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Molecular sorting during endocytosis
... In case of COVID-19 infection, low pH in intra-cytoplasmic vesicles is necessary for viral entrance. The entry of the virus inside the host cell is the first and the most critical stage in virus infection [57,58]. Growth factor receptors such as the keratinocyte growth factor receptor, epidermal growth factor receptor (EGFR) [56] and transferrin receptor [57] have been used to study clathrin-dependent endocytosis. ...
... The entry of the virus inside the host cell is the first and the most critical stage in virus infection [57,58]. Growth factor receptors such as the keratinocyte growth factor receptor, epidermal growth factor receptor (EGFR) [56] and transferrin receptor [57] have been used to study clathrin-dependent endocytosis. Endosomes transport endocytosed receptors to the cell surface, destroying or recycling them. ...
... When early endosomes mature to become late endosomes, they become more acidic (pH 6.0 to 5.5). Endosomes must be acidified for integrated viruses to establish an infection [57,58]. The clathrin-independent pathways, on the other hand, include a caveola-dependent route. ...
Background
Coronaviruses such as Severe Acute Respiratory Syndrome coronavirus (SARS), Middle Eastern Respiratory Syndrome (MERS) and Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) are associated with critical illnesses, including severe respiratory disorders. SARS-CoV-2 is the causative agent of the deadly COVID-19 illness, which has spread globally as a pandemic. SARS-CoV-2 may enter the human body through olfactory lobes and interact with the angiotensin-converting enzyme2 (ACE2) receptor, further facilitating cell binding and entry into the cells. Reports have shown that the virus can pass through the blood–brain barrier (BBB) and enter the central nervous system (CNS), resulting in various disorders. Cell entry by SARS-CoV-2 largely relies on TMPRSS2 and cathepsin L, which activate S protein. TMPRSS2 is found on the cell surface of respiratory, gastrointestinal and urogenital epithelium, while cathepsin-L is a part of endosomes.
Aim
The current review aims to provide information on how SARS-CoV-2 infection affects brain function.. Furthermore, CNS disorders associated with SARS-CoV-2 infection, including ischemic stroke, cerebral venous thrombosis, Guillain–Barré syndrome, multiple sclerosis, meningitis, and encephalitis, are discussed. The many probable mechanisms and paths involved in developing cerebrovascular problems in COVID patients are thoroughly detailed.
Main body
There have been reports that the SARS-CoV-2 virus can cross the blood–brain barrier (BBB) and enter the central nervous system (CNS), where it could cause a various illnesses. Patients suffering from COVID-19 experience a range of neurological complications, including sleep disorders, viral encephalitis, headaches, dysgeusia, and cognitive impairment. The presence of SARS-CoV-2 in the cerebrospinal fluid (CSF) of COVID-19 patients has been reported. Health experts also reported its presence in cortical neurons and human brain organoids. The possible mechanism of virus infiltration into the brain can be neurotropic, direct infiltration and cytokine storm-based pathways. The olfactory lobes could also be the primary pathway for the entrance of SARS-CoV-2 into the brain.
Conclusions
SARS-CoV-2 can lead to neurological complications, such as cerebrovascular manifestations, motor movement complications, and cognitive decline. COVID-19 infection can result in cerebrovascular symptoms and diseases, such as strokes and thrombosis. The virus can affect the neural system, disrupt cognitive function and cause neurological disorders. To combat the epidemic, it is crucial to repurpose drugs currently in use quickly and develop novel therapeutics.
... Lang et al., 1994) and S. typhimurium (Rathman et al., 1996(Rathman et al., , 1997 phagosomes undergo rapid fusion with acidic compartments to produce mature phagolysosomes. In M. avium-and M. tuberculosis-infected macrophages, maturation of the phagosome is arrested in an early transitional stage, which prevents or delays the formation of a mature phagolysosome (Sturgill-Koszycki et al., 1994, 1996. A more detailed analysis of the localisation of the protein during activation and infection would assist in defining Nramp1 function in relation to these two important processes. ...
... Anti-Nramp1 polyclonal and mAbs confirmed Nramp1 expression at the immunogold-EM level in bone marrow-derived macrophages from wild-type ( Fig. 2A,B) and mutant (Fig. 2C) congenic mouse strains. Gold particles were localised to the membranes of electron-lucent ( Fig. 2A,C) and electron-dense (Fig. 2B) organelles in both control (data not shown) and interferon-γ/LPS activated macrophages, with morphological characteristics of endosomes and lysosomes (Geuze et al., 1988;Griffiths et al., 1988;Mellman, 1996). This is consistent with the localisation of Nramp1 to intravesicular compartments demonstrated by confocal microscopy. ...
... donovani and Mycobacterium species) or macropinocytosis (S. typhimurium), both of which are driven by actin and result in the formation of phagosomes. As they mature, these phagosomes have been shown to acquire characteristics of late endosomes and lysosomes (de Chastellier et al., 1993;Lang et al., 1994;Sturgill-Koszycki et al., 1994, 1996Rathman et al., 1996Rathman et al., , 1997. However, the rate at which this occurs varies between micro-organisms and, for M. avium and M. tuberculosis in particular, phagosome maturation has been shown to be arrested in an early transitional stage, which acquires an immature form of cathepsin D from the TGN but remains accessible to internalized transferrin (Sturgill-Koszycki et al., 1996). ...
The murine natural resistance-associated macrophage protein, Nramp1, has multiple pleiotropic effects on macrophage activation and regulates survival of intracellular pathogens including Leishmania, Salmonella and Mycobacterium species. Nramp1 acts as an iron transporter, but precisely how this relates to macrophage activation and/or pathogen survival remains unclear. To gain insight into function, anti-Nramp1 monoclonal and polyclonal antibodies are used here to localise Nramp1 following activation and infection. Confocal microscope analysis in uninfected macrophages demonstrates that both the mutant (infection-susceptible) and wild-type (infection-resistant) forms of the protein localise to the membranes of intracellular vesicular compartments. Gold labelling and electron microscopy defines these compartments more precisely as electron-lucent late endosomal and electrondense lysosomal compartments, with Nramp1 colocalizing with Lamp1 and cathepsins D and L in both compartments, with macrosialin in late endosomes, and with BSA-5 nm gold in pre-loaded lysosomes. Nramp1 is upregulated with interferon-γ and lipopolysaccaride treatment, coinciding with an increase in labelling in lysosomes relative to late endosomes and apparent dispersion of Nramp1-positive vesicles from a perinuclear location towards the periphery of the cytoplasm along the microtubular network. In both control and activated macrophages, expression of the protein is 3-to 4-fold higher in wild-type compared to mutant macrophages. In Leishmania major-infected macrophages, Nramp1 is observed in the membrane of the pathogen-containing phagosomes, which retain a perinuclear localization in resting macrophages. In Mycobacterium avium-infected resting and activated macrophages, Nramp1-positive vesicles migrated to converge, but not always fuse, with pathogen-containing phagosomes. The Nramp1 protein is thus located where it can have a direct influence on phagosome fusion and the microenvironment of the pathogen, as well as in the more general regulation of endosomal/lysosomal function in macrophages.
... Early endosomes consist of vacuolar and tubular structures, which helps facilitate the protein sorting processes by providing different surface area-tovolume ratios at different points. The high surface area-to-volume ratio at the tubular 8 domain allows transmembrane receptors to cluster and accumulate before either recycling back to the plasma membrane or retrogradely towards to the TGN (Mellman, 1996;Geuze et al., 1987). In contrast, the low surface area-to-volume ratio at the vacuolar domains of early endosomes allow the accumulation of ligands in the lumen destined for lysosomal degradation (Mellman, 1996;Geuze et al., 1983). ...
... The high surface area-to-volume ratio at the tubular 8 domain allows transmembrane receptors to cluster and accumulate before either recycling back to the plasma membrane or retrogradely towards to the TGN (Mellman, 1996;Geuze et al., 1987). In contrast, the low surface area-to-volume ratio at the vacuolar domains of early endosomes allow the accumulation of ligands in the lumen destined for lysosomal degradation (Mellman, 1996;Geuze et al., 1983). The pH of early endosomes is slightly acidic and the weakly acidic environment allows the dissociation of the receptor-ligand complex (Mellman, 1996;Sipe and Murphy, 1987;Mellman et al., 1986). ...
... In contrast, the low surface area-to-volume ratio at the vacuolar domains of early endosomes allow the accumulation of ligands in the lumen destined for lysosomal degradation (Mellman, 1996;Geuze et al., 1983). The pH of early endosomes is slightly acidic and the weakly acidic environment allows the dissociation of the receptor-ligand complex (Mellman, 1996;Sipe and Murphy, 1987;Mellman et al., 1986). The free membrane embedded receptors are then recycled back to the plasma membrane or TGN, while the released soluble ligands and the lysosomal hydrolases remain in the endosomes which mature before delivery to the lysosome (Elkin et al., 2016;Scott et al., 2014). ...
Charcot-Marie-Tooth disease (CMT) is the commonest inherited neuromuscular disorder, which affects the peripheral nervous system leading to nerve degeneration. CMT is categorised into two forms, ‘axonal’ and ‘demyelinating’, which reflects the main site of pathology as the axon or Schwann cells respectively. Over 90 genes have been identified associated with the disease. Among the genes associated with demyelinating CMT, the focus of my thesis is LITAF, mutations in which lead to an autosomal dominant demyelinating CMT known as CMT type 1C. LITAF is a 17 kDa protein likely to be involved in endocytic degradation and trafficking of specific cargo proteins. It contains an N-terminal proline-rich region mediating protein-protein interactions and a C-terminal LITAF domain consisting of a zinc-ribbon structure with a hydrophobic region incorporated. Most of the CMT1C mutations are clustered in this highly conserved LITAF domain. LITAF was predicted to play roles in recruiting ESCRT components and exosome formation, but the precise function remains unclear. Furthermore, why mutations in LITAF lead to CMT is not known. My work therefore focused on characterising the function of the LITAF protein both in health and disease. In my thesis, I first tried to determine the subcellular localisation of LITAF proteins and also investigated the function of the highly conserved C-terminal LITAF domain in targeting protein to the membrane. With regards to the function of LITAF, potential binding partners were screened using the traditional GST pull-down assay and the in-situ proximity labelling assay, BioID. A number of novel potential binding partners were identified in both assays. Among the list of potential binding partners, BAG3 was captured in both pull-down assays and was chosen for further studies. The interaction with LITAF was characterised and the potential role of this interaction in autophagy was investigated. Integrin, which was also captured in the BioID assay, was another protein chosen for further studies. Internalisation and recycling assay were developed to investigate the function of LITAF in integrin trafficking. The potential of CMT mutations in impairing the internalisation of integrin in A431 cells and the difficulty in performing the assays were discussed. Lastly, with patient fibroblasts available in our lab, disease phenotypes were analysed using two types of imaging technique: transmission electron microscopy (TEM) (in collaboration with J. Edgar) and immunofluorescence microscopy. Swollen vacuoles were observed in the TEM images of the patient fibroblasts only, and various uptake assays were performed to identify these enlarged compartments. In summary, this work offers an insight into the function of LITAF both in health and disease as well as identifying potential intracellular binding partners that might shed light on pathogenesis. Furthermore, the modified trafficking assays described in this thesis can be applied to Schwann cell and patient fibroblasts, providing further tools to probe the underlying membrane trafficking pathways that are dysfunctional in CMT.
... As in other cell types in ECs, endocytosis is involved in the internalization of molecules from the plasma membrane and extracellular environment, and plasma membrane recycling, including uptake and the degradation of signal molecules [46,47]. The development of endosomes depends on the role they play in transcytosis through the central zone of EC. (Figure 4A,B). ...
... The main components of the endocytic system are clathrin-dependent endocytic buds ("vesicles"), and early and late endosomes and their derivatives [46]. Additionally, ECs exhibit a clathrin-independent endocytosis with its own less characterized components. ...
The main component of blood and lymphatic vessels is the endothelium covering their luminal surface. It plays a significant role in many cardiovascular diseases. Tremendous progress has been made in deciphering of molecular mechanisms involved into intracellular transport. However, molecular machines are mostly characterized in vitro. It is important to adapt this knowledge to the situation existing in tissues and organs. Moreover, contradictions have accumulated within the field related to the function of endothelial cells (ECs) and their trans-endothelial pathways. This has induced necessity for the re-evaluation of several mechanisms related to the function of vascular ECs and intracellular transport and transcytosis there. Here, we analyze available data related to intracellular transport within ECs and re-examine several hypotheses about the role of different mechanisms in transcytosis across ECs. We propose a new classification of vascular endothelium and hypotheses related to the functional role of caveolae and mechanisms of lipid transport through ECs.
... Lysosomes are membrane-bound organelles and contain various acid hydrolases to degrade macromolecules including proteins, lipids and nucleotides 17,18 . The degradative substrates in the extracellular Article https://doi.org/10.1038/s41556-023-01098-9 ...
... REs are organelles that function in recycling molecules back to the plasma membrane 17,42 . Besides their classical roles in endocytic recycling, it has been shown that REs have a role in exocytic and retrograde membrane traffic 43 and clathrin adaptor AP-1 function at REs for the retrograde membrane traffic 46 . ...
Stimulator of interferon genes (STING) is essential for the type I interferon response against a variety of DNA pathogens. Upon emergence of cytosolic DNA, STING translocates from the endoplasmic reticulum to the Golgi where STING activates the downstream kinase TBK1, then to lysosome through recycling endosomes (REs) for its degradation. Although the molecular machinery of STING activation is extensively studied and defined, the one underlying STING degradation and inactivation has not yet been fully elucidated. Here we show that STING is degraded by the endosomal sorting complexes required for transport (ESCRT)-driven microautophagy. Airyscan super-resolution microscopy and correlative light/electron microscopy suggest that STING-positive vesicles of an RE origin are directly encapsulated into Lamp1-positive compartments. Screening of mammalian Vps genes, the yeast homologues of which regulate Golgi-to-vacuole transport, shows that ESCRT proteins are essential for the STING encapsulation into Lamp1-positive compartments. Knockdown of Tsg101 and Vps4, components of ESCRT, results in the accumulation of STING vesicles in the cytosol, leading to the sustained type I interferon response. Knockdown of Tsg101 in human primary T cells leads to an increase the expression of interferon-stimulated genes. STING undergoes K63-linked ubiquitination at lysine 288 during its transit through the Golgi/REs, and this ubiquitination is required for STING degradation. Our results reveal a molecular mechanism that prevents hyperactivation of innate immune signalling, which operates at REs.
... MVBs are part of the pleiomorphic endosomal system, which consists of primary endocytic vesicles, early endosomes (EEs), late endosomes (LEs) and lysosomes (reviewed by Geuze, 1998;Mellman, 1996). Within the endocytic tract, EEs are the major entry site for endocytosed material, whereas LEs are thought to receive newly synthesized lysosomal hydrolases directly from the trans-Golgi network. ...
... Lysosomes are the next station in the endocytic tract and, together with LEs, constitute the main site of protein and lipid degradation. The position of different subcompartments along the endocytic route can be determined on the basis of morphological characteristics, kinetics of fluid-phase marker uptake and differential distribution of marker proteins (Geuze, 1998;Mellman, 1996). The recycling plasma membrane protein transferrin receptor (TfR) is frequently used to identify early sorting and recycling endosomes. ...
Exosomes are small membrane vesicles that are secreted by a multitude of cell types as a consequence of fusion of multivesicular late endosomes/lysosomes with the plasma membrane. Depending on their origin, exosomes can play roles in different physiological processes. Maturing reticulocytes externalize obsolete membrane proteins such as the transferrin receptor by means of exosomes, whereas activated platelets release exosomes whose function is not yet known. Exosomes are also secreted by cytotoxic T cells, and these might ensure specific and efficient targeting of cytolytic substances to target cells. Antigen presenting cells, such as B lymphocytes and dendritic cells, secrete MHC class-I- and class-II-carrying exosomes that stimulate T cell proliferation in vitro. In addition, dendritic-cell-derived exosomes, when used as a cell-free vaccine, can eradicate established murine tumors. Although the precise physiological target(s) and functions of exosomes remain largely to be resolved, follicular dendritic cells (accessory cells in the germinal centers of secondary lymphoid organs) have recently been shown to bind B-lymphocyte-derived exosomes at their cell surface, which supports the notion that exosomes play an immunoregulatory role. Finally, since exosomes are derived from multivesicular bodies, their molecular composition might provide clues to the mechanism of protein and lipid sorting in endosomes.
... Endocytosis serves to maintain cellular homeostasis by recovering protein and lipid components that are inserted into the plasma membrane by ongoing secretory activity (Mellman 1996). It is an essential process for sustaining organismal homeostasis, enabling the control of an extraordinary array of activities required for existence as part of a multicellular community. ...
... Transfer of material from early endosomes to late endosomes involves the dissociation of vacuolar elements from the early endosome network, and their subsequent migration on microtubules to the perinuclear cytoplasm where they fuse with late endosomes (Mellman et al., 1996). Carrier vesicles may be formed from late endosomes. ...
p>In this study, the enhanced cytotoxic effect of the anti-CD7 IT HB2-Saporin and the anti-CD38 IT OKT10-Saporin used in combination when compared to their use individually was demonstrated on the human T-ALL cell line HSB-2 both in vivo and in vitro. In an in vitro cell proliferation assay and an in vivo SCID mouse model, OKT10-Saporin was shown to be more effective than HB2-Saporin, but neither individual IT was as potent as their combined use. In contrast, HB2-Saporin performed best in the short term protein synthesis assay (PSI) and the combination of two ITs demonstrated an intermediate potency between that of the two individual ITs. Three explanations were proposed for the improved efficacy of using two ITs simultaneously: 1) co-ligation of CD7 and CD38 might alter the individual internationalisation characteristics; 2) targeting against two molecules overcomes the heterogeneity of antigen expression on tumour cells or 3) using two immunotoxins increases the amount of toxin delivered to the target cell. It is possible that all three explanations are valid.
Flow cytometry and confocal microscopy were used to determine the internalisation and intracellular routing characteristics of CD7 and CD38 on the T-ALL cell line HSB-2, when ligated by antibody individually or in combination. These studies indicate that CD7 and CD38 have very different internalisation kinetics. CD7 clears very rapidly from the cell surface following ligation by antibody, whereas only 50% of CD38 molecules internalise over a 24 hr duration. When CD7 and CD38 were ligated by antibody simultaneously, no change to the internalisation characteristics or intracellular routing of either antibody appeared altered. In these studies it has become clear that there is no direct correlation between internationalisation, intracellular routing and cytotoxic potency.
Brefeldin A was used as a tool to investigate what intracellular routes these ITs may follow within the cell. These were preliminary studies and further work is required.
These studies have revealed that no single factor determines IT potency. Internalisation rate is only important if degradation is avoided and trafficking occurs to an appropriate intracellular compartment from which the toxin component can translocate.</p
... Phagocytosis can be defined as the process by which specialised cells such as macrophages engulf foreign matter for processing, usually by the RES, and is related to large particles (>500nm diameter). Pinocytosis can be defined as the process of engulfing small portions of the surrounding fluid and processing it through endosomes and into lysosomes and is usually initiated by clathrin-coated pits and is common to almost all cell types (reviewed by Mellman, 1996). Figure 1.9 shows the differences in subcellular localisation of low molecular weight and a polymer-drug conjugate. ...
... Polymer-drug conjugates have to go through a number of stages before the cytotoxic agent can get to its site of action, if that site is intracellular which in the case of many anticancer agents is the nucleus. They firstly need to be taken up by the cell and therefore need to be of a physical diameter which is not too large (Mellman, 1996). The attached anticancer agent needs to be stable in the acidic milieu of the intracellular compartment (Ohkuma & Poole, 1978) and also needs to be not broken down by the enzymes it encounters, of which the lysosome is thought to contain up to 70 types (Dean, 1977). ...
Over the past two decades cancer chemotherapy has resulted in a small number of previously fatal cancers becoming curable. Many cancers, particularly the so-called solid tumours, do not respond well to conventional chemotherapy. To maximise tumour targeting and minimize host tissue toxicity a large number of drug delivery systems have been proposed. Polymer anticancer conjugates based on N-(2- hydroxypropyl) methacrylamide (HPMA) have recently entered early clinical trial. It has been shown that HPMA copolymer conjugates preferentially extravasate into solid tumours and are retained there by a process known as the 'enhanced permeability and retention' (EPR) effect. A natural anticancer agent, derived from ellipticine, namely 6-(3-aminopropyl) ellipticine (APE) was selected for conjugation to HPMA copolymers. In this study a series of HPMA copolymer-APE conjugates were synthesised, containing a variety of drug loadings (1.07-6.10%w/w) conjugated via the tetrapeptide linker (Gly-Phe-Leu- Gly). These conjugates were designed to be localised in tumours following injection and to be taken up by tumour cells via the process of endocytosis before liberating APE, mediated by cathepsin B present in the lysosome. These conjugates were shown to form complex intramolecular micelles in solution which resulted in the conjugates of a high drug loading showing reduced APE release in vitro (20%/5h) compared to the medium and low drug loading conjugates (55%/5h), suggesting hindered enzyme access. All conjugates showed a marked reduction in haemolysis, a common problem with ellipticines, compared to APE alone. Anti-tumour activity was observed in the s.c. B16F10 murine melanoma model and also in the CORL-23 human non small cell lung carcinoma xenograft in mice, particularly for the conjugate of medium APE loading. This thesis also examined the extravasation and intratumoural distribution of HPMA copolymer-anticancer conjugates using HPMA copolymer doxorubicin (PK1) as a model conjugate in the rat dorsal window chamber model.
... In general, lysosomes are intracellular digestive organelles that are considered to be the end-point of endocytosis and autophagy. 7 Several studies have shown that various cellular stimuli can induce lysosomes to undergo exocytosis, which involves the fusion of lysosomes with the plasma membrane and the discharge of their contents into the extracellular space. 8,9 Lysosomal exocytosis is a fundamental process that plays many important roles in the body: for example, it mediates antigen presentation by antigen-presenting cells, 10 neurotransmitter release by neurons, 11 and, as mentioned above, bone resorption by osteoclasts. ...
Mature osteoclasts degrade bone matrix by exocytosis of active proteases from secretory lysosomes through a ruffled border. However, the molecular mechanisms underlying lysosomal trafficking and secretion in osteoclasts remain largely unknown. Here, we show with GeneChip analysis that RUN and FYVE domain-containing protein 4 (RUFY4) is strongly upregulated during osteoclastogenesis. Mice lacking Rufy4 exhibited a high trabecular bone mass phenotype with abnormalities in osteoclast function in vivo. Furthermore, deleting Rufy4 did not affect osteoclast differentiation, but inhibited bone-resorbing activity due to disruption in the acidic maturation of secondary lysosomes, their trafficking to the membrane, and their secretion of cathepsin K into the extracellular space. Mechanistically, RUFY4 promotes late endosome-lysosome fusion by acting as an adaptor protein between Rab7 on late endosomes and LAMP2 on primary lysosomes. Consequently, Rufy4-deficient mice were highly protected from lipopolysaccharide- and ovariectomy-induced bone loss. Thus, RUFY4 plays as a new regulator in osteoclast activity by mediating endo-lysosomal trafficking and have a potential to be specific target for therapies against bone-loss diseases such as osteoporosis.
... During maturation, the pH within the endosomal lumen decreases, leading to the release of siRNA from its delivery vehicle into the cytoplasm. However, siRNA efficacy can be limited by degradation caused by endosomal nucleases and sequestration in non-productive compartments [39,40]. Larger siRNA molecules are more likely to become entrapped, while cationic materials used for delivery can bind to the endosomal membrane, resulting in entrapment [41]. ...
Small interfering RNA (siRNA) is a potential method of gene silencing to target specific genes. Although the U.S. Food and Drug Administration (FDA) has approved multiple siRNA-based therapeutics, many biological barriers limit their use for treating diseases. Such limitations include challenges concerning systemic or local administration, short half-life, rapid clearance rates, nonspecific binding, cell membrane penetration inability, ineffective endosomal escape, pH sensitivity, endonuclease degradation, immunological responses, and intracellular trafficking. To overcome these barriers, various strategies have been developed to stabilize siRNA, ensuring their delivery to the target site. Chemical modifications implemented with nucleotides or the phosphate backbone can reduce off-target binding and immune stimulation. Encapsulation or formulation can protect siRNA from endonuclease degradation and enhance cellular uptake while promoting endosomal escape. Additionally, various techniques such as viral vectors, aptamers, cell-penetrating peptides, liposomes, and polymers have been developed for delivering siRNA, greatly improving their bioavailability and therapeutic potential.
... Endocytosis is a multicomponent series of processes involving internalization of cell surface proteins, trafficking of these factors for recycling or degradation, and sorting to distinct cellular sites (17,18). ...
Glioblastoma is the most malignant primary brain tumor for which the prognosis remains dismal even with aggressive surgical, medical, and radiation therapies. Glioblastoma stem cells (GSCs) promote therapeutic resistance and cellular heterogeneity due to their self-renewal properties and capacity for plasticity. To understand the molecular processes essential for maintaining GSCs, we performed an integrative analysis comparing active enhancer landscapes, transcriptional profiles, and functional genomics profiles of GSCs and non-neoplastic neural stem cells (NSCs). We identified sorting nexin 10 (SNX10), an endosomal protein sorting factor, as selectively expressed in GSCs compared to NSCs and essential for GSC survival. Targeting SNX10 impaired GSC viability and proliferation, induced apoptosis, and reduced self-renewal capacity. Mechanistically, GSCs utilized endosomal protein sorting to promote platelet-derived growth factor receptor β (PDGFRβ) proliferative and stem cell signaling pathways through post-transcriptional regulation of the PDGFR tyrosine kinase. Targeting SNX10 expression extended survival of orthotopic xenograft-bearing mice, and high SNX10 expression correlated with poor glioblastoma patient prognosis, suggesting its potential clinical importance. Thus, our study reveals an essential connection between endosomal protein sorting and oncogenic receptor tyrosine kinase signaling and suggests that targeting endosomal sorting may represent a promising therapeutic approach for glioblastoma treatment.
... It was significantly down-regulated in lysosomal pathways in AF vs. CP and ME vs. CP comparisons. Cathepsins are a class of proteolytic enzymes that mainly exist in lysosomes and participate in lysosome-mediated protein digestion and transformation together with other proteases (Mellman, 1996). CTSL is widely distributed in the lysosome or cytoplasm of various organisms and plays an important role in the process of proteolysis (Turk et al., 2000). ...
Neptunea cumingii is a carnivorous snail with a very high market value, but it cannot be cultivated on a large scale at present due to the lack of an appropriate artificial feed. In this study, we fed N. cumingii four kinds of diets (Cristaria plicata meat, Chlamys farreri mantle, Mytilus edulis meat, and artificial feed) for 90 days and then measured growth indexes and digestive and immune enzyme indexes. We also subjected liver samples to transcriptome sequencing to detect significant differences among the four groups at the transcriptome level. The survival rate of N. cumingii fed with each type of feed was > 90%. The weight, shell length, shell height, and weight gain of the N. cumingii in the C. plicata meat group were significantly higher than those of the other treatment groups, while the values in the artificial feed group were significantly lower than those of the other treatment groups. The superoxide dismutase, catalase, amylase, protease, and lipase activities were higher in the stomach, intestine, and liver of snails in the C. plicata meat feeding group than in those of the other treatment groups, and they were significantly higher than those of the artificial feed group (p < 0.05); the opposite was true for malondialdehyde content. A total of 3407 differentially expressed genes (DEGs) were found in the M. edulis meat group vs. C. plicata meat group comparison. A total of 3422 DEGs genes were identified in the artificial feed group vs. C. plicata meat group comparison. A total of 3,929 DEGs were found in the C. plicata meat group vs. C. farreri mantle group comparison. Through Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis, we found the DEGs in the liver of N. cumingii fed with the different diets were mainly enriched in the energy metabolism, growth, and immunity pathways. Our results indicated that among the four diets tested, C. plicata meat had the best effect on N. cumingii, and they provided an important reference for identification of suitable diets for the development of N. cumingii artificial breeding.
... Nonetheless, even with bioactive polypeptides that target PPIs in hand, the challenge of delivering these into mammalian cells where they would be most useful remains. Exogenous reagents-usually proteins and larger assemblies-presented to cells can enter actively through endocytosis 17,18 . In this case, however, the reagents end up in the endolysosomal system, which can inhibit release into the cytosol 19 . ...
Increasingly, it is possible to design peptide and protein assemblies de novo from first principles or computationally. This approach provides new routes to functional synthetic polypeptides, including designs to target and bind proteins of interest. Much of this work has been developed in vitro. Therefore, a challenge is to deliver de novo polypeptides efficiently to sites of action within cells. Here we describe the design, characterisation, intracellular delivery, and subcellular localisation of a de novo synthetic peptide system. This system comprises a dual-function basic peptide, programmed both for cell penetration and target binding, and a complementary acidic peptide that can be fused to proteins of interest and introduced into cells using synthetic DNA. The designs are characterised in vitro using biophysical methods and X-ray crystallography. The utility of the system for delivery into mammalian cells and subcellular targeting is demonstrated by marking organelles and actively engaging functional protein complexes. The de novo design of a pair of complementary peptides, one basic for cell penetration and target binding and one acidic that can be fused to proteins of interest, provides an approach for delivery into mammalian cells and subcellular targeting.
... It has already been suggested that the initial endosomes could be partitioned into functional regions, the tubular regions being preferred for the storage of material that would be destined for recycling. Simultaneously, the large vesicles would concentrate the materials destined for the degradation pathway (Mellman 1996a). The material intended for recycling back to the plasma membrane can follow two routes: direct recycling from the initial endosomes or directing to the recycling endosomes (Grant and Donaldson 2009). ...
Trypanosoma cruzi is the agent of Chagas disease with a high prevalence in Latin America. This protozoan presents a complex life cycle in the invertebrate (hematophagous triatominae) and the vertebrate hosts. Here we review the most relevant information on essential aspects of maintaining the parasite in vitro (axenic medium and cell cultures) and mammals, describing the most appropriate animal models. The diversity of the parasite is discussed based on biochemical and molecular information. Particular emphasis is given to the structural organization of the various developmental stages of the parasite, putting together morphological, biochemical, and molecular data on structures such as (a) the plasma membrane-cell surface complex, (b) the flagellum (including flagellar membrane, axoneme, paraflagellar rod and adhesion of the flagellum to the cell body), and other structures such as (c) the kinetoplast-mitochondrion complex, (d) the sub-pellicular and cytoplasmic microtubules, (e) endoplasmic reticulum-Golgi complex system, (f) contractile vacuole, (g) acidocalcisomes, (h) the kinetoplast-mitochondrion complex, (i) glycosomes, (j) lipid bodies, (k) P- bodies, (l) the endocytic pathway, (m) the exocytic pathway, including the release of microvesicles and exosomes by the parasite, and (n) the nucleus. Finally, we also analyze the basic information on the process of interaction of T. cruzi with host cells.Keywords
T. cruzi
Life cycleUltrastructureChagas disease
... Eukaryotic cells uptake components of the plasma membrane, including lipids (phospholipids and cholesterol) and proteins through multiple endocytic pathways (Grant and Donaldson, 2009). Cargo molecules that enter the cell converge in early/sorting endosomes, where they are sorted and delivered to various destinations (Mellman, 1996). ...
The disruption of endosomal actin architecture negatively affects endocytic recycling. However, the underlying homeostatic mechanisms that regulate actin organization during recycling remain unclear. In this study, we identified a synergistic endosomal actin assembly restricting mechanism in C. elegans involving WTS-1/LATS kinase, which is a core component of the Hippo pathway. WTS-1 resides on the sorting endosomes and colocalizes with the actin polymerization regulator PTRN-1/CAMSAPs. We observed an increase in PTRN-1-labeled structures in WTS-1-deficient cells, indicating that WTS-1 can limit the endosomal localization of PTRN-1. Accordingly, the actin overaccumulation phenotype in WTS-1-depleted cells was mitigated by the associated PTRN-1 loss. We further demonstrated that recycling defects and actin overaccumulation in WTS-1-deficient cells were reduced by the overexpression of constitutively active UNC-60A/cofilin(S3A), which aligns with the role of LATS as a positive regulator of cofilin activity. Altogether, our data confirmed previous findings, and we proposed an additional model: WTS-1 acts alongside the UNC-60A/cofilin-mediated actin disassembly to restrict the assembly of endosomal F-actin by curbing PTRN-1 dwelling on endosomes, preserving recycling transport.
... We next assessed trafficking of the PDGFR homodimers through late endosomes, which precedes trafficking to the lysosome for degradation (Mellman, 1996), by examining colocalization of the Venus signal with signal from an antibody recognizing the late endosome . CC-BY-NC-ND 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. ...
Signaling through the platelet-derived growth factor receptors (PDGFRs) plays a critical role in multiple cellular processes during development. The two PDGFRs, PDGFRα and PDGFRβ, dimerize to form homodimers and/or heterodimers. Here, we overcome previous limitations in studying PDGFR dimer-specific dynamics by generating cell lines stably expressing C-terminal fusions of each PDGFR with bimolecular fluorescence complementation (BiFC) fragments corresponding to the N-terminal or C-terminal regions of the Venus fluorescent protein. We find that PDGFRβ receptors homodimerize more quickly than PDGFRα receptors in response to PDGF ligand, with increased levels of autophosphorylation. Further, we demonstrate that PDGFRα homodimers are trafficked and degraded more quickly, while PDGFRβ homodimers are more likely to be recycled back to the cell membrane. We show that PDGFRβ homodimer activation results in a greater amplitude of phospho-ERK1/2 and phospho-AKT signaling, as well as increased proliferation and migration. Collectively, our findings provide significant insight into how biological specificity is introduced to generate unique responses downstream of PDGFR engagement.
Summary
The authors utilize a novel bimolecular fluorescence complementation approach to investigate PDGFR homodimer-specific dynamics. They uncover differences in the timing and extent of receptor dimerization, activation and trafficking, which lead to changes in downstream signaling and cellular activity.
... 72 Indeed, this process is the primary method for absorption of many immunoglobulins 73 and proteins. 74 In addition to villi and microvilli, other microstructures of epithelial cells are to be taken into consideration when designing DDS for therapeutic delivery. For example, tight junctions within the epithelial barrier make it difficult for large drug molecules (≥6 nanometers) to pass through the epithelial layer. ...
The systemic pharmacotherapeutic efficacy of immunomodulatory drugs is heavily influenced by its route of administration. A few common routes for the systemic delivery of immunotherapeutics are intravenous, intraperitoneal, and intramuscular injections. However, the development of novel biomaterials, in adjunct to current progress in immunoengineering, is providing an exciting area of interest for oral drug delivery for systemic targeting. Oral immunotherapeutic delivery is a highly preferred route of administration due to its ease of administration, higher patient compliance, and increased ability to generate specialized immune responses. However, the harsh environment and slow systemic absorption, due to various biological barriers, reduces the immunotherapeutic bioavailability, and in turn prevents widespread use of oral delivery. Nonetheless, cutting edge biomaterials are being synthesized to combat these biological barriers within the gastrointestinal (GI) tract for the enhancement of drug bioavailability and targeting the immune system. For example, advancements in biomaterials and synthesized drug agents have provided distinctive methods to promote localized drug absorption for the modulation of local or systemic immune responses. Additionally, novel breakthroughs in the immunoengineering field show promise in the development of vaccine delivery systems for disease prevention as well as combating autoimmune diseases, inflammatory diseases, and cancer. This review will discuss current progress made within the field of biomaterials and drug delivery systems to enhance oral immunotherapeutic availability, and how these new delivery platforms can be utilized to deliver immunotherapeutics for resolution of immune‐related diseases.
... The fluidity of the membrane is paramount to understanding cellular dynamics, as it supports membrane protein diffusion and reorganization (1). Hydrodynamic shear stresses at the interface between lipid bilayers and their aqueous environment influence the biochemical responses underlying critical functions (3), such as dictating cell shape (4), sensing (5), adhesion and locomotion (6,7), cell division (8), and transport of molecules and ions (9)(10)(11)(12). ...
Significance
Lipid bilayers constitute the matrix of cellular membranes and synthetic vesicles used in drug delivery. This self-assembled structure is only a few nanometers thick but provides an effective barrier between aqueous fluids. The response of lipid bilayers to shear stresses induced by surrounding fluid flows can trigger biophysical processes in cells and influence the efficacy of drug delivery by synthetic vesicles. Here, we use optical tweezers to apply and measure local hydrodynamic shear stresses on both sides of a freestanding lipid bilayer. With this method, we determine the rheological properties of bilayers and capture a previously unreported phenomenon when the intermonolayer friction is so low that the monolayers slip past each other and hydrodynamic shear is not transmitted through the bilayer.
... Lysosomes play key roles in maintaining cellular homeostasis by degrading and recycling substrates received from the endocytic and autophagic pathways (Klionsky and Emr, 2000; Levine and Klionsky, 2004;Mellman, 1996). Degradation of endocytic and autophagic cargos depends on their dynamic interactions with lysosomes, which provide the acid hydrolases required for substrate degradation (Luzio et al., 2007). ...
Niemann-Pick disease type C (NPC) is a neurodegenerative lysosomal storage disorder characterized by lipid accumulation in endolysosomes. An early pathologic hallmark is axonal dystrophy occurring at presymptomatic stages in NPC mice. However, the mechanisms underlying this pathologic change remain obscure. Here, we demonstrate that endocytic-autophagic organelles accumulate in NPC dystrophic axons. Using super-resolution and live-neuron imaging, we reveal that elevated cholesterol on NPC lysosome membranes sequesters kinesin-1 and Arl8 independent of SKIP and Arl8-GTPase activity, resulting in impaired lysosome transport into axons, contributing to axonal autophagosome accumulation. Pharmacologic reduction of lysosomal membrane cholesterol with 2-hydroxypropyl-β-cyclodextrin (HPCD) or elevated Arl8b expression rescues lysosome transport, thereby reducing axonal autophagic stress and neuron death in NPC. These findings demonstrate a pathological mechanism by which altered membrane lipid composition impairs lysosome delivery into axons and provide biological insights into the translational application of HPCD in restoring axonal homeostasis at early stages of NPC disease.
... Cells internalize plasma membrane components and extracellular material, such as receptors, transporters, growth factors and solute molecules, by endocytosis. Endocytic vesicles pinch off from the plasma membrane and deliver their cargoes to early endosomes [31]. From there, they can be channeled into several trafficking routes: They can recycle back to the plasma membrane [32][33][34], move to juxtanuclear endocytic recycling compartments, head for the trans-Golgi network [35], or they can be sorted into multivesicular bodies and degradative lysosomal compartments [36][37][38]. ...
Autophagosome formation requires PROPPIN/WIPI proteins and monophosphorylated phosphoinositides, such as phosphatidylinositol-3-phosphate (PtdIns3P) or PtdIns5P. This process occurs in association with mammalian endosomes, where the PROPPIN WIPI1 has additional, undefined roles in vesicular traffic. To explore whether these functions are interconnected, we dissected routes and subreactions of endosomal trafficking requiring WIPI1. WIPI1 specifically acts in the formation and fission of tubulo-vesicular endosomal transport carriers. This activity supports the PtdIns(3,5)P2-dependent transport of endosomal cargo toward the plasma membrane, Golgi, and lysosomes, suggesting a general role of WIPI1 in endosomal protein exit. Three features differentiate the endosomal and macroautophagic/autophagic activities of WIPI1: phosphoinositide binding site II, the requirement for PtdIns(3,5)P2, and bilayer deformation through a conserved amphipathic α-helix. Their inactivation preserves autophagy but leads to a strong enlargement of endosomes, which accumulate micrometer-long endosomal membrane tubules carrying cargo proteins. WIPI1 thus supports autophagy and protein exit from endosomes by different modes of action. We propose that the type of phosphoinositides occupying its two lipid binding sites, the most unusual feature of PROPPIN/WIPI family proteins, switches between these effector functions.
Abbreviations
EGF: epidermal growth factorEGFR: epidermal growth factor receptorKD: knockdownKO: knockoutPtdIns3P: phosphatidylinositol-3-phosphatePtdIns5P: phosphatidylinositol-5-phosphatePtdIns(3,5)P2: phosphatidylinositol-3,5-bisphosphateTF: transferrinTFRC: transferrin receptorWT: wildtype
... Line profiles of vesicles derived from raw confocal and STED data confirm the improvement in resolution gained by STED. The full width half maxima (FWHM) of the Lorentzian fits suggest a spatial resolution significantly below the diffraction limit ( Table S6), which enabled the fusion with early endosomes and thus vesicle growth (33,34). Co-incubation with the clathrin inhibitor Pitstop2 Division duration (min) div freq (div Fig. S8B and S8C). ...
Targeted imaging and therapy approaches based on novel prostate-specific membrane antigen (PSMA) inhibitors have fundamentally changed the treatment regimen of prostate cancer. However, the exact mechanism of PSMA inhibitor internalization has not yet been studied, and the inhibitors' subcellular fate remains elusive. Here, we investigated the intracellular distribution of peptidomimetic PSMA inhibitors and of PSMA itself by stimulated emission depletion (STED) nanoscopy, applying a novel nonstandard live cell staining protocol. Imaging analysis confirmed PSMA cluster formation at the cell surface of prostate cancer cells and clathrin-dependent endocytosis of PSMA inhibitors. Following the endosomal pathway, PSMA inhibitors accumulated in prostate cancer cells at clinically relevant time points. In contrast with PSMA itself, PSMA inhibitors were found to eventually distribute homogeneously in the cytoplasm, a molecular condition that promises benefits for treatment as cytoplasmic and in particular perinuclear enrichment of the radionuclide carriers may better facilitate the radiation-mediated damage of cancerous cells. This study is the first to reveal the subcellular fate of PSMA/PSMA inhibitor complexes at the nanoscale and aims to inspire the development of new approaches in the field of prostate cancer research, diagnostics, and therapeutics.
Significance
This study uses STED fluorescence microscopy to reveal the subcellular fate of PSMA/PSMA inhibitor complexes near the molecular level, providing insights of great clinical interest and suggestive of advantageous targeted therapies.
... R eceptors a re th e m selv e s regulated an d sw itched off by internalisation a n d dow nregulation (Mellman, 1996) or dephosphorylation, by recep to r a sso c ia te d p h o s p h a ta s e s . This w as initially discovered in Drosophila for receptor tyrosine kinase regulation (corkscrew an d torso (Perkins et al., 1996) or m ore recently, S H P (Lanier, 1998;M yers et al., 1998;R oach et al., 1998;Stein et al., 1998;Yu et al., 1998) ...
Protein phosphorylation plays an essential role in a diverse array of signalling cascades and regulates many cellular processes. Protein kinase Cs (PKCs) constitute one of the families of kinases involved in phosphorylating substrates on serine or threonine residues. These kinases were initially identified as being receptors for tumour promoters (phorbol esters) and the conditions required to activate different isoforms determine the subgroup classification of the 10 isoforms. Classical PKCs (α,β1, βII and γ) depend upon Ca2+ and lipids (DAG, PS- phospholipids.) Novel PKCs (δ, ϵ, η, μ and θ) are insensitive to Ca2+ but are activated by lipids, DAG and phospholipids. The atypical PKCs (ζ, i and λ) differ greatly. These proteins are insensitive to Ca2+ and phorbol ester binding. The lack of knowledge on the control of the atypical PKCs has made the role of the atypical PKCs more elusive. Nevertheless, PKC ζ has been implicated in cell growth and differentiation. Moreover, PKC ζ is thought to be involved in a plethora of signal transduction pathways, including the Ras and MEK/MAPK pathways. The related atypical PKC i may be involved in UV induced apoptosis and insulin signalling. The aims of this thesis are to define the control and biological role of the atypical PKCs - primarily focusing on PKC ζ. As one approach, the project attempted to create a knockout mouse. This would help define a biological end point and therefore permit elucidation of the inputs. This study led to the identification of a pseudogene and its origin is described. As a second approach to investigate PKC ζ control, various direct paths were followed - ranging from searching for potential binding proteins and cellular localisation, to analysis of activation by lipids and phosphorylation. These studies have provided evidence for the dynamic control of PKC ζ (and PKC i) through a kinase cascade involving the lipid kinase PI3-kinase, the lipid responsive PDK1 and finally phosphorylation of PKC ζ at a site defined as threonine 410. The operation of this pathway and its influence on PKC ζ autophosphorylation (in vivo) and activity (in vitro) are presented.
... Endocytosis is the process by which extracellular material is internalised into the cell (Okamoto, 1998) and all eucaryotic cells exhibit one or more forms of endocytosis (reviewed in Mellman, 1996). The term 'endocytosis' has been used to encompass two different mechanisms o f uptake. ...
Water soluble polymers, including natural polymers (e.g. polyamino acids and polysaccharides) and synthetic polymers (e.g. polyethyleneglycol (PEG)) and N-(2- hydroxypropyl)methacrylamide (HPMA) copolymers) are finding increasing use as polymer therapeutics. Dextrin and dextrin-2-Sulphate (D-2-S) are poly(1-4 glucose) polymers that have entered into clinical use: dextrin as a peritoneal dialysis solution and D-2-S as an anti-HIV treatment. The aim of this study was (1) to quantitate the biodistribution of dextrin and D-2-S and (2) to evaluate the potential of these polymers for use as drug carriers. First, pendant groups were introduced by succinoylation (1-60 mol%). To study biodistribution (after s.c., i.v. or i.p. administration) probes were then synthesised containing either -TyrNH2 or -DTPA (~1 mol%) to allow labelling with 125I iodine or 111In indium respectively. After i.p. administration 125I-labelled D-2-S remained longer in the peritoneal cavity (~27 times) than 125I-labelled dextrin (t1/2 = 2.3 h and 5 min respectively). Maximum tissue accumulation was seen in the liver, for 125I-labelled dextrin approximately 20% administered dose (2 min) and for 125I-labelled D-2-S (15.3% administered dose (24 h). Gamma camera images obtained using 111In-labelled polymers were consistent with the data obtained using 125I-labelled compounds. Using the succinoylated intermediate, dextrin- and D-2-S-amphotericin B conjugates (AmpB) were prepared containing 0.01-3.50 wt% and 0.01-16.0 wt% AmpB respectively. Conjugation increased drug solubility approximately 10 fold. Preliminary in vitro testing showed IC50 values for AmpB, dextrin- and D-2-S-AmpB (IC50 of 11.0 >50, and >50 μg/ml respectively) and haemolytic activity at 24 h (Hb50 of 0.06 mg/ml, >50 μg/ml and 8.0 μg/ml respectively). Additionally, experiments were carried out with dextrin-doxorubicin (Dox) (9 wt%, Dox-equiv) to ascertain its tumour targeting potential and antitumour activity. Whereas free Dox was inactive (T/C= 105%) in a s.c. B16F10 tumour model dextrin- Dox had a T/C = 144%. Dextrin and D-2-S have shown that they have potential for further development as water soluble drug carriers.
... Wg is degraded by autophagy following internalization into photoreceptors To elucidate the physiological importance of Wg endocytosis, we sought to identify the pathway that follows endocytosis. The main processes that occur after endocytosis are degradation and recycling pathways (Mellman, 1996;Maxfield and McGraw, 2004). Since autophagy reportedly works in an optical stimulus-dependent manner in Drosophila (Midorikawa et al., 2010;Weiss and Minke, 2015), we investigated the involvement of autophagy. ...
Neural activity contributes to synaptic regulation in sensory systems, which allows organisms to adjust to changing environments. However, little is known about how synaptic molecular components are regulated to achieve activity-dependent plasticity at central synapses. Previous studies have shown that following prolonged exposure to natural ambient light, the presynaptic active zone (AZ), an area associated with presynaptic neurotransmitter release in Drosophila photoreceptors, undergoes reversible remodeling. Other studies suggest that the secretory protein Wingless (Wg; an ortholog of Wnt-1) can mediate communication between synaptic cells to achieve synaptic remodeling. However, the source of Wg and the mechanism of Wg signal modulation by neuronal activity remained unclear. Here, we found that Wg secreted from glial cells regulates synaptic remodeling in photoreceptors. In addition, antibody staining revealed that Wg changes its localization depending on light conditions. Although Wg is secreted from glial cells, Wg appeared inside photoreceptor axons when flies were kept under light conditions, suggesting that an increase in neuronal activity causes Wg internalization into photoreceptors by endocytosis. Indeed, by blocking endocytosis in photoreceptors, the localization of Wg in photoreceptors disappeared. Interestingly, Wg accumulation was higher in axons with disassembled AZ structure than in axons whose AZ structure was stabilized at the single-cell level, indicating that Wg endocytosis may trigger AZ disassembly. Furthermore, when we genetically activated Wg signaling, Wg accumulation in photoreceptors decreased. Conversely, when we suppressed Wg signaling there was an increase in Wg accumulation. Through RNAi screening of Ca²⁺-binding proteins in photoreceptors, we found that Calcineurin is a key molecule that triggers Wg endocytosis. Overall, we propose that Wg signaling is regulated by a negative feedback loop driven by Wg endocytosis. The increase in neuronal activity is transmitted via calcium signaling, which leads to a decrease in Wg signaling and thereby promotes presynaptic remodeling.
MicroRNAs (miRNAs) control 60% of genes expressed in the human body, but their role in malaria pathogenesis is incompletely understood. For the first time, we demonstrate cell type-specific alterations to the miRNA profiles during the early response to malaria infection in brain and lung endothelial cells (ECs). In brain ECs, incubation with Plasmodium falciparum-infected red blood cells in the ring stage (iRBCs) most significantly affected endocytosis-related miRNAs and mRNAs. Contrastingly, in lung ECs, iRBCs altered electron transport chain-related miRNAs and mRNAs. We also present a novel dataset of inherent differences between microRNA profiles in brain and lung ECs and their secreted extracellular vesicles (EVs). We demonstrated that shear stress affected multiple pathways in brain ECs, which were controlled by numerous human miRNAs. Together, these findings demonstrate that host miRNAs respond to parasite exposure; this is accompanied by stimulation of downstream signaling pathways within the ECs. Therefore, we consider miRNAs to be the initial spark (Code of duty) for the early host-parasite interaction events.
In B cells, antigen processing and peptide-antigen (pAg) presentation is essential to ignite high-affinity antibody responses with the help of cognate T cells. B cells efficiently internalize and direct specific antigens for processing and loading onto MHCII. This critical step, which enables pAg presentation, occurs in MHCII compartments (MIICs) which possess the enzymatic machinery for pAg loading on MHCII. The intracellular transport systems that guide antigen and maintain this unique compartment remain enigmatic. Here, we probed the possible functional role of two known endosomal proteins, the Rab family small GTPases Rab7 and Rab9, that are both reported to colocalize with internalized antigen. As compared to Rab9, we found Rab7 to exhibit a higher overlap with antigen and MIIC components. Rab7 also showed a higher association with antigen degradation. The inhibition of Rab7 drastically decreased pAg presentation. Additionally, we detected the strong colocalization of perinuclearly clustered and presumably MIIC-associated antigen with autophagy protein LC3. When we pharmacologically inhibited autophagy, pAg presentation was inhibited. Together, our data promote Rab7 as an important regulator of antigen processing and, considering the previously reported functions of Rab7 in autophagy, this also raises the possibility of the involvement of autophagy-related machinery in this process.
Clathrin-mediated vesicle trafficking plays central roles in the post-Golgi transport. In yeast, AP-1 complex and GGA adaptors (GGAs) are predicted to generate distinct transport vesicles at the TGN, and epsin-related Ent3p/Ent5p act as accessories for these adaptors. Recently, we showed that vesicle transport from the TGN is crucial for yeast Rab5 (Vps21p)-mediated endosome formation, and that Ent3p/5p are crucial for this process, whereas AP-1 and GGAs are dispensable. However, these observations were incompatible with previous studies showing that these adaptors are required for Ent3p/5p recruitment to the TGN, and thus the overall mechanism responsible for regulation of Vps21p activity remains ambiguous. Here we investigated the functional relationships between clathrin adaptors in post-Golgi-mediated Vps21p activation. We were able to show that AP-1 disruption in ent3Δ/5Δ mutant impairs Vps21p-GEF Vps9p transport to the Vps21p compartment, and severely reduces Vps21p activity. Additionally, GGAs, PI4 kinase Pik1p and Rab11 GTPases Ypt31p/32p were found to have partially overlapping functions for recruitment of AP-1 and Ent3p/5p to the TGN. These findings suggest a distinct role of clathrin adaptors for Vps21p activation in the TGN-endosome trafficking pathway.
Endocytic recycling regulates the cell surface receptor composition of the plasma membrane. The surface expression levels of the T cell receptor (TCR), in concert with signal transducing co-receptors, regulate T cell responses, such as proliferation, differentiation, and cytokine production. Altered TCR expression contributes to pro-inflammatory skewing, which is a hallmark of autoimmune diseases, such as systemic lupus erythematosus (SLE), defined by a reduced function of regulatory T cells (Tregs) and the expansion of CD4+ helper T (Th) cells. The ensuing secretion of inflammatory cytokines, such as interferon-γ and interleukin (IL)-4, IL-17, IL-21, and IL-23, trigger autoantibody production and tissue infiltration by cells of the adaptive and innate immune system that induce organ damage. Endocytic recycling influences immunological synapse formation by CD4+ T lymphocytes, signal transduction from crosslinked surface receptors through recruitment of adaptor molecules, intracellular traffic of organelles, and the generation of metabolites to support growth, cytokine production, and epigenetic control of DNA replication and gene expression in the cell nucleus. This review will delineate checkpoints of endosome traffic that can be targeted for therapeutic interventions in autoimmune and other disease conditions.
Fusarium pseudograminearum is one of the major fungal pathogens that cause Fusarium crown rot (FCR) worldwide and can lead to a substantially reduced grain yield and quality. Transcription factors play an important role in regulating growth and pathogenicity in plant pathogens. In this study, we identified a putative Zn(II)2Cys6 fungal-type domain-containing transcription factor and named it FpUme18. The expression of FpUME18 was induced during the infection of wheat by F. pseudograminearum. The ΔFpume18 deletion mutant showed defects in growth, conidial production, and conidial germination. In the responses to the cell wall, salt and oxidative stresses, the ΔFpume18 mutant inhibited the rate of mycelial growth at a higher rate compared with the wild type. The staining of conidia and mycelia with lipophilic dye FM4-64 revealed a delay in endocytosis when FpUME18 was deleted. FpUME18 also positively regulated the expression of phospholipid-related synthesis genes. The deletion of FpUME18 attenuated the pathogenicity of wheat coleoptiles. FpUME18 also participated in the production of the DON toxin by regulating the expression of TRI genes. Collectively, FpUme18 is required for vegetative growth, conidiation, stress response, endocytosis, and full virulence in F. pseudograminearum.
Drug therapy for retinal diseases (e.g., age-related macular degeneration, the leading cause of blindness) is generally performed by invasive intravitreal injection because of poor drug delivery caused by the blood–retinal barrier (BRB). This study aimed to develop a nanocarrier for the non-invasive delivery of small interfering RNA (siRNA) to the posterior segment of the eye (i.e., the retina) by eyedrops. To this end, we prepared a hybrid nanocarrier based on a multifunctional peptide and liposomes, and the composition was optimized. A cytoplasm-responsive stearylated peptide (STR-CH2R4H2C) was used as the multifunctional peptide because of its superior ability to enhance the complexation, cell permeation, and intracellular dynamics of siRNA. By adding STR-CH2R4H2C to the surface of liposomes, intracellular uptake increased regardless of the liposome surface charge. The STR-CH2R4H2C-modified cationic nanocarrier demonstrated significant siRNA transfection efficiency with no cytotoxicity, enhanced siRNA release from endosomes, and effectively suppressed vascular endothelial growth factor expression in rat retinal pigment epithelium cells. The 2.0 mol% STR-CH2R4H2C-modified cationic nanocarrier enhanced intraocular migration into the retina after instillation into rat eyes.
Lysosomes play a central role in cellular homeostasis and alterations in this compartment associate with many diseases. The most studied example is that of lysosomal storage disorders (LSDs), a group of 60+ maladies due to genetic mutations affecting lysosomal components, mostly enzymes. This leads to aberrant intracellular storage of macromolecules, altering normal cell function and causing multiorgan syndromes, often fatal within the first years of life. Several treatment modalities are available for a dozen LSDs, mostly consisting of enzyme replacement therapy (ERT) strategies. Yet, poor biodistribution to main targets such as the central nervous system, musculoskeletal tissue, and others, as well as generation of blocking antibodies and adverse effects hinder effective LSD treatment. Drug delivery systems are being studied to surmount these obstacles, including polymeric constructs and nanoparticles that constitute the focus of this article. We provide an overview of the formulations being tested, the diseases they aim to treat, and the results observed from respective in vitro and in vivo studies. We also discuss the advantages and disadvantages of these strategies, the remaining gaps of knowledge regarding their performance, and important items to consider for their clinical translation. Overall, polymeric nanoconstructs hold considerable promise to advance treatment for LSDs.
Cells have the ability to communicate with their immediate and distant neighbors through the release of extracellular vesicles (EVs). EVs facilitate intercellular signaling through the packaging of specific cargo in all type of cells, and perturbations of EV biogenesis, sorting, release and uptake is the basis of a number of disorders. In this review, we summarize recent advances of the complex roles of the sphingolipid ceramide and lysosomes in the journey of EV biogenesis to uptake.
Red claw crayfish ( Cherax quadricarinatus ) is an economic freshwater shrimp with great commercial potential. However, the suitable stocking density of C. quadricarinatus is still unclear in integrated rice–crayfish farming system. Thus, this study aimed to investigate the effects of stocking density on growth performance, mitophagy, endocytosis and metabolism of C. quadricarinatus. The C. quadricarinatus was reared at low density (LD, 35.73 g/m ² ), middle density (MD, 71.46 g/m ² ) and high density (HD, 107.19 g/m ² ) in an integrated rice–crayfish farming system. After 90 days of farming, the growth performance of C. quadricarinatus significantly decreased in the MD and HD groups relative to that in the LD group. The HD treatment caused oxidative stress and lipid peroxidation at the end of the experiment in hepatopancreas. Transcriptome analysis showed that there were 1,531 DEGs (differently expressed genes) between the LD group and HD group, including 1,028 upregulated genes and 503 downregulated genes. KEGG (Kyoto Encyclopedia of Genes and Genomes) enrichment analysis indicated that the DEGs were significantly enriched in endocytosis and mitophagy pathways. Meanwhile, four lipid metabolism pathways, including biosynthesis of unsaturated fatty acids, fatty acid biosynthesis, glycerolipid metabolism and glycerophospholipid metabolism, exhibited an upregulated tendency in the HD group. In conclusion, our data showed that when the stocking density reached up to 207.15 g/m ² in HD group, the growth performance of C. quadricarinatus was significantly inhibited in this system. Meanwhile, the data indicated that C. quadricarinatus may respond to the stressful condition via activating antioxidant defense system, endocytosis, mitophagy and metabolism-related pathways in hepatopancreas.
Signaling through the platelet-derived growth factor receptors (PDGFRs) plays a critical role in multiple cellular processes during development. The two PDGFRs, PDGFRα and PDGFRβ, dimerize to form homodimers and/or heterodimers. Here, we overcome previous limitations in studying PDGFR dimer-specific dynamics by generating cell lines stably expressing C-terminal fusions of each PDGFR with bimolecular fluorescence complementation (BiFC) fragments corresponding to the N-terminal or C-terminal regions of the Venus fluorescent protein. We find that PDGFRβ receptors homodimerize more quickly than PDGFRα receptors in response to PDGF ligand, with increased levels of autophosphorylation. Furthermore, we demonstrate that PDGFRα homodimers are trafficked and degraded more quickly, whereas PDGFRβ homodimers are more likely to be recycled back to the cell membrane. We show that PDGFRβ homodimer activation results in a greater amplitude of phospho-ERK1/2 and phospho-AKT signaling, as well as increased proliferation and migration. Finally, we demonstrate that inhibition of clathrin-mediated endocytosis leads to changes in cellular trafficking and downstream signaling, particularly for PDGFRα homodimers. Collectively, our findings provide significant insight into how biological specificity is introduced to generate unique responses downstream of PDGFR engagement.
This article has an associated First Person interview with the first author of the paper.
Early endosomes is one of the main compartments of endocytic pathway. Its vesicular nature causes the discussions regarding the biogenesis of early endosomes and the mechanisms of their functioning. Advances in the study of the early endosomal molecular mechanisms have led to a significant convergence of different points of view. This article discusses the main problems and concepts associated with early endosomes both in the historical aspect and the present state of arts in this field.
Ca²⁺ signalling is of prime importance in controlling numerous cell functions in the brain. Endolysosomes are acidic organelles currently emerging as important Ca²⁺ stores in astrocytes, microglia, endothelial cells, and neurons. These acidic Ca²⁺ stores in neurons are found in axons, soma, dendrites, and axon endings and could provide local sources of Ca²⁺ to control synaptic transmission, neuronal plasticity, and autophagy to name a few. This review will address how acidic Ca²⁺ stores are recruited in response to cell stimulation. We will focus on the role of the endolysosomal two-pore channels (TPCs) and their physiological agonist nicotinic acid adenine dinucleotide phosphate (NAADP) and how they interact with cyclic ADP-ribose and ryanodine receptors from the endoplasmic reticulum. Finally, this review will describe new pharmacological tools and animal mutant models now available to explore acidic Ca²⁺ stores as key elements in brain function and dysfunction.
Ticks are blood-sucking arthropods that have developed myriad of strategies to get a blood meal from the vertebrate host. They first attach to the host skin, select a bite site for a blood meal, create a feeding niche at the bite site, secrete plethora of molecules in its saliva and then starts feeding. On the other side, host defenses will try to counter-attack and stop tick feeding at the bite site. In this constant battle between ticks and the host, arthropods successfully pacify the host and completes a blood meal and then replete after full engorgement. In this review, we discuss some of the known and emerging roles for arthropod components such as cement, salivary proteins, lipocalins, HSP70s, OATPs, and extracellular vesicles/exosomes in facilitating successful blood feeding from ticks. In addition, we discuss how tick-borne pathogens modulate(s) these components to infect the vertebrate host. Understanding the biology of arthropod blood feeding and molecular interactions at the tick-host interface during pathogen transmission is very important. This information would eventually lead us in the identification of candidates for the development of transmission-blocking vaccines to prevent diseases caused by medically important vector-borne pathogens.
Early endosomes (EEs) are part of the endocytic transport pathway and resemble the earliest class of transport vesicles between the internalization of extracellular material, their cellular distribution or vacuolar degradation. In filamentous fungi, EEs fulfill important functions in long distance transport of cargoes as mRNAs, ribosomes and peroxisomes. Formation and maturation of early endosomes is controlled by the specific membrane-bound Rab-GTPase Rab5 and tethering complexes as CORVET (class C core vacuole/endosome tethering). In the basidiomycete Ustilago maydis, Rab5a is the prominent GTPase to recruit CORVET to EEs; in rab5a deletion strains, this function is maintained by the second EE-associated GTPase Rab5b. The tethering- and core- subunits of CORVET are essential, buttressing a central role for EE transport in U. maydis. The function of EEs in long distance transport is supported by the Nma1 protein that interacts with the Vps3 subunit of CORVET. The interaction stabilizes the binding of Vps3 to the CORVET core complex that is recruited to Rab5a via Vps8. Deletion of nma1 leads to a significantly reduced number of EEs, and an increased conversion rate of EEs to late endosomes. Thus, Nma1 modulates the lifespan of EEs to ensure their availability for the various long distance transport processes.
Membrane contact sites (MCSs) serve as a zone for nonvesicular lipid transport by oxysterol-binding protein (OSBP)-related proteins (ORPs). ORPs mediate lipid countertransport, in which two distinct lipids are transported counterdirectionally. How such lipid countertransport controls specific biological functions, however, remains elusive. We report that lipid countertransport by ORP10 at ER–endosome MCSs regulates retrograde membrane trafficking. ORP10, together with ORP9 and VAP, formed ER–endosome MCSs in a phosphatidylinositol 4-phosphate (PI4P)-dependent manner. ORP10 exhibited a lipid exchange activity toward its ligands, PI4P and phosphatidylserine (PS), between liposomes in vitro, and between the ER and endosomes in situ. Cell biological analysis demonstrated that ORP10 supplies a pool of PS from the ER, in exchange for PI4P, to endosomes where the PS-binding protein EHD1 is recruited to facilitate endosome fission. Our study highlights a novel lipid exchange at ER–endosome MCSs as a nonenzymatic PI4P-to-PS conversion mechanism that organizes membrane remodeling during retrograde membrane trafficking.
Exosomes are secreted from a variety of cells and transmit parental cell-derived biomolecules, such as nucleic acids and proteins, to recipient cells in distant organs. In addition to their important roles in both physiological and pathological conditions, exosomes are expected to serve as natural drug carriers without any cytotoxicity, immunogenicity, or tumorigenicity. However, the use of exosomes as drug delivery tools is limited due to the low uptake efficiency of the target cells, insufficient release of the contents from the endosome to the cytosol, and possible adverse effects caused by the delivery to non-target cells. In the present study, we examined the effects of the modification of exosomes with carbonate apatite or a lactose-carrying polymer. Using newly generated monitoring exosomes that contain either firefly luciferase or fused mCherry/enhanced green fluorescent protein, we demonstrated that the modification of exosomes with carbonate apatite improved their release from the endosome into the cytosol in recipient cells. Meanwhile, the modification of exosomes with a lactose-carrying polymer enhanced the selective delivery to parenchymal hepatocytes. These modified exosomes may provide an efficient strategy for macromolecule therapy for incurable diseases that cannot be treated with conventional small-molecule compounds.
With recent advances in nanotechnology and therapeutic nucleic acids (TNAs), various nucleic acid nanoparticles (NANPs) have demonstrated great promise in diagnostics and therapeutics. However, the full realization of NANPs’ potential necessitates the development of a safe, efficient, biocompatible, stable, tissue-specific, and non-immunogenic delivery system. Exosomes, the smallest extracellular vesicles and an endogenous source of nanocarriers, offer these advantages while avoiding complications associated with manufactured agents. The lipid membranes of exosomes surround a hydrophilic core, allowing for the simultaneous incorporation of hydrophobic and hydrophilic drugs, nucleic acids, and proteins. Additional capabilities for post-isolation exosome surface modifications with imaging agents, targeting ligands, and covalent linkages also pave the way for their diverse biomedical applications. This review focuses on exosomes: their biogenesis, intracellular trafficking, transportation capacities, and applications with emphasis on the delivery of TNAs and programmable NANPs. We also highlight some of the current challenges and discuss opportunities related to the development of therapeutic exosome-based formulations and their clinical translation.
The objective of this study was to report that lysosome extracted from egg white could be used as a drug through oral administration for treating diseases by using pH sensitive alginate oligosaccharides. Lysosome-alginate oligosaccharides composite were formulated for oral administration of lysosomes. The dissolution test confirmed the availability of the oral dosage form. When lysosome were used as an independent drug, the activity of protein was lost due to influence of low pH. Its antibacterial activity was also remarkably reduced. However, when lysosome-alginate oligosaccharides composite form was used, antimicrobial activity of lysozyme was maintained. At low pH, a gel-like matrix was formed by alginate oligosaccharides to protect the lysosome. When the pH was increased, alginate oligosaccharides were dissolved and the lysosome was released. SDS–polyacrylamide gel electrophoresis analysis of released lysosomes revealed that alginate oligosaccharide could effectively protect the lysosome from degradation or hydrolysis under acidic conditions for at least 2 h. The results of this study are important for application of lysosomes as therapeutic agents, and also it was confirmed that alginate oligosaccharides have potential as direct delivery system for the oral application of protein derived therapies.
Nephrotic syndrome is in adult patients mainly due to membranous nephropathy (MN) characterized by thickening of the glomerular basement membrane (GBM) and immune complex formation between podocytes and the GBM. Autoantibodies directed against the M-type phospholipase A2 receptor (PLA2R) and thrombospondin 1 domain-containing 7 A (THSD7A) can be used as diagnostic biomarkers. THSD7A seems to be of direct pathogenic significance as is suggested by experimental models and plasmapheresis in humans. Recently, further antigens like NELL-1 (neural tissue encoding protein with EGF-like repeats-1), exostosin 1 and 2 have been discovered. Thus, MN should be classified into antibody positive and antibody negative MN. More specific immunosuppressive treatments directed against B-cells and antibody production like rituximab have been introduced in addition to already existing immunosuppressive protocols including steroids, chlorambucil, cyclophosphamide, and calcineurin inhibitors. Antibody removal using immunoadsorption or plasmapheresis leads to short-term reduction in proteinuria and might be indicated only in patients with very severe proteinuria and complications. Studies are needed to identify a more specific immunosuppression directed against the production and effects of autoantibodies in order to protect the kidneys from autoimmune mediated tissue damage and to identify patients who require an immunosuppressive treatment, as the remission rate is high in patients with MN.
Delivering anticancer drugs to tumor tissues with high specificity to realize minimal adverse effects is currently the main research focus of cancer treatment. Nε-acyl lysine methyl esters (Nε-lys) have the potential to serve as novel and safe anticancer agents owing to their pH-responsive membrane-disruptive activity and non-toxic metabolic products. In this study, we synthesized three Nε-lys with different hydrophobic chain lengths and evaluated their cytotoxicity to cancer and normal cells. One of the compounds with an acyl chain of 14 carbon atoms (MKM) showed moderate cytotoxicity and was delivered to cancer cells encapsulated in liposomes (MKM-LP). We additionally modified the liposomes with folate and PEG as a cancer-targeting drug delivery system (FA-PEG-MKM-LP) to enhance the cell selectivity. We tested the difference in the cellular uptake of liposomes between a folate receptor-positive human ovarian cancer cell line (SKOV3) and a healthy human ovary epithelial cell line (IOSE). The experimental results showed that FA-PEG-MKM-LP had low cytotoxicity to IOSE and higher cancer-targeting ability than other tested liposomes. Fluorescence imaging of dead cells and a LDH release assay further demonstrated that the cytotoxicity of FA-PEG-MKM-LP possessed time- and dose-dependent activity and specificity to cancer cells. These results lay a foundation for the development of Nε-lys as a promising anticancer drug.
Oral cancer constitutes approximately 2% of all cancers, while the most common type, oral squamous cell carcinoma (OSCC) represents 90% of oral cancers. Although the treatment of OSCC has improved recently, it still has a high rate of local recurrence and poor prognosis, with a 5-year survival rate of only 50%. Advanced stage OSCC tends to metastasize to lymph nodes. Thus, exploring new therapeutic strategies for OSCC is therefore an urgent priority. Exosomes, the small membrane vesicles derived from endosomes, have been detected in a wide array of bodily fluids. Exosomes contain a diversity of proteins, mRNAs, and non-coding RNAs, including microRNAs, long non-coding RNAs, piRNAs, circular RNAs, tsRNAs, and ribosomal RNAs, which are delivered to neighboring cells or even transported to distant sites. Exosomes have been associated with the tumorigenesis of OSCC, promote the proliferation, colonization, and metastasis of OSCC by transferring their contents to the target cells. Furthermore, exosomes are involved in the regulation of the tumor microenvironment to transform conditions favoring cancer progression in vivo. In this review, we summarize the crucial role of exosomes in the tumorigenesis and progression of OSCC and discuss the potential clinical application of exosomes in OSCC treatment.
The rapid outbreak of coronavirus disease 2019 (COVID-19) has demonstrated the need for development of new vaccine candidates and therapeutic drugs to fight against the underlying virus, severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2). Currently, no antiviral treatment is available to treat COVID-19 as treatment is mostly directed to only relieving the symptoms. Retrospectively, herbal medicinal plants have been used for thousands of years as a medicinal alternative including for the treatment of various viral illnesses. However, a comprehensive description using various medicinal plants in treating coronavirus infection has not to date been described adequately, especially their modes of action. Most other reports and reviews have also only focused on selected ethnobotanical herbs such as Traditional Chinese Medicine, yet more plants can be considered to enrich the source of the anti-viral compounds. In this review, we have screened and identified potential herbal medicinal plants as anti-coronavirus medication across major literature databases without being limited to any regions or ethnobotanic criteria. As such we have successfully gathered experimentally validated in vivo, in vitro, or in silico findings of more than 30 plants in which these plant extracts or their related compounds, such as those of Artemisia annua L., Houttuynia cordata Thunb., and Sambucus formosana Nakai, are described through their respective modes of action against specific mechanisms or pathways during the viral infection. This includes inhibition of viral attachment and penetration, inhibition of viral RNA and protein synthesis, inhibition of viral key proteins such as 3-chymotrypsin-like cysteine protease (3CLpro) and papain-like protease 2 (PLpro), as well as other mechanisms including inhibition of the viral release and enhanced host immunity. We hope this compilation will help researchers and clinicians to identify the source of appropriate anti-viral drugs from plants in combating COVID-19 and, ultimately, save millions of affected human lives.
Eukaryotic cells employ diverse uptake mechanisms depending on their specialized functions. While such mechanisms vary widely in their defining criteria: scale, molecular machinery utilized, cargo selection, and cargo destination, to name a few, they all result in the internalization of extracellular solutes and fluid into membrane-bound endosomes. Upon scission from the plasma membrane, this compartment is immediately subjected to extensive remodeling which involves tubulation and vesiculation/budding of the limiting endomembrane. This is followed by a maturation process involving concomitant retrograde transport by microtubule-based motors and graded fusion with late endosomes and lysosomes, organelles that support the degradation of the internalized content. Here we review an important determinant for sorting and trafficking in early endosomes and in lysosomes; the control of tension on the endomembrane. Remodeling of endomembranes is opposed by high tension (caused by high hydrostatic pressure) and supported by the relief of tension. We describe how the timely and coordinated efflux of major solutes along the endocytic pathway affords the cell control over such tension. The channels and transporters that expel the smallest components of the ingested medium from the early endocytic fluid are described in detail as these systems are thought to enable endomembrane deformation by curvature-sensing/generating coat proteins. We also review similar considerations for the lysosome where resident hydrolases liberate building blocks from luminal macromolecules and transporters flux these organic solutes to orchestrate trafficking events. How the cell directs organellar trafficking based on the luminal contents of organelles of the endocytic pathway is not well-understood, however, we propose that the control over membrane tension by solute transport constitutes one means for this to ensue.
Extracellular vesicles, phospholipid bilayer-membrane vesicles of cellular origin, are emerging as nanocarriers of biological information between cells. Extracellular vesicles transport virtually all biologically active macromolecules (e.g., nucleotides, lipids, and proteins), thus eliciting phenotypic changes in recipient cells. However, we only partially understand the cellular mechanisms driving the encounter of a soluble ligand transported in the lumen of extracellular vesicles with its cytosolic receptor: a step required to evoke a biologically relevant response. In this context, we review herein current evidence supporting the role of two well-described cellular transport pathways: the endocytic pathway as the main entry route for extracellular vesicles and the autophagic pathway driving lysosomal degradation of cytosolic proteins. The interplay between these pathways may result in the target engagement between an extracellular vesicle cargo protein and its cytosolic target within the acidic compartments of the cell. This mechanism of cell-to-cell communication may well own possible implications in the pathogenesis of neurodegenerative disorders.
Vacuolar protein sorting 45 homolog (VPS45), a member of the Sec1/Munc18 (SM) family, has been implicated in the regulation of endosomal trafficking. VPS45 deficiency in human patients results in congenital neutropenia, bone marrow fibrosis, and extramedullary renal hematopoiesis. Detailed mechanisms of the VPS45 function are unknown. Here, we show an essential role of mammalian VPS45 in maintaining the intracellular organization of endolysosomal vesicles and promoting recycling of cell-surface receptors. Loss of VPS45 causes defective Rab5-to-Rab7 conversion resulting in trapping of cargos in early endosomes and impaired delivery to lysosomes. In this context, we demonstrate aberrant trafficking of the G-CSF receptor (G-CSFR) in the absence of VPS45. Furthermore, we find that lack of VPS45 in mice is not compatible with embryonic development. Thus, we identify mammalian VPS45 as a critical regulator of trafficking through the endosomal system and early embryogenesis of mice.
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