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Eukaryotic cells undergo shape changes during their division and growth. This involves flow of material both in the cell membrane and in the cytoskeletal layer beneath the membrane. Such flows result in redistribution of phospholipid at the cell surface and actomyosin in the cortex. Here we focus on the growth of the intercellular surface during cell division in a Caenorhabditis elegans embryo. The growth of this surface leads to the formation of a double-layer of separating membranes between the two daughter cells. The division plane typically has a circular periphery and the growth starts from the periphery as a membrane invagination, which grows radially inward like the shutter of a camera. The growth is typically not concentric, in the sense that the closing internal ring is located off-center. Cytoskeletal proteins anillin and septin have been found to be responsible for initiating and maintaining the asymmetry of ring closure but the role of possible asymmetry in the material flow into the growing membrane has not been investigated yet. Motivated by experimental evidence of such flow asymmetry, here we explore the patterns of internal ring closure in the growing membrane in response to asymmetric boundary fluxes. We highlight the importance of the flow asymmetry by showing that many of the asymmetric growth patterns observed experimentally can be reproduced by our model, which incorporates the viscous nature of the membrane and contractility of the associated cortex.

Synthetic membrane systems, such as giant unilamellar vesicles and solid supported lipid bilayers, have widened our understanding of biological processes occurring at or through membranes. Artificial systems are particularly suited to study the inherent properties of membranes with regard to their components and characteristics. This review critically reflects the emerging molecular mechanism of lipid-driven endocytosis and the impact of model membrane systems in elucidating the complex interplay of biomolecules within this process. Lipid receptor clustering induced by binding of several toxins, viruses and bacteria to the plasma membrane leads to local membrane bending and formation of tubular membrane invaginations. Here, lipid shape, and protein structure and valency are the essential parameters in membrane deformation. Combining observations of complex cellular processes and their reconstitution on minimal systems seems to be a promising future approach to resolve basic underlying mechanisms. This article is part of a Special Issue entitled: Mechanobiology.

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 nano-scale template on the membrane to spatially control actin nucleation during vesicle formation. Mathematical modeling of actin polymerization showed that this WASP nano-template creates sufficient force 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.

Clathrin-mediated endocytosis (CME) underlies intra- and extracellular material trafficking in eukaryotes, and is essential to protein metabolism, intercellular signaling, membrane remodeling and other cell regulatory processes. Although CME is usually driven by F-actin polymerization, membrane invagination can also occur through actin independent mechanisms. Here, we show that viscoelastic protein condensates that form via liquid-liquid phase separation at the sites of endocytosis initiation facilitate actin independent CME. Binding energies of the condensate with the membrane and surrounding cytosol provides work required to drive membrane invagination. Our findings expand the repertoire of functions associated with protein condensates that form via liquid-liquid phase separation to include their ability to do work at soft interfaces, thus shaping and organizing cellular matter.

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.

Summary Although many of the cellular and molecular mechanisms of angiogenesis have been intensely studied [1], little is known about the processes that underlie vascular anastomosis. We have generated transgenic fish lines expressing an EGFP-tagged version of the junctional protein zona occludens 1 (ZO1) to visualize individual cell behaviors that occur during vessel fusion and lumen formation in vivo. These life observations show that endothelial cells (ECs) use two distinct morphogenetic mechanisms, cell membrane invagination and cord hollowing to generate different types of vascular tubes. During initial steps of anastomosis, cell junctions that have formed at the initial site of cell contacts expand into rings, generating a cellular interface of apical membrane compartments, as defined by the localization of the apical marker podocalyxin-2 (Pdxl2). During the cord hollowing process, these apical membrane compartments are brought together via cell rearrangements and extensive junctional remodeling, resulting in lumen coalescence and formation of a multicellular tube. Vessel fusion by membrane invagination occurs adjacent to a preexisting lumen in a proximal to distal direction and is blood-flow dependent. Here, the invaginating inner cell membrane undergoes concomitant apicobasal polarization and the vascular lumen is formed by the extension of a transcellular lumen through the EC, which forms a unicellular or seamless tube.

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.

SummaryEndosomal sorting complexes required for transport (ESCRTs) regulate several events involving membrane invagination, including multivesicular body (MVB) biogenesis, viral budding, and cytokinesis [1]. In each case, upstream ESCRTs combine with additional factors, such as Bro1 proteins [2–4], to recruit ESCRT-III and the ATPase VPS4 in order to drive membrane scission [5]. A clue to understanding how such diverse cellular processes might be controlled independently of each other has been the identification of ESCRT isoforms. Mammalian ESCRT-I comprises TSG101, VPS28, VPS37A–D [6–8], and MVB12A/B [9]. These could generate several ESCRT-I complexes, each targeted to a different compartment and able to recruit distinct ESCRT-III proteins. Here we identify a novel ESCRT-I component, ubiquitin-associated protein 1 (UBAP1), which contains a region conserved in MVB12 [10]. UBAP1 binds the endosomal Bro1 protein His domain protein tyrosine phosphatase (HDPTP) [4], but not Alix, a Bro1 protein involved in cytokinesis [11, 12]. UBAP1 is required for sorting EGFR to the MVB and for endosomal ubiquitin homeostasis, but not for cytokinesis. UBAP1 is part of a complex that contains a fraction of total cellular TSG101 and that also contains VPS37A but not VPS37C. Hence, the presence of UBAP1, in combination with VPS37A, defines an endosome-specific ESCRT-I complex.

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.

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.

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.

Endosomes along the degradation pathway exhibit a characteristic multivesicular organization, resulting from the accumulation of small vesicles or other membranous structures within the endosomal lumen. After endocytosis and transport to early endosomes, activated signaling receptors are incorporated within lumenal vesicles through the action of Hrs and ESCRT complexes, a process that contributes to terminate signaling. Then, vesicles and their protein cargo are transported towards lysosomes for degradation. However, evidence also shows that lumenal vesicles can undergo "back-fusion" with the late endosome limiting membrane, a route presumably followed by proteins and lipids that need to be recycled from within the endosomal lumen. Vesicular stomatitis virus and anthrax toxin exploit this lumenal transport pathway to reach the cytoplasm. The back-fusion process depends on the late endosomal lipid lysobisphosphatidic acid or bis (monoacylglycero) phosphate and its putative effector Alix/AIP1. In this review, we will discuss intra-endosomal transport routes in mammalian cells, and in particular the mechanisms that are presumably involved in membrane invagination, vesicle formation and fusion in a space inaccessible to proteins known to control intracellular membrane traffic.

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.

Cortical actin patches are dynamic structures required for endocytosis in yeast. Recent studies have shown that components of cortical patches localize to the plasma membrane in a precisely orchestrated manner, and their movements at and away from the plasma membrane may define the endocytic membrane invagination and vesicle scission events, respectively. Here, through live-cell imaging, we analyze the dynamics of the highly conserved class I unconventional myosin, Myo5, which also localizes to cortical patches and is known to be involved in endocytosis and actin nucleation. Myo5 exhibits a pattern of dynamic localization different from all cortical patch components analyzed to date. Myo5 associates with cortical patches only transiently and remains stationary during its brief cortical lifespan. The peak of Myo5 association with cortical patches immediately precedes the fast movement of Arp2/3 complex-associated structures away from the plasma membrane, thus correlating precisely with the proposed vesicle scission event. To further test the role of Myo5, we generated a temperature-sensitive mutant myo5 allele. In the myo5 mutant cells, Myo5 exhibits a significantly extended cortical lifespan as a result of a general impairment of Myo5 function, and Arp2 patches exhibit an extended slow-movement phase prior to the fast movement toward the cell interior. The myo5 mutant cells are defective in fluid-phase endocytosis and exhibit an increased number of invaginations on the membrane. Based on these results, we hypothesize that the myosin I motor protein facilitates the membrane fusion/vesicle scission event of endocytosis.

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.

Sphingomyelin is an abundant component of eukaryotic membranes. A specific enzyme, sphingomyelinase can convert this lipid to ceramide, a central second messenger in cellular signaling for apoptosis (programmed cell death), differentiation, and senescence. We used microinjection and either Hoffman modulation contrast or fluorescence microscopy of giant liposomes composed of 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC), N-palmitoyl-sphingomyelin (C16:0-SM), and Bodipy-sphingomyelin as a fluorescent tracer (molar ratio 0.75:0.20:0.05, respectively) to observe changes in lipid lateral distribution and membrane morphology upon formation of ceramide. Notably, in addition to rapid domain formation (capping), vectorial budding of vesicles, i.e., endocytosis and shedding, can be induced by the asymmetrical sphingomyelinase-catalyzed generation of ceramide in either the outer or the inner leaflet, respectively, of giant phosphatidylcholine/sphingomyelin liposomes. These results are readily explained by 1) the lateral phase separation of ceramide enriched domains, 2) the area difference between the adjacent monolayers, 3) the negative spontaneous curvature, and 4) the augmented bending rigidity of the ceramide-containing domains, leading to membrane invagination and vesiculation of the bilayer.

The sorting of specific proteins into clathrin-coated pits and the mechanics of membrane invagination are determined by assembly of the clathrin lattice. Recent structures of a six-fold barrel clathrin coat at 21 A resolution by electron cryomicroscopy and of the clathrin terminal domain and linker at 2.6 A by X-ray crystallography together show how domains of clathrin interact and orient within the coat and reveal the strongly puckered shape and conformational variability of individual triskelions. The β propeller of the terminal domain faces the membrane so that recognition segments from adaptor proteins can extend along its lateral grooves. Clathrin legs adapt to different coat environments in the barrel by flexing along a segment at the knee that is free of contacts with other molecules.

Clathrin and adaptors are components of clathrin-coated pits and vesicles. The AP-1 adaptor complex is associated with clathrin-coated vesicles budding from the TGN, while the AP-2 adaptor complex is associated with clathrin-coated vesicles budding from the plasma membrane. The clathrin forms a polyhedral lattice and is believed to be the driving force behind membrane invagination leading to vesicle budding. The adaptors attach the clathrin to the membrane and also interact with the cytoplasmic domains of selected transmembrane proteins, causing these proteins to become concentrated in clathrin-coated vesicles. Clathrin-coated vesicles budding from the TGN have been implicated in the sorting of newly synthesised lysosomal enzymes, while clathrin-coated vesicles budding from the plasma membrane facilitate the receptor-mediated endocytosis of ligands, such as low density lipoproteins and transferrin. A novel adaptor-related complex, AP-3, has recently been identified, which is recruited onto membranes of the TGN and a more peripheral compartment but does not appear to be associated with clathrin. Genetic studies indicate that AP-3 plays a role in the sorting of proteins to lysosomes and lysosome-related organelles.

Cardiomyocytes are characterized by a complex network of membrane invagination (the transverse axial tubular system, TATS) that propagates the action potential to the cell core guarantying a synchronous and uniform cell contraction. Pathological settings are commonly associated with structural remodelling of TATS that may cause failures of action potential propagation at single T-tubular level. Here, we investigate whether structural alterations occurring in TATS are linked to overall changes in its electrical conductivity which in turn hinder the regular propagation of action potentials across the network. Exploiting the formal analogy between diffusion and electrical conductivity we linked the conductivity with the diffusion properties of TATS. Fluorescence recovery after photo-bleaching (FRAP) microscopy is used to probe the diffusion properties of the tubular system in isolated cardiomyocytes: the fluorescent dextran inside TATS lumen is photo-bleached and the diffusion of unbleached dextran from extracellular space to TATS is monitored. We designed a mathematical model that correlate the time constant of fluorescence recovery with the apparent diffusion coefficient of the dextran. Then, taken advantage of the analogy between diffusion and conductivity, the apparent diffusion is used to assess TATS conductivity. This value is used to evaluate the efficiency of the passive spread of voltage changes along TATS. To probe how TATS conductivity is influenced by geometrical features, we used a model of acutely detubulated cells. We found that the overall reduction of TATS halves tubular conductivity. Then we tested our method in a pathological setting characterized by structural alteration, i.e. the heart failure rat model. We found that the tubular conductivity is not changed as compared to control. Actually, this result should not be surprising since heart failure is characterized by compound alterations that potentially compensate each other in their individual effect on global conductivity.

The recent invention of super-resolution fluorescence microscopy allows nanoscopic investigation of cellular structures. Among these techniques, the Stochastic Optical Reconstruction Microscopy (STORM) is based on precise single molecule localization of photoswitchable fluorescent probes. By stochastically activating, imaging and deactivating subsets of fluorophores, it makes their images optically resolvable and determines their positions with nanometer precision. A super-resolution image is then reconstructed using these localizations. We now extend this approach to three-dimensional (3D) microscopy by determining the 3D coordinates of activated probes through astigmatism imaging: a cylindrical lens is inserted into the imaging optical path such that the image of individual molecules appear elliptical with the ellipticity depending on its z-position. Using this approach, we have achieved an optical resolution of 20-30 nm in the x-y direction and 50-60 nm in the z direction, representing an order of magnitude improvement over conventional fluorescence microscopy in all three dimensions. We have resolved the nanoscopic morphology of cellular structures that was previously deemed impossible by light microscopy.As a specific application, we use STORM to study the mechanism of clathrin mediated endocytosis in an in vitro reconstituted system. Proteins of interest in this system are directly labeled with photoswitchable fluorescent probes. This procedure is facilitated by covalently linking the two components of the probe, an activator dye and a photoswitchable reporter fluorophore, to form a single chemical unit prior to protein labeling. Using multicolor 3D STORM, we have characterized the spatial organization of plasma membrane, clathrin, actin and tubule forming proteins dynamin at the site of clathrin mediated endocytosis. These results reveal the molecular architecture of the nascent clathrin-coated pits at the nanometer scale and help to establish the role of actin and dynamin in membrane invagination, scission and vesicle formation.

One of the pathological hallmarks of Alzheimer's disease (AD) is extracellular plaques, in which the 42aa Amyloid beta peptide (Aβ 1-42) is the main component. Aβ 1-42 is produced at cholesterol-rich regions of neuronal membranes by endoproteolysis of the parental amyloid precursor protein and is secreted into the extracellular space. Although Aβ 1-42 accumulates extracellularly, neurons internalize the Aβ 1-42 peptide which could contribute to disease progression and which may be the first step to both cytotoxicity and propagation of misfolded Aβ between cells.Interaction with membrane bilayer is likely the first step in the molecular mechanism of neuronal Aβ 1-42 uptake. Therefore, we studied interaction of Aβ 1-42 with the lipid bilayer in a giant unilamellar vesicles (GUVs) model system. We found that the Aβ 1-42 bound to the lipid bilayer and, after a lag phase induced invagination of the membrane into small vesicular structures. Only early oligomeric structures of Aβ and stabilized the negative curvature of membrane, whereas both monomeric and fibrillar forms of the peptide were unable to induce or sustain membrane invagination.Our results suggest that Aβ may facilitate vesicle formation in lipid bilayer membranes, which may be a factor in cellular Aβ internalization but may also hint at a possible physiological function of the Aβ peptide at the synaptic membrane.

In atrial myocytes lacking t-tubules, fluid pressure (FP, ∼16 dyn/cm2) has been found to elicit global Ca2+ waves: one with longitudinal propagation (L-wave) and the other with transverse propagation (T-wave). T-wave is associated with action potential, whereas L-wave often, but not always, triggers action potential involving T-wave during its propagation. Here we examined spatiotemporal characteristics, and structural and molecular basis for the FP-induced L-wave using two-dimensional (2-D) confocal Ca2+ imaging, combined with three-dimensional (3-D) visualizations of cell membrane and type 2 ryanodine receptors (RyR2), in rat atrial myocytes. We found that FP-induced L-waves generally originated from central focal Ca2+ release sites (“L-wave core”) located at 15-25% of the cell length. The core releases had 3∼6 signal peaks and grew faster in one-direction in 2-D measurements. They were more prolonged and much larger compared with the Ca2+ sparks. Tetracaine (1 mM, 10 s) reversibly inhibited the FP-induced core release as well as the L-wave, suggesting both involve the RyRs. Immediately after recording FP-induced L-wave, the cell's membrane was further visualized with wheat germ agglutinin (WGA, 1.25 μg/ml). Overlapping the L-wave core and the cell membrane, and reconstructing the WGA confocal image stacks in 3-D, revealed membrane invagination at the centrally localized core or in the vicinity of the core site. Interestingly, in some atrial cells we found denser (≈0.62-μm intervals) disoriented RyR alignments in the cell interior at ∼20% of the cell length and nearby the cell end. We conclude that the crumpled invaginated surface membrane, associated with dense RyRs, may play an important role in transducing fluid pressure into the core Ca2+ signal, resulting in arrhythmogenic L-wave.

Previous pulse-chase studies have shown that bacteriochlorophyll a-protein complexes destined eventually for the photosynthetic (chromatophore) membrane of Rhodopseudomonas sphaeroides appear first in a distinct pigmented fraction. This rapidly labeled material forms an upper band when extracts of phototrophically grown cells are subjected directly to rate-zone sedimentation. In the present investigation, flash-induced absorbance changes at 605 nm have demonstrated that the upper fraction is enriched two-fold in photochemical reaction center activity when compared to chromotophores; a similar enrichment in the reaction center-associated B-875 antenna bacteriochlorophyll complex was also observed. Although b- and c-type cytochromes were present in the upper pigmented band, no photoreduction of the b-type components could be demonstrated. The endogenous c-type cytochrome (Em = +345 mV) was photooxidized slowly upon flash illumination. The extent of the reaction was increased markedly with excess exogenous ferrocytochrome c but only slightly in chromatophores. Only a small light-induced carotenoid band shift was observed. These results indicate that the rapidly labeled fraction contains photochemically competent reaction centers associated loosely with c-type and unconnected to b-type cytochrome. It is suggested that this fraction arises from new sites of cytoplasmic membrane invagination which fragment to form leaky vesicles upon cell disruption.

The extent of membrane invagination or endocytosis in intact erythrocytes was quantified by measuring the loss of acetylcholinesterase activity. Primaquine-induced endocytosis was completely inhibited in ATP-depleted cells. However, chlorpromazine and vinblastine were capable of inducing membrane invagination in depleted cells. With both drugs, the loss of enzyme activity was less than that measured in fresh cells. We conclude that drug-induced endocytosis is not necessarily an energy-dependent process.

During the morphogenesis of small plasmodia developing from endoplasmic drops, an extended plasmalemma invagination system is formed. This system is a characteristic constituent of the ectoplasm. The invaginations have different cytophysiological functions. The transition from the initial very irregular plasmalemma indentations in protoplasmic drops to the highly organized circular invagination ring of protoplasmic strands, i.e., the differentiation as well as the disassembly of this circular invagination system in retracting endings of strands was investigated with the aid of the semithin- and ultrathin-sectioning technique. Live observation of protoplasmic drops revealed that simultaneously with the onset of initially irregular oscillating contractions, small endoplasmic streamlets are generated. Subsequently, an improvement of the coordination of contraction activities leads to an oriented mass transport of protoplasm and thereby to locomotion. The growing endoplasmic channels continuously develop into the well-known structure of protoplasmic strands. Differentiation and disassembly of circular plasmalemma invaginations are based on processes of membrane invagination in combination with intracytosis and exocytosis. The importance and correlations of the following phenomena for morphogenesis and differentiation are discussed: 1) the formation and distribution of the contractile apparatus, i.e., the system of cytoplasmic actomyosin fibrils, 2) plasmalemma invaginations, 3) the generation of oscillating contractions, and 4) the endoplasmic streaming.

In this paper the origin of the membrane investing the newly formed elongating organelles during mammalian spermiogenesis is studied. According to previous authors, the beginning axoneme is hollowed in a deep membrane invagination. We demonstrate that in man, rat, and bull this new surface is formed by several clusters of Golgi-originated vesicles which form a periaxonemal double cylinder which finally fuses at its end with the old plasma membrane. So the new periaxonemal plasma membrane is preformed in the spermatid body. The membrane surrounding the elongating head is, on the contrary, simply an extension of the old one, because migration of Golgi vesicles and preformed new membranes have not been observed in this region. Con A properties of new and old membranes are the same and will change only after the transit through the epididymis.

A case of vitiligo with inflammatory raised borders was observed electron microsocopically, resulting in an interesting view of the formation of Birbeck granules in Langerhans cells. Following the formation of larger coated vesicles, which perform adsorptive pinocytosis from the cell membrane, membrane invagination of the cell occurred shown as the tubular infolding, resulting in the observation of the characteristic Birbeck granule band pattern in its interior. This phenomenon supports the theory of Hashimoto and Tarnowski (1968) that Birbeck granules are formed from the infolding of the cell membrane. In addition, our study shows the involvement of adsorptive pinocytosis in the formation of the granules. It was suggested that when the coated Birbeck granule shifts into the cell, possibly its coat is detached and the vesicle portion forms the globule of the Birbeck granule.