De novo formation of early endosomes during Rab5 to Rab7 transition

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.

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

10.1101/2020.07.06.189050 ◽

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.

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Mycobacterium's Arrest of Phagosome Maturation in Macrophages Requires Rab5 Activity and Accessibility to Iron

Molecular Biology of the Cell ◽

10.1091/mbc.e02-12-0780 ◽

2003 ◽

Vol 14 (8) ◽

pp. 3366-3377 ◽

Cited By ~ 78

Author(s):

Victoria A. Kelley ◽

Jeffrey S. Schorey

Keyword(s):

Mycobacterium Avium ◽

Dominant Negative ◽

Late Endosome ◽

Maturation Process ◽

Phagosome Maturation ◽

Retroviral Transduction ◽

Murine Macrophages ◽

Late Endosomes ◽

Early Endosomes ◽

Insight Into

Many mycobacteria are intramacrophage pathogens that reside within nonacidified phagosomes that fuse with early endosomes but do not mature to phagolysosomes. The mechanism by which mycobacteria block this maturation process remains elusive. To gain insight into whether fusion with early endosomes is required for mycobacteria-mediated inhibition of phagosome maturation, we investigated how perturbing the GTPase cycles of Rab5 and Rab7, GTPases that regulate early and late endosome fusion, respectively, would affect phagosome maturation. Retroviral transduction of the constitutively activated forms of both GTPases into primary murine macrophages had no effect on Mycobacterium avium retention in an early endosomal compartment. Interestingly, expression of dominant negative Rab5, Rab5(S34N), but not dominant negative Rab7, resulted in a significant increase in colocalization of M. avium with markers of late endosomes/lysosomes and increased mycobacterial killing. This colocalization was specific to mycobacteria since Rab5(S34N) expressing cells showed diminished trafficking of endocytic tracers to lysosomes. We further demonstrated that maturation of M. avium phagosomes was halted in Rab5(S34N) expressing macrophages supplemented with exogenous iron. These findings suggest that fusion with early endosomes is required for mycobacterial retention in early phagosomal compartments and that an inadequate supply of iron is one factor in mycobacteria's inability to prevent the normal maturation process in Rab5(S34N)-expressing macrophages.

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

10.1101/169185 ◽

2017 ◽

Cited By ~ 1

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.

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

The Journal of Cell Biology ◽

10.1083/jcb.133.1.29 ◽

1996 ◽

Vol 133 (1) ◽

pp. 29-41 ◽

Cited By ~ 249

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.

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The delivery of endocytosed cargo to lysosomes

Biochemical Society Transactions ◽

10.1042/bst0371019 ◽

2009 ◽

Vol 37 (5) ◽

pp. 1019-1021 ◽

Cited By ~ 82

Author(s):

J. Paul Luzio ◽

Michael D.J. Parkinson ◽

Sally R. Gray ◽

Nicholas A. Bright

Keyword(s):

Plasma Membrane ◽

Mammalian Cells ◽

Late Endosome ◽

Snare Complex ◽

Coated Pits ◽

Free System ◽

Endosomal Sorting ◽

Protein Receptor ◽

Late Endosomes ◽

Early Endosomes

In mammalian cells, endocytosed cargo that is internalized through clathrin-coated pits/vesicles passes through early endosomes and then to late endosomes, before delivery to lysosomes for degradation by proteases. Late endosomes are MVBs (multivesicular bodies) with ubiquitinated membrane proteins destined for lysosomal degradation being sorted into their luminal vesicles by the ESCRT (endosomal sorting complex required for transport) machinery. Cargo is delivered from late endosomes to lysosomes by kissing and direct fusion. These processes have been studied in live cell experiments and a cell-free system. Late endosome–lysosome fusion is preceded by tethering that probably requires mammalian orthologues of the yeast HOPS (homotypic fusion and vacuole protein sorting) complex. Heterotypic late endosome–lysosome membrane fusion is mediated by a trans-SNARE (soluble N-ethylmaleimide-sensitive factor-attachment protein receptor) complex comprising Syntaxin7, Vti1b, Syntaxin8 and VAMP7 (vesicle-associated membrane protein 7). This differs from the trans-SNARE complex required for homotypic late endosome fusion in which VAMP8 replaces VAMP7. VAMP7 is also required for lysosome fusion with the plasma membrane and its retrieval from the plasma membrane to lysosomes is mediated by its folded N-terminal longin domain. Co-ordinated interaction of the ESCRT, HOPS and SNARE complexes is required for cargo delivery to lysosomes.

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Fusion between Phagosomes, Early and Late Endosomes: A Role for Actin in Fusion between Late, but Not Early Endocytic Organelles

Molecular Biology of the Cell ◽

10.1091/mbc.e03-05-0334 ◽

2004 ◽

Vol 15 (1) ◽

pp. 345-358 ◽

Cited By ~ 70

Author(s):

Rune Kjeken ◽

Morten Egeberg ◽

Anja Habermann ◽

Mark Kuehnel ◽

Pascale Peyron ◽

...

Keyword(s):

Actin Polymerization ◽

De Novo ◽

Light And Electron Microscopy ◽

Early Endosome ◽

Latex Bead ◽

Fusion Partner ◽

Late Endosomes ◽

Early Endosomes ◽

Membrane Bound

Actin is implicated in membrane fusion, but the precise mechanisms remain unclear. We showed earlier that membrane organelles catalyze the de novo assembly of F-actin that then facilitates the fusion between latex bead phagosomes and a mixture of early and late endocytic organelles. Here, we correlated the polymerization and organization of F-actin with phagosome and endocytic organelle fusion processes in vitro by using biochemistry and light and electron microscopy. When membrane organelles and cytosol were incubated at 37°C with ATP, cytosolic actin polymerized rapidly and became organized into bundles and networks adjacent to membrane organelles. By 30-min incubation, a gel-like state was formed with little further polymerization of actin thereafter. Also during this time, the bulk of in vitro fusion events occurred between phagosomes/endocytic organelles. The fusion between latex bead phagosomes and late endocytic organelles, or between late endocytic organelles themselves was facilitated by actin, but we failed to detect any effect of perturbing F-actin polymerization on early endosome fusion. Consistent with this, late endosomes, like phagosomes, could nucleate F-actin, whereas early endosomes could not. We propose that actin assembled by phagosomes or late endocytic organelles can provide tracks for fusion-partner organelles to move vectorially toward them, via membrane-bound myosins, to facilitate fusion.

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

Biochemical Journal ◽

10.1042/bcj20170588 ◽

2017 ◽

Vol 474 (21) ◽

pp. 3615-3626 ◽

Cited By ~ 11

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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On the fate of early endosomes

Biological Chemistry ◽

10.1515/bc.2009.056 ◽

2009 ◽

Vol 390 (8) ◽

Cited By ~ 40

Author(s):

Anne Spang

Keyword(s):

Molecular Mechanisms ◽

Signaling Molecules ◽

Early Endosome ◽

Late Endosome ◽

Late Endosomes ◽

Early Endosomes ◽

Logistic Problem ◽

Intellectual Level ◽

Intense Research

Abstract Proteins are endocytosed by various pathways into the cell. All these pathways converge at the level of the early endosome. The fate of the early endosome and how proteins are sorted into recycling and late endosomes/multi-vesicular body is a matter of debate and intense research. Obviously, the transition from early to late endosome poses an interesting logistic problem and would merit attention on an intellectual level. Numerous diseases are also caused by defects in turning off/over signaling molecules or mis-sorting of proteins at the level of the early endosome. This brief review aims to discuss different molecular mechanisms whereby early-to-late endosome transition could be achieved.

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Annexin A6 modulates TBC1D15/Rab7/StARD3 axis to control endosomal cholesterol export in NPC1 cells

Cellular and Molecular Life Sciences ◽

10.1007/s00018-019-03330-y ◽

2019 ◽

Vol 77 (14) ◽

pp. 2839-2857 ◽

Cited By ~ 4

Author(s):

Elsa Meneses-Salas ◽

Ana García-Melero ◽

Kristiina Kanerva ◽

Patricia Blanco-Muñoz ◽

Frederic Morales-Paytuvi ◽

...

Keyword(s):

Late Endosome ◽

Dependent Manner ◽

Lipid Transfer ◽

Cholesterol Accumulation ◽

Membrane Contact Sites ◽

Late Endosomes ◽

Membrane Contact ◽

Domain 3 ◽

Niemann Pick ◽

Mutant Cells

Abstract Cholesterol accumulation in late endosomes is a prevailing phenotype of Niemann-Pick type C1 (NPC1) mutant cells. Likewise, annexin A6 (AnxA6) overexpression induces a phenotype reminiscent of NPC1 mutant cells. Here, we demonstrate that this cellular cholesterol imbalance is due to AnxA6 promoting Rab7 inactivation via TBC1D15, a Rab7-GAP. In NPC1 mutant cells, AnxA6 depletion and eventual Rab7 activation was associated with peripheral distribution and increased mobility of late endosomes. This was accompanied by an enhanced lipid accumulation in lipid droplets in an acyl-CoA:cholesterol acyltransferase (ACAT)-dependent manner. Moreover, in AnxA6-deficient NPC1 mutant cells, Rab7-mediated rescue of late endosome-cholesterol export required the StAR-related lipid transfer domain-3 (StARD3) protein. Electron microscopy revealed a significant increase of membrane contact sites (MCS) between late endosomes and ER in NPC1 mutant cells lacking AnxA6, suggesting late endosome-cholesterol transfer to the ER via Rab7 and StARD3-dependent MCS formation. This study identifies AnxA6 as a novel gatekeeper that controls cellular distribution of late endosome-cholesterol via regulation of a Rab7-GAP and MCS formation.

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INPP4B promotes PI3Kα-dependent late endosome formation and Wnt/β-catenin signaling in breast cancer

Nature Communications ◽

10.1038/s41467-021-23241-6 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Samuel J. Rodgers ◽

Lisa M. Ooms ◽

Viola M. J. Oorschot ◽

Ralf B. Schittenhelm ◽

Elizabeth V. Nguyen ◽

...

Keyword(s):

Breast Cancer ◽

Tumor Growth ◽

Breast Cancer Cells ◽

Triple Negative ◽

Akt Signaling ◽

Late Endosome ◽

Breast Cancers ◽

Lysosomal Degradation ◽

Late Endosomes ◽

Mrna And Protein Expression

AbstractINPP4B suppresses PI3K/AKT signaling by converting PI(3,4)P2 to PI(3)P and INPP4B inactivation is common in triple-negative breast cancer. Paradoxically, INPP4B is also a reported oncogene in other cancers. How these opposing INPP4B roles relate to PI3K regulation is unclear. We report PIK3CA-mutant ER+ breast cancers exhibit increased INPP4B mRNA and protein expression and INPP4B increased the proliferation and tumor growth of PIK3CA-mutant ER+ breast cancer cells, despite suppression of AKT signaling. We used integrated proteomics, transcriptomics and imaging to demonstrate INPP4B localized to late endosomes via interaction with Rab7, which increased endosomal PI3Kα-dependent PI(3,4)P2 to PI(3)P conversion, late endosome/lysosome number and cargo trafficking, resulting in enhanced GSK3β lysosomal degradation and activation of Wnt/β-catenin signaling. Mechanistically, Wnt inhibition or depletion of the PI(3)P-effector, Hrs, reduced INPP4B-mediated cell proliferation and tumor growth. Therefore, INPP4B facilitates PI3Kα crosstalk with Wnt signaling in ER+ breast cancer via PI(3,4)P2 to PI(3)P conversion on late endosomes, suggesting these tumors may be targeted with combined PI3K and Wnt/β-catenin therapies.

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