-1) cells, but we show that this Cav-1 was secreted by the LNCaP(Cav-1) tumors. We demonstrate that conditioned media from LNCaP(Cav-1) cells contained Cav-1 that was associated with a lipoprotein particle ranging in size from 15 to 30 nm and a density similar to high density lipoprotein particle. These results suggest that LNCaP(Cav-1) cells secreting Cav-1 particle produce an endocrine factor that stimulates tumor growth.

Rab-regulated membrane traffic between adiposomes and multiple endomembrane systems.

Lipid droplets play a critical role in a variety of metabolic diseases. Numerous proteomic studies have provided detailed information about the protein composition of the droplet, which has revealed that they are functional organelles involved in many cellular processes, including lipid storage and metabolism, membrane traffic, and signal transduction. Thus, the droplet proteome indicates that lipid accumulation is only one of a constellation of organellar functions critical for normal lipid metabolism in the cell. As a result of this new understanding, we suggested the name adiposome for this organelle. The trafficking ability of the adiposome is likely to be very important for lipid uptake, retention, and distribution, as well as membrane biogenesis and lipid signaling. We have taken advantage of the ease of purifying lipid-filled adiposomes to develop a cell-free system for studying adiposome-mediated traffic. Using this approach, we have determined that the interaction between adiposomes and endosomes is dependent on Rab GTPases but is blocked by ATPase. These methods also allowed us to identify multiple proteins that dynamically associate with adiposomes in a nucleotide-dependent manner. An adiposome-endosome interaction in vitro occurs in the absence of cytosolic factors, which simplifies the assay dramatically. This assay will enable researchers to dissect the molecular mechanisms of interaction between these two organelles. This chapter provides a detailed account of the methods developed.

Morphologically and Functionally Distinct Lipid Droplet Subpopulations.

Lipid droplet (LD), a multi-functional organelle, is often found to associate with other cellular membranous structures and vary in size in a given cell, which may be related to their functional diversity. Here we established a method to separate LD subpopulations from isolated CHO K2 LDs into three different size categories. The subpopulation with smallest LDs was nearly free of ER and other membranous structures while those with larger LDs contained intact ER. These distinct subpopulations of LDs differed in their protein composition and ability to recruit proteins. This method was also applicable to LDs obtained from other sources, such as Huh7 cells, mouse liver and brown adipose tissue, et al. We developed an in vitro assay requiring only isolated LDs, Coenzyme A, and ATP to drive lipid synthesis. The LD subpopulation nearly depleted of ER was able to incorporate fatty acids into triacylglycerol and phospholipids. Together, our data demonstrate that LDs in a given cell are heterogeneous in size and function, and suggest that LDs are one of cellular lipid synthetic organelles.

Sites of Ca(2+) wave initiation move with caveolae to the trailing edge of migrating cells.

The caveola is a membrane domain that compartmentalizes signal transduction at the cell surface. Normally in endothelial cells, groups of caveolae are found clustered along stress fibers or at the lateral margins in all regions of the cell. Subsets of these clusters appear to contain the signaling machinery for initiating Ca(2+) wave formation. Here we report that induction of cell migration, either by wounding a cell monolayer or by exposing cells to laminar shear stress, causes caveolae to move to the trailing edge of the cell. Concomitant with the relocation of the caveolae, sites of Ca(2+) wave initiation move to the same location. In as much as the relocated caveolae contain elements of the signaling machinery required for ATP-stimulated release of Ca(2+) from the ER, these results suggest that caveolae function as containers that carry this machinery to different cellular locations.

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.

Mechanism of caveolin filament assembly.

Caveolin-1 was the first protein identified that colocalizes with the approximately 10-nm filaments found on the inside surface of caveolae membranes. We have used a combination of electron microscopy (EM), circular dichroism, and analytical ultracentrifugation to determine the structure of the oligomers that form when the first 101 aa of caveolin-1 (Cav(1-101)) are allowed to associate. We determined that amino acids 79-96 in this caveolin-1 fragment are arranged in an alpha-helix. Cav(1-101) oligomers are approximately 11 nm in diameter and contain seven molecules of Cav(1-101). These subunits, in turn, are able to assemble into 50 nm long x 11 nm diameter filaments that closely match the morphology of the filaments in the caveolae filamentous coat. We propose that the heptameric subunit forms in part through lateral interactions between the alpha-helices of the seven Cav(1-101) units. Caveolin-1, therefore, appears to be the structural molecule of the caveolae filamentous coat.

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.

Chinese hamster ovary K2 cell lipid droplets appear to be metabolic organelles involved in membrane traffic.

The principal lipids in animal cell lipid droplets are cholesterol, cholesterol ester, and triglyceride, but the protein composition of this compartment is largely unknown. Here we report on the proteomic analysis of lipid droplets. Using a combination of mass spectrometry and immunoblotting, we identify nearly 40 specifically associated proteins in droplets isolated from Chinese hamster ovary K2 cells grown in normal medium. The proteins fall in to five groups: structural molecules of the droplet-like adipose differentiation-related protein; multiple enzymes involved in the synthesis, storage, utilization, and degradation of cholesterol esters and triglycerides; multiple, different Rab GTPases known to be involved in regulating membrane traffic; signaling molecules such as p50RhoGAP; and a group of proteins that do not fit any classification but include proteins often found in caveolae/rafts such as caveolin-1 and 2 and flotillin-1. The proteome of droplets isolated from cells grown in the presence of oleate is largely the same except for an increase in the amount of adipose differentiation-related protein, caveolin-1, and a protein thought to be involved in phospholipid recycling called CGI-58. Based on the protein profile, the lipid droplet appears to be a complex, metabolically active organelle that is directly involved in membrane traffic and possibly phospholipid recycling. We propose the name adiposome for this organelle.