Koert N.J. Burger

CtBP/BARS induces fission of Golgi membranes by acylating lysophosphatidic acid

Membrane fission is essential in intracellular transport. Acyl-coenzyme As (acyl-CoAs) are important in lipid remodelling and are required for fission of COPI-coated vesicles. Here we show that CtBP/BARS, a protein that functions in the dynamics of Golgi tubules, is an essential component of the fission machinery operating at Golgi tubular networks, including Golgi compartments involved in protein transport and sorting. CtBP/BARS-induced fission was preceded by the formation of constricted sites in Golgi tubules, whose extreme curvature is likely to involve local changes in the membrane lipid composition. We find that CtBP/BARS uses acyl-CoA to selectively catalyse the acylation of lysophosphatidic acid to phosphatidic acid both in pure lipidic systems and in Golgi membranes, and that this reaction is essential for fission. Our results indicate a key role for lipid metabolic pathways in membrane fission.

Modulation of Membrane Curvature by Phosphatidic Acid and Lysophosphatidic Acid

The local generation of phosphatidic acid plays a key role in the regulation of intracellular membrane transport through mechanisms which are largely unknown. Phosphatidic acid may recruit and activate downstream effectors, or change the biophysical properties of the membrane and directly induce membrane bending and/or destabilization. To evaluate these possibilities, we determined the phase properties of phosphatidic acid and lysophosphatidic acid at physiological conditions of pH and ion concentrations. In single‐lipid systems, unsaturated phosphatidic acid behaved as a cylindrical, bilayer‐preferring lipid at cytosolic conditions (37 °C, pH 7.2, 0.5 mm free Mg2+), but acquired a type‐II shape at typical intra‐Golgi conditions, a mildly acidic pH and submillimolar free Ca2+ (pH 6.6–5.9, 0.3 mm Ca2+). Lysophosphatidic acid formed type‐I lipid micelles in the absence of divalent cations, but anhydrous cation‐lysophosphatidic acid bilayer complexes in their presence. These data suggest a similar molecular shape for phosphatidic acid and lysophosphatidic acid at cytosolic conditions; however, experiments in mixed‐lipid systems indicate that their shape is not identical. Lysophosphatidic acid stabilized the bilayer phase of unsaturated phosphatidylethanolamine, while the opposite effect was observed in the presence of phosphatidic acid. These results support the hypothesis that a conversion of lysophosphatidic acid into phosphatidic acid by endophilin or BARS (50 kDa brefeldin A ribosylated substrate) may induce negative spontaneous monolayer curvature and regulate endocytic and Golgi membrane fission. Alternative models for the regulation of membrane fission based on the strong dependence of the molecular shape of (lyso)phosphatidic acid on pH and divalent cations are also discussed.

Secretory traffic triggers the formation of tubular continuities across Golgi sub-compartments

The organization of secretory traffic remains unclear, mainly because of the complex structure and dynamics of the secretory pathway. We have thus studied a simplified system, a single synchronized traffic wave crossing an individual Golgi stack, using electron tomography. Endoplasmic-reticulum-to-Golgi carriers join the stack by fusing with cis cisternae and induce the formation of intercisternal tubules, through which they redistribute their contents throughout the stack. These tubules seem to be pervious to Golgi enzymes, whereas Golgi vesicles are depleted of both enzymes and cargo. Cargo then traverses the stack without leaving the cisternal lumen. When cargo exits the stack, intercisternal connections disappear. These findings provide a new view of secretory traffic that includes dynamic intercompartment continuities as key players.

ER-to-Golgi Carriers Arise through Direct En Bloc Protrusion and Multistage Maturation of Specialized ER Exit Domains

Protein transport between the ER and the Golgi in mammalian cells occurs via large pleiomorphic carriers, and most current models suggest that these are formed by the fusion of small ER-derived COPII vesicles. We have examined the dynamics and structural features of these carriers during and after their formation from the ER by correlative video/light electron microscopy and tomography. We found that saccular carriers containing either the large supramolecular cargo procollagen or the small diffusible cargo protein VSVG arise through cargo concentration and direct en bloc protrusion of specialized ER domains in the vicinity of COPII-coated exit sites. This formation process is COPII dependent but does not involve budding and fusion of COPII-dependent vesicles. Fully protruded saccules then move centripetally, evolving into one of two types of carriers (with distinct kinetic and structural features). These findings provide an alternative framework for analysis of ER-to-Golgi traffic.

Small cargo proteins and large aggregates can traverse the Golgi by a common mechanism without leaving the lumen of cisternae

Procollagen (PC)-I aggregates transit through the Golgi complex without leaving the lumen of Golgi cisternae. Based on this evidence, we have proposed that PC-I is transported across the Golgi stacks by the cisternal maturation process. However, most secretory cargoes are small, freely diffusing proteins, thus raising the issue whether they move by a transport mechanism different than that used by PC-I. To address this question we have developed procedures to compare the transport of a small protein, the G protein of the vesicular stomatitis virus (VSVG), with that of the much larger PC-I aggregates in the same cell. Transport was followed using a combination of video and EM, providing high resolution in time and space. Our results reveal that PC-I aggregates and VSVG move synchronously through the Golgi at indistinguishable rapid rates. Additionally, not only PC-I aggregates (as confirmed by ultrarapid cryofixation), but also VSVG, can traverse the stack without leaving the cisternal lumen and without entering Golgi vesicles in functionally relevant amounts. Our findings indicate that a common mechanism independent of anterograde dissociative carriers is responsible for the traffic of small and large secretory cargo across the Golgi stack.

TIP47 functions in the biogenesis of lipid droplets

TIP47 (tail-interacting protein of 47 kD) was characterized as a cargo selection device for mannose 6-phosphate receptors (MPRs), directing their transport from endosomes to the trans-Golgi network. In contrast, our current analysis shows that cytosolic TIP47 is not recruited to organelles of the biosynthetic and endocytic pathways. Knockdown of TIP47 expression had no effect on MPR distribution or trafficking and did not affect lysosomal enzyme sorting. Therefore, our data argue against a function of TIP47 as a sorting device. Instead, TIP47 is recruited to lipid droplets (LDs) by an amino-terminal sequence comprising 11-mer repeats. We show that TIP47 has apolipoprotein-like properties and reorganizes liposomes into small lipid discs. Suppression of TIP47 blocked LD maturation and decreased the incorporation of triacylglycerol into LDs. We conclude that TIP47 functions in the biogenesis of LDs.

Quantitative label-free imaging of lipid composition and packing of individual cellular lipid droplets using multiplex CARS microscopy

Lipid droplets (LDs) are highly dynamic organelles that perform multiple functions, including the regulated storage and release of cholesterol and fatty acids. Information on the molecular composition of individual LDs within their cellular context is crucial in understanding the diverse biological functions of LDs, as well as their involvement in the development of metabolic disorders such as obesity, type II diabetes, and atherosclerosis. Although ensembles of LDs isolated from cells and tissues were analyzed in great detail, quantitative information on the heterogeneity in lipid composition of individual droplets, and possible variations within single lipid droplets, is lacking. Therefore, we used a label-free quantitative method to image lipids within LDs in 3T3-L1 cells. The method combines submicron spatial resolution in three dimensions, using label-free coherent anti-Stokes Raman scattering microscopy, with quantitative analysis based on the maximum entropy method. Our method allows quantitative imaging of the chemistry (level of acyl unsaturation) and physical state (acyl chain order) of individual LDs. Our results reveal variations in lipid composition and physical state between LDs contained in the same cell, and even within a single LD.

An electrostatic/hydrogen bond switch as the basis for the specific interaction of phosphatidic acid with proteins

Phosphatidic acid (PA) is a minor but important phospholipid that, through specific interactions with proteins, plays a central role in several key cellular processes. The simple yet unique structure of PA, carrying just a phosphomonoester head group, suggests an important role for interactions with the positively charged essential residues in these proteins. We analyzed by solid-state magic angle spinning 31P NMR and molecular dynamics simulations the interaction of low concentrations of PA in model membranes with positively charged side chains of membrane-interacting peptides. Surprisingly, lysine and arginine residues increase the charge of PA, predominantly by forming hydrogen bonds with the phosphate of PA, thereby stabilizing the protein-lipid interaction. Our results demonstrate that this electrostatic/hydrogen bond switch turns the phosphate of PA into an effective and preferred docking site for lysine and arginine residues. In combination with the special packing properties of PA, PA may well be natures preferred membrane lipid for interfacial insertion of positively charged membrane protein domains.

Nanocapsules: lipid-coated aggregates of cisplatin with high cytotoxicity

Cisplatin is one of the most widely used agents in the treatment of solid tumors, but its clinical utility is limited by toxicity. The development of less toxic, liposomal formulations of cisplatin has been hampered by the low water solubility and low lipophilicity of cisplatin, resulting in very low encapsulation efficiencies. We describe a novel method allowing the efficient encapsulation of cisplatin in a lipid formulation; it is based on repeated freezing and thawing of a concentrated solution of cisplatin in the presence of negatively charged phospholipids. The method is unique in that it generates nanocapsules, which are small aggregates of cisplatin covered by a single lipid bilayer. The nanocapsules have an unprecedented drug-to-lipid ratio and an in vitro cytotoxicity up to 1000-fold higher than the free drug. Analysis of the mechanism of nanocapsule formation suggests that the method may be generalized to other drugs showing low water solubility and lipophilicity.

Greasing Membrane Fusion and Fission Machineries

Biological membrane fusion is a local‐point event, extremely fast, and under strict control. Proteins are responsible for the mutual recognition of the fusion partners and for the initiation of biomembrane fusion, and thus determine where and when fusion occurs. However, the central event during membrane fusion is the merger of two membranes, which requires a transient reorganization of membrane lipids into highly curved fusion intermediates. This review focuses on the potential role of lipids in the generation of membrane curvature, and thus in the regulation of membrane fusion and fission.