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The GTPase Rab26 links synaptic vesicles to the autophagy pathway.

Binotti B, Pavlos NJ, Riedel D, Wenzel D, Vorbrüggen G, Schalk AM, Kühnel K, Boyken J, Erck C, Martens H, Chua JJ, Jahn R - Elife (2015)

Bottom Line: Overexpression of active but not of GDP-preferring Rab26 enhances vesicle clustering, which is particularly conspicuous for the EGFP-tagged variant, resulting in a massive accumulation of synaptic vesicles in neuronal somata without altering the distribution of other organelles.Both endogenous and induced clusters co-localize with autophagy-related proteins such as Atg16L1, LC3B and Rab33B but not with other organelles.Furthermore, Atg16L1 appears to be a direct effector of Rab26 and binds Rab26 in its GTP-bound form, albeit only with low affinity.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurobiology, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.

ABSTRACT
Small GTPases of the Rab family not only regulate target recognition in membrane traffic but also control other cellular functions such as cytoskeletal transport and autophagy. Here we show that Rab26 is specifically associated with clusters of synaptic vesicles in neurites. Overexpression of active but not of GDP-preferring Rab26 enhances vesicle clustering, which is particularly conspicuous for the EGFP-tagged variant, resulting in a massive accumulation of synaptic vesicles in neuronal somata without altering the distribution of other organelles. Both endogenous and induced clusters co-localize with autophagy-related proteins such as Atg16L1, LC3B and Rab33B but not with other organelles. Furthermore, Atg16L1 appears to be a direct effector of Rab26 and binds Rab26 in its GTP-bound form, albeit only with low affinity. We propose that Rab26 selectively directs synaptic and secretory vesicles into preautophagosomal structures, suggesting the presence of a novel pathway for degradation of synaptic vesicles.

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Rab26 overexpression causes vesicle clusters in HeLa.Endogenous ATG16L1 is recruited to vesicle clusters induced by transient overexpression of EGFP-tagged Rab26WT (A) or Rab26QL (B) in HeLa cells. Although GFP-Rab26TN forms small clusters in these cells, endogenous Atg16L1 is not recruited to these structures (C). (D) Immunogold electron microscopy analysis revealed that HeLa cells transfected with EGFP-Rab26WT exhibited formation of huge clusters of small vesicle (left panel, inset) that are sometimes surrounded by external membranes (right panel, inset) and large vacuole autolysosome-like compartments whose membrane were labeled with EGFP-Rab26WT (center and right panel). Rab26 was visualized by anti-GFP antibody coupled with 10 nm gold particles.DOI:http://dx.doi.org/10.7554/eLife.05597.014
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fig6s1: Rab26 overexpression causes vesicle clusters in HeLa.Endogenous ATG16L1 is recruited to vesicle clusters induced by transient overexpression of EGFP-tagged Rab26WT (A) or Rab26QL (B) in HeLa cells. Although GFP-Rab26TN forms small clusters in these cells, endogenous Atg16L1 is not recruited to these structures (C). (D) Immunogold electron microscopy analysis revealed that HeLa cells transfected with EGFP-Rab26WT exhibited formation of huge clusters of small vesicle (left panel, inset) that are sometimes surrounded by external membranes (right panel, inset) and large vacuole autolysosome-like compartments whose membrane were labeled with EGFP-Rab26WT (center and right panel). Rab26 was visualized by anti-GFP antibody coupled with 10 nm gold particles.DOI:http://dx.doi.org/10.7554/eLife.05597.014

Mentions: Therefore, to test whether the Rab26 containing clusters are linked to the autophagy pathway, we next checked for association with autophagosome-related proteins. First, we assessed for colocalization between Rab26 and Atg16L1, a component of pre-autophagosomes (Mizushima et al., 2003; Ravikumar et al., 2010). For this purpose, hippocampal neurons transiently expressing EGFP-Rab26WT were immunostained for endogenous Atg16L1. Indeed, an almost perfect colocalization between Atg16L1 and Rab26-positive clusters was detected in neuronal cell bodies (Figure 6A), thereby identifying these clusters as autophagosomal precursors. Next, we stained untransfected neurons for endogenous Rab26 and Atg16L1. Again, a high degree of overlap was observed between Rab26 and Atg16L1 in clusters decorating neurites (Figure 6B) but not in cell bodies which remained largely unstained (not shown). Strong overlap with Atg16L1 was also observed when neurons were transfected with FLAG-Rab26WT and/or FLAG-Rab26QL, but not with FLAG-Rab26TN (Figure 6C). This indicated that the association of Rab26 with autophagosomes depends on the GTP-form of the protein. This GTP-dependency was similarly noted in HeLa cells following ectopic expression of Rab26. In this instance, overexpression of GTP-bound forms (WT and QL), but not GDP-bound (TN) form, of EGFP-Rab26 led to the formation of large Atg16L1-positive clusters (Figure 6—figure supplement 1A–C). Analysis by immunogold-TEM again revealed that these clusters consisted of small but often heterogeneous vesicles, partially surrounded by membranes, with EGFP labeling detected both on vesicles within clusters as well as on their encapsulating membrane(s) (Figure 6—figure supplement 1D).10.7554/eLife.05597.013Figure 6.Clusters containing GTP-Rab26 colocalize with autophagosome-specific proteins both in cell bodies and dendrites of cultured hippocampal neurons.


The GTPase Rab26 links synaptic vesicles to the autophagy pathway.

Binotti B, Pavlos NJ, Riedel D, Wenzel D, Vorbrüggen G, Schalk AM, Kühnel K, Boyken J, Erck C, Martens H, Chua JJ, Jahn R - Elife (2015)

Rab26 overexpression causes vesicle clusters in HeLa.Endogenous ATG16L1 is recruited to vesicle clusters induced by transient overexpression of EGFP-tagged Rab26WT (A) or Rab26QL (B) in HeLa cells. Although GFP-Rab26TN forms small clusters in these cells, endogenous Atg16L1 is not recruited to these structures (C). (D) Immunogold electron microscopy analysis revealed that HeLa cells transfected with EGFP-Rab26WT exhibited formation of huge clusters of small vesicle (left panel, inset) that are sometimes surrounded by external membranes (right panel, inset) and large vacuole autolysosome-like compartments whose membrane were labeled with EGFP-Rab26WT (center and right panel). Rab26 was visualized by anti-GFP antibody coupled with 10 nm gold particles.DOI:http://dx.doi.org/10.7554/eLife.05597.014
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4337689&req=5

fig6s1: Rab26 overexpression causes vesicle clusters in HeLa.Endogenous ATG16L1 is recruited to vesicle clusters induced by transient overexpression of EGFP-tagged Rab26WT (A) or Rab26QL (B) in HeLa cells. Although GFP-Rab26TN forms small clusters in these cells, endogenous Atg16L1 is not recruited to these structures (C). (D) Immunogold electron microscopy analysis revealed that HeLa cells transfected with EGFP-Rab26WT exhibited formation of huge clusters of small vesicle (left panel, inset) that are sometimes surrounded by external membranes (right panel, inset) and large vacuole autolysosome-like compartments whose membrane were labeled with EGFP-Rab26WT (center and right panel). Rab26 was visualized by anti-GFP antibody coupled with 10 nm gold particles.DOI:http://dx.doi.org/10.7554/eLife.05597.014
Mentions: Therefore, to test whether the Rab26 containing clusters are linked to the autophagy pathway, we next checked for association with autophagosome-related proteins. First, we assessed for colocalization between Rab26 and Atg16L1, a component of pre-autophagosomes (Mizushima et al., 2003; Ravikumar et al., 2010). For this purpose, hippocampal neurons transiently expressing EGFP-Rab26WT were immunostained for endogenous Atg16L1. Indeed, an almost perfect colocalization between Atg16L1 and Rab26-positive clusters was detected in neuronal cell bodies (Figure 6A), thereby identifying these clusters as autophagosomal precursors. Next, we stained untransfected neurons for endogenous Rab26 and Atg16L1. Again, a high degree of overlap was observed between Rab26 and Atg16L1 in clusters decorating neurites (Figure 6B) but not in cell bodies which remained largely unstained (not shown). Strong overlap with Atg16L1 was also observed when neurons were transfected with FLAG-Rab26WT and/or FLAG-Rab26QL, but not with FLAG-Rab26TN (Figure 6C). This indicated that the association of Rab26 with autophagosomes depends on the GTP-form of the protein. This GTP-dependency was similarly noted in HeLa cells following ectopic expression of Rab26. In this instance, overexpression of GTP-bound forms (WT and QL), but not GDP-bound (TN) form, of EGFP-Rab26 led to the formation of large Atg16L1-positive clusters (Figure 6—figure supplement 1A–C). Analysis by immunogold-TEM again revealed that these clusters consisted of small but often heterogeneous vesicles, partially surrounded by membranes, with EGFP labeling detected both on vesicles within clusters as well as on their encapsulating membrane(s) (Figure 6—figure supplement 1D).10.7554/eLife.05597.013Figure 6.Clusters containing GTP-Rab26 colocalize with autophagosome-specific proteins both in cell bodies and dendrites of cultured hippocampal neurons.

Bottom Line: Overexpression of active but not of GDP-preferring Rab26 enhances vesicle clustering, which is particularly conspicuous for the EGFP-tagged variant, resulting in a massive accumulation of synaptic vesicles in neuronal somata without altering the distribution of other organelles.Both endogenous and induced clusters co-localize with autophagy-related proteins such as Atg16L1, LC3B and Rab33B but not with other organelles.Furthermore, Atg16L1 appears to be a direct effector of Rab26 and binds Rab26 in its GTP-bound form, albeit only with low affinity.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurobiology, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.

ABSTRACT
Small GTPases of the Rab family not only regulate target recognition in membrane traffic but also control other cellular functions such as cytoskeletal transport and autophagy. Here we show that Rab26 is specifically associated with clusters of synaptic vesicles in neurites. Overexpression of active but not of GDP-preferring Rab26 enhances vesicle clustering, which is particularly conspicuous for the EGFP-tagged variant, resulting in a massive accumulation of synaptic vesicles in neuronal somata without altering the distribution of other organelles. Both endogenous and induced clusters co-localize with autophagy-related proteins such as Atg16L1, LC3B and Rab33B but not with other organelles. Furthermore, Atg16L1 appears to be a direct effector of Rab26 and binds Rab26 in its GTP-bound form, albeit only with low affinity. We propose that Rab26 selectively directs synaptic and secretory vesicles into preautophagosomal structures, suggesting the presence of a novel pathway for degradation of synaptic vesicles.

Show MeSH
Related in: MedlinePlus