scholarly journals An endosomal beta COP is involved in the pH-dependent formation of transport vesicles destined for late endosomes.

1996 ◽  
Vol 133 (1) ◽  
pp. 29-41 ◽  
Author(s):  
F Aniento ◽  
F Gu ◽  
R G Parton ◽  
J Gruenberg
Keyword(s):  
Ph Sensor ◽  
Membrane Association ◽  
Acidic Properties ◽  
Endosomal Membrane ◽  
Transport Vesicles ◽  
Late Endosomes ◽  
Early Endosomes ◽  
Endosomal Membranes ◽  
Transport Vesicle ◽  
Ph Dependent

In this paper, we show that beta COP is present on endosomes and is required for the formation of vesicles which mediate transport from early to late endosomes. Both the association of beta COP to endosomal membranes as well as transport vesicle formation depend on the lumenal pH. We find that epsilon COP, but not gamma COP, is also associated to endosomes, and that this association is also lumenal pH dependent. Our data, thus, indicate that a subset of COPs is part of the mechanism regulating endosomal membrane transport, and that membrane association of these COPs is controlled by the acidic properties of early endosomes, presumably via a trans-membrane pH sensor.

scholarly journals Exosomes induce endolysosomal permeabilization as a gateway by which exosomal tau seeds escape into the cytosol

2021 ◽  
Author(s):  
Juan Carlos Polanco ◽  
Gabriel Rhys Hand ◽  
Adam Briner ◽  
Chuanzhou Li ◽  
Jürgen Götz
Keyword(s):  
Critical Role ◽  
Membrane Integrity ◽  
Lysosomal Degradation ◽  
Escape Route ◽  
Cellular Invasion ◽  
Endosomal Membrane ◽  
Late Endosomes ◽  
Endosomal Membranes ◽  
Endosomal Pathway ◽  
Tau Aggregation

AbstractThe microtubule-associated protein tau has a critical role in Alzheimer’s disease and other tauopathies. A proposed pathomechanism in the progression of tauopathies is the trans-synaptic spreading of tau seeds, with a role for exosomes which are secretory nanovesicles generated by late endosomes. Our previous work demonstrated that brain-derived exosomes isolated from tau transgenic rTg4510 mice encapsulate tau seeds with the ability to induce tau aggregation in recipient cells. We had also shown that exosomes can hijack the endosomal pathway to spread through interconnected neurons. Here, we reveal how tau seeds contained within internalized exosomes exploit mechanisms of lysosomal degradation to escape the endosome and induce tau aggregation in the cytosol of HEK293T-derived ‘tau biosensor cells’. We found that the majority of the exosome-containing endosomes fused with lysosomes to form endolysosomes. Exosomes induced their permeabilization, irrespective of the presence of tau seeds, or whether the exosomal preparations originated from mouse brains or HEK293T cells. We also found that permeabilization is a conserved mechanism, operating in both non-neuronal tau biosensor cells and primary neurons. However, permeabilization of endolysosomes only occurred in a small fraction of cells, which supports the notion that permeabilization occurs by a thresholded mechanism. Interestingly, tau aggregation was only induced in cells that exhibited permeabilization, presenting this as an escape route of exosomal tau seeds into the cytosol. Overexpression of RAB7, which is required for the formation of endolysosomes, strongly increased tau aggregation. Conversely, inhibition of lysosomal function with alkalinizing agents, or by knocking-down RAB7, decreased tau aggregation. Together, we conclude that the enzymatic activities of lysosomes permeabilize exosomal and endosomal membranes, thereby facilitating access of exosomal tau seeds to cytosolic tau to induce its aggregation. Our data underscore the importance of endosomal membrane integrity in mechanisms of cellular invasion by misfolded proteins that are resistant to lysosomal degradation.

scholarly journals De novo formation of an early endosome during Rab5 to Rab7 transition

2020 ◽  
Author(s):  
Frode Miltzow Skjeldal ◽  
Linda Hofstad Haugen ◽  
Duarte Mateus ◽  
Dominik Frei ◽  
Oddmund Bakke
Keyword(s):  
De Novo ◽  
Early Endosome ◽  
Late Endosome ◽  
Second Phase ◽  
Specific Domain ◽  
Endosomal Membrane ◽  
Late Endosomes ◽  
Early Endosomes ◽  
Main Determinants ◽  
Insight Into

AbstractRab5 and Rab7a are the main determinants of early and late endosomes and are important regulators of endosomal progression. The transport from early endosomes to late endosome seems to be regulated through an endosomal maturation switch where Rab5 is exchanged with Rab7a on the same endosome. Here we provide new insight into the mechanism of endosomal maturation where we have discovered a stepwise Rab5 detachment, sequentially regulated by Rab7a. The initial detachment of Rab5 is Rab7a independent and demonstrate a diffusion-like exchange between cytosol and endosomal membrane, and the second phase is slower where Rab5 converges into a specific domain that specifically detaches as a Rab5 indigenous endosome. Consequently, we show that early endosomal maturation regulated through the Rab5 to Rab7a switch induce the formation of a new fully functional early endosome. Progression through a stepwise early endosomal maturation regulates the direction of the transport and concomitantly regulates the homeostasis of early endosomes.

scholarly journals Rab5-mediated endosome formation is regulated at the trans-Golgi network

2019 ◽  
Vol 2 (1) ◽  
Author(s):  
Makoto Nagano ◽  
Junko Y. Toshima ◽  
Daria Elisabeth Siekhaus ◽  
Jiro Toshima
Keyword(s):  
Plasma Membrane ◽  
Early Endosome ◽  
Vesicle Transport ◽  
Nucleotide Exchange ◽  
Trafficking Pathway ◽  
Transport Vesicles ◽  
Late Endosomes ◽  
Early Endosomes ◽  
Critical Location ◽  
Golgi Network

AbstractEarly endosomes, also called sorting endosomes, are known to mature into late endosomes via the Rab5-mediated endolysosomal trafficking pathway. Thus, early endosome existence is thought to be maintained by the continual fusion of transport vesicles from the plasma membrane and the trans-Golgi network (TGN). Here we show instead that endocytosis is dispensable and post-Golgi vesicle transport is crucial for the formation of endosomes and the subsequent endolysosomal traffic regulated by yeast Rab5 Vps21p. Fittingly, all three proteins required for endosomal nucleotide exchange on Vps21p are first recruited to the TGN before transport to the endosome, namely the GEF Vps9p and the epsin-related adaptors Ent3/5p. The TGN recruitment of these components is distinctly controlled, with Vps9p appearing to require the Arf1p GTPase, and the Rab11s, Ypt31p/32p. These results provide a different view of endosome formation and identify the TGN as a critical location for regulating progress through the endolysosomal trafficking pathway.

Endosome fusion in living cells overexpressing GFP-rab5

1999 ◽  
Vol 112 (21) ◽  
pp. 3667-3675 ◽  
Author(s):  
R.L. Roberts ◽  
M.A. Barbieri ◽  
K.M. Pryse ◽  
M. Chua ◽  
J.H. Morisaki ◽  
...  
Keyword(s):  
Time Lapse ◽  
Confocal Imaging ◽  
Fusion Event ◽  
Vesicle Docking ◽  
Endosomal Membrane ◽  
Early Endosomes ◽  
Cytoplasmic Vesicles ◽  
Confocal Fluorescence ◽  
Endosomal Membranes ◽  
Fluorescent Spot

CHO and BHK cells which overexpress either wild-type rab5 or rab5:Q79L, a constitutively active rab5 mutant, develop enlarged cytoplasmic vesicles that exhibit many characteristics of early endosomes including immunoreactivity for rab5 and transferrin receptor. Time-lapse video microscopy shows the enlarged endosomes arise primarily by fusion of smaller vesicles. These fusion events occur mostly by a ‘bridge’ fusion mechanism in which the initial opening between vesicles does not expand; instead, membrane flows slowly and continuously from the smaller to the larger endosome in the fusing pair, through a narrow, barely perceptible membranous ‘bridge’ between them. The unique aspect of rab5 mediated ‘bridge’ fusion is the persistence of a tight constriction at the site where vesicles merge and we hypothesize that this constriction results from the relatively slow disassembly of a putative docking/fusion complex. To determine the relation of rab5 to the fusion ‘bridge’, we used confocal fluorescence microscopy to monitor endosome fusion in cells overexpressing GFP-rab5 fusion proteins. Vesicle docking in these cells is accompanied by recruitment of the GFP-rab5 into a brightly fluorescent spot in the ‘bridge’ region between fusing vesicles that persists throughout the entire length of the fusion event and which often persist for minutes following endosome fusion. Other endosomal membrane markers, including FM4-64, are not concentrated in fusion ‘bridges’. These results support the idea that the GFP-rab5 spots represent the localized accumulation of GFP-rab5 between fusing endosomes and not simply overlap of adjacent membranes. The idea that the GFP-rab5 spots do not represent membrane overlap is further supported by experiments using photobleaching techniques and confocal imaging which show that GFP-rab5 localized in spots between fusion couplets is resistant to diffusion while GFP-rab5 on endosomal membranes away from these spots rapidly diffuses with a rate constant of about 1.0 (+/-0.3) x10(-)(9)cm(2)/second.

scholarly journals VPS18 recruits VPS41 to the human HOPS complex via a RING-RING interaction

2017 ◽  
Author(s):  
Morag R. Hunter ◽  
Edward J. Scourfield ◽  
Edward Emmott ◽  
Stephen C. Graham
Keyword(s):  
Zinc Finger ◽  
Terminal Segment ◽  
Ring Domain ◽  
Core Complex ◽  
Endosomal Membrane ◽  
Late Endosomes ◽  
Early Endosomes ◽  
Ring Domains ◽  
Membrane Tethering ◽  
Ring Interaction

ABSTRACTEukaryotic cells use conserved multisubunit membrane tethering complexes, including CORVET and HOPS, to control the fusion of endomembranes. These complexes have been extensively studied in yeast, but to date there have been far fewer studies of metazoan CORVET and HOPS. Both of these complexes comprise six subunits: a common four-subunit core and two unique subunits. Once assembled, these complexes function to recognise specific endosomal membrane markers and facilitate SNARE-mediated membrane fusion. CORVET promotes the homotypic fusion of early endosomes, while HOPS promotes the fusion of lysosomes to late endosomes and autophagosomes. Many of the subunits of both CORVET and HOPS contain putative C-terminal zinc-finger domains. Here, the contribution of these domains to the assembly of the human CORVET and HOPS complexes has been examined. Using biochemical techniques, we demonstrate that the zinc-containing RING domains of human VPS18 and VPS41 interact directly to form a stable heterodimer. In cells, these RING domains are able to integrate into endogenous HOPS, showing that the VPS18 RING domain is required to recruit VPS41 to the core complex subunits. Importantly, this mechanism is not conserved throughout eukaryotes, as yeast Vps41 does not contain a C-terminal zinc-finger motif. The subunit analogous to VPS41 in human CORVET is VPS8, in which the RING domain has an additional C-terminal segment that is predicted to be disordered. Both the RING and disordered C-terminal domains are required for integration of VPS8 into endogenous CORVET complexes, suggesting that HOPS and CORVET recruit VPS41 and VPS8 via distinct molecular interactions.

scholarly journals GCC185 plays independent roles in Golgi structure maintenance and AP-1–mediated vesicle tethering

2011 ◽  
Vol 194 (5) ◽  
pp. 779-787 ◽  
Author(s):  
Frank C. Brown ◽  
Carmel H. Schindelhaim ◽  
Suzanne R. Pfeffer
Keyword(s):  
Coiled Coil ◽  
Retrograde Transport ◽  
Vesicle Tethering ◽  
Transport Vesicles ◽  
Late Endosomes ◽  
Transport Vesicle ◽  
Golgi Structure ◽  
Clathrin Adaptor ◽  
Golgi Network

GCC185 is a long coiled-coil protein localized to the trans-Golgi network (TGN) that functions in maintaining Golgi structure and tethering mannose 6-phosphate receptor (MPR)–containing transport vesicles en route to the Golgi. We report the identification of two distinct domains of GCC185 needed either for Golgi structure maintenance or transport vesicle tethering, demonstrating the independence of these two functions. The domain needed for vesicle tethering binds to the clathrin adaptor AP-1, and cells depleted of GCC185 accumulate MPRs in transport vesicles that are AP-1 decorated. This study supports a previously proposed role of AP-1 in retrograde transport of MPRs from late endosomes to the Golgi and indicates that docking may involve the interaction of vesicle-associated AP-1 protein with the TGN-associated tethering protein GCC185.

scholarly journals De novo formation of early endosomes during Rab5 to Rab7 transition

2021 ◽  
pp. jcs.254185
Author(s):  
Frode Miltzow Skjeldal ◽  
Linda Hofstad Haugen ◽  
Duarte Mateus ◽  
Dominik M. Frei ◽  
Anna Vik Rødseth ◽  
...  
Keyword(s):  
De Novo ◽  
Late Endosome ◽  
Second Phase ◽  
Endosomal Membrane ◽  
Late Endosomes ◽  
Early Endosomes ◽  
Main Determinants ◽  
Insight Into

Rab5 and Rab7a are the main determinants of early and late endosomes and are important regulators of endosomal progression. The transport from early endosomes to late endosome seems to be regulated through an endosomal maturation switch where Rab5 is gradually exchanged with Rab7a on the same endosome. Here we provide new insight into the mechanism of endosomal maturation where we have discovered a stepwise Rab5 detachment, sequentially regulated by Rab7a. The initial detachment of Rab5 is Rab7a independent and demonstrate a diffusion-like exchange between cytosol and endosomal membrane, and a second phase where Rab5 converges into specific domains that detaches as a Rab5 indigenous endosome. Consequently, we show that early endosomal maturation regulated through the Rab5 to Rab7a switch induce the formation of new fully functional rab5 positive early endosomes. Progression through a stepwise early endosomal maturation regulates the direction of the transport and concomitantly regulates the homeostasis of early endosomes.

NSF is required for transport from early to late endosomes

1997 ◽  
Vol 110 (17) ◽  
pp. 2079-2087 ◽  
Author(s):  
L.J. Robinson ◽  
F. Aniento ◽  
J. Gruenberg
Keyword(s):  
Membrane Trafficking ◽  
Protein Transport ◽  
Degradation Pathway ◽  
Biochemical Properties ◽  
Endocytic Pathway ◽  
Late Endosomes ◽  
Early Endosomes ◽  
Sensitive Factor ◽  
Endosomal Membranes

Protein transport between early and late endosomes is a major membrane trafficking pathway in the cell followed by many proteins, including all down-regulated receptors. Yet, little is known at the molecular level about the mechanisms regulating membrane interactions in the endocytic pathway beyond early endosomes. In this study, we have used an in vitro transport assay to study the biochemical properties of endosome docking/fusion events. Our data demonstrate that N-ethylmaleimide (NEM) sensitive factor (NSF) and its soluble associated proteins (SNAPs) are required for transport from early to late endosomes, as well as at all other steps of endosomal membrane transport. We also find that these proteins are enriched on endosomal membranes. In addition, our studies suggest that besides NSF/SNAPs, another NEM-sensitive component may also be involved in docking/fusion at this late stage of the pathway. Finally, we find that, in contrast to Golgi membranes, NSF association to both early and late endosomal membranes occurs via an ATP-independent mechanism, indicating that the binding properties of endosomal and biosynthetic NSF are different. Our data thus show that NSF/SNAPs, perhaps together with another NEM-sensitive factor, are part of the basic molecular machinery which controls docking/fusion events during transport from early to late endosomes, along the lysosomal degradation pathway.

scholarly journals Functional Dissection of COP-I Subunits in the Biogenesis of Multivesicular Endosomes

1997 ◽  
Vol 139 (5) ◽  
pp. 1183-1195 ◽  
Author(s):  
Feng Gu ◽  
Fernando Aniento ◽  
Robert G. Parton ◽  
Jean Gruenberg
Keyword(s):  
Restrictive Temperature ◽  
Cho Cell ◽  
Independent Manner ◽  
Late Endosomes ◽  
Early Endosomes ◽  
Endosomal Membranes ◽  
Endosomal Ph ◽  
And Function ◽  
Multivesicular Endosomes

In the present paper, we show that transport from early to late endosomes is inhibited at the restrictive temperature in a mutant CHO cell line (ldlF) with a ts-defect in ε coatomer protein (εCOP), although internalization and recycling continue. Early endosomes then appear like clusters of thin tubules devoid of the typical multivesicular regions, which are normally destined to become vesicular intermediates during transport to late endosomes. We also find that the in vitro formation of these vesicles from BHK donor endosomes is inhibited in cytosol prepared from ldlF cells incubated at the restrictive temperature. Although εCOP is rapidly degraded in ldlF cells at the restrictive temperature, cellular amounts of the other COP-I subunits are not affected. Despite the absence of εCOP, we find that a subcomplex of β, β′, and ζCOP is still recruited onto BHK endosomes in vitro, and this binding exhibits the characteristic properties of endosomal COPs with respect to stimulation by GTPγS and sensitivity to the endosomal pH. Previous studies showed that γ and δCOP are not found on endosomes. However, αCOP, which is normally present on endosomes, is no longer recruited when εCOP is missing. In contrast, all COP subunits, except obviously εCOP itself, still bind BHK biosynthetic membranes in a pH-independent manner in vitro. Our observations thus indicate that the biogenesis of multivesicular endosomes is coupled to early endosome organization and depends on COP-I proteins. Our data also show that membrane association and function of endosomal COPs can be dissected: whereas β, β′, and ζCOP retain the capacity to bind endosomal membranes, COP function in transport appears to depend on the presence of α and/or εCOP.

scholarly journals VPS18 recruits VPS41 to the human HOPS complex via a RING–RING interaction

2017 ◽  
Vol 474 (21) ◽  
pp. 3615-3626 ◽  
Author(s):  
Morag R. Hunter ◽  
Edward J. Scourfield ◽  
Edward Emmott ◽  
Stephen C. Graham
Keyword(s):  
Zinc Finger ◽  
Terminal Segment ◽  
Ring Domain ◽  
Core Complex ◽  
Endosomal Membrane ◽  
Late Endosomes ◽  
Early Endosomes ◽  
Membrane Tethering ◽  
New Gene ◽  
Ring Interaction

Eukaryotic cells use conserved multisubunit membrane tethering complexes, including CORVET (class C core vacuole/endosome tethering) and HOPS (homotypic fusion and vacuole protein sorting), to control the fusion of endomembranes. These complexes have been extensively studied in yeast, but to date there have been far fewer studies of metazoan CORVET and HOPS. Both of these complexes comprise six subunits: a common four-subunit core and two unique subunits. Once assembled, these complexes function to recognise specific endosomal membrane markers and facilitate SNARE-mediated membrane fusion. CORVET promotes the homotypic fusion of early endosomes, while HOPS promotes the fusion of lysosomes to late endosomes and autophagosomes. Many of the subunits of both CORVET and HOPS contain putative C-terminal zinc-finger domains. Here, the contribution of these domains to the assembly of the human CORVET and HOPS complexes has been examined. Using biochemical techniques, we demonstrate that the zinc-containing RING (really interesting new gene) domains of human VPS18 and VPS41 interact directly to form a stable heterodimer. In cells, these RING domains are able to integrate into endogenous HOPS, showing that the VPS18 RING domain is required to recruit VPS41 to the core complex subunits. Importantly, this mechanism is not conserved throughout eukaryotes, as yeast Vps41 does not contain a C-terminal zinc-finger motif. The subunit analogous to VPS41 in human CORVET is VPS8, in which the RING domain has an additional C-terminal segment that is predicted to be disordered. Both the RING and disordered C-terminal domains are required for integration of VPS8 into endogenous CORVET complexes, suggesting that HOPS and CORVET recruit VPS41 and VPS8 via distinct molecular interactions.

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