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

2004 ◽  
Vol 15 (1) ◽  
pp. 345-358 ◽  
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.

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 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 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 Endosomal trafficking regulates receptor-mediated transcytosis of antibodies across the blood brain barrier

2017 ◽  
Vol 38 (4) ◽  
pp. 727-740 ◽  
Author(s):  
Arsalan S Haqqani ◽  
Christie E Delaney ◽  
Eric Brunette ◽  
Ewa Baumann ◽  
Graham K Farrington ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Blood Brain Barrier ◽  
Early Endosome ◽  
Brain Barrier ◽  
Endosomal Trafficking ◽  
Brain Endothelial Cells ◽  
Density Fractions ◽  
Blood Brain ◽  
Late Endosomes ◽  
Early Endosomes

Current methods for examining antibody trafficking are either non-quantitative such as immunocytochemistry or require antibody labeling with tracers. We have developed a multiplexed quantitative method for antibody ‘tracking’ in endosomal compartments of brain endothelial cells. Rat brain endothelial cells were co-incubated with blood-brain barrier (BBB)-crossing FC5, monovalent FC5Fc or bivalent FC5Fc fusion antibodies and control antibodies. Endosomes were separated using sucrose-density gradient ultracentrifugation and analyzed using multiplexed mass spectrometry to simultaneously quantify endosomal markers, receptor-mediated transcytosis (RMT) receptors and the co-incubated antibodies in each fraction. The quantitation showed that markers of early endosomes were enriched in high-density fractions (HDF), whereas markers of late endosomes and lysosomes were enriched in low-density fractions (LDF). RMT receptors, including transferrin receptor, showed a profile similar to that of early endosome markers. The in vitro BBB transcytosis rates of antibodies were directly proportional to their partition into early endosome fractions of brain endothelial cells. Addition of the Fc domain resulted in facilitated antibody ‘redistribution’ from LDF into HDF and additionally into multivesicular bodies (MVB). Sorting of various FC5 antibody formats away from late endosomes and lysosomes and into early endosomes and a subset of MVB results in increased antibody transcytosis at the abluminal side of the BBB.

scholarly journals EGF-induced PIP2 hydrolysis releases and activates cofilin locally in carcinoma cells

2007 ◽  
Vol 179 (6) ◽  
pp. 1247-1259 ◽  
Author(s):  
Jacco van Rheenen ◽  
Xiaoyan Song ◽  
Wies van Roosmalen ◽  
Michael Cammer ◽  
Xiaoming Chen ◽  
...  
Keyword(s):  
Actin Polymerization ◽  
Carcinoma Cells ◽  
Actin Binding ◽  
Temporal Regulation ◽  
Vitro Binding ◽  
Epidermal Growth ◽  
Membrane Bound ◽  
Regulation Of Actin Cytoskeleton

Lamellipodial protrusion and directional migration of carcinoma cells towards chemoattractants, such as epidermal growth factor (EGF), depend upon the spatial and temporal regulation of actin cytoskeleton by actin-binding proteins (ABPs). It is generally hypothesized that the activity of many ABPs are temporally and spatially regulated by PIP2; however, this is mainly based on in vitro–binding and structural studies, and generally in vivo evidence is lacking. Here, we provide the first in vivo data that directly visualize the spatial and temporal regulation of cofilin by PIP2 in living cells. We show that EGF induces a rapid loss of PIP2 through PLC activity, resulting in a release and activation of a membrane-bound pool of cofilin. Upon release, we find that cofilin binds to and severs F-actin, which is coincident with actin polymerization and lamellipod formation. Moreover, our data provide evidence for how PLC is involved in the formation of protrusions in breast carcinoma cells during chemotaxis and metastasis towards EGF.

Virotoxins polymerize actin and induce membrane fragmentation in cytoplasmic preparations of Amoeba proteus

1992 ◽  
Vol 70 (8) ◽  
pp. 719-723 ◽  
Author(s):  
Claude Gicquaud ◽  
Michel Paré
Keyword(s):  
Actin Polymerization ◽  
Amoeba Proteus ◽  
Cytoplasmic Actin ◽  
Membrane Bound ◽  
Spherical Vesicles

Virotoxins and phalloidin are peptides that induce actin polymerization in vitro. We have compared the effect of five virotoxins and phalloidin on the ultrastructure of spread preparations of Amoeba proteus cytoplasm. Like phalloidin, the five virotoxins induce polymerization of cytoplasmic actin. Moreover, the virotoxins, but not phalloidin, induce membrane fragmentation in small spherical vesicles. We, therefore, conclude that these virotoxins may have another membrane-bound target besides actin.Key words: phalloidin, virotoxin, motility, actin, membrane.

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.

The endocytic pathway in animal cells

1990 ◽  
Vol 48 (3) ◽  
pp. 41-41
Author(s):  
Gareth Griffiths
Keyword(s):  
Early Endosome ◽  
Endocytic Pathway ◽  
Three Dimensions ◽  
Cell Periphery ◽  
Animal Cells ◽  
The Third ◽  
Negative Stain ◽  
Late Endosomes ◽  
Work Done

Our recent data have provided evidence that the endocytic pathway comprises four structurally and functionally distinct compartments in addition to the clathrin coated vesicles. The most proximal of these compartments, the early endosome, is structurally complex, consists of cisternal, tubular, as well as vesicular domains and is predominantly localized to the cell periphery. We have proposed that the vesicular domains bud off the early endosome to form the spherical endosome (carrier) vesicles that transport material from early to late endosomes, a process that requires microtubules. These endosome vesicles are spherical (0.2-0.4μm diameter) structures whose lumen is packed with thin tubules. The structure of the latter has recently become more evident in negative stain preparations. These vesicles can also be shown to bind microtubules in vitro (work done in collaboration with Jean Gruenberg's group, EMBL). The third compartment, the late endosome or prelysosomal compartment (PLC) is, like the early endosome, complex in three-dimensions.

Movement of spotted fever rickettsiae through tick host cells in vitro

1996 ◽  
Vol 54 ◽  
pp. 810-811
Author(s):  
U. G. Munderloh ◽  
S. F. Hayes ◽  
J. Cummings ◽  
T. J. Kurtti
Keyword(s):  
Mammalian Cells ◽  
Actin Polymerization ◽  
Spotted Fever ◽  
Shigella Flexneri ◽  
Light And Electron Microscopy ◽  
Host Cells ◽  
Spotted Fever Group ◽  
Symbiotic Association ◽  
Obligate Intracellular

Spotted fever group (SFG) rickettsiae are obligate intracellular prokaryotes that include tick-borne pathogens of animals and man as well as organisms that live in symbiotic association with their tick hosts. A striking feature of the behavior of pathogenic rickettsiae in the vertebrate is their ability to quickly disseminate between cells from the original site of entry shortly after infection, and before severe lesions are detected. Similarly, ticks become systemically infected with SFG rickettsiae, indicating that an efficient mechanism of dispersal also exists in the vector. This is accomplished despite the fact that rickettsiae are not motile.Kadurugamuwa et al. (1991) have used light and electron microscopy to show that Shigella flexneri utilize host cytoskeletal components to travel through cytoplasmic extensions and penetrate into neighboring cells. Using mammalian cells cultured in vitro, Heinzen et al. (1993) have demonstrated that SFG rickettsiae cause host cell actin polymerization at one rickettsial pole causing them to be propelled through the cytoplasm, and to transfer rapidly from cell to cell.

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