Fusion of microsomal vesicles

Studies which indicate the fusion of rat liver microsomal vesicles show that the rate of fusion of microsomal vesicles, as revealed by electron microscopic examinations, is dependent on the fusion temperature and the amount of detergent present in the microsomal suspension.     

Molecular machinery mediating vesicle budding,docking and fusion

A general machinery buds and fuses transport vesicles which connect intracellular compartments with each other and allow communication with the extracellular environment. Cytoplasmic coat proteins deform membranes to bud vesicles and interact directly or indirectly with cargo molecules. Compartment-specific SNAREs (SNAP receptors) on vesicles and target membranes dock vesicles and provide a scaffolding for the general fusion machinery to initiate lipid bilayer fusion.     

Incorporation of phospholipids in Ehrlich ascites tumor cells

Ehrlich ascites tumor cells (EATC) were incubated with unilamellar vesicles (UV) or multilamellar vesicles (MV). UV and MV were incorporated differently into EATC. The increase in 32P-phospholipid in EATC in the presence of UV was 12% in 300 min. Absorption of phospholipid from MV could account for only 3%. About 50% of the UV incorporation of 32P was by endocytosis and/or fusion.     

Multiple roles for the actin cytoskeleton during regulated exocytosis

Regulated exocytosis is the main mechanism utilized by specialized secretory cells to deliver molecules to the cell surface by virtue of membranous containers (i.e., secretory vesicles). The process involves a series of highly coordinated and sequential steps, which include the biogenesis of the vesicles, their delivery to the cell periphery, their fusion with the plasma membrane, and the release of their content into the extracellular space. Each of these steps is regulated by the actin cytoskeleton. In this review, we summarize the current knowledge regarding the involvement of actin and its associated molecules during each of the exocytic steps in vertebrates, and suggest that the overall role of the actin cytoskeleton during regulated exocytosis is linked to the architecture and the physiology of the secretory cells under examination. Specifically, in neurons, neuroendocrine, endocrine, and hematopoietic cells, which contain small secretory vesicles that undergo rapid exocytosis (on the order of milliseconds), the actin cytoskeleton plays a role in pre-fusion events, where it acts primarily as a functional barrier and facilitates docking. In exocrine and other secretory cells, which contain large secretory vesicles that undergo slow exocytosis (seconds to minutes), the actin cytoskeleton plays a role in post-fusion events, where it regulates the dynamics of the fusion pore, facilitates the integration of the vesicles into the plasma membrane, provides structural support, and promotes the expulsion of large cargo molecules.     

A trapper keeper for TRAPP, its structures and functions

During biosynthesis many membrane and secreted proteins are transported from the endoplasmic reticulum, through the Golgi and on to the plasma membrane in small transport vesicles. These transport vesicles have to undergo budding, movement, tethering, docking, and fusion at each organelle of the biosynthetic pathway. The transport protein particle (TRAPP) complex was initially identified as the tethering factor for endoplasmic reticulum (ER)—derived COPII vesicles, but the functions of TRAPP may extend to other areas of biology. Three forms of TRAPP complexes have been discovered to date, and recent advances in research have provided new insights on the structures and functions of TRAPP. Here we provide a comprehensive review of the recent findings in TRAPP biology.     

Cytomorphologische Voraussetzung des Vesikulardrüsenepithels für die Wirkung von Testosteron

Summary Studies were carried out on the influence of testosterone on the growth of seminal vesicles in relation to age. Moreover, the postnatal development of the secretory cells in seminal vesicles was investigated by electron microscopy, and the functional morphology of the epithelial cells at the moment of first effect of testosterone was discussed.     

Incorporation of phospholipids in Ehrlich ascites tumor cells

Summary Ehrlich ascites tumor cells (EATC) were incubated with unilamellar vesicles (UV) or multilamellar vesicles (MV). UV and MV were incorporated differently into EATC. The increase in³²P-phospholipid in EATC in the presence of UV was 12% in 300 min. Absorption of phospholipid from MV could account for only 3%. About 50% of the UV incorporation of³²P was by endocytosis and/or fusion.This work was supported by a grant from the Consejo Nacional de Investigaciones Científicas y Técnicas, Argentina. J. L. Corchs is an investigator from the Consejo Nacional de Investigaciones Científicas y Técnicas.     

SNAREs and SNARE regulators in membrane fusion and exocytosis

Eukaryotes have a remarkably well-conserved apparatus for the trafficking of proteins between intracellular compartments and delivery to their target organelles. This apparatus comprises the secretory (or ‘protein export’) pathway, which is responsible for the proper processing and delivery of proteins and lipids, and is essential for the derivation and maintenance of those organelles. Protein transport between intracellular compartments is mediated by carrier vesicles that bud from one organelle and fuse selectively with another. Therefore, organelle-specific trafficking of vesicles requires specialized proteins that regulate vesicle transport, docking and fusion. These proteins are generically termed SNAREs and comprise evolutionarily conserved families of membrane-associated proteins (i.e. the synaptobrevin/VAMP, syntaxin and SNAP-25 families) which mediate membrane fusion. SNAREs act at all levels of the secretory pathway, but individual family members tend to be compartment-specific and, thus, are thought to contribute to the specificity of docking and fusion events. In this review, we describe the different SNARE families which function in exocytosis, as well as discuss the role of possible negative regulators (e.g. ‘SNARE-masters’) in mediating events leading to membrane fusion. A model to illustrate the dynamic cycling of SNAREs between fusion-incompetent and fusion-competent states, called the SNARE cycle, is presented. Received 8 October 1998; received after revision 26 November 1998; accepted 26 November 1998     

Confocal microscopy and biochemical analysis reveal spatial and functional separation between anandamide uptake and hydrolysis in human keratinocytes

The signaling activity of anandamide (AEA) is terminated by its uptake across the cellular membrane and subsequent intracellular hydrolysis by the fatty acid amide hydrolase (FAAH). To date, the existence of an AEA membrane transporter (AMT) independent of FAAH activity remains questionable, although it has been recently corroborated by pharmacological and genetic data. We performed confocal microscopy and biochemical analysis in human HaCaT keratinocytes, in order to study the cellular distribution of AMT and FAAH. We found that FAAH is intracellularly localized as a punctate staining partially overlapping with the endoplasmic reticulum. Consistently, subcellular fractionation and reconstitution of vesicles from membranes of different compartments demonstrated that FAAH activity was localized mainly in microsomal fractions, whereas AMT activity was almost exclusively in plasma membranes. These results provide the first morphological and biochemical evidence to support the view that transport and hydrolysis are two spatially and functionally distinct processes in AEA degradation.Received 11 October 2004; received after revision 24 November 2004; accepted 7 December 2004     

Sequential biogenesis of host cell membrane rearrangements induced by hepatitis C virus infection

Like most positive-strand RNA viruses, hepatitis C virus (HCV) forms a membrane-associated replication complex consisting of replicating RNA, viral and host proteins anchored to altered cell membranes. We used a combination of qualitative and quantitative electron microscopy (EM), immuno-EM, and the 3D reconstruction of serial EM sections to analyze the host cell membrane alterations induced by HCV. Three different types of membrane alteration were observed: vesicles in clusters (ViCs), contiguous vesicles (CVs), and double-membrane vesicles (DMVs). The main ultrastructural change observed early in infection was the formation of a network of CVs surrounding the lipid droplets. Later stages in the infectious cycle were characterized by a large increase in the number of DMVs, which may be derived from the CVs. These DMVs are thought to constitute the membranous structures harboring the viral replication complexes in which viral replication is firmly and permanently established and to protect the virus against double-stranded RNA-triggered host antiviral responses.     

The molecular machinery of synaptic vesicle exocytosis

At the synapse, neurotransmitters are released via Ca(2+)-triggered exocytotic fusion of synaptic vesicles with the presynaptic plasma membrane. Synaptic vesicle exocytosis seems to share many basic principles and homologous proteins with other membrane fusion events. Conserved components of the general fusion machinery that participate in synaptic vesicle exocytosis include soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs), ATPase N-ethylmaleimide-sensitive factor, Munc18/nSec1, Rab3 GTPase, and the exocyst proteins. In addition, synaptic vesicle exocytosis uses a set of unique components, such as synaptotagmin, complexin, Munc13, and RIM, to meet the special needs of fast Ca(2+)-triggered neurotransmitter release. This review summarizes present knowledge about the molecular mechanisms by which these components mediate and/or regulate synaptic vesicle exocytosis.     

Analysis of nuclear apoptotic process in a cell-free system

We report an analysis of the apoptotic process of mouse liver nuclei induced in a cell-free carrot cytosol system by cytochrome c. Typical characteristics of apoptosis were observed, such as chromatin condensation, margination, apoptotic bodies and DNA ladders. Furthermore, transmission and scanning electron microscope analysis of the apoptotic nuclei detected chromatin-free nuclear vesicles before apoptotic bodies appeared at a comparatively late phase. When AC-YVAD-CHO, an inhibitor of caspase 6, was introduced into the system, these vesicles and apoptotic bodies disappeared completely within our study sections. We confirmed the results using whole-mount electron microscopy, and found that although the nuclear lamina was destroyed early, the nuclear matrix largely remained intact during the course of apoptosis. The nuclear matrix played an important role in maintaining the integrity of apoptotic cells and connecting the apoptotic bodies and apoptotic nucleus. Received 29 September 2000; revised 10 December 2000; accepted 13 December 2000     

Types of nerve terminals in fetal and neonatal rabbit myocardium

Summary With the use of electron microscopy 4 types of axonal profiles were observed in the developing myocardium of rabbits: 1) adrenergic axons which contained mainly small dense-core vesicles and which presumably can store 5-hydroxydopamine; 2) cholinergic axons which contained small clear synaptic vesicles and which were acetylcholinesterase-positive; 3) axons which contained large vesicles filled with moderately electron-dense material and which resembled purinergic axons; and 4) profiles filled with mitochondria, vesicles of various sizes, lysosome-like bodies, and microtubules and which resembled sensory terminals.Supported in part by the Kentucky Heart Association and in part by the Human Development Studies Program, University of Kentucky. Excellent technical assistance of Mrs Merle Wekstein is gratefully acknowledged.     

Types of nerve terminals in fetal and neonatal rabbit myocardium

With the use of electron microscopy 4 types of axonal profiles were observed in the developing myocardium of rabbits: 1) adrenergic axons which contained mainly small dense-core vesicles and which presumably can store 5-hydroxy-dopamine; 2) cholinergic axons which contained small clear synaptic vesicles and which were acetylcholinesterase-positive; 3) axons which contained large vesicles filled with moderately electron-dense material and which resembled purinergic axons; and 4) profiles filled with mitochondria, vesicles of various sizes, lysosome-like bodies, and microtubules and which resembled sensory terminals.     

High-voltage electron microscopy of adrenal medulla: Direct connections between mitochondria and catecholamine-storage vesicles

Summary A high-voltage electron microscopic study of adrenal medullary cells from hypoglycemia-stressed rats revealed the existence of tubular channels which create a luminal continuity between the mitochondrial compartment and the catecholamine-storage vesicles. It is suggested that these channels allow for the transfer of materials such as high-energy nucleotides between the mitochondria and the catecholamine-storage vesicles without an intervening membrane.We thank Barbara Coalgate, Lin Cooper, Joann Cox, and the staff at the US Steel HVEM Laboratory for their assistance. This work was supported by NIH-DRR-70-4136, WVU Medical Corporation Research Grants, WVU Senate Research Grant, and West Virginia Heart Association Research Grant.     

Electron probe x-ray microanalysis and cryoultramicrotomy of unstained myocardial sarcoplasmic reticulum,in situ and fragmented

Summary Myocardial sarcoplasmic reticulum of cats in situ and fragmented sarcoplasmic reticulum (FSR) were analysed using X-ray microanalysis, cryoultramicrotomy and scanning transmission electron microscopy. 2 types of FSR vesicles can be distinguished morphologically and by their different elemental composition especially by different Ca loading. The Ca content of the sarcoplasmic reticulum can also be detected in situ.Acknowledgments. We thank Drs A.V. Somlyo and A.P. Somlyo for helpful advice and valuable discussion.This work was supported by the Deutsche Forschungsgemeinschaft.     

Current knowledge on exosome biogenesis and release

Exosomes are nanosized membrane vesicles released by fusion of an organelle of the endocytic pathway, the multivesicular body, with the plasma membrane. This process was discovered more than 30 years ago, and during these years, exosomes have gone from being considered as cellular waste disposal to mediate a novel mechanism of cell-to-cell communication. The exponential interest in exosomes experienced during recent years is due to their important roles in health and disease and to their potential clinical application in therapy and diagnosis. However, important aspects of the biology of exosomes remain unknown. To explore the use of exosomes in the clinic, it is essential that the basic molecular mechanisms behind the transport and function of these vesicles are better understood. We have here summarized what is presently known about how exosomes are formed and released by cells. Moreover, other cellular processes related to exosome biogenesis and release, such as autophagy and lysosomal exocytosis are presented. Finally, methodological aspects related to exosome release studies are discussed.     

The membrane domain of vacuolar H<Superscript>+</Superscript>ATPase: a crucial player in neurotransmitter exocytotic release

V-ATPases are multimeric enzymes made of two sectors, a V1 catalytic domain and a V0 membrane domain. They accumulate protons in various intracellular organelles. Acidification of synaptic vesicles by V-ATPase energizes the accumulation of neurotransmitters in these storage organelles and is therefore required for efficient synaptic transmission. In addition to this well-accepted role, functional studies have unraveled additional hidden roles of V0 in neurotransmitter exocytosis that are independent of the transport of protons. V0 interacts with SNAREs and calmodulin, and perturbing these interactions affects neurotransmitter release. Here, we discuss these data in relation with previous results obtained in reconstituted membranes and on yeast vacuole fusion. We propose that V0 could be a sensor of intra-vesicular pH that controls the exocytotic machinery, probably regulating SNARE complex assembly during the synaptic vesicle priming step, and that, during the membrane fusion step, V0 might favor lipid mixing and fusion pore stability.     

14-3-2 protein in rat brain synapses

Summary The distribution of the 14-3-2 protein in rat brain synapses was studied by immuno electron microscopy. The protein was localized to the postsynaptic web and to the postsynaptic membrane, but was also prominent both in the presynaptic membrane and in the presynaptic densities. No significant activity was observed in the synaptic vesicles.Acknowledgments. Our sincere thanks to Mrs Ulla Svedin, Eng., for skilled technical assistance. This work was supported by grants from the Swedish Medical Research Council, the Medical Faculty of Göteborg, Tore Nilson's Foundation for Medical Research and by the Swedish-Italian Group of Neurobiology. A. G. received financial support from Consiglio Nazionale delle Ricerche (CNR, ROME).     

Genetic and molecular analysis of the synaptotagmin family

Secretion is a fundamental cellular process used by all eukaryotes to insert proteins into the plasma membrane and transport signaling molecules and intravesicular proteins into the extracellular space. Secretion requires the fusion of two phospholipid bilayers within the cell, an energetically unfavorable process. A conserved repertoire of vesicle-trafficking proteins has evolved that function to overcome this energy barrier and temporally and spatially control membrane fusion within the cell. Within neurons, opening of synaptic calcium channels and subsequent calcium entry triggers synchronous synaptic vesicle exocytosis and neurotransmitter release into the synaptic cleft. After fusion, synaptic vesicles undergo endocytosis, are refilled with neurotransmitter, and return to the vesicle pool for further rounds of cycling. It is within this local synaptic trafficking pathway that the synaptotagmin family of calcium-binding synaptic vesicle proteins has been postulated to function. Here we review the current literature on the function of the synaptotagmin family and discuss the implications for synaptic transmission and membrane trafficking. Received 14 August 2000; received after revision 20 September 2000, accepted 14 October 2000