Evidence for Early Endosome‐like Fusion of Recently Endocytosed Synaptic Vesicles

Rizzoli, Silvio O.; Bethani, Ioanna; Zwilling, Daniel; Wenzel, Dirk; Siddiqui, Tabrez J.; Brandhorst, Dorothea; Jahn, Reinhard

doi:10.1111/j.1600-0854.2006.00466.x

Cited by 72 publications

(103 citation statements)

References 45 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

Section: A New Microscopy-based Assay For Early Endosomal Dockingsupporting

confidence: 76%

“…The second peak spans between ϳ150 -500 nm, thus revealing a population of endosomes that are not fused but closely associated with each other. We conclude that the first peak of the endosome distance distribution ( Figure 1C) represents fused endosomes (in agreement with previous investigations on the subject, Rizzoli et al, 2006;Bethani et al, 2007), whereas the second peak represents docked endosomes, thus allowing for differentiating docking from fusion.An alternative method for monitoring in vitro docking of organelles was previously described for yeast vacuoles. According to this procedure, clusters of organelles are differentiated from single organelles simply by addition of a fluorescent membrane probe (e.g., a styryl dye) at the end of the docking reaction, which allows for visualizing all membranes in the reaction mixture Wang et al, 2002).…”

supporting

confidence: 76%

“…As shown in Figure 4A, this is indeed the case: In the presence of BAPTA the pool of fused endosomes is virtually abolished, whereas the pool of docked vesicles is largely unaffected. As expected, the slower calcium chelator EGTA, which is known not to affect fusion in this system (Holroyd et al, 1999;Rizzoli et al, 2006) had no significant effect on docking (three independent experiments, data not shown).Being able to prevent docked endosomes to progress to fusion allowed for investigating whether not only initiation but also maintenance of docking is dependent on ATP. We tested this by incubating the endosomes for 15 min in the presence of BAPTA.…”

mentioning

confidence: 94%

See 2 more Smart Citations

SNARE Function Is Not Involved in Early Endosome Docking

Geumann

Barysch

Hoopmann

et al. 2008

MBoC

Self Cite

View full text Add to dashboard Cite

Docking and fusion of transport vesicles constitute elementary steps in intracellular membrane traffic. While docking is thought to be initiated by Rab-effector complexes, fusion is mediated by SNARE (N-ethylmaleimide-sensitive factor[NSF] attachment receptor) proteins. However, it has been recently debated whether SNAREs also play a role in the establishment or maintenance of a stably docked state. To address this question, we have investigated the SNARE dependence of docking and fusion of early endosomes, one of the central sorting compartments in the endocytic pathway. A new, fluorescence-based in vitro assay was developed, which allowed us to investigate fusion and docking in parallel. Similar to homotypic fusion, docking of early endosomes is dependent on the presence of ATP and requires physiological temperatures. Unlike fusion, docking is insensitive to the perturbation of SNARE function by means of soluble SNARE motifs, SNARE-specific F ab fragments, or by a block of NSF activity. In contrast, as expected, docking is strongly reduced by interfering with the synthesis of phosphatidyl inositol (PI)-3 phosphate, with the function of Rab-GTPases, as well as with early endosomal autoantigen 1 (EEA1), an essential tethering factor. We conclude that docking of early endosomes is independent of SNARE function. INTRODUCTIONThe function of the secretory pathway requires transport of material between different intracellular compartments or organelles. This is achieved by organelle budding and fusion, two processes that are highly controlled within the cell. In order for two organelles to fuse, they need to first undergo docking and then priming, an ATP-dependent process that sets up the fusion machinery (Kawasaki et al., 1998;Klenchin and Martin, 2000). Docking, defined as the close contact of two membranes in preparation of fusion (Schikorski and Stevens, 2001), is thought to be controlled by Rab/Ypt GTPases and initialized by specialized tethering molecules bridging the organelles (Sztul and Lupashin, 2006). Several such tethers (generally large coiled-coil proteins or multisubunit complexes) are known (Waters and Hughson, 2000). Tethers may be directly recruited by activated GTPases, as for example the GARP (Golgi-associated retrograde protein) complex in retrograde trafficking. Alternatively, they may contain a guanine nucleotide exchange factor (GEF) activity such as the transport protein particle (TRAPP 1) complex, which activates the GTPase Ypt1p and functions in ER-to-Golgi traffic. Both activities may also be combined, such as in the vacuolar HOPS (homotypic fusion and vacuole protein sorting) complex, which does not only act as a GEF but also as an effector for Ypt7p. Some of these tethers like the HOPS and Dsl1 complex have also been implicated in SNARE (N-ethylmaleimide-sensitive factor [NSF] attachment receptor) binding, which would provide a link to the core fusion machinery (for review see Cai et al., 2007).SNARE proteins deliver the energy necessary for membrane fusion through the interaction of SNA...

show abstract

Section: A New Microscopy-based Assay For Early Endosomal Dockingsupporting

confidence: 76%

supporting

confidence: 76%

mentioning

confidence: 94%

See 1 more Smart Citation

SNARE Function Is Not Involved in Early Endosome Docking

Geumann

Barysch

Hoopmann

et al. 2008

MBoC

Self Cite

View full text Add to dashboard Cite

show abstract

“…We then examined the subcellular localization of TI-VAMP in mocha hippocampal granule cells from which MFs originate. We found that TI-VAMP accumulated in the cell bodies of granule cells, as shown by colocalization with VAMP4, a vesicular SNARE located in early endosomes (11,12) and the trans-Golgi network (ref. 13; Fig.…”

Section: Asynchronous Release Evoked At Mossy Fiber (Mf) Terminals Ismentioning

confidence: 99%

Loss of AP-3 function affects spontaneous and evoked release at hippocampal mossy fiber synapses

Scheuber

Rudge

Danglot

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

117

View full text Add to dashboard Cite

Synaptic vesicle (SV) exocytosis mediating neurotransmitter release occurs spontaneously at low intraterminal calcium concentrations and is stimulated by a rise in intracellular calcium. Exocytosis is compensated for by the reformation of vesicles at plasma membrane and endosomes. Although the adaptor complex AP-3 was proposed to be involved in the formation of SVs from endosomes, whether its function has an indirect effect on exocytosis remains unknown. Using mocha mice, which are deficient in functional AP-3, we identify an AP-3-dependent tetanus neurotoxin-resistant asynchronous release that can be evoked at hippocampal mossy fiber (MF) synapses. Presynaptic targeting of the tetanus neurotoxin-resistant vesicle soluble N -ethylmaleimide-sensitive factor attachment protein receptor (SNARE) tetanus neurotoxin-insensitive vesicle-associated membrane protein (TI-VAMP) is lost in mocha hippocampal MF terminals, whereas the localization of synaptobrevin 2 is unaffected. In addition, quantal release in mocha cultures is more frequent and more sensitive to sucrose. We conclude that lack of AP-3 results in more constitutive secretion and loss of an asynchronous evoked release component, suggesting an important function of AP-3 in regulating SV exocytosis at MF terminals.

show abstract

“…When the endosome-like structure was first observed, it was proposed to result from the fusion of multiple small vesicles newly retrieved from the plasma membrane (4,7). Recent electron microscopic studies reveal deep invaginations of plasma membrane resembling endosome-like structures in conditions that block or slow down endocytosis (5,(8)(9)(10).…”

mentioning

confidence: 99%

Rapid bulk endocytosis and its kinetics of fission pore closure at a central synapse

2007

Proc. Natl. Acad. Sci. U.S.A.

120

View full text Add to dashboard Cite

Upon exocytosis, fused vesicles must be retrieved for recycling. One route of retrieval is to generate endosome-like structures, from which small vesicles bud off. Endosome-like structures are widely thought to be generated slowly (Ϸ1 min) from the plasma membrane, a process called bulk endocytosis. Although the concept of bulk endocytosis seems established, the kinetic evidence showing the instant of the bulk membrane fission at synapses is still missing. The present work provides this missing piece of evidence at a calyx-type synapse. We used the capacitance measurement technique, which offers a high time resolution (Ϸ1 ms) to resolve the fission process. The instant of bulk membrane fission was reflected as a brief downward capacitance shift (DCS) of Ϸ20 -500 fF (mean ‫؍‬ 123 fF) with 10 -90% decay time of Ϸ30 -500 ms. At least 8.6 -11.0% of exocytosed vesicles were retrieved by DCSs. During a DCS, the decrease in the fission pore conductance was detected, from which we found that the fission pore diameter decreased at Ϸ39 nm/s. This provided the measurement of the rate of fission during bulk endocytosis at synapses. The DCS frequency peaked (Ϸ0.021 Hz) in <10 s after stimulation and decayed with a half time <20 s, indicating that the time course of bulk endocytosis is much faster than previously estimated with low time-resolution techniques. Our results also suggest that bulk endocytosis was composed of two kinetically different steps: the DCS that reflected the fission process and the time between stimulation and the DCS, during which membrane invagination led to the fission pore formation.calyx of Held ͉ capacitance measurement ͉ membrane invagination ͉ synaptic transmission

show abstract

Evidence for Early Endosome‐like Fusion of Recently Endocytosed Synaptic Vesicles

Cited by 72 publications

References 45 publications

SNARE Function Is Not Involved in Early Endosome Docking

SNARE Function Is Not Involved in Early Endosome Docking

Loss of AP-3 function affects spontaneous and evoked release at hippocampal mossy fiber synapses

Rapid bulk endocytosis and its kinetics of fission pore closure at a central synapse

Contact Info

Product

Resources

About