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.

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 The Inhibitory Role of Rab11b in Osteoclastogenesis through Triggering Lysosome-Induced Degradation of c-Fms and RANK Surface Receptors

2020 ◽  
Vol 21 (24) ◽  
pp. 9352
Author(s):  
Manh Tien Tran ◽  
Yuka Okusha ◽  
Yunxia Feng ◽  
Masatoshi Morimatsu ◽  
Penggong Wei ◽  
...  
Keyword(s):  
Osteoclast Differentiation ◽  
Activated T Cells ◽  
Surface Receptors ◽  
Late Endosomes ◽  
Early Endosomes ◽  
D Cell ◽  
D Cells ◽  
Vitro Model

Rab11b, abundantly enriched in endocytic recycling compartments, is required for the establishment of the machinery of vesicle trafficking. Yet, no report has so far characterized the biological function of Rab11b in osteoclastogenesis. Using in vitro model of osteoclasts differentiated from murine macrophages like RAW-D cells or bone marrow-derived macrophages, we elucidated that Rab11b served as an inhibitory regulator of osteoclast differentiation sequentially via (i) abolishing surface abundance of RANK and c-Fms receptors; and (ii) attenuating nuclear factor of activated T-cells c1 (NFATc-1) upstream signaling cascades, following RANKL stimulation. Rab11b was localized in early and late endosomes, Golgi complex, and endoplasmic reticulum; moreover, its overexpression enlarged early and late endosomes. Upon inhibition of lysosomal function by a specific blocker, chloroquine (CLQ), we comprehensively clarified a novel function of lysosomes on mediating proteolytic degradation of c-Fms and RANK surface receptors, drastically ameliorated by Rab11b overexpression in RAW-D cell-derived osteoclasts. These findings highlight the key role of Rab11b as an inhibitor of osteoclastogenesis by directing the transport of c-Fms and RANK surface receptors to lysosomes for degradation via the axis of early endosomes-late endosomes-lysosomes, thereby contributing towards the systemic equilibrium of the bone resorption phase.

scholarly journals The R-SNARE Endobrevin/VAMP-8 Mediates Homotypic Fusion of Early Endosomes and Late Endosomes

2000 ◽  
Vol 11 (10) ◽  
pp. 3289-3298 ◽  
Author(s):  
Wolfram Antonin ◽  
Claudia Holroyd ◽  
Ritva Tikkanen ◽  
Stefan Höning ◽  
Reinhard Jahn
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Subcellular Fractionation ◽  
Immunogold Electron Microscopy ◽  
Fusion Reactions ◽  
Coated Pits ◽  
Late Endosomes ◽  
Early Endosomes ◽  
Golgi Network

Endobrevin/VAMP-8 is an R-SNARE localized to endosomes, but it is unknown in which intracellular fusion step it operates. Using subcellular fractionation and quantitative immunogold electron microscopy, we found that endobrevin/VAMP-8 is present on all membranes known to communicate with early endosomes, including the plasma membrane, clathrin-coated pits, late endosomes, and membranes of thetrans-Golgi network. Affinity-purified antibodies that block the ability of endobrevin/VAMP-8 to form SNARE core complexes potently inhibit homotypic fusion of both early and late endosomes in vitro. Fab fragments were as active as intact immunoglobulin Gs. Recombinant endobrevin/VAMP-8 inhibited both fusion reactions with similar potency. We conclude that endobrevin/VAMP-8 operates as an R-SNARE in the homotypic fusion of early and late endosomes.

scholarly journals ARHGEF3 regulates skeletal muscle regeneration and strength through autophagy

2020 ◽  
Author(s):  
Jae-Sung You ◽  
Nilmani Singh ◽  
Adriana Reyes-Ordonez ◽  
Nidhi Khanna ◽  
Zehua Bao ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Muscle Regeneration ◽  
Mammalian Target Of Rapamycin ◽  
Akt Signaling ◽  
Skeletal Muscle Regeneration ◽  
Myoblast Differentiation ◽  
Independent Manner ◽  
And Function ◽  
Old Mice

SummarySkeletal muscle regeneration is essential for restoring muscle function upon injury and for the maintenance of muscle health with aging. ARHGEF3, a Rho-specific GEF, negatively regulates myoblast differentiation via mammalian target of rapamycin complex 2 (mTORC2)-Akt signaling in a GEF-independent manner in vitro. Here, we investigated ARHGEF3’s role in skeletal muscle regeneration by creating ARHGEF3 KO mice. These mice exhibited no discernible phenotype under normal conditions. Upon injury, however, ARHGEF3 deficiency enhanced the mass, fiber size and function of regenerating muscles in both young and aged mice. Surprisingly, these effects were not mediated by mTORC2-Akt signaling, but by the GEF activity of ARHGEF3. Furthermore, ARHGEF3 KO promoted muscle regeneration through activation of autophagy, a process that is also critical for maintaining muscle strength. Accordingly, in old mice, ARHGEF3 depletion prevented muscle weakness by restoring autophagy flux. Collectively, our findings identify an unexpected link between ARHGEF3 and autophagy-related muscle pathophysiology.

scholarly journals Effect of Bafilomycin A1 and Nocodazole on Endocytic Transport in HeLa Cells: Implications for Viral Uncoating and Infection

1998 ◽  
Vol 72 (12) ◽  
pp. 9645-9655 ◽  
Author(s):  
Nora Bayer ◽  
Daniela Schober ◽  
Elisabeth Prchla ◽  
Robert F. Murphy ◽  
Dieter Blaas ◽  
...  
Keyword(s):  
Viral Infection ◽  
Conformational Change ◽  
Hela Cells ◽  
Specific Inhibitor ◽  
Fluid Phase ◽  
Inhibitory Effect ◽  
Bafilomycin A1 ◽  
Late Endosomes ◽  
Early Endosomes ◽  
Endosomal Ph

ABSTRACT Bafilomycin A1 (baf), a specific inhibitor of vacuolar proton ATPases, is commonly employed to demonstrate the requirement of low endosomal pH for viral uncoating. However, in certain cell types baf also affects the transport of endocytosed material from early to late endocytic compartments. To characterize the endocytic route in HeLa cells that are frequently used to study early events in viral infection, we used 35S-labeled human rhinovirus serotype 2 (HRV2) together with various fluid-phase markers. These virions are taken up via receptor-mediated endocytosis and undergo a conformational change to C-antigenic particles at a pH of <5.6, resulting in release of the genomic RNA and ultimately in infection (E. Prchla, E. Kuechler, D. Blaas, and R. Fuchs, J. Virol. 68:3713–3723, 1994). As revealed by fluorescence microscopy and subcellular fractionation of microsomes by free-flow electrophoresis (FFE), baf arrests the transport of all markers in early endosomes. In contrast, the microtubule-disrupting agent nocodazole was found to inhibit transport by accumulating marker in endosomal carrier vesicles (ECV), a compartment intermediate between early and late endosomes. Accordingly, lysosomal degradation of HRV2 was suppressed, whereas its conformational change and infectivity remained unaffected by this drug. Analysis of the subcellular distribution of HRV2 and fluid-phase markers in the presence of nocodazole by FFE revealed no difference from the control incubation in the absence of nocodazole. ECV and late endosomes thus have identical electrophoretic mobilities, and intraluminal pHs of <5.6 and allow uncoating of HRV2. As bafilomycin not only dissipates the low endosomal pH but also blocks transport from early to late endosomes in HeLa cells, its inhibitory effect on viral infection could in part also be attributed to trapping of virus in early endosomes which might lack components essential for uncoating. Consequently, inhibition of viral uncoating by bafilomycin cannot be taken to indicate a low pH requirement only.

scholarly journals Annexin A8 Regulates Late Endosome Organization and Function

2008 ◽  
Vol 19 (12) ◽  
pp. 5267-5278 ◽  
Author(s):  
Verena Goebeler ◽  
Michaela Poeter ◽  
Dagmar Zeuschner ◽  
Volker Gerke ◽  
Ursula Rescher
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Actin Filaments ◽  
Late Endosome ◽  
Endocytic Pathway ◽  
Mitogen Activated Protein Kinase ◽  
Late Endosomes ◽  
Endosomal Membranes ◽  
Epidermal Growth ◽  
And Function

Different classes of endosomes exhibit a characteristic intracellular steady-state distribution governed by interactions with the cytoskeleton. Late endosomes, organelles of the degradative lysosomal route, seem to require associated actin filaments for proper localization and function. We show here that the F-actin and phospholipid binding protein annexin A8 is associated specifically with late endosomes. Altering intracellular annexin A8 levels drastically affected the morphology and intracellular distribution of late endosomes. Trafficking through the degradative pathway was delayed in the absence of annexin A8, resulting in attenuated ligand-induced degradation of the epidermal growth factor receptor and prolonged epidermal growth factor-induced activation of mitogen-activated protein kinase. Depletion of annexin A8 reduced the association of late endosomal membranes with actin filaments. These results indicate that the defective cargo transport through the late endocytic pathway and the imbalanced signaling of activated receptors observed in the absence of annexin A8 results from the disturbed association of late endosomal membranes with the actin network, resulting in impaired actin-based late endosome motility.

Cyclic AMP-independent ATF family members interact with NF-kappa B and function in the activation of the E-selectin promoter in response to cytokines

1993 ◽  
Vol 13 (11) ◽  
pp. 7180-7190 ◽  
Author(s):  
W Kaszubska ◽  
R Hooft van Huijsduijnen ◽  
P Ghersa ◽  
A M DeRaemy-Schenk ◽  
B P Chen ◽  
...  
Keyword(s):  
Cyclic Amp ◽  
Consensus Sequence ◽  
Direct Interaction ◽  
Nf Kappa B ◽  
Independent Manner ◽  
Protein Protein Interaction ◽  
Responsive Element ◽  
And Function

We previously reported that NF-kappa B and a complex we referred to as NF-ELAM1 play a central role in cytokine-induced expression of the E-selectin gene. In this study we identify cyclic AMP (cAMP)-independent members of the ATF family binding specifically to the NF-ELAM1 promoter element. The NF-ELAM1 element (TGACATCA) differs by a single nucleotide substitution from the cAMP-responsive element consensus sequence. We demonstrate that this sequence operates in a cAMP-independent manner to induce transcription and thus define it as a non-cAMP-responsive element (NCRE). We show that ATFa is a component of the NF-ELAM1 complex and its overexpression activates the E-selectin promoter. In addition, ATFa, ATF2, and ATF3 interact directly with NF-kappa B in vitro, linking two unrelated families of transcription factors in a novel protein-protein interaction. Furthermore, we demonstrate that the ability of overexpressed NF-kappa B to transactivate the E-selectin promoter in vivo is dependent on the NF-ELAM1 complex. Our results suggest that a direct interaction between ATFs and NF-kappa B is, at least in part, the mechanism by which these factors specifically regulate E-selectin promoter activity.

scholarly journals Acidification of endosome subpopulations in wild-type Chinese hamster ovary cells and temperature-sensitive acidification-defective mutants.

1989 ◽  
Vol 108 (4) ◽  
pp. 1291-1300 ◽  
Author(s):  
S Schmid ◽  
R Fuchs ◽  
M Kielian ◽  
A Helenius ◽  
I Mellman
Keyword(s):  
Chinese Hamster Ovary ◽  
Cho Cells ◽  
Chinese Hamster ◽  
Temperature Sensitive ◽  
Cell Fractionation ◽  
Permissive Temperature ◽  
Wild Type ◽  
Late Endosomes ◽  
Early Endosomes

During endocytosis in Chinese hamster ovary (CHO) cells, Semliki Forest virus (SFV) passes through two distinct subpopulations of endosomes before reaching lysosomes. One subpopulation, defined by cell fractionation using free flow electrophoresis as "early endosomes," constitutes the major site of membrane and receptor recycling; while "late endosomes," an electrophoretically distinct endosome subpopulation, are involved in the delivery of endosomal content to lysosomes. In this paper, the pH-sensitive conformational changes of the SFV E1 spike glycoprotein were used to study the acidification of these defined endosome subpopulations in intact wild-type and acidification-defective CHO cells. Different virus strains were used to measure the kinetics at which internalized SFV was delivered to endosomes of pH less than or equal to 6.2 (the pH at which wild-type E1 becomes resistant to trypsin digestion) vs. endosomes of pH less than or equal to 5.3 (the threshold pH for E1 of the SFV mutant fus-1). By correlating the kinetics of acquisition of E1 trypsin resistance with the transfer of SFV among distinct endosome subpopulations defined by cell fractionation, we found that after a brief residence in vesicles of relatively neutral pH, internalized virus encountered pH less than or equal to 6.2 in early endosomes with a t1/2 of 5 min. Although a fraction of the virus reached a pH of less than or equal to 5.3 in early endosomes, most fus-1 SFV did not exhibit the acid-induced conformational change until arrival in late endosomes (t1/2 = 8-10 min). Thus, acidification of both endosome subpopulations was heterogeneous. However, passage of SFV through a less acidic early endosome subpopulation always preceded arrival in the more acidic late endosome subpopulation. In mutant CHO cells with temperature-sensitive defects in endosome acidification in vitro, acidification of both early and late endosomes was found to be impaired at the restrictive temperature (41 degrees C). The acidification defect was also found to be partially penetrant at the permissive temperature, resulting in the inability of any early endosomes in these cells to attain pH less than or equal to 5.3. In vitro studies of endosomes isolated from mutant cells suggested that the acidification defect is most likely in the proton pump itself. In one mutant, this defect resulted in increased sensitivity of the electrogenic H+ pump to fluctuations in the endosomal membrane potential.

scholarly journals Plasticity of early endosomes

1992 ◽  
Vol 103 (2) ◽  
pp. 335-348 ◽  
Author(s):  
R.G. Parton ◽  
P. Schrotz ◽  
C. Bucci ◽  
J. Gruenberg
Keyword(s):  
Fluid Phase ◽  
Early Endosome ◽  
Cross Linking ◽  
High Plasticity ◽  
Late Endosomes ◽  
Low Concentrations ◽  
Cell Surface Biotinylation ◽  
Early Endosomes

We observed that the structural organization of early endosomes was significantly modified after cell surface biotinylation followed by incubation in the presence of low concentrations of avidin. Under these conditions early endosomes increased in size to form structures which extended over several micrometers and which had an intra-luminal content with a characteristic electron-dense appearance. The modified early endosomes were not formed when either avidin or biotinylation was omitted, suggesting that they resulted from the cross-linking of internalized biotinylated proteins by avidin. Accumulation of a fluid-phase tracer was increased after the avidin-biotin treatment (145% after 45 min). Both recycling and transport to the late endosomes still occurred, albeit to a somewhat lower extent than in control cells. Quantitative electron microscopy showed that the volume of the endosomal compartment was increased approximately 1.5-fold but that the surface area of the compartment decreased relative to its volume after avidin-biotin treatment. Finally, overexpression of a rab5 mutant, which is known to inhibit early endosome fusion in vitro, prevented the formation of these structures in vivo and caused early endosome fragmentation. Altogether, our data suggest that early endosomes exhibit a high plasticity in vivo. Cross-linking appears to interfere with this dynamic process but does not arrest membrane traffic to/from early endosomes.

scholarly journals Isolation and characterization of mutant CHO cell lines with compartment-specific resistance to brefeldin A.

1994 ◽  
Vol 126 (1) ◽  
pp. 65-75 ◽  
Author(s):  
J P Yan ◽  
M E Colon ◽  
L A Beebe ◽  
P Melançon
Keyword(s):  
Growth Inhibition ◽  
Cell Lines ◽  
Brefeldin A ◽  
Cho Cell ◽  
Isolation And Characterization ◽  
Early Endosomes ◽  
Intracellular Target ◽  
And Function ◽  
Mutant Lines

22 CHOBFY (BFY) cell lines were isolated at a frequency 2-30 x 10(-7) from mutagenized populations on the basis of their ability to grow in the presence of 1 microgram/ml brefeldin A (BFA). Four of the five mutant lines tested are genetically stable and none of the mutant lines characterized degrade this drug. Immunofluorescence studies reveal that whereas early endosomes and the Golgi complex have nearly identical BFA sensitivities in the parent CHO line, the relative sensitivities of these two organelles were dramatically altered in all six mutant lines tested. Four cell lines maintain normal Golgi appearance at a BFA concentration as high as 10 micrograms/ml. Mutant lines show wide variation in the level of resistance to growth inhibition by BFA, but none of the mutant lines characterized grow above 2 micrograms/ml BFA. This specific growth inhibition is observed under conditions where Golgi morphology and function remain unaffected, suggesting that some factor(s) unrelated to Golgi function remains sensitive to BFA in BFY mutant lines. These observations provide strong evidence for the presence of multiple, organelle-specific targets for BFA. Cell-free measurements with membrane extracts establish that resistance to BFA in BFY-1 cells involves a membrane-associated factor distinct from ARFs and coatomers. This collection of mutant lines may prove valuable for the identification of intracellular target(s) for BFA and/or of effectors that interact upstream or downstream with these targets, thereby uncovering the cascade which regulates assembly of organelle-specific coats.

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