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The origin of the eukaryotic cell is a major open question in biology. Asgard archaea are the closest known prokaryotic relatives of eukaryotes, and their genomes encode various eukaryotic signature proteins, indicating some elements of cellular complexity prior to the emergence of the first eukaryotic cell. Yet, microscopic evidence to demonstrate the cellular structure of uncultivated Asgard archaea in the environment is thus far lacking. We used primer-free sequencing to retrieve 715 almost full-length Loki- and Heimdallarchaeota 16S rRNA sequences and designed novel oligonucleotide probes to visualize their cells in marine sediments (Aarhus Bay, Denmark) using catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH). Super-resolution microscopy revealed 1–2 µm large, coccoid cells, sometimes occurring as aggregates. Remarkably, the DNA staining was spatially separated from ribosome-originated FISH signals by 50–280 nm. This suggests that the genomic material is condensed and spatially distinct in a particular location and could indicate compartmentalization or membrane invagination in Asgard archaeal cells.

A P2X1-eYFP knock-in mouse was generated to study receptor expression and mobility in smooth muscle and blood cells. eYFP was added to the C-terminus of the P2X1R and replaced the native P2X1R. Fluorescence corresponding to P2X1-eYFPR was detected in urinary bladder smooth muscle, platelets and megakaryocytes. ATP-evoked currents from wild type and P2X1-eYFP isolated urinary bladder smooth muscle cells had the same peak current amplitude and time-course showing that the eYFP addition had no obvious effect on properties. Fluorescence recovery after photobleaching (FRAP) in bladder smooth muscle cells demonstrated that surface P2X1Rs are mobile and their movement is reduced following cholesterol depletion. Compared to the platelet and megakaryocyte, P2X1-eYFP fluorescence was negligible in red blood cells and the majority of smaller marrow cells. The spatial pattern of P2X1-eYFP fluorescence in the megakaryocyte along with FRAP assessment of mobility suggested that P2X1Rs are expressed extensively throughout the membrane invagination system of this cell type. The current study highlights that the spatiotemporal properties of P2X1R expression can be monitored in real time in smooth muscle cells and megakaryocytes/platelets using the eYFP knock-in mouse model. Electronic supplementary material The online version of this article (10.1007/s11302-019-09666-1) contains supplementary material, which is available to authorized users.

Endocytosis is a fundamental cellular trafficking pathway, which requires an organized assembly of the multiprotein endocytic coat to pull the plasma membrane into the cell. Although the protein composition of the endocytic coat is known, its functional architecture is not well understood. Here, we determine the nanoscale organization of the endocytic coat by FRET microscopy in yeast Saccharomyces cerevisiae. We assessed pairwise proximities of 18 conserved coat‐associated proteins and used clathrin subunits and protein truncations as molecular rulers to obtain a high‐resolution protein map of the coat. Furthermore, we followed rearrangements of coat proteins during membrane invagination and their binding dynamics at the endocytic site. We show that the endocytic coat proteins are not confined inside the clathrin lattice, but form distinct functional layers above and below the lattice. Importantly, key endocytic proteins transverse the clathrin lattice deeply into the cytoplasm connecting thus the membrane and cytoplasmic parts of the coat. We propose that this design enables an efficient and regulated function of the endocytic coat during endocytic vesicle formation., FRET‐based protein proximity mapping reveals the nanoscale organization of the conserved endocytic coat: Coat proteins form several functional layers above and below the clathrin lattice with key endocytic factors transversing through it.

Chromosomal rearrangements that facilitate tumor formation and progression through activation of oncogenic tyrosine kinases are frequently observed in cancer. The ETV6-NTRK3 (EN) fusion has been implicated in various cancers, including infantile fibrosarcoma, secretory breast carcinoma, and acute myeloblastic leukemia, and has exhibited in vivo and in vitro transforming ability. In the present study, we analyzed transcriptome alterations using DNA microarray and RNA-Seq in EN-transduced NIH3T3 fibroblasts to identify the mechanisms that are involved in EN-mediated tumorigenesis. Through functional profile assessment of EN-regulated transcriptome alterations, we found that upregulated genes by EN were mainly associated with cell motion, membrane invagination, and cell proliferation, while downregulated genes were involved in cell adhesion, which correlated with the transforming potential and increased proliferation in EN-transduced cells. KEGG pathway analysis identified the JAK-STAT signaling pathway with the highest statistical significance. Moreover, Ingenuity Pathway Analysis and gene regulatory network analysis identified the STAT1 transcription factor and its target genes as top EN-regulated molecules. We further demonstrated that EN enhanced STAT1 phosphorylation but attenuated STAT1 acetylation, eventually inhibiting the interaction between the NF-κB p65 subunit and acetylated STAT1. Consequently, nuclear translocation of NF-κB p65 and subsequent NF-κB activity were increased by EN. Notably, inhibition of STAT1 phosphorylation attenuated tumorigenic ability of EN in vitro and in vivo. Taken together, here we report, for the first time, STAT1 as a significant EN-regulated transcription factor and a crucial mediator of EN-induced tumorigenesis.

The aggregation of the tau protein into neurofibrillary tangles is believed to correlate with cognitive decline in several neurodegenerative disorders, including Alzheimer's disease. Recent studies suggest that tau's interactions with the cell membrane could serve as a toxicity pathway and also enhance fibrillation into paired helical filaments (PHFs). Conformational changes associated with tau-membrane interactions are poorly understood, and their characterization could improve our understanding of tau pathogenicity. In this study, we investigated the molecular level structural changes associated with the interaction of the tau hexapeptide PHF6 with model lipid membranes and characterized the effects of these interactions on membrane stability and peptide fibrillation. We used two PHF6 forms, the aggregation-prone PHF6 with N-terminal acetylation (Ac-PHF6) and the non-aggregation prone PHF6 with a standard N terminus (NH(3)(+)-PHF6). We found that both PHF6 peptides are neurotoxic and exhibit similar membrane-mediated changes, consisting of: 1) favorable interactions with anionic membranes, 2) membrane destabilization through lipid extraction, and 3) membrane-mediated fibrillation. The rate at which these changes occurred was the main difference between the two peptides. NH(3)(+)-PHF6 displayed slow membrane-mediated fibrillation after 6 days of incubation, whereas Ac-PHF6 adopted a β-sheet conformation at the surface of the membrane within hours. Ac-PHF6 interactions with the membrane were also accompanied by membrane invagination and rapid membrane destabilization. Overall, our results reveal that membrane interactions could play a critical role in tau toxicity and fibrillation, and highlight that unraveling these interactions is important for significantly advancing the development of therapeutic strategies to manage tau-associated neurodegenerative diseases.

Inhomogeneous structure occurring in biological membranes being rich in glycosphingolipids (GSL) has been proposed as an important phenomenon involved in the cellular endocytosis process. However, little is known about the correlation between the formation of microdomains and the GSL-dependent biogenesis for tubular endocytic pits occurred on the surface of the cellular membrane. In the present work, the interaction between the bacterial Shiga toxin from Escherichia coli (STxB) and its cellular receptor GSL globotriaosylceramide (Gb3) were studied using small unilamellar vesicle (SUV). The model membrane invagination induced by STxB was determined by the contrast variation small-angle neutron scattering (SANS) and the synchrotron radiation facility based small-angle X-ray scattering (SR-SAXS). The results revealed that Gb3 molecules provided the binding sites for STxB, inducing increased membrane fluctuation. The formation of protein–lipid complex (STxB-Gb3) apparently induced the thinning of model membrane with the thickness decreased from 3.10 nm to 2.50 nm. It is the first time to successfully characterize the mesoscopic change on membrane thickness upon GSL-dependent endocytic process using a small-angle scattering technique. Overall, this paper provided a practical method to quantify the inhomogeneous biological membrane structures, which is important to understand the cellular endocytosis process. © 2021 by the authors. Licensee MDPI, Basel, Switzerland.

Bacteria must divide to increase in number and colonize their niche. Binary fission is the most widespread means of bacterial cell division, but even this relatively simple mechanism has many variations on a theme. In most bacteria, the tubulin homologue FtsZ assembles into a ring structure, termed the Z ring, at the site of cytokinesis and recruits additional proteins to form a large protein machine - the divisome - that spans the membrane. In this Review, we discuss current insights into the regulation of the assembly of the Z ring and how the divisome drives membrane invagination and septal cell wall growth while flexibly responding to various cellular inputs.

Viruses that generate double-stranded RNA (dsRNA) during replication must overcome host defense systems designed to detect this infection intermediate. All positive-sense RNA viruses studied to date modify host membranes to help facilitate the sequestration of dsRNA from host defenses and concentrate replication factors to enhance RNA production. Flock House virus (FHV) is an attractive model for the study of these processes since it is well characterized and infects Drosophila cells, which are known to have a highly effective RNA silencing system. During infection, FHV modifies the outer membrane of host mitochondria to form numerous membrane invaginations, called spherules, that are ∼50 nm in diameter and known to be the site of viral RNA replication. While previous studies have outlined basic structural features of these invaginations, very little is known about the mechanism underlying their formation. Here we describe the optimization of an experimental system for the analysis of FHV host membrane modifications using crude mitochondrial preparations from infected Drosophila cells. These preparations can be programmed to synthesize both single- and double-stranded FHV RNA. The system was used to demonstrate that dsRNA is protected from nuclease digestion by virus-induced membrane invaginations and that spherules play an important role in stimulating RNA replication. Finally, we show that spherules generated during FHV infection appear to be dynamic as evidenced by their ability to form or disperse based on the presence or absence of RNA synthesis. IMPORTANCE It is well established that positive-sense RNA viruses induce significant membrane rearrangements in infected cells. However, the molecular mechanisms underlying these rearrangements, particularly membrane invagination and spherule formation, remain essentially unknown. How the formation of spherules enhances viral RNA synthesis is also not understood, although it is assumed to be partly a result of evading host defense pathways. To help interrogate some of these issues, we optimized a cell-free replication system consisting of mitochondria isolated from Flock House virus-infected Drosophila cells for use in biochemical and structural studies. Our data suggest that spherules generated during Flock House virus replication are dynamic, protect double-stranded RNA, and enhance RNA replication in general. Cryo-electron microscopy suggests that the samples are amenable to detailed structural analyses of spherules engaged in RNA synthesis. This system thus provides a foundation for understanding the molecular mechanisms underlying spherule formation, maintenance, and function during positive-sense viral RNA replication.

Endocytosis, the process whereby the plasma membrane invaginates to form vesicles, is essential for bringing many substances into the cell and for membrane turnover. The mechanism driving clathrin-mediated endocytosis (CME) involves > 50 different protein components assembling at a single location on the plasma membrane in a temporally ordered and hierarchal pathway. These proteins perform precisely choreographed steps that promote receptor recognition and clustering, membrane remodeling, and force-generating actin-filament assembly and turnover to drive membrane invagination and vesicle scission. Many critical aspects of the CME mechanism are conserved from yeast to mammals and were first elucidated in yeast, demonstrating that it is a powerful system for studying endocytosis. In this review, we describe our current mechanistic understanding of each step in the process of yeast CME, and the essential roles played by actin polymerization at these sites, while providing a historical perspective of how the landscape has changed since the preceding version of the YeastBook was published 17 years ago (1997). Finally, we discuss the key unresolved issues and where future studies might be headed.

How various macromolecules are exchanged between cells and how they gain entry into recipient cells are fundamental questions in cell biology with important implications e.g. non-viral drug delivery, infectious disease, metabolic disorders, and cancer. The role of heparan sulfate proteoglycan (HSPG) as a cell-surface receptor of diverse macromolecular cargo has recently been manifested. Exosomes, cell penetrating peptides, polycation-nucleic acid complexes, viruses, lipoproteins, growth factors and morphogens among other ligands enter cells through HSPG-mediated endocytosis. Key questions that partially have been unraveled over recent years include the respective roles of HSPG core protein and HS chain structure specificity for macromolecular cargo endocytosis, the down-stream intracellular signaling events involved in HSPG-dependent membrane invagination and vesicle formation, and the biological significance of the HSPG transport pathway. Here, we discuss the intriguing role of HSPGs as a major entry pathway of macromolecules in mammalian cells with emphasis on recent in vitro and in vivo data that provide compelling evidence of HSPG as an autonomous endocytosis receptor.

The model photosynthetic bacterium Rhodobacter sphaeroides uses a network of bacteriochlorophyll (BChl)-protein complexes embedded in spherical intracytoplasmic membranes (ICM) to collect and utilise solar energy. We studied the effects of high- and low-light growth conditions, where BChl levels increased approximately four-fold from 1.6×10(6) to 6.5×10(6) molecules per cell. Most of this extra pigment is accommodated in the proliferating ICM system, which increases from approximately 274 to 1468 vesicles per cell. Thus, 16×10(6)nm(2) of specialised membrane surface area is made available for harvesting and utilising solar energy compared to 3×10(6)nm(2) under high-light conditions. Membrane mapping using atomic force microscopy revealed closely packed dimeric and monomeric reaction centre-light harvesting 1-PufX (RC-LH1-PufX) complexes in high-light ICM with room only for small clusters of LH2, whereas extensive LH2-only domains form during adaptation to low light, with the LH2/RC ratio increasing three-fold. The number of upper pigmented band (UPB) sites where membrane invagination is initiated hardly varied; 704 (5.8×10(5) BChls/cell) and 829 (4.9×10(5) BChls/cell) UPB sites per cell were estimated under high- and low-light conditions, respectively. Thus, the lower ICM content in high-light cells is a consequence of fewer ICM invaginations reaching maturity. Taking into account the relatively poor LH2-to-LH1 energy transfer in UPB membranes it is likely that high-light cells are relatively inefficient at energy trapping, but can grow well enough without the need to fully develop their photosynthetic membranes from the relatively inefficient UPB to highly efficient mature ICM.

In this review, we elucidate the mechanisms of Aβ oligomer toxicity which may contribute to Alzheimer’s disease (AD). In particular, we discuss on the interaction of Aβ oligomers with the membrane through the process of adsorption and insertion. Such interaction gives rises to phase transitions in the sub-structures of the Aβ peptide from α-helical to β-sheet structure. By means of a coarse-grained model, we exhibit the tendency of β-sheet structures to aggregate, thus providing further insights to the process of membrane induced aggregation. We show that the aggregated oligomer causes membrane invagination, which is a precursor to the formation of pore structures and ion channels. Other pathological progressions to AD due to Aβ oligomers are also covered, such as their interaction with the membrane receptors, and their direct versus indirect effects on oxidative stress and intraneuronal accumulation. We further illustrate that the molecule curcumin is a potential Aβ toxicity inhibitor as a β-sheet breaker by having a high propensity to interact with certain Aβ residues without binding to them. The comprehensive understanding gained from these current researches on the various toxicity mechanisms show promises in the provision of better therapeutics and treatment strategies in the near future.

Eisosomes, large protein complexes that are predominantly composed of BAR-domain-containing proteins Pil1 and its homologs, are situated under the plasma membrane of ascomycetes. A successful targeting of Pil1 onto the future site of eisosome accompanies maturation of eisosome. During or after recruitment, Pil1 undergoes self-assembly into filaments that can serve as scaffolds to induce membrane furrows or invaginations. Although a consequence of the invagination is likely to redistribute particular proteins and lipids to a different location, the precise physiological role of membrane invagination and eisosome assembly awaits further investigation. The present review summarizes recent research findings within the field regarding the detailed structural and functional significance of Pil1 on eisosome organization.

Synaptic transmission relies on effective and accurate compensatory endocytosis. F-BAR proteins may serve as membrane curvature sensors and/or inducers and thereby support membrane remodelling processes; yet, their in vivo functions urgently await disclosure. We demonstrate that the F-BAR protein syndapin I is crucial for proper brain function. Syndapin I knockout (KO) mice suffer from seizures, a phenotype consistent with excessive hippocampal network activity. Loss of syndapin I causes defects in presynaptic membrane trafficking processes, which are especially evident under high-capacity retrieval conditions, accumulation of endocytic intermediates, loss of synaptic vesicle (SV) size control, impaired activity-dependent SV retrieval and defective synaptic activity. Detailed molecular analyses demonstrate that syndapin I plays an important role in the recruitment of all dynamin isoforms, central players in vesicle fission reactions, to the membrane. Consistently, syndapin I KO mice share phenotypes with dynamin I KO mice, whereas their seizure phenotype is very reminiscent of fitful mice expressing a mutant dynamin. Thus, syndapin I acts as pivotal membrane anchoring factor for dynamins during regeneration of SVs.

In the model photosynthetic bacterium Rhodobacter sphaeroides domains of light-harvesting 2 (LH2) complexes surround and interconnect dimeric reaction centre–light-harvesting 1–PufX (RC–LH1–PufX) ‘core’ complexes, forming extensive networks for energy transfer and trapping. These complexes are housed in spherical intracytoplasmic membranes (ICMs), which are assembled in a stepwise process where biosynthesis of core complexes tends to dominate the early stages of membrane invagination. The kinetics of LH2 assembly were measured in PufX mutants that assemble monomeric core complexes, as a consequence of either a twelve-residue N-terminal truncation of PufX (PufXΔ12) or the complete removal of PufX (PufX−). Lower rates of LH2 assembly and retarded maturation of membrane invagination were observed for the larger and less curved ICM from the PufX− mutant, consistent with the proposition that local membrane curvature, initiated by arrays of bent RC–LH1–PufX dimers, creates a favourable environment for stable assembly of LH2 complexes. Transmission electron microscopy and high-resolution atomic force microscopy were used to examine ICM morphology and membrane protein organisation in these mutants. Some partitioning of core and LH2 complexes was observed in PufX− membranes, resulting in locally ordered clusters of monomeric RC–LH1 complexes. The distribution of core and LH2 complexes in the three types of membrane examined is consistent with previous models of membrane curvature and domain formation (Frese et al., 2008), which demonstrated that a combination of crowding and asymmetries in sizes and shapes of membrane protein complexes drives membrane organisation.

Osteoblastic and osteocytic cells are highly responsive to the lipid growth factor lysophosphatidic acid (LPA) but the mechanisms by which LPA alters bone cell functions are largely unknown. A major effect of LPA on osteocytic cells is the stimulation of dendrite membrane outgrowth, a process that we predicted to require changes in gene expression and protein distribution. We employed DNA microarrays for global transcriptional profiling of MLO-Y4 osteocytic cells grown for 6 and 24 hours in the presence or absence of LPA. We identified 932 transcripts that displayed statistically significant changes in abundance of at least 1.25-fold in response to LPA treatment. Gene ontology (GO) analysis revealed that the regulated gene products were linked to diverse cellular processes, including DNA repair, response to unfolded protein, ossification, protein-RNA complex assembly, and amine biosynthesis. Gene products associated with the regulation of actin microfilament dynamics displayed the most robust expression changes, and LPA-induced dendritogenesis in vitro was blocked by the stress fiber inhibitor cytochalasin D. Mass spectrometry-based proteomic analysis of MLO-Y4 cells revealed significant LPA-induced changes in the abundance of 284 proteins at 6 hours and 844 proteins at 24 hours. GO analysis of the proteomic data linked the effects of LPA to cell processes that control of protein distribution and membrane outgrowth, including protein localization, protein complex assembly, Golgi vesicle transport, cytoskeleton-dependent transport, and membrane invagination/endocytosis. Dendrites were isolated from LPA-treated MLO-Y4 cells and subjected to proteomic analysis to quantitatively assess the subcellular distribution of proteins. Sets of 129 and 36 proteins were enriched in the dendrite fraction as compared to whole cells after 6 hours and 24 hours of LPA exposure, respectively. Protein markers indicated that membranous organelles were largely excluded from the dendrites. Highly represented among the proteins with elevated abundances in dendrites were molecules that regulate cytoskeletal function, cell motility and membrane adhesion. Our combined transcriptomic/proteomic analysis of the response of MLO-Y4 osteocytic cells to LPA indicates that dendritogenesis is a membrane- and cytoskeleton-driven process with actin dynamics playing a particularly critical role.

Transformation (i.e., genetic modification of a cell by the incorporation of exogenous DNA) is indispensable for manipulating fungi. Here, we review the transformation methods for Saccharomyces cerevisiae, Schizosaccharomyces pombe, Candida albicans, Pichia pastoris and Aspergillus species and discuss some common modifications to improve transformation efficiency. We also present a model of the mechanism underlying S. cerevisiae transformation, based on recent reports and the mechanism of transfection in mammalian systems. This model predicts that DNA attaches to the cell wall and enters the cell via endocytotic membrane invagination, although how DNA reaches the nucleus is unknown. Polyethylene glycol is indispensable for successful transformation of intact cells and the attachment of DNA and also possibly acts on the membrane to increase the transformation efficiency. Both lithium acetate and heat shock, which enhance the transformation efficiency of intact cells but not that of spheroplasts, probably help DNA to pass through the cell wall.

Summary Clathrin-mediated endocytosis is an essential cellular function in all eukaryotes that is driven by a self-assembled macromolecular machine of over 50 different proteins in tens to hundreds of copies. How these proteins are organized to produce endocytic vesicles with high precision and efficiency is not understood. Here, we developed high-throughput superresolution microscopy to reconstruct the nanoscale structural organization of 23 endocytic proteins from over 100,000 endocytic sites in yeast. We found that proteins assemble by radially ordered recruitment according to function. WASP family proteins form a circular nanoscale template on the membrane to spatially control actin nucleation during vesicle formation. Mathematical modeling of actin polymerization showed that this WASP nano-template optimizes force generation for membrane invagination and substantially increases the efficiency of endocytosis. Such nanoscale pre-patterning of actin nucleation may represent a general design principle for directional force generation in membrane remodeling processes such as during cell migration and division., Graphical Abstract, Highlights • High-throughput superresolution imaging of 23 proteins at thousands of endocytic sites • Endocytic proteins organize into nanoscale radial zones according to their function • A circular WASP nano-template patterns actin filament nucleation • The WASP nano-template is crucial for high efficiency of endocytosis, Superresolution analysis of endocytic vesicle assembly identifies a patterned actin template that orchestrates protein recruitment.

Plants maintained in high soil salinity generally develop particular structures to either tolerate or survive such adverse environments. Excretion of excess ions by special salt glands or other similar structures is a well-known phenomenon for regulating the mineral content of many halophytes. However, the three chenopod halophytes of Suaeda inhabit high saline soils, yet they exhibit no signs of salt excretion structures. The current study has been undertaken to assess the structural attributes of these halophytes to reveal their cellular characteristics during growth in salt tolerance. Transmission and scanning electron microscopy, as well as ion chromatography, have been employed for the study. One of the most noticeable features uncovered was the epidermal cutinization shown to be heavy on the outer epidermis and characterized externally by thick wax plates. Numerous vesicles and membranous invagination in the vacuoles were common features within the mesophyll cytoplasm. Invaginations of the vacuolar and/or plasma membrane frequently formed secondary vacuoles which later became distinct, membrane-bound compartments. Significant accumulation of solid sodium chloride salts was well demonstrated in the vacuoles of air-dried epidermis. Finally, salt tolerance mechanisms in these Suaeda have been discussed with respect to other halophyte modifications that improve salt tolerance in various ways.

The purple phototrophic bacteria synthesize an extensive system of intracytoplasmic membranes (ICM) in order to increase the surface area for absorbing and utilizing solar energy. Rhodobacter sphaeroides cells contain curved membrane invaginations. In order to study the biogenesis of ICM in this bacterium mature (ICM) and precursor (upper pigmented band – UPB) membranes were purified and compared at the single membrane level using electron, atomic force and fluorescence microscopy, revealing fundamental differences in their morphology, protein organization and function. Cryo-electron tomography demonstrates the complexity of the ICM of Rba. sphaeroides. Some ICM vesicles have no connection with other structures, others are found nearer to the cytoplasmic membrane (CM), often forming interconnected structures that retain a connection to the CM, and possibly having access to the periplasmic space. Near-spherical single invaginations are also observed, still attached to the CM by a ‘neck’. Small indents of the CM are also seen, which are proposed to give rise to the UPB precursor membranes upon cell disruption. ‘Free-living’ ICM vesicles, which possess all the machinery for converting light energy into ATP, can be regarded as bacterial membrane organelles.

SummaryBAR superfamily domains shape membranes through poorly understood mechanisms. We solved structures of F-BAR modules bound to flat and curved bilayers using electron (cryo)microscopy. We show that membrane tubules form when F-BARs polymerize into helical coats that are held together by lateral and tip-to-tip interactions. On gel-state membranes or after mutation of residues along the lateral interaction surface, F-BARs adsorb onto bilayers via surfaces other than their concave face. We conclude that membrane binding is separable from membrane bending, and that imposition of the module's concave surface forces fluid-phase bilayers to bend locally. Furthermore, exposure of the domain's lateral interaction surface through a change in orientation serves as the crucial trigger for assembly of the helical coat and propagation of bilayer bending. The geometric constraints and sequential assembly of the helical lattice explain how F-BAR and classical BAR domains segregate into distinct microdomains, and provide insight into the spatial regulation of membrane invagination.

Internalization of nanoparticles by biomembranes is critical for nanomedicine development; however, this process, especially its dynamics aspect, is still not well understood. Using coarse-grained molecular modeling combined with free energy calculations, we studied the endocytic process for spherical, prolate and oblate particles with varied aspect ratios, volumes and interaction strengths. Rich dynamic wrapping behaviors have been observed. Small ellipsoids follow a pathway that includes particle laying-down, membrane invagination and wrapping, and then disruption of the membrane neck. However, the step of particle laying-down is skipped for large ellipsoids. Because of the significantly decreased local mean curvature at the side edge (oblate ellipsoid) or tips (prolate ellipsoid), the rotation is less favorable for particles with larger volume. Given the existence of a local minimum and an energy barrier during the endocytic process presented by our free energy calculations, the oblate particle provides longer endocytic time than the corresponding prolate particle. For large particles, the free energy surfaces are smooth, with no local minimum. When we increase the interaction strength between the membrane and the particle, the endocytic process is greatly affected. Moreover, a “sandwiched structure”, in which the particle lays between the two membrane layers, was observed for both prolate and oblate particles.

Clathrin seems to be dispensable for some endocytic processes and, in several instances, no cytosolic coat protein complexes could be detected at sites of membrane invagination. Hence, new principles must in these cases be invoked to account for the mechanical force driving membrane shape changes. Here we show that the Gb3 (glycolipid)-binding B-subunit of bacterial Shiga toxin induces narrow tubular membrane invaginations in human and mouse cells and model membranes. In cells, tubule occurrence increases on energy depletion and inhibition of dynamin or actin functions. Our data thus demonstrate that active cellular processes are needed for tubule scission rather than tubule formation. We conclude that the B-subunit induces lipid reorganization that favours negative membrane curvature, which drives the formation of inward membrane tubules. Our findings support a model in which the lateral growth of B-subunit–Gb3 microdomains is limited by the invagination process, which itself is regulated by membrane tension. The physical principles underlying this basic cargo-induced membrane uptake may also be relevant to other internalization processes, creating a rationale for conceptualizing the perplexing diversity of endocytic routes. An imaging study of an early step of bacterial toxin intake into cells — membrane invagination — reveals a cargo-induced mechanism that may also apply to other pathogens and more generally to other endocytosis events. The B subunit of Shiga toxin (from Shigella dysenteriae) is seen to enter cells via narrow tubular membrane invaginations. The toxin induces membrane reorganization prior to formation of tubular invaginations, which occurs independently of protein complexes (like clathrin) that have been ascribed membrane deforming capacities, and also when cellular energy is depleted. So membrane invagination relies on physical principles and can occur spontaneously, without the need for sophisticated cellular machinery. A study of endocytosis of Shigella toxin shows that it enters cells via narrow tubular membrane invaginations, with similar properties on cell and model membranes. The toxin induces membrane reorganisation before the formation of tubular invaginations.

Here, we investigated the role of EhVps32 protein (a member of the endosomal-sorting complex required for transport) in endocytosis of Entamoeba histolytica, a professional phagocyte. Confocal microscopy, TEM and cell fractionation revealed EhVps32 in cytoplasmic vesicles and also located adjacent to the plasma membrane. Between 5 to 30 min of phagocytosis, EhVps32 was detected on some erythrocytes-containing phagosomes of acidic nature, and at 60 min it returned to cytoplasmic vesicles and also appeared adjacent to the plasma membrane. TEM images revealed it in membranous structures in the vicinity of ingested erythrocytes. EhVps32, EhADH (an ALIX family member), Gal/GalNac lectin and actin co-localized in the phagocytic cup and in some erythrocytes-containing phagosomes, but EhVps32 was scarcely detected in late phagosomes. During dextran uptake, EhVps32, EhADH and Gal/GalNac lectin, but not actin, co-localized in pinosomes. EhVps32 recombinant protein formed oligomers composed by rings and filaments. Antibodies against EhVps32 monomers stained cytoplasmic vesicles but not erythrocytes-containing phagosomes, suggesting that in vivo oligomers are formed on phagosome membranes. The involvement of EhVps32 in phagocytosis was further study in pNeoEhvps32-HA-transfected trophozoites, which augmented almost twice their rate of erythrophagocytosis as well as the membranous concentric arrays built by filaments, spirals and tunnel-like structures. Some of these structures apparently connected phagosomes with the phagocytic cup. In concordance, the EhVps32-silenced G3 trophozoites ingested 80% less erythrocytes than the G3 strain. Our results suggest that EhVps32 participates in E. histolytica phagocytosis and pinocytosis. It forms oligomers on erythrocytes-containing phagosomes, probably as a part of the scission machinery involved in membrane invagination and intraluminal vesicles formation., Author Summary Trophozoites of E. histolytica represent an excellent model to study endosomal-sorting complex required for transport components due to their high endocytic activity and vesicle trafficking. The key role of EhVps32 on phagocytosis is supported by: i) its presence on phagosomes, ii) its interaction with EhADH (an erythrocytes receptor), Gal/GalNac lectin and actin, iii) the higher rate of erythrophagocytosis showed by EhVps32 overexpressing trophozoites, iv) the diminish rate of phagocytosis in EhVps32-silenced G3 trophozoites, and v) its location in erythrocytes-containing acidic phagosomes. Here, we discovered the presence of membranous concentric helicoidally and tunnel-like structures constituted by EhVps32 and EhADH that may have a dynamic role in membrane remodeling and in the generation of intraluminal vesicles in the phagosomes. Elucidating molecular mechanisms of endocytosis-exocytosis pathways will help us to better understand the pathogenic process of E. histolytica and develop new drugs for diagnosis and vaccine methods.

SummaryEndocytosis depends on an extensive network of interacting proteins that execute a series of distinct subprocesses. Previously, we used live-cell imaging of six budding-yeast proteins to define a pathway for association of receptors, adaptors, and actin during endocytic internalization. Here, we analyzed the effects of 61 deletion mutants on the dynamics of this pathway, revealing functions for 15 proteins, and we analyzed the dynamics of 8 of these proteins. Our studies provide evidence for four protein modules that cooperate to drive coat formation, membrane invagination, actin-meshwork assembly, and vesicle scission during clathrin/actin-mediated endocytosis. We found that clathrin facilitates the initiation of endocytic-site assembly but is not needed for membrane invagination or vesicle formation. Finally, we present evidence that the actin-meshwork assembly that drives membrane invagination is nucleated proximally to the plasma membrane, opposite to the orientation observed for previously studied actin-assembly-driven motility processes.

During infection many bacteriophages deliver only their nucleic acid, leaving the virus capsid outside (for a review, see reference 42). Double-stranded RNA (dsRNA) viruses must also bring virion-associated RNA-dependent RNA polymerases into the cell, since cells do not possess enzymes that are capable of transcribing and replicating viral dsRNA templates. Accordingly, the infection mechanisms of dsRNA bacteriophages differ considerably from bacteriophages that bring only their genome into the host cytosol. The RNA-dependent RNA polymerase of φ6, a dsRNA bacteriophage belonging to the Cystoviridae family, is a component of the viral polyhedral inner capsid (core or polymerase complex), which is surrounded by a shell of protein P8 making the nucleocapsid (NC) (10). φ6 has a lipid-protein envelope that surrounds the NC (28, 50). Bacteriophage φ6 was initially isolated by using Pseudomonas syringae pathovar phaseolicola (50), but it is able to infect several other P. syringae pathovars (14). The primary receptor of φ6 is a chromosomally encoded type IV pilus of P. syringae (2, 50). The host bacteria use these pili to adsorb to the leaf surface of the target plant (44, 45). In contrast to filamentous single-stranded DNA phages that attach to the pilus tip, many RNA phages, such as bacteriophage φ6, attach to the sides of the pilus. φ6 attaches to the pilus with its spike protein P3 (34, 44, 45). To carry the viral polymerase complex into the host cytoplasm, φ6 must traverse the gram-negative cell's envelope, a multilayer barrier composed of the outer membrane (OM), the peptidoglycan layer in the periplasm, and the plasma membrane (PM). The integral phage envelope protein P6 mediates the fusion between the viral membrane and the OM. The fusion leads to the release of the phage NC into the periplasm without leakage of periplasmic markers (4). The NC-associated peptidoglycan-digesting endopeptidase P5 is required to deliver the viral core across the peptidoglycan layer (12, 35). During the final stage of entry, NC penetrates the PM to release the viral core into the host cytosol. This event is assisted by the NC surface protein P8 (2, 25, 41, 46). Based on electron microscopic data, penetration of the NC particle into the cytosol occurs via a membrane invagination and an intracellular vesicle, a process similar to endocytic entry of animal viruses (40, 48). The actual mechanisms of how the NC-containing PM vesicle is pinched off into the cytosol and how the NC is uncoated are currently not known. However, the membrane voltage (Δψ) plays a key role in this process (41). The φ6 entry mechanism is unique among prokaryotes and in many aspects resembles the penetration mechanisms used by both enveloped and nonenveloped animal viruses (20, 40, 48), such as φ6 membrane fusion with the host OM (4), and endocytosis, as the pathway for NC penetration (41, 46). Bacteriophage φ13 was isolated from the leaves of the radish plant (Raphanus sativum) (36). Based on cryo-electron microscopy, the φ13 virion is very similar to that of φ6 (51). The diameter of both virions is ∼86 nm, and the NCs appear as ∼58 nm-diameter particles. Spikes protrude from the enveloped surface of both virions. The genome organization of φ13 is also similar to bacteriophage φ6, and there are similarities in the amino acid sequences of some proteins. However, the products of genes 3, 5, 9, and 10 have no detectable similarity to the corresponding proteins in φ6 (43). The preliminary morphological analysis of cells infected with φ13 also indicates that it uses an entry mechanism similar to that of φ6. However, the Mindich laboratory (36) showed that these viruses use an lipopolysaccharide (LPS) receptor. Phage φ13 does not infect the normal φ6 host but is able to infect a strain that is resistant to φ6 with no type IV pilus but with a truncated O chain of LPS (36). In addition to φ6-mediated envelope fusion, many gram-negative bacteria release OM vesicles packed with periplasmic components (6, 23). These vesicles are able to fuse with the OM of the target cell and introduce their content into the periplasm. In this case two LPS-covered membrane surfaces come into contact and fuse. Small unilamellar phospholipid vesicles are also able to fuse with the surface of gram-negative bacteria but only after the cells have been treated with EDTA (33). Lipid bilayer patches in the OM and bacterial OM proteins seem to be involved in this type of fusion. The enveloped dsRNA phage fusion is unique since the viral phospholipid membrane fuses with the LPS-containing bacterial OM. A number of fluorescent probes have been used to monitor membrane fusion events in animal virus infections (7, 49). Currently, the lipophilic fluorescent dye octadecyl rhodamine B (R18), developed by Keller et al. (27), is the most popular fluorophore used. It has been shown that R18 can be incorporated into intact virions to concentrations inducing self-quenching without redistribution of the probe into the target membrane under conditions where no fusion occurs. After fusion, the virion-bound fluorophore diffuses into the cell membrane, resulting in relief of self-quenching (24, 30, 37), and an increase in fluorescence signal is subsequently observed. We developed an R18-based fusion assay to monitor the entry of phages φ6 and φ13. In addition, membrane-associated events during the initial stages of infection were monitored by potentiometric studies of lipophilic ion distribution to gain information on the changes in P. syringae envelope characteristics during φ6 and φ13 entry.

Summary Bacterial division requires the co-ordination of membrane invagination, driven by the constriction of the FtsZ-ring, and concomitant cell wall synthesis, performed by the high-molecular-weight penicillin-binding proteins (HMW PBPs). Using immunofluorescence techniques, we show in Streptococcus pneumoniae that this co-ordination requires PBP3, a d,d-carboxypeptidase that degrades the substrate of the HMW PBPs. In a mutant deprived of PBP3, the apparent rings of HMW PBPs and that of FtsZ are no longer co-localized. In wild-type cells, PBP3 is absent at the future division site and present over the rest of the cell surface, implying that the localization of the HMW PBPs at mid-cell depends on the availability of their substrate. FtsW, a putative translocase of the substrate of the PBPs, forms an apparent ring that is co-localized with the septal HMW PBPs throughout the cell cycle of wild-type cells. In particular, the constriction of the FtsW-ring occurs after that of the FtsZ-ring, with the same delay as the constriction of the septal PBP-rings. However, in the absence of PBP3, FtsW remains co-localized with FtsZ in contrast to the HMW PBPs. Our work reveals an unexpected complexity in the relationships between the division proteins. The consequences of the absence of PBP3 indicate that the peptidoglycan composition is central to the co-ordination of the division process.

In budding yeast, many proteins involved in endocytic internalization, including adaptors and actin cytoskeletal proteins, are localized to cortical patches of differing protein composition. Using multicolor real-time fluorescence microscopy and particle tracking algorithms, we define an early endocytic pathway wherein an invariant sequence of changes in cortical patch protein composition correlates with changes in patch motility. Three Arp2/3 activators each showed a distinct behavior, suggesting distinct patch-related endocytic functions. Actin polymerization occurs late in the endocytic pathway and is required both for endocytic internalization and for patch disassembly. In cells lacking the highly conserved endocytic protein Sla2p, patch motility was arrested and actin comet tails associated with endocytic patch complexes. Fluorescence recovery after photobleaching of the actin comet tails revealed that endocytic complexes are nucleation sites for rapid actin polymerization. Attention is now focused on the mechanisms by which the order and timing of events in this endocytic pathway are achieved.

Endophilin-1, a cytoplasmic Src homology 3 (SH3) domain-containing protein, localises in brain presynaptic nerve termini. Endophilin dimerises through its N-terminus, and participates at multiple stages in clathrin-coated endocytosis, from early membrane invagination to synaptic vesicle uncoating. Both its C-terminal SH3 domain and N-terminus are required for endocytosis. Through its SH3 domain, endophilin bound to proline-rich domains (PRDs) in other endocytic proteins, including synaptojanin and dynamin. The N-terminal region possesses unique functions affecting lipid membrane curvature, through lysophosphatidic acid acyl transferase (LPAAT) activity and direct binding and tubulating activity. In addition to synaptic vesicle formation, endophilin-1 complexes with signalling molecules, including cell surface receptors, metalloprotease disintegrins and germinal centre kinase-like kinase (GLK). Therefore, endophilin-1 may serve to couple vesicle biogenesis with intracellular signalling cascades.

A major feature that distinguishes prokaryotic organisms from eukaryotes is their less complex internal structure, in which all membrane-associated functions are thought to be present within a continuous lipid-protein bilayer, rather than with distinct organelles. Contrary to this notion, as described by Tucker and co-workers in this issue of Molecular Microbiology, the application of cryo-electron tomography to the purple bacterium Rhodobacter sphaeroides has demonstrated a heretofore unrecognized ultrastructural complexity within the intracytoplasmic membrane (ICM) housing the photosynthetic apparatus. In addition to distinguishing invaginations of the cytoplasmic membrane (CM) and interconnected vesicular structures still attached to the CM, a eukaryote-like ICM budding process was revealed, which results in the formation of fully detached vesicular structures. These bacterial organelles are able to carry out both the light-harvesting and light-driven energy transduction activities necessary for the cells to assume a photosynthetic lifestyle. Their formation is shown to represent the final stage in a membrane invagination and growth process, originating with small CM indentations, which after cell disruption give rise to a membrane fraction that can be separated from mature ICM vesicles by rate-zone sedimentation.

Clathrin-coated vesicles mediate diverse processes such as nutrient uptake, downregulation of hormone receptors, formation of synaptic vesicles, virus entry, and transport of biosynthetic proteins to lysosomes. Cycles of coat assembly and disassembly are integral features of clathrin-mediated vesicular transport (Fig. 1a). Coat assembly involves recruitment of clathrin triskelia, adaptor complexes and other factors that influence coat assembly, cargo sequestration, membrane invagination and scission1,2,3 (Fig. 1a). Coat disassembly is thought to be essential for fusion of vesicles with target membranes and for recycling components of clathrin coats to the cytoplasm for further rounds of vesicle formation. In vitro, cytosolic heat-shock protein 70 (Hsp70) and the J-domain co-chaperone auxilin catalyse coat disassembly4. However, a specific function of these factors in uncoating in vivo has not been demonstrated, leaving the physiological mechanism and significance of uncoating unclear. Here we report the identification and characterization of a Saccharomyces cerevisiae J-domain protein, Aux1. Inactivation of Aux1 results in accumulation of clathrin-coated vesicles, impaired cargo delivery, and an increased ratio of vesicle-associated to cytoplasmic clathrin. Our results demonstrate an in vivo uncoating function of a J domain co-chaperone and establish the physiological significance of uncoating in transport mediated by clathrin-coated vesicles.

The hemolytic activity of beta-cyclodextrin (beta-CyD) on rabbit erythrocytes was reduced by the introduction of negatively-charged groups onto the hydroxyls of beta-CyD; the membrane disrupting abilities decreased in the order of beta-CyD > 2-hydroxypropyl-beta-CyD (HP-beta-CyD) > sulfobutyl-beta-CyD (SB-beta-CyD) >> beta-CyD sulfate (S-beta-CyD). Under pre-hemolytic concentrations, both beta-CyD and SB-beta-CyD induced shape changes of membrane invagination on the erythrocytes. In sharp contrast, S-beta-CyD showed biphasic effect on the shape of the erythrocytes; i.e. the crenation at relatively low concentrations and the invagination at higher concentrations. The S-beta-CyD-induced membrane crenation arose from a direct action on the membranes rather than cell metabolism-mediated effects. Unlike beta-CyD, S-beta-CyD was found to bind to the erythrocytes and may be confined to the outer surface of the membrane bilayer, which may expand the exterior layer relative to the cytoplasmic half, thereby inducing the cells to crenate. On the other hand, the membrane invagination mediated by the three beta-CyDs was initiated by extracting specific membrane lipids from the cells, depending upon their inclusion abilities, subsequently leading to the lysis of the cells. These results indicate that SB-beta-CyD and S-beta-CyD interact with the erythrocyte membranes in a differential manner and possess lower membrane disrupting abilities than the parent beta-CyD and HP-beta-CyD.

During endocytic vesicle formation, distinct subdomains along the membrane invagination are specified by different proteins, which bend the membrane and drive scission. Bin-Amphiphysin-Rvs (BAR) and Fer-CIP4 homology-BAR (F-BAR) proteins can induce membrane curvature and have been suggested to facilitate membrane invagination and scission. Two F-BAR proteins, Syp1 and Bzz1, are found at budding yeast endocytic sites. Syp1 arrives early but departs from the endocytic site before formation of deep membrane invaginations and scission. Using genetic, spatiotemporal, and ultrastructural analyses, we demonstrate that Bzz1, the heterodimeric BAR domain protein Rvs161/167, actin polymerization, and the lipid phosphatase Sjl2 cooperate, each through a distinct mechanism, to induce membrane scission in yeast. Additionally, actin assembly and Rvs161/167 cooperate to drive formation of deep invaginations. Finally, we find that Bzz1, acting at the invagination base, stabilizes endocytic sites and functions with Rvs161/167, localized along the tubule, to achieve proper endocytic membrane geometry necessary for efficient scission. Together, our results reveal that dynamic interplay between a lipid phosphatase, actin assembly, and membrane-sculpting proteins leads to proper membrane shaping, tubule stabilization, and scission.

A continuum model based on the contact mechanics theory was developed and used for evaluating virus indentation forces at the early stage of membrane invagination, as well as the work of the virus indentation forces and virus-cell contact pressures in a receptor-mediated endocytosis, depending on the virus size and virus/cell stiffnesses. The model indicated that early virus indentation forces are in the order of 1–10 pN and for a given extent of virus engulfment, they increase linearly with the elastic modulus of the host cell and also with the square of the virus radius. The work of invagination at the initial phase of virus endocytosis is in the order of tens of zeptojoules and peak virus-cell contact pressures at this stage are in the order of hundreds of Pascals to several kPa. For a given extent of virus engulfment, peak and average contact pressures increase linearly with the elastic modulus of the host cell but interestingly, they are negligibly affected by the virus size. The present model may be useful in the fields of cellular biomechanics, virology and nanodrug delivery to evaluate mechanical factors during the early phase of membrane invagination.

Glomerulopathy and nephrotic syndrome were induced in rats by intravenous puromycin aminonucleoside. Ten days after the injection of puromycin, the animals have developed heavy proteinuria. During this phase, glomerular epithelial cell endocytosis was studied by injecting a conjugate of horseradish peroxidase and poly-L-lysine. This conjugate has been shown to be endocytosed by glomerular epithelial cells. The rats were serially sacrificed from 1 min to 24 h after this injection. Peroxidase was localised cytochemically and observed at light and electron microscopy. The early events of endocytosis in glomerulopathy (namely the binding to the plasma membrane, the membrane invagination and the formation of the early vesicles) were qualitatively similar to those in the normal. The later events (the fusion of the vesicles and their movement within the cells) were inhibited. The results show that puromycin aminonucleoside damages epithelial cell endocytotic activity and affects the later processing of the conjugate within the cells.

During this process, cargo mol-ecules are clustered and concentrated within the emerging coated pit. After coat assembly and membrane invagination, which is facilitated by BAR domain-containing proteins, vesicle fission is catalyzed by the recruitment and helical assembly of the GTPase dynamin at the neck of the burgeoning vesicle. Membrane scission requires the cooperative activity of dynamin with SH3 domain-containing proteins, many of which possess BAR domains and regulate the actin cytoskeleton. Actin is required for endocytosis in yeast and may have a regulatory role in mammalian cells. The actin-associated motor proteins myosin 1E and myosin VI are linked to the endocytic machinery via dynamin or synaptojanin and Dab2, respectively. During vesicle uncoating, primary endocytic vesicles may acquire Rab5, a small GTPase involved in transport to early endosomes and in early endosome docking and fusion. Primary endocytic vesicles are targeted to and fuse with early endosomes expressing phosphatidylinositol(3)-phosphate, PI(3)P, in a process dependent on Rab5 effector proteins and early endosomal SNAREs. Differential sorting of cargo destined for recycling (requiring Rab11 and possibly Rab4) or for late endosome/lysosomal degradation (requiring Rab7) can occur at the level of clathrin-coated pits and be com-pleted in early endosomal subdomains. Transport of cargo from early endosomes to the trans-Golgi network requires the retromer protein complex.

The heat-pretreated amoebae (hyalospheres) are well suited cell models to study several manifestations of endocytosis: invagination of initial funnels, formation of pinocytotic channels, their activity and disintegration, production of microand macroendosomes directly from the surface membrane. All these phenomena are rhythmically reproduced (with periods ranging from 9 to 27 s) at the same active spots on the cell surface and accompanied by pulsation of the adjacent peripheral cytoplasmic layers. Successive portions of the contractile cortical network are serially detached from the plasma membrane and retracted inwards (on average 1 detachment per 15 s). They are suggested to be responsible for the traction component of endocytotic movements, i.e., for pulling the initial invagination funnels, elongation of channels, and inward transport of macroendosomes which are embedded in them. On the other hand, retraction of the cortical network squeezes the hyaloplasm outwards and thus the pressure component of endocytosic is produced. This results in cell surface expansion around the orifice of endocytotic channels or formation of macroendosomes by constriction at the mouth of large surface invaginations. Moreover, the retracting cortical network produces various radial transhyaline strands which seem to play a, not fully understood, role in membrane invagination and inward transport of microendosomes, and to accompany cytoplasmic pulsation around channels. The contractile network lining the walls of the channels may be detected in vivo, when some old channels are destroyed and their membrane dissociates from the cytoskeletal sleeve. The central role of the rhythmic detachment of the contractile network from the plasma membrane is common to the locomotory and endocytotic movements.

International audience; How invasive bacteria exploit mammalian host cell components to induce their entry into cells has received a lot of attention in the last two decades. Model organisms have emerged and helped understanding the various mechanisms that are used. Among those, Listeria monocytogenes is one of the most documented organisms. It enters into cells via two bacterial proteins, internalin (also called InlA) and InlB, which interact with cell surface receptors, Ecadherin and the hepatocyte growth factor receptor, Met, respectively. These interactions initiate a series of events that leads to actin polymerization, membrane invagination and bacterial internalization. Investigations on internalin- and InlB-mediated entries have repeatedly shown that Listeria fully usurps the host cell machinery. Moreover, they have also shown that previously unknown components discovered during the study of Listeria invasion play a role either in E-cadherin-mediated cell–cell adhesion or Met signalling. Unexpectedly, recent studies have highlighted a role for clathrin in Listeria InlB-mediated actin polymerization and entry, revealing a new role for this endocytic protein, i.e. in bacterial-induced internalization. Furthermore, comparative studies have demonstrated that the clathrin-mediated endocytosis machinery is also used in the internalin–Ecadherin pathway, and for the entry of other bacteria that enter by a ‘zipper’ mechanism. By contrast, the clathrinmediated endocytic machinery is not used by bacteria that inject effectors into mammalian cells via the type III secretion systemandenter bythe so-called triggermechanism, characterized by enormous membrane ruflles that result in the macropinocytosis of the corresponding bacteria. Finally, adherent bacteria, for example enteropathogenic Escherichia coli (EPEC), also co-opt clathrin to induce the formation of actin-rich pedestals. Together, these new data illuminate our view on how actin rearrangements may be coupled to clathrin recruitment during bacterial infection. They also shed light on a new function for clathrin in mammalian cells,i.e. internalization of objects much larger than previously accepted.

SummaryClathrin-mediated endocytosis is the main path for receptor internalization in metazoans and is essential for controlling cell integrity and signaling. It is driven by a large array of protein and lipid interactions that have been deciphered mainly by biochemical and genetic means. To place these interactions into context, and ultimately build a fully operative model of endocytosis at the molecular level, it is necessary to know the kinetic details of the role of each protein in this process. In this review, we describe the recent efforts made, by using live cell imaging, to define clear steps in the formation of endocytic vesicles and to observe the recruitment of key proteins during membrane invagination, the scission of a newly formed vesicle, and its movement away from the plasma membrane.