Lipid phase separation in vesicles enhances TRAIL-mediated cytotoxicity

Ligand spatial presentation and density play important roles in many signaling pathways mediated by cell receptors and are critical parameters when designing protein-conjugated therapeutic nanoparticles. Currently, Janus particles are most often used to spatially control ligand conjugation, but the technological challenge of manufacturing Janus particles limits adoption for translational applications. Here, we demonstrate that lipid phase separation can be used to spatially control protein presentation onto lipid vesicles. We used this system to study the density dependence of TNF-related apoptosis inducing ligand (TRAIL), a model therapeutic protein that exhibits greater cytotoxicity to cancer cells when conjugated onto a vesicle surface than when administered as a soluble protein. Using assays for apoptosis and caspase activity, we show that phase separated TRAIL vesicles induced higher cytotoxicity to Jurkat cancer cells than uniformly-conjugated TRAIL vesicles, and enhanced cytotoxicity was dependent on the TRAIL domain density. We then assessed this relationship in other cancer cell lines and demonstrated that phase separated TRAIL vesicles only enhanced cytotoxicity through one TRAIL receptor, DR5, while another TRAIL receptor, DR4, was unaffected by the TRAIL density. These results indicate unique signaling requirements for each TRAIL receptor and how TRAIL therapy could be tailored depending on the relative levels of expression for cancer receptors of interest. Overall, this work demonstrates a readily adoptable method to control protein conjugation and density on bilayer vesicles that can be easily adopted to other therapeutic nanoparticle systems to improve receptor signaling of nanoparticles targeted to cancer and diseased cells.

Interactions of Lipid Droplets with the Intracellular Transport Machinery

Historically, studies of intracellular membrane trafficking have focused on the secretory and endocytic pathways and their major organelles. However, these pathways are also directly implicated in the biogenesis and function of other important intracellular organelles, the best studied of which are peroxisomes and lipid droplets. There is a large recent body of work on these organelles, which have resulted in the introduction of new paradigms regarding the roles of membrane trafficking organelles. In this review, we discuss the roles of membrane trafficking in the life cycle of lipid droplets. This includes the complementary roles of lipid phase separation and proteins in the biogenesis of lipid droplets from endoplasmic reticulum (ER) membranes, and the attachment of mature lipid droplets to membranes by lipidic bridges and by more conventional protein tethers. We also discuss the catabolism of neutral lipids, which in part results from the interaction of lipid droplets with cytosolic molecules, but with important roles for both macroautophagy and microautophagy. Finally, we address their eventual demise, which involves interactions with the autophagocytotic machinery. We pay particular attention to the roles of small GTPases, particularly Rab18, in these processes.

Nanoscale membrane curvature sorts lipid phases and alters lipid diffusion

ABSTRACTCellular homeostasis requires the precise spatial and temporal control of membrane shape and composition. Membrane regions of high curvature, such as endocytic pits and viral buds, contain distinct lipids and proteins. However, the interplay between membrane curvature and local membrane composition is poorly understood at the nanoscale. Here, we employed single-molecule localization microscopy to observe single-lipid diffusion in model bilayers with varying lipid compositions, phase, temperature, and membrane curvature. Engineered membrane buds were observed for the creation of lateral compositional heterogeneity in otherwise homogeneous membranes. Membrane curvature recruited liquid-disordered lipid phases in phase-separated membranes and altered the diffusion of the lipids. Supported lipid bilayers were created over 50-nm radius nanoparticles to engineer nanoscale membrane curvature that mimics the size of naturally occurring endocytic pits and viral buds. The disorder-preferring lipids sorted to the nanoscale curvature at all temperatures, but only when embedded in a membrane capable of sustaining liquid-liquid phase separation at low temperatures. This result suggests that lipid sorting by the membrane curvature was only possible when coupled with lipid phase separation. The curvature affected the local membrane composition most strongly when the curvature was locally surrounded by a liquid-ordered phase typically associated with a stiffer bending modulus. The curvature-induced sorting of lipid phases was quantified by the sorting of disorder-preferring fluorescent lipids, single-lipid diffusion measurements, and simulations that couple the lipid phase separation to the membrane shape. Unlike single-component membranes, lipids in phase-separated membranes demonstrated faster diffusion on curved membranes than the surrounding, flat membrane. These results demonstrate that curvature-induced membrane compositional heterogeneity can be achieved by collective behavior with lipid phase separation when single-molecule properties (i.e., packing parameter) are insufficient. These results support the hypothesis that the coupling of lipid phases and membrane shape may yield lateral membrane composition heterogeneities with functional consequences.STATEMENT OF SIGNIFICANCENanoscopic membrane organization and dynamics are critical for cellular function but challenging to experimentally measure. This work brings together super-resolution optical methods with engineered substrates to reveal the interplay between curvature, composition, phase, and diffusion in model membranes. We report that curvature can induce phase separation in otherwise homogeneous membranes and that the phase-curvature coupling has a direct implication on lipid mobility. In sum, this discovery advances our understanding of the fundamental membrane biophysics that regulate membrane activities such as endocytosis and viral budding.

Coupling between lipid miscibility and phosphotyrosine driven protein condensation at the membrane

Lipid miscibility phase separation has long been considered to be a central element of cell membrane organization. More recently, protein condensation phase transitions, into three-dimensional droplets or in two-dimensional lattices on membrane surfaces, have emerged as another important organizational principle within cells. Here, we reconstitute the LAT:Grb2:SOS protein condensation on the surface of giant unilamellar vesicles capable of undergoing lipid phase separations. Our results indicate that assembly of the protein condensate on the membrane surface can drive lipid phase separation. This phase transition occurs isothermally and is governed by tyrosine phosphorylation on LAT. Furthermore, we observe that the induced lipid phase separation drives localization of the SOS substrate, K-Ras, into the LAT:Grb2:SOS protein condensate.Statement of SignificanceProtein condensation phase transitions are emerging as an important organizing principles in cells. One such condensate plays a key role in T cell receptor signaling. Immediately after receptor activation, multivalent phosphorylation of the adaptor protein LAT at the plasma membrane leads to networked assembly of a number of signaling proteins into a two-dimensional condensate on the membrane surface. In this study, we demonstrate that LAT condensates in reconstituted vesicles are sufficient to drive lipid phase separation. This lipid reorganization drives another key downstream signaling molecule, Ras, into the LAT condensates. These results show that the LAT condensation phase transition, which is actively controlled by phosphorylation reactions, extends its influence to control lipid phase separation in the underlying membrane.

Low membrane fluidity triggers lipid phase separation and protein segregation in vivo

SummaryImportant physicochemical properties of cell membranes such as fluidity sensitively depend on fluctuating environmental factors including temperature, pH or diet. To counteract these disturbances, living cells universally adapt their lipid composition in return. In contrast to eukaryotic cells, bacteria tolerate surprisingly drastic changes in their lipid composition while retaining viability, thus making them a more tractable model to study this process. Using the model organisms Escherichia coli and Bacillus subtilis, which regulate their membrane fluidity with different fatty acid types, we show here that inadequate membrane fluidity interferes with essential cellular processes such as morphogenesis and maintenance of membrane potential, and triggers large-scale lipid phase separation that drives protein segregation into the fluid phase. These findings illustrate why lipid homeostasis is such a critical cellular process. Finally, our results provide direct in vivo support for current in vitro and in silico models regarding lipid phase separation and associated protein segregation.