It is now clear that membrane association of intrinsically disordered proteins or intrinsically disordered regions regulates many cellular processes, such as membrane targeting of Src family kinases and ion channel gating. Residue-specific characterization by nuclear magnetic resonance spectroscopy, molecular dynamics simulations, and other techniques has shown that polybasic motifs and amphipathic helices are the main drivers of membrane association; sequence-based prediction of residue-specific membrane association propensity has become possible. Membrane association facilitates protein-protein interactions and protein aggregation-these effects are due to reduced dimensionality but are similar to those afforded by condensate formation via liquid-liquid phase separation (LLPS). LLPS at the membrane surface provides a powerful means for recruiting and clustering proteins, as well as for membrane remodeling.

Cancer encompasses a range of severe diseases characterized by uncontrolled cell growth and the potential for metastasis. Understanding the mechanism underlying tumorigenesis has been a central focus of cancer research. Self-propagating protein aggregates, known as prions, are linked to various biological functions and diseases, particularly those related to mammalian neurodegeneration. However, it remains unclear whether prion-like mechanisms contribute to tumorigenesis and cancer. Using a combined approach of algorithmic predictions, alongside genetic and biochemical experimentation, we identified numerous cancer-associated proteins prone to aggregation, many of which contain prion-like domains (PrLDs). These predictions were experimentally validated for both aggregation and prion-formation. We demonstrate that several PrLDs undergo nucleation-limited amyloid formation, which can alter protein activity in a mitotically heritable fashion. These include SSXT, a subunit of the chromatin-remodeling BAF (hSWI/SNF) complexes; CLOCK, a core component of the circadian clock; and EPN4, a clathrin-interacting protein involved in protein trafficking between the trans-Golgi network and endosomes. The prions formed by these PrLDs occurred in multiple variants and depended on Hsp104, a molecular chaperone with disaggregase activity. Our results reveal an inherent tendency for prion-like aggregation in human cancer-associated proteins, suggesting a potential role for such aggregation in the epigenetic changes driving tumorigenesis.

Mycobacterium tuberculosis (Mtb) evades host defense by hijacking and rupturing the phagosome. ESAT-6, a secreted virulence protein of Mtb, is known to be critical for phagosome rupture. However, the mechanism of ESAT-6-mediated disruption of the phagosomal membrane remains unknown. Using in vitro reconstitution, live-cell imaging, and numerical simulations, we discover that ESAT-6 polymerization forces remodeling and vesiculation of the phagosome-like compartment both in vitro and in vivo. Shallow insertion of ESAT-6 leads to tubular and bud-like deformations on the membrane facilitated by a reduction in membrane tension. Growing fibrils generate both radial and tangential forces causing local remodeling and shape transition of the membrane into buds. The ESAT-6-bound tensed membrane undergoes local changes in membrane curvature and lipid phase separation that assist the subsequent fission. Overall, the findings provide mechanistic insights into the long-standing question of phagosome disruption by Mtb for its escape.

In recent years, interventional surgery has become a treatment for ischemic stroke due to its low risk of injury. However, the occurrence of restenosis hinders the long-term effectiveness and safety of stent implantation. At present, drug-eluting stents mainly prevent the stenosis of drug-eluting stents by inhibiting the proliferation of smooth muscle cells (SMCs). However, these drugs cause damage to endothelial cells (ECs), prevent timely re endothelialization of blood vessels, and increase the risk of late thrombosis and late restenosis. EPS-15-interacting protein 1 (Epsin1)- EPS-15-interacting protein 2 (Epsin2)-shrna coated stents have the potential to promote early endothelialization and inhibit restenosis, which contributes to the candidate development of novel drug coated stents. We found that the expression of Epsin was elevated in the mouse carotid artery ligation model, and the intimal hyperplasia(IH) could be reduced by intervening Epsin. Epsin in cultured endothelial cells was interfered to study proliferation and migration functions, and its role in cocultured endothelial cells and smooth muscle cells was evaluated. In addition, we explored the potential therapeutic benefits of inhibiting Epsin in a porcine model using scaffolds coated with plasmids containing Epsin short hairpin RNA (shRNA). Our study showed that the expression of Epsin1 and Epsin2 was elevated in the proliferative intima of mice, and the inhibition of Epsin reduced the proliferation of neointima in mice. The inhibition of Epsin led to enhanced proliferation and migration of endothelial cells, and maintained a healthy cell membrane potential. In cocultured cells, inhibition of Epsin resulted in reduced proliferation and migration of smooth muscle cells. In a porcine carotid artery model, Epsin shRNA coated scaffolds promoted early re endothelialization and reduced IH. These results suggest that Epsin plays a crucial role in endothelial and smooth muscle cell proliferation and migration functions, and its inhibition may be a potentially effective therapeutic strategy to prevent in stent stenosis.

Atherosclerosis is a multifaceted disease characterised by chronic inflammation and vascular remodelling, leading to plaque formation and cardiovascular complications. Recent evidence highlights the critical role of epsins, a family of endocytic proteins, in the pathogenesis of atherosclerosis. This manuscript explores the multifarious functions of epsins in atherosclerosis, focusing on their involvement in angiogenesis, lymphangiogenesis, and the modulation of key signalling pathways. We discuss how epsins facilitate EndoMT through their interaction with the TGFβ signalling pathway, which contributes to vascular smooth muscle cell-like phenotypes and plaque instability. Additionally, we examine the therapeutic potential of targeting epsins, elucidating their interactions with crucial partners such as LDLR, LRP-1, and TLR 2/4, among others, in mediating lipid metabolism and inflammation. Furthermore, we highlight the promising prospects of epsin-targeting peptides and small interfering RNAs as therapeutic agents for atherosclerosis treatment. Despite these advancements, the research faces limitations, including a reliance on specific mouse models and a need for comprehensive studies on the long-term effects of epsin modulation. Therefore, future investigations should focus on elucidating the detailed mechanisms of epsin function and their implications in cardiovascular health, fostering collaborations to translate basic research into innovative therapeutic strategies. This work underscores the necessity for further exploration of epsins to unlock their full therapeutic potential in combating atherosclerosis and related cardiovascular diseases.

Clathrin-mediated endocytosis internalizes proteins and lipids from the cell surface, supporting nutrient uptake, signaling, and membrane trafficking. Recent work has demonstrated that a flexible, liquid-like network of initiator proteins is responsible for catalyzing assembly of clathrin-coated vesicles in diverse organisms including yeast, mammals, and plants. How do cells regulate the assembly of this dynamic network to produce cargo-loaded vesicles? Here we reveal the ability of an endocytic adaptor protein, Epsin1, to conditionally stabilize the initiator protein network, creating a cargo-dependent checkpoint during clathrin-mediated endocytosis. Epsin1 is known to recruit ubiquitylated transmembrane proteins to endocytic sites. Using in vitro assays, we demonstrate that Epsin1 uses competitive binding and steric repulsion to destabilize condensation of initiator proteins in the absence of ubiquitin. However, when polyubiquitin is present, Epsin1 binds to both ubiquitin and initiator proteins, creating attractive interactions that stabilize condensation. Similarly, in mammalian cells, endocytic dynamics and ligand uptake are disrupted by removal of either ubiquitin or Epsin1. Surprisingly, when Epsin1 and ubiquitin are removed simultaneously, endocytic defects are rescued to near wildtype levels, although endocytic sites lose the ability to distinguish between ubiquitylated and non-ubiquitylated cargos. Taken together, these results suggest that Epsin1 tunes protein condensation to ensure the presence of ubiquitylated cargo during assembly of clathrin-coated vesicles. More broadly, these findings illustrate how a balance of attractive and repulsive molecular interactions controls the stability of liquid-like protein networks, providing dynamic control over key cellular events.

Adaptor protein complex-3 (AP-3) mediates cargo sorting from endosomes to lysosomes and lysosome-related organelles. Recently, it was shown that AP-3 adopts a constitutively open conformation compared to the related AP-1 and AP-2 coat complexes, which are inactive until undergoing large conformational changes upon membrane recruitment. How AP-3 is regulated is therefore an open question. To understand the mechanism of AP-3 membrane recruitment and activation, we reconstituted human AP-3 and determined multiple structures in the soluble and membrane-bound states using electron cryo-microscopy. Similar to yeast AP-3, human AP-3 is in a constitutively open conformation. To reconstitute AP-3 activation by adenosine di-phosphate (ADP)-ribosylation factor 1 (Arf1), a small guanosine tri-phosphate (GTP)ase, we used lipid nanodiscs to build Arf1-AP-3 complexes on membranes and determined three structures showing the stepwise conformational changes required for formation of AP-3 coated vesicles. First, membrane recruitment is driven by one of two predicted Arf1 binding sites, which flexibly tethers AP-3 to the membrane. Second, cargo binding causes AP-3 to adopt a fixed position and rigidifies the complex, which stabilizes binding for a second Arf1 molecule. Finally, binding of the second Arf1 molecule provides the template for AP-3 dimerization, providing a glimpse into the first step of coat polymerization. We propose coat polymerization only occurs after cargo engagement, thereby linking cargo sorting with assembly of higher-order coat structures. Additionally, we provide evidence for two amphipathic helices in AP-3, suggesting that AP-3 contributes to membrane deformation during coat assembly. In total, these data provide evidence for the first stages of AP-3-mediated vesicle coat assembly.

The MARTINI force field is one of the most used coarse-grained models for biomolecular simulations. Many limitations of the model including the protein-protein overaggregation have been improved in its latest version, MARTINI-3. In this study, we investigate the efficacy of the MARTINI-3 parameters for capturing the interactions of peripheral proteins with model plasma membranes. Particularly, we consider two classes of proteins, namely, annexin and epsin, which are known to generate negative and positive membrane curvatures, respectively. We find that current MARTINI-3 parameters are not able to correctly describe the protein-membrane interface and the protein-induced membrane curvatures for any of these proteins. The problem arises due to the lack of proper hydrophobic interactions between the protein residues and lipid tails. Making systematic adjustments, we show that a combination of reduction in the protein-water interactions and enhancement of protein-lipid hydrophobic interactions is essential for accurate prediction of the interfacial structure including the protein-induced membrane curvature. Next, we apply our model to a couple of other peripheral proteins, namely, Snf7, a core component of the ESCRT-III complex, and the PH domain of evectin-2. We find that our model captures the protein-membrane interfacial structure much more accurately than the MARTINI-3 model for all of the peripheral proteins considered in this study. However, the strategy described in this study may not be suitable for oligomeric transmembrane proteins where protein-protein hydrophobic interactions should be increased instead of protein-lipid hydrophobic interactions.

Clathrin forms a triskelion, or three-legged, network that regulates cellular processes by facilitating cargo internalization and trafficking in eukaryotes. Its N-terminal domain is crucial for interacting with adaptor proteins, which link clathrin to the membrane and engage with specific cargo. The N-terminal domain contains up to four adaptor-binding sites, though their role in preferential occupancy by adaptor proteins remains unclear. In this study, we examine the binding hierarchy of adaptors for clathrin, using integrative biophysical and structural approaches, along with in vivo functional experiments. We find that yeast epsin Ent5 has the highest affinity for clathrin, highlighting its key role in cellular trafficking. Epsins Ent1 and Ent2, crucial for endocytosis but thought to have redundant functions, show distinct binding patterns. Ent1 exhibits stronger interactions with clathrin than Ent2, suggesting a functional divergence toward actin binding. These results offer molecular insights into adaptor protein selectivity, suggesting they competitively bind clathrin while also targeting three different clathrin sites.

In biology, shape and function are related. Therefore, it is important to understand how membrane shape is generated, stabilised and sensed by proteins and how this relates to organelle function. Here, we present an assay that can detect curvature preference and membrane remodelling with free-floating liposomes using protein concentrations in physiologically relevant ranges. The assay reproduced known curvature preferences of BAR domains and allowed the discovery of high-curvature preference for the PH domain of AKT and the FYVE domain of HRS (also known as HGS). In addition, our method reproduced the membrane vesiculation activity of the ENTH domain of epsin-1 (EPN1) and showed similar activity for the ANTH domains of PiCALM and Hip1R. Finally, we found that the curvature sensitivity of the N-BAR domain of endophilin inversely correlates to membrane charge and that deletion of its N-terminal amphipathic helix increased its curvature specificity. Thus, our method is a generally applicable qualitative method for assessing membrane curvature sensing and remodelling by proteins.

Ubiquitin (Ub) signals are recognized and decoded into cellular responses by Ub-receptors, proteins that tether the Ub-binding domain(s) (UBDs) with response elements. Typically, UBDs bind mono-Ub in highly dynamic and weak affinity manners, presenting challenges in identifying and characterizing their binding interfaces. Here, we report the development of a new approach to facilitate the detection of these weak interactions using split-reporter systems where two interacting proteins are proximally co-translated from a single mRNA. This proximity significantly enhances the readout signals of weak protein-protein interactions (PPIs). We harnessed this system to characterize the ultra-weak UBD and ENTH (Epsin N-terminal Homology) and discovered that the yeast Ent1-ENTH domain contains two Ub-binding patches. One is similar to a previously characterized patch on STAM1(signal-transducing adaptor molecule)-VHS (Vps27, Hrs, and STAM), and the other was predicted by AlphaFold. Using a split-CAT selection system that co-translates Ub and ENTH in combination with mutagenesis, we assessed and confirmed the existence of a novel binding patch around residue F53 on ENTH. Co-translation in the split-CAT system provides an effective tool for studying weak PPIs and offers new insights into Ub-receptor interactions.

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.

Metabolic enzymes can adapt during energy stress, but the consequences of these adaptations remain understudied. Here, we discovered that hexokinase 1 (HK1), a key glycolytic enzyme, forms rings around mitochondria during energy stress. These HK1-rings constrict mitochondria at contact sites with the endoplasmic reticulum (ER) and mitochondrial dynamics protein (MiD51). HK1-rings prevent mitochondrial fission by displacing the dynamin-related protein 1 (Drp1) from mitochondrial fission factor (Mff) and mitochondrial fission 1 protein (Fis1). The disassembly of HK1-rings during energy restoration correlated with mitochondrial fission. Mechanistically, we identified that the lack of ATP and glucose-6-phosphate (G6P) promotes the formation of HK1-rings. Mutations that affect the formation of HK1-rings showed that HK1-rings rewire cellular metabolism toward increased TCA cycle activity. Our findings highlight that HK1 is an energy stress sensor that regulates the shape, connectivity, and metabolic activity of mitochondria. Thus, the formation of HK1-rings may affect mitochondrial function in energy-stress-related pathologies.

Ubiquitination plays an essential role in protein stability, subcellular localization, and interactions. Crosstalk between different types of ubiquitination results in distinct biological outcomes for proteins. However, the role of ubiquitination-related crosstalk in lymph node (LN) metastasis and the key regulatory factors controlling this process have not been determined. Using high-throughput sequencing, we found that ubiquitin-conjugating enzyme E2 C (UBE2C) was overexpressed in bladder cancer (BCa) and was strongly associated with an unfavorable prognosis. Overexpression of UBE2C increased BCa lymphangiogenesis and promoted LN metastasis both in vitro and in vivo. Mechanistically, UBE2C mediated sodium-coupled neutral amino acid transporter 2 (SNAT2) monoubiquitination at lysine 59 to inhibit K63-linked polyubiquitination at lysine 33 of SNAT2. Crosstalk between monoubiquitination and K63-linked polyubiquitination increased SNAT2 membrane protein levels by suppressing epsin 1-mediated (EPN1-mediated) endocytosis. SNAT2 facilitated glutamine uptake and metabolism to promote VEGFC secretion, ultimately leading to lymphangiogenesis and LN metastasis in patients with BCa. Importantly, inhibition of UBE2C significantly attenuated BCa lymphangiogenesis in a patient-derived xenograft model. Our results reveal the mechanism by which UBE2C mediates crosstalk between the monoubiquitination and K63-linked polyubiquitination of SNAT2 to promote BCa metastasis and identify UBE2C as a promising target for treating LN-metastatic BCa.

Atherosclerosis, a chronic systemic inflammatory condition, is implicated in most cardiovascular ischemic events. The pathophysiology of atherosclerosis involves various cell types and associated processes, including endothelial cell activation, monocyte recruitment, smooth muscle cell migration, involvement of macrophages and foam cells, and instability of the extracellular matrix. The process of endothelial-to-mesenchymal transition (EndoMT) has recently emerged as a pivotal process in mediating vascular inflammation associated with atherosclerosis. This transition occurs gradually, with a significant portion of endothelial cells adopting an intermediate state, characterized by a partial loss of endothelial-specific gene expression and the acquisition of "mesenchymal" traits. Consequently, this shift disrupts endothelial cell junctions, increases vascular permeability, and exacerbates inflammation, creating a self-perpetuating cycle that drives atherosclerotic progression. While endothelial cell dysfunction initiates the development of atherosclerosis, autophagy, a cellular catabolic process designed to safeguard cells by recycling intracellular molecules, is believed to exert a significant role in plaque development. Identifying the pathological mechanisms and molecular mediators of EndoMT underpinning endothelial autophagy, may be of clinical relevance. Here, we offer new insights into the underlying biology of atherosclerosis and present potential molecular mechanisms of atherosclerotic resistance and highlight potential therapeutic targets.

Extracellular vesicles (EVs), transporting diverse cellular components, play a crucial role in intercellular communication in numerous physiological and pathological processes. EVs have also been recognized as a drug delivery platform for therapeutic purposes and cell-free regenerative medicine. While various approaches have focused on increasing EV production for efficient use therapeutic use of EVs, enhancing the quality of EVs, such as ensuring efficient uptake by their target cells, has not been widely explored. In this study, we linked a negative membrane curvature-forming inverse BAR (IBAR) domain with an integrin β tail-binding talin F3 domain to create the IBAR-F3 fusion protein. We observed that IBAR-F3 can trigger filopodia-like membrane protrusions and attract integrins to those protrusion-rich regions, when expressed in Chinese hamster ovary cells expressing integrin αIIbβ3. Surprisingly, the expression of IBAR-F3 also induced a robust production of EVs, which were then efficiently taken up by nearby cells in an integrin-dependent manner. Moreover, IBAR triggered integrin activation, presumably by inducing negative membrane curvature that likely disrupts the interaction between the integrin α and β transmembrane domain. Therefore, we suggest that IBAR-F3 should be utilized to promote both EV production and efficient uptake mediated by integrins. Furthermore, the negative curvature-inducing integrin activation suggests that integrins on EVs can be activated by the nanoscale change in the curvature of the EV without the need for conventional machinery to activate integrin inside the EVs.

Endocytosis requires a coordinated framework of molecular interactions that ultimately lead to the fission of nascent endocytic structures. How cytosolic proteins such as dynamin concentrate at discrete sites that are sparsely distributed across the plasma membrane remains poorly understood. Two dynamin-1 major splice variants differ by the length of their C-terminal proline-rich region (short-tail and long-tail). Using sptPALM in PC12 cells, neurons and MEF cells, we demonstrate that short-tail dynamin-1 isoforms ab and bb display an activity-dependent recruitment to the membrane, promptly followed by their concentration into nanoclusters. These nanoclusters are sensitive to both Calcineurin and dynamin GTPase inhibitors, and are larger, denser, and more numerous than that of long-tail isoform aa. Spatiotemporal modelling confirms that dynamin-1 isoforms perform distinct search patterns and undergo dimensional reduction to generate endocytic nanoclusters, with short-tail isoforms more robustly exploiting lateral trapping in the generation of nanoclusters compared to the long-tail isoform.

Evaluating the heterogeneity of extracellular vesicles (EVs) is crucial for unraveling their complex actions and biodistribution. Here, we identify consistent architectural heterogeneity of EVs using cryogenic transmission electron microscopy (cryo-TEM), which has an inherent ability to image biological samples without harsh labeling methods while preserving their native conformation. Imaging EVs isolated using different methodologies from distinct sources, such as cancer cells, normal cells, immortalized cells, and body fluids, we identify a structural atlas of their dominantly consistent shapes. We identify EV architectural attributes by utilizing a segmentation neural network model. In total, 7,576 individual EVs were imaged and quantified by our computational pipeline. Across all 7,576 independent EVs, the average eccentricity was 0.5366 ± 0.2, and the average equivalent diameter was 132.43 ± 67 nm. The architectural heterogeneity was consistent across all sources of EVs, independent of purification techniques, and compromised of single spherical, rod-like or tubular, and double shapes. This study will serve as a reference foundation for high-resolution images of EVs and offer insights into their potential biological impact.

Biological membranes are integral cellular structures that can be curved into various geometries. These curved structures are abundant in cells as they are essential for various physiological processes. However, curved membranes are inherently unstable, especially on nanometer length scales. To stabilize curved membranes, cells can utilize proteins that sense and generate membrane curvature. In this review, we summarize recent research that has advanced our understanding of interactions between proteins and curved membrane surfaces, as well as work that has expanded our ability to study curvature sensing and generation. Additionally, we look at specific examples of cellular processes that require membrane curvature, such as neurotransmission, clathrin-mediated endocytosis (CME), and organelle biogenesis.

In circulation, T cells are spherical with selectin enriched dynamic microvilli protruding from the surface. Following extravasation, these microvilli serve another role, continuously surveying their environment for antigen in the form of peptide-MHC (pMHC) expressed on the surface of antigen presenting cells (APCs). Upon recognition of their cognate pMHC, the microvilli are initially stabilized and then flatten into F-actin dependent microclusters as the T cell spreads over the APC. Within 1-5 min, clathrin is recruited by the ESCRT-0 component Hrs to mediate release of T cell receptor (TCR) loaded vesicles directly from the plasma membrane by clathrin and ESCRT-mediated ectocytosis (CEME). After 5-10 min, Hrs is displaced by the endocytic clathrin adaptor epsin-1 to induce clathrin-mediated trans-endocytosis (CMTE) of TCR-pMHC conjugates. Here we discuss some of the functional properties of the clathrin machinery which enables it to control these topologically opposite modes of membrane transfer at the immunological synapse, and how this might be regulated during T cell activation.

Influenza A virus (IAV) is the primary causative agent of influenza, colloquially called the flu. Each year, it infects up to a billion people, resulting in hundreds of thousands of human deaths, and causes devastating avian outbreaks with worldwide losses worth billions of dollars. Always present is the possibility that a highly pathogenic novel subtype capable of direct human-to-human transmission will spill over into humans, causing a pandemic as devastating if not more so than the 1918 influenza pandemic. While antiviral drugs for influenza do exist, they target very few aspects of IAV replication and risk becoming obsolete due to antiviral resistance. Antivirals targeting other areas of IAV replication are needed to overcome this resistance and combat the yearly epidemics, which exact a serious toll worldwide. This review aims to summarise the key steps in the IAV replication cycle, along with highlighting areas of research that need more focus.

Many membrane peripheral proteins have evolved to transiently interact with the surface of (curved) lipid bilayers. Currently, methods to quantitatively predict sensing and binding free energies for protein sequences or structures are lacking, and such tools could greatly benefit the discovery of membrane-interacting motifs, as well as their de novo design.

Here, we trained a transformer neural network model on molecular dynamics data for >50 000 peptides that is able to accurately predict the (relative) membrane-binding free energy for any given amino acid sequence. Using this information, our physics-informed model is able to classify a peptide's membrane-associative activity as either non-binding, curvature sensing, or membrane binding. Moreover, this method can be applied to detect membrane-interaction regions in a wide variety of proteins, with comparable predictive performance as state-of-the-art data-driven tools like DREAMM, PPM3, and MODA, but with a wider applicability regarding protein diversity, and the added feature to distinguish curvature sensing from general membrane binding.

We made these tools available as a web server, coined Protein-Membrane Interaction predictor (PMIpred), which can be accessed at https://pmipred.fkt.physik.tu-dortmund.de.

Apicomplexan parasites possess specialized secretory organelles called rhoptries, micronemes, and dense granules that play a vital role in host infection. In this study, we demonstrate that TgREMIND, a protein found in Toxoplasma gondii, is necessary for the biogenesis of rhoptries and dense granules. TgREMIND contains a Fes-CIP4 homology-Bin/Amphiphysin/Rvs (F-BAR) domain, which binds to membrane phospholipids, as well as a novel uncharacterized domain that we have named REMIND (regulator of membrane-interacting domain). Both the F-BAR domain and the REMIND are crucial for TgREMIND functions. When TgREMIND is depleted, there is a significant decrease in the abundance of dense granules and abnormal transparency of rhoptries, leading to a reduction in protein secretion from these organelles. The absence of TgREMIND inhibits host invasion and parasite dissemination, demonstrating that TgREMIND is essential for the proper function of critical secretory organelles required for successful infection by Toxoplasma.

This work studied the membrane curvature generated by anchored proteins lacking amphipathic helices and intrinsic morphologies, including the Epsin N-terminal homology domain, intrinsically disordered C-terminal domain, and truncated C-terminal fragments, by using coarse-grained molecular dynamics simulations. We found that anchored proteins can stabilize the thermal undulation of membranes at a wavelength five times the protein's binding size. This proportional connection is governed by the membrane bending rigidity and protein density. Extended intrinsically disordered proteins with relatively high hydrophobicity favor colliding with the membrane, leading to a much larger binding size, and show superiority in generating membrane curvature at low density over folded proteins.

Circulating vascular endothelial growth factor (VEGF) ligands and receptors are central regulators of vasculogenesis, angiogenesis, and lymphangiogenesis. In response to VEGF ligand binding, VEGF receptor tyrosine kinases initiate the chain of events that transduce extracellular signals into endothelial cell responses such as survival, proliferation, and migration. These events are controlled by intricate cellular processes that include the regulation of gene expression at multiple levels, interactions of numerous proteins, and intracellular trafficking of receptor-ligand complexes. Endocytic uptake and transport of macromolecular complexes through the endosome-lysosome system helps fine-tune endothelial cell responses to VEGF signals. Clathrin-dependent endocytosis remains the best understood means of macromolecular entry into cells, although the importance of non-clathrin-dependent pathways is increasingly recognized. Many of these endocytic events rely on adaptor proteins that coordinate internalization of activated cell-surface receptors. In the endothelium of both blood and lymphatic vessels, epsins 1 and 2 are functionally redundant adaptors involved in receptor endocytosis and intracellular sorting. These proteins are capable of binding both lipids and proteins and are important for promoting curvature of the plasma membrane as well as binding ubiquitinated cargo. Here, we discuss the role of epsin proteins and other endocytic adaptors in governing VEGF signaling in angiogenesis and lymphangiogenesis and discuss their therapeutic potential as molecular targets.

Evaluating the heterogeneity of extracellular vesicles (EVs) is crucial for unraveling their complex actions and biodistribution. Here, we identify consistent architectural heterogeneity of EVs using cryogenic transmission electron microscopy (cryo-TEM) which has an inherent ability to image biological samples without harsh labeling methods and while preserving their native conformation. Imaging EVs isolated using different methodologies from distinct sources such as cancer cells, normal cells, and body fluids, we identify a structural atlas of their dominantly consistent shapes. We identify EV architectural attributes by utilizing a segmentation neural network model. In total, 7,576 individual EVs were imaged and quantified by our computational pipeline. Across all 7,576 independent EVs, the average eccentricity was 0.5366, and the average equivalent diameter was 132.43 nm. The architectural heterogeneity was consistent across all sources of EVs, independent of purification techniques, and compromised of single spherical (S. Spherical), rod-like or tubular, and double shapes. This study will serve as a reference foundation for high-resolution EV images and offer insights into their potential biological impact.

The pathogenesis of various diseases often involves an intricate interplay between membrane proteins and membrane curvature. Understanding the underlying mechanisms of this interaction could offer novel perspectives on disease treatment. In this review, we provide an introduction to membrane curvature and its association with membrane proteins. Furthermore, we delve into the impact and potential implications of this interaction in the context of disease treatment. Lastly, we discuss the prospects and challenges associated with harnessing these interactions for effective disease management, aiming to provide fresh insights into therapeutic strategies.

Liquid-liquid phase separation of proteins occurs on both surfaces of cellular membranes during diverse physiological processes. In vitro reconstitution could provide insight into the mechanisms underlying these events. However, most existing reconstitution techniques provide access to only one membrane surface, making it difficult to probe transmembrane phenomena. To study protein phase separation simultaneously on both membrane surfaces, we developed an array of freestanding planar lipid membranes. Interestingly, we observed that liquid-like protein condensates on one side of the membrane colocalized with those on the other side, resulting in transmembrane coupling. Our results, based on lipid probe partitioning and mobility of lipids, suggest that protein condensates locally reorganize membrane lipids, a process which could be explained by multiple effects. These findings suggest a mechanism by which signals originating on one side of a biological membrane, triggered by protein phase separation, can be transferred to the opposite side.

Proteins and peptides with hydrophobic and amphiphilic segments are responsible for many biological functions. The sensing and generation of membrane curvature are the functions of several protein domains or motifs. While some specific membrane proteins play an essential role in controlling the curvature of distinct intracellular membranes, others participate in various cellular processes such as clathrin-mediated endocytosis, where several proteins sort themselves at the neck of the membrane bud. A few membrane-inserting proteins form nanopores that permeate selective ions and water to cross the membrane. In addition, many natural and synthetic small peptides and protein toxins disrupt the membrane by inducing nonspecific pores in the membrane. The pore formation causes cell death through the uncontrolled exchange between interior and exterior cellular contents. In this article, we discuss the insertion depth and orientation of protein/peptide helices, and their role as a sensor and inducer of membrane curvature as well as a pore former in the membrane. We anticipate that this extensive review will assist biophysicists to gain insight into curvature sensing, generation, and pore formation by membrane insertion.

Membrane curvature sensing is essential for a diverse range of biological processes. Recent experiments have revealed that a single nanometer-sized septin protein has different binding rates to membrane-coated glass beads of 1-µm and 3-µm diameters, even though the septin is orders of magnitude smaller than the beads. This sensing ability is especially surprising since curvature-sensing proteins must deal with persistent thermal fluctuations of the membrane, leading to discrepancies between the bead's curvature and the local membrane curvature sensed instantaneously by a protein. Using continuum models of fluctuating membranes, we investigate whether it is feasible for a protein acting as a perfect observer of the membrane to sense micron-scale curvature either by measuring local membrane curvature or by using bilayer lipid densities as a proxy. To do this, we develop algorithms to simulate lipid density and membrane shape fluctuations. We derive physical limits to the sensing efficacy of a protein in terms of protein size, membrane thickness, membrane bending modulus, membrane-substrate adhesion strength, and bead size. To explain the experimental protein-bead association rates, we develop two classes of predictive models: (i) for proteins that maximally associate to a preferred curvature and (ii) for proteins with enhanced association rates above a threshold curvature. We find that the experimentally observed sensing efficacy is close to the theoretical sensing limits imposed on a septin-sized protein. Protein-membrane association rates may depend on the curvature of the bead, but the strength of this dependence is limited by the fluctuations in membrane height and density.

Compartmentalization by membranes is a common feature of eukaryotic cells and serves to spatiotemporally confine biochemical reactions to control physiology. Membrane-bound organelles such as the endoplasmic reticulum (ER), the Golgi complex, endosomes and lysosomes, and the plasma membrane, continuously exchange material via vesicular carriers. In addition to vesicular trafficking entailing budding, fission, and fusion processes, organelles can form membrane contact sites (MCSs) that enable the nonvesicular exchange of lipids, ions, and metabolites, or the secretion of neurotransmitters via subsequent membrane fusion. Recent data suggest that biomolecule and information transfer via vesicular carriers and via MCSs share common organizational principles and are often mediated by proteins with intrinsically disordered regions (IDRs). Intrinsically disordered proteins (IDPs) can assemble via low-affinity, multivalent interactions to facilitate membrane tethering, deformation, fission, or fusion. Here, we review our current understanding of how IDPs drive the formation of multivalent protein assemblies and protein condensates to orchestrate vesicular and nonvesicular transport with a special focus on presynaptic neurotransmission. We further discuss how dysfunction of IDPs causes disease and outline perspectives for future research.

Clathrin-mediated endocytosis (CME), the major cellular entry pathway, starts when clathrin assembles on the plasma membrane into clathrin-coated pits (CCPs). Two populations of CCPs are detected within the same cell: 'productive' CCPs that invaginate and pinch off, forming clathrin-coated vesicles (CCVs) [1, 2], and 'abortive' CCPs [3, 4, 5] that prematurely disassemble. The mechanisms of gating between these two populations and their relations to the functions of dozens of early-acting endocytic accessory proteins (EAPs) [5, 6, 7, 8, 9] have remained elusive. Here, we use experimentally-guided modeling to integrate the clathrin machinery and membrane mechanics in a single dynamical system. We show that the split between the two populations is an emergent property of this system, in which a switch between an Open state and a Closed state follows from the competition between the chemical energy of the clathrin basket and the mechanical energy of membrane bending. In silico experiments revealed an abrupt transition between the two states that acutely depends on the strength of the clathrin basket. This critical strength is lowered by membrane-bending EAPs [10, 11, 12]. Thus, CME is poised to be shifted between abortive and productive events by small changes in membrane curvature and/or coat stability. This model clarifies the workings of a putative endocytic checkpoint whose existence was previously proposed based on statistical analyses of the lifetime distributions of CCPs [4, 13]. Overall, a mechanistic framework is established to elucidate the diverse and redundant functions of EAPs in regulating CME progression.

In the late stages of the HIV-1 life cycle, membrane localization and self-assembly of Gag polyproteins induce membrane deformation and budding. Release of the virion requires direct interaction between immature Gag lattice and upstream ESCRT machinery at the viral budding site, followed by assembly of downstream ESCRT-III factors, culminating in membrane scission. However, molecular details of upstream ESCRT assembly dynamics at the viral budding site remain unclear. In this work, using coarse-grained (CG) molecular dynamics (MD) simulations, we investigated the interactions between Gag, ESCRT-I, ESCRT-II, and membrane to delineate the dynamical mechanisms by which upstream ESCRTs assemble templated by late-stage immature Gag lattice. We first systematically derived "bottom-up" CG molecular models and interactions of upstream ESCRT proteins from experimental structural data and extensive all-atom MD simulations. Using these molecular models, we performed CG MD simulations of ESCRT-I oligomerization and ESCRT-I/II supercomplex formation at the neck of the budding virion. Our simulations demonstrate that ESCRT-I can effectively oligomerize to higher-order complexes templated by the immature Gag lattice both in the absence of ESCRT-II and when multiple copies of ESCRT-II are localized at the bud neck. The ESCRT-I/II supercomplexes formed in our simulations exhibit predominantly columnar structures, which has important implications for the nucleation pathway of downstream ESCRT-III polymers. Importantly, ESCRT-I/II supercomplexes bound to Gag initiate membrane neck constriction by pulling the inner edge of the bud neck closer to the ESCRT-I headpiece ring. Our findings serve to elucidate a network of interactions between upstream ESCRT machinery, immature Gag lattice, and membrane neck that regulate protein assembly dynamics at the HIV-1 budding site.

The mechanisms by which peripheral membrane proteins generate curvature is currently an active area of research. One of the proposed mechanisms is amphipathic insertion or the 'wedge' mechanism in which the protein shallowly inserts an amphipathic helix inside the membrane to drive the curvature. However, recent experimental studies have challenged the efficiency of the 'wedge' mechanism as it requires unusual protein densities. These studies proposed an alternative mechanism, namely 'protein-crowding', in which the lateral pressure generated by the random collisions among the membrane bound proteins drives the bending. In this study, we employ atomistic and coarse-grained molecular dynamics simulations to investigate the effects of amphipathic insertion and protein crowding on the membrane surface. Considering epsin N-terminal homology (ENTH) domain as a model protein, we show that amphipathic insertion is not essential for membrane bending. Our results suggest that ENTH domains can aggregate on the membrane surface by employing another structured region (H₃ helix). And this protein crowding decreases the cohesive energy of the lipid tails which causes a significant decrease in the membrane bending rigidity. The ENTH domain can generate a similar degree of membrane curvature irrespective of the activity of its H₀ helix. Our results are consistent with the recent experimental results.

Endocytosis is a complex process whereby cell surface proteins, lipids and fluid from the extracellular environment are packaged, sorted and internalized into cells. Endocytosis is also a mechanism of drug internalization into cells. There are multiple routes of endocytosis that determine the fate of molecules, from degradation in the lysosomes to recycling back to the plasma membrane. The overall rates of endocytosis and temporal regulation of molecules transiting through endocytic pathways are also intricately linked with signalling outcomes. This process relies on an array of factors, such as intrinsic amino acid motifs and post-translational modifications. Endocytosis is frequently disrupted in cancer. These disruptions lead to inappropriate retention of receptor tyrosine kinases on the tumour cell membrane, changes in the recycling of oncogenic molecules, defective signalling feedback loops and loss of cell polarity. In the past decade, endocytosis has emerged as a pivotal regulator of nutrient scavenging, response to and regulation of immune surveillance and tumour immune evasion, tumour metastasis and therapeutic drug delivery. This Review summarizes and integrates these advances into the understanding of endocytosis in cancer. The potential to regulate these pathways in the clinic to improve cancer therapy is also discussed.

Compensatory endocytosis keeps the membrane surface area of secretory cells constant following exocytosis. At chemical synapses, clathrin-independent ultrafast endocytosis maintains such homeostasis. This endocytic pathway is temporally and spatially coupled to exocytosis; it initiates within 50 ms at the region immediately next to the active zone where vesicles fuse. However, the coupling mechanism is unknown. Here, we demonstrate that filamentous actin is organized as a ring, surrounding the active zone at mouse hippocampal synapses. Assuming the membrane area conservation is due to this actin ring, our theoretical model suggests that flattening of fused vesicles exerts lateral compression in the plasma membrane, resulting in rapid formation of endocytic pits at the border between the active zone and the surrounding actin-enriched region. Consistent with model predictions, our data show that ultrafast endocytosis requires sufficient compression by exocytosis of multiple vesicles and does not initiate when actin organization is disrupted, either pharmacologically or by ablation of the actin-binding protein Epsin1. Our work suggests that membrane mechanics underlie the rapid coupling of exocytosis to endocytosis at synapses.

The higher-order assembly of Bin-amphiphysin-Rvs (BAR) domain proteins, including the FCH-BAR (F-BAR) domain proteins, into lattice on the membrane is essential for the formation of subcellular structures. However, the regulation of their ordered assembly has not been elucidated. Here, we show that the higher ordered assembly of growth-arrested specific 7 (GAS7), an F-BAR domain protein, is regulated by the multivalent scaffold proteins of Wiskott-Aldrich syndrome protein (WASP)/neural WASP, that commonly binds to the BAR domain superfamily proteins, together with WISH, Nck, the activated small guanosine triphosphatase Cdc42, and a membrane-anchored phagocytic receptor. The assembly kinetics by fluorescence resonance energy transfer monitoring indicated that the GAS7 assembly on liposomes started within seconds and was further increased by the presence of these proteins. The regulated GAS7 assembly was abolished by Wiskott-Aldrich syndrome mutations both in vitro and in cellular phagocytosis. Therefore, Cdc42 and the scaffold proteins that commonly bind to the BAR domain superfamily proteins promoted GAS7 assembly.

Autophagy is a highly conserved recycling process of eukaryotic cells that degrades protein aggregates or damaged organelles with the participation of autophagy-related proteins. Membrane bending is a key step in autophagosome membrane formation and nucleation. A variety of autophagy-related proteins (ATGs) are needed to sense and generate membrane curvature, which then complete the membrane remodeling process. The Atg1 complex, Atg2-Atg18 complex, Vps34 complex, Atg12-Atg5 conjugation system, Atg8-phosphatidylethanolamine conjugation system, and transmembrane protein Atg9 promote the production of autophagosomal membranes directly or indirectly through their specific structures to alter membrane curvature. There are three common mechanisms to explain the change in membrane curvature. For example, the BAR domain of Bif-1 senses and tethers Atg9 vesicles to change the membrane curvature of the isolation membrane (IM), and the Atg9 vesicles are reported as a source of the IM in the autophagy process. The amphiphilic helix of Bif-1 inserts directly into the phospholipid bilayer, causing membrane asymmetry, and thus changing the membrane curvature of the IM. Atg2 forms a pathway for lipid transport from the endoplasmic reticulum to the IM, and this pathway also contributes to the formation of the IM. In this review, we introduce the phenomena and causes of membrane curvature changes in the process of macroautophagy, and the mechanisms of ATGs in membrane curvature and autophagosome membrane formation.

Conventionally, myo-D-inositol 1, 4,5-trisphosphate (IP₃) is thought to exert its second messenger effects through the gating of IP₃R Ca²⁺ release channels, located in Ca²⁺-storage organelles like the endoplasmic reticulum. However, there is considerable indirect evidence to support the concept that IP₃ might interact with other, non-IP₃R proteins within cells. To explore this possibility further, the Protein Data Bank was searched using the term "IP3". This resulted in the retrieval of 203 protein structures, the majority of which were members of the IP₃R/ryanodine receptor superfamily of channels. Only 49 of these structures were complexed with IP₃. These were inspected for their ability to interact with the carbon-1 phosphate of IP₃, since this is the least accessible phosphate group of its precursor, phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂). This reduced the number of structures retrieved to 35, of which 9 were IP₃Rs. The remaining 26 structures represent a diverse range of proteins, including inositol-lipid metabolizing enzymes, signal transducers, PH domain containing proteins, cytoskeletal anchor proteins, the TRPV4 ion channel, a retroviral Gag protein and fibroblast growth factor 2. Such proteins may impact on IP₃ signalling and its effects on cell-biology. This represents an area open for exploration in the field of IP₃ signalling.

Proteins can specifically bind to curved membranes through curvature-induced hydrophobic lipid packing defects. The chemical diversity among such curvature "sensors" challenges our understanding of how they differ from general membrane "binders" that bind without curvature selectivity. Here, we combine an evolutionary algorithm with coarse-grained molecular dynamics simulations (Evo-MD) to resolve the peptide sequences that optimally recognize the curvature of lipid membranes. We subsequently demonstrate how a synergy between Evo-MD and a neural network (NN) can enhance the identification and discovery of curvature sensing peptides and proteins. To this aim, we benchmark a physics-trained NN model against experimental data and show that we can correctly identify known sensors and binders. We illustrate that sensing and binding are phenomena that lie on the same thermodynamic continuum, with only subtle but explainable differences in membrane binding free energy, consistent with the serendipitous discovery of sensors.

Epsin endocytic adaptor proteins are implicated in the progression of atherosclerosis; however, the underlying molecular mechanisms have not yet been fully defined. In this study, we determined how epsins enhance endothelial-to-mesenchymal transition (EndoMT) in atherosclerosis and assessed the efficacy of a therapeutic peptide in a preclinical model of this disease.

Using single-cell RNA sequencing combined with molecular, cellular, and biochemical analyses, we investigated the role of epsins in stimulating EndoMT using knockout in Apoe-/- and lineage tracing/proprotein convertase subtilisin/kexin type 9 serine protease mutant viral-induced atherosclerotic mouse models. The therapeutic efficacy of a synthetic peptide targeting atherosclerotic plaques was then assessed in Apoe-/- mice.

Single-cell RNA sequencing and lineage tracing revealed that epsins 1 and 2 promote EndoMT and that the loss of endothelial epsins inhibits EndoMT marker expression and transforming growth factor-β signaling in vitro and in atherosclerotic mice, which is associated with smaller lesions in the Apoe-/- mouse model. Mechanistically, the loss of endothelial cell epsins results in increased fibroblast growth factor receptor-1 expression, which inhibits transforming growth factor-β signaling and EndoMT. Epsins directly bind ubiquitinated fibroblast growth factor receptor-1 through their ubiquitin-interacting motif, which results in endocytosis and degradation of this receptor complex. Consequently, administration of a synthetic ubiquitin-interacting motif-containing peptide atheroma ubiquitin-interacting motif peptide inhibitor significantly attenuates EndoMT and progression of atherosclerosis.

We conclude that epsins potentiate EndoMT during atherogenesis by increasing transforming growth factor-β signaling through fibroblast growth factor receptor-1 internalization and degradation. Inhibition of EndoMT by reducing epsin-fibroblast growth factor receptor-1 interaction with a therapeutic peptide may represent a novel treatment strategy for atherosclerosis.

Endocytosis in mammalian cells is a fundamental cellular machinery that regulates vital physiological processes, such as the absorption of metabolites, release of neurotransmitters, uptake of hormone cellular defense, and delivery of biomolecules across the plasma membrane. A remarkable characteristic of the endocytic machinery is the sequential assembly of the complex proteins at the plasma membrane, followed by internalization and fusion of various biomolecules to different cellular compartments. In all eukaryotic cells, functional characterization of endocytic pathways is based on dynamics of the protein complex and signal transduction modules. To coordinate the assembly and functions of the numerous parts of the endocytic machinery, the endocytic proteins interact significantly within and between the modules. Clathrin-dependent and -independent endocytosis, caveolar pathway, and receptor mediated endocytosis have been attributed to a greater variety of physiological and pathophysiological roles such as, autophagy, metabolism, cell division, apoptosis, cellular defense, and intestinal permeabilization. Notably, any defect or alteration in the endocytic machinery results in the development of pathological consequences associated with human diseases such as cancer, cardiovascular diseases, neurological diseases, and inflammatory diseases. In this review, an in-depth endeavor has been made to illustrate the process of endocytosis, and associated mechanisms describing pathological manifestation associated with dysregulated endocytosis machinery.

Reactive oxygen species (ROS) as well-known endogenous stimuli has been widely used to activate drug delivery systems (DDSs) for tumor-specific therapy. Unfortunately, endogenous ROS in the tumor microenvironment (TME) is not enough to achieve effective therapeutic efficacy and cancer cells have adapted to high oxidative stress by upregulating glutathione (GSH) level. Herein, we devised a novel ROS-activable self-immolative prodrug CASDB with both GSH-depletion ability and ROS self-supply competence. Then, an stimuli-responsive nanoplatform integrating CASDB with clinical chemotherapeutics mitoxantrone (MTO) and PLGA was fabricated (denoted as CMPs) through nanoprecipitation method. The CMPs could achieve desired accumulation at tumor tissues through enhanced permeability and retention (EPR) effects. Then the accumulated CMPs could induce tumor cell apoptosis efficiently. Especially, ROS in tumor sites could trigger the immolation of CASDB to generate CA and quinone methide (QM). Then CA and QM cooperatively promoted damage of mitochondria due to oxidative stress and led to cancer cells more sensitive to MTO. Accordingly, MTO could perturb cellular microenvironment of cancer cells then promote the degradation of CASDB. The experiment results demonstrated that CMPs were ideal for desirable synergetic tumor-specific anticancer therapy with negligible systemic toxicity. The half-maximal inhibitory concentrations (IC50) value of CMPs was 6.53 μM, while the IC50 values of MTO was 14.76 μM. And the CMPs group showed the strongest tumor suppressor effect with the tumor sizes increased to 1.2-fold (Control group: 20.6-fold, MTO only: 3.0-fold). This study should be inspirational for designing efficient prodrugs to overcome the handicaps of traditional chemotherapy.