Lipid rafts: at a crossroad between cell biology and physics.

Membrane lateral heterogeneity is accepted as a requirement for the function of biological membranes and the notion of lipid rafts gives specificity to this broad concept. However, the lipid raft field is now at a technical impasse because the physical tools to study biological membranes as a liquid that is ordered in space and time are still being developed. This has lead to a disconnection between the concept of lipid rafts as derived from biochemical and biophysical assays and their existence in the cell. Here, we compare the concept of lipid rafts as it has emerged from the study of synthetic membranes with the reality of lateral heterogeneity in biological membranes. Further application of existing tools and the development of new tools are needed to understand the dynamic heterogeneity of biological membranes.

SRBC/cavin-3 is a caveolin adapter protein that regulates caveolae function.

Caveolae are a major membrane domain common to most cells. One of the defining features of this domain is the protein caveolin. The exact function of caveolin, however, is not clear. One possible function is to attract adapter molecules to caveolae in a manner similar to how clathrin attracts molecules to coated pits. Here, we characterize a candidate adapter molecule called SRBC. SRBC binds PKCdelta and is a member of the STICK (substrates that interact with C-kinase) superfamily of PKC-binding proteins. We also show it co-immunoprecipitates with caveolin-1. A leucine zipper in SRBC is essential for both co-precipitation with caveolin and localization to caveolae. SRBC remains associated with caveolin when caveolae bud to form vesicles (cavicles) that travel on microtubules to different regions of the cell. In the absence of SRBC, intracellular cavicle traffic is markedly impaired. We conclude that SRBC (sdr-related gene product that binds to c-kinase) and two other family members [PTRF (Pol I and transcription release factor) and SDPR] function as caveolin adapter molecules that regulate caveolae function.

Sterol-dependent nuclear import of ORP1S promotes LXR regulated trans-activation of apoE.

Oxysterol binding protein related protein 1S (ORP1S) is a member of a family of sterol transport proteins. Here we present evidence that ORP1S translocates from the cytoplasm to the nucleus in response to sterol binding. The sterols that best promote nuclear import of ORP1S also activate the liver X receptor (LXR) transcription factors and we show that ORP1S binds to LXRs, promotes binding of LXRs to LXR response elements (LXREs) and specifically enhances LXR-dependent transcription via the ME.1 and ME.2 enhancer elements of the apoE gene. We propose that ORP1S is a cytoplasmic sterol sensor, which transports sterols to the nucleus and promotes LXR-dependent gene transcription through select enhancer elements.

Disruption of the Arabidopsis CGI-58 homologue produces Chanarin-Dorfman-like lipid droplet accumulation in plants.

CGI-58 is the defective gene in the human neutral lipid storage disease called Chanarin-Dorfman syndrome. This disorder causes intracellular lipid droplets to accumulate in nonadipose tissues, such as skin and blood cells. Here, disruption of the homologous CGI-58 gene in Arabidopsis thaliana resulted in the accumulation of neutral lipid droplets in mature leaves. Mass spectroscopy of isolated lipid droplets from cgi-58 loss-of-function mutants showed they contain triacylglycerols with common leaf-specific fatty acids. Leaves of mature cgi-58 plants exhibited a marked increase in absolute triacylglycerol levels, more than 10-fold higher than in wild-type plants. Lipid levels in the oil-storing seeds of cgi-58 loss-of-function plants were unchanged, and unlike mutations in ß-oxidation, the cgi-58 seeds germinated and grew normally, requiring no rescue with sucrose. We conclude that the participation of CGI-58 in neutral lipid homeostasis of nonfat-storing tissues is similar, although not identical, between plant and animal species. This unique insight may have implications for designing a new generation of technologies that enhance the neutral lipid content and composition of crop plants.

Phospho-caveolin-1 mediates integrin-regulated membrane domain internalization.

Growth of normal cells is anchorage dependent because signalling through multiple pathways including Erk, phosphatidylinositol-3-OH kinase (PI(3)K) and Rac requires integrin-mediated cell adhesion. Components of these pathways localize to low-density, cholesterol-rich domains in the plasma membrane named 'lipid rafts' or 'cholesterol-enriched membrane microdomains' (CEMM). We previously reported that integrin-mediated adhesion regulates CEMM transport such that cell detachment from the extracellular matrix triggers CEMM internalization and clearance from the plasma membrane. We now report that this internalization is mediated by dynamin-2 and caveolin-1. Internalization requires phosphorylation of caveolin-1 on Tyr 14. A shift in localization of phospho-caveolin-1 from focal adhesions to caveolae induces CEMM internalization upon cell detachment, which mediates inhibition of Erk, PI(3)K and Rac. These data define a novel molecular mechanism for growth and tumour suppression by caveolin-1.

Sterol-induced dislocation of 3-hydroxy-3-methylglutaryl coenzyme A reductase from endoplasmic reticulum membranes into the cytosol through a subcellular compartment resembling lipid droplets.

Sterol-induced binding to Insigs in the endoplasmic reticulum (ER) allows for ubiquitination of 3-hydroxy-3-methylglutaryl coenzyme A reductase, the rate-limiting enzyme in cholesterol synthesis. This ubiquitination marks reductase for recognition by the ATPase VCP/p97, which mediates extraction and delivery of reductase from ER membranes to cytosolic 26 S proteasomes for degradation. Here, we report that reductase becomes dislocated from ER membranes into the cytosol of sterol-treated cells. This dislocation exhibits an absolute requirement for the actions of Insigs and VCP/p97. Reductase also appears in a buoyant fraction of sterol-treated cells that co-purifies with lipid droplets, cytosolic organelles traditionally regarded as storage depots for neutral lipids such as triglycerides and cholesteryl esters. Genetic, biochemical, and localization studies suggest a model in which reductase is dislodged into the cytosol from an ER subdomain closely associated with lipid droplets.

Targeting sequences of UBXD8 and AAM-B reveal that the ER has a direct role in the emergence and regression of lipid droplets.

Lipid droplets are sites of neutral lipid storage thought to be actively involved in lipid homeostasis. A popular model proposes that droplets are formed in the endoplasmic reticulum (ER) by a process that begins with the deposition of neutral lipids between the membrane bilayer. As the droplet grows, it becomes surrounded by a monolayer of phospholipid derived from the outer half of the ER membrane, which contains integral membrane proteins anchored by hydrophobic regions. This model predicts that for an integral droplet protein inserted into the outer half of the ER membrane to reach the forming droplet, it must migrate in the plane of the membrane to sites of lipid accumulation. Here, we report the results of experiments that directly test this hypothesis. Using two integral droplet proteins that contain unique hydrophobic targeting sequences (AAM-B and UBXD8), we present evidence that both proteins migrate from their site of insertion in the ER to droplets that are forming in response to fatty acid supplementation. Migration to droplets occurs even when further protein synthesis is inhibited or dominant-negative Sar1 blocks transport to the Golgi complex. Surprisingly, when droplets are induced to disappear from the cell, both proteins return to the ER as the level of neutral lipid declines. These data suggest that integral droplet proteins form from and regress to the ER as part of a cyclic process that does not involve traffic through the secretory pathway.

An intimate collaboration between peroxisomes and lipid bodies.

Although peroxisomes oxidize lipids, the metabolism of lipid bodies and peroxisomes is thought to be largely uncoupled from one another. In this study, using oleic acid-cultured Saccharomyces cerevisiae as a model system, we provide evidence that lipid bodies and peroxisomes have a close physiological relationship. Peroxisomes adhere stably to lipid bodies, and they can even extend processes into lipid body cores. Biochemical experiments and proteomic analysis of the purified lipid bodies suggest that these processes are limited to enzymes of fatty acid beta oxidation. Peroxisomes that are unable to oxidize fatty acids promote novel structures within lipid bodies ("gnarls"), which may be organized arrays of accumulated free fatty acids. However, gnarls are suppressed, and fatty acids are not accumulated in the absence of peroxisomal membranes. Our results suggest that the extensive physical contact between peroxisomes and lipid bodies promotes the coupling of lipolysis within lipid bodies with peroxisomal fatty acid oxidation.

The lipodystrophy protein seipin is found at endoplasmic reticulum lipid droplet junctions and is important for droplet morphology.

Lipodystrophy is a disorder characterized by a loss of adipose tissue often accompanied by severe hypertriglyceridemia, insulin resistance, diabetes, and fatty liver. It can be inherited or acquired. The most severe inherited form is Berardinelli-Seip Congenital Lipodystrophy Type 2, associated with mutations in the BSCL2 gene. BSCL2 encodes seipin, the function of which has been entirely unknown. We now report the identification of yeast BSCL2/seipin through a screen to detect genes important for lipid droplet morphology. The absence of yeast seipin results in irregular lipid droplets often clustered alongside proliferated endoplasmic reticulum (ER); giant lipid droplets are also seen. Many small irregular lipid droplets are also apparent in fibroblasts from a BSCL2 patient. Human seipin can functionally replace yeast seipin, but a missense mutation in human seipin that causes lipodystrophy, or corresponding mutations in the yeast gene, render them unable to complement. Yeast seipin is localized in the ER, where it forms puncta. Almost all lipid droplets appear to be on the ER, and seipin is found at these junctions. Therefore, we hypothesize that seipin is important for droplet maintenance and perhaps assembly. In addition to detecting seipin, the screen identified 58 other genes whose deletions cause aberrant lipid droplets, including 2 genes encoding proteins known to activate lipin, a lipodystrophy locus in mice, and 16 other genes that are involved in endosomal-lysosomal trafficking. The genes identified in our screen should be of value in understanding the pathway of lipid droplet biogenesis and maintenance and the cause of some lipodystrophies.

Evidence that mono-ADP-ribosylation of CtBP1/BARS regulates lipid storage.

Mono-ADP-ribosylation is emerging as an important posttranslational modification that modulates a variety of cell signaling pathways. Here, we present evidence that mono-ADP-ribosylation of the transcriptional corepressor C terminal binding protein, brefeldin A (BFA)-induced ADP-ribosylated substrate (CtBP1/BARS) regulates neutral lipid storage in droplets that are surrounded by a monolayer of phospholipid and associated proteins. CtBP1/BARS is an NAD-binding protein that becomes ribosylated when cells are exposed to BFA. Both endogenous lipid droplets and droplets enlarged by oleate treatment are lost after 12-h exposure to BFA. Lipid loss requires new protein synthesis, and it is blocked by multiple ribosylation inhibitors, but it is not stimulated by disruption of the Golgi apparatus or the endoplasmic reticulum unfolded protein response. Small interfering RNA knockdown of CtBP1/BARS mimics the effect of BFA, and mouse embryonic fibroblasts derived from embryos that are deficient in CtBP1/BARS seem to be defective in lipid accumulation. We conclude that mono-ADP-ribosylation of CtBP1/BARS inactivates its repressor function, which leads to the activation of genes that regulate neutral lipid storage.

A role for lipid droplets in inter-membrane lipid traffic.

All cells have the capacity to accumulate neutral lipids and package them into lipid droplets. Recent proteomic analyses indicate that lipid droplets are not simple lipid storage depots, but rather complex organelles that have multiple cellular functions. One of these proposed functions is to distribute neutral lipids as well as phospholipids to various membrane-bound organelles within the cell. Here, we summarize the lipid droplet-associated membrane-trafficking proteins and review the evidence that lipid droplets interact with endoplasmic reticulum, endosomes, peroxisomes, and mitochondria. Based on this evidence, we present a model for how lipid droplets can distribute lipids to specific membrane compartments.

Joe Goldstein and Mike Brown: from cholesterol homeostasis to new paradigms in membrane biology.

Joe Goldstein and Mike Brown have worked for over 30 years on the molecular basis of cholesterol homeostasis. Through the systematic use of genetics, biochemistry, molecular biology and cell biology, they have identified a complex set of interacting molecules that work coordinately to regulate cholesterol import and synthesis. Not only did they identify the crucial proteins in this pathway but also determined their function. An unexpected outcome of their work has been a new understanding of the structure and function of cell membranes. From the low-density lipoprotein receptor to sterol regulatory element binding protein (SREBP) to SREBP cleavage-activating protein to Insig-1, each protein has provided a new and fundamentally novel insight into how membranes function as molecular sensors that respond to changes in the metabolic condition of the cell by moving molecules between cellular compartments.*

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.

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.

Does external pneumatic compression treatment between bouts of overreaching resistance training sessions exert differential effects on molecular signaling and performance-related variables compared to passive recovery? An exploratory study.

PURPOSE: We sought to compare the effects of external pneumatic compression (EPC) and sham when used concurrently with resistance training on performance-related outcomes and molecular measures related to recovery. METHODS: Twenty (N = 20) resistance-trained male participants (aged 21.6±2.4 years) were randomized to balanced sham or EPC intervention groups. The protocol consisted of 3 consecutive days of heavy, voluminous back squat exercise followed by EPC/sham treatment (Days2-4) and 3 consecutive days of recovery (Days5-7) with EPC/sham only on Days5-6. On Day1 (PRE), and Days3-7, venipuncture, flexibility and pressure-to-pain threshold (PPT) measures were performed. Vastsus lateralis muscle tissue was biopsied at PRE, 1-h post-EPC/sham treatment on Day2 (POST1) and 24-h post-EPC/sham treatment on Day7 (POST2). Isokinetic peak torque was assessed at PRE and POST2. RESULTS: Peak isokinetic strength did not change from PRE to POST2 in either group. The PPT was significantly lower on Days3-6 with sham, indicating greater muscle soreness, though this was largely abolished in the EPC group. A significant decrease in flexibility with sham was observed on Day3 (+16.2±4.6% knee joint angle; P<0.01) whereas there was no change with EPC (+2.8±3.8%; P>0.01). Vastus lateralis poly-ubiquitinated proteins significantly increased at the POST2 time point relative to PRE with sham (+66.6±24.6%; P<0.025) and were significantly greater (P<0.025) than those observed with EPC at the same time point (-18.6±8.5%). 4-hydroxynonenal values were significantly lower at POST2 relative to PRE with EPC (-16.2±5.6%; P<0.025) and were significantly lower (P<0.025) than those observed with sham at the same time point (+11.8±5.9%). CONCLUSION: EPC mitigated a reduction in flexibility and PPT that occurred with sham. Moreover, EPC reduced select skeletal muscle oxidative stress and proteolysis markers during recovery from heavy resistance exercise.

Aging in Rats Differentially Affects Markers of Transcriptional and Translational Capacity in Soleus and Plantaris Muscle.

Alterations in transcriptional and translational mechanisms occur during skeletal muscle aging and such changes may contribute to age-related atrophy. Herein, we examined markers related to global transcriptional output (i.e., myonuclear number, total mRNA and RNA pol II levels), translational efficiency [i.e., eukaryotic initiation and elongation factor levels and muscle protein synthesis (MPS) levels] and translational capacity (ribosome density) in the slow-twitch soleus and fast-twitch plantaris muscles of male Fischer 344 rats aged 3, 6, 12, 18, and 24 months (n = 9-10 per group). We also examined alterations in markers of proteolysis and oxidative stress in these muscles (i.e., 20S proteasome activity, poly-ubiquinated protein levels and 4-HNE levels). Notable plantaris muscle observations included: (a) fiber cross sectional area (CSA) was 59% (p < 0.05) and 48% (p < 0.05) greater in 12 month vs. 3 month and 24 month rats, respectively, suggesting a peak lifetime value near 12 months and age-related atrophy by 24 months, (b) MPS levels were greatest in 18 month rats (p < 0.05) despite the onset of atrophy, (c) while regulators of ribosome biogenesis [c-Myc and upstream binding factor (UBF) protein levels] generally increased with age, ribosome density linearly decreased from 3 months of age and RNA polymerase (Pol) I protein levels were lowest in 24 month rats, and d) 20S proteasome activity was robustly up-regulated in 6 and 24 month rats (p < 0.05). Notable soleus muscle observations included: (a) fiber CSA was greatest in 6 month rats and was maintained in older age groups, and (b) 20S proteasome activity was modestly but significantly greater in 24 month vs. 3/12/18 month rats (p < 0.05), and (c) total mRNA levels (suggestive of transcriptional output) trended downward in older rats despite non-significant between-group differences in myonuclear number and/or RNA Pol II protein levels. Collectively, these findings suggest that plantaris, not soleus, atrophy occurs following 12 months of age in male Fisher rats and this may be due to translational deficits (i.e., changes in MPS and ribosome density) and/or increases in proteolysis rather than increased oxidative stress and/or alterations in global transcriptional mechanisms.

Effects of Whey, Soy or Leucine Supplementation with 12 Weeks of Resistance Training on Strength, Body Composition, and Skeletal Muscle and Adipose Tissue Histological Attributes in College-Aged Males.

We sought to determine the effects of L-leucine (LEU) or different protein supplements standardized to LEU (~3.0 g/serving) on changes in body composition, strength, and histological attributes in skeletal muscle and adipose tissue. Seventy-five untrained, college-aged males (mean ± standard error of the mean (SE); age = 21 ± 1 years, body mass = 79.2 ± 0.3 kg) were randomly assigned to an isocaloric, lipid-, and organoleptically-matched maltodextrin placebo (PLA, n = 15), LEU (n = 14), whey protein concentrate (WPC, n = 17), whey protein hydrolysate (WPH, n = 14), or soy protein concentrate (SPC, n = 15) group. Participants performed whole-body resistance training three days per week for 12 weeks while consuming supplements twice daily. Skeletal muscle and subcutaneous (SQ) fat biopsies were obtained at baseline (T1) and ~72 h following the last day of training (T39). Tissue samples were analyzed for changes in type I and II fiber cross sectional area (CSA), non-fiber specific satellite cell count, and SQ adipocyte CSA. On average, all supplement groups including PLA exhibited similar training volumes and experienced statistically similar increases in total body skeletal muscle mass determined by dual X-ray absorptiometry (+2.2 kg; time p = 0.024) and type I and II fiber CSA increases (+394 µm² and +927 µm²; time p < 0.001 and 0.024, respectively). Notably, all groups reported increasing Calorie intakes ~600-800 kcal/day from T1 to T39 (time p < 0.001), and all groups consumed at least 1.1 g/kg/day of protein at T1 and 1.3 g/kg/day at T39. There was a training, but no supplementation, effect regarding the reduction in SQ adipocyte CSA (-210 µm²; time p = 0.001). Interestingly, satellite cell counts within the WPC (p < 0.05) and WPH (p < 0.05) groups were greater at T39 relative to T1. In summary, LEU or protein supplementation (standardized to LEU content) does not provide added benefit in increasing whole-body skeletal muscle mass or strength above PLA following 3 months of training in previously untrained college-aged males that increase Calorie intakes with resistance training and consume above the recommended daily intake of protein throughout training. However, whey protein supplementation increases skeletal muscle satellite cell number in this population, and this phenomena may promote more favorable training adaptations over more prolonged periods.

Molecular, neuromuscular, and recovery responses to light versus heavy resistance exercise in young men.

Recent evidence suggests that resistance training with light or heavy loads to failure results in similar adaptations. Herein, we compared how both training modalities affect the molecular, neuromuscular, and recovery responses following exercise. Resistance-trained males (mean ± SE: 22 ± 2 years, 84.8 ± 9.0 kg, 1.79 ± 0.06 m; n = 15) performed a crossover design of four sets of leg extensor exercise at 30% (light RE) or 80% (heavy RE) one repetition maximum (1RM) to repetition failure, and heavy RE or light RE 1 week later. Surface electromyography (EMG) was monitored during exercise, and vastus lateralis muscle biopsies were collected at baseline (PRE), 15 min (15mPOST), and 90 min following RE (90mPOST) for examination of molecular targets and fiber typing. Isokinetic dynamometry was also performed before (PRE), immediately after (POST), and 48 h after (48hPOST) exercise. Dependent variables were analyzed using repeated measures ANOVAs and significance was set at P ≤ 0.05. Repetitions completed were greater during light RE (P < 0.01), while EMG amplitude was greater during heavy RE (P ≤ 0.01). POST isokinetic torque was reduced following light versus heavy RE (P < 0.05). Postexercise expression of mRNAs and phosphoproteins associated with muscle hypertrophy were similar between load conditions. Additionally, p70s6k (Thr389) phosphorylation and fast-twitch fiber proportion exhibited a strong relationship after both light and heavy RE (r > 0.5). While similar mRNA and phosphoprotein responses to both modalities occurred, we posit that heavy RE is a more time-efficient training method given the differences in total repetitions completed, lower EMG amplitude during light RE, and impaired recovery response after light RE.

ERbeta has nongenomic action in caveolae.

ERalpha and ERbeta serve classically as transcription factors, and ERalpha also mediates nongenomic responses to E2 such as the activation of endothelial nitric oxide synthase (eNOS). In contrast, the nongenomic capacities of endogenous ERbeta are poorly understood. We evaluated eNOS activation by E2 in cultured endothelial cells that express endogenous ERbeta to determine whether the ERbeta isoform has nongenomic action and to reveal the subcellular locale of that function. A subpopulation of ERbeta was localized to the endothelial cell plasma membrane, overexpression of ERbeta enhanced rapid eNOS stimulation by E2, and the response to endogenous ER activation was inhibited by the ERbeta-selective antagonist RR-tetrahydrochrysene (THC). eNOS activation through ERbeta was reconstituted and shown to occur independent of ERalpha in COS-7 cells, and ERbeta protein in COS-7 was directed to the plasma membrane. THC also blunted E2 activation of eNOS in isolated endothelial cell plasma membranes. Furthermore, ERbeta protein was detected and THC attenuated E2 stimulation of eNOS in isolated endothelial cell caveolae, and functional ERbeta-eNOS coupling was recapitulated in caveolae from transfected COS-7 cells. These findings in the ER-eNOS signaling paradigm indicate that endogenous ERbeta has nongenomic action in caveolae.

Alterations in membrane cholesterol that affect structure and function of caveolae.

Most of the available methods for modifying caveolae structure and function depend on altering the cholesterol content of caveolae. The most important aspect of each method is to ensure the reagents are working in the cells that are being studied. The idiosyncrasies of each method are such that they cannot be universally applied without carefully optimizing the conditions. When used correctly, these methods are accepted as a specific way to perturb the structure and function of caveolae.