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Eukaryotic cells assemble an actomyosin ring during cytokinesis to function as a force-generating machine to drive membrane invagination, and to counteract the intracellular pressure and the cell surface tension. It is unclear whether additional factors such as the extracellular matrix (cell wall in yeasts and fungi) affect the actomyosin ring contraction. While studying the fission yeast β-glucan synthase mutant cps1-191, which is defective in division septum synthesis and actomyosin ring contraction, we found that significantly weakening of the extracellular glycan matrix caused the spheroplasts to divide at the non-permissive condition. This division was dependent on a functional actomyosin ring and vesicular trafficking, but independent of normal septum synthesis. cps1-191 cells with weakened extracellular glycan matrix divide non-medially with a much slower ring contraction rate compared to wild type cells under similar conditions, which we term as cytofission. Interestingly, the high turgor pressure appears to play minimal roles in inhibiting ring contraction in cps1-191 mutants as decreasing the turgor pressure alone does not enable cytofission. We propose that during cytokinesis, the extracellular glycan matrix restricts actomyosin ring contraction and membrane ingression, and remodeling of the extracellular components through division septum synthesis relieves the inhibition and facilitates actomyosin ring contraction.

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.

Mitochondrial cristae contain electron transport chain complexes and are distinct from the inner boundary membrane (IBM). While many details regarding the regulation of mitochondrial structure are known, the relationship between cristae structure and function during organelle development is not fully described. Here, we used serial-section tomography to characterize the formation of lamellar cristae in immature mitochondria during a period of dramatic mitochondrial development that occurs after Drosophila emergence as an adult. We found that the formation of lamellar cristae was associated with the gain of cytochrome c oxidase (COX) function, and the COX subunit, COX4, was localized predominantly to organized lamellar cristae. Interestingly, 3D tomography showed some COX-positive lamellar cristae were not connected to IBM. We hypothesize that some lamellar cristae may be organized by a vesicle germination process in the matrix, in addition to invagination of IBM. OXA1 protein, which mediates membrane insertion of COX proteins, was also localized to cristae and reticular structures isolated in the matrix additional to the IBM, suggesting that it may participate in the formation of vesicle germination-derived cristae. Overall, our study elaborates on how cristae morphogenesis and functional maturation are intricately associated. Our data support the vesicle germination and membrane invagination models of cristae formation.

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.

Invadopodia are integrin-mediated adhesions with abundant PI(3,4)P2 However, the functional role of PI(3,4)P2 in adhesion signaling remains unclear. Here, we find that the PI(3,4)P2 biogenesis regulates the integrin endocytosis at invadopodia. PI(3,4)P2 is locally produced by PIK3CA and SHIP2 and is concentrated at the trailing edge of the invadopodium arc. The PI(3,4)P2-rich compartment locally forms small puncta (membrane buds) in a SNX9-dependent manner, recruits dynein activator Hook1 through AKTIP, and rearranges into micrometer-long tubular invaginations (membrane tubes). The uncurving membrane tube extends rapidly, follows the retrograde movement of dynein along microtubule tracks, and disconnects from the plasma membrane. Activated integrin-beta3 is locally internalized through the pathway of PI(3,4)P2-mediated membrane invagination and is then actively recycled. Blockages of PI3K, SHIP2, and SNX9 suppress integrin-beta3 endocytosis, delay adhesion turnover, and impede transwell invasion of MEF-Src and MDA-MB-231 cells. Thus, the production of PI(3,4)P2 promotes invasive cell migration by stimulating the trafficking of integrin receptor at the invadopodium.

Clostridium botulinum C2 toxin is a clostridial binary toxin consisting of actin ADP-ribosyltransferase (C2I) and C2II binding components. Activated C2II (C2IIa) binds to cellular receptors and forms oligomer in membrane rafts. C2IIa oligomer assembles with C2I and contributes to the transport of C2I into the cytoplasm of host cells. C2IIa induces Ca2+-induced lysosomal exocytosis, extracellular release of the acid sphingomyelinase (ASMase), and membrane invagination and endocytosis through generating ceramides in the membrane by ASMase. Here, we reveal that C2 toxin requires the lysosomal enzyme cathepsin B (CTSB) during endocytosis. Lysosomes are a rich source of proteases, containing cysteine protease CTSB and cathepsin L (CTSL), and aspartyl protease cathepsin D (CTSD). Cysteine protease inhibitor E64 blocked C2 toxin-induced cell rounding, but aspartyl protease inhibitor pepstatin-A did not. E64 inhibited the C2IIa-promoted extracellular ASMase activity, indicating that the protease contributes to the activation of ASMase. C2IIa induced the extracellular release of CTSB and CTSL, but not CTSD. CTSB knockdown by siRNA suppressed C2 toxin-caused cytotoxicity, but not siCTSL. These findings demonstrate that CTSB is important for effective cellular entry of C2 toxin into cells through increasing ASMase activity.

Exosomes are a group of nanosized extracellular vesicles that include various bioactive nucleic acids, lipids, and proteins. They originate from membrane invagination and are released by exocytosis, which can transmit signals to target cells to achieve cell-to-cell communication and maintain homeostasis. The heart is a complex multicellular organ that contains resident cell types such as fibroblasts, endothelial cells, and smooth muscle cells. Communication between different cell types and immune systems is essential for the dynamic equilibrium of the cardiac internal environment. Intercellular communication is a universal phenomenon mediated by exosomes and their contents during several pathological processes in cardiovascular diseases, such as cardiomyocyte hypertrophy, apoptosis, and angiogenesis. Therefore, exosomes can be used as novel invasive diagnostic biomarkers in multiple diseases, including atherosclerosis, myocardial ischemia, cardiac fibrosis, and ischemia-reperfusion injury. In addition, the biocompatible nature and low immunogenicity of exosomes make them high-quality nanoparticle drug carriers with potential applications in translational medicine and therapeutic strategies. Here, we focus on the biogenesis, isolation, biological functions, and future application prospects of exosomes in cardiovascular disease.

Endocytosis is the process of actively transporting materials into a cell by membrane engulfment. Traditionally, endocytosis was divided into three forms: phagocytosis (cell eating), pinocytosis (cell drinking), and the more selective receptor-mediated endocytosis (clathrin-mediated endocytosis); however, other important endocytic pathways (e.g., caveolin-dependent endocytosis) contribute to the uptake of extracellular substances. In each, the plasma membrane changes shape to allow the ingestion and internalization of materials, resulting in the formation of an intracellular vesicle. While receptor-mediated endocytosis remains the best understood pathway, mammalian cells utilize each form of endocytosis to respond to their environment. Receptor-mediated endocytosis permits the internalization of cell surface receptors and their ligands through a complex membrane invagination process that is facilitated by clathrin and adaptor proteins. Internalized vesicles containing these receptor-ligand cargoes fuse with early endosomes, which can then be recycled back to the plasma membrane, delivered to other cellular compartments, or destined for degradation by fusing with lysosomes. These intracellular fates are largely determined by the interaction of specific cargoes with adaptor proteins, such as the epsins, disabled-homolog 2 (Dab2), the stonin proteins, epidermal growth factor receptor substrate 15, and adaptor protein 2 (AP-2). In this review, we focus on the role of epsins and Dab2 in controlling these sorting processes in the context of cardiovascular disease. In particular, we will focus on the function of epsins and Dab2 in inflammation, cholesterol metabolism, and their fundamental contribution to atherogenicity.

Mitosis and endocytosis are two fundamental cellular processes essential for maintaining a eukaryotic life. Mitosis partitions duplicated chromatin enveloped in the nuclear membrane into two new cells, whereas endocytosis takes in extracellular substances through membrane invagination. These two processes are spatiotemporally separated and seemingly unrelated. However, recent studies have uncovered that endocytic proteins have moonlighting functions in mitosis, and mitotic complexes manifest additional roles in endocytosis. In this review, we summarize important proteins or protein complexes that participate in both processes, compare their mechanism of action, and discuss the rationale behind this multifunctionality. We also speculate on the possible origin of the functional reciprocity from an evolutionary perspective.

Insect epithelial cells contain cellular extensions such as bristles, hairs, and scales. These cellular extensions are homologous structures that differ in morphology and function. They contain actin bundles that dictate their cellular morphology. While the organization, function, and identity of the major actin-bundling proteins in bristles and hairs are known, this information on scales is unknown. In this study, we characterized the development of scales and the role of actin bundles in the mosquito, Aedes aegypti. We show that scales undergo drastic morphological changes during development, from a cylindrical to flat shape with longer membrane invagination. Scale actin-bundle distribution changes from the symmetrical organization of actin bundles located throughout the bristle membrane to an asymmetrical organization. By chemically inhibiting actin polymerization and by knocking out the forked gene in the mosquito (Ae-Forked; a known actin-bundling protein) by CRISPR-Cas9 gene editing, we showed that actin bundles are required for shaping bristle, hair, and scale morphology. We demonstrated that actin bundles and Ae-Forked are required for bristle elongation, but not for that of scales. In scales, actin bundles are required for width formation. In summary, our results reveal, for the first time, the developmental process of mosquito scale formation and also the role of actin bundles and actin-bundle proteins in scale morphogenesis. Moreover, our results reveal that although scale and bristle are thought to be homologous structures, actin bundles have a differential requirement in shaping mosquito scales compared to bristles.

Positive-strand RNA viruses, such as dengue virus (DENV), induce the extensive rearrangement of intracellular membranes that serve as a scaffold for the assembly of the viral replication machinery. In the case of DENV, the main endomembrane ultrastructure produced in infected cells consists of invaginations of the endoplasmic reticulum, designated vesicle packets (VPs), which are the assumed sites of viral RNA replication. VPs are observed as arrays of vesicles surrounded by an outer membrane, the formation of which is induced by the viral nonstructural proteins, presumably in conjunction with specific host factors. However, little is known about the mechanisms governing VP formation, which is mainly due to the lack of a replication-independent system supporting the biogenesis of these membranous structures. Here we describe an expression-based, viral RNA replication-independent, DENV polyprotein system, designated as pIRO (plasmid-induced replication organelle), which is sufficient to induce VP formation. We show that VPs induced by pIRO expression are morphologically indistinguishable from those found in infected cells, suggesting that DENV replication organelle formation does not require RNA replication. We conclude that the pIRO system is a novel and valuable tool that can be used to dissect the mechanisms underlying DENV replication organelle formation.

The enteric bacterial pathogens Listeria monocytogenes (Listeria) and enteropathogenic Escherichia coli (EPEC) remodel the eukaryotic actin cytoskeleton during their disease processes. Listeria generate slender actin-rich comet/rocket tails to move intracellularly, and later, finger-like membrane protrusions to spread amongst host cells. EPEC remain extracellular, but generate similar actin-rich membranous protrusions (termed pedestals) to move atop the host epithelia. These structures are crucial for disease as diarrheal (and systemic) infections are significantly abrogated during infections with mutant strains that are unable to generate the structures. The current repertoire of host components enriched within these structures is vast and diverse. In this protein catalog, we and others have found that host actin crosslinkers, such as palladin and α-actinin-1, are routinely exploited. To expand on this list, we set out to investigate the distribution of PDLIM1, a scaffolding protein and binding partner of palladin and α-actinin-1, during bacterial infections. We show that PDLIM1 localizes to the site of initial Listeria entry into cells. Following this, PDLIM1 localizes to actin filament clouds surrounding immotile bacteria, and then colocalizes with actin once the comet/rocket tails are generated. Unlike palladin or α-actinin-1, PDLIM1 is maintained within the actin-rich core of membrane protrusions. Conversely, α-actinin-1, but not PDLIM1 (or palladin), is enriched at the membrane invagination that internalizes the Listeria-containing membrane protrusion. We also show that PDLIM1 is a component of the EPEC pedestal core and that its recruitment is dependent on the bacterial effector Tir. Our findings highlight PDLIM1 as another protein present within pathogen-induced actin-rich structures.

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.

Pseudomonas aeruginosa is a ubiquitous environmental bacterium that is one of the leading causes of nosocomial infections. P. aeruginosa is able to switch between planktonic, intracellular, and biofilm-based lifestyles, which allows it to evade the immune system as well as antibiotic treatment. Hence, alternatives to antibiotic treatment are urgently required to combat P. aeruginosa infections. Lectins, like the fucose-specific LecB, are promising targets, because removal of LecB resulted in decreased virulence in mouse models. Currently, several research groups are developing LecB inhibitors. However, the role of LecB in host-pathogen interactions is not well understood. The significance of our research is in identifying cellular mechanisms of how LecB facilitates P. aeruginosa infection. We introduce LecB as a new member of the list of bacterial molecules that bind integrins and show that P. aeruginosa can move forward underneath attached epithelial cells by loosening cell-basement membrane attachment in a LecB-dependent manner., The opportunistic bacterium Pseudomonas aeruginosa produces the fucose-specific lectin LecB, which has been identified as a virulence factor. LecB has a tetrameric structure with four opposing binding sites and has been shown to act as a cross-linker. Here, we demonstrate that LecB strongly binds to the glycosylated moieties of β1-integrins on the basolateral plasma membrane of epithelial cells and causes rapid integrin endocytosis. Whereas internalized integrins were degraded via a lysosomal pathway, washout of LecB restored integrin cell surface localization, thus indicating a specific and direct action of LecB on integrins to bring about their endocytosis. Interestingly, LecB was able to trigger uptake of active and inactive β1-integrins and also of complete α3β1-integrin–laminin complexes. We provide a mechanistic explanation for this unique endocytic process by showing that LecB has the additional ability to recognize fucose-bearing glycosphingolipids and causes the formation of membrane invaginations on giant unilamellar vesicles. In cells, LecB recruited integrins to these invaginations by cross-linking integrins and glycosphingolipids. In epithelial wound healing assays, LecB specifically cleared integrins from the surface of cells located at the wound edge and blocked cell migration and wound healing in a dose-dependent manner. Moreover, the wild-type P. aeruginosa strain PAO1 was able to loosen cell-substrate adhesion in order to crawl underneath exposed cells, whereas knockout of LecB significantly reduced crawling events. Based on these results, we suggest that LecB has a role in disseminating bacteria along the cell-basement membrane interface.

Insect epithelial cells contain cellular extensions such as bristles, hairs and scales. It has been suggested that these cellular extensions are homologous structures that differ in morphology and function. These cellular extensions contain actin bundles that dictate their cellular morphology; bristle and hair are cylindrical in shape, while scales are wider and flattened. While the organization, function and identity of the major actin bundling protein in bristles and hairs is known, this information in scales is unknown. In this study, we characterized the development of scales and the role of actin bundles in the mosquito,Aedes aegypti. We show that scales undergo drastic morphological changes during development, from cylindrical shape to flat shape with longer membrane invagination. Scale actin bundle distribution changes during development, from symmetrical organization of actin bundles located throughout the bristle membrane, to asymmetrical organization of the actin bundles. By chemically inhibiting actin polymerization and by knocking-out theforkedgene in the mosquito (Ae-Forked;a known actin bundling protein), by CRISPR-Cas9 gene editing, we showed that actin bundles are required for shaping bristle, hair and scale morphology. We demonstrated that actin bundles andAe-Forkedare required for bristle elongation, but not that of scales. In scales, actin bundles are required for width formation. Our results reveal a differential requirement of actin bundles in shaping mosquito scales compared to bristles.

Virus infection has drawn extensive attention since it causes serious or even deadly diseases, consequently inducing a series of social and public health problems. Caveolin-1 is the most important structural protein of caveolae, a membrane invagination widely known for its role in endocytosis and subsequent cytoplasmic transportation. Caveolae/caveolin-1 is tightly associated with a wide range of biological processes, including cholesterol homeostasis, cell mechano-sensing, tumorigenesis, and signal transduction. Intriguingly, the versatile roles of caveolae/caveolin-1 in virus infections have increasingly been appreciated. Over the past few decades, more and more viruses have been identified to invade host cells via caveolae-mediated endocytosis, although other known pathways have been explored. The subsequent post-entry events, including trafficking, replication, assembly, and egress of a large number of viruses, are caveolae/caveolin-1-dependent. Deprivation of caveolae/caveolin-1 by drug application or gene editing leads to abnormalities in viral uptake, viral protein expression, or virion release, whereas the underlying mechanisms remain elusive and must be explored holistically to provide potential novel antiviral targets and strategies. This review recapitulates our current knowledge on how caveolae/caveolin-1 functions in every step of the viral infection cycle and various relevant signaling pathways, hoping to provide a new perspective for future viral cell biology research.

Branched and cross-linked actin networks mediate cellular processes that move and shape membranes. To understand how actin contributes during the different stages of endocytic membrane reshaping, we analyzed deletion mutants of yeast actin network components using a hybrid imaging approach that combines live imaging with correlative microscopy. We could thus temporally dissect the effects of different actin network perturbations, revealing distinct stages of actin-based membrane reshaping. Our data show that initiation of membrane bending requires the actin network to be physically linked to the plasma membrane and to be optimally cross-linked. Once initiated, the membrane invagination process is driven by nucleation and polymerization of new actin filaments, independent of the degree of cross-linking and unaffected by a surplus of actin network components. A key transition occurs 2 s before scission, when the filament nucleation rate drops. From that time point on, invagination growth and vesicle scission are driven by an expansion of the actin network without a proportional increase of net actin amounts. The expansion is sensitive to the amount of filamentous actin and its cross-linking. Our results suggest that the mechanism by which actin reshapes the membrane changes during the progress of endocytosis, possibly adapting to varying force requirements.

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.

Conserved proteins drive clathrin-mediated endocytosis (CME), which universally involves a burst of actin assembly. To gain fundamental mechanistic insights into this process, a side-by-side quantitative comparison of CME was performed on two distantly related yeast species. Though endocytic protein abundance in S. pombe and S. cerevisiae are more similar than previously thought, membrane invagination speed and depth are two-fold greater in fission yeast than in budding yeast. In both yeasts, Arp2/3 complex activation drives membrane invagination when triggered by the accumulation of ∼70 WASP molecules. In contrast to budding yeast, WASP-mediated actin nucleation activity plays an essential role in fission yeast endocytosis. Genetics and live-cell imaging revealed core CME spatiodynamic similarities between the two yeasts, though two-zone actin assembly is a fission yeast-specific mechanism, which is not essential for CME. These studies identified conserved CME mechanisms and species-specific adaptations and have broad implications that extend from yeast to humans.

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.

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.

SummaryIn animals, cellularization of a coenocyte is a specialized form of cytokinesis that results in the formation of a polarized epithelium during early embryonic development. It is characterized by coordinated assembly of an actomyosin network, which drives inward membrane invaginations. However, whether coordinated cellularization driven by membrane invagination exists outside animals is not known. To that end, we investigate cellularization in the ichthyosporean Sphaeroforma arctica, a close unicellular relative of animals. We show that the process of cellularization involves coordinated inward plasma membrane invaginations dependent on an actomyosin network, and reveal the temporal order of its assembly. This leads to the formation of a polarized layer of cells resembling an epithelium. We show that this epithelium-like stage is associated with tightly regulated transcriptional activation of genes involved in cell adhesion. Hereby we demonstrate the presence of a selforganized, clonally-generated, polarized layer of cells in a unicellular relative of animals.

In animals, cellularization of a coenocyte is a specialized form of cytokinesis that results in the formation of a polarized epithelium during early embryonic development. It is characterized by coordinated assembly of an actomyosin network, which drives inward membrane invaginations. However, whether coordinated cellularization driven by membrane invagination exists outside animals is not known. To that end, we investigate cellularization in the ichthyosporean Sphaeroforma arctica, a close unicellular relative of animals. We show that the process of cellularization involves coordinated inward plasma membrane invaginations dependent on an actomyosin network and reveal the temporal order of its assembly. This leads to the formation of a polarized layer of cells resembling an epithelium. We show that this stage is associated with tightly regulated transcriptional activation of genes involved in cell adhesion. Hereby we demonstrate the presence of a self-organized, clonally-generated, polarized layer of cells in a unicellular relative of animals., This work was funded by European Research Council Consolidator Grant (ERC-2012-Co −616960) to IR-T; MEXT KAKENHI (grant Nos. 221S0002 to AT; grants no 26891021 and 16K07468 to HS); and a Young Research Talents grant from the Research Council of Norway (grant no. 240284) to JB. OD was supported by a Swiss National Science Foundation Early PostDoc Mobility fellowship (P2LAP3_171815) and a Marie Sklodowska-Curie individual fellowship (MSCA-IF 746044). AO was supported by a Marie Sklodowska-Curie individual fellowship (MSCA-IF 747086).

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. We assessed 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 is stratified into several functional layers situated above and below the clathrin lattice with key proteins transversing through the lattice deeply into the cytoplasm. We propose that this conserved design enables an efficient and regulated function of the endocytic coat during endocytosis.

Over the last 20 years, extracellular vesicles (EVs) have been established as an additional way to transmit signals outside the cell. They are membrane-surrounded structures of nanometric size that can either originate from the membrane invagination of multivesicular bodies of the late endosomal compartment (exosomes) or bud from the plasma membrane (microvesicles). They contain proteins, lipids, and nucleic acids—namely miRNA, but also mRNA and lncRNA—which are derived from the parental cell, and have been retrieved in every fluid of the body. As carriers of antigens, either alone or in association with major histocompatibility complex (MHC) class II and class I molecules, their immunomodulatory properties have been extensively investigated. Moreover, recent studies have shown that EVs may carry and deliver membrane-derived bioactive lipids that play an important function in the immune system and related pathologies, such as prostaglandins, leukotrienes, specialized pro-resolving mediators, and lysophospholipids. EVs protect bioactive lipids from degradation and play a role in the transcellular synthesis of prostaglandins and leukotrienes. Here, we summarized the role of EVs in the regulation of immune response, specifically focusing our attention on the emerging role of EVs as carriers of bioactive lipids, which is important for immune system function.

Cell division in most prokaryotes is mediated by the well-studied fts genes, with FtsZ as the principal player. In many archaeal species, however, division is orchestrated differently. The Crenarchaeota phylum of archaea features the action of the three proteins, CdvABC. This Cdv system is a unique and less-well-studied division mechanism that merits closer inspection. In vivo, the three Cdv proteins form a composite band that contracts concomitantly with the septum formation. Of the three Cdv proteins, CdvA is the first to be recruited to the division site, while CdvB and CdvC are thought to participate in the active part of the Cdv division machinery. Interestingly, CdvB shares homology with a family of proteins from the eukaryotic ESCRT-III complex, and CdvC is a homolog of the eukaryotic Vps4 complex. These two eukaryotic complexes are key factors in the endosomal sorting complex required for transport (ESCRT) pathway, which is responsible for various budding processes in eukaryotic cells and which participates in the final stages of division in Metazoa. There, ESCRT-III forms a contractile machinery that actively cuts the membrane, whereas Vps4, which is an ATPase, is necessary for the turnover of the ESCRT membrane-abscission polymers. In contrast to CdvB and CdvC, CdvA is unique to the archaeal Crenarchaeota and Thaumarchaeota phyla. The Crenarchaeota division mechanism has often been suggested to represent a simplified version of the ESCRT division machinery thus providing a model system to study the evolution and mechanism of cell division in higher organisms. However, there are still many open questions regarding this parallelism and the division mechanism of Crenarchaeota. Here, we review the existing data on the role of the Cdv proteins in the division process of Crenarchaeota as well as concisely review the ESCRT system in eukaryotes. We survey the similarities and differences between the division and abscission mechanisms in the two cases. We suggest that the Cdv system functions differently in archaea than ESCRT does in eukaryotes, and that, unlike the eukaryotic case, the Cdv system's main function may be related to surplus membrane invagination and cell-wall synthesis.

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.

The vasculature of the zebrafish trunk is composed of tubes with different cellular architectures. Unicellular tubes form their lumen through membrane invagination and transcellular cell hollowing, whereas multicellular vessels become lumenized through a chord hollowing process. Endothelial cell proliferation is essential for the subsequent growth and maturation of the blood vessels. However, how cell division, lumen formation and cell rearrangement are coordinated during angiogenic sprouting has so far not been investigated at detailed cellular level. Reasoning that different tubular architectures may impose discrete mechanistic constraints on endothelial cell division, we analyzed and compared the sequential steps of cell division, namely mitotic rounding, cytokinesis, actin re-distribution and adherence junction formation, in different blood vessels. In particular, we characterized the interplay between cell rearrangement, mitosis and lumen dynamics within unicellular and multicellular tubes. The lumen of unicellular tubes becomes constricted and is ultimately displaced from the plane of cell division, where a de novo junction forms through the recruitment of junctional proteins at the site of abscission. By contrast, the new junctions separating the daughter cells within multicellular tubes form through the alteration of pre-existing junctions, and the lumen is retained throughout mitosis. We also describe variations in the progression of cytokinesis: while membrane furrowing between daughter cells is symmetric in unicellular tubes, we found that it is asymmetric in those multicellular tubes that contained a taut intercellular junction close to the plane of division. Our findings illustrate that during the course of normal development, the cell division machinery can accommodate multiple tube architectures, thereby avoiding disruptions to the vascular network.

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.

Escherichia coli FtsN is a bitopic membrane protein that is essential for triggering active cell constriction. A small periplasmic subdomain (EFtsN) is required and sufficient for function, but its mechanism of action is unclear. We isolated extragenic EFtsN*-suppressing mutations that restore division in cells producing otherwise non-functional variants of FtsN. These mapped to the IC domain of FtsA in the cytoplasm and to small subdomains of the FtsB and FtsL proteins in the periplasm. All FtsB and FtsL variants allowed survival without EFtsN, but many then imposed a new requirement for interaction between the cytoplasmic domain of FtsN (NFtsN) with FtsA. Alternatively, variants of FtsA, FtsB or FtsL acted synergistically to allow cell division in the complete absence of FtsN. Strikingly, moreover, substitution of a single residue in FtsB (E56) proved sufficient to rescue ΔftsN cells as well. In FtsN+ cells, EFtsN*-suppressing mutations promoted cell fission at an abnormally small cell size, and caused cell shape and integrity defects under certain conditions. This and additional evidence support a model in which FtsN acts on either side of the membrane to induce a conformational switch in both FtsA and the FtsBLQ subcomplex to derepress septal peptidoglycan synthesis and membrane invagination.

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.

Actin-related protein 2/3 (Arp2/3) complex activation by nucleation promoting factors (NPFs) such as WASP, plays an important role in many actin-mediated cellular processes. In yeast, Arp2/3-mediated actin filament assembly drives endocytic membrane invagination and vesicle scission. Here we used genetics and quantitative live-cell imaging to probe the mechanisms that concentrate NPFs at endocytic sites, and to investigate how NPFs regulate actin assembly onset. Our results demonstrate that SH3 (Src homology 3) domain-PRM (proline-rich motif) interactions involving multivalent linker proteins play central roles in concentrating NPFs at endocytic sites. Quantitative imaging suggested that productive actin assembly initiation is tightly coupled to accumulation of threshold levels of WASP and WIP, but not to recruitment kinetics or release of autoinhibition. These studies provide evidence that WASP and WIP play central roles in establishment of a robust multivalent SH3 domain-PRM network in vivo, giving actin assembly onset at endocytic sites a switch-like behavior. DOI: http://dx.doi.org/10.7554/eLife.29140.001, eLife digest Actin is one of the most abundant proteins in yeast, mammalian and other eukaryotic cells. It assembles into long chains known as filaments that the cell uses to generate forces for various purposes. For example, actin filaments are needed to pull part of the membrane surrounding the cell inwards to bring molecules from the external environment into the cell by a process called endocytosis. In yeast, a member of the WASP family of proteins promotes the assembly of actin filaments around the site where endocytosis will occur. To achieve this, WASP interacts with several other proteins including WIP and myosin, a motor protein that moves along actin filaments to generate mechanical forces. However, it was not clear how these proteins work together to trigger actin filaments to assemble at the right place and time. Sun et al. addressed this question by studying yeast cells with genetic mutations affecting one or more of these proteins. The experiments show that WASP, myosin and WIP are recruited to sites where endocytosis is about to occur through specific interactions with other proteins. For example, a region of WASP known as the proline-rich domain can bind to proteins that contain an “SH3” domain. WASP and WIP arrive first, stimulating actin to assemble in an “all and nothing” manner and attracting myosin to the actin. Further experiments indicate that WASP and WIP need to reach a threshold level before actin starts to assemble. The findings of Sun et al. suggest that WASP and WIP play key roles in establishing the network of proteins needed for actin filaments to assemble during endocytosis. These proteins are needed for many other processes in yeast and other cells, including mammalian cells. Therefore, the next steps will be to investigate whether WASP and WIP use the same mechanism to operate in other situations. DOI: http://dx.doi.org/10.7554/eLife.29140.002

Many viruses that replicate in the cytoplasm compartmentalise their genome replication and transcription in specific subcellular microenvironments or organelle-like structures, to increase replication efficiency and protect against host cell defences. Recent studies have investigated the complex membrane rearrangements induced by diverse positive-strand RNA viruses, which are of two morphotypes : membrane invagination towards the lumen of the endoplasmic reticulum (ER) or other specifically targeted organelles and double-membrane vesicles (DMVs) formed by extrusion of the ER membrane. DMVs resemble small autophagosomes and the viruses inducing these intriguing organelles are known to promote autophagy, suggesting a potential link between DMVs and the autophagic pathway. In this review, we summarise recent findings concerning the biogenesis, architecture and role of DMVs in the life cycle of viruses from different families, and discuss their possible connection to autophagy or other related pathways.

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.

Endocytosis is a complex and dynamic process that involves dozens of different proteins to define the site of endocytosis, form a membrane invagination, and pinch off a membrane vesicle into the cytoplasm. Fluorescent light microscopy is a powerful tool to visualize the dynamic behaviors of the proteins taking part in the endocytic process. The resolution of light microscopy is, however, a serious limitation. Here, we detail a fluorescence microscope method that we have developed to visualize the dynamics of the clathrin-mediated endocytic protein machinery in yeast cells. This method is based on subpixel centroid tracking of endocytic proteins. For each endocytic protein, the centroid trajectories obtained from multiple endocytic events are used to compute an average trajectory that describes, at nanometer scale, the assembly and movement of the protein during endocytosis. The average trajectories of the different endocytic proteins are then aligned together in space and time to reconstruct how the different proteins behave relative to each other during the endocytic process.

Vertebrate cells can initiate ciliogenesis from centrioles at the cell center, near the Golgi, forming primary cilia confined or submerged in a deep narrow pit created by membrane invagination. How or why cells maintain submerged cilia is unclear. Here, by characterizing centriole sub-distal appendages (sDAP) in cells exclusively growing submerged cilia, we found that a group of sDAP components localize to the centriole proximal end through the cohesion factor C-Nap1, and that sDAP functions redundantly with C-Nap1 for submerged cilia maintenance. Loss of sDAP and C-Nap1 has no effect on cilia assembly, but disrupts stable Golgi-cilia association, and allows normally submerged cilia to fully surface, losing the deep membrane invagination. Intriguingly, unlike submerged cilia (stationary), surfaced cilia actively respond to mechanical stimuli with motions, and can ectopically recruit hedgehog signaling components in the absence of agonist. We propose that spatial control of ciliogenesis uncouples or specifies sensory properties of cilia.

Efficient digestion and absorption of nutrients by the intestine requires a very large apical surface area, a feature that is enhanced by the presence of villi, fingerlike epithelial projections that extend into the lumen. Prior to villus formation, the epithelium is a thick pseudostratified layer. In mice, villus formation begins at embryonic day (E)14.5, when clusters of mesenchymal cells form just beneath the thick epithelium. At this time, analysis of the flat lumenal surface reveals a regular pattern of short apical membrane invaginations that form in regions of the epithelium that lie in between the mesenchymal clusters. Apical invaginations begin in the proximal intestine and spread distally, deepening with time. Interestingly, mitotically rounded cells are frequently associated with these invaginations. These mitotic cells are located at the tips of the invaginating membrane (internalized within the epithelium), rather than adjacent to the apical surface. Further investigation of epithelial changes during membrane invagination reveals that epithelial cells located between mesenchymal clusters experience a circumferential compression, as epithelial cells above each cluster shorten and widen. Using a computational model, we examined whether such forces are sufficient to cause apical invaginations. Simulations and in vivo data reveal that proper apical membrane invagination involves intraepithelial compressive forces, mitotic cell rounding in the compressed regions and apico-basal contraction of the dividing cell. Together, these data establish a new model that explains how signaling events intersect with tissue forces to pattern apical membrane invaginations that define the villus boundaries.

The source of constriction required for division of a bacterial cell remains enigmatic. FtsZ is widely believed to be a key player, because in vitro experiments indicate that it can deform liposomes when membrane tethered. However in vivo evidence for such a role has remained elusive as it has been challenging to distinguish the contribution of FtsZ from that of peptidoglycan-ingrowth. To differentiate between these two possibilities we studied the early stages of division in Escherichia coli, when FtsZ is present at the division site but peptidoglycan synthesizing enzymes such as FtsI and FtsN are not. Our approach was to use correlative cryo-fluorescence and cryo-electron microscopy (cryo-CLEM) to monitor the localization of fluorescently labeled FtsZ, FtsI or FtsN correlated with the septal ultra-structural geometry in the same cell. We noted that the presence of FtsZ at the division septum is not sufficient to deform membranes. This observation suggests that, although FtsZ can provide a constrictive force, the force is not substantial at the onset of division. Conversely, the presence of FtsN always correlated with membrane invagination, indicating that allosteric activation of peptidoglycan ingrowth is the trigger for constriction of the cell envelope during cell division in E. coli.

In a previous paper (Picco et al., 2015), the dynamic architecture of the protein machinery during clathrin-mediated endocytosis was visualized using a new live imaging and particle tracking method. Here, by combining this approach with correlative light and electron microscopy, we address the role of clathrin in this process. During endocytosis, clathrin forms a cage-like coat around the membrane and associated protein components. There is growing evidence that clathrin does not determine the membrane morphology of the invagination but rather modulates the progression of endocytosis. We investigate how the deletion of clathrin heavy chain impairs the dynamics and the morphology of the endocytic membrane in budding yeast. Our results show that clathrin is not required for elongating or shaping the endocytic membrane invagination. Instead, we find that clathrin contributes to the regularity of vesicle scission and thereby to controlling vesicle size.

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.

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.

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.

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.

Against the odds of membrane resistance, members of the BIN/Amphiphysin/Rvs (BAR) domain superfamily shape membranes and their activity is indispensable for a plethora of life functions. While crystal structures of different BAR dimers advanced our understanding of membrane shaping by scaffolding and hydrophobic insertion mechanisms considerably, especially life-imaging techniques and loss-of-function studies of clathrin-mediated endocytosis with its gradually increasing curvature show that the initial idea that solely BAR domain curvatures determine their functions is oversimplified. Diagonal placing, lateral lipid-binding modes, additional lipid-binding modules, tilde shapes and formation of macromolecular lattices with different modes of organisation and arrangement increase versatility. A picture emerges, in which BAR domain proteins create macromolecular platforms, that recruit and connect different binding partners and ensure the connection and coordination of the different events during the endocytic process, such as membrane invagination, coat formation, actin nucleation, vesicle size control, fission, detachment and uncoating, in time and space, and may thereby offer mechanistic explanations for how coordination, directionality and effectiveness of a complex process with several steps and key players can be achieved.

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.

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.

Cells sense and respond to their environment largely through the function of receptors, transporters, and channels within the plasma membrane. For cells to appropriately respond to environmental cues, they must maintain the proper protein complement at the cell surface. This involves both delivering proteins to the cell surface and removing them when necessary. The multivesicular body (MVB) sorting reaction within the endocytic pathway provides an important cellular mechanism for terminating the function of integral membrane proteins destined for degradation within the lysosome (reviewed in 1–3). The MVB sorting machinery recognizes a subset of endocytic cargoes and concentrates them into regions within the endosomal membrane. These sections then bud as small vesicles into the lumen of the endosome, giving the endosome a multivesicular appearance by electron microscopy. Subsequent fusion of the MVB with the lysosome delivers these intralumenal vesicles to the hydrolytic environment of the lysosome, where the lipid and protein contents of the vesicles are degraded (reviewed in 4). The topology of membrane invagination into the endosomal lumen (exvagination from the cytosol) during MVB sorting is similar to the budding process whereby enveloped viruses, including HIV-1 and Ebola virus, egress from the cell (Figure 1). Viral structural proteins, such as Gag from HIV-1 and VP40 from Ebola virus, utilize the MVB sorting machinery to bud either from the plasma membrane or into an intracellular compartment for subsequent release from the cell (reviewed in 5,6). Therefore, the MVB sorting machinery is important both for viral replication as well as for lysosomal delivery of endocytosed proteins. In addition, a number of recent studies have shown a role for the MVB sorting machinery in late steps of cytokinesis 7–13, emphasizing the importance of this machinery for diverse cellular processes of similar membrane topology. Figure 1 Roles for cellular machinery in MVB sorting and viral particle formation MVB sorting is conserved throughout eukaryotes, and studies in both yeast and mammalian systems have identified a series of trans-acting factors that mediate this reaction (reviewed in 2,14,15). The endosomal sorting complexes required for transport (ESCRTs) and associated proteins constitute the majority of this machinery. Cargo recognition is mediated by interactions with the Vps27/Hrs-Hse1/STAM complex as well as with ESCRT-I (Vps23/Tsg101, Vps28, Vps37 and Mvb12) 16–20. ESCRT-II (Vps22, Vps25, Vps36) appears to function downstream or in parallel to ESCRT-I, and is also believed to interact with cargo based on ubiquitin-binding domain activity within Vps36 21–25. ESCRT-II additionally serves to facilitate the recruitment and assembly of ESCRT-III subunits (Vps20/CHMP6, Snf7/CHMP4, Vps2/CHMP2, Vps24/CHMP3, Did2/CHMP1, Vps60/CHMP5) into a polymer 25–28. Interactions between ESCRT-III and the Vps4-Vta1 complex then promote disassembly of ESCRT-III and may be linked to the completion of intralumenal vesicle (ILV) formation 29–35. The concerted actions of these multimeric complexes serve to concentrate and deliver MVB cargoes into ILVs of the endosome for their eventual degradation within the lysosome. While lysosomal degradation provides a mechanism for the long-term attenuation of transmembrane proteins (such as cell surface receptors, transporters, and channels), sorting into the MVB appears to abrogate their functions prior to degradation. Sorting activated receptors into the MVB sequesters their cytoplasmic kinase domains into the endosomal lumen and away from cytoplasmic substrates, effectively terminating signaling from activated receptors. A well-studied example illustrating the importance of this reaction is the sorting of activated epidermal growth factor receptor (EGFR) (reviewed in 36,37). Defects that prevent EGFR sorting into intralumenal vesicles are oncogenic due to receptor hyperactivity, and this oncogenic activity is attributed to deficiencies in both receptor sequestration and lysosomal degradation (reviewed in 38,39). While MVB sorting can have an important impact on signaling, signaling can also impact MVB sorting. EGFR delivery into the MVB pathway is accelerated in response to EGF stimulation; moreover, EGF stimulation actually promotes MVB biogenesis 40,41. The Hrs-STAM complex is phosphorylated after stimulation of receptor tyrosine kinases 42–44, and this phosphorylation impacts machinery levels and receptor degradation 45. Therefore, it is important to understand the mechanisms by which signaling and MVB sorting impact each other and how flux through the MVB pathway is regulated.

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.