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The road taken: Past and future foundations of membrane traffic
... alleles (ie Fcmr −/− > Fcmr +/-> Fcmr +/+ ) [4]. Since the affinity of human FcµR for pentameric IgM is ~10 nM, which is much (~100-fold) lower than the concentration of plasma IgM (~1 µM), we initially* thought that FcµR on murine B cells in vivo must always be occupied with plasma IgM, and the IgM-bound FcµR should be internalized, rapidly retrieved from early endosomes and returned to the plasma membrane, thereby maintaining the plasma IgM concentration to a constant level, as do neonatal IgG FcR (FcRn) on endothelial cells and FcαR on blood phagocytes [20][21][22]. [*Later, we learned that the IgM ligand binding activity between human and mouse FcµRs was distinct (see below).] However, the half-life of exogenously administered IgM was comparable in both Fcmr KO and WT mice, hence ruling out the above receptor recycling possibility [3]. ...
Unlike Fc receptors for switched immunoglobulin (Ig) isotypes, Fc receptor for IgM (FcµR) is selectively expressed by lymphocytes. The ablation of the FcµR gene in mice impairs B cell tolerance as evidenced by concomitant production of autoantibodies of IgM and IgG isotypes. In this essay, we reiterate the autoimmune phenotypes observed in mutant mice, ie IgM homeostasis, dysregulated humoral immune responses including autoantibodies, and Mott cell formation. We also propose the potential phenotypes in individuals with FCMR deficiency and the model for FcµR-mediated regulation of self-reactive B cells.
... It also induces apoptosis. Fluoride exposure decreases protein synthesis by affecting the vesicular traffic in Golgi bodies and endoplasmic reticulum (ER) in cells [47]. ...
Fluoride (F¯) in groundwater is one such ion that makes the groundwater unsuitable for drinking purposes and responsible for serious human health problems such as dental, skeletal, and non-skeletal fluorosis in more than 25 countries worldwide. Recent studies estimated that cases of fluorosis are prevalent in 67 million people in 19 out of the 32 states of India due to intake of fluoride-enriched groundwater ranging from 0.01 to 37.0 mg/l affecting 8 and 18 million people among them by skeletal and dental fluorosis, respectively. T he distribution of fluoride in groundwater varies due to geogenic or anthropogenic sources or combined factors. The various geological and artificial factors that govern the F¯ prevalence in aquifers have been discussed. Different types of adverse health hazards of fluoride on humans and plants have also been enlightened. Since fluoride occurrence above 1.0 mg/l (BIS) or 1.5 mg/l (WHO) in groundwater can produce endemic health issues, there is an essential requirement to monitor groundwater fluoride levels frequently. Higher concentration in water needs treatment to bring them under the guideline value. Some available fluoride removal techniques from groundwater have been deliberated in study. Groundwater fluoride contamination is generally natural and unpreventable. However, most people unintentionally consume the untreated groundwater in developing countries leading to a widespread problem worldwide. A prominent solution is to educate and aware people about this issue. Frequent monitoring and defluoridation of the contaminated groundwa ter must be needed before consumption for a healthy world. The present review work is a step toward this fluoride hazard awareness with compact information about its various sources, status, removal technologies, and their relation with the environment.
... The coat proteins COPI, COPII, and Clathrin, are responsible for the transportation of vesicles inside the cell, especially to and from the Golgi apparatus. COP I coats vesicles traveling from the cis-Golgi to the RER (retrograde transport).COP II coats vesicles traveling from the RER to the cis-Golgi (anterograde transport), and Clathrin coats vesicles traveling from the Golgi to the lysosomes and from the plasma membrane to the lysosomes (10)(11)(12)(13)(14). ...
With the discovery of novel diseases and pathways, as well as a new outlook on certain existing diseases, cellular trafficking disorders attract a great deal of interest and focus. Understanding the function of genes and their products in protein and lipid synthesis, cargo sorting, packaging, and delivery has allowed us to appreciate the intricate pathophysiology of these biological processes at the molecular level and the multi-system disease manifestations of these disorders. This article focuses primarily on lymphocyte intracellular trafficking diseases from a clinician’s perspective. Familial hemophagocytic lymphohistiocytosis is the prototypical disease of abnormal vesicular transport in the lymphocytes. In this review, we highlight other mechanisms involved in cellular trafficking, including membrane contact sites, autophagy, and abnormalities of cytoskeletal structures affecting the immune cell function, based on a newer classification system, along with management aspects of these conditions.
... Ultrasensitive detection of molecular interactions is essential for widespread practice ranging from environmental monitoring, drug discovery to medical diagnostics and fundamental research such as cell signaling, cellular metabolism and growth, DNA replication and repair, and regulation of gene expression [1][2][3][4][5]. Nanomechanical sensors can convert a weak force into a displacement or resonant frequency drift that is measurable by electrical or optical means, and have become valuable tools for the understanding and recognition of molecular interactions in microcosms [6][7][8][9][10]. ...
Unlabelled:
Ultrasensitive molecular detection and quantization are crucial for many applications including clinical diagnostics, functional proteomics, and drug discovery; however, conventional biochemical sensors cannot satisfy the stringent requirements, and this has resulted in a long-standing dilemma regarding sensitivity improvement. To this end, we have developed an ultrasensitive relay-type nanomechanical sensor based on a magneto lever. By establishing the link between very weak molecular interaction and five orders of magnitude larger magnetic force, analytes at ultratrace level can produce a clearly observable mechanical response. Initially, proof-of-concept studies showed an improved detection limit up to five orders of magnitude when employing the magneto lever, as compared with direct detection using probe alone. In this study, we subsequently demonstrated that the relay-type sensing mode was universal in application ranging from micromolecule to macromolecule detection, which can be easily extended to detect enzymes, DNA, proteins, cells, viruses, bacteria, chemicals, etc. Importantly, we found that, sensitivity was no longer subject to probe affinity when the magneto lever was sufficiently high, theoretically, even reaching single-molecule resolution.
Electronic supplementary material:
Supplementary material (experimental section) is available in the online version of this article at 10.1007/s12274-022-5049-0.
... The methods based on chromogenic media are easy to perform and useful in preliminary tests, but due to the possible misidentification of closely related species, they require confirmations by more advanced methods. Several different commercially available biochemical systems based on fungal carbohydrate assimilation profiles are also often used to distinguish Candida species in addition with methods based on spectroscopy, including mass spectrometry characterized by significant precision [196][197][198][199]. The application of other spectroscopic methods in yeast identification, including rapid evaporative ionization MS (REIMS), vibrational spectroscopy, or surface-enhanced resonance Raman spectroscopy (SERRS) was described in detail in the review of Arastehfar et al. [200]. ...
Although Candida species are widespread commensals of the microflora of healthy individuals, they are also among the most important human fungal pathogens that under certain conditions can cause diseases (candidiases) of varying severity ranging from mild superficial infections of the mucous membranes to life-threatening systemic infections. So far, the vast majority of research aimed at understanding the molecular basis of pathogenesis has been focused on the most common species—Candida albicans. Meanwhile, other closely related species belonging to the CTG clade, namely, Candida tropicalis and Candida dubliniensis, are becoming more important in clinical practice, as well as a relatively newly identified species, Candida auris. Despite the close relationship of these microorganisms, it seems that in the course of evolution, they have developed distinct biochemical, metabolic, and physiological adaptations, which they use to fit to commensal niches and achieve full virulence. Therefore, in this review, we describe the current knowledge on C. tropicalis, C. dubliniensis, and C. auris virulence factors, the formation of a mixed species biofilm and mutual communication, the environmental stress response and related changes in fungal cell metabolism, and the effect of pathogens on host defense response and susceptibility to antifungal agents used, highlighting differences with respect to C. albicans. Special attention is paid to common diagnostic problems resulting from similarities between these species and the emergence of drug resistance mechanisms. Understanding the different strategies to achieve virulence, used by important opportunistic pathogens of the genus Candida, is essential for proper diagnosis and treatment.
... In mammals, neurons, endocrine cells and exocrine cells all secrete proteins along the secretory pathway [17]. Genetics and in vitro experiments have revealed the molecular mechanism of exocytosis from neurons and from endocrine and exocrine cells. ...
Objective:
Secretory carrier membrane proteins (SCAMPs) constitute a group of membrane transport proteins in plants, insects and mammals. The mammalian genome contains five types of SCAMP genes, namely, SCAMP1-SCAMP5. SCAMPs participate in the vesicle cycling fusion of vesicles and cell membranes and play roles in regulating exocytosis and endocytosis, activating synaptic function and transmitting nerve signals. Among these proteins, SCAMP5 is highly expressed in the brain and has direct or indirect effects on the function of the central nervous system. This paper may allow us to better understand the role of SCAMP5 in the central nervous system diseases. SCAMP5 regulates membrane transport, controls the exocytosis of SVs and is related to secretion carrier and membrane function. In addition, SCAMP5 plays a major role in the normal maintenance of the physiological functions of nerve cells. This article summarizes the effects of SCAMP5 on nerve cell exocytosis, endocytosis and synaptic function, as well as the relationship between SCAMP5 and various neurological diseases, to better understand the role of SCAMP5 in the pathogenesis of neurological diseases.
Methods:
Through PubMed, this paper examined and analyzed the role of SCAMP5 in the central nervous system, as well as the relationship between SCAMP5 and various neurological diseases using the key terms "secretory carrier membrane proteins"," SCAMP5"," exocytosis"," endocytosis", "synaptic function", "central nervous system diseases" up to 01 March 2022.
Results:
SCAMP5 regulates membrane transport, controls the exocytosis of SVs and is related to secretion carrier and membrane function. In addition, SCAMP5 plays a major role in the normal maintenance of the physiological functions of nerve cells.
Conclusion:
This article summarizes the effects of SCAMP5 on nerve cell exocytosis, endocytosis and synaptic function, as well as the relationship between SCAMP5 and various neurological diseases, to better understand the role of SCAMP5 in the pathogenesis of neurological diseases.
... Endocytosis spans a number mechanistically varied pathways including phagocytosis, micropinocytosis, caveolae-mediated endocytosis, and CME (McMahon and Boucrot, 2011;Kaksonen and Roux, 2018). CME is heavily implicated in nutrient uptake, signal transduction, synaptic vesicle recycling, maintenance of cell polarity, and antigen presentation (Mellman and Warren, 2000). CME can be broken up into five major stages and can be explained by examining the mechanism of cargo internalisation (Figure 4). ...
Endocytosis is a process essential to the health and well-being of cell. It is required for the internalisation and sorting of “cargo”—the macromolecules, proteins, receptors and lipids of cell signalling. Clathrin mediated endocytosis (CME) is one of the key processes required for cellular well-being and signalling pathway activation. CME is key role to the recycling of synaptic vesicles [synaptic vesicle recycling (SVR)] in the brain, it is pivotal to signalling across synapses enabling intracellular communication in the sensory and nervous systems. In this review we provide an overview of the general process of CME with a particular focus on two key proteins: clathrin and dynamin that have a central role to play in ensuing successful completion of CME. We examine these two proteins as they are the two endocytotic proteins for which small molecule inhibitors, often of known mechanism of action, have been identified. Inhibition of CME offers the potential to develop therapeutic interventions into conditions involving defects in CME. This review will discuss the roles and the current scope of inhibitors of clathrin and dynamin, providing an insight into how further developments could affect neurological disease treatments.
... Amongst these effectors, we can include tethering proteins (such as golgins and MTCs) or regulatory factors (such as GEFs and GAPs, that help modulate and control the GTP cycle). Rab-associated tethering factors mainly belong to two classes: those of the long, fibrous family of coiled-coil proteins named golgins (including members such as GM130, giantin, p115, EEA1) [55,65]; and large MTCs, such as the TRAPP, COG, exocyst, CORVET, and HOPS complexes. Amongst the latter, the TRAPP, COG, and GARP complexes localize to the Golgi membranes and are involved in the direct or indirect tethering and docking of intra-Golgi, ER-Golgi, or endosome-to-TGN transport carriers [61]. ...
The Golgi complex is the membrane-bound organelle that lies at the center of the secretory pathway. Its main functions are to maintain cellular lipid homeostasis, to orchestrate protein processing and maturation, and to mediate protein sorting and export. These functions are not independent of one another, and they all require that the membranes of the Golgi complex have a well-defined biochemical composition. Importantly, a finely-regulated spatiotemporal organization of the Golgi membrane components is essential for the correct performance of the organelle. In here, we review our current mechanistic and molecular understanding of how Golgi membranes are spatially organized in the lateral and axial directions to fulfill their functions. In particular, we highlight the current evidence and proposed models of intra-Golgi transport, as well as the known mechanisms for the retention of Golgi residents and for the sorting and export of transmembrane cargo proteins. Despite the controversies, conflicting evidence, clashes between models, and technical limitations, the field has moved forward and we have gained extensive knowledge in this fascinating topic. However, there are still many important questions that remain to be completely answered. We hope that this review will help boost future investigations on these issues.
... Eukaryotic cells contain diverse membranes, each with a characteristic and carefully regulated protein and lipid content. Most membrane proteins are first inserted into the ER and then traffic among cellular compartments in vesicles (Mellman and Warren, 2000). The same is true of lipids-most are synthesized in the ER and are then exchanged between organelles in vesicles (Vance, 2015). ...
We have long known that lipids traffic between cellular membranes via vesicles but have only recently appreciated the role of nonvesicular lipid transport. Nonvesicular transport can be high volume, supporting biogenesis of rapidly expanding membranes, or more targeted and precise, allowing cells to rapidly alter levels of specific lipids in membranes. Most such transport probably occurs at membrane contact sites, where organelles are closely apposed, and requires lipid transport proteins (LTPs), which solubilize lipids to shield them from the aqueous phase during their transport between membranes. Some LTPs are cup like and shuttle lipid monomers between membranes. Others form conduits allowing lipid flow between membranes. This review describes what we know about nonvesicular lipid transfer mechanisms while also identifying many remaining unknowns: How do LTPs facilitate lipid movement from and into membranes, do LTPs require accessory proteins for efficient transfer in vivo, and how is directionality of transport determined?
... SNAP-25 family proteins lack a transmembrane domain: two SNARE motifs are linked by a linker region and then inserted into the membrane by palmitoylation modification [23][24][25]. SNAREs appear to lie at the center of the membrane fusion mediating vesicle fusion within the endomembrane system and the vesicle exocytosis, which includes the ER, the Golgi, endosomes, and lysosomes [26,27]. SNAREs mediate fusion through the trans-SNARE complex that, "zipping" from the distal N-terminal region to the proximal C-terminal region, brings the two opposing membranes closer and eventually completing the fusion of the membranes [28][29][30]. ...
From lifeless viral particles to complex multicellular organisms, membrane fusion is inarguably the important fundamental biological phenomena. Sitting at the heart of membrane fusion are protein mediators known as fusogens. Despite the extensive functional and structural characterization of these proteins in recent years, scientists are still grappling with the fundamental mechanisms underlying membrane fusion. From an evolutionary perspective, fusogens follow divergent evolutionary principles in that they are functionally independent and do not share any sequence identity; however, they possess structural similarity, raising the possibility that membrane fusion is mediated by essential motifs ubiquitous to all. In this review, we particularly emphasize structural characteristics of small-molecular-weight fusogens in the hope of uncovering the most fundamental aspects mediating membrane–membrane interactions. By identifying and elucidating fusion-dependent functional domains, this review paves the way for future research exploring novel fusogens in health and disease.
... The core structure is surrounded by a variety of vesicles. The Golgi is a sorting hub and modification compartment in cells [137]. Normally, it receives and modifies proteins and lipids that exit from the ER. ...
Membrane contact sites (MCSs) are defined as regions where two organelles are closely apposed, and most MCSs associated with each other via protein-protein or protein-lipid interactions. A number of key molecular machinery systems participate in mediating substance exchange and signal transduction, both of which are essential processes in terms of cellular physiology and pathophysiology. The endoplasmic reticulum (ER) is the largest reticulum network within the cell and has extensive communication with other cellular organelles, including the plasma membrane (PM), mitochondria, Golgi, endosomes and lipid droplets (LDs). The contacts and reactions between them are largely mediated by various protein tethers and lipids. Ions, lipids and even proteins can be transported between the ER and neighboring organelles or recruited to the contact site to exert their functions. This review focuses on the key molecules involved in the formation of different contact sites as well as their biological functions. © The author(s). This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/). See http://ivyspring.com/terms for full terms and conditions.
... Vesicular transport is a cellular transport process by vesicle membranes. The inner membrane system refers to organelles surrounded by membrane structures including autophagosomes and lysosomes (Mellman and Warren, 2000;Bonifacino and Glick, 2004;Maxfield and van Meer, 2010). In addition, a GO analysis was used to assign DEGs into three main categories, in which "biological process" and "cellular component" were closely related to autophagy. ...
Porphyromonas gingivalis (P. gingivalis), one of the most important pathogens of periodontitis, is closely associated with the aggravation and recurrence of periodontitis and systemic diseases. Antibacterial peptide LL-37, transcribed from the cathelicidin antimicrobial peptide (CAMP) gene, exhibits a broad spectrum of antibacterial activity and regulates the immune system. In this study, we demonstrated that LL-37 reduced the number of live P. gingivalis (ATCC 33277) in HaCaT cells in a dose-dependent manner via an antibiotic-protection assay. LL-37 promoted autophagy of HaCaT cells internalized with P. gingivalis. Inhibition of autophagy with 3-methyladenine (3-MA) weakened the inhibitory effect of LL-37 on the number of intracellular P. gingivalis. A cluster of orthologous groups (COGs) and a gene ontology (GO) functional analysis were used to individually assign 65 (10%) differentially expressed genes (DEGs) to an “Intracellular trafficking, secretion, and vesicular transport” cluster and 306 (47.08%) DEGs to metabolic processes including autophagy. Autophagy-related genes, a tripartite motif-containing 22 (TRIM22), and lysosomal-associated membrane protein 3 (LAMP3) were identified as potentially involved in LL-37-induced autophagy. Finally, bioinformatics software was utilized to construct and predict the protein–protein interaction (PPI) network of CAMP-TRIM22/LAMP3-Autophagy. The findings indicated that LL-37 can reduce the quantity of live P. gingivalis internalized in HaCaT cells by promoting autophagy in these cells. The transcriptome sequencing and analysis also revealed the potential molecular pathway of LL-37-induced autophagy.
... Le transport des protéines selon la voie classique de sécrétion dépend de la formation de vésicules intermédiaires tapissées de protéine COPI (ou COPI-coated vesicles). Parmi ces protéines COPI, on retrouve des protéines particulières telles que la protéine GTPase ADP-ribosylation factor 1 (ARF1) ou [Pelham, 2001 ;Mellman and Warren, 2000]. ...
Le secteur de l'élevage bovin est en attente d'outils permettant de prédire et piloter les qualités sensorielles de la viande, notamment la tendreté qui est une priorité pour les consommateurs et pour la filière. La qualité de la viande est un phénotype complexe qui ne peut être évalué que tardivement après l'abattage des animaux et maturation de la viande. Depuis plus de 15ans, des recherches ont permis d'identifier des marqueurs de la qualité de la viande utilisables sur échantillons musculaires prélevés par biopsie ou sur carcasse. Cependant, des travaux récents ont rapporté des relations parfois inverses entre l’abondance de certains biomarqueurs et la tendreté de la viande selon le muscle ou le type d’animal considéré. Ainsi, l'identification de biomarqueurs « génériques » et peu-invasifs s’impose comme un enjeu pour le secteur bovin. Dans ce contexte, l’objectif de ma thèse a été d’identifier de potentiels biomarqueurs plasmatiques pour prédire la tendreté de la viande bovine. Afin de répondre à cet objectif, mes travaux de thèse se sont articulés autour de deux approches: (i) une approche d’intégration de données moléculaires combinée à une analyse bio-informatique (approche in-silico) pour prédire le sécrétome plasmatique ; (ii) une approche expérimentale, basée sur la stratégie protéomique Shotgun LC MS/MS, pour identifier des candidats plasmatiques et des candidats musculaires (muscles Longissimus et Rectus abdominis) à partir de groupes de génisses IGP Fleur d’Aubrac contrastées pour la tendreté. Afin de caractériser ce phénotype complexe, les groupes extrêmes analysés ont été déterminés selon trois systèmes d’évaluation de la tendreté (Warner Brazler Shear Force, sensoriel et index synthétique combinant ces deux systèmes). La combinaison de ces deux approches a permis de proposer pour la première fois une liste de 107 protéines plasmatiques candidates pour évaluer la tendreté de la viande bovine, parmi lesquelles 32 sont retrouvées dans des QTL de tendreté. Cette stratégie a également permis de compléter la liste de candidats musculaires déjà connus. Les données agrégées dans la littérature sont majoritairement associées à des bovins mâles (taurillons et bouvillons), ainsi ces travaux permettent de proposer pour la première fois une liste de candidats musculaires et plasmatiques pour la tendreté de génisses. Ces travaux de thèse ont également permis de compléter les connaissances sur le déterminisme de la tendreté, notamment via l’implication des voies de sécrétion vésiculaires (micro-vésiculaire et macro-vésiculaire (exosome)) et les cils primaires. Ces résultats pourraient permettre de proposer des outils de phénotypage pour prédire « le potentiel tendreté » du vivant de l’animal, mais aussi de valoriser des filières de qualité, d’orienter/segmenter les marchés et de proposer des conduites d’élevage adaptées au potentiel des animaux.
... Une fois dans l'endosome tardif ces dernières peuvent également se diriger vers le Golgi, la membrane plasmique ou l'endosome précoce mais aussi vers le lysosome où elles seront dégradées. Au niveau de la membrane plasmique, les protéines peuvent être endocytées par différents mécanismes puis sont dirigées vers l'endosome précoce où elles peuvent alors soit se diriger vers l'endosome de recyclage pour être recyclées à la membrane plasmique, soit dirigées vers l'endosome tardif pour être dégradées ou encore vers le Golgi (Mellman and Warren, 2000). Au delà de ces voies canoniques, d'autres voies apparaissent régulièrement dans la littérature telles la possibilité pour les protéines et les lipides d'être échangés directement entre les organelles via des points de contact. ...
La polarité cellulaire est un processus fondamental qui contrôle les spécificités fonctionnelle et physiologique de la plupart des cellules eucaryotes. Cette asymétrie intracellulaire repose sur l’existence de compartiments membranaires distincts, à la fois dans leur composition en protéines mais également en phosphatidyl-inositols (PIs). Ainsi, la mise en place et le maintien de la localisation asymétrique de modules multi-protéiques associés notamment aux protéines PAR sont essentiels pour l’élaboration des domaines de polarité cellulaire. Durant ma thèse, j’ai étudié les relations entre les protéines de polarité et les PIs dans le contrôle de la polarité cellulaire. Plus particulièrement, en utilisant la chambre ovarienne de Drosophile, j’ai cherché à caractériser la suite d’évènements qui en amont régule l’activité de la PIP5K, Skittles (SKTL), qui produit le PI(4,5)P2 et à caractériser les mécanismes moléculaires qui lient le PI(4,5)P2, SKTL et les protéines PAR dans le contrôle et le maintien de la polarité cellulaire. J’ai contribué à caractériser l’importance de PI(4,5)P2 majoritairement produit par SKTL, dans le maintien de la polarité apico-basale et lors de la morphogenèse des cellules folliculaires de la chambre ovarienne. Le PI(4,5)P2 assure la localisation apicale de PAR3 et le maintien des jonctions adhérentes, sans affecter la localisation de PAR1. Par une méthode de quantification précise, j’ai ensuite démontré dans l’ovocyte que SKTL et le PI(4,5)P2, probablement grâce au trafic vésiculaire, étaient requis pour à la fois l’accumulation à l’antérieur de PAR3 et son exclusion au postérieur qui se fait à partir du stade 9B. L’accumulation antérieure de PAR3 est également dépendante d’un transport Dynéine dépendant et de la kinase IKKε tandis que son exclusion postérieure dépendant des phosphorylations par PAR1. Enfin, j’ai également étudié les modifications post traductionnelles de SKTL et leur importance dans la polarité cellulaire. J’ai identifié la présence de palmitoylation et de phosphorylations dont certaines impliquent la kinase PAR1 et la phosphatase PP1. Ces phosphorylations pourraient avoir un lien avec le rôle de SKTL dans le trafic vésiculaire. Ces résultats permettent donc d’élucider certains mécanismes cellulaires qui contrôlent la mise en place et le maintien de la polarité des cellules en liant les PIs et les protéines PAR
... Soluble secreted proteins commonly contain N-terminal signal peptide or hydrophobic sequence that drive them to endoplasmic reticulum/Golgi network to be released extracellularly after fusion of Golgi derived vesicle with plasma membrane [42,43]. Beside this classical secretion pathway [44,45], several other cytoplasmic proteins follow alternative mechanisms of secretion collectively defined as non-classical or unconventional secretory pathways (UPS) [42,44,46] commonly activated under stressed conditions [46,47]. Here, we reported that the probability (SignalP 5.0 [13]) of NGB sequence to contain any N-terminal signal that could direct it toward the classical secretory pathway is very low. ...
Components of tumor microenvironment, including tumor and/or stromal cells-derived factors, exert a critical role in breast cancer (BC) progression. Here we evaluated the possible role of neuroglobin (NGB), a monomeric globin that acts as a compensatory protein against oxidative and apoptotic processes, as part of BC microenvironment. The extracellular NGB levels were evaluated by immunofluorescence of BC tissue sections and by Western blot of the culture media of BC cell lines. Moreover, reactive oxygen species (ROS) generation, cell apoptosis, and cell migration were evaluated in different BC cells and non-tumorigenic epithelial mammary cells treated with BC cells (i.e., Michigan Cancer Foundation-7, MCF-7) conditioned culture media and extracellular NGB. Results demonstrate that NGB is a component of BC microenvironment. NGB is released in tumor microenvironment by BC cells only under oxidative stress conditions where it can act as autocrine/paracrine factor able to communicate cell resilience against oxidative stress and chemotherapeutic treatment.
... In eukaryotic cells, secretory and membrane proteins undergo synthesis, proper folding and delivery through the secretory pathway. Since the experiments of Palade (Palade, 1975), it is admitted that this pathway includes the ER, the Golgi complex (the intermediary ERGIC was discovered more recently), and the plasma membrane, and that transport from one compartment to another is mediated by transport vesicles (Mellman and Warren, 2000). Interestingly, there is both anterograde transport, from ER to Golgi to plasma membrane, and retrograde transports from the surface to the Golgi or from the Golgi back to the ER. ...
Coronaviruses are an important family of emerging pathogens, as shown by therecent emergence of pathogenic SARS-CoV (Severe acute respiratory syndromecoronavirus) and MERS-CoV (Middle-East respiratory syndrome coronavirus) in the lasttwo decades. There are still some knowledge gaps concerning the biology ofcoronaviruses and we do not have any specific treatment or vaccine.Among the four structural proteins of the virus, the M protein is considered to bethe motor of viral assembly. Expressed alone in cells, M proteins can go beyond theassembly site of the virus (Endoplasmic reticulum-Golgi intermediate compartment,ERGIC) in the secretory pathway. We confirmed MERS-M localization in the Trans-Golginetwork (TGN) and identified two signals involved in its intracellular trafficking in its Cterminaldomain: a DxE ER export signal, and a KxGxYR TGN retention signal. The DxEsignal was already identified on another viral protein, whereas the KxGxYR signal is anew motif. To confirm the role of KxGxYR signal in TGN retention, we constructedchimeras between MERS-M and the protein M of the Infectious bronchitis virus (IBV),located in the ERGIC. Our results suggest that for both MERS-M and IBV-M the Cterminaldomain is determinant for the specific localization of the proteins.We also initiated a project on the characterization of the antiviral activity ofdigoxigenin against HCoV-229E. Our results demonstrated that it inhibits HCoV-229E ata post-entry step, with an IC50 of 250nM, and that it is not toxic at this concentration.Digoxigenin also inhibits hepatitis C virus (HCV) and likely has an effect on an early stepof replication of RNA (+) viruses.
... Throughout their journey, cargo proteins are processed by specialized reactions (folding, followed by post-translational modifications like glycosylation, and then sorting) in successive stations, each of which resuts in the generation of intermediate products, such as unfolded or partially modified proteins, which are further processed and transported across the successive secretory stations (Mellman and Warren, 2000). ...
The membrane transport apparatus comprises a series of separate membrane bound compartments, or transport stations, that are responsible for the synthesis, processing, transport, sorting and delivery to their final cellular destinations of most transmembrane and soluble lumenal proteins. Over the last decades the membrane transport system has been shown to be extensively regulated both by environmental inputs and by internal homeostatic signalling systems, or control systems, that operate to maintain the homeostasis and optimal functionality of the main transport stations, such as the endoplasmic reticulum and the Golgi, in the face of internal and external perturbations. The trans-Golgi network (TGN) is a major transport and processing station and the main sorting compartment of the transport apparatus. However, the mechanisms that control cargo export and sorting at the TGN have so far remained elusive. Here we focus on the sorting of basolateral cargo proteins and show that these proteins bind to the TGN localized orphan receptor GPRC5A. The cargo-GPRC5A complex triggers the activation of a signaling pathway that involves the G subunits dependent activation of the phospholipase C beta 3 (PLCb3), which inturn induces diacyl glycerol (DAG) production. DAG recruits and activates protein kinase D (PKD) and the phosphorylation of its substrates. This step results in the formation of basolateral carriers for delivery of these cargoes to the basolateral plasma membrane domain. We term this mechanism ARTG (AutoRegulation of TGN export). Remarkably, the impairment of ARTG pathway components, and in particular of GPRC5A, causes defects in the polarized organization of epithelial cells.
... This transient elevation of Ca 2+ activates membrane fusion machinery that is mediated by evolutionary conserved families of soluble NSF (N-ethylmaleimide-sensitive factor) attachment protein receptors (SNAREs) (Jahn and Fasshauer 2012). Vesicle fusion leads to release of its contained soluble messengers into the extracellular environment; in addition, fusion intercalates membrane-integral proteins and signaling lipids into the plasma membrane (Mellman and Warren 2000). Finally, disassembly and recycling of SNARE core complexes after fusion is mediated by the ATPase NSF in conjunction with soluble NSF adaptor proteins (SNAPs) (Whiteheart and Kubalek 1995). ...
The neurosecretion of chemical messengers via Ca²⁺-dependent exocytosis is fundamental to the survival of multicellular organisms and to the induction of physiological changes that may underlie learning and behavior. These processes involve ensembles of molecular interactions or ‘nanomachines’, which continuously modulate vesicle cycling and membrane fusion. The formation and disassembly of molecular complexes directing the secretory pathway principally define the (1) the number of exocytotic sites for release-ready vesicles at the plasma membrane; (2) the relative occupancy of the sites; (3) the probability of stimulus-dependent exocytotic release at each site; and (4) the degree to which renewal processes are engaged to support highly repetitive and/or sustained secretion. In this chapter, we focus on classes of proteins that have been considered “clamps” within the Ca²⁺-regulated release process.
... From the point of view of the immune response, macropinocytosis is an important event for the processing and presentation of antigens by antigen-presenting cells (APC) such as macrophages and dendritic cells. Activated and mature dendritic cells downregulate macropinocytosis favoring their migration to the lymph nodes for the presentation of these antigens to the T cells [6]. Antigenic peptides derived from internalized material are loaded onto major histocompatibility complex (MHC) molecules; of class I (MHC-I, usually self or endogenous antigens) or class II (MHC-II, foreign or extracellular antigens) for presentation to T cells to induce adaptive immune responses, besides, macropinocytosis can also result www.edoriumjournalofcellbiology.com ...
In the last century, several contributions were made on the organization and structure of the basic components of the cell, such as the plasma membrane. The fluid mosaic model proposed that the lipid bilayer functioned as a two-dimensional solvent, of neutral character and with little influence on the function of membrane proteins [1]. However, we now know that the plasma membrane contains a large variety of lipids that differ in their properties and that the interactions between these lipids originate heterogeneous structures in the plane of the plasma membrane. In this context, cell migration is a process associated with the plasma membrane and the rearrangement of cytoskeletal proteins that is essential for many processes such as embryonic development, healing, immune responses and tissue development; which like the motility are processes that require the reorganization of cytoskeletal and are able by the entry of extracellular calcium allowing cell adhesion [2]. These mechanisms, until some years ago were understood of an individual form, since the knowledge was not available on the way where the proteins that participate in the mechanisms mentioned above are coordinated spatially and temporally to carry out these functions.
... The secretory pathway is an alternative means of peripheral transport for macromolecules and it encompasses the rough-ER, the Golgi, and post-Golgi carrier vesicles [164,165]. The organelles of the secretory pathway respond to a wide array of exogenous and endogenous stimuli and maintain a distinct, cell-type specific organisation [165,166]. ...
Influenza viruses are respiratory pathogens that represent a significant threat to public health, despite the large-scale implementation of vaccination programs. It is necessary to understand the detailed and complex interactions between influenza virus and its host cells in order to identify successful strategies for therapeutic intervention. During viral entry, the cellular microenvironment presents invading pathogens with a series of obstacles that must be overcome to infect permissive cells. Influenza hijacks numerous host cell proteins and associated biological pathways during its journey into the cell, responding to environmental cues in order to successfully replicate. The cellular cytoskeleton and its constituent microtubules represent a heavily exploited network during viral infection. Cytoskeletal filaments provide a dynamic scaffold for subcellular viral trafficking, as well as virus-host interactions with cellular machineries that are essential for efficient uncoating, replication, and egress. In addition, influenza virus infection results in structural changes in the microtubule network, which itself has consequences for viral replication. Microtubules, their functional roles in normal cell biology, and their exploitation by influenza viruses will be the focus of this review.
... The first method is traditionally considered as the one which is mostly employed for the secretion of proteins. Majority of the cytokines, cell surface receptors and ECM derived components opt for this method of secretion [50]. The Golgi-independent pathway is characterized by secretion in the form of micro-vesicles such as exosomes. ...
In Breast cancer, Lung is the second most common site of metastasis after the bone. Various
factors are responsible for Lung metastasis occurring secondary to Breast cancer. Cancer cellderived
secretory factors are commonly known as ‘Cancer Secretomes’. They exhibit a prompt role
in the mechanism of Breast cancer lung metastasis. They are also major constituents of hostassociated
tumor microenvironment. Through cross-talk between cancer cells and the extracellular
matrix components, cancer cell-derived extracellular matrix components (CCECs) such as
hyaluronan, collagens, laminin and fibronectin cause ECM remodeling at the primary site (breast) of
cancer. However, at the secondary site (lung), tenascin C, periostin and lysyl oxidase, along with
pro-metastatic molecules Coco and GALNT14, contribute to the formation of pre-metastatic niche
(PMN) by promoting ECM remodeling and lung metastatic cells colonization. Cancer cell-derived
secretory factors by inducing cancer cell proliferation at the primary site, their invasion through the
tissues and vessels and early colonization of metastatic cells in the PMN, potentiate the mechanism
of Lung metastasis in Breast cancer.
On the basis of biochemical structure, these secretory factors are broadly classified into proteins and
non-proteins. This is the first review that has highlighted the role of cancer cell-derived secretory
factors in Breast cancer Lung metastasis (BCLM). It also enumerates various researches that have
been conducted to date in breast cancer cell lines and animal models that depict the prompt role of
various types of cancer cell-derived secretory factors involved in the process of Breast cancer lung
metastasis. In the future, by therapeutically targeting these cancer driven molecules, this specific
type of organ-tropic metastasis in breast cancer can be successfully treated.
... Unfortunately, the term "glycosome" has been used far more frequently to refer to the glycolytic enzyme-containing peroxisomes of trypanosomatids, a family of unicellular parasites (Michels et al. 2006), which creates confusion. Also, in the strict definition, organelles are membrane-bound entities (Klausner et al. 1992;Mellman and Warren 2000), and glycogen is primarily cytosolic. Perhaps it is safest to simply recognize that a host of proteins are associated with the glycogen granule, its structure is complex and highly regulated, and it is a dynamic participant in cellular metabolism. ...
This chapter reviews the history of glycogen-related research and discusses in detail the structure, regulation, chemical properties and subcellular distribution of glycogen and its associated proteins, with particular focus on these aspects in brain tissue.
... Proteins secreted via the classical pathway, are synthesized in the endoplasmic reticulum (ER) presenting a signal peptide at their N-terminus [7]. They are transported to the Golgi apparatus in a coat protein complex II (COPII)-coated vesicles and then, within secretory vesicles, to the plasma membrane and cell exterior [8,9]. During their passage through the Golgi apparatus, proteins are modified by different processes such as glycosylation [10], phosphorylation [11] and palmitoylation [12,13]. ...
Over the past years, it has become evident that cancer initiation and progression depends on several components of the tumor microenvironment, including inflammatory and immune cells, fibroblasts, endothelial cells, adipocytes, and extracellular matrix. These components of the tumor microenvironment and the neoplastic cells interact with each other providing pro and antitumor signals. The tumor-stroma communication occurs directly between cells or via a variety of molecules secreted, such as growth factors, cytokines, chemokines and microRNAs. This secretome, which derives not only from tumor cells but also from cancer-associated stromal cells, is an important source of key regulators of the tumorigenic process. Their screening and characterization could provide useful biomarkers to improve cancer diagnosis, prognosis, and monitoring of treatment responses.
... The secretory pathway is composed of the rough endoplasmic reticulum (rough ER), ER exit sites (ERESs) the ER-to-Golgi intermediate compartment (ERGIC), the Golgi complex and post-Golgi carriers en route to their final destination. Each organelle in the secretory pathway has a precise structure, organization and function in order to provide appropriate protein folding and post-translation modifications (Mellman and Warren 2000;Spang 2009). Synthesis of proteins, intracellular transport and storage was first discovered in the pancreas cells of guinea pig during the study of digestive enzyme pathway (Caro and Palade 1964). ...
COPI vesicles mediate retrograde Golgi to ER transport and intra-Golgi transport within the secretory pathway. COPI vesicles are Golgi derived vesicles, which are coated with heptameric complex known as coatomer. Coatomer is made up of seven subunits alpha, beta, beta prime,delta, gamma,zeta and epsilon-COP. COPI coatomer is recruited to the Golgi membrane with the help of small GTPases Arf to stimulate the vesicle formation and capture cargo proteins to deliver them to the targeted membrane. In mammals the -COP subunit has two paralogs. Whereas in the related COPII system, paralogs of coat subunits were shown to expand the cargo repertoire of COPII vesicles, no paralog specific function had been described to date for COPI paralog subunits. In this work we have set out to investigate such specific functions. Guided by RNAseq data in differentiating mES that showed that Copg1 is upregulated during neuronal differentiation. We generated Copg1 and Copg2 KO P19 pluripotent cells and studied if they could differentiate. Strikingly Copg1 KO cells fail to form tight embryonic bodies (EBs) and to form long neurites though they could differentiate into neurons. This work shows for the first time strong evidence for paralog-specific function of the COPI pathway in mammalian cells.
... Results have shown that exposure to high F − concentration can alter the appearance of apoptotic genes in peripheral blood mononuclear cells (PBMC) in the human populations of Mexico (Salgado-Bustamante et al., 2010). F − exposure also affects the vesicular traffic in Golgi bodies and endoplasmic reticulum (ER) in biological systems, through the intervention with protein synthesis and secretion (Mellman and Warren, 2000). Neurotransmitter secretion is also influenced by F − exposure (Flora et al., 2009;Borasio et al., 2004). ...
... Results have shown that exposure to high F − concentration can alter the appearance of apoptotic genes in peripheral blood mononuclear cells (PBMC) in the human populations of Mexico (Salgado-Bustamante et al., 2010). F − exposure also affects the vesicular traffic in Golgi bodies and endoplasmic reticulum (ER) in biological systems, through the intervention with protein synthesis and secretion (Mellman and Warren, 2000). Neurotransmitter secretion is also influenced by F − exposure (Flora et al., 2009;Borasio et al., 2004). ...
... As stress granules are linked to the inhibition of translation initiation, Sec bodies are linked to the inhibition of secretory pathway function, especially at the level of ER exit. In growth conditions, the secretory pathway transports proteins (and lipids) from the ER to the Golgi from where they are dispatched to the plasma membrane, the extracellular environment or other intracellular membrane-bound organelles.2,127 One of the first steps of this pathway is the exit of newly synthesized proteins from the ER at specific ER exit sites, characterized by the concentration of COPII (coat protein II)-coated buds and vesicles in which these proteins are packaged for transport to the Golgi. ...
In cells at steady state, two forms of cell compartmentalization co‐exist: membrane‐bound organelles and phase‐separated membraneless organelles. Membraneless organelles are present in both the nucleus and the cytoplasm. Strikingly, cellular stress is a strong inducer of the reversible membraneless compartments refered to as stress assemblies. Stress assemblies play key roles in survival during cell stress and in thriving of cells upon stress relief. The two best studied stress assemblies are the RNA based P‐bodies and stress granules that form in response to oxidative, ER, osmotic and nutrient stress as well as many others. Both Processing‐bodies (P‐bodies) and stress granules are heterogeneous with respect to both the pathways that lead to their formation and their protein and RNA content. Strikingly, in yeast and Drosophila, nutrient stress also leads to the formation of many other types of prosurvival cytoplasmic stress assemblies, some of which are not RNA based. Nutrient stress leads to a drop in cytoplasmic pH, which combined with posttranslational modifications of granule contents, induces phase separation.
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... In the secretory pathway, secretory cargo proteins newly synthesized in the ER are delivered to the Golgi apparatus, where they are processed and glycosylated before being sorted to their final destinations (Mellman and Warren, 2000;Emr et al., 2009). The Golgi apparatus is the central station of membrane traffic and is usually in the form of ordered stacks of several cisternae. ...
To describe trafficking of secretory cargo within the Golgi apparatus, the cisternal maturation model predicts that Golgi cisternae change their properties from cis to trans while cargo remains in the cisternae. Cisternal change has been demonstrated in living yeast Saccharomyces cerevisiae ; however, the behavior of cargo has yet to be examined directly. In this study, we conducted simultaneous three-color and four-dimensional visualization of secretory transmembrane cargo together with early and late Golgi resident proteins. We show that cargo stays in a Golgi cisterna during maturation from cis-Golgi to trans-Golgi and further to the trans-Golgi network (TGN), which involves dynamic mixing and segregation of two zones of the earlier and later Golgi resident proteins. The location of cargo changes from the early to the late zone within the cisterna during the progression of maturation. In addition, cargo shows an interesting behavior during the maturation to the TGN. After most cargo has reached the TGN zone, a small amount of cargo frequently reappears in the earlier zone.
... They leave the endoplasmic reticulum as a part of the vesicles and then get to the Golgi apparatus; then they move to cell membrane, where they are released into extracellular space by the fusion of Golgi-derived ves-icles with the plasma membrane. This process of protein secretion has been well characterized and is called classical pattern of protein secretion [42][43][44]. ...
Although understanding of the molecular biology of cancer has advanced and medicine has an impressive arsenal of chemotherapeutic drugs, the problem of tumor resistance to individual drugs and drug combinations has not yet been resolved. Known mechanisms of cancer chemoresistance do not explain the reason for such a phenomenon as “apoptosis-induced proliferation,” where cells dying under the effect of the therapy secrete some signaling molecules into the extracellular medium to promote proliferation, survival, and acquisition of a more aggressive phenotype of neighboring cancer cells. The nature of this unexpected phenomenon is only now beginning to be partially clarified, but the nature of such signaling between apoptotic cancer cells and their neighboring cells remains largely unknown. For this reason, in this review we discuss currently known types of intercellular communication of tumor cells, give specific examples of important secreted molecules involved in signaling between cancer cells, and describe possible cell interactions contributing to the progress of cancer outgrowth.
... These proteins are transported to the Golgi apparatus and subsequently, to the cell surface where they are liberated into the microenvironment by fusion of the Golgi-derived vesicles with the plasma membrane. This well-characterized protein secretion pathway is known as the classical Secretory Pathway (SP) (Mellman and Warren, 2000;Walter et al., 1984). Other evidence shows that, in addition to this mechanism, proteins can be exported by ER/Golgi-independent pathways through the so-called non-classical SP (Nickel, 2003). ...
In recent years, the discovery of “tumor niche”, a microenvironment that favors tumor development has changed our perspective of cancer. This microenvironment generated by the tumor cells itself and surrounding cells, is capable of providing essential elements for its growth. Consequently, the homeostasis of the Secretory Pathway (SP) has become an essential player in cancer development. The SP not only promotes cellular adaptation to protein misfolding due to oncogenic transformation or challenging tumor niche but also allows tumor cells to produce specific secretomes. This impacts tumor cells in cis‐ or trans‐ as well as stromal cells in the tumor niche. In this context, the Anterior GRadient 2 (AGR2) protein has been identified as a key player. AGR2 is a protein disulfide isomerase that resides in the Endoplasmic Reticulum (ER) and mediates the formation of disulfide bonds, catalyzes the cysteine‐based redox reactions and assists the quality control of proteins. AGR2 not only plays an essential role in the homeostasis of the SP but also exerts pro‐oncogenic gain‐of‐function due to its reported mislocalization in the tumor niche microenvironment. In this review, we summarize the dual role of AGR2, inside and outside the ER, on the tumor niche and its microenvironment.
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... The newly synthesized proteins then exit the ER at an ER-exit site (ERES) through coat protein complex II (COPII)-coated vesicles and, so, reach the Golgi network before being dispatched to the plasma membrane, lysosomes, endosomes or peroxisomes (Gee et al., 2018;Viotti, 2016). Fusion of vesicular intermediates and organelles is mediated by soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein (SNAP) receptor proteins (SNAREs), Rab proteins and their regulators (Mellman and Warren, 2000). ...
Most secretory proteins travel through a well-documented conventional secretion pathway involving the endoplasmic reticulum (ER) and the Golgi complex. However, recently, it has been shown that a significant number of proteins reach the plasma membrane or extracellular space via unconventional routes. Unconventional protein secretion (UPS) can be divided into two types: (i) the extracellular secretion of cytosolic proteins that do not bear a signal peptide (i.e. leaderless proteins) and (ii) the cell-surface trafficking of signal-peptide-containing transmembrane proteins via a route that bypasses the Golgi. Understanding the UPS pathways is not only important for elucidating the mechanisms of intracellular trafficking pathways but also has important ramifications for human health, because many of the proteins that are unconventionally secreted by mammalian cells and microorganisms are associated with human diseases, ranging from common inflammatory diseases to the lethal genetic disease of cystic fibrosis. Therefore, it is timely and appropriate to summarize and analyze the mechanisms of UPS involvement in disease pathogenesis, as they may be of use for the development of new therapeutic approaches. In this Review, we discuss the intracellular trafficking pathways of UPS cargos, particularly those related to human diseases. We also outline the disease mechanisms and the therapeutic potentials of new strategies for treating UPS-associated diseases.
... Moreover, this enzyme, through the activation of a calcium dependent pathway, can participate in various biological functions (Mondola et al., 2002;Viggiano et al., 2012). However, the mechanism involved in the molecular secretory pathway of SOD1 is not yet clear (Mellman and Warren, 2000;Bosco et al., 2010;Forsberg et al., 2010). ...
The constitutive secretion of antioxidant Cu-Zn Superoxide dismutase (SOD1) has been widely demonstrated in many cellular lines. In addition, we showed that as well as the basal SOD1 secretion, this enzyme is also exported through depolarization of excitable cells by high extracellular K concentration. Recent data showed that SOD1 was able to activate muscarinic M1 receptor producing the activation, via phospholipase C, of ERK1-2 and AKT pathways. It is also known that about 20% of familial amyotrophic lateral sclerosis (fALS) is due to mutations in the gene coding for SOD1. The aim of the present research is to evaluate whether, analogously to wild type SOD1 (SOD1wt), the mutated form of SOD1G93A is able to activate ERK1-2 and AKT through muscarinic M1 receptor in SK-N-BE as well as in motoneuron like NSC-34. Our results demonstrated that in NSC-34 and SK-N-BE cells mutated SOD1G93A carried out a more evident activation of ERK1-2 and AKT and a stronger increase of intracellular calcium levels compared to SOD1WT; we also demonstrated that these effects are mediated by the M1 receptor as shown using pirenzepine, a specific M1 inhibitor and the calcium chelator BAPTA. Of note, M1 receptor pathway activation by SOD1G93A, but not by SOD1WT, is associated with both an increase of reactive oxygen species and a cytotoxic effect.
... Soluble proteins can be secreted in a constitutive manner or in a regulated way, upon different neural or hormonal stimuli. The secretory organelles, consisting of the rough ER, ER exit sites, ERGIC (ER-to Golgi intermediate compartment) and the Golgi complex each have a distinct organization and structure and provide a proper environment for protein folding and protein modification, which includes the addition of sugars and lipids [104][105][106]. Proteins require an ER signal sequence, generally at the N-terminus, to enter the ER during or after translation. ...
... Thereafter, the newly synthesized proteins exit the ER at ER exit site via coat protein complex II (COPII)-coated vesicles, reach the Golgi and the trans Golgi network before being dispatched to the plasma membrane [2]. Fusion between vesicular intermediates and organelles is mediated by soluble N-ethylmaleimide (NEM)-sensitive factor attachment protein receptors (SNAREs), Ras-like GTPases of the Rab protein family, and their regulators [3]. This secretory pathway is also responsible for the intracellular transport to other organelles such as lysosomes, endosomes, and peroxisomes. ...
Over the past 20 years it has become evident that eukaryotic cells utilize both conventional and unconventional pathways to deliver proteins to their target sites. Most proteins with a signal peptide and/or a transmembrane domain are conventionally transported through the endoplasmic reticulum to the Golgi apparatus and then to the plasma membrane. However, an increasing number of both soluble cargos (Type I, II, and III) and integral membrane proteins (Type IV) have been found to reach the plasma membrane via unconventional protein secretion (UPS) pathways that bypass the Golgi apparatus under certain conditions, such as cellular stress or development. Well-known examples of transmembrane proteins that undergo Type IV UPS pathways are position-specific antigen subunit alpha 1 integrin, cystic fibrosis transmembrane conductance regulator, myeloproliferative leukemia virus oncogene, and pendrin. Although we collectively refer to all Golgi-bypassing routes as UPS, individual trafficking pathways are diverse compared to the conventional pathways, and the molecular mechanisms of UPS pathways are not yet completely defined. This review summarizes the intracellular trafficking pathways of UPS cargo proteins, particularly those with transmembrane domains, and discusses the molecular machinery involved in the UPS of transmembrane proteins.
Background:
Vacuolar protein sorting (VPS) plays a crucial role in intracellular molecular transport between organelles. However, studies have indicated a correlation between VPSs and tumorigenesis and the development of several cancers. Nevertheless, the association between VPSs and hepatocellular carcinoma (HCC) remains unclear.
Methods:
By analyzing databases such as The Cancer Genome Atlas (TCGA) and The International Cancer Genome Consortium (ICGC), we investigated the differences in VPSs expression between normal tissue and HCC transcriptomes. Furthermore, we examined the relationship between VPSs expression and overall survival (OS) in patients with HCC. Univariate and multivariate Cox analyses were employed to assess the prognostic value of VPS72 as an independent factor, and the correlation between VPS72 and the tumor immune microenvironment was also analyzed.
Results:
We observed significant overexpression of 28 VPSs in HCC tissues compared to normal tissues. The mRNA expression of VPSs displayed a negative correlation with OS, while exhibiting a positive correlation with tumor grade and stage. Additionally, both univariate and multivariate Cox analyses identified VPS72 as a potential independent risk factor for HCC prognosis. Overexpression of VPS72 demonstrated a positive correlation with various clinicopathological factors associated with poor prognosis, as well as the infiltration levels of immune cells.
Conclusion:
Therefore, our research shows that VPSs participate in HCC occurrence and development, especially VPS72, which may act as a potential target for HCC treatment and prognosis biomarker.
Unlabelled:
In the human body, proteins secreted into peripheral blood vessels are known as the secretome, and they represent the physiological or pathological status of cells. The unique response of cells to toxin exposure can be confirmed via secretome analysis, which can be used to discover toxic mechanisms or exposure markers. Alpha-amanitin (α-AMA) is the most widely studied amatoxin and inhibits transcription and protein synthesis by directly interacting with RNA polymerase II. However, secretory proteins released during hepatic failure caused by α-AMA have not been fully characterized. In this study, we analyzed the secretome of α-AMA-treated Huh-7 cells and mice using a comparative proteomics technique. Overall, 1440 and 208 proteins were quantified in cell media and mouse serum, respectively. Based on the bioinformatics results for the commonly downregulated proteins in cell media and mouse serum, we identified complement component 3 (C3) as a marker for α-AMA-induced hepatotoxicity. Through western blot in cell secretome and C3 ELISA assays in mouse serum, we validated α-AMA-induced downregulation of C3. In conclusion, using comparative proteomics and molecular biology techniques, we found that α-AMA-induced hepatotoxicity reduced C3 levels in the secretome. We expect that this study will aid in identifying new toxic mechanisms, therapeutic targets, and exposure markers of α-AMA-induced hepatotoxicity.
Supplementary information:
The online version contains supplementary material available at 10.1007/s43188-022-00163-z.
The protozoan parasite, Trypanosoma brucei, offers a simple system to study the growth and duplication of the Golgi. Cell lines stably expressing a photoactivatable GFP attached to an endogenous Golgi protein are permeabilized using digitonin. Photoactivation followed by imaging can then be used to follow the formation of the new Golgi.Key wordsGolgiBiogenesis
Trypanosoma brucei
ParasitePermeabilizationImagingPhotoactivation
Rab proteins are a family of small GTPases that function as molecular switches of intracellular vesicle formation and membrane trafficking. As a key factor, Rab GTPase participates in autophagy and protein transport and acts as the central hub of membrane trafficking in eukaryotes. The role of Rab GTPase in neurodegenerative disorders, such as Alzheimer's and Parkinson's, has been extensively investigated; however, its implication in cardiovascular embryogenesis and diseases remains largely unknown. In this review, we summarize previous findings and reveal their importance in the onset and progression of cardiac diseases, as well as their emergence as potential therapeutic targets for cardiovascular disease.
Secretagogues bearing a 3-arylquinoline scaffold-induced secretory events in normal cells that released the tumor suppressor protein, prostate apoptosis response-4 protein (Par-4) sequestered by the intermediary filament protein, vimentin. The secretion of the Par-4 protein and its binding to a selective, cell-surface receptor GRP78 subsequently triggered paracrine apoptosis in cancer cells. These findings provided a rationale for the study of Par-4 secretagogues as potential agents for the inhibition of tumor growth. Developing secretagogues with these scaffolds, determining vimentin as the biomolecular target, using molecular dynamics to model arylquin binding to vimentin, and understanding the secretion of Par-4 and its apoptotic effects held promise as a new approach for small-molecule interventions as potential treatments for cancer.
Coordinated AP-1 and clathrin coat assembly mediate secretory sorting on the trans-Golgi network (TGN) during conventional secretion. Here we found that SMAP-1/SMAPs deficiency caused the apical protein ERM-1 to accumulate on the basolateral side of the TGN. In contrast, the basolateral protein SLCF-1 appeared abnormally on the apical membrane. SMAP-1 colocalized with AP-1 on the TGN. The integrity of AP-1 is required for the subcellular presence of SMAP-1. Moreover, we found that the loss of SMAP-1 reduced clathrin-positive structures in the cytosol, suggesting that SMAP-1 has a regulatory role in clathrin assembly on the TGN. Functional experiments showed that overexpressing clathrin effectively alleviated exocytic defects due to the lack of SMAP-1, corroborating the role of SMAP-1 in promoting the assembly of clathrin on the TGN. Together, our results suggested that the AP-1 complex regulates the TGN localization of SMAP-1, promoting clathrin assembly to ensure polarized conventional secretion in C. elegans intestinal epithelia.
As a cellular interface between the blood and tissues, the endothelial cell (EC) monolayer is involved in the control of key functions including vascular tone, permeability and homeostasis, leucocyte trafficking and hemostasis. EC regulatory functions require long-distance communications between ECs, circulating hematopoietic cells and other vascular cells for efficient adjusting thrombosis, angiogenesis, inflammation, infection and immunity. This intercellular crosstalk operates through the extracellular space and is orchestrated in part by the secretory pathway and the exocytosis of Weibel Palade Bodies (WPBs), secretory granules and extracellular vesicles (EVs). WPBs and secretory granules allow both immediate release and regulated exocytosis of messengers such as cytokines, chemokines, extracellular membrane proteins, coagulation or growth factors. The ectodomain shedding of transmembrane protein further provide the release of both receptor and ligands with key regulatory activities on target cells. Thin tubular membranous channels termed tunneling nanotubes (TNTs) may also connect EC with distant cells. EVs, in particular exosomes, and TNTs may contain and transfer different biomolecules (e.g., signaling mediators, proteins, lipids, and microRNAs) or pathogens and have emerged as a major triggers of horizontal intercellular transfer of information.
The sections in this article are
Introduction
Localization in the Cell
Ultrastructure and Organization
The G olgi Apparatus and Cell Division
Acknowledgements
The sections in this article are
Introduction
Clathrin‐Coated Vesicles
COPI Vesicles
COPII Vesicles
Acknowledgements
Green fluorescent protein (GFP)-based video microscopy can provide profound insight into biological processes by generating information on the ‘history,’ or dynamics, of the cellular structures involved in such processes in live cells.
A crucial limitation of this approach, however, is that many such structures may not be resolved by light microscopy. Like more recent super-resolution techniques, correlative video-light–electron microscopy (CLEM) was developed to overcome this limitation. CLEM integrates GFP-based video microscopy and electron microscopy through a series of ancillary techniques, such as proper fixation, hybrid labeling and retracing, and so provides sufficient resolution as well as, crucially, cellular ‘context’ to the fluorescent dynamic structures of interest. CLEM ‘multiplies’ the power of video microscopy and is having an important impact in several areas cell and developmental biology. Here, we discuss potential, limitations and perspectives of correlative approaches aimed at integrating the unique insight generated by video microscopy with information from other forms of imaging.
γ-Aminobutyric acid type A (GABAA) receptors are members of the ligand gated ion channel superfamily and are the major sites of fast synaptic inhibition in the brain. GABAA receptors are clustered at inhibitory synapses, however, precisely how these receptors are inserted into postsynaptic membranes and stabilised at synapses remains unknown. The focus of this thesis is to further understand the mechanisms important for the targeting and cell surface stability of ionotropic GABAA receptors. To examine the membrane trafficking of these receptors a fusion protein of the γ2L subunit with green fluorescent protein (GFP) was produced. This fusion protein was functional and allowed the visualisation of receptor membrane targeting and endocytosis in live cells. Interestingly, the targeting of this construct to GABAergic synapses in cultured hippocampal neurones was dependent upon coassembly with receptor α and β subunits. This suggests that the assembly and membrane targeting of GABAA receptors containing the γ2 subunit follow similar itineraries in heterologous systems and neurones. GABAA receptor β and γ subunits were also found to associate with the adaptin complex AP2, a protein implicated in the recruitment of transmembrane proteins into clathrin coated pits. Furthermore, in cultured hippocampal neurones blocking dynamin-dependant endocytosis caused a gradual increase in the amplitude of mIPSCs that reached a plateau at 1.7 to 2.3-fold of it's control value within 40-50 min. These results suggest there must be a relatively fast turnover of GABAA receptors between the cell surface and intracellular compartments. To further study the role of the GABAA receptor binding partner, GABARAP, we compared the subcellular distribution of this protein with that of gephyrin, a protein important for GABAA receptor synaptic clustering. In cultured hippocampal neurones GABARAP associated weakly with synaptic GABAA receptor and gephyrin clusters. However, GABARAP was found at high levels on intracellular membrane compartments including the Golgi and subsynaptic cisternae. Furthermore, GABARAP was found to associate with N-ethylmaleimide sensitive factor (NSF), a protein essential for intracellular membrane transport events and important for receptor trafficking. This suggests that GABARAP may act to facilitate the trafficking of GABAA receptors via it's ability to interact with NSF. The results presented in this thesis suggest that GABAA receptors cycle into and out of the plasma membrane in a process that may be regulated by their association with adaptin complexes and recruitment of NSF via GABARAP. This may allow neurones to specifically modify cell surface receptor levels which may have a critical role in controlling the efficacy of inhibitory synaptic transmission.
1. Human ABCG2 is a half transporter implicated in drug efflux and development of multidrug resistance (MDR) in cancer cells. Here we present the regulatory effects of early endocytic Rab GTPases, Rab5A and Rab21 on ABCG2.
2. ABCG2 was stably expressed in MCF-7 cells (MCF-7/G2). Rab5A and Rab21 was manipulated in MCF-7/G2 cells by co expression or siRNA knockdown and their effect on ABCG2 mediated drug efflux was quantified using fluorescence microscopy.
3. The ectopically expressed ABCG2 was predominantly confined to the plasma membrane and was capable of drug efflux. Expression of constitutively active Rab5A-Q79L mutant in MCF-7/G2 cells decreased the cell surface expression of ABCG2, resulting in the reduction of ABCG2 mediated drug efflux. In contrast, expression of inactive Rab5A-S34N mutant enhanced cell surface expression of ABCG2 and drug efflux. Moreover, reduction in endogenous Rab21 levels in MCF-7/G2 cells by siRNA knockdown, increased the surface localisation of ABCG2. Consequently, efflux ability of cells increased and intracellular retention of doxorubicin and Hoechst 33342; substrates of ABCG2, decreased significantly.
4. These findings suggest that Rab5A and Rab21 play important roles in regulating ABCG2 surface localisation and turnover and can be exploited as a potential strategy to overcome MDR in cancer cells.
At the center of the secretory pathway, the Golgi complex ensures correct processing and sorting of cargos towards their final destination sites which are the cytoplasm, plasma membrane, and endosome-lysosome system. Recent technological developments have discovered new morphological and functional charcteristics of the Golgi apparatus. In this review, I emphasize that the Golgi apparatus is not a stable organelle instead a highly dynamic organelle changing its morphological and functional characteristics in different physiological and pathological conditions. It is not composed of a simple discontinuous parallel stacks but is composed of various domains such as cis-Golgi network, trans-Golgi network, and intercisternal network including various coated vesicles, vacuoles and tubules. Furthermore, the Golgi apparatus is not only involved in the secretory pathway, but also involved in the storage of Ca2+ very likely to smooth endoplasmic reticulum. Additionally, the Golgi apparatus might serve as a microtubule organizing center which is especially important to participate in Golgi reassembly after cell division.
In designing bioassay systems for low-abundance biomolecule detection, most research focuses on improving transduction mechanisms while ignoring the intrinsically fundamental limitations in solution: mass transfer and binding affinity. We demonstrate enhanced biomolecular surface binding using an acoustic nano-electromechanical system (NEMS) resonator, as an on-chip biomolecular concentrator which breaks both mass transfer and binding affinity limitations. As a result, a concentration factor of 10⁵ has been obtained for various biomolecules. The resultantly enhanced surface binding between probes on the absorption surface and analytes in solution enables us to lower the limit of detection for representative proteins. We also integrated the biomolecular concentrator into an optoelectronic bioassay platform to demonstrate delivery of proteins from buffer/serum to the absorption surface. Since the manufacture of the resonator is CMOS-compatible, we expect it to be readily applied to further analysis of biomolecular interactions in molecular diagnostics.
We previously identified BET3 by its genetic interactions with BET1, a gene whose SNARE-like product acts in endoplasmic reticulum (ER)-to-Golgi transport. To gain insight into the function of Bet3p, we added three c-myc tags to its C-terminus and immunopurified this protein from a clarified detergent extract. Here we report that Bet3p is a member of a large complex (800 kDa) that we call TRAPP (transport protein particle). We propose that TRAPP plays a key role in the targeting and/or fusion of ER-to-Golgi transport vesicles with their acceptor compartment. The localization of Bet3p to the cis-Golgi complex, as well as biochemical studies showing that Bet3p functions on this compartment, support this hypothesis. TRAPP contains at least nine other constituents, five of which have been identified and shown to be highly conserved novel proteins.
Rab proteins have been primarily implicated in vesicle docking as regulators of SNARE pairing. Recent findings, however, indicate that their function in vesicle trafficking can go beyond this role, and a number of proteins, unrelated to each other, have been identified as putative Rab effectors. Although the GTPase switch of Rab proteins is highly conserved, functional mechanisms may be highly diversified among members of the Rab family.
We have prepared a conjugate of epidermal growth factor (EGF) and ferritin that retains substantial binding affinity for cell receptors and is biologically active. Glutaraldehyde-activated EGF was covalently linked to ferritin to produce a conjugate that contained EGF and ferritin in a 1:1 molar ratio. The conjugate was separated from free ferritin by affinity chromatography using antibodies to EGF. Monolayers of human epithelioid carcinoma cells (A-431) were incubated with EGF:ferritin at 4 degrees C and processed for transmission electron microscopy. Under these conditions, approximately 6 X 10(5) molecules of EGF:ferritin bound to the plasma membrane of each cell. In the presence of excess native EGF, the number of bound ferritin particles was reduced by 99%, indicating that EGF:ferritin binds specifically to cellular EGF receptors. At 37 degrees C, cell-bound EGF:ferritin rapidly redistributed in the plane of the plasma membrane to form small groups that were subsequently internalized into pinocytic vesicles. By 2.5 min at 37 degrees C, 32% of the cell-bound EGF:ferritin was localized in vesicles. After 2.5 min, there was a decrease in the proportion of conjugate in vesicles with a concomitant accumulation of EGF:ferritin in multivesicular bodies. By 30 min, 84% of the conjugate was located in structures morphologically identified as multivesicular bodies or lysosomes. These results are consistent with other morphological and biochemical studies utilizing 125I-EGF and fluorescein-conjugated EGF.
Fractionation of MOPC 41 DL-1 tumors revealed that the mRNA for the light chain of immunoglobulin is localized exclusively in membrane-bound ribosomes. It was shown that the translation product of isolated light chain mRNA in a heterologous protein-synthesizing system in vitro is larger than the authentic secreted light chain; this confirms similar results from several laboratories. The synthesis in vitro of a precursor protein of the light chain is not an artifact of translation in a heterologous system, because it was shown that detached polysomes, isolated from detergent-treated rough microsomes, not only contain nascent light chains which have already been proteolytically processed in vivo but also contain unprocessed nascent light chains. In vitro completion of these nascent light chains thus resulted in the synthesis of some chains having the same mol wt as the authentic secreted light chains, because of completion of in vivo proteolytically processed chains and of other chains which, due to the completion of unprocessed chains, have the same mol wt as the precursor of the light chain. In contrast, completion of the nascent light chains contained in rough microsomes resulted in the synthesis of only processed light chains. Taken together, these results indicate that the processing activity is present in isolated rough microsomes, that it is localized in the membrane moiety of rough microsomes, and, therefore, that it was most likely solubilized during detergent treatment used for the isolation of detached polysomes. Furthermore, these results established that processing in vivo takes place before completion of the nascent chain. The data also indicate that in vitro processing of nascent chains by rough microsomes is dependent on ribosome binding to the membrane. If the latter process is interfered with by aurintricarboxylic acid, rough microsomes also synthesize some unprocessed chains. The data presented in this paper have been interpreted in the light of a recently proposed hypothesis. This hypothesis, referred to as the signal hypothesis, is described in greater detail in the Discussion section.
HRP has been used as a cytochemical marker for a sterelogic analysis of pinocytic vesicles and secondary lysosomes in cultivated macrophages and L cells. Evidence is presented that the diaminobenzidine technique (a) detects all vaculoes containing encyme and (b) distinguishes between incoming pinocytic vesicles and those which have fused with pre-existing lysosomes to form secondary lososomes. The HRP reactive pinocytic vesicle spaces fills completely within 5 min after exposure to enzyme, while the secondary lysosome compartment is saturated in 45--60 min. The size distribution of sectioned (profile) vaculoe diameters was measured at equilibrium and converted to actual (spherical) dimensions using a technique modified from Dr. S. D. Wicksell. The most important findings in this study have to do with the rate at which pinocytosed fluid and surface membrane move into the cell and on their subsequent fate. Each minute macrophages form at least 125 pinocytic vesicles having a fractional vol of 0.43% of the cell's volume and a fractional area of 3.1% of the cell's surface area. The fractional volume and surface area flux rates for L cells were 0.05% and 0.8% per minute respectively. Macrophages and L cells thus interiorize the equivalent of their cell surface area every 33 and 125 min. During a 3-period, the size of the secondary lysosome compartment remains constant and represents 2.5% of the cell volume and 18% of the surface area. Each hour, therefore, the volume and surface area of incoming vesicles is 10 times greater than the dimensions of the secondary lysosomes in both macrophages and L cells. This implies a rapid reduction in vesicle size during the formation of the secondary lysosome and the egress of pinocytosed fluid from the vacuole and the cell. In addition, we postulate that membrane components of the vacuole are subsequently recycled back to the cell surface.
A set of 11 clones encoding putative GTP binding proteins highly homologous to the yeast YPT1/SEC4 gene products have been isolated from an MDCK cell cDNA library. We localized three of the corresponding proteins in mammalian cells by using affinity-purified antibodies in immunofluorescence and immunoelectron microscopy studies. One, the MDCK homolog of rab2, is associated with a structure having the characteristics of an intermediate compartment between the endoplasmic reticulum and the Golgi apparatus. The second, rab5, is located at the cytoplasmic surface of the plasma membrane and on early endosomes, while the third, rab7, is found on late endosomes. These findings provide evidence that members of the YPT1/SEC4 subfamily of GTP binding proteins are localized to specific exocytic and endocytic subcompartments in mammalian cells.
A protein with an apparent mol. wt of 18,000 daltons (synaptobrevin) was identified in synaptic vesicles from rat brain. Some of its properties were studied using monoclonal and polyclonal antibodies. Synaptobrevin is an integral membrane protein with an isoelectric point of approximately 6.6. During subcellular fractionation, synaptobrevin followed the distribution of small synaptic vesicles, with the highest enrichment in the purified vesicle fraction. Immunogold electron microscopy of subcellular particles revealed that synaptobrevin is localized in nerve endings where it is concentrated in the membranes of virtually all small synaptic vesicles. No significant labeling was observed on the membranes of peptide-containing large dense core vesicles. In agreement with these results, synaptobrevin immunoreactivity has a widespread distribution in nerve terminal-containing regions of the central and peripheral nervous system as shown by light microscopy immunocytochemistry. Outside the nervous system, synaptobrevin immunoreactivity was found in endocrine cells and cell lines (endocrine pancreas, adrenal medulla, PC12 cells, insulinoma cells) but not in other cell types, for example smooth muscle, skeletal muscle and exocrine pancreas. Thus, the distribution of synaptobrevin is similar to that of synaptophysin, a well-characterized membrane protein of small vesicles in neurons and endocrine cells.
The Golgi complex is a series of membrane compartments through which proteins destined for the plasma membrane, secretory
vesicles, and lysosomes move sequentially. A model is proposed whereby these three different classes of proteins are sorted
into different vesicles in the last Golgi compartment, the trans Golgi network. This compartment corresponds to a tubular
reticulum on the trans side of the Golgi stack, previously called Golgi endoplasmic reticulum lysosomes (GERL).
We have studied by electron microscopy and immunocytochemistry the formation of secretory granules containing adrenocorticotropic hormone (ACTH) in murine pituitary cells of the AtT20 line. The first compartment in which condensed secretory protein appears is a complex reticular network at the extreme trans side of the Golgi stacks beyond the TPPase-positive cisternae. Condensed secretory protein accumulates in dilated regions of this trans Golgi network. Examination of en face and serial sections revealed that "condensing vacuoles" are in fact dilations of the trans Golgi network and not detached vacuoles. Only after presumptive secretory granules have reached an advanced stage of morphological maturation do they detach from the trans Golgi network. Frequently both the dilations of the trans Golgi network containing condensing secretory protein and the detached immature granules in the peri-Golgi region have surface coats which were identified as clathrin by immunocytochemistry. Moreover both are the site of budding (or fusion) of coated vesicles, some of which contain condensed secretory protein. The mature granules below the plasma membrane do not, however, have surface coats. Immunoperoxidase labeling with an antiserum specific for ACTH and its precursor polypeptide confirmed that many of the coated vesicles associated with the trans Golgi network contain ACTH. The involvement of the trans Golgi network and coated vesicles in the formation of secretory granules is discussed.
N-Ethylmaleimide (NEM) inhibits protein transport between successive compartments of the Golgi stack in a cell-free system. After inactivation of the Golgi membranes by NEM, transport can be rescued by adding back an appropriately prepared cytosol fraction. This complementation assay has allowed us to purify the NEM-sensitive factor, which we term NSF. The NEM-sensitive factor is a tetramer of 76-kDa subunits, and appears to act catalytically, one tetramer leading to the metabolism of numerous transport vesicles.
Cells from established tissue culture lines of mouse (cIID) and human (VA-2) origin were fused together with Sendai virus, producing heterokaryons bearing both mouse and human surface antigens which were then followed by the indirect fluorescent antibody method. Within 40 mm following fusion, total mixing of both parental antigens occurred in over 90% of the heterokaryons.
Mouse H-2 (histocompatibility) and human surface antigens were visualized by successive treatment of the heterokaryons with a mixture of mouse alloantiserum and rabbit anti-VA-2 antiserum, followed by a mixture of fluorescein-labelled goat anti-mouse IgG and tetramethyl-rhodamine-labelled goat anti-rabbit IgG(Fc).
The cIIDxVA-2 fusions were carried Out in suspension and maintained at 37°C in a shaking water bath; aliquots were removed at various intervals and stained with the above reagents. The heterokaryon population was observed to change from an initial one (5-min post-fusion) of non-mosaics (unmixed cell surfaces of red and green fluorescence) to one of over 90% mosaics (total intermixing of the 2 fluorochromes) by 40 min after fusion. Mouse-human hybrid lines, derived from similar fusions, gave fluorescence patterns identical to those of the mosaic heterokaryons.
Four possible mechanisms would yield such results: (i) a very rapid metabolic turnover of the antigens; (ii) integration of units into the membrane from a cytoplasmic precursor pool; (iii) movement, or ‘diffusion’of antigen in the plane of the membrane; or (iv) movement of existing antigen from one membrane site into the cytoplasm and its emergence at a new position on the membrane.
In an effort to distinguish among these possibilities, the following inhibitor treatments were carried out: (1) both short- and long-term (6-h pre-treatment) inhibition of protein synthesis by puromycin, cycloheximide, and chloramphenicol; (2) short-term inhibition of ATP formation by dinitrophenol (DNP) and NaF; (3) short- and long-term inhibition of glutamine dependent pathways with the glutamine analogue 6-diazo-5-oxonorleucine; and (4) general metabolic suppression by lowered temperature.
The only treatment found effective in preventing the mosaicism was lowered temperature, from which resulted a sigmoidal curve for per cent mosaics versus incubation temperature. These results would be consistent with mechanisms iii and/or iv but appear to rule out i and ii.
From the speed with which the antigen markers can be seen to propagate across the cell membrane, and from the fact that the treatment of parent cells with a variety of metabolic inhibitors does not inhibit antigen spreading, it appears that the cell surface of heterokaryons is not a rigid structure, but is ‘fluid’ enough to allow free ‘diffusion’ of surface antigens resulting in their intermingling within minutes after the initiation of fusion.
Five points are discussed regarding the vesicular structure isolated by fractionation techniques from the brain and liver of the guinea pig. 1. One type of vesicle, fixed by OsO(4) and shown in thin sections, is identified with the coated vesicle that has been observed in many varieties of tissues. 2. The vesicle contained in a spherical polygonal "basketwork" shown by the negative-staining techniques is identical with the coated vesicle shown in sections. 3. Despite minute observation of this basketwork we could not confirm the existence of "hairlike projections" extending from the convex cytoplasmic surface of the vesicle. We are inclined to believe, therefore, that the hairlike projections are actually the superimposed visual images of the regular hexagons and pentagons of the network composing the basketwork. 4. We repeat the hypothesis originally advanced by Roth and Porter (1) that the "coating" of the coated vesicle plays a role in the mechanism of the infolding and fission of the membrane; we suggest that these events are caused by the transformation of the regular hexagons (of the coating) into regular pentagons. 5. Finally, we make a suggestion as to the nature of those vesicles which have on their surface subparticles which look like "elementary particles (2)."
A fluid mosaic model is presented for the gross organization and structure of the proteins and lipids of biological membranes. The model is consistent with the restrictions imposed by thermodynamics. In this model, the proteins that are integral to the membrane are a heterogeneous set of globular molecules, each arranged in an amphipathic structure, that is, with the ionic and highly polar groups protruding from the membrane into the aqueous phase, and the nonpolar groups largely buried in the hydrophobic interior of the membrane. These globular molecules are partially embedded in a matrix of phospholipid. The bulk of the phospholipid is organized as a discontinuous, fluid bilayer, although a small fraction of the lipid may interact specifically with the membrane proteins. The fluid mosaic structure is therefore formally analogous to a two-dimensional oriented solution of integral proteins (or lipoproteins) in the viscous phospholipid bilayer solvent. Recent experiments with a wide variety of techniqes and several different membrane systems are described, all of which abet consistent with, and add much detail to, the fluid mosaic model. It therefore seems appropriate to suggest possible mechanisms for various membrane functions and membrane-mediated phenomena in the light of the model. As examples, experimentally testable mechanisms are suggested for cell surface changes in malignant transformation, and for cooperative effects exhibited in the interactions of membranes with some specific ligands.
When the nerves of isolated frog sartorius muscles were stimulated at 10 Hz, synaptic vesicles in the motor nerve terminals became transiently depleted. This depletion apparently resulted from a redistribution rather than disappearance of synaptic vesicle membrane, since the total amount of membrane comprising these nerve terminals remained constant during stimulation. At 1 min of stimulation, the 30% depletion in synaptic vesicle membrane was nearly balanced by an increase in plasma membrane, suggesting that vesicle membrane rapidly moved to the surface as it might if vesicles released their content of transmitter by exocytosis. After 15 min of stimulation, the 60% depletion of synaptic vesicle membrane was largely balanced by the appearance of numerous irregular membrane-walled cisternae inside the terminals, suggesting that vesicle membrane was retrieved from the surface as cisternae. When muscles were rested after 15 min of stimulation, cisternae disappeared and synaptic vesicles reappeared, suggesting that cisternae divided to form new synaptic vesicles so that the original vesicle membrane was now recycled into new synaptic vesicles. When muscles were soaked in horseradish peroxidase (HRP), this tracerfirst entered the cisternae which formed during stimulation and then entered a large proportion of the synaptic vesicles which reappeared during rest, strengthening the idea that synaptic vesicle membrane added to the surface was retrieved as cisternae which subsequently divided to form new vesicles. When muscles containing HRP in synaptic vesicles were washed to remove extracellular HRP and restimulated, HRP disappeared from vesicles without appearing in the new cisternae formed during the second stimulation, confirming that a one-way recycling of synaptic membrane, from the surface through cisternae to new vesicles, was occurring. Coated vesicles apparently represented the actual mechanism for retrieval of synaptic vesicle membrane from the plasma membrane, because during nerve stimulation they proliferated at regions of the nerve terminals covered by Schwann processes, took up peroxidase, and appeared in various stages of coalescence with cisternae. In contrast, synaptic vesicles did not appear to return directly from the surface to form cisternae, and cisternae themselves never appeared directly connected to the surface. Thus, during stimulation the intracellular compartments of this synapse change shape and take up extracellular protein in a manner which indicates that synaptic vesicle membrane added to the surface during exocytosis is retrieved by coated vesicles and recycled into new synaptic vesicles by way of intermediate cisternae.
We have examined, in the pancreatic exocrine cell, the metabolic requirements for the conversion of condensing vacuoles into zymogen granules and for the discharge of the contents of zymogen granules. To study condensing vacuole conversion, we pulse labeled guinea pig pancreatic slices for 4 min with leucine-(3)H and incubated them in chase medium for 20 min to allow labeled proteins to reach condensing vacuoles. Glycolytic and respiratory inhibitors were then added and incubation continued for 60 min to enable labeled proteins to reach granules in control slices. Electron microscope radioautography of cells or of zymogen granule pellets from treated slices showed that a large proportion of prelabeled condensing vacuoles underwent conversion in the presence of the combined inhibitors. Osmotic fragility studies on zymogen granule suspensions suggest that condensation may result from the aggregation of secretory proteins in an osmotically inactive form. Discharge was studied using an in vitro radioassay based on the finding that prelabeled zymogen granules can be induced to release their labeled contents to the incubation medium by carbamylcholine or pancreozymin. Induced discharge is not affected if protein synthesis is blocked by cycloheximide for up to 2 hr, but is strictly dependent on respiration. The data indicate that transport and discharge do not require the pari passu synthesis of secretory or nonsecretory proteins (e.g. membrane proteins), suggesting that the cell may reutilize its membranes during the secretory process. The energy requirements for zymogen discharge may be related to the fusion-fission of the granule membrane with the apical plasmalemma.
In the previous paper we described an in vitro system of guinea pig pancreatic slices whose secretory proteins can be pulse-labeled with radioactive amino acids. From kinetic experiments performed on smooth and rough microsomes isolated by gradient centrifugation from such slices, we obtained direct evidence that secretory proteins are transported from the cisternae of the rough endoplasmic reticulum to condensing vacuoles of the Golgi complex via small vesicles located in the periphery of the complex. Since condensing vacuoles ultimately become zymogen granules, it was of interest to study this phase of the secretory cycle in pulse-labeled slices. To this intent, a zymogen granule fraction was isolated by differential centrifugation from slices at the end of a 3-min pulse with leucine-(14)C and after varying times of incubation in chase medium. At the end of the pulse, few radioactive proteins were found in this fraction; after +17 min in chaser, its proteins were half maximally labeled; they became maximally labeled between +37 and +57 min. Parallel electron microscopic radioautography of intact cells in slices pulse labeled with leucine-(3)H showed, however, that zymogen granules become labeled, at the earliest, +57 min post-pulse. We assumed that the discrepancy between the two sets of results was due to the presence of rapidly labeled condensing vacuoles in the zymogen granule fraction. To test this assumption, electron microscopic radioautography was performed on sections of zymogen granule pellets isolated from slices pulse labeled with leucine-(3)H and subsequently incubated in chaser. The results showed that the early labeling of the zymogen granule fractions was, indeed, due to the presence of highly labeled condensing vacuoles among the components of these fractions.
Temperature-induced structural changes in the cortical region of the garland cell, which is considered to be active in endocytosis, were investigated in a temperature-sensitive, single gene mutant of Drosophila melanogaster, shibirets1 (shi) and wild-type (Oregon-R). At 19 degrees C, both shi and wild type showed similar structural features: an irregularly extended network of labyrinthine channels, coated pits and vesicles, tubular elements and alpha vacuoles. Tannic acid (TA) impregnation showed that coated pits comprised approximately 20-25% of the total coated profiles at 19 degrees C in both shi and wild-type. When flies were incubated in a horseradish peroxidase (HRP) solution for 5 min, organelles such as coated profiles, tubular elements, and alpha vacuoles were labeled. In wild-type at 30 degrees C, minor changes were observed--mainly a decrease in the distribution of the labyrinthine channels and an increase in HRP uptake. On the other hand, in shi at 30 degrees C, the labyrinthine channels were much elongated and their network became far more complex, indicating the expansion of the surface area of the cell. Also, the coated profiles were increased in number while the number of tubular elements was decreased considerably. The TA method showed that almost all of the coated profiles were coated pits, coated vesicles being almost completely absent at 30 degrees C in shi. Furthermore, HRP uptake activity was considerably decreased at 30 degrees C. These structural changes, as well as the reduced HRP uptake activity, were reversible when the temperature was lowered to 19 degrees C. The observations suggest that in the garland cell of shi the conversion of coated pits to coated vesicles, that is, membrane pinch-off, is blocked at high temperature.
Using immunogold labeling of ultrathin cryosections, we have studied the localization of secretory, lysosomal, and membrane proteins in the Golgi complexes of several cell types. All proteins were present in the stacks of Golgi cisternae, illustrating that the cisternae comprise a ubiquitous way station for proteins with multiple destinations. The labeling patterns support the concept that peripheral Golgi vesicles represent the main site of secretory protein concentration. Of the membrane proteins studied, the Golgi enzyme galactosyltransferase was confined to the trans-most few cisternae, whereas the receptors for asialoglycoproteins and for polymeric immunoglobulin A occurred in most cisternae, with increasing concentration approaching the trans side. The findings are discussed in relation to a cisternal cis to trans progression of Golgi cisternae and membrane specificity.
Fibroblasts apparently ingest low density lipoproteins (LDL) by a selective mechanism of receptor-mediated endocytosis involving the formation of coated vesicles from the plasma membrane. However, it is not known exactly how coated vesicles collect LDL receptors and pinch off from the plasma membrane. In this report, the quick-freeze, deep-etch, rotary-replication method has been applied to fibroblasts; it displays with unusual clarity the coats that appear under the plasma membrane at the start of receptor-mediated endocytosis. These coats appear to be polygonal networks of 7-nm strands or struts arranged into 30-nm polygons, most of which are hexagons but some of which are 5- and 7-sided rings. The proportion of pentagons in each network increases as the coated area of the plasma membrane puckers up from its planar configuration (where the network is mostly hexagons) to its most sharply curved condition as a pinched-off coated vesicle. Coats around the smallest vesicles (which are icosahedrons of hexagons and pentagons) appear only slightly different from "empty coats" purified from homogenized brain, which are less symmetrical baskets containing more pentagons than hexagons. A search for structural intermediates in this coat transformation allows a test of T. Kanaseki and K. Kadota's (1969. J. Cell Biol. 42:202--220.) original idea that an internal rearrangement in this basketwork from hexagons to pentagons could "power" coated vesicle formation. The most noteworthy variations in the typical hexagonal honeycomb are focal juxtapositions of 5- and 7-sided polygons at points of partial contraction and curvature in the basketwork. These appear to precede complete contraction into individual pentagons completely surrounded by hexagons, which is the pattern that characterizes the final spherical baskets around coated vesicles.
The aldehydes introduced in this paper and the more appropriate concentrations for their general use as fixatives are: 4 to 6.5 per cent glutaraldehyde, 4 per cent glyoxal, 12.5 per cent hydroxyadipaldehyde, 10 per cent crotonaldehyde, 5 per cent pyruvic aldehyde, 10 per cent acetaldehyde, and 5 per cent methacrolein. These were prepared as cacodylate- or phosphate-buffered solutions (0.1 to 0.2 M, pH 6.5 to 7.6) that, with the exception of glutaraldehyde, contained sucrose (0.22 to 0.55 M). After fixation of from 0.5 hour to 24 hours, the blocks were stored in cold (4°C) buffer (0.1 M) plus sucrose (0.22 M). This material was used for enzyme histochemistry, for electron microscopy (both with and without a second fixation with 1 or 2 per cent osmium tetroxide) after Epon embedding, and for the combination of the two techniques. After fixation in aldehyde, membranous differentiations of the cell were not apparent and the nuclear structure differed from that commonly observed with osmium tetroxide. A postfixation in osmium tetroxide, even after long periods of storage, developed an image that—notable in the case of glutaraldehyde—was largely indistinguishable from that of tissues fixed under optimal conditions with osmium tetroxide alone. Aliesterase, acetylcholinesterase, alkaline phosphatase, acid phosphatase, 5-nucleotidase, adenosine triphosphatase, and DPNH and TPNH diaphorase activities were demonstrable histochemically after most of the fixatives. Cytochrome oxidase, succinic dehydrogenase, and glucose-6-phosphatase were retained after hydroxyaldipaldehyde and, to a lesser extent, after glyoxal fixation. The final product of the activity of several of the above-mentioned enzymes was localized in relation to the fine structure. For this purpose the double fixation procedure was used, selecting in each case the appropriate aldehyde.
Recombinant v- and t-SNARE proteins reconstituted into separate lipid bilayer vesicles assemble into SNAREpins-SNARE complexes linking two membranes. This leads to spontaneous fusion of the docked membranes at physiological temperature. Docked unfused intermediates can accumulate at lower temperatures and can fuse when brought to physiological temperature. A supply of unassembled v- and t-SNAREs is needed for these intermediates to form, but not for the fusion that follows. These data imply that SNAREpins are the minimal machinery for cellular membrane fusion.
We have examined the blood of man and of the rabbit, dog, guinea pig, sheep, and goat. There exists a great difference in the size of the red blood cells of these animals, but the total surfaces of the chromocytes See PDF for Structure from 0.1 cc. blood do not show a similarly great divergence, because animals having very small cells (goat and sheep) have much greater quantities of these cells in their blood than animals with blood cells of larger dimensions (dog and rabbit). We give all the results of our experiments, omitting only those in which we were unable to avoid losses in the procedure of evaporation of the acetone. It is clear that all our results fit in well with the supposition that the chromocytes are covered by a layer of fatty substances that is two molecules thick.
Proteins and peptides can enter cells by receptor-mediated endocytosis, a coupled process by which selected extracellular proteins or peptides are first bound to specific cell surface receptors and then rapidly internalised by the cell. Internalisation follows clustering of the receptors in specialised regions of the cell surface called coated pits that invaginate to form intracellular coated vesicles. It is now recognised that receptor-mediated endocytosis has a fundamental role in the growth, nutrition and differentiation of animal cells.
The data presented in this paper demonstrate that native small ribosomal subunits from reticulocytes (containing initiation factors) and large ribosomal subunits derived from free polysomes of reticulocytes by the puromycin-KCl procedures can function with stripped microsomes derived from dog pancreas rough microsomes in a protein-synthesizing system in vitro in response to added IgG light chain mRNA so as to segregate the translation product in a proteolysis-resistant space. No such segregation took place for the translation product of globin mRNA. In addition to their ability to segregate the translation product of a specific heterologous mRNA, native dog pancreas rough microsomes as well as derived stripped microsomes were able to proteolytically process the larger, primary translation product in an apparently correct manner, as evidenced by the identical mol wt of the segregated translation product and the authentic secreted light chain. Segregation as well as proteolytic processing by native and stripped microsomes occurred only during ongoing translation but not after completion of translation. Attempts to solubilize the proteolytic processing activity, presumably localized in the microsomal membrane by detergent treatment, and to achieve proteolytic processing of the completed light chain precursor protein failed. Taken together, these results establish unequivocally that the information for segregation of a translation product is encoded in the mRNA itself, not in the protein-synthesizing apparatus; this provides strong evidence in support of the signal hypothesis.
1975 is the fiftieth anniversary of the proposal, by Gorter and Grendel,
that biological membranes are based on a lipid bilayer. Now well
established, this proposal, like the DNA double helix, was a major
breakthrough in molecular cell biology. Here we discuss current concepts
about the molecular composition, organisation and behaviour of the
plasma membranes of mammalian erythrocytes and nucleated cells.
Electron microscopy of various tissues has revealed the existence of intracellular vesicles with “coats” on their cytoplasmic surfaces. Such vesicles have been seen associated with micropinocytosis. A simple and rapid procedure for the purification of coated vesicles from pig brain has been developed. The structures obtained have an external diameter of about 600 Å and a remarkable network-like appearance. The most striking observation about these vesicles, reported here, is that they contain essentially just one protein species, having an apparent molecular weight on sodium dodecyl sulphate gels of about 180,000. Digestion of the coats with trypsin or Pronase shows that most of this protein is located on the external surface (i.e. facing the cytoplasm) of these structures. The lipid vesicles thus liberated appear to have an external diameter of about 350 Å. An estimate of the number of protein molecules per vesicle is consistent with the coat having an icosahedral arrangement of subunits.
Synaptic vesicles store neurotransmitters that are released during calcium-regulated exocytosis. The specificity of neurotransmitter
release requires the localization of both synaptic vesicles and calcium channels to the presynaptic active zone. Two 35-kilodalton
proteins (p35 or syntaxins) were identified that interact with the synaptic vesicle protein p65 (synaptotagmin). The p35 proteins
are expressed only in the nervous system, are 84 percent identical, include carboxyl-terminal membrane anchors, and are concentrated
on the plasma membrane at synaptic sites. An antibody to p35 immunoprecipitated solubilized N-type calcium channels. The p35
proteins may function in docking synaptic vesicles near calcium channels at presynaptic active zones.
Three new and likely related components of the cellular fusion machinery have been purified from bovine brain cytosol, termed alpha-SNAP (35 kd), beta-SNAP (36 kd), and gamma-SNAP (39 kd). Transport between cisternae of the Golgi complex measured in vitro requires SNAP activity during the membrane fusion stage, and each SNAP is capable of binding the general cellular fusion protein NSF to Golgi membranes. The SNAP-NSF-membrane complex may be an early stage in the assembly of a proposed multisubunit "fusion machine" on the target membrane. SNAP transport factor activity is also found in yeast. Yeast cytosol prepared from a secretion mutant defective in export from the endoplasmic reticulum (sec17) lacks SNAP activity, which can be restored in vitro by the addition of pure alpha-SNAP, but not beta- or gamma-SNAPs. These data suggest that the mechanism of action of SNAPs in membrane fusion is conserved in evolution.
Cultured hippocampal neurons were infected with a temperature-sensitive mutant of vesicular stomatitis virus (VSV) and a wild-type strain of the avian influenza fowl plague virus (FPV). The intracellular distribution of viral glycoproteins was monitored by immunofluorescence microscopy. In mature, fully polarized neurons the VSV glycoprotein (a basolateral protein in epithelial MDCK cells) moved from the Golgi complex to the dendritic domain, whereas the hemagglutinin protein of FPV (an apically sorted protein in MDCK cells) was targeted preferentially, but not exclusively, to the axon. The VSV glycoprotein appeared in clusters on the dendritic surface, while the hemagglutinin was distributed uniformly along the axonal membrane. Based on the finding that the same viral glycoproteins are sorted in a polarized fashion in both neuronal and epithelial cells, we propose that the molecular mechanisms of surface protein sorting share common features in the two cell types.
We compared the internalization and intracellular sorting of epidermal growth factor receptor (EGF-R) and point mutant kinase-negative EGF-R separately expressed in NIH 3T3 cells lacking endogenous receptor. Both EGF-Rs internalized rapidly, but kinase-negative receptor was surface down-regulated only with monensin or at 20 degrees C. Furthermore, EGF internalized by mutant receptor alone was, in significant proportion, returned to the cell surface undegraded. Hence unlike wild-type receptor, kinase-negative EGF-R recycles. By electron microscopy the early pathways of endocytosis for the two receptors were identical; however, after 10-20 min the pathways diverged at the multivesicular body (MVB). Wild-type EGF-R, destined for degradation, localized to internal vesicles, while kinase-negative EGF-R, destined for recycling, localized to surface membranes of the MVBs and moved to small tubulovesicles. We conclude that sorting of internalized receptor for degradation or recycling can occur through spatial segregation within the MVB, and sorting of EGF-R is controlled by tyrosine kinase activity.
Guanosine 5'-triphosphate (GTP)-binding proteins have been implicated in the transport of newly synthesized proteins along the secretory pathway of yeast and mammalian cells. Early vesicle fusion events that follow receptor-mediated endocytosis as measured by three in vitro assays were blocked by guanosine 5'-O-(3-thiotriphosphate) and aluminum fluoride. The effect was specific for guanosine nucleotides and depended on the presence of cytosolic factors. Thus, GTP-binding proteins may also have a role in the transport of molecules along the endocytic pathway.
Article de synthese sur les lysosomes: biogenese, definition, role biologique. Les enzymes lysosomales sont decrites ainsi que leur role dans l'exo et l'endocytose. La structure et le biogenese de la membrane lysosomale sont etudiees
We describe a scheme for the purification of the nonclathrin-coated vesicles that mediate transport of proteins between Golgi cisternae and probably from ER to Golgi. These "Golgi-derived coated vesicles" accumulate when Golgi membranes are incubated with ATP and cytosol in the presence of GTP gamma S, a compound that blocks vesicle fusion. The coated vesicles dissociate from the Golgi cisternae in high salt and can then be purified by employing differential and density gradient centrifugation. Golgi-derived coated vesicles have a putative polypeptide composition that is distinct from both cytosol and Golgi membranes, as well as from that of clathrin-coated vesicles.
The interaction between membrane proteins and cytoplasmic structural proteins is thought to be one mechanism for maintaining the spatial order of proteins within functional domains on the plasma membrane. Such interactions have been characterized extensively in the human erythrocyte, where a dense, cytoplasmic matrix of proteins comprised mainly of spectrin and actin, is attached through a linker protein, ankyrin, to the anion transporter (Band 3). In several nonerythroid cell types, including neurons, exocrine cells and polarized epithelial cells homologues of ankyrin and spectrin (fodrin) are localized in specific membrane domains. Although these results suggest a functional linkage between ankyrin and fodrin and integral membrane proteins in the maintenance of membrane domains in nonerythroid cells, there has been little direct evidence of specific molecular interactions. Using a direct biological and chemical approach, we show here that ankyrin binds to the ubiquitous (Na+ + K+)ATPase, which has an asymmetrical distribution in polarized cells.
Several proteins are associated with, or are integral components of, the lipid bilayer that forms the delineating membrane of neuronal synaptic vesicles. To characterize these molecules, we used a polyclonal antiserum raised against purified cholinergic synaptic vesicles from Torpedo to screen a cDNA expression library constructed from mRNA of the electromotor nucleus. One clone encodes VAMP-1 (vesicle-associated membrane protein 1), a nervous-system-specific protein of 120 amino acids whose primary sequence can be divided into three domains: a proline-rich amino terminus, a highly charged internal region, and a hydrophobic carboxyl-terminal domain that is predicted to comprise a membrane anchor. Tryptic digestion of intact and lysed vesicles suggests that the protein faces the cytoplasm, where it may play a role in packaging, transport, or release of neurotransmitters.
Secretion is blocked at the post-Golgi stage within 5 min of a shift of sec4-8 cells from 25 degrees C to 37 degrees C. Analysis of SEC4 predicts a protein product of 23.5 kd molecular weight that shares 32% homology with ras proteins and is essential for growth. The regions of best homology are those involved in the binding and hydrolysis of GTP. Duplication of SEC4 suppresses post-Golgi-blocked mutations in three sec genes. These mutations are lethal when combined with sec4-8 at 25 degrees C. Mutations that block elsewhere on the pathway are not suppressed by the SEC4 duplication and are not lethal when combined with sec4-8. We propose that the SEC4 product is a GTP-binding protein that plays an essential role in controlling a late stage of the secretory pathway.
An extensive reorganization of the Golgi complex (GC) was found in the acinar cells of pancreatic lobules incubated in vitro under conditions which inhibited ATP synthesis and thereby blocked the intracellular transport of secretory proteins. After 15-min incubation under N2 or in the presence of 1 mM dinitrophenol (DNP), transitional elements of the endoplasmic reticulum (ER) lost their protrusions, small peripheral Golgi vesicles decreased drastically in number, and fibrillar aggregates approximately 0.2 to 0.5 micron in diameter appeared on the cis side of the stacks of Golgi cisternae. These aggregates often contained vesicle-free, small (approximately 40 nm), globular cages and, occasionally, vesicle-free, clathrin-like cages. Fibrillar aggregates were also observed on the trans side of Golgi stacks. Other changes included the proliferation of GERL-elements ("rigid lamellae") and a striking increase in the population of coated vesicles trans to the Golgi complex and throughout the apical region of the anoxic acinar cells. All changes were found to be reversible provided the cells were incubated for less than 1 h under N2. These observations suggest that the fibrillar elements and the associated cages described in this study may play a role in the vesicular transport of newly synthesized proteins from the ER to the Golgi complex. Further work is needed to explore this possibility.
X-ray diffraction patterns from intact, isolated Mycoplasma laidlawii membranes at temperatures above and below the thermal phase transition of the membrane lipids reveal important features of the molecular structure of the membrane.(1) The presence of the close hexagonal fatty acid chain packing giving rise to a sharp 4.15 Å diffraction line indicates that the membrane lipids are in a bilayer below the thermal phase transition. (2) The characteristics predicted for the diffraction from a lipid bilayer can be identified in the low-angle patterns from intact membranes both above and below the thermal phase transition, and the differences between the patterns are as expected from the changes in the wideangle pattern. (3) The interpretation of the low-angle patterns is confirmed by changing the average fatty acid chain length and observing the expected changes in the pattern produced by changes in the width of the bilayer. (4) The membrane profile diffraction can be largely accounted for by the lipid bilayer, so the majority of the protein is not located in uniform layers on either side of the bilayer regions of the membrane. (5) The area per lipid molecule is 40 to 45 Å2 in the membrane bilayer below the transition and 60 to 70 Å2 above it. Since the lipids are very closely packed, protein cannot penetrate the head group layers below the transition to make hydrophobic contact without disrupting the packing. Furthermore, the lipid-protein interaction does not greatly constrain the spacing of the lipid head groups.
We describe a cell-free system in which the membrane glycoprotein of vesicular stomatitis virus is rapidly and efficiently transported to membranes of the Golgi complex by a process resembling intracellular protein transport. Transport in vitro is energy-dependent and is accompanied by terminal glycosylation of the membrane glycoprotein (dependent upon UDP-GlcNAc and resulting in resistance to endo-beta-N-acetylglucosaminidase H).
The intracellular pathway of biogenesis of the vesicular stomatitis virus transmembrane glycoprotein was investigated in situ by using indirect immunofluorescence of whole infected Chinese hamster ovary cells and immunoelectron microscopy of ultrathin frozen sections of infected cells. Transport of the glycoprotein was synchronized by using the temperature-sensitive virus mutant Orsay-45 and a temperature shift-down protocol. Sequential appearance of the glycoprotein in the rough endoplasmic reticulum, Golgi apparatus, and plasmalemma was demonstrated. The potential of this system for further studies is discussed.
When the intracellular transit of 3H-labeled (pro)-insulin polypeptides is perturbed by monensin in the pancreatic B-cell, proinsulin conversion is impaired and the radioactive peptides accumulate in a clathrin-coated membrane compartment related to the Golgi apparatus. Clathrin was demonstrated by immunocytochemistry using the postembedding protein A-gold technique. The coated compartment, which is dilated by monensin, comprises Golgi cisternae with condensing secretory material and newly formed secretory granules; under monensin block, the noncoated (storage) secretory granules do not become significantly labeled. These data suggest that an unperturbed passage through a Golgi-related, clathrin-coated membrane compartment which subsequently matures into noncoated secretory granules is needed for the normal processing of (pro)insulin polypeptides.
Concurrently with or shortly after their synthesis on ribosomes, numerous specific proteins are unidirectionally translocated across or asymmetrically integrated into distinct cellular membranes. Thereafter, subpopulations of these proteins need to be sorted from each other and routed for export or targeted to other intracellular membranes or compartments. It is hypothesized here that the information for these processes, termed "protein topogenesis," is encoded in discrete "topogenic" sequences that constitute a permanent or transient part of the polypeptide chain. The repertoire of distinct topogenic sequences is predicted to be relatively small because many different proteins would be topologically equivalent-i.e., targeted to the same intracellular address. The information content of topogenic sequences would be decoded and processed by distinct effectors. Four types of topogenic sequences could be distinguished: signal sequences, stop-transfer sequences, sorting sequences, and insertion sequences. Signal sequences initiate translocation of proteins across specific membranes. They would be decoded and processed by protein translocators that, by virtue of their signal sequence-specific domain and their unique location in distinct cellular membranes, effect unidirectional translocation of proteins across specific cellular membranes. Stop-transfer sequences interrupt the translocation process that was previously initiated by a signal sequence and, by excluding a distinct segment of the polypeptide chain from translocation, yield asymmetric integration of proteins into translocation-competent membranes. Sorting sequences would act as determinants for posttranslocational traffic of subpopulations of proteins, originating in translocation-competent donor membranes (and compartments) and going to translocation-incompetent receiver membranes (and compartments). Finally, insertion sequences initiate unilateral integration of proteins into the lipid bilayer without the mediation of a distinct protein effector. Examples are given for topogenic sequences, either alone or in combination, to provide the information for the location of proteins in any of the intracellular compartments or for the asymmetric orientation of proteins and their location in any of the cellular membranes. Proposals are made concerning the evolution of topogenic sequences and the relationship of protein topogenesis to the precellular evolution of membranes and compartments.
Cells of a Saccharomyces cerevisiae mutant that is temperature-sensitive for secretion and cell surface growth become dense during incubation at the non-permissive temperature (37 degrees C). This property allows the selection of additional secretory mutants by sedimentation of mutagenized cells on a Ludox density gradient. Colonies derived from dense cells are screened for conditional growth and secretion of invertase and acid phosphatase. The sec mutant strains that accumulate an abnormally large intracellular pool of invertase at 37 degrees C (188 mutant clones) fall into 23 complementation groups, and the distribution of mutant alleles suggests that more complementation groups could be found. Bud emergence and incorporation of a plasma membrane sulfate permease activity stop quickly after a shift to 37 degrees C. Many of the mutants are thermoreversible; upon return to the permissive temperature (25 degrees C) the accumulated invertase is secreted. Electron microscopy of sec mutant cells reveals, with one exception, the temperature-dependent accumulation of membrane-enclosed secretory organelles. We suggest that these structures represent intermediates in a pathway in which secretion and plasma membrane assembly are colinear.
The mechanisms through which synaptic vesicle membranes are reinternalized after exocytosis remain a matter of debate. Because several vesicular transport steps require GTP hydrolysis, GTP-gamma S may help identify intermediates in synaptic vesicle recycling. In GTP-gamma S-treated nerve terminals, we observed tubular invaginations of the plasmalemma that were often, but not always, capped by a clathrin-coated bud. Strikingly, the walls of these tubules were decorated by transverse electron-dense rings that were morphologically similar to structures formed by dynamin around tubular templates. Dynamin is a GTPase implicated in synaptic vesicle endocytosis and here we show that the walls of these membranous tubules, but not their distal ends, were positive for dynamin immunoreactivity. These findings demonstrate that dynamin and clathrin act at different sites in the formation of endocytic vesicles. They strongly support a role for dynamin in the fission reaction and suggest that stabilization of the GTP-bound conformation of dynamin leads to tubule formation by progressive elongation of the vesicle stalk.