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X-force Fusion Lifecycle 2010 Download: How to Install and Activate Autodesk Products



Endosome maturation involves a conversion from Rab5 to Rab7 (Rink et al, 2005; Vonderheit and Helenius, 2005; Poteryaev et al, 2010). The conversion can be blocked by expressing a constitutively active mutant of Rab5 (Q79L), and by depletion of VPS39, a subunit of the HOPS complex (Rink et al, 2005). This results in the formation of hybrid endosomal compartments with markers for both EEs and LEs. These abnormal hybrid compartments seem to arise from homotypic fusions of EEs and heterotypic fusions with LEs or lysosomes, and are found to accumulate ILVs (Rosenfeld et al, 2001; Hirota et al, 2007; Wegner et al, 2010). Expression of the Rab5(Q79L) mutant results in sorting defects of both recycling and degradative cargo. Lysosome biogenesis is also affected as the amount of lysosomes in dense Percoll gradient fractions is greatly reduced (Rosenfeld et al, 2001).




X-force Fusion Lifecycle 2010 Download



Additional key players in the Rab5/Rab7 conversion are starting to emerge. According to recent data, these include SAND-1/Mon1 and Ccz1, two factors recruited from the cytosol to endosomal membranes. They were first identified as factors required for vacuole fusion in yeast, and in transport of yolk proteins from EEs to LEs in C. elegans (Wang et al, 2003; Poteryaev et al, 2007). Recent work focusing on phagosomes in C. elegans have provided evidence that SAND-1/Mon1 and Ccz1 are involved in the Rab conversion in endosomes and phagosomes (Kinchen and Ravichandran, 2010; Poteryaev et al, 2010).


To terminate Rab5 activation on endosomes, a GAP has to activate Rab5-mediated hydrolysis of bound GTP into GDP. Several GAPs have been assigned to Rab5, including RN-Tre and RAB GAP-5 (Lanzetti et al, 2000; Haas et al, 2005). However, the target of RN-Tre may actually be Rab41 (Haas et al, 2005). A recent addition to the list of Rab5 GAPs is TBC-2, which in C. elegans was shown to be required both during endosome and phagosome maturation (Li et al, 2009; Chotard et al, 2010). It is recruited to endosomes only when Rab7 is already present, thereby allowing coordination of Rab5 inactivation with Rab7 activation. Confusion is caused by the observation that the closest human homologue of TBC-2, Armus, possesses GAP activity towards Rab7 (Frasa et al, 2010). Earlier studies have recognized a role for the yeast Gyp7p and its human orthologue TBC1D15 as Ypt7p/Rab7 GAPs (Vollmer et al, 1999; Zhang et al, 2005; Peralta et al, 2010).


Once established as a domain in the hybrid endosome, Rab7-GTP recruits its own effectors. These include factors such as RILP, a protein that connects LEs to dynein motors; components of the retromer complex that support vesicle traffic to the TGN; components of the HOPS complex serving as a tether for LE fusion; and Rabring7, a RING-type E3 ligase (Zhang et al, 2009). In contrast to these, Rubicon and PLEKHM1, recently identified Rab7 effectors, act as negative regulators of endosome maturation (Sun et al, 2010; Tabata et al, 2010).


PIKfyve has also been linked to the regulation of ion channels, mostly by controlling their localization at the plasma membrane by exocytosis (Shisheva, 2008). In addition, PIKfyve may directly regulate the activity of channels in LEs. The calcium permeable cation channel TRPML1 was recently demonstrated to be directly activated by the presence of PtdIns(3,5)P(2) enabling the efflux of Ca2+ (Dong et al, 2010). Ca2+ is known to have an important regulatory function in homotypic and heterotypic fusions of LEs and lysosomes/vacuoles as well as in reformation of lysosomes from endolysosomes (Luzio et al, 2007a). The localized production of PtdIns(3,5)P(2) on endosomal membranes thus allows the control of channel activity in confined regions and may enable regulation of LE fusion/fission in a spatio-temporal manner. It is plausible that the highly vacuolated phenotype of PIKfyve/Fab1p mutants is partially the result of osmotic swelling or deregulated fusion/fission processes induced by the misregulation of endosomal ion channels (Shisheva, 2008).


Recent findings indicate a dynactin independent linkage between LEs and dynein through a protein called Snapin, originally identified as a neuronal SNARE-binding protein and implicated in synaptic vesicle fusion (Ilardi et al, 1999; Pan et al, 2009). Snapin links LE cargo by binding directly to dynein intermediate chains. It has a crucial role in controlling LE retrograde transport and maturation in neurons (Cai et al, 2010). Deletion of Snapin in neurons results in accumulation of immature LEs, further highlighting the role of motility in coordinating endosomal maturation. The relationship between Snapin- and the dynactin/RILP-mediated LE transport remains unclear.


The formation of exosomes depends on ESCRT function, although it is apparent that ESCRT-independent mechanisms may also exist. In immature DCs, MHC II is ubiquitinated, enters ILVs, and is targeted for degradation, while in mature DCs the MHC II containing ILVs are secreted as exosomes (Buschow et al, 2009). How the LEs fuse with the plasma membrane has been the focus of recent studies. In HeLa cells, Rab27a, Rab27b and their effectors Slp4 and Slac2b were found to promote fusion (Ostrowski et al, 2010). In oligodendrocytes, Rab35 and its GAPs, TBC1D10A-C have been implicated (Hsu et al, 2010). 2ff7e9595c


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