Understanding amphisomes

2021 ◽  
Vol 478 (10) ◽  
pp. 1959-1976
Author(s):  
Dhasarathan Ganesan ◽  
Qian Cai
Keyword(s):  
Retrograde Transport ◽  
Autophagic Flux ◽  
Rab Gtpases ◽  
Cellular Mechanisms ◽  
Fusion Event ◽  
Late Endosomes ◽  
Multiple Protein ◽  
Unconventional Secretion ◽  
Recent Developments ◽  
Neurotrophic Signaling

Amphisomes are intermediate/hybrid organelles produced through the fusion of endosomes with autophagosomes within cells. Amphisome formation is an essential step during a sequential maturation process of autophagosomes before their ultimate fusion with lysosomes for cargo degradation. This process is highly regulated with multiple protein machineries, such as SNAREs, Rab GTPases, tethering complexes, and ESCRTs, are involved to facilitate autophagic flux to proceed. In neurons, autophagosomes are robustly generated in axonal terminals and then rapidly fuse with late endosomes to form amphisomes. This fusion event allows newly generated autophagosomes to gain retrograde transport motility and move toward the soma, where proteolytically active lysosomes are predominantly located. Amphisomes are not only the products of autophagosome maturation but also the intersection of the autophagy and endo-lysosomal pathways. Importantly, amphisomes can also participate in non-canonical functions, such as retrograde neurotrophic signaling or autophagy-based unconventional secretion by fusion with the plasma membrane. In this review, we provide an updated overview of the recent discoveries and advancements on the molecular and cellular mechanisms underlying amphisome biogenesis and the emerging roles of amphisomes. We discuss recent developments towards the understanding of amphisome regulation as well as the implications in the context of major neurodegenerative diseases, with a comparative focus on Alzheimer's disease and Parkinson's disease.

Evidence for retrograde traffic between terminal lysosomes and the prelysosomal/late endosome compartment

1994 ◽  
Vol 107 (1) ◽  
pp. 145-157 ◽  
Author(s):  
A. Jahraus ◽  
B. Storrie ◽  
G. Griffiths ◽  
M. Desjardins
Keyword(s):  
Light Microscope ◽  
Retrograde Transport ◽  
Late Endosome ◽  
Endocytic Pathway ◽  
Fusion Event ◽  
Microtubule Network ◽  
Cell Biol ◽  
Late Endosomes ◽  
Latex Beads ◽  
Retrograde Traffic

We have investigated the interactions occurring between the prelysosomal compartment, PLC/late endosome, and terminal lysosomes using an approach that allowed us to internalize and deliver specific tracers to these compartments, and look for evidence of their meeting. After internalization of sucrose, the lysosomes (sucrosomes), but not the PLC/late endosomes, became significantly swollen and visible in the light microscope. If invertase is then added to the medium it reaches the lysosomes where it cleaves sucrose into fructose and glucose. These sugars, unlike sucrose, can be transported into the cytosol, resulting in the disappearance of the sucrosomes. We previously showed that phagocytosed latex beads are delivered specifically to, and reside in, the PLC/late endosome, a stage earlier than the lysosomes in the endocytic pathway (Rabinowitz et al. (1992) J. Cell Biol. 116, 95–112). In the present study, we demonstrate that invertase conjugated to the latex beads, and thus immobilized in late endosomes, has access to the sucrose present in the more distal lysosomes. Experiments using nocodazole indicate that this retrograde fusion event requires the presence of an intact microtubule network. The simplest interpretation of our results is that the two compartments fuse, allowing for a retrograde transport of sucrose from the lysosomes to the PLC/late endosomes.

scholarly journals Targeting the sAC-Dependent cAMP Pool to Prevent SARS-Cov-2 Infection

Cells ◽  
2020 ◽  
Vol 9 (9) ◽  
pp. 1962
Author(s):  
Muhammad Aslam ◽  
Yury Ladilov
Keyword(s):  
Endocytic Pathway ◽  
Host Cells ◽  
The Novel ◽  
Cellular Mechanisms ◽  
Late Endosomes ◽  
Proven Efficacy ◽  
Potential Impact ◽  
Potential Strategy ◽  
Novel Coronavirus ◽  
High Level

An outbreak of the novel coronavirus (CoV) SARS-CoV-2, the causative agent of COVID-19 respiratory disease, infected millions of people since the end of 2019, led to high-level morbidity and mortality and caused worldwide social and economic disruption. There are currently no antiviral drugs available with proven efficacy or vaccines for its prevention. An understanding of the underlying cellular mechanisms involved in virus replication is essential for repurposing the existing drugs and/or the discovery of new ones. Endocytosis is the important mechanism of entry of CoVs into host cells. Endosomal maturation followed by the fusion with lysosomes are crucial events in endocytosis. Late endosomes and lysosomes are characterized by their acidic pH, which is generated by a proton transporter V-ATPase and required for virus entry via endocytic pathway. The cytoplasmic cAMP pool produced by soluble adenylyl cyclase (sAC) promotes V-ATPase recruitment to endosomes/lysosomes and thus their acidification. In this review, we discuss targeting the sAC-specific cAMP pool as a potential strategy to impair the endocytic entry of the SARS-CoV-2 into the host cell. Furthermore, we consider the potential impact of sAC inhibition on CoV-induced disease via modulation of autophagy and apoptosis.

scholarly journals The yeast Batten disease orthologue Btn1 controls endosome–Golgi retrograde transport via SNARE assembly

2011 ◽  
Vol 195 (2) ◽  
pp. 203-215 ◽  
Author(s):  
Rachel Kama ◽  
Vydehi Kanneganti ◽  
Christian Ungermann ◽  
Jeffrey E. Gerst
Keyword(s):  
Disease Gene ◽  
Transmembrane Domain ◽  
Batten Disease ◽  
Retrograde Transport ◽  
Attachment Protein ◽  
Protein Receptor ◽  
Late Endosomes ◽  
Target Membrane ◽  
Opposing Effects ◽  
Protein Attachment

The human Batten disease gene CLN3 and yeast orthologue BTN1 encode proteins of unclear function. We show that the loss of BTN1 phenocopies that of BTN2, which encodes a retromer accessory protein involved in the retrieval of specific cargo from late endosomes (LEs) to the Golgi. However, Btn1 localizes to Golgi and regulates soluble N-ethyl-maleimide sensitive fusion protein attachment protein receptor (SNARE) function to control retrograde transport. Specifically, BTN1 overexpression and deletion have opposing effects on phosphorylation of the Sed5 target membrane SNARE, on Golgi SNARE assembly, and on Golgi integrity. Although Btn1 does not interact physically with SNAREs, it regulates Sed5 phosphorylation by modulating Yck3, a palmitoylated endosomal kinase. This may involve modification of the Yck3 lipid anchor, as substitution with a transmembrane domain suppresses the deletion of BTN1 and restores trafficking. Correspondingly, deletion of YCK3 mimics that of BTN1 or BTN2 with respect to LE–Golgi retrieval. Thus, Btn1 controls retrograde sorting by regulating SNARE phosphorylation and assembly, a process that may be adversely affected in Batten Disease patients.

scholarly journals Herpes simplex virus entry by a non-conventional endocytic pathway

2020 ◽  
Author(s):  
Giulia Tebaldi ◽  
Suzanne M. Pritchard ◽  
Anthony V. Nicola
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Epithelial Cells ◽  
Retrograde Transport ◽  
Endocytic Pathway ◽  
Host Cells ◽  
Rab Gtpases ◽  
Transport Pathway ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACTHerpes simplex virus 1 (HSV-1) causes significant morbidity and mortality in humans worldwide. HSV-1 enters epithelial cells via an endocytosis mechanism that is low pH-dependent. However, the precise intracellular pathway has not been identified, including the compartment where fusion occurs. In this study, we utilized a combination of molecular and pharmacological approaches to better characterize HSV entry by endocytosis. HSV-1 entry was unaltered in both cells treated with siRNA to Rab5 or Rab7 and cells expressing dominant-negative forms of these GTPases, suggesting entry is independent of the conventional endo-lysosomal network. The fungal metabolite brefeldin A (BFA) and the quinoline compound Golgicide A (GCA) inhibited HSV-1 entry via beta-galactosidase reporter assay and impaired incoming virus transport to the nuclear periphery, suggesting a role for trans Golgi network (TGN) functions and retrograde transport in HSV entry. Silencing of Rab9 or Rab11 GTPases, which are involved in the retrograde transport pathway, resulted in only a slight reduction in HSV infection. Together these results suggest that HSV enters host cells by an intracellular route independent of the lysosome-terminal endocytic pathway.IMPORTANCEHSV-1, the prototype alphaherpesvirus, is ubiquitous in the human population and causes lifelong infection that can be fatal in neonatal and immunocompromised individuals. HSV enters many cell types by endocytosis, including epithelial cells, the site of primary infection in the host. The intracellular itinerary for HSV entry remains unclear. We probed the potential involvement of several Rab GTPases in HSV-1 entry, and suggest that endocytic entry of HSV-1 is independent of the canonical lysosome-terminal pathway. A non-traditional endocytic route may be employed, such as one that intersects with the TGN. These results may lead to novel targets for intervention.

scholarly journals FgRab5 and FgRab7 are essential for endosomes biogenesis and non-redundantly recruit the retromer complex to the endosomes in Fusarium graminearum

2021 ◽  
Vol 1 (1) ◽  
Author(s):  
Yakubu Saddeeq Abubakar ◽  
Han Qiu ◽  
Wenqin Fang ◽  
Huawei Zheng ◽  
Guodong Lu ◽  
...  
Keyword(s):  
Fusarium Graminearum ◽  
Retrograde Transport ◽  
Small Gtpases ◽  
Rab Proteins ◽  
Rab Gtpases ◽  
Wild Type ◽  
Retromer Complex ◽  
Cargo Proteins ◽  
Endosomal Membranes ◽  
In The Wild

AbstractThe retromer complex, composed of the cargo-selective complex (CSC) Vps35-Vps29-Vps26 in complex with the sorting nexin dimer Vps5-Vps17, mediates the sorting and retrograde transport of cargo proteins from the endosomes to the trans-Golgi network in eukaryotic cells. Rab proteins belong to the Ras superfamily of small GTPases and regulate many trafficking events including vesicle formation, budding, transport, tethering, docking and fusion with target membranes. Herein, we investigated the potential functional relationship between the retromer complex and the 11 Rab proteins that exist in Fusarium graminearum using genetic and high-resolution laser confocal microscopic approaches. We found that only FgRab5 (FgRab5A and FgRab5B) and FgRab7 associate with the retromer complex. Both FgVps35-GFP and FgVps17-GFP are mis-localized and appear diffused in the cytoplasm of ΔFgrab5A, ΔFgrab5B and ΔFgrab7 mutants as compared to their punctate localization within the endosomes of the wild-type. FgRab7 and FgRab5B were found to co-localize with the retromer on endosomal membranes. Most strikingly, we found that these three Rab GTPases are indispensable for endosome biogenesis as both early and late endosomes could not be detected in the cells of the mutants after FM4-64 staining of the cells, while they were very clearly seen in the wild-type PH-1. Furthermore, FgRab7 was found to recruit FgVps35 but not FgVps17 to the endosomal membranes, whereas FgRab5B recruits both FgVps35 and FgVps17 to the membranes. Thus, we conclude that the Rab proteins FgRab5A, FgRab5B and FgRab7 play critical roles in the biogenesis of endosomes and in regulating retromer-mediated trafficking in F. graminearum.

scholarly journals Generation of GLA-Knockout Human Embryonic Stem Cell Lines to Model Autophagic Dysfunction and Exosome Secretion in Fabry Disease-Associated Hypertrophic Cardiomyopathy

Cells ◽  
2019 ◽  
Vol 8 (4) ◽  
pp. 327 ◽  
Author(s):  
Song ◽  
Chien ◽  
Yarmishyn ◽  
Chou ◽  
Yang ◽  
...  
Keyword(s):  
Hypertrophic Cardiomyopathy ◽  
Fabry Disease ◽  
Disease Model ◽  
Embryonic Stem ◽  
Treatment Strategies ◽  
Autophagic Flux ◽  
Rab Gtpases ◽  
Pharmacological Chaperone ◽  
Enzyme Replacement ◽  
Wide Range

Fabry disease (FD) is a rare inherited disorder characterized by a wide range of systemic symptoms; it is particularly associated with cardiovascular and renal problems. Enzyme replacement therapy and pharmacological chaperone migalastat are the only approved and effective treatment strategies for FD patients. It is well documented that alpha-galactosidase A (GLA) enzyme activity deficiency causes globotriaosylceramide (Gb3) accumulation, which plays a crucial role in the etiology of FD. However, the detailed mechanisms remain unclear, and the lack of a reliable and powerful disease model is an obstacle. In this study, we created such a model by using CRISPR/Cas9-mediated editing of GLA gene to knockout its expression in human embryonic stem cells (hESCs). The cardiomyocytes differentiated from these hESCs (GLA-null CMs) were characterized by the accumulation of Gb3 and significant increases of cell surface area, the landmarks of FD-associated cardiomyopathy. Furthermore, we used mass spectrometry to compare the proteomes of GLA-null CMs and parental wild type CMs and found that the Rab GTPases involved in exocytotic vesicle release were significantly downregulated. This caused impairment of autophagic flux and protein turnover, resulting in an increase of reactive oxygen species and apoptosis. To summarize, we established a FD model which can be used as a promising tool to study human hypertrophic cardiomyopathy in a physiologically and pathologically relevant manner and to develop new therapies by targeting Rab GTPases signaling-related exosomal vesicles transportation.

scholarly journals Highlights and recent developments in allergic diseases in EAACI journals (2019)

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
J. Bousquet ◽  
C. E. Grattan ◽  
C. A. Akdis ◽  
P. A. Eigenmann ◽  
K. Hoffmann-Sommergruber ◽  
...  
Keyword(s):  
Critical Role ◽  
Allergic Diseases ◽  
New Drugs ◽  
Cellular Mechanisms ◽  
Pediatric Allergy ◽  
Allergy And Immunology ◽  
Recent Developments ◽  
Translational Allergy ◽  
Substantial Progress ◽  
Meta Analyses

AbstractThe European Academy of Allergy and Clinical Immunology (EAACI) owns three journals: Allergy, Pediatric Allergy and Immunology and Clinical and Translational Allergy. One of the major goals of EAACI is to support health promotion in which prevention of allergy and asthma plays a critical role and to disseminate the knowledge of allergy to all stakeholders including the EAACI junior members. There was substantial progress in 2019 in the identification of basic mechanisms of allergic and respiratory disease and the translation of these mechanisms into clinics. Better understanding of molecular and cellular mechanisms, efforts for the development of biomarkers for disease prediction, novel prevention and intervention studies, elucidation of mechanisms of multimorbidities, entrance of new drugs in the clinics as well as recently completed phase three clinical studies and publication of a large number of allergen immunotherapy studies and meta-analyses have been the highlights of the last year.

Endocytosis and retrograde axonal traffic in motor neurons.

2005 ◽  
Vol 72 ◽  
pp. 139-150 ◽  
Author(s):  
Katrin Deinhardt ◽  
Giampietro Schiavo
Keyword(s):  
Spinal Cord ◽  
Axonal Transport ◽  
Motor Neurons ◽  
Molecular Basis ◽  
Retrograde Transport ◽  
Membrane Dynamics ◽  
Transport Pathway ◽  
Precise Control ◽  
Recent Developments ◽  
Botulinum Neurotoxins

Spinal cord motor neurons control voluntary movement by relaying messages that arrive from upper brain centres to the innervated muscles. Despite the importance of motor neurons in human health and disease, the precise control of their membrane dynamics and its effect on motor neuron homoeostasis and survival are poorly understood. In particular, the molecular basis of the co-ordination of specific endocytic events with the axonal retrograde transport pathway is largely unknown. To study these important vesicular trafficking events, we pioneered the use of atoxic fragments of tetanus and botulinum neurotoxins to follow endocytosis and retrograde axonal transport in motor neurons. These neurotoxins bind specifically to pre-synaptic nerve terminals, where they are internalized. Whereas botulinum neurotoxins remain at the neuromuscular junction, tetanus toxin is retrogradely transported along the axon to the cell body, where it is released into the intersynaptic space and is internalized by adjacent inhibitory interneurons. The high neurospecificity and the differential intracellular sorting make tetanus and botulinum neurotoxins ideal tools to study neuronal physiology. In the present review, we discuss recent developments in our understanding of the internalization and trafficking of these molecules in spinal cord motor neurons. Furthermore, we describe the development of a reliable transfection method for motor neurons based on microinjection, which will be extremely useful for dissecting further the molecular basis of membrane dynamics and axonal transport in these cells.

Abstract TP478: Mechanisms of Brain Endothelial Erythrophagocytosis: Insights Into the Pathogenesis of Cerebral Microbleeds

Stroke ◽  
2020 ◽  
Vol 51 (Suppl_1) ◽  
Author(s):  
Jiahong Sun ◽  
Prema Vyas ◽  
Samar Mann ◽  
Sriyansh Yarlagadda ◽  
Mark J Fisher ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Vitamin C ◽  
Annexin V ◽  
Cerebral Microbleeds ◽  
Endosomal Trafficking ◽  
Brain Endothelial Cells ◽  
Brain Endothelium ◽  
Cellular Mechanisms ◽  
Microvascular Endothelium ◽  
Late Endosomes

Background: Cerebral microbleeds, MRI signatures of cerebral microhemorrhage, are thought to arise from brain microvessel disruption. Our prior work suggests an alternate origin for some microbleeds, emphasizing brain endothelial erythrophagocytosis (BEE) producing microhemorrhage-like lesions (“pseudo-microbleeds”) in absence of vascular disruption (Frontiers Cell Neurosci 9-18; PMID: 30237761). We demonstrated an erythrophagocytic phenotype of brain microvascular endothelium for oxidatively-stressed red blood cells (RBC), and migration of iron-rich hemoglobin across the brain endothelium. However, cellular mechanisms underlying BEE and hemoglobin passage across brain endothelium are not defined. Herein we investigated a) role of oxidative stress and RBC phosphatidylserine (PS) exposure in BEE and b) intracellular endosomal trafficking of RBC within brain endothelial cells. Methods: Murine brain endothelial cells (bEnd.3 cells) were incubated with 2x10 6 mouse RBC treated with 3mM tert-butylhydroperoxide (t-BHP, an oxidative stressor) or sterile PBS (control) for 48h at 37°C, in the presence of 7.5 μg annexin V to cloak PS, or vitamin C (15μM-1500μM) to reduce reactive oxygen species (ROS). BEE was evaluated by hematoxylin & eosin stain and diaminofluorene assay for hemoglobin. RBC ROS levels, PS exposure, and cell viability were measured. bEnd.3 cells were immunostained to visualize RBC in early and late endosomes, the latter being the presumed site of RBC degradation post-erythrophagocytosis. Results: tBHP induced both ROS production and PS exposure in RBC. There was a 9-fold increase (p<0.001) in BEE of t-BHP-RBC compared with control. Vitamin C reduced RBC ROS levels (70%, p<0.001) and PS exposure (27%, p<0.001), while annexin V blocked RBC PS exposure (65%, p<0.001). BEE was significantly attenuated by annexin V (63%, p<0.001) and vitamin C (39%, p<0.001). No change in bEnd.3 viability was observed and t-BHP-RBC localized to both early and late endosomes. Conclusions: These data demonstrate the importance of RBC PS exposure in BEE, and describe the intracellular trafficking of RBC in brain endothelial cells. These findings provide insights into the development of microhemorrhage-like lesions and cerebral microbleeds.

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