ABSTRACT
In this chapter, we aim to bridge basic molecular and cellular principles surrounding membrane curvature generation with rewiring of cellular signals in cancer through multiscale models. We describe a general framework that integrates signaling with other cellular functions like trafficking, cell-cell and cell-matrix adhesion, and motility. The guiding question in our approach is: how does a physical change in cell membrane configuration caused by external stimuli (including those by the extracellular microenvironment) alter trafficking, signaling and subsequent cell fate? We answer this question by constructing a modeling framework based on stochastic spatial continuum models of cell membrane deformations. We apply this framework to explore the link between trafficking, signaling in the tumor microenvironment, and cell fate. At each stage, we aim to connect the results of our predictions with cellular experiments.
Subject(s)
Cell Membrane , Models, Biological , Neoplasms , Tumor Microenvironment , Humans , Cell Membrane/metabolism , Neoplasms/pathology , Neoplasms/metabolism , Tumor Microenvironment/physiology , Signal Transduction , Thermodynamics , AnimalsABSTRACT
Biomolecular condensates play a major role in numerous cellular processes, including several that occur on the surface of lipid bilayer membranes. There is increasing evidence that cellular membrane trafficking phenomena, including the internalization of the plasma membrane through endocytosis, are mediated by multivalent protein-protein interactions that can lead to phase separation. We have recently found that proteins involved in the clathrin-independent endocytic pathway named Fast Endophilin Mediated Endocytosis can undergo liquid-liquid phase separation (LLPS) in solution and on lipid bilayer membranes. Here, the protein solution concentrations required for phase separation to be observed are significantly smaller compared to those required for phase separation in solution. LLPS is challenging to systematically characterize in cellular systems in general, and on biological membranes in particular. Model membrane approaches are more suitable for this purpose as they allow for precise control over the nature and amount of the components present in a mixture. Here we describe a method that enables the imaging of LLPS domain formation on solid supported lipid bilayers. These allow for facile imaging, provide long-term stability, and avoid clustering of vesicles and vesicle-attached features (such as buds and tethers) in the presence of multi-valent membrane interacting proteins.
Subject(s)
Lipid Bilayers , Lipid Bilayers/chemistry , Lipid Bilayers/metabolism , Biomolecular Condensates/chemistry , Biomolecular Condensates/metabolism , Acyltransferases/metabolism , Acyltransferases/chemistry , Optical Imaging/methods , Cell Membrane/metabolism , Cell Membrane/chemistry , Endocytosis , Humans , Membrane Proteins/chemistry , Membrane Proteins/metabolismABSTRACT
A definitive understanding of the interplay between protein binding/migration and membrane curvature evolution is emerging but needs further study. The mechanisms defining such phenomena are critical to intracellular transport and trafficking of proteins. Among trafficking modalities, exosomes have drawn attention in cancer research as these nano-sized naturally occurring vehicles are implicated in intercellular communication in the tumor microenvironment, suppressing anti-tumor immunity and preparing the metastatic niche for progression. A significant question in the field is how the release and composition of tumor exosomes are regulated. In this perspective article, we explore how physical factors such as geometry and tissue mechanics regulate cell cortical tension to influence exosome production by co-opting the biophysics as well as the signaling dynamics of intracellular trafficking pathways and how these exosomes contribute to the suppression of anti-tumor immunity and promote metastasis. We describe a multiscale modeling approach whose impact goes beyond the fundamental investigation of specific cellular processes toward actual clinical translation. Exosomal mechanisms are critical to developing and approving liquid biopsy technologies, poised to transform future non-invasive, longitudinal profiling of evolving tumors and resistance to cancer therapies to bring us one step closer to the promise of personalized medicine.
ABSTRACT
Lipid asymmetry - that is, a nonuniform lipid distribution between the leaflets of a bilayer - is a ubiquitous feature of biomembranes and is implicated in several cellular phenomena. Differential tension - that is, unequal lateral monolayer tensions comparing the leaflets of a bilayer- is closely associated with lipid asymmetry underlying these varied roles. Because differential tension is not directly measurable in combination with the fact that common methods to adjust this quantity grant only semi-quantitative control over it, a detailed understanding of lipid asymmetry and differential tension are impeded. To overcome these challenges, we leveraged reversible complexation of phospholipid by methyl-ß-cyclodextrin (mbCD) to tune the direction and magnitude of lipid asymmetry in synthetic vesicles. Lipid asymmetry generated in our study induced (i) vesicle shape changes and (ii) gel-liquid phase coexistence in 1-component vesicles. By applying mass-action considerations to interpret our findings, we discuss how this approach provides access to phospholipid thermodynamic potentials in bilayers containing lipid asymmetry (which are coupled to the differential tension of a bilayer). Because lipid asymmetry yielded by our approach is (i) tunable and (ii) maintained over minute to hour timescales, we anticipate that this approach will be a valuable addition to the experimental toolbox for systematic investigation into the biophysical role(s) of lipid asymmetry (and differential tension).
Subject(s)
Lipid Bilayers , Phospholipids , beta-Cyclodextrins , Lipid Bilayers/chemistry , Lipid Bilayers/metabolism , beta-Cyclodextrins/chemistry , Phospholipids/chemistry , ThermodynamicsABSTRACT
Bin/Amphiphysin/Rvs (BAR) proteins are known as classical membrane curvature generators during endocytosis. Amphiphysin, a member of the N-BAR sub-family of proteins that contain a characteristic amphipathic sequence at the N-terminus of the BAR domain, is involved in clathrin-mediated endocytosis. Full-length amphiphysin contains a ~ 400 amino acid long disordered linker connecting the N-BAR domain and a C-terminal Src homology 3 (SH3) domain. We express and purify recombinant amphiphysin and its N-BAR domain along with an N-terminal glutathione-S-transferase (GST) tag. The GST tag allows extraction of the protein of interest using affinity chromatography and is removed in the subsequent protease treatment and ion-exchange chromatography steps. In the case of the N-BAR domain, cleavage of the GST tag was found to cause precipitation. This issue can be minimized by adding glycerol to the protein purification buffers. In the final step, size exclusion chromatography removes any potential oligomeric species. This protocol has also been successfully used to purify other N-BAR proteins, such as endophilin, Bin1, and their corresponding BAR domains. Graphical overview.
ABSTRACT
Proteins can organize into dynamic, functionally important assemblies on fluid membrane surfaces. Phase separation has emerged as an important mechanism for forming such protein assemblies on the membrane during cell signaling, endocytosis, and cytoskeleton regulation. Protein-protein phase separation thus adds novel fluid mosaics to the classical Singer and Nicolson model. Protein condensates formed in this process can modulate membrane morphologies. This is evident from recent reports of protein condensate-driven membrane reshaping in processes such as endocytosis, autophagosome formation, and protein storage vacuole morphogenesis in plants. Lateral phase separation (on the membrane surface) of peripheral curvature coupling proteins can modulate such membrane morphological transitions. Additionally, three-dimensional protein phase separation can result in droplets that through adhesion can affect membrane shape changes. How do these condensate-driven curvature generation mechanisms contrast with the classically recognized scaffolding and amphipathic helix insertion activities of specific membrane remodeling proteins? A salient feature of these condensate-driven membrane activities is that they depend upon both macroscopic features (such as interfacial energies of the condensate, membrane, and cytosol) as well as microscopic, molecular-level interactions (such as protein-lipid binding). This review highlights the current understanding of the mechanisms underlying curvature generation by protein condensates in various biological pathways.
Subject(s)
Membrane Proteins , Membrane Proteins/metabolism , Membranes/metabolismABSTRACT
A specific group of transmembrane receptors, including the ß1-adrenergic receptor (ß1-AR), is internalized through a non-clathrin pathway known as Fast Endophilin Mediated Endocytosis (FEME). A key question is: how does the endocytic machinery assemble and how is it modulated by activated receptors during FEME. Here we show that endophilin, a major regulator of FEME, undergoes a phase transition into liquid-like condensates, which facilitates the formation of multi-protein assemblies by enabling the phase partitioning of endophilin binding proteins. The phase transition can be triggered by specific multivalent binding partners of endophilin in the FEME pathway such as the third intracellular loop (TIL) of the ß1-AR, and the C-terminal domain of lamellipodin (LPD). Other endocytic accessory proteins can either partition into, or target interfacial regions of, these condensate droplets, and LPD also phase separates with the actin polymerase VASP. On the membrane, TIL promotes protein clustering in the presence of endophilin and LPD C-terminal domain. Our results demonstrate how the multivalent interactions between endophilin, LPD, and TIL regulate protein assembly formation on the membrane, providing mechanistic insights into the priming and initiation steps of FEME.
Subject(s)
Actins , Endocytosis , Carrier Proteins , Signal TransductionABSTRACT
BAR (Bin/Amphiphysin/Rvs) domain containing proteins function as lipid bilayer benders and curvature sensors, and they contribute to membrane shaping involved in cell signaling and metabolism. The mechanism for their membrane shape sensing has been investigated by both equilibrium binding and kinetic studies. In prior research, stopped-flow spectroscopy has been used to deduce a positive dependence on membrane curvature for the binding rate constant, kon, of a BAR protein called endophilin. However, the impact of bulk diffusion of endophilin, on the kinetic binding parameters has not been thoroughly considered. Employing similar methods, and using lipid vesicles of multiple sizes, we obtained a linear dependence of kon on vesicle curvature. However, we found that the observed relation can be explained without considering the local curvature sensing ability of endophilin in the membrane association process. In contrast, the diffusion-independent unbinding rate constant (koff) obtained from stopped-flow measurements shows a negative dependence on membrane curvature, which is controlled/mediated by endophilin-membrane interactions. This latter dependency, in addition to protein-protein interactions on the membrane, explains the selective binding of BAR proteins to highly curved membranes in equilibrium binding experiments.
Subject(s)
Acyltransferases , Lipid Bilayers , Acyltransferases/metabolism , Cell Membrane/metabolism , Kinetics , Lipid Bilayers/chemistry , Membranes/metabolismABSTRACT
Lipid bilayer membranes undergo rapid bending undulations with wavelengths from tens of nanometers to tens of microns due to thermal fluctuations. Here, we probe such undulations and the membranes' mechanics by measuring the time-varying orientation of single gold nanorods (GNRs) adhered to the membrane, using high-speed dark field microscopy. In a lipid vesicle, such measurements allow the determination of the membrane's viscosity, bending rigidity, and tension as well as the friction coefficient for sliding of the monolayers over one another. The in-plane rotation of the GNR is hindered by undulations in a tension dependent manner, consistent with simulations. The motion of single GNRs adhered to the plasma membrane of living cultured cells similarly reveals the membrane's complex physics and coupling to the cell's actomyosin cortex.
ABSTRACT
Endophilin, which is a member of the Bin-amphiphysin-Rvs (BAR) domain protein superfamily, contains a homodimeric N-BAR domain of a characteristic crescent shape. The N-BAR domain comprises a six-helix bundle and is known to sense and generate membrane curvature. Here, we characterize aspects of the unfolding mechanism of the endophilin A1 N-BAR domain during thermal denaturation and examine factors that influence the thermal stability of this domain. Far-UV circular dichroism (CD) spectroscopy was applied to monitor changes in the secondary structure above room temperature. The protein's conformational changes were further characterized through Foerster resonance energy transfer and cross-linking experiments at varying temperatures. Our results indicate that thermal unfolding of the endophilin N-BAR is (minimally) a two-step process, with a dimeric intermediate that displays partial helicity loss. Furthermore, a thermal shift assay and temperature-dependent CD were applied to compare the unfolding processes of several truncated versions of endophilin. The melting temperature of the N-BAR domain decreased when we deleted either the N-terminal H0 helix or the unstructured linker of endophilin. This result suggests that these intrinsically disordered domains may play a role in structurally stabilizing the functional N-BAR domain in vivo. Finally, we show that single-site mutations can also compromise endophilin's thermal stability.
ABSTRACT
Lipid oxidation, detected in metabolic processes, is induced in excess when the cellular membrane suffers extra oxidative stress. Lipid oxidation can compromise biomembrane function in part through perturbations of lipid packing, membrane permeability, and morphology. Two major types of oxidation products, one with a partially truncated lipid tail with a hydrophilic group at the tail-end, and secondly, a lysolipid (with one of the chains completely truncated) can disturb the membrane bilayer packing significantly. However, they also have an increased tendency to desorb from the membrane. In this study we investigated desorption kinetics of two characteristic lipid oxidation products (PAzePC and 18 : 1 LysoPC) from a model membrane system, and we evaluated the consequences of this process on membrane shape transitions. Using a microfluidic chamber coupled with micropipette aspiration, we observed the incorporation of the two lipids into the membrane of a giant unilamellar vesicle (GUV) and further determined their desorption rates, association rates and flip-flop rates. For both lipids, the desorption is on the time scale of seconds, one to two orders of magnitude faster than their flipping rates. Dilution of the outer solution of the GUVs allowed asymmetric desorption of these two lipids from the GUVs. This process induced lipid number asymmetry and charge asymmetry, specifically for PAzePC containing GUVs, and caused membrane tubulation. Our results indicate that the desorption of lipid oxidation products can alter the local structure of biomembranes and result in morphological changes that may relate to membrane function.
Subject(s)
Lipid Bilayers , Unilamellar Liposomes , Cell Membrane/metabolism , Kinetics , Lipid Bilayers/metabolism , Oxidation-Reduction , Unilamellar Liposomes/metabolismABSTRACT
Liquid-liquid phase separation has recently emerged as an important fundamental organizational phenomenon in biological settings. Most studies of biological phase separation have focused on droplets that "condense" from solution above a critical concentration, forming so-called "membraneless organelles" suspended in solution. However, membranes are ubiquitous throughout cells, and many biomolecular condensates interact with membrane surfaces. Such membrane-associated phase-separated systems range from clusters of integral or peripheral membrane proteins in the plane of the membrane to free, spherical droplets wetting membrane surfaces to droplets containing small lipid vesicles. In this review, we consider phase-separated liquids that interact with membrane surfaces and we discuss the consequences of those interactions. The physical properties of distinct liquid phases in contact with bilayers can reshape the membrane, and liquid-liquid phase separation can construct membrane-associated protein structures, modulate their function, and organize collections of lipid vesicles dynamically. We summarize the common phenomena that arise in these systems of liquid phases and membranes.
ABSTRACT
Endophilin is an N-BAR protein, which is characterized by a crescent-shaped BAR domain and an amphipathic helix that contributes to the membrane binding of these proteins. The exact function of that H0 helix has been a topic of debate. In mammals, there are five different endophilin isoforms, grouped into A (three members) and B (two members) subclasses, which have been described to differ in their subcellular localization and function. We asked to what extent molecular properties of the H0 helices of these members affect their membrane targeting behavior. We found that all H0 helices of the endophilin isoforms display a two-state equilibrium between disordered and α-helical states in which the helical secondary structure can be stabilized through trifluoroethanol. The helicities in high TFE were strikingly different among the H0 peptides. We investigated H0-membrane partitioning by the monitoring of secondary structure changes via CD spectroscopy. We found that the presence of anionic phospholipids is critical for all H0 helices partitioning into membranes. Membrane partitioning is found to be sensitive to variations in membrane complexity. Overall, the H0 B subfamily displays stronger membrane partitioning than the H0 A subfamily. The H0 A peptide-membrane binding occurs predominantly through electrostatic interactions. Variation among the H0 A subfamily may be attributed to slight alterations in the amino acid sequence. Meanwhile, the H0 B subfamily displays greater specificity for certain membrane compositions, and this may link H0 B peptide binding to endophilin B's cellular function.
Subject(s)
Acyltransferases/metabolism , Isoenzymes/metabolism , Acyltransferases/chemistry , Amino Acid Sequence , Cell Membrane/metabolism , Circular Dichroism , Hydrophobic and Hydrophilic Interactions , Isoenzymes/chemistry , Protein Structure, SecondaryABSTRACT
The eukaryotic plasma membrane's lipid composition is found to be ubiquitously asymmetric comparing inner and outer leaflets. This membrane lipid asymmetry plays a crucial role in diverse cellular processes critical for cell survival. A specialized set of transmembrane proteins called translocases, or flippases, have evolved to maintain this membrane lipid asymmetry in an energy-dependent manner. One potential consequence of local variations in membrane lipid asymmetry is membrane remodeling, which is essential for cellular processes such as intracellular trafficking. Recently, there has been a surge in the identification and characterization of flippases, which has significantly advanced the understanding of their functional mechanisms. Furthermore, there are intriguing possibilities for a coupling between membrane curvature and flippase activity. In this review we highlight studies that link membrane shape and remodeling to differential stresses generated by the activity of lipid flippases with an emphasis on data obtained through model membrane systems. We review the common mechanistic models of flippase-mediated lipid flipping and discuss common techniques used to test lipid flippase activity. We then compare the existing data on lipid translocation rates by flippases and conclude with potential future directions for this field.
Subject(s)
Carrier Proteins/metabolism , Cell Membrane/metabolism , Lipid Bilayers/metabolism , Membrane Lipids/metabolism , Animals , Humans , Protein TransportABSTRACT
Endophilin plays key roles during endocytosis of cellular receptors, including generating membrane curvature to drive internalization. Electrostatic interactions between endophilin's BIN/Amphiphysin/Rvs domain and anionic membrane lipids have been considered the major driving force in curvature generation. However, the SH3 domain of endophilin also interacts with the proline-rich third intracellular loop (TIL) of various G-protein-coupled receptors (GPCRs), and it is unclear whether this interaction has a direct role in generating membrane curvature during endocytosis. To examine this, we designed model membranes with a membrane density of 1400 receptors per µm2 represented by a covalently conjugated TIL region from the ß1-adrenergic receptor. We observed that TIL recruits endophilin to membranes composed of 95 mol% of zwitterionic lipids via the SH3 domain. More importantly, endophilin recruited via TIL tubulates vesicles and gets sorted onto highly curved membrane tubules. These observations indicate that the cellular membrane bending and curvature sensing activities of endophilin can be facilitated through detection of the TIL of activated GPCRs in addition to binding to anionic lipids. Furthermore, we show that TIL electrostatically interacts with membranes composed of anionic lipids. Therefore, anionic lipids can modulate TIL/SH3 domain binding. Overall, our findings imply that an interplay between TIL, charged membrane lipids, BAR domain, and SH3 domain could exist in the biological system and that these components may act in coordination to regulate the internalization of cellular receptors.
Subject(s)
Acyltransferases/metabolism , Cell Membrane/metabolism , Endocytosis , Lipids/chemistry , Proline-Rich Protein Domains , Receptors, Adrenergic, beta/metabolism , Acyltransferases/chemistry , Acyltransferases/genetics , Humans , Protein Interaction Domains and Motifs , Protein Transport , Receptors, Adrenergic, beta/genetics , src Homology DomainsABSTRACT
Phosphatidylinositol-4,5-bisphosphate (PIP2) is an important signaling lipid in eukaryotic cell plasma membranes, playing an essential role in diverse cellular processes. The headgroup of PIP2 is highly negatively charged, and this lipid displays a high critical micellar concentration compared to housekeeping phospholipid analogs. Given the crucial role of PIP2, it is imperative to study its localization, interaction with proteins, and membrane-shaping properties. Biomimetic membranes have served extensively to elucidate structural and functional aspects of cell membranes including protein-lipid and lipid-lipid interactions, as well as membrane mechanics. Incorporation of PIP2 into biomimetic membranes, however, has at times resulted in discrepant findings described in the literature. With the goal to elucidate the mechanical consequences of PIP2 incorporation, we studied the desorption of PIP2 from biomimetic giant unilamellar vesicles by means of a fluorescent marker. A decrease in fluorescence intensity with the age of the vesicles suggested that PIP2 lipids were being desorbed from the outer leaflet of the membrane. To evaluate whether this desorption was asymmetric, the vesicles were systematically diluted. This resulted in an increase in the number of internally tubulated vesicles within minutes after dilution, suggesting that the desorption was asymmetric and also generated membrane curvature. By means of a saturated chain homolog of PIP2, we showed that the fast desorption of PIP2 is facilitated by presence of an arachidonic lipid tail and is possibly due to its oxidation. Through measurements of the pulling force of membrane tethers, we quantified the effect of this asymmetric desorption on the spontaneous membrane curvature. Furthermore, we found that the spontaneous curvature could be modulated by externally increasing the concentration of PIP2 micelles. Given that the local concentration of PIP2 in biological membranes is variable, spontaneous curvature generated by PIP2 may affect the formation of highly curved structures that can serve as initiators for signaling events.
Subject(s)
Cell Membrane/metabolism , Phosphatidylinositol 4,5-Diphosphate/metabolism , Animals , Humans , Micelles , SwineABSTRACT
Self-assembling dendrimers have facilitated the discovery of periodic and quasiperiodic arrays of supramolecular architectures and the diverse functions derived from them. Examples are liquid quasicrystals and their approximants plus helical columns and spheres, including some that disregard chirality. The same periodic and quasiperiodic arrays were subsequently found in block copolymers, surfactants, lipids, glycolipids, and other complex molecules. Here we report the discovery of lamellar and hexagonal periodic arrays on the surface of vesicles generated from sequence-defined bicomponent monodisperse oligomers containing lipid and glycolipid mimics. These vesicles, known as glycodendrimersomes, act as cell-membrane mimics with hierarchical morphologies resembling bicomponent rafts. These nanosegregated morphologies diminish sugar-sugar interactions enabling stronger binding to sugar-binding proteins than densely packed arrangements of sugars. Importantly, this provides a mechanism to encode the reactivity of sugars via their interaction with sugar-binding proteins. The observed sugar phase-separated hierarchical arrays with lamellar and hexagonal morphologies that encode biological recognition are among the most complex architectures yet discovered in soft matter. The enhanced reactivity of the sugar displays likely has applications in material science and nanomedicine, with potential to evolve into related technologies.
Subject(s)
Biomimetic Materials/chemistry , Cell Membrane/chemistry , Biomimetics/methods , Dendrimers/chemistry , Glycolipids/chemistry , Lipids/chemistry , Nanomedicine/methods , Sugars/chemistry , Surface-Active Agents/chemistryABSTRACT
Natural, including plant, and synthetic phenolic acids are employed as building blocks for the synthesis of constitutional isomeric libraries of self-assembling dendrons and dendrimers that are the simplest examples of programmed synthetic macromolecules. Amphiphilic Janus dendrimers are synthesized from a diversity of building blocks including natural phenolic acids. They self-assemble in water or buffer into vesicular dendrimersomes employed as biological membrane mimics, hybrid and synthetic cells. These dendrimersomes are predominantly uni- or multilamellar vesicles with size and polydispersity that is predicted by their primary structure. However, in numerous cases, unilamellar dendrimersomes completely free of multilamellar assemblies are desirable. Here, we report the synthesis and structural analysis of a library containing 13 amphiphilic Janus dendrimers containing linear and branched alkyl chains on their hydrophobic part. They were prepared by an optimized iterative modular synthesis starting from natural phenolic acids. Monodisperse dendrimersomes were prepared by injection and giant polydisperse by hydration. Both were structurally characterized to select the molecular design principles that provide unilamellar dendrimersomes in higher yields and shorter reaction times than under previously used reaction conditions. These dendrimersomes are expected to provide important tools for synthetic cell biology, encapsulation, and delivery.