Formation Of Internal Vesicles Research Articles

Interaction Mechanisms of Giant Unilamellar Vesicles with Hydrophobic Glass Surfaces and Silicone Oil-Water Interfaces: Adsorption, Deformation, Rupture, Dynamic Shape Changes, Internal Vesicle Formation, and Desorption.

Phospholipid monolayers at oil-water interfaces are often obtained via vesicle adsorption. However, the interaction mechanisms of vesicles with these oil-water interfaces remain unclear. Herein, we studied the adsorption of giant unilamellar vesicles (GUVs) of approximately 2-5 μm diameter onto silicone oil-water interfaces and glass surfaces modified with hexamethyldisilazane (HMDS) and octadecyltrimethoxysilane (ODTMS) using fluorescence microscopy. The GUVs exhibited various modes of interaction, adsorbing on the silanized glass surfaces without sizable deformation, whereas GUVs bound to the silicone oil-water interface exhibited large deformation. After adsorption, GUV rupture occurred within 350, 110, and 3 ms on HMDS-modified glass, ODTMS-modified glass, and silicone oil-water interface, respectively. On glass surfaces, GUV rupture was often initiated and proceeded with pore formation near the surface. The monolayer patches formed by GUV rupture on HMDS-modified glass remained for at least 1 h over an area approximately twice of that estimated from the original GUV. On the ODTMS-modified glass and silicone oil surfaces, the monolayer patch structures disappeared in milliseconds owing to lipid diffusion across the interface. When adsorbed on the oil-water interface, the GUVs spontaneously underwent dynamic shape changes, internal vesicle formation, and desorption without rupture. Thus, it can be concluded that these different pathways arose from different lipid-surface affinities.

Cell-free reconstitution of multivesicular body (MVB) cargo sorting.

The signaling activity of cell surface localized membrane proteins occurs primarily while these proteins are located on the plasma membrane but is, in some cases, not terminated until the proteins are degraded. Following internalization and movement through the endocytic pathway en route to lysosomes, membrane proteins transit a late endosomal organelle called the multivesicular body (MVB). MVBs are formed by invagination of the limiting membrane of endosomes, resulting in an organelle possessing a limiting membrane and containing internal vesicles. The fate of an internalized membrane protein depends on whether it buds outwardly from the endosomal membrane, promoting recycling and continued signaling, or is internalized into internal MVB vesicles and is ultimately degraded upon MVB-lysosome fusion. The molecular machinery that regulates the separation of membrane proteins destined for degradation from those resulting in surface expression is not well understood.To elucidate the molecular mechanisms that underlie membrane protein sorting, we have reconstituted an endosomal sorting event under cell-free conditions. We took advantage of the itinerary of a prototypical membrane protein, the epidermal growth factor receptor (EGFR) and designed a biochemical monitor for cargo movement into internal MVB vesicles that is generally modifiable for other membrane proteins. Since is it not known how internal vesicle formation is related to cargo sorting, morphological examination using transmission electron microscopy (TEM) allows separate monitoring of vesicle formation. We have determined that MVB sorting is dependent on cytosolic components, adenosine triphosphate (ATP), time, temperature, and an intact proton gradient. This assay reconstitutes the maturation of late endosomes and allows the morphological and biochemical examination of vesicle formation and membrane protein sorting.

The Arabidopsis Endosomal Sorting Complex Required for Transport III Regulates Internal Vesicle Formation of the Prevacuolar Compartment and Is Required for Plant Development.

We have established an efficient transient expression system with several vacuolar reporters to study the roles of endosomal sorting complex required for transport (ESCRT)-III subunits in regulating the formation of intraluminal vesicles of prevacuolar compartments (PVCs)/multivesicular bodies (MVBs) in plant cells. By measuring the distributions of reporters on/within the membrane of PVC/MVB or tonoplast, we have identified dominant negative mutants of ESCRT-III subunits that affect membrane protein degradation from both secretory and endocytic pathways. In addition, induced expression of these mutants resulted in reduction in luminal vesicles of PVC/MVB, along with increased detection of membrane-attaching vesicles inside the PVC/MVB. Transgenic Arabidopsis (Arabidopsis thaliana) plants with induced expression of ESCRT-III dominant negative mutants also displayed severe cotyledon developmental defects with reduced cell size, loss of the central vacuole, and abnormal chloroplast development in mesophyll cells, pointing out an essential role of the ESCRT-III complex in postembryonic development in plants. Finally, membrane dissociation of ESCRT-III components is important for their biological functions and is regulated by direct interaction among Vacuolar Protein Sorting-Associated Protein20-1 (VPS20.1), Sucrose Nonfermenting7-1, VPS2.1, and the adenosine triphosphatase VPS4/SUPPRESSOR OF K+ TRANSPORT GROWTH DEFECT1.

Extensive Vesicle Formation by a Monotopic Membrane Glycosyltransferase

Over-expression of the foreign monotopic glycosyltransferase MGS in Escherichia coli triggers the formation of a large amount of internal vesicles (J. Biol. Chem. 284:33904, 2009). This phenomenon seems to be directly related with the interactions between the protein and the inner membrane lipids. It has been shown that this massive vesiculation process is not induced by the enzymatic activity of the protein, but rather due to the structural and mechanical properties of MGS. In this work we study the specific interactions of MGS with the lipid membrane and we unveil the biophysical mechanisms that lead to the massive formation of internal vesicles in E. coli.We use both molecular dynamics (MD) simulations and analytical modeling to understand the molecular basis for this phenomenon and the relative importance of various potential driving forces that may induce membrane deformations, such as lipid-protein interactions and protein crowding. First we studied the specific interactions between a single MGS and the lipid membrane. By using MD simulations we show that the N-domain (of the double Rossmann fold) has a leading role in modulating the bonds with the lipid bilayer, and that the connection between MGS and lipidic membranes is modulated by both hydrophobic and electrostatic interactions. These conclusions are in perfect agreement with chemical experiments run in parallel.Furthermore, we are using coarse-grained MD simulations to study the effects of asymmetric crowding and determine how several MGS molecules bound to the same bilayer leaflet may induce membrane deformations and trigger the formation of internal vesicles. Both experiments and simulations allow us to gain a deeper insight into the mechanism of vesicle formation by MGS, and potentially by other monotopic proteins.

Disruption of the annexin A1/S100A11 complex increases the migration and clonogenic growth by dysregulating epithelial growth factor (EGF) signaling

Endocytosis of activated growth factor receptors regulates spatio-temporal cellular signaling. In the case of the EGF receptor, sorting into multivesicular bodies (MVBs) controls signal termination and subsequently leads to receptor degradation in lysosomes. Annexin A1, a Ca2+-regulated membrane binding protein often deregulated in human cancers, interacts with the EGF receptor and is phosphorylated by internalized EGF receptor on endosomes. Most relevant for EGF receptor signal termination, annexin A1 is required for the formation of internal vesicles in MVBs that sequester ligand-bound EGF receptor away from the limiting membrane. To elucidate the mechanism underlying annexin A1-dependent EGF receptor trafficking we employed an N-terminally truncated annexin A1 mutant that lacks the EGF receptor phosphorylation site and the site for interaction with its protein ligand S100A11. Overexpression of this dominant-negative mutant induces a delay in EGF-induced EGF receptor transport to the LAMP1-positive late endosomal/lysosomal compartment and impairs ligand-induced EGF receptor degradation. Consistent with these findings, EGF-stimulated EGF receptor and MAP kinase pathway signaling is prolonged. Importantly, depletion of S100A11 also results in a delayed EGF receptor transport and prolonged MAP kinase signaling comparable to the trafficking defect observed in cells expressing the N-terminally truncated annexin A1 mutant. These results strongly suggest that the function of annexin A1 as a regulator of EGF receptor trafficking, degradation and signaling is critically mediated through an N-terminal interaction with S100A11 in the endosomal compartment. This interaction appears to be essential for lysosomal targeting of the EGF receptor, possibly by providing a physical scaffold supporting inward vesiculation in MVBs. This article is part of a Special Issue entitled: 12th European Symposium on Calcium.

Perforin activity at membranes leads to invaginations and vesicle formation

The cytotoxic cell granule secretory pathway is essential for immune defence. How the pore-forming protein perforin (PFN) facilitates the cytosolic delivery of granule-associated proteases (granzymes) remains enigmatic. Here we show that PFN is able to induce invaginations and formation of complete internal vesicles in giant unilamellar vesicles. Formation of internal vesicles depends on native PFN and calcium and antibody labeling shows the localization of PFN at the invaginations. This vesiculation is recapitulated in large unilamellar vesicles and in this case PFN oligomers can be seen associated with the necks of the invaginations. Capacitance measurements show PFN is able to increase a planar lipid membrane surface area in the absence of pore formation, in agreement with the ability to induce invaginations. Finally, addition of PFN to Jurkat cells causes the formation of internal vesicles prior to pore formation. PFN is capable of triggering an endocytosis-like event in addition to pore formation, suggesting a new paradigm for its role in delivering apoptosis-inducing granzymes into target cells.

Cell-Free Reconstitution of Multivesicular Body Formation and Receptor Sorting

The number of surface membrane proteins and their residence time on the plasma membrane are critical determinants of cellular responses to cues that can control plasticity, growth and differentiation. After internalization, the ultimate fate of many plasma membrane proteins is dependent on whether they are sorted for internalization into the lumenal vesicles of multivesicular bodies (MVBs), an obligate step prior to lysosomal degradation. To help to elucidate the mechanisms underlying MVB sorting, we have developed a novel cell-free assay that reconstitutes the sorting of a prototypical membrane protein, the epidermal growth factor receptor, with which we have probed some of its molecular requirements. The sorting event measured is dependent on cytosol, ATP, time, temperature and an intact proton gradient. Depletion of Hrs inhibited biochemical and morphological measures of sorting that were rescued by inclusion of recombinant Hrs in the assay. Moreover, depletion of signal-transducing adaptor molecule (STAM), or addition of mutated ATPase-deficient Vps4, also inhibited sorting. This assay reconstitutes the maturation of late endosomes, including the formation of internal vesicles and the sorting of a membrane protein, and allows biochemical investigation of this process.

HIV budding, a target for innate antiviral response

The curvature of host cell membranes during the budding of HIV viruses occurs by an opposite topology, as compared to endocytosis and phagocytosis phenomena. This topology of vesicle formation is indeed observed during the formation of internal vesicles of late endosomal compartments called multivesicular bodies (MVB). Formation of these vesicles is controlled by ubiquitylation and the recruitment of the endosomal sorting complex required for transport (ESCRT). The sorting of HIV-1 structural protein Pr55gag into the viral bud requires the recruitment of the ESCRT complex, potentiated by Pr55gag ubiquitylation. Interestingly, in response to viral infection, type I interferon triggers the expression of the ubiquitin-like polypeptide ISG15 which then inhibits the recruitment of the ESCRT machinery by inhibiting Pr55gag ubiquitylation.

There goes the neighborhood. Focus on “Localized PtdIns 3,5-P2 synthesis to regulate early endosome dynamics and fusion”

the membranes of eukaryotic cells contain small amounts of phosphoinositides, which are phosphorylated forms of the membrane lipid phosphatidylinositol (PtdIns). Together, PtdIns and its phosphoinositide derivatives constitute ∼10% of the total cellular lipid in most cells. Although

Gene silencing reveals a specific function of hVps34 phosphatidylinositol 3-kinase in late versus early endosomes

The human type III phosphatidylinositol 3-kinase, hVps34, converts phosphatidylinositol (PtdIns) to phosphatidylinositol 3-phosphate [PtdIns(3)P]. Studies using inhibitors of phosphatidylinositide 3-kinases have indicated that production of PtdIns(3)P is important for a variety of vesicle-mediated trafficking events, including endocytosis, sorting of receptors in multivesicular endosomes, and transport of lysosomal enzymes from the trans-Golgi network (TGN) to the endosomes and lysosomes. This study utilizes small interfering (si)RNA-mediated gene silencing to define the specific trafficking pathways in which hVps34 functions in human U-251 glioblastoma cells. Suppression of hVps34 expression reduced the cellular growth rate and caused a striking accumulation of large acidic phase-lucent vacuoles that contain lysosomal membrane proteins LAMP1 and LGP85. Analysis of these structures by electron microscopy suggests that they represent swollen late endosomes that have lost the capacity for inward vesiculation but retain the capacity to fuse with lysosomes. Morphological perturbation of the late endosome compartment was accompanied by a reduced rate of processing of the endosomal intermediate form of cathepsin D to the mature lysosomal form. There was also a reduction in the rate of epidermal growth factor receptor (EGFR) dephosphorylation and degradation following ligand stimulation, consistent with the retention of the EGFR on the limiting membranes of the enlarged late endosomes. By contrast, the suppression of hVps34 expression did not block trafficking of cathepsin D between the TGN and late endosomes, or endocytic uptake of fluid-phase markers, or association of a PtdIns(3)P-binding protein, EEA1, with early endosomes. LAMP1-positive vacuoles were depleted of PtdIns(3)P in the hVps34-knockdown cells, as judged by their inability to bind the PtdIns(3)P probe GFP-2xFYVE. By contrast, LAMP1-negative vesicles continued to bind GFP-2xFYVE in the knockdown cells. Overall, these findings indicate that hVps34 plays a major role in generating PtdIns(3)P for internal vesicle formation in multivesicular/late endosomes. The findings also unexpectedly suggest that other wortmannin-sensitive kinases and/or polyphosphoinositide phosphatases may be able to compensate for the loss of hVps34 and maintain PtdIns(3)P levels required for vesicular trafficking in the early endocytic pathway or the TGN.

ATPase-deficient hVPS4 impairs formation of internal endosomal vesicles and stabilizes bilayered clathrin coats on endosomal vacuoles.

Epidermal growth factor receptors (EGFRs) destined for lysosomal degradation are sorted in the early endosomal vacuole into small, lumenal vesicles that arise by inward budding of the limiting membrane. We have previously shown that, before their incorporation into internal vesicles, EGFRs are concentrated in flat bilayered-clathrin coats on the endosomal vacuole. Here, we show that an ATPase-deficient mutant of hVPS4 (hVPS4(EQ)) increases the association of bilayered coats with endosomal vacuoles. In addition, hVPS4(EQ) leads to a reduction in the number of internal vesicles in early and late endosomal vacuoles, and retention of EGFRs at the limiting membrane. Interestingly, hVPS4(EQ) was predominantly found on non-coated regions of endosomal vacuoles, often at the rim of a coated area. In line with published data on Vps4p function in yeast, these results suggest that hVPS4 is involved in the release of components of the bilayered coat from the endosomal membrane. Moreover, our data suggest that disassembly of the coat is required for the formation of internal vesicles.

The UIM domain of Hrs couples receptor sorting to vesicle formation.

Hepatocyte growth factor regulated tyrosine kinase substrate (Hrs), a main component of the 'bilayered' clathrin coat on sorting endosomes, was originally identified as a substrate of activated tyrosine kinase receptors. We have analysed Hrs phosphorylation in response to epidermal growth factor (EGF) stimulation and show that the evolutionary conserved tyrosines Y329 and Y334 provide the principal phosphorylation sites. Hrs is proposed to concentrate ubiquitinated receptors within clathrin-coated regions via direct interaction with its UIM (ubiquitin interaction motif) domain. We show that the same UIM domain is necessary for EGF-stimulated tyrosine phosphorylation of Hrs. Over-expression of wild-type Hrs or a double mutant, Y329/334F, defective in EGF-dependent phosphorylation, both substantially retard EGF receptor (EGFR) degradation by inhibiting internal vesicle formation and thereby preventing EGFR incorporation into lumenal vesicles of the multivesicular bodies. In contrast, mutation or deletion of the Hrs-UIM domain strongly suppresses this effect. In addition the UIM-deletion and point mutants are also observed on internal membranes, indicating a failure to dissociate from the endosomal membrane prior to incorporation of the receptor complex into lumenal vesicles. Our data suggest a role for the UIM-domain of Hrs in actively retaining EGFR at the limiting membrane of endosomes as a prelude to lumenal vesicle formation.

Digital Image Analysis of Internal Light Spots of Appressoria ofColletotrichum acutatum

ABSTRACT The initial penetration process of appressoria of Colletotrichum acutatum on almond leaves was studied using digital image analysis of light micrographs and scanning electron microscopy. For image analysis, a series of sequential, partially focused digital micrographs of appressoria was analyzed to generate a single, completely focused montage image with a continuous in-focus depth of field. In studies on the development of the internal light spot (ILS), we observed that 50.4% of the appressoria formed an ILS after leaves were inoculated and incubated for 12 h at 20 degrees C, and that this increased to 95.8% after 24 h. Comparative image analyses of appressoria with and without ILSs using depth relief mapping and line profile software options showed that the ILS had a depth relief that was below that of the leaf surface. Depth relief analysis in the ILS region during incubation revealed an increase in depth in this area of up to 1.8 mum in some of the appressoria. A comparative morphological study of the ILS in montage images and the penetration pore of appressoria in scanning electron micrographs showed similar shapes and dimensions of the two structures in the appressorium. Light micrographs of histological sections confirmed fungal penetration and internal vesicle formation in almond leaves within 36 h after inoculation and incubation at 20 degrees C. This study represents the first direct evidence that the ILS in appressoria corresponds to the penetration pore and the developing penetration peg using a rapid, digital image analysis technique.

Human VPS34 is required for internal vesicle formation within multivesicular endosomes.

After internalization from the plasma membrane, activated EGF receptors (EGFRs) are delivered to multivesicular bodies (MVBs). Within MVBs, EGFRs are removed from the perimeter membrane to internal vesicles, thereby being sorted from transferrin receptors, which recycle back to the plasma membrane. The phosphatidylinositol (PI) 3′-kinase inhibitor, wortmannin, inhibits internal vesicle formation within MVBs and causes EGFRs to remain in clusters on the perimeter membrane. Microinjection of isotype-specific inhibitory antibodies demonstrates that the PI 3′-kinase required for internal vesicle formation is hVPS34. In the presence of wortmannin, EGFRs continue to be delivered to lysosomes, showing that their removal from the recycling pathway and their delivery to lysosomes does not depend on inward vesiculation. We showed previously that tyrosine kinase-negative EGFRs fail to accumulate on internal vesicles of MVBs but are recycled rather than delivered to lysosomes. Therefore, we conclude that selection of EGFRs for inclusion on internal vesicles requires tyrosine kinase but not PI 3′-kinase activity, whereas vesicle formation requires PI 3′-kinase activity. Finally, in wortmannin-treated cells there is increased EGF-stimulated tyrosine phosphorylation when EGFRs are retained on the perimeter membrane of MVBs. Therefore, we suggest that inward vesiculation is involved directly with attenuating signal transduction.