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Curvature-generating proteins that direct membrane trafficking assemble on the surface of lipid bilayers to bud transport intermediates, which move protein and lipid cargoes from one cellular compartment to another. However, it remains unclear what controls the overall shape of the membrane bud once curvature induction has begun. In vitro experiments showed that excessive concentrations of the COPII protein Sar1 promoted the formation of membrane tubules from synthetic vesicles, while COPII-coated transport intermediates in cells are generally more spherical or lobed in shape. To understand the origin of these morphological differences, we employ atomistic, coarse-grained (CG), and continuum mesoscopic simulations of membranes in the presence of multiple curvature-generating proteins. We first characterize the membrane-bending ability of amphipathic peptides derived from the amino terminus of Sar1, as a function of interpeptide angle and concentration using an atomistic bicelle simulation protocol. Then, we employ CG simulations to reveal that Sec23 and Sec24 control the relative spacing between Sar1 protomers and form the inner-coat unit through an attachment with Sar1. Finally, using dynamical triangulated surface simulations based on the Helfrich Hamiltonian, we demonstrate that the uniform distribution of spacer molecules among curvature-generating proteins is crucial to the spherical budding of the membrane. Overall, our analyses suggest a new role for Sec23, Sec24, and cargo proteins in COPII-mediated membrane budding process in which they act as spacers to preserve a dispersed arrangement of Sar1 protomers and help determine the overall shape of the membrane bud.

 

Synaptotagmin (syt) 1, a Ca²⁺sensor for synaptic vesicle exocytosis, functions in vivo as a multimer. Syt1 senses Ca²⁺via tandem C2-domains that are connected to a single transmembrane domain via a juxtamembrane linker. Here, we show that this linker segment harbors a lysine-rich, intrinsically disordered region that is necessary and sufficient to mediate liquid-liquid phase separation (LLPS). Interestingly, condensate formation negatively regulates the Ca²⁺-sensitivity of syt1. Moreover, Ca²⁺and anionic phospholipids facilitate the observed phase separation, and increases in [Ca²⁺]_ipromote the fusion of syt1 droplets in living cells. Together, these observations suggest a condensate-mediated feedback loop that serves to fine-tune the ability of syt1 to trigger release, via alterations in Ca²⁺binding activity and potentially through the impact of LLPS on membrane curvature during fusion reactions. In summary, the juxtamembrane linker of syt1 emerges as a regulator of syt1 function by driving self-association via LLPS.

 

Biomolecular phase separation has emerged as an essential mechanism for cellular organization. How cells respond to environmental stimuli in a robust and sensitive manner to build functional condensates at the proper time and location is only starting to be understood. Recently, lipid membranes have been recognized as an important regulatory center for biomolecular condensation. However, how the interplay between the phase behaviors of cellular membranes and surface biopolymers may contribute to the regulation of surface condensation remains to be elucidated. Using simulations and a mean-field theoretical model, we show that two key factors are the membrane’s tendency to phase-separate and the surface polymer’s ability to reorganize local membrane composition. Surface condensate forms with high sensitivity and selectivity in response to features of biopolymer when positive co-operativity is established between coupled growth of the condensate and local lipid domains. This effect relating the degree of membrane–surface polymer co-operativity and condensate property regulation is shown to be robust by different ways of tuning the co-operativity, such as varying membrane protein obstacle concentration, lipid composition, and the affinity between lipid and polymer. The general physical principle emerged from the current analysis may have implications in other biological processes and beyond. 

Development of effective strategies for the internalization of nanoparticles is essential in many applications, such as drug delivery. Most, if not all, previous studies are based on equilibrium considerations. In this work, inspired by the recent development of a pro-drug delivery strategy based on reversible esterification, we consider a non-equilibrium transport mechanism for nanoparticles of a 6 nm diameter across the lipid membrane. We divide the transport process into insertion and ejection steps, which are studied with coarse-grained models using free energy and reactive Monte Carlo simulations, respectively. The simulations show that the non-equilibrium transport efficiency is relatively insensitive to the fraction of reactive surface ligands once a modest threshold is surpassed, while the distribution pattern of different (hydrophilic, reactive and permanent hydrophobic) ligands on the nanoparticle surface has a notable impact on both the insertion and ejection steps. Our study thus supports a novel avenue for designing nanoparticles that are able to be efficiently internalized and provides a set of relevant guidelines for surface functionalization. 

Many intrinsically disordered peptides have been shown to undergo liquid–liquid phase separation and form complex coacervates, which play various regulatory roles in the cell. Recent experimental studies found that such phase separation processes may also occur at the lipid membrane surface and help organize biomolecules during signaling events; in some cases, phase separation of proteins at the membrane surface was also observed to lead to significant remodeling of the membrane morphology. The molecular mechanisms that govern the interactions between complex coacervates and lipid membranes and the impacts of such interactions on their structure and morphology, however, remain unclear. Here we study the coacervation of poly-glutamate (E 30 ) and poly-lysine (K 30 ) in the presence of lipid bilayers of different compositions. We carry out explicit-solvent coarse-grained molecular dynamics simulations by using the MARTINI (v3.0) force-field. We find that more than 20% anionic lipids are required for the coacervate to form stable contact with the bilayer. Upon wetting, the coacervate induces negative curvature to the bilayer and facilitates local lipid demixing, without any peptide insertion. The magnitude of negative curvature, extent of lipid demixing, and asphericity of the coacervate increase with the concentration of anionic lipids. Overall, we observe a decrease in the number of contacts among the polyelectrolytes as the droplet spreads over the bilayer. Therefore, unlike previous suggestions, interactions among polyelectrolytes do not constitute a driving force for the membrane bending upon wetting by the coacervate. Rather, analysis of interaction energy components suggests that bending of the membrane is favored by enhanced interactions between polyelectrolytes with lipids as well as with counterions. Kinetic studies reveal that, at the studied polyelectrolyte concentrations, the coacervate formation precedes bilayer wetting. 

Carbon dots (CDs) are emerging as the material of choice in a range of applications due to their excellent photoluminescence properties, ease of preparation from inexpensive precursors, and low toxicity. However, the precise nature of the mechanism for the fluorescence is still under debate, and several molecular fluorophores have been reported. In this work, a new blue fluorophore, 5-oxopyrrolidine-3-carboxylic acid, was discovered in carbon dots synthesized from the most commonly used precursors: citric acid and urea. The molecular product alone has demonstrated interesting aggregation-enhanced emission (AEE), making it unique compared to other fluorophores known to be generated in CDs. We propose that this molecular fluorophore is associated with a polymer backbone within the CDs, and its fluorescence behavior is largely dependent on intermolecular interactions with the polymers or other fluorophores. Thus, a new class of non-traditional fluorophores is now relevant to the consideration of the CD fluorescence mechanism, providing both an additional challenge to the community in resolving the mechanism and an opportunity for a greater range of CD design schemes and applications.