Selective Endocytic Uptake of Targeted Liposomes Occurs within a Narrow Range of Liposome Diameters.

Cell surface receptors facilitate signaling and nutrient uptake. These processes are dynamic, requiring receptors to be actively recycled by endocytosis. Due to their differential expression in disease states, receptors are often the target of drug-carrier particles, which are adorned with ligands that bind specifically to receptors. These targeted particles are taken into the cell by multiple routes of internalization, where the best-characterized pathway is clathrin-mediated endocytosis. Most studies of particle uptake have utilized bulk assays rather than observing individual endocytic events. As a result, the detailed mechanisms of particle uptake remain obscure. To address this gap, we employed a live-cell imaging approach to study the uptake of individual liposomes as they interact with clathrin-coated structures. By tracking individual internalization events, we find that the size of liposomes rather than the density of the ligands on their surfaces primarily determines their probability of uptake. Interestingly, targeting has the greatest impact on endocytosis of liposomes of intermediate diameters, with the smallest and largest liposomes being internalized or excluded, respectively, regardless of whether they are targeted. These findings, which highlight a previously unexplored limitation of targeted delivery, can be used to design more effective drug carriers.

α-Synuclein colocalizes with AP180 and affects the size of clathrin lattices.

α-Synuclein and family members ß- and γ-synuclein are presynaptic proteins that sense and generate membrane curvature, properties important for synaptic vesicle (SV) cycling. αßγ-synuclein triple knockout neurons exhibit SV endocytosis deficits. Here, we investigated if α-synuclein affects clathrin assembly in vitro. Visualizing clathrin assembly on membranes using a lipid monolayer system revealed that α-synuclein increases clathrin lattices size and curvature. On cell membranes, we observe that α-synuclein is colocalized with clathrin and its adapter AP180 in a concentric ring pattern. Clathrin puncta that contain both α-synuclein and AP180 were significantly larger than clathrin puncta containing either protein alone. We determined that this effect occurs in part through colocalization of α-synuclein with the phospholipid PI(4,5)P2 in the membrane. Immuno-electron microscopy (EM) of synaptosomes uncovered that α-synuclein relocalizes from SVs to the presynaptic membrane upon stimulation, positioning α-synuclein to function on presynaptic membranes during or after stimulation. Additionally, we show that deletion of synucleins impacts brain-derived clathrin-coated vesicle size. Thus, α-synuclein affects the size and curvature of clathrin structures on membranes and functions as an endocytic accessory protein.

Capturing the mechanics of clathrin-mediated endocytosis.

Clathrin-mediated endocytosis enables selective uptake of molecules into cells in response to changing cellular needs. It occurs through assembly of coat components around the plasma membrane that determine vesicle contents and facilitate membrane bending to form a clathrin-coated transport vesicle. In this review we discuss recent cryo-electron microscopy structures that have captured a series of events in the life cycle of a clathrin-coated vesicle. Both single particle analysis and tomography approaches have revealed details of the clathrin lattice structure itself, how AP2 may interface with clathrin within a coated vesicle and the importance of PIP2 binding for assembly of the yeast adaptors Sla2 and Ent1 on the membrane. Within cells, cryo-electron tomography of clathrin in flat lattices and high-speed AFM studies provided new insights into how clathrin morphology can adapt during CCV formation. Thus, key mechanical processes driving clathrin-mediated endocytosis have been captured through multiple techniques working in partnership.

Mercury-instructed assembly (MiA): architecting clathrin triskelion-inspired highly functional C3-symmetric triskelion nanotorus functional structures into microtorus structures.

To detect heavy metal toxicity using self-assembled nanostructures, a clathrin triskelion-inspired highly functional C3-symmetric trimerized biotinylated di-tryptophan peptide was used. This triskelion peptide is known to self-assemble into nanotorus-like structures and can therefore act as a nanocage for various analytes. In this work, in addition to spectroscopy, force and electron microscopy were successfully used to detect the effect of toxic metal ions such as zinc, cadmium, and mercury by exploiting the change in the nanotorus morphology. Different concentrations of mercury led to the expansion of nanotorus structures into microtori. Therefore, we provide a unique application of heavy metal toxicity by utilizing "material nanoarchitectonics" to architect nanotorus structures into higher-order microtorus structures, as instructed by mercury. Such a strategy can make heavy metal sensing easier for materials scientists and open new avenues for biomedical/environmental science applications.

Large self-assembled clathrin lattices spontaneously disassemble without sufficient adaptor proteins.

Clathrin-coated structures must assemble on cell membranes to internalize receptors, with the clathrin protein only linked to the membrane via adaptor proteins. These structures can grow surprisingly large, containing over 20 clathrin, yet they often fail to form productive vesicles, instead aborting and disassembling. We show that clathrin structures of this size can both form and disassemble spontaneously when adaptor protein availability is low, despite high abundance of clathrin. Here, we combine recent in vitro kinetic measurements with microscopic reaction-diffusion simulations and theory to differentiate mechanisms of stable vs unstable clathrin assembly on membranes. While in vitro conditions drive assembly of robust, stable lattices, we show that concentrations, geometry, and dimensional reduction in physiologic-like conditions do not support nucleation if only the key adaptor AP-2 is included, due to its insufficient abundance. Nucleation requires a stoichiometry of adaptor to clathrin that exceeds 1:1, meaning additional adaptor types are necessary to form lattices successfully and efficiently. We show that the critical nucleus contains ~25 clathrin, remarkably similar to sizes of the transient and abortive structures observed in vivo. Lastly, we quantify the cost of bending the membrane under our curved clathrin lattices using a continuum membrane model. We find that the cost of bending the membrane could be largely offset by the energetic benefit of forming curved rather than flat structures, with numbers comparable to experiments. Our model predicts how adaptor density can tune clathrin-coated structures from the transient to the stable, showing that active energy consumption is therefore not required for lattice disassembly or remodeling during growth, which is a critical advance towards predicting productive vesicle formation.

Dual clathrin and integrin signaling systems regulate growth factor receptor activation.

The crosstalk between growth factor and adhesion receptors is key for cell growth and migration. In pathological settings, these receptors are drivers of cancer. Yet, how growth and adhesion signals are spatially organized and integrated is poorly understood. Here we use quantitative fluorescence and electron microscopy to reveal a mechanism where flat clathrin lattices partition and activate growth factor signals via a coordinated response that involves crosstalk between epidermal growth factor receptor (EGFR) and the adhesion receptor ß5-integrin. We show that ligand-activated EGFR, Grb2, Src, and ß5-integrin are captured by clathrin coated-structures at the plasma membrane. Clathrin structures dramatically grow in response to EGF into large flat plaques and provide a signaling platform that link EGFR and ß5-integrin through Src-mediated phosphorylation. Disrupting this EGFR/Src/ß5-integrin axis prevents both clathrin plaque growth and dampens receptor signaling. Our study reveals a reciprocal regulation between clathrin lattices and two different receptor systems to coordinate and enhance signaling. These findings have broad implications for the regulation of growth factor signaling, adhesion, and endocytosis.

Docosahexaenoic acid-containing phosphatidic acid interacts with clathrin coat assembly protein AP180 and regulates its interaction with clathrin.

The clathrin coat assembly protein AP180 drives endocytosis, which is crucial for numerous physiological events, such as the internalization and recycling of receptors, uptake of neurotransmitters and entry of viruses, including SARS-CoV-2, by interacting with clathrin. Moreover, dysfunction of AP180 underlies the pathogenesis of Alzheimer's disease. Therefore, it is important to understand the mechanisms of assembly and, especially, disassembly of AP180/clathrin-containing cages. Here, we identified AP180 as a novel phosphatidic acid (PA)-binding protein from the mouse brain. Intriguingly, liposome binding assays using various phospholipids and PA species revealed that AP180 most strongly bound to 1-stearoyl-2-docosahexaenoyl-PA (18:0/22:6-PA) to a comparable extent as phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), which is known to associate with AP180. An AP180 N-terminal homology domain (1-289 aa) interacted with 18:0/22:6-PA, and a lysine-rich motif (K38-K39-K40) was essential for binding. The 18:0/22:6-PA in liposomes in 100 nm diameter showed strong AP180-binding activity at neutral pH. Notably, 18:0/22:6-PA significantly attenuated the interaction of AP180 with clathrin. However, PI(4,5)P2 did not show such an effect. Taken together, these results indicate the novel mechanism by which 18:0/22:6-PA selectively regulates the disassembly of AP180/clathrin-containing cages.

Clathrin packets move in slow axonal transport and deliver functional payloads to synapses.

In non-neuronal cells, clathrin has established roles in endocytosis, with clathrin cages enclosing plasma membrane infoldings, followed by rapid disassembly and reuse of monomers. However, in neurons, clathrin is conveyed in slow axonal transport over days to weeks, and the underlying transport/targeting mechanisms, mobile cargo structures, and even its precise presynaptic localization and physiologic role are unclear. Combining live imaging, photobleaching/conversion, mass spectrometry, electron microscopy, and super-resolution imaging, we found that unlike in dendrites, where clathrin cages rapidly assemble and disassemble, in axons, clathrin and related proteins organize into stable "transport packets" that are unrelated to endocytosis and move intermittently on microtubules, generating an overall slow anterograde flow. At synapses, multiple clathrin packets abut synaptic vesicle (SV) clusters, and clathrin packets also exchange between synaptic boutons in a microtubule-dependent "superpool." Within synaptic boundaries, clathrin is surprisingly dynamic, continuously exchanging between local clathrin assemblies, and its depletion impairs SV recycling. Our data provide a conceptual framework for understanding clathrin trafficking and presynaptic targeting that has functional implications.

TXNIP interaction with GLUT1 depends on PI(4,5)P2.

GLUT1 is a major glucose facilitator expressed ubiquitously among tissues. Upregulation of its expression plays an important role in the development of many types of cancer and metabolic diseases. Thioredoxin-interacting protein (TXNIP) is an α-arrestin that acts as an adaptor for GLUT1 in clathrin-mediated endocytosis. It regulates cellular glucose uptake in response to both intracellular and extracellular signals via its control on GLUT1-4. In order to understand the interaction between GLUT1 and TXNIP, we generated GLUT1 lipid nanodiscs and carried out isothermal titration calorimetry and single-particle electron microscopy experiments. We found that GLUT1 lipid nanodiscs and TXNIP interact in a 1:1 ratio and that this interaction requires phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2 or PIP2).

Multi-modal adaptor-clathrin contacts drive coated vesicle assembly.

Clathrin-coated pits are formed by the recognition of membrane and cargo by the AP2 complex and the subsequent recruitment of clathrin triskelia. A role for AP2 in coated-pit assembly beyond initial clathrin recruitment has not been explored. Clathrin binds the ß2 subunit of AP2, and several binding sites have been identified, but our structural knowledge of these interactions is incomplete and their functional importance during endocytosis is unclear. Here, we analysed the cryo-EM structure of clathrin cages assembled in the presence of ß2 hinge-appendage (ß2HA). We find that the ß2-appendage binds in at least two positions in the cage, demonstrating that multi-modal binding is a fundamental property of clathrin-AP2 interactions. In one position, ß2-appendage cross-links two adjacent terminal domains from different triskelia. Functional analysis of ß2HA-clathrin interactions reveals that endocytosis requires two clathrin interaction sites: a clathrin-box motif on the hinge and the "sandwich site" on the appendage. We propose that ß2-appendage binding to more than one triskelion is a key feature of the system and likely explains why assembly is driven by AP2.

Clathrin: the molecular shape shifter.

Clathrin is best known for its contribution to clathrin-mediated endocytosis yet it also participates to a diverse range of cellular functions. Key to this is clathrin's ability to assemble into polyhedral lattices that include curved football or basket shapes, flat lattices or even tubular structures. In this review, we discuss clathrin structure and coated vesicle formation, how clathrin is utilised within different cellular processes including synaptic vesicle recycling, hormone desensitisation, spermiogenesis, cell migration and mitosis, and how clathrin's remarkable 'shapeshifting' ability to form diverse lattice structures might contribute to its multiple cellular functions.

Computational modeling suggests binding-induced expansion of Epsin disordered regions upon association with AP2.

Intrinsically disordered regions (IDRs) are prevalent in the eukaryotic proteome. Common functional roles of IDRs include forming flexible linkers or undergoing allosteric folding-upon-binding. Recent studies have suggested an additional functional role for IDRs: generating steric pressure on the plasma membrane during endocytosis, via molecular crowding. However, in order to accomplish useful functions, such crowding needs to be regulated in space (e.g., endocytic hotspots) and time (e.g., during vesicle formation). In this work, we explore binding-induced regulation of IDR steric volume. We simulate the IDRs of two proteins from Clathrin-mediated endocytosis (CME) to see if their conformational spaces are regulated via binding-induced expansion. Using Monte-Carlo computational modeling of excluded volumes, we generate large conformational ensembles (3 million) for the IDRs of Epsin and Eps15 and dock the conformers to the alpha subunit of Adaptor Protein 2 (AP2α), their CME binding partner. Our results show that as more molecules of AP2α are bound, the Epsin-derived ensemble shows a significant increase in global dimensions, measured as the radius of Gyration (RG) and the end-to-end distance (EED). Unlike Epsin, Eps15-derived conformers that permit AP2α binding at one motif were found to be more likely to accommodate binding of AP2α at other motifs, suggesting a tendency toward co-accessibility of binding motifs. Co-accessibility was not observed for any pair of binding motifs in Epsin. Thus, we speculate that the disordered regions of Epsin and Eps15 perform different roles during CME, with accessibility in Eps15 allowing it to act as a recruiter of AP2α molecules, while binding-induced expansion of the Epsin disordered region could impose steric pressure and remodel the plasma membrane during vesicle formation.

High-Content Drug Discovery Screening of Endocytosis Pathways.

Endocytosis is the dynamic internalization of cargo (receptors, hormones, viruses) for cellular signaling or processing. It involves multiple mechanisms, classified depending on critical proteins involved, speed, morphology of the derived intracellular vesicles, or substance trafficked. Pharmacological targeting of specific endocytosis pathways has a proven utility for diverse clinical applications from epilepsy to cancer. A multiplexable, high-content screening assay has been designed and implemented to assess various forms of endocytic trafficking and the associated impact of potential small molecule modulators. The applications of this assay include (1) drug discovery in the search for specific, cell-permeable endocytosis pathway inhibitors (and associated analogues from structure-activity relationship studies), (2) deciphering the mechanism of internalization for a novel ligand (using pathway-specific inhibitors), (3) assessment of the importance of specific proteins in the trafficking process (using CRISPR-Cas9 technology, siRNA treatment, or transfection), and (4) identifying whether endocytosis inhibition is an off-target for novel compounds designed for alternative purposes. We describe this method in detail and provide a range of troubleshooting options and alternatives to modify the protocol for lab-specific applications.

vLUME: 3D virtual reality for single-molecule localization microscopy.

vLUME is a virtual reality software package designed to render large three-dimensional single-molecule localization microscopy datasets. vLUME features include visualization, segmentation, bespoke analysis of complex local geometries and exporting features. vLUME can perform complex analysis on real three-dimensional biological samples that would otherwise be impossible by using regular flat-screen visualization programs.

Barbadin selectively modulates FPR2-mediated neutrophil functions independent of receptor endocytosis.

FPR2, a member of the family of G protein-coupled receptors (GPCRs), mediates neutrophil migration, a response that has been linked to ß-arrestin recruitment. ß-Arrestin regulates GPCR endocytosis and can also elicit non-canonical receptor signaling. To determine the poorly understood role of ß-arrestin in FPR2 endocytosis and in NADPH-oxidase activation in neutrophils, Barbadin was used as a research tool in this study. Barbadin has been shown to bind the clathrin adaptor protein (AP2) and thereby prevent ß-arrestin/AP2 interaction and ß-arrestin-mediated GPCR endocytosis. In agreement with this, AP2/ß-arrestin interaction induced by an FPR2-specific agonist was inhibited by Barbadin. Unexpectedly, however, Barbadin did not inhibit FPR2 endocytosis, indicating that a mechanism independent of ß-arrestin/AP2 interaction may sustain FPR2 endocytosis. This was confirmed by the fact, that FPR2 also underwent agonist-promoted endocytosis in ß-arrestin deficient cells, albeit at a diminished level as compared to wild type cells. Dissection of the Barbadin effects on FPR2-mediated neutrophil functions including NADPH-oxidase activation mediated release of reactive oxygen species (ROS) and chemotaxis revealed that Barbadin had no effect on chemotactic migration whereas the release of ROS was potentiated/primed. The effect of Barbadin on ROS production was reversible, independent of ß-arrestin recruitment, and similar to that induced by latrunculin A. Taken together, our data demonstrate that endocytic uptake of FPR2 occurs independently of ß-arrestin, while Barbadin selectively augments FPR2-mediated ROS production independently of receptor endocytosis. Given that Barbadin binds to AP2 and prevents the AP2/ß-arrestin interaction, our results indicate a role for AP2 in FPR2-mediated ROS release from neutrophils.

Protamine sulphate coated poly (lactide-co-glycolide) nanoparticles of MUC-1 peptide improved cellular uptake and cytokine release in mouse antigen presenting cells.

AIM: MUC-1-peptide (M-1-pep) loaded poly (lactide-co-glycolide) nanoparticles were coated with protamine sulphate (PS), M-1-pep-PS-P-NPs for targeting antigen presenting cells (APCs) to evoke cytokine release. METHODS AND RESULTS: M-1-pep-PS-P-NPs were tailored by emulsion-diffusion evaporation method and characterised in vitro under a set of rigorous parameters. The average particle size and zeta potential of optimised M-1-pep-PS-P-B-NPs was measured to be 132.21 ± 30.71 nm and 6.29 ± 0.71 mV, significantly (p < 0.01) higher than 71.24 ± 17.76-nm and -43.41 ± 3.37 mV of M-1-pep-P-NPs. Further, 50-µg/ml concentration of M-1-pep-PS-P-B-NPs displayed 82.4% cellular uptake in RAW 264.7 cells calculated in setting of fluorescence intensity significantly (p < 0.05) elevated than 63.1% of M-1-pep-P-NPs. Consistent to quantitative results, M-1-pep-PS-P-B-NPs also confirmed advanced cellular uptake (CU) in RAW 264.7 cells in contrast to M-1-pep-P-NPs suppose to be through multiple mechanisms including phagocytosis and clathrin mediated endocytosis. CONCLUSION: M-1-pep-PS-P-B-NPs must be evaluated in vivo through inhalation route of administration for antitumor prospective in lung cancer xenograft model.

Clathrin's life beyond 40: Connecting biochemistry with physiology and disease.

Understanding of the range and mechanisms of clathrin functions has developed exponentially since clathrin's discovery in 1975. Here, newly established molecular mechanisms that regulate clathrin activity and connect clathrin pathways to differentiation, disease and physiological processes such as glucose metabolism are reviewed. Diversity and commonalities of clathrin pathways across the tree of life reveal species-specific differences enabling functional plasticity in both membrane traffic and cytokinesis. New structural information on clathrin coat formation and cargo interactions emphasises the interplay between clathrin, adaptor proteins, lipids and cargo, and how this interplay regulates quality control of clathrin's function and is compromised in infection and neurological disease. Roles for balancing clathrin-mediated cargo transport are defined in stem cell development and additional disease states.

Clathrin mediated endocytosis is involved in the uptake of exogenous double-stranded RNA in the white mold phytopathogen Sclerotinia sclerotiorum.

RNA interference (RNAi) technologies have recently been developed to control a growing number of agronomically significant fungal phytopathogens, including the white mold pathogen, Sclerotinia sclerotiorum. Exposure of this fungus to exogenous double-stranded RNA (dsRNA) results in potent RNAi-mediated knockdown of target genes' transcripts, but it is unclear how the dsRNA can enter the fungal cells. In nematodes, specialized dsRNA transport proteins such as SID-1 facilitate dsRNA uptake, but for many other eukaryotes in which the dsRNA uptake mechanisms have been examined, endocytosis appears to mediate the uptake process. In this study, using live cell imaging, transgenic fungal cultures and endocytic inhibitors, we determined that the uptake mechanism in S. sclerotiorum occurs through clathrin-mediated endocytosis. RNAi-mediated knockdown of several clathrin-mediated endocytic genes' transcripts confirmed the involvement of this cellular uptake process in facilitating RNAi in this fungus. Understanding the mode of dsRNA entry into the fungus will prove useful in designing and optimizing future dsRNA-based control methods and in anticipating possible mechanisms by which phytopathogens may develop resistance to this novel category of fungicides.

Microtubule polymerization state and clathrin-dependent internalization regulate dynamics of cardiac potassium channel: Microtubule and clathrin control of KV1.5 channel.

Ion channel trafficking powerfully influences cardiac electrical activity as it regulates the number of available channels at the plasma membrane. Studies have largely focused on identifying the molecular determinants of the trafficking of the atria-specific KV1.5 channel, the molecular basis of the ultra-rapid delayed rectifier current IKur. Besides, regulated KV1.5 channel recycling upon changes in homeostatic state and mechanical constraints in native cardiomyocytes has been well documented. Here, using cutting-edge imaging in live myocytes, we investigated the dynamics of this channel in the plasma membrane. We demonstrate that the clathrin pathway is a major regulator of the functional expression of KV1.5 channels in atrial myocytes, with the microtubule network as the prominent organizer of KV1.5 transport within the membrane. Both clathrin blockade and microtubule disruption result in channel clusterization with reduced membrane mobility and internalization, whereas disassembly of the actin cytoskeleton does not. Mobile KV1.5 channels are associated with the microtubule plus-end tracking protein EB1 whereas static KV1.5 clusters are associated with stable acetylated microtubules. In human biopsies from patients in atrial fibrillation associated with atrial remodeling, drastic modifications in the trafficking balance occurs together with alteration in microtubule polymerization state resulting in modest reduced endocytosis and increased recycling. Consequently, hallmark of atrial KV1.5 dynamics within the membrane is clathrin- and microtubule- dependent. During atrial remodeling, predominance of anterograde trafficking activity over retrograde trafficking could result in accumulation ok KV1.5 channels in the plasma membrane.