scholarly journals Axonal and dendritic endocytic pathways in cultured neurons.

1992 ◽  
Vol 119 (1) ◽  
pp. 123-137 ◽  
Author(s):  
R G Parton ◽  
K Simons ◽  
C G Dotti
Keyword(s):  
Cell Body ◽  
Hippocampal Neurons ◽  
Multivesicular Body ◽  
Nerve Terminals ◽  
Multivesicular Bodies ◽  
Late Endosomes ◽  
Distal Side ◽  
Early Endosomes ◽  
Fast Transport ◽  
Endocytic Pathways

The endocytic pathways from the axonal and dendritic surfaces of cultured polarized hippocampal neurons were examined. The dendrites and cell body contained extensive networks of tubular early endosomes which received endocytosed markers from the somatodendritic domain. In axons early endosomes were confined to presynaptic terminals and to varicosities. The somatodendritic but not the presynaptic early endosomes were labeled by internalized transferrin. In contrast to early endosomes, late endosomes and lysosomes were shown to be predominantly located in the cell body. Video microscopy was used to follow the transport of internalized markers from the periphery of axons and dendrites back to the cell body. Labeled structures in both domains moved unidirectionally by retrograde fast transport. Axonally transported organelles were sectioned for EM after video microscopic observation and shown to be large multivesicular body-like structures. Similar structures accumulated at the distal side of an axonal lesion. Multivesicular bodies therefore appear to be the major structures mediating transport of endocytosed markers between the nerve terminals and the cell body. Late endocytic structures were also shown to be highly mobile and were observed moving within the cell body and proximal dendritic segments. The results show that the organization of the endosomes differs in the axons and dendrites of cultured rat hippocampal neurons and that the different compartments or stages of the endocytic pathways can be resolved spatially.

scholarly journals Hrs regulates multivesicular body formation via ESCRT recruitment to endosomes

2003 ◽  
Vol 162 (3) ◽  
pp. 435-442 ◽  
Author(s):  
Kristi G. Bache ◽  
Andreas Brech ◽  
Anja Mehlum ◽  
Harald Stenmark
Keyword(s):  
Membrane Proteins ◽  
Rna Viruses ◽  
Multivesicular Body ◽  
Viral Proteins ◽  
Multivesicular Bodies ◽  
Body Formation ◽  
Endosomal Sorting ◽  
Late Endosomes ◽  
Early Endosomes ◽  
Endosomal Vesicles

Hrs and the endosomal sorting complexes required for transport, ESCRT-I, -II, and -III, are involved in the endosomal sorting of membrane proteins into multivesicular bodies and lysosomes or vacuoles. The ESCRT complexes are also required for formation of intraluminal endosomal vesicles and for budding of certain enveloped RNA viruses such as HIV. Here, we show that Hrs binds to the ESCRT-I subunit Tsg101 via a PSAP motif that is conserved in Tsg101-binding viral proteins. Depletion of Hrs causes a reduction in membrane-associated ESCRT-I subunits, a decreased number of multivesicular bodies and an increased size of late endosomes. Even though Hrs mainly localizes to early endosomes and Tsg101 to late endosomes, the two proteins colocalize on a subpopulation of endosomes that contain lyso-bisphosphatidic acid. Overexpression of Hrs causes accumulation of Tsg101 on early endosomes and prevents its localization to late endosomes. We conclude that Hrs mediates the initial recruitment of ESCRT-I to endosomes and, thereby, indirectly regulates multivesicular body formation.

scholarly journals EEA1, a Tethering Protein of the Early Sorting Endosome, Shows a Polarized Distribution in Hippocampal Neurons, Epithelial Cells, and Fibroblasts

2000 ◽  
Vol 11 (8) ◽  
pp. 2657-2671 ◽  
Author(s):  
Jean M. Wilson ◽  
Meltsje de Hoop ◽  
Natasha Zorzi ◽  
Ban-Hock Toh ◽  
Carlos G. Dotti ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Mammalian Cells ◽  
Hippocampal Neurons ◽  
Cell Types ◽  
Early Endosomes ◽  
Filamentous Material ◽  
Endocytic Vesicles ◽  
Fibroblastic Cells ◽  
Darby Canine Kidney ◽  
Endocytic Pathways

EEA1 is an early endosomal Rab5 effector protein that has been implicated in the docking of incoming endocytic vesicles before fusion with early endosomes. Because of the presence of complex endosomal pathways in polarized and nonpolarized cells, we have examined the distribution of EEA1 in diverse cell types. Ultrastructural analysis demonstrates that EEA1 is present on a subdomain of the early sorting endosome but not on clathrin-coated vesicles, consistent with a role in providing directionality to early endosomal fusion. Furthermore, EEA1 is associated with filamentous material that extends from the cytoplasmic surface of the endosomal domain, which is also consistent with a tethering/docking role for EEA1. In polarized cells (Madin-Darby canine kidney cells and hippocampal neurons), EEA1 is present on a subset of “basolateral-type” endosomal compartments, suggesting that EEA1 regulates specific endocytic pathways. In both epithelial cells and fibroblastic cells, EEA1 and a transfected apical endosomal marker, endotubin, label distinct endosomal populations. Hence, there are at least two distinct sets of early endosomes in polarized and nonpolarized mammalian cells. EEA1 could provide specificity and directionality to fusion events occurring in a subset of these endosomes in polarized and nonpolarized cells.

In Vivo Multivesicular Body and Exosome Secretion in the Intestinal Epithelial Cells of Turtles During Hibernation

2019 ◽  
Vol 25 (6) ◽  
pp. 1341-1351
Author(s):  
Waseem Ali Vistro ◽  
Yufei Huang ◽  
Xuebing Bai ◽  
Ping Yang ◽  
Abdul Haseeb ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Intestinal Epithelial Cells ◽  
Susceptibility Gene ◽  
Multivesicular Body ◽  
Intestinal Epithelial ◽  
Transmission Electron ◽  
Late Endosomes ◽  
Early Endosomes ◽  
Cluster Of Differentiation

AbstractThe present study was designed to investigate the in vivo biological processes of multivesicular bodies (MVBs) and exosomes in mitochondria-rich cells (MRCs), goblet cells (GCs), and absorptive cells (ACs) in turtle intestines during hibernation. The exosome markers, cluster of differentiation 63 (CD63) and tumor susceptibility gene 101 (TSG101), were positively expressed in intestinal villi during turtle hibernation. The distribution and formation processes of MVBs and exosomes in turtle MRCs, GCs, and ACs were further confirmed by transmission electron microscopy. During hibernation, abundantly secreted early endosomes (ees) were localized in the luminal and basal cytoplasm of the MRCs and ACs, and late endosomes (les) were dispersed with the supranuclear parts of the MRCs and ACs. Many “heterogeneous” MVBs were identified throughout the cytoplasm of the MRCs and ACs. Interestingly, the ees, les, and MVBs were detected in the cytoplasm of the GCs during hibernation; however, they were absent during nonhibernation. Furthermore, the exocytosis pathways of exosomes and autophagic vacuoles were observed in the MRCs, GCs, and ACs during hibernation. In addition, the number of different MVBs with intraluminal vesicles (ILVs) and heterogeneous endosome–MVB–exosome complexes was significantly increased in the MRCs, GCs, and ACs during hibernation. All these findings indicate that intestinal epithelial cells potentially perform a role in the secretion of MVBs and exosomes, which are essential for mucosal immunity, during hibernation.

scholarly journals DRAM1 requires PI(3,5)P2 generation by PIKfyve to deliver vesicles and their cargo to endolysosomes

2020 ◽  
Author(s):  
Michiel van der Vaart ◽  
Adrianna Banducci-Karp ◽  
Gabriel Forn-Cuní ◽  
Philip M.M. Witt ◽  
Joost J. Willemse ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Transmembrane Protein ◽  
Vesicle Fusion ◽  
Multivesicular Bodies ◽  
Potential Therapeutic Target ◽  
Zebrafish Model ◽  
Lipid Kinase ◽  
Late Endosomes ◽  
Early Endosomes ◽  
Chemical Inhibition

Endolysosomal vesicle trafficking and autophagy are crucial degradative pathways in maintenance of cellular homeostasis. The transmembrane protein DRAM1 is a potential therapeutic target that primarily localises to endolysosomal vesicles and promotes autophagy and vesicle fusion with lysosomes. However, the molecular mechanisms underlying DRAM1-mediated vesicle fusion events remain unclear. Using high-resolution confocal microscopy in the zebrafish model, we show that mCherry-Dram1 labelled vesicles interact and fuse with early endosomes marked by PI(3)P. Following these fusion events, early endosomes mature into late endosomes in a process dependent on the conversion of PI(3)P into PI(3,5)P2 by the lipid kinase PIKfyve. Chemical inhibition of PIKfyve reduces the targeting of Dram1 to acidic endolysosomal vesicles, arresting Dram1 in multivesicular bodies, early endosomes, or non-acidified vesicles halted in their fusion with early endosomes. In conclusion, Dram1-mediated vesicle fusion requires the formation of PI(3,5)P2 to deliver vesicles and their cargo to the degradative environment of the lysosome.

scholarly journals Rap1-PDZ-GEF1 interacts with a neurotrophin receptor at late endosomes, leading to sustained activation of Rap1 and ERK and neurite outgrowth

2007 ◽  
Vol 178 (5) ◽  
pp. 843-860 ◽  
Author(s):  
Shu Hisata ◽  
Toshiaki Sakisaka ◽  
Takeshi Baba ◽  
Tomohiro Yamada ◽  
Kazuhiro Aoki ◽  
...  
Keyword(s):  
Neurite Outgrowth ◽  
Hippocampal Neurons ◽  
Feedback Mechanism ◽  
Neurotrophin Receptor ◽  
Dependent Manner ◽  
Late Endosomes ◽  
Early Endosomes ◽  
Positive Feedback Mechanism ◽  
Membrane Spanning ◽  
Sustained Activation

Neurotrophins, such as NGF and BDNF, induce sustained activation of Rap1 small G protein and ERK, which are essential for neurite outgrowth. We show involvement of a GDP/GTP exchange factor (GEF) for Rap1, PDZ-GEF1, in these processes. PDZ-GEF1 is activated by GTP-Rap1 via a positive feedback mechanism. Upon NGF binding, the TrkA neurotrophin receptor is internalized from the cell surface, passes through early endosomes, and arrives in late endosomes. A tetrameric complex forms between PDZ-GEF1, synaptic scaffolding molecule and ankyrin repeat-rich membrane spanning protein which interacts directly with the TrkA receptor. At late endosomes, the complex induces sustained activation of Rap1 and ERK, resulting in neurite outgrowth. In cultured rat hippocampal neurons, PDZ-GEF1 is recruited to late endosomes in a BDNF-dependent manner involved in BDNF-induced neurite outgrowth. Thus, the interaction of PDZ-GEF1 with an internalized neurotrophin receptor transported to late endosomes induces sustained activation of both Rap1 and ERK and neurite outgrowth.

scholarly journals Dynamic Dissection of the Endocytosis of Porcine Epidemic Diarrhea Coronavirus Cooperatively Mediated by Clathrin and Caveolae as Visualized by Single-Virus Tracking

mBio ◽  
2021 ◽  
Vol 12 (2) ◽  
Author(s):  
Yangyang Li ◽  
Jian Wang ◽  
Wei Hou ◽  
Yanke Shan ◽  
Shouyu Wang ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cell Membrane ◽  
Viral Fusion ◽  
Endosome Trafficking ◽  
Late Endosomes ◽  
Early Endosomes ◽  
Membrane Barrier ◽  
Porcine Epidemic Diarrhea ◽  
Kinetics Of ◽  
Endocytic Pathways

ABSTRACT Coronaviruses (CoVs) have caused severe diseases in humans and animals. Endocytic pathways, such as clathrin-mediated endocytosis (CME) and caveolae-mediated endocytosis (CavME), play an important role for CoVs to penetrate the cell membrane barrier. In this study, a novel CoV entry manner is unraveled in which clathrin and caveolae can cooperatively mediate endocytosis of porcine epidemic diarrhea coronavirus (PEDV). Using multicolor live-cell imaging, the dynamics of the fluorescently labeled clathrin structures, caveolae structures, and PEDV were dissected. During CavME of PEDV, we found that clathrin structures can fuse with caveolae near the cell plasma membrane, and the average time of PEDV penetrating the cell membrane was within ∼3 min, exhibiting a rapid course of PEDV entry. Moreover, based on the dynamic recruitment of clathrin and caveolae structures and viral motility, the direct evidence also shows that about 20% of PEDVs can undergo an abortive entry via CME and CavME. Additionally, the dynamic trafficking of PEDV from clathrin and caveolae structures to early endosomes, and from early endosomes to late endosomes, and viral fusion were directly dissected, and PEDV fusion mainly occurred in late endosomes within ∼6.8 min after the transport of PEDV to late endosomes. Collectively, this work systematically unravels the early steps of PEDV infection, which expands our understanding of the mechanism of CoV infection. IMPORTANCE Emerging and re-emerging coronaviruses cause serious human and animal epidemics worldwide. For many enveloped viruses, including coronavirus, it is evident that breaking the plasma membrane barrier is a pivotal and complex process, which contains multiple dynamic steps. Although great efforts have been made to understand the mechanisms of coronavirus endocytic pathways, the direct real-time imaging of individual porcine epidemic diarrhea coronavirus (PEDV) internalization has not been achieved yet. In this study, we not only dissected the kinetics of PEDV entry via clathrin-mediated endocytosis and caveolae-mediated endocytosis and the kinetics of endosome trafficking and viral fusion but also found a novel productive coronavirus entry manner in which clathrin and caveolae can cooperatively mediate endocytosis of PEDV. Moreover, we uncovered the existence of PEDV abortive endocytosis. In summary, the productive PEDV entry via the cooperation between clathrin and caveolae structures and the abortive endocytosis of PEDV provide new insights into coronavirus penetrating the plasma membrane barrier.

scholarly journals Endosomal maturation by Rab conversion in Aspergillus nidulans is coupled to dynein-mediated basipetal movement

2012 ◽  
Vol 23 (10) ◽  
pp. 1889-1901 ◽  
Author(s):  
Juan F. Abenza ◽  
Antonio Galindo ◽  
Mario Pinar ◽  
Areti Pantazopoulou ◽  
Vivian de los Ríos ◽  
...  
Keyword(s):  
Aspergillus Nidulans ◽  
Multivesicular Body ◽  
Endocytic Pathway ◽  
Dependent Manner ◽  
Long Distance ◽  
Genetic Studies ◽  
Late Endosomes ◽  
Early Endosomes ◽  
A Minor ◽  
Long Distance Movement

We exploit the ease with which highly motile early endosomes are distinguished from static late endosomes in order to study Aspergillus nidulans endosomal traffic. RabSRab7 mediates homotypic fusion of late endosomes/vacuoles in a homotypic fusion- and vacuole protein sorting/Vps41–dependent manner. Progression across the endocytic pathway involves endosomal maturation because the end products of the pathway in the absence of RabSRab7 are minivacuoles that are competent in multivesicular body sorting and cargo degradation but retain early endosomal features, such as the ability to undergo long-distance movement and propensity to accumulate in the tip region if dynein function is impaired. Without RabSRab7, early endosomal Rab5s—RabA and RabB—reach minivacuoles, in agreement with the view that Rab7 homologues facilitate the release of Rab5 homologues from endosomes. RabSRab7 is recruited to membranes already at the stage of late endosomes still lacking vacuolar morphology, but the transition between early and late endosomes is sharp, as only in a minor proportion of examples are RabA/RabB and RabSRab7 detectable in the same—frequently the less motile—structures. This early-to-late endosome/vacuole transition is coupled to dynein-dependent movement away from the tip, resembling the periphery-to-center traffic of endosomes accompanying mammalian cell endosomal maturation. Genetic studies establish that endosomal maturation is essential, whereas homotypic vacuolar fusion is not.

scholarly journals A Super-Resolved View of the Alzheimer’s Disease-Related Amyloidogenic Pathway in Hippocampal Neurons

2021 ◽  
pp. 1-20
Author(s):  
Yang Yu ◽  
Yang Gao ◽  
Bengt Winblad ◽  
Lars Tjernberg ◽  
Sophia Schedin Weiss
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Hippocampal Neurons ◽  
Three Dimensional ◽  
Amyloid Β ◽  
Stimulated Emission ◽  
Amyloid Β Peptide ◽  
Primary Neurons ◽  
Amyloid Β Protein ◽  
Early Endosomes

Background: Processing of the amyloid-β protein precursor (AβPP) is neurophysiologically important due to the resulting fragments that regulate synapse biology, as well as potentially harmful due to generation of the 42 amino acid long amyloid β-peptide (Aβ 42), which is a key player in Alzheimer’s disease. Objective: Our aim was to clarify the subcellular locations of the amyloidogenic AβPP processing in primary neurons, including the intracellular pools of the immediate substrate, AβPP C-terminal fragment (APP-CTF) and the product (Aβ 42). To overcome the difficulties of resolving these compartments due to their small size, we used super-resolution microscopy. Methods: Mouse primary hippocampal neurons were immunolabelled and imaged by stimulated emission depletion (STED) microscopy, including three-dimensional, three-channel imaging and image analyses. Results: The first (β-secretase) and second (γ-secretase) cleavages of AβPP were localized to functionally and distally distinct compartments. The β-secretase cleavage was observed in early endosomes, where we were able to show that the liberated N- and C-terminal fragments were sorted into distinct vesicles budding from the early endosomes in soma. Lack of colocalization of Aβ 42 and APP-CTF in soma suggested that γ-secretase cleavage occurs in neurites. Indeed, APP-CTF was, in line with Aβ 42 in our previous study, enriched in the presynapse but absent from the postsynapse. In contrast, full-length AβPP was not detected in either the pre- or the postsynaptic side of the synapse. Furthermore, we observed that endogenously produced and endocytosed Aβ 42 were localized in different compartments. Conclusion: These findings provide critical super-resolved insight into amyloidogenic AβPP processing in primary neurons.

scholarly journals Relationship between invariant chain expression and major histocompatibility complex class II transport into early and late endocytic compartments.

1993 ◽  
Vol 177 (3) ◽  
pp. 583-596 ◽  
Author(s):  
P Romagnoli ◽  
C Layet ◽  
J Yewdell ◽  
O Bakke ◽  
R N Germain
Keyword(s):  
Major Histocompatibility Complex ◽  
Mhc Class Ii ◽  
Class Ii ◽  
Endocytic Pathway ◽  
Invariant Chain ◽  
Major Histocompatibility ◽  
Histocompatibility Complex ◽  
Late Endosomes ◽  
Early Endosomes ◽  
Endocytic Compartments

Invariant chain (Ii), which associates with major histocompatibility complex (MHC) class II molecules in the endoplasmic reticulum, contains a targeting signal for transport to intracellular vesicles in the endocytic pathway. The characteristics of the target vesicles and the relationship between Ii structure and class II localization in distinct endosomal subcompartments have not been well defined. We demonstrate here that in transiently transfected COS cells expressing high levels of the p31 or p41 forms of Ii, uncleaved Ii is transported to and accumulates in transferrin-accessible (early) endosomes. Coexpressed MHC class II is also found in this same compartment. These early endosomes show altered morphology and a slower rate of content movement to later parts of the endocytic pathway. At more moderate levels of Ii expression, or after removal of a highly conserved region in the cytoplasmic tail of Ii, coexpressed class II molecules are found primarily in vesicles with the characteristics of late endosomes/prelysosomes. The Ii chains in these late endocytic vesicles have undergone proteolytic cleavage in the lumenal region postulated to control MHC class II peptide binding. These data indicate that the association of class II with Ii results in initial movement to early endosomes. At high levels of Ii expression, egress to later endocytic compartments is delayed and class II-Ii complexes accumulate together with endocytosed material. At lower levels of Ii expression, class II-Ii complexes are found primarily in late endosomes/prelysosomes. These data provide evidence that the route of class II transport to the site of antigen processing and loading involves movement through early endosomes to late endosomes/prelysosomes. Our results also reveal an unexpected ability of intact Ii to modify the structure and function of the early endosomal compartment, which may play a role in regulating this processing pathway.

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