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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.

International audience; The delivery of active molecules into cells requires the efficient translocation of the plasma membrane barrier. Penetratin is a promising cell penetrating peptide is which crosses the cell membrane by a receptor and metabolic energy-independent mechanism. In previous work, we have shown that basic peptides induce membrane invaginations (i.e., tubes formation by induction of negative curvature of membranes) suggesting a new mechanism for cellular uptake of cell penetrating peptides: "physical endo-cytosis". These effects on membrane curvature are favoured in pure liquid disordered but not in pure liquid ordered (raft-like) membrane domains. Herein, we present experiments in heterogeneous membranes composed of mixed domains. The results show that Penetratin is able to induce invaginations in membranes in which liquid ordered and liquid disordered membranes coexist. We suggest that Penetratin is able to recruit specific lipids locally forming fluid membrane patches dispersed inside a liquid ordered membrane zone resulting in the invagination of tubes composed of heterogeneous membrane domains.

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

We analyzed the development of multiple septa in elongated multinucleated cells (hyphae) of the filamentous ascomycete Ashbya gossypii in which septation is apparently uncoupled from nuclear cycles. A key player for this compartmentalization is the PCH protein Hof1. Hyphae that are lacking this protein form neither actin rings nor septa but still elongate at wild-type speed. Using in vivo fluorescence microscopy, we present for the first time the coordination of cytokinesis and septation in multiseptated and multinucleated cells. Hof1, the type II myosin Myo1, the landmark protein Bud3, and the IQGAP Cyk1 form collars of cortical bars already adjacent to hyphal tips, thereby marking the sites of septation. While hyphae continue to elongate, these proteins gradually form cortical rings. This bar-to-ring transition depends on Hof1 and Cyk1 but not Myo1 and is required for actin ring assembly. The Fes/CIP4 homology (FCH) domain of Hof1 ensures efficient localization of Hof1, whereas ring integrity is conferred by the Src homology 3 (SH3) domain. Up to several hours after site selection, actin ring contraction leads to membrane invagination and subsequent cytokinesis. Simultaneously, a septum forms between the adjacent hyphal compartments, which do not separate. During evolution, A. gossypii lost the homologs of two enzymes essential for cell separation in Saccharomyces cerevisiae.

Along the degradation pathway, endosomes exhibit a characteristic multivesicular organization, resulting from the budding of vesicles into the endosomal lumen. After endocytosis and transport to early endosomes, activated signaling receptors are incorporated into these intralumenal vesicles through the action of the ESCRT machinery, a process that contributes to terminate signaling. Then, the vesicles and their protein cargo are further transported towards lysosomes for degradation. Evidence also shows that intralumenal vesicles can undergo "back-fusion" with the late endosome limiting membrane, a route exploited by some pathogens and presumably followed by proteins and lipids that need to be recycled from within the endosomal lumen. This process depends on the late endosomal lipid lysobisphosphatidic acid and its putative effector Alix/AIP1, and is presumably coupled to the invagination of the endosomal limiting membrane at the molecular level via ESCRT proteins. In this review, we discuss the intra-endosomal transport routes in mammalian cells, and in particular the different mechanisms involved in membrane invagination, vesicle formation and fusion in a space inaccessible to proteins known to control intracellular membrane traffic.

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

The expulsion of material from a cell by fusion of vesicles at the plasma membrane, and the entry of a virus by membrane invagination are complex membrane-associatedprocesses whose control is crucial to cell survival. Our ability to visualize the dynamics of such processes experimentally is limited by spatial resolution and the speed of molecular rearrangements. The increase in computing power of the last few decades enables the construction of computational tools for observing cellular processes in silico. As experiments yield increasing amounts of data on the protein and lipid constituents of the cell, computer simulations parametrized using this data are beginning to allow models of cellular processes to be interrogated in ways unavailable in the laboratory. Mesoscopic simulations retain only those molecular features that are believed to be relevant to the processes of interest. This allows the dynamics of spatially heterogeneous membranes and the crowded cytoplasmic environment to be followed at a modest computational cost. The price for such power is that the atomic detail of the constituents is much lower than in atomistic Molecular Dynamics simulations. We argue that this price is worth paying because mesoscopic simulations can generate new insight into the complex, dynamic life of a cell.

Trypanosoma brucei is the causative agent of African sleeping sickness. It possesses a single flagellum, which additional to its mobility role, is an important sensory and signaling organelle [1,2]. The flagellum exits the cell from a membrane invagination, called the flagellar pocket (FP), region where endo- and exocytosis occur. A ring-shaped structure, called the flagellar pocket collar (FPC), encloses the FP defining the flagellum exit point. BILBO1 is the first FPC protein identified and is essential for the FPC and FP biogenesis [2]. A T. brucei gDNA yeast two-hybrid (Y2H) screen identified several BILBO1 protein partners, including FPC4 a protein we are characterizing at the molecular and functional level. Our anti-FPC4 antibody and endogenous FPC4-myc cell confirmed that FPC4 localizes both on, and close to the FPC. RNAi down-regulation of FPC4 has no impact on cell proliferation. However, over-expression of FPC4 leads to filament formation and to a mild, but reduced growth defect. Co-immunolocalization demonstrated that BILBO1 relocalizes to the FP4 filaments indicating BILBO1-FPC4 interaction and the role of FPC4 in FPC structure. Y2H shows that BILBO1 interacts with the C-terminal domain of FPC4 and we are currently identifying the minimal interaction domains, with the long-term objective of a drug screen to block this interaction. Expression of FPC4 in a heterologous system (human cells) suggests that the N-terminal domain binds to microtubules (MT). This was confirmed by in vitro MT binding assays and is ongoing. We are also characterizing FPC4 function in bloodstream form. Our preliminary data suggest that FPC4 could be a FPC-microtubule linker involved in FPC segregation during the cell cycle.

Co-existing paracellular and transcellular barrier defect in intestinal epithelium was documented in inflammatory bowel disease, celiac disease, and intestinal obstruction. Mechanisms regarding tight junction disruption have been extensively studied; however, limited progress has been made in research on bacterial transcytosis. Densely packed brush border (BB), with cholesterol-based lipid rafts in the intermicrovillous membrane invagination, serves as an ultrastructural barrier to prevent direct contact of luminal microbes with the cellular soma. Evidence in in vitro epithelial cell cultures and in vivo animal models of bowel obstruction and antibiotic-resistant bacterial infection had indicated that nonpathogenic, noninvasive enteric bacteria may hijack the lipid raft-mediated endocytic pathways. Our studies have shown that low dose interferon-gamma (IFNγ) causes long myosin light chain kinase (MLCK)-dependent terminal web (TW) contraction and BB fanning, allowing bacteria to pass through the consequently widened intermicrovillous cleft to be endocytosed via caveolin-associated lipid rafts. Activation of intracellular innate immune receptors by bacteria-containing endosomes may further induce inflammatory and oxidative stress, leading to secondary tight junction damage. The finding of bacterial internalization preceding tight junction damage suggests that abnormal bacterial uptake by epithelial cells may contribute to the initiation or relapse of chronic intestinal inflammation.

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.

Magnetosome chains are the intracellular structures that allow magnetotactic bacteria to align in and navigate along geomagnetic fields (Bazylinski and Frankel 2004). These organelles are typically defined as a unit consisting of a magnetite or greigite crystal surrounded by a lipid bilayer membrane (Balkwill et al. 1980). Although these magnetic minerals are the usual targets of most studies of magnetotactic bacteria it is the magnetosome membrane that fascinates cell biologists. One of the cornerstones of cell biology has been that membrane-bound organelles are unique to eukaryotes. However, it is now known that membranous organelles exist in many prokaryotes raising the possibility that the endo-membrane system of eukaryotic cells might have originated in prokaryotes (Jetten et al. 2003; Seufferheld et al. 2003; Fuerst 2005). Magnetosomes are one of the best-studied examples of these prokaryotic organelles with the potential to be an ideal system for the study of organelle development in prokaryotes. This work provides a review of the current knowledge of magnetosomes from a cell biological perspective focusing on the composition and formation of the magnetosome membrane and the cytoskeletal framework organizing individual magnetosomes into chains.

In the paper, we address the problem of the temporal characterization of Drosophila embryos. We have developed a method for automated staging of an embryo on the basis of a confocal image of its gene expression pattern. Phases of spectral Fourier coefficients were used as the features characterizing temporal changes in expression patterns. The age detection is implemented by applying support vector regression, which is a machine learning method for creating regression functions of arbitrary type from a set of training data. The training set is composed of embryos for which the precise developmental age was determined by measuring the degree of membrane invagination. Testing the quality of regression on the training set showed a good prediction accuracy.

SummaryActin polymerization essential for endocytic internalization in budding yeast is controlled by four nucleation promoting factors (NPFs) that each exhibits a unique dynamic behavior at endocytic sites. How each NPF functions and is regulated to restrict actin assembly to late stages of endocytic internalization is not known. Quantitative analysis of NPF biochemical activities, and genetic analysis of recruitment and regulatory mechanisms, defined a linear pathway in which protein composition changes at endocytic sites control actin assembly and function. We show that yeast WASP initiates actin assembly at endocytic sites and that this assembly and the recruitment of a yeast WIP-like protein by WASP recruit a type I myosin with both NPF and motor activities. Importantly, type I myosin motor and NPF activities are separable, and both contribute to endocytic coat inward movement, which likely represents membrane invagination. These results reveal a mechanism in which actin nucleation and myosin motor activity cooperate to promote endocytic internalization.

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

The Saccharomyces cerevisiae synaptojanin-like proteins (Sjl1, Sjl2, and Sjl3) are phosphoinositide (PI) phosphatases that regulate PI metabolism in the control of actin organization and membrane trafficking. However, the primary sites of action for each of the yeast synaptojanin-like proteins remain unclear. In this study, we show that Sjl2 is localized to cortical actin patches, sites of endocytosis. Cortical recruitment of Sjl2 requires the actin patch component Abp1. Consistent with this, the SH3 domain-containing protein Abp1 physically associates with Sjl2 through its proline-rich domain. Furthermore, abp1Delta mutations confer defects resembling loss of SJL2; sjl1Delta abp1Delta double-mutant cells exhibit invaginated plasma membranes and impaired endocytosis, findings similar to those for sjl1Delta sjl2Delta mutant cells. Thus, Abp1 acts as an adaptor protein in the localization or concentration of Sjl2 during late stages of endocytic vesicle formation. Overexpression of the Hip1-related protein Sla2 delayed the formation of extended plasma membrane invaginations in sjl2ts cells, indicating that Sla2 may become limiting or misregulated in cells with impaired PI phosphatase activity. Consistent with this, the cortical actin patch protein Sla2 is mislocalized in sjl1Delta sjl2Delta mutant cells. Together, our studies suggest that PI metabolism by the synaptojanin-like proteins coordinately directs actin dynamics and membrane invagination, in part by regulation of Sla2.

Dividing animal and plant cells maintain a constant chromosome content through temporally separated rounds of replication and segregation. Until recently, the mechanisms by which animal and plant cells maintain a constant surface area have been considered to be distinct. The prevailing view was that surface area was maintained in dividing animal cells through temporally separated rounds of membrane expansion and membrane invagination. The latter event, known as cytokinesis, produces two physically distinct daughter cells and has been thought to be primarily driven by actomyosin-based constriction. By contrast, membrane addition seems to be the primary mechanism that drives cytokinesis in plants and, thus, the two events are linked mechanistically and temporally. In this article (which is part of the Cytokinesis series), we discuss recent studies of a variety of organisms that have made a convincing case for membrane trafficking at the cleavage furrow being a key component of both animal and plant cytokinesis.

The physical interaction of the plasma membrane with the associated cortical cytoskeleton is important in many morphogenetic processes during development. At the end of the syncytial blastoderm of Drosophila the plasma membrane begins to fold in and forms the furrow canals in a regular hexagonal pattern. Every furrow canal leads the invagination of membrane between adjacent nuclei. Concomitantly with furrow canal formation, actin filaments are assembled at the furrow canal. It is not known how the regular pattern of membrane invagination and the morphology of the furrow canal is determined and whether actin filaments are important for furrow canal formation. We show that both the guanyl-nucleotide exchange factor RhoGEF2 and the formin Diaphanous (Dia) are required for furrow canal formation. In embryos from RhoGEF2 or dia germline clones, furrow canals do not form at all or are considerably enlarged and contain cytoplasmic blebs. Both Dia and RhoGEF2 proteins are localised at the invagination site prior to formation of the furrow canal. Whereas they localise independently of F-actin,Dia localisation requires RhoGEF2. The amount of F-actin at the furrow canal is reduced in dia and RhoGEF2 mutants,suggesting that RhoGEF2 and Dia are necessary for the correct assembly of actin filaments at the forming furrow canal. Biochemical analysis shows that Rho1 interacts with both RhoGEF2 and Dia, and that Dia nucleates actin filaments. Our results support a model in which RhoGEF2 and dia control position, shape and stability of the forming furrow canal by spatially restricted assembly of actin filaments required for the proper infolding of the plasma membrane.

Endosomes, which comprise a diverse population of membrane vesicles and tubules, sort proteins and lipids to various cellular destinations. The organization and functions of these pleiomorphic cellular organelles have been extensively studied. Matsuo et al.1 now provide new exciting evidence on the role of lysobiphosphatidic acid (LBPA), a resident phospholipid of internal vesicles of the late endosome, in the control of membrane invagination and endosome biogenesis. In vivo, LBPA functions are controlled by the adaptor protein Alix and depend on pH gradients along the endosomal compartments. BioEssays 26:604–607, 2004. © 2004 Wiley Periodicals, Inc.

Formation of the Drosophila cellular blastoderm involves both membrane invagination and cytoskeletal regulation. Mutations in src64and tec29 reveal a novel role for these genes in controlling contraction of the actin-myosin microfilament ring during this process. Although membrane invagination still proceeds in mutant embryos, its depth is not uniform, and basal closure of the cells does not occur during late cellularization. Double-mutant analysis between scraps, a mutation in anillin that eliminates microfilament rings, and bottleneck suggests that microfilaments can still contract even though they are not organized into rings. However, the failure of rings to contract in the src64 bottleneck double mutant suggests that src64 is required for microfilament ring contraction even in the absence of Bottleneck protein. Our results suggest that src64-dependent microfilament ring contraction is resisted by Bottleneck to create tension and coordinate membrane invagination during early cellularization. The absence of Bottleneck during late cellularization allows src64-dependent microfilament ring constriction to drive basal closure.

Microautophagy is the transfer of cytosolic components into the lysosome by direct invagination of the lysosomal membrane and subsequent budding of vesicles into the lysosomal lumen. This process is topologically equivalent to membrane invagination during multivesicular body formation and to the budding of enveloped viruses. Vacuoles are lysosomal compartments of yeasts. Vacuolar membrane invagination can be reconstituted in vitro with purified yeast vacuoles, serving as a model system for budding of vesicles into the lumen of an organelle. Using this in vitro system, we defined different reaction states. We identified inhibitors of microautophagy in vitro and used them as tools for kinetic analysis. This allowed us to characterize four biochemically distinguishable steps of the reaction. We propose that these correspond to sequential stages of vacuole invagination and vesicle scission. Formation of vacuolar invaginations was slow and temperature-dependent, whereas the final scission of the vesicle from a preformed invagination was fast and proceeded even on ice. Our observations suggest that the formation of invaginations rather than the scission of vesicles is the rate-limiting step of the overall reaction.

Caveolae are the sites in the cell membrane responsible for concentrating an array of signaling molecules critical for cell function. Recent studies have begun to identify the functions of caveolin-1, the 22-kDa caveolar protein that oligomerizes and inserts into the cytoplasmic face of the plasma membrane. Caveolin-1 appears to regulate caveolar internalization by stabilizing caveolae at the plasma membrane rather than controlling the shape of the membrane invagination. Because caveolin-1 is a scaffolding protein, it has also been hypothesized to function as a “master regulator” of signaling molecules in caveolae. Deletion of the caveolin-1 gene in mice resulted in cardiac hypertrophy and lung fibrosis, indicating its importance in cardiac and lung development. In the endothelium, caveolin-1 regulates nitric oxide signaling by binding to and inhibiting endothelial nitric oxide synthase (eNOS). Increased cytosolic Ca2+or activation of the kinase Akt leads to eNOS activation and its dissociation from caveolin-1. Caveolae have also been proposed as the vesicle carriers responsible for transcellular transport (transcytosis) in endothelial cells. Transcytosis, the primary means of albumin transport across continuous endothelia, occurs by fission of caveolae from the membrane. This event is regulated by tyrosine phosphorylation of caveolin-1 and dynamin. As Ca2+influx channels and pumps are localized in caveolae, caveolin-1 is also an important determinant of Ca2+signaling in endothelial cells. Many of these findings were presented in San Diego, CA, at the 2003 Experimental Biology symposium “Caveolin Regulation of Endothelial Function” and are reviewed in this summary.

Polyoxyethyleneglycolalkylether (CmEn, m=12, n=8) can induce a large torocyte-like endovesicle in human erythrocytes. The present study aimed to examine how variations in the molecular structure of CmEn (m=10,12,14,16,18; n=1‐10,23) affect the occurrence of torocyte endovesicles. Our results show that torocytes occur most frequently when m=12,14 and n=8,9. At this molecular configuration the detergents induce inward membrane bending (stomatocytic S1‐S2 shapes) resulting in the formation of a large membrane invagination. These detergents have a strong membrane perturbing, i.e., haemolytic, effect. Theoretical calculations indicate that a torocyte-shaped inside-out membrane vesicle can be created from a large membrane invagination due to the impact of laterally mobile anisotropic membrane inclusions. Such inclusions may be detergent‐membrane component complexes or unanchored integral membrane proteins. It is shown that a nonhomogeneous lateral distribution of anisotropic membrane inclusions may stabilise the torocyte endovesicle shape, characterised by having opposite membranes in the thin central region of the vesicles separated by a certain distance. Tubular, conical or inverted conical isotropic inclusions cannot do so. D 2004 Elsevier B.V. All rights reserved.

The hydrogenosome is an unusual organelle found in several trichomonad species and other protists living in oxygen poor or anoxic environments. The hydrogenosome behavior in the protist Tritrichomonas foetus, parasite of the urogenital tract of cattle, is reported here. The hydrogenosomes were followed by light and transmission electron microscopy during the whole cell cycle. Videomicroscopy, immunofluorescence microscopy, and immunocytochemistry were also used. It is shown that the hydrogenosomes divide at any phase of the cell cycle and that the organellar division is not synchronized. During the interphase the hydrogenosomes are distributed mainly along the axostyle and costa, and at the beginning of mitosis migrate to around the nucleus. Three forms of hydrogenosome division were seen: (1). segmentation, where elongated hydrogenosomes are further separated by external membranous profiles; (2). partition, where rounded hydrogenosomes, in a bulky form, are further separated by a membranous internal septum and, (3). a new dividing form: heart-shaped hydrogenosomes, which gradually present a membrane invagination leading to the organelle division. The hydrogenosomes divide at any phase of the cell cycle. A necklace of intramembranous particles delimiting the outer hydrogenosomal membrane in the region of organelle division was observed by freeze-etching. Similarities between hydrogenosomes and mitochondria behavior during the cell cycle are discussed.

Proteins of the annexin family are believed to be involved in membrane-related processes, but their precise functions remain unclear. Here, we have made use of several experimental approaches, including pathological conditions, RNA interference and in vitro transport assays, to study the function of annexin II in the endocytic pathway. We find that annexin II is required for the biogenesis of multivesicular transport intermediates destined for late endosomes, by regulating budding from early endosomes-but not the membrane invagination process. Hence, the protein appears to be a necessary component of the machinery controlling endosomal membrane dynamics and multivesicular endosome biogenesis. We also find that annexin II interacts with cholesterol and that its subcellular distribution is modulated by the subcellular distribution of cholesterol, including in cells from patients with the cholesterol-storage disorder Niemann-Pick C. We conclude that annexin II forms cholesterol-containing platforms on early endosomal membranes, and that these platforms regulate the onset of the degradation pathway in animal cells.

Clathrin-mediated vesicle budding is essential for a variety of intracellular membrane transport reactions in the secretory and endocytic pathways (1, 2). This process can be dissected in several sequential steps that include the recruitment of the clathrin coat, membrane invagination and concomitant propagation of the clathrin lattice, constriction of the vesicle neck, and finally fission of the coated vesicle, followed by its rapid uncoating (1, 3, 4). Although clathrin and clathrin adaptors, assisted by other accessory factors, drive this reaction up to the stage of a deeply invaginated coated pit, a distinct factor, the GTPase dynamin, is thought to play a critical role in fission (5, 6). To provide new clues concerning all sites where clathrin functions in mammalian cell physiology and on the vesicular traffic pathways that are dependent on clathrin, Iversen et al. (7) have engineered a mammalian cell line in which expression of clathrin can be suppressed by an inducible antisense RNA. Unexpectedly, this study has suggested a role for clathrin heavy chain in the regulation of dynamin expression and function. This study suggests a role for clathrin heavy chain in the regulation of dynamin expression and function.

The hemolytic behavior of a novel cytoprotective agent, DY-9760e (3-[2-[4-(3-chloro-2-methylphenyl)-1-piperazinyl]ethyl]-5,6-dimethoxy-1-(4-imidazolylmethyl)-1H-indazole dihydrochloride 3.5 hydrate) was investigated using rabbit erythrocytes. Further, the effects of water-soluble cyclodextrin derivatives, such as 2-hydroxypropyl-beta-cyclodextrin (HP-beta-CyD) and sulfobutyl ether of beta-cyclodextrin (SBE-beta-CyD), on the hemolytic activity of DY-9760e were studied. DY-9760e induced hemolysis at concentrations >0.2-0.3 mM in phosphate buffered saline (PBS) of pH 4.0 and 6.0, where DY-9760e is predominantly in dicationic and monocationic forms, respectively. The hemolytic activity of the monocationic DY-9760e was higher than that of the dicationic species, and the hemolysis at pH 4.0 involved the formation of methemoglobin. DY9760e induced the morphological change of erythrocytes towards membrane invagination at both pH 4.0 and 6.0. SBE7-beta-CyD significantly suppressed the DY-9760e-induced hemolysis and morphological change at both pH 4.0 and 6.0, as well as the formation of methemoglobin at pH 4.0. On the other hand, HP-beta-CyD suppressed only the hemolysis, but neither the morphological change nor the formation of methemoglobin. In addition, the inhibitory effect of SBE7-beta-CyD on the hemolysis was greater than that of HP-beta-CyD. The superior inhibitory effect of SBE7-beta-CyD on the DY-9760-induced hemolysis, the morphological change, and the formation of methemoglobin may be attributable to the formation of a stable inclusion complex with DY-9760e and to the weaker hemolytic activity of SBE7beta-CyD than HP-beta-CyD. These results suggest potential use of SBE7-beta-CyD as a parenteral carrier for DY-9760e.

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.

The shape of the newly described torocyte red blood cell endovesicles induced by octaethyleneglycol dodecylether (C12E8) is characterized. A possible explanation for the origin and stability of the observed torocyte endovesicles is suggested. Three partly complementary mechanisms are outlined, all originating from the interaction of C12E8 molecules with the membrane. The first is a preferential intercalation of the C12E8 molecule into the inner membrane layer, resulting in a membrane invagination which may finally close, forming an inside-out endovesicle. The second is a preference of the C12E8-induced membrane inclusions (clusters) for small local curvature which would favour torocyte endovesicle shape with large regions of small or even negative membrane mean curvatures, the C12E8 membrane inclusion being defined as a complex composed of the embedded C12E8 molecule and some adjacent phospholipid molecules which are significantly distorted due to the presence of the embedded C12E8 molecule. The preference of the C12E8 inclusions for zero or negative local curvature may also lead to the nonhomogeneous lateral distribution of the C12E8 inclusions resulting in their accumulation in the membrane of torocyte endovesicles. The third possible mechanism is orientational ordering of the C12E8-induced inclusions in the regions of torocyte endovesicles with high local membrane curvature deviator.

The invasion of the erythrocyte by Plasmodium falciparum depends on the ability of the merozoite to move through the membrane invagination. This ability is probably mediated by actin dependent motors. Using affinity columns with G-actin and F-actin we isolated actin binding proteins from the parasite. By immunoblotting and immunoprecipitation with specific antibodies we identified the presence of tropomyosin, myosin, a-actinin, and two different actins in the eluate corresponding to F-actin binding proteins. In addition to these, a 240-260 kDa doublet, different in size from the erythrocyte spectrin, reacted with an antibody against human spectrin. All the above mentioned proteins were metabolically radiolabeled when the parasite was cultured with 35S-methionine. The presence of these proteins in P. falciparum is indicative of a complex cytoskeleton and supports the proposed role for an actin-myosin motor during invasion.

We have characterized the role of the penicillin-binding protein PBP 2B in cell division of Bacillus subtilis. We have shown that depletion of the protein results in an arrest in division, but that this arrest is slow, probably because the protein is relatively stable. PBP 2B-depleted filaments contained, at about their mid-points, structures resembling partially formed septa, into which most, if not all, of the division proteins had assembled. Although clearly deficient in wall material, membrane invagination seemed to continue, indicating that membrane and wall ingrowth can be uncoupled. At other potential division sites along the filaments, no visible ingrowths were observed, although FtsZ rings assembled at regular intervals. Thus, PBP 2B is apparently required for both the initiation of division and continued septal ingrowth. Immunofluorescence microscopy showed that the protein is recruited to the division site. The pattern of localization suggested that this recruitment occurs continually during septal ingrowth. During sporulation, PBP 2B was present transiently in the asymmetrical septum of sporulating cells, and its availability may play a role in the regulation of sporulation septation.

For more than two decades, investigators who have been studying signal transduction in the heart (and other tissues) have struggled to explain data implicating the existence of “compartmentation.” The data have included evidence for selective effects of certain types of hormonal and neurotransmitter agonists on “downstream” events, in spite of their utilization of common “upstream” second messenger pathways.1 2 The concept of compartmentation arose, at least partially, as an explanation for such phenomena. Yet progress in defining the physical nature of these compartments has, until recently, been rather slow. One problem has been that the biochemical paradigm, which typically involves destroying cells, isolating subcellular fractions, and assaying enzymatic or other activity, is not particularly well suited for precise identification of signaling domains responsible for compartmentation. Consider the folly of this type of approach if one were to use it to identify each of the parts of an appliance, such as a television set. One would smash the appliance, breaking it down into smaller pieces and then attempt to assemble these “fractions” in order to try and learn the function of each component. Nevertheless, progress has recently accelerated in this area, in part because of evolving notions regarding the existence of specialized regions of the sarcolemmal membrane. Early work in a variety of cell types focused on the role of clathrin-coated pit regions that were involved in receptor-mediated endocytosis, in particular of transport proteins and certain receptors.3 More recently, such coated pit regions have been implicated in receptor-promoted internalization of G protein–coupled receptors (GPCRs).4 5 Another specialized region of the plasma membrane that has attracted increasing attention is the caveola, a specialized membrane invagination (“little cave”), whose morphology was originally described in the 1950s. These structures were first identified in endothelial cells and were described as having an …

Previously, we showed caveolae contain a population of protein kinase Calpha (PKCalpha) that appears to regulate membrane invagination. We now report that multiple PKC isoenzymes are enriched in caveolae of unstimulated fibroblasts. To understand the mechanism of PKC targeting, we prepared caveolae lacking PKCalpha and measured the interaction of recombinant PKCalpha with these membranes. PKCalpha bound with high affinity and specificity to caveolae membranes. Binding was calcium dependent, did not require the addition of factors that activate the enzyme, and involved the regulatory domain of the molecule. A 68-kD PKCalpha-binding protein identified as sdr (serum deprivation response) was isolated by interaction cloning and localized to caveolae. Antibodies against sdr inhibited PKCalpha binding. A 100-amino acid sequence from the middle of sdr competitively blocked PKCalpha binding while flanking sequences were inactive. Caveolae appear to be a membrane site where PKC enzymes are organized to carry out essential regulatory functions as well as to modulate signal transduction at the cell surface.

Substances destined for export from eukaryotic cells are packed into membrane vesicles (or granules). Exocytosis is the process of fusion of these vesicles with the plasma membrane whereby soluble components from the vesicle interior are released outside and the membrane of the vesicle is integrated into the plasma membrane. Thus, exocytosis acts not only as an outwardly directed activity of the cell, but also fulfils an important role in the intracellular exchange of membranes. Exocytosis increases the area of the plasma membrane. The opposite process, endocytosis involves a membrane invagination and fission of a vesicle into the cell with a consequent decrease in the plasma membrane area.

A dramatic reorganization of the cytoskeleton underlies the cellularisation of the syncytial Drosophila embryo. Formation of a regular network of acto-myosin filaments, providing a structural framework, and possibly a contractile force as well, appears essential for the synchronous invagination of the plasma membrane between adjacent nuclei. The serendipity alpha (sry α) gene is required for this complete reorganization of the microfilaments at the onset of membrane invagination. We compare here the structure and expression of sry α between D. pseudoobscura, D. subobscura and D. melanogaster. Interspersion of evolutionarily highly conserved and divergent regions is observed in the protein. One such highly conserved region shows sequence similarities to a motif found in proteins of the ezrin-radixinmoesin (ERM) family. Four 7-13 bp motifs are conserved in the 5 promoter region; two of these are also found, and at the same position relative to the TATA box, in nullo, another zygotic gene recently shown to be involved in cellularisation. The compared patterns of expression of D. melanogaster sry and nullo, and D. pseudoobscura sry reveal a complex regulation of the spatiotemporal accumulation of their transcripts. The D. pseudoobscura sry gene is able to rescue the cellularisation defects associated with a complete loss of sry α function in D. melanogaster embryos, even though species-specific aspects of its expression are maintained. Despite their functional homologies, the D. melanogaster and D. pseudoobscura sry RNAs have different subcellular localisations, suggesting that this specific localization has no conserved role in targeting the sry α protein to the apical membranes.

Receptor-mediated endocytosis is initiated by the accumulation of receptor-ligand complexes in clathrin-coated pits on the plasma membrane. The clustering of receptors in these domains is followed by membrane invagination and the subsequent formation of coated vesicles. Ligand binding induces the clustering of some receptors—for example, epidermal growth factor (EGF) receptor—in coated pits (Beginout et al.,1985). Alternatively, many other receptors—low density lipoprotein receptor (LDL), transferrtin receptor—are internalized constitutively in the absence of ligand (Anderson et al., 1977; Harding et al.,1983). Numerous studies suggest the signals for clustering and internalization are encoded in the cytoplasmic tail of endocytosed receptors (Davis et al., 1987; Lobel et al., 1989). Proteins associated with clathrin in the vesicle coat, such as adaptins, may interact directly with receptor tails to regulate these early steps in endocytosis (Pearse, 1988; Glickman et al.,1989). Shortly after their formation, coated vesicles containing receptor-ligand complexes lose their clathrin lattice in an ATP-dependent reaction (Rothman and Schmid, 1986). The resulting smooth vesicles (early endosomes) may retain some peripheral proteins and membrane components that function in intracellular transport and sorting reactions.

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.

The mechanism by which platelets endocytose or release particulate or soluble substances is poorly understood. Engulfed materials enter the open canalicular system (OCS) by a process akin to phagocytosis, but fusion of platelet granules with the OCS is rarely observed. Secretion of granule contents, a concomitant of the "release reaction" which occurs during platelet aggregation, does not take place by extrusion at the surface membrane as is true for other secretory cells. Some substances may be secreted without obvious granule loss. To examine whether structural properties of the platelet membrane could account for this unusual behavior, thin section and freeze-fracture analyses were performed on platelets which had undergone endocytosis under a variety of experimental conditions. After freeze-cleavage, most of the intramembranous particles (IMP) remain associated with the outer leaflet of the platelet plasma membrane. The sites where the OCS reaches the surface membrane are marked by pits on the cytoplasmic leaflet (P face) and by complementary protrusions on the outer leaflet (E face) of the membrane. Endocytosis of small particles and solutes takes place via these structures. This process is not energy dependent but arrested at 4 degrees C. Distension of the OCS does not appear to affect the size or number of the pits. On the other hand, large particles are taken up by membrane invagination without redistribution of IMP's and independent of the pits. This process is sensitive to metabolic inhibition. Thus, the studies have demonstrated the existence of two different pathways for platelet endocytosis which are postulated to be also involved in secretion. The selective release of substances contained in different granules may be related to the "inside-out" structure of the plasma and OCS membranes.

Elongating caulonemal apical cells of the mossPhyscomitrium turbinatum were cultivatedin vitro and observed during successive stages of cell elongation and division. Actively-growing cells which had completed approximately half of their growth in length were examined by electron microscopy. The distribution of many organelles changes progressively from the cell tip to the distal edge of the large basal vacuole, establishing an apical-basal gradient in organization. Whereas the vacuoles become progressively more extensive in more mature parts of the cell, the dictyosomes, chloroplasts and smooth endoplasmic reticulum are more numerous in younger regions. Some mitochondria in the younger regions of the cell contain localized areas of membrane invagination. Attempts were made to clarify the origin and growth of vacuoles, which become increasingly prominent as the apical cell elongates.

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.

Digestion of macromolecules inside secondary lysosomes results in small molecules leaving the lysosome by permeation. This may cause a drop in osmotic pressure and a consequent shrinkage of the lysosome. A membrane invagination may be formed which becomes internalized and then digested. This digestion causes new shrinkage. Minimal-sized lysosomes may ensue. The later are again engaged in digestive events when fusing with larger lysosomes. Through consecutive shrinkage and fusion cycles of many lysosomes residues slowly accumulate. Shrinkage may be counteracted by adding digestible material without increasing the lysosomal membrane surface. Transfer tubules can do so. They deliver their content to multivesicular bodies. The other lysosome types, however, are subject to shrinkage since they fuse with organelles (phagosomes, primary lysosomes) which insert at least part of their membrane.

Drug-induced endocytosis in intact human erythrocytes is a membrane-mediated event involving membrane invagination and fusion. The drugs capable of inducing endocytosis are amphipathic cations and it was thought that they intercalate themselves into the relatively electronegative cytosolic portion of the phospholipid bilayer. The current studies show that at concentrations that produce endocytosis vinblastine and chlorpromazine are not only concentrated in the red cell membrane compartment but reach high levels in the cytosol as well. The suspending medium can produce impressive effects on the extent of endocytosis. Sodium lactate enhances chlorpromazine and vinblastine endocytosis without altering the incorporation or distribution of these agents. Sucrose enhances chlorpromazine and inhibits vinblastine endocytosis, and while it produces no effects on chlorpromazine incorporation it inhibits vinblastine incorporation, thus in part accounting for its effects. It is proposed that the amphipathic dr...

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.

SummaryEndocytosis, like many dynamic cellular processes, requires precise temporal and spatial orchestration of complex protein machinery to mediate membrane budding. To understand how this machinery works, we directly correlated fluorescence microscopy of key protein pairs with electron tomography. We systematically located 211 endocytic intermediates, assigned each to a specific time window in endocytosis, and reconstructed their ultrastructure in 3D. The resulting virtual ultrastructural movie defines the protein-mediated membrane shape changes during endocytosis in budding yeast. It reveals that clathrin is recruited to flat membranes and does not initiate curvature. Instead, membrane invagination begins upon actin network assembly followed by amphiphysin binding to parallel membrane segments, which promotes elongation of the invagination into a tubule. Scission occurs on average 9 s after initial bending when invaginations are ∼100 nm deep, releasing nonspherical vesicles with 6,400 nm2 mean surface area. Direct correlation of protein dynamics with ultrastructure provides a quantitative 4D resource.

After slow progress in the efforts to develop phase plates for electron microscopes, functional phase plate using thin carbon film has been reported recently. It permits collecting high-contrast images of close-to-life biological structures with cryo-fixation and without staining. This report reviews the state of the art for phase plates and what is innovated with them in biological electron microscopy. The extension of thin-film phase plates to the material-less type using electrostatic field or magnetic field is also addressed. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s12551-008-0006-z) contains supplementary material, which is available to authorized users.

Pombe Cdc15 homology (PCH) proteins play an important role in a variety of actin-based processes, including clathrin-mediated endocytosis (CME). The defining feature of the PCH proteins is an evolutionarily conserved EFC/F-BAR domain for membrane association and tubulation. In the present study, we solved the crystal structures of the EFC domains of human FBP17 and CIP4. The structures revealed a gently curved helical-bundle dimer of approximately 220 A in length, which forms filaments through end-to-end interactions in the crystals. The curved EFC dimer fits a tubular membrane with an approximately 600 A diameter. We subsequently proposed a model in which the curved EFC filament drives tubulation. In fact, striation of tubular membranes was observed by phase-contrast cryo-transmission electron microscopy, and mutations that impaired filament formation also impaired membrane tubulation and cell membrane invagination. Furthermore, FBP17 is recruited to clathrin-coated pits in the late stage of CME, indicating its physiological role.

Ligand–receptor interactions, which represent the core for cell signaling and internalization processes are largely affected by the spatial configuration of host cell receptors. There is a growing piece of evidence that receptors are not homogeneously distributed within the plasma membrane, but are rather pre-clustered in nanodomains, or clusters are formed upon ligand binding. Pathogens have evolved many strategies to evade the host immune system and to ensure their survival by hijacking plasma membrane receptors that are most often associated with lipid rafts. In this review, we discuss the early stage molecular and physiological events that occur following ligand binding to host cell glycolipids. The ability of various biological ligands (e.g. toxins, lectins, viruses or bacteria) that bind to glycolipids to induce their own uptake into mammalian cells by creating negative membrane curvature and membrane invaginations is explored. We highlight recent trends in understanding nanoscale plasma membrane (re-)organization and present the benefits of using synthetic membrane systems. This article is part of a Special Issue entitled: Nanoscale membrane organisation and signalling.