Molecular determinants of selective clearance of protein inclusions by autophagy.

Protein quality control is essential for cellular survival. Failure to eliminate pathogenic proteins leads to their intracellular accumulation in the form of protein aggregates. Autophagy can recognize protein aggregates and degrade them in lysosomes. However, some aggregates escape the autophagic surveillance. Here we analyse the autophagic degradation of different types of aggregates of synphilin-1, a protein often found in pathogenic protein inclusions. We show that small synphilin-1 aggregates and large aggresomes are differentially targeted by constitutive and inducible autophagy. Furthermore, we identify a region in synphilin-1, necessary for its own basal and inducible aggrephagy and sufficient for the degradation of other pro-aggregating proteins. Although the presence of this peptide is sufficient for basal aggrephagy, inducible aggrephagy requires its ubiquitination, which diminishes protein mobility on the surface of the aggregate and favours the recruitment and assembly of the protein complexes required for autophagosome formation. Our study reveals different mechanisms for cells to cope with aggregate proteins via autophagy and supports the idea that autophagic susceptibility of prone-to-aggregate proteins may not depend on the nature of the aggregating proteins per se, but on their dynamic properties in the aggregate.

Characterization of MSB synapses in dissociated hippocampal culture with simultaneous pre- and postsynaptic live microscopy.

Multisynaptic boutons (MSBs) are presynaptic boutons in contact with multiple postsynaptic partners. Although MSB synapses have been studied with static imaging techniques such as electron microscopy (EM), the dynamics of individual MSB synapses have not been directly evaluated. It is known that the number of MSB synapses increases with synaptogenesis and plasticity but the formation, behavior, and fate of individual MSB synapses remains largely unknown. To address this, we developed a means of live imaging MSB synapses to observe them directly over time. With time lapse confocal microscopy of GFP-filled dendrites in contact with VAMP2-DsRed-labeled boutons, we recorded both MSBs and their contacting spines hourly over 15 or more hours. Our live microscopy showed that, compared to spines contacting single synaptic boutons (SSBs), MSB-contacting spines exhibit elevated dynamic behavior. These results are consistent with the idea that MSBs serve as intermediates in synaptic development and plasticity.

A variable cytoplasmic domain segment is necessary for γ-protocadherin trafficking and tubulation in the endosome/lysosome pathway.

Clustered protocadherins (Pcdhs) are arranged in gene clusters (α, ß, and γ) with variable and constant exons. Variable exons encode cadherin and transmembrane domains and ~90 cytoplasmic residues. The 14 Pcdh-αs and 22 Pcdh-γs are spliced to constant exons, which, for Pcdh-γs, encode ~120 residues of an identical cytoplasmic moiety. Pcdh-γs participate in cell-cell interactions but are prominently intracellular in vivo, and mice with disrupted Pcdh-γ genes exhibit increased neuronal cell death, suggesting nonconventional roles. Most attention in terms of Pcdh-γ intracellular interactions has focused on the constant domain. We show that the variable cytoplasmic domain (VCD) is required for trafficking and organelle tubulation in the endolysosome system. Deletion of the constant cytoplasmic domain preserved the late endosomal/lysosomal trafficking and organelle tubulation observed for the intact molecule, whereas deletion or excision of the VCD or replacement of the Pcdh-γA3 cytoplasmic domain with that from Pcdh-α1 or N-cadherin dramatically altered trafficking. Truncations or internal deletions within the VCD defined a 26-amino acid segment required for trafficking and tubulation in the endolysosomal pathway. This active VCD segment contains residues that are conserved in Pcdh-γA and Pcdh-γB subfamilies. Thus the VCDs of Pcdh-γs mediate interactions critical for Pcdh-γ trafficking.

Persistence of excitatory shaft synapses adjacent to newly emerged dendritic protrusions.

In the early postnatal hippocampus, the first synapses to appear on excitatory pyramidal neurons are formed directly on dendritic shafts. Very few dendritic spines are present at this time. By adulthood, however, the overwhelming majority of synapses are located at the tips of dendritic spines. Several models have been proposed to account for the transition from mostly shaft to mostly spinous synapses but none has been demonstrated conclusively. To investigate the cellular mechanism underlying the shaft-to-spinous synapse transition, we designed live imaging experiments to directly observe the dynamics of shaft and spinous synapses on developing dendrites. Immunofluorescent synaptic labeling of GFP-filled neurons showed that the shaft-to-spinous synapse transition in dissociated culture mirrors that in vivo. Along with electron microscopy, the fluorescent labeling also showed that veritable shaft synapses are abundant in dissociated culture and that shaft synapses are frequently adjacent to spines or other dendritic protrusions, a configuration previously observed in vivo by others. We used live long-term time lapse confocal microscopy of GFP-filled dendrites and VAMP2-DsRed-labeled boutons to record the fate of shaft synapses and associated dendritic protrusions and boutons with images taken hourly for up to 31 continuous hours. Inspection of the time lapse imaging series revealed that shaft synapses can persist adjacent to either existing or newly grown dendritic protrusions. Alternatively, a shaft synapse bouton can redistribute to contact an adjacent dendritic protrusion. However, we never observed shaft synapses transforming themselves into spines or any type of dendritic protrusions. We conclude that repeated iterations of dendritic protrusion or spine outgrowth adjacent to shaft synapses is very likely to be a critical component of the shaft-to-spinous synapse transition during CNS development.

Streamlined embedding of cell monolayers on gridded glass-bottom imaging dishes for correlative light and electron microscopy.

Correlative light and electron microscopy (CLEM) has facilitated study of intracellular trafficking. Routine application of CLEM would be advantageous for many laboratories but previously described techniques are particularly demanding, even for those with access to laser scanning confocal microscopy (LSCM) and transmission electron microscopy (TEM). We describe streamlined methods for TEM of green fluorescent protein (GFP)-labeled organelles after imaging by LSCM using gridded glass bottom imaging dishes. GFP-MAP 1A/1B LC3 (GFP-LC3) transfected cells were treated with rapamycin, fixed and imaged by LSCM. Confocal image stacks were acquired enabling full visualization of each GFP-LC3 labeled organelle. After LSCM, cells were embedded for TEM using a simplified two step method that stabilizes the glass bottom such that the block can be separated from the glass by mild heating. All imaging and TEM processing are performed in the same dish. The LSCM imaged cells were relocated on the block and serial sectioned. Correlation of LSCM, DIC, and TEM images was facilitated by cellular landmarks. All GFP labeled structures were successfully reidentified and imaged by serial section TEM. This method could make CLEM more accessible to nonspecialized laboratories with basic electron microscopy expertise and could be used routinely to confirm organelle localization of fluorescent puncta.

γ-protocadherins are enriched and transported in specialized vesicles associated with the secretory pathway in neurons.

Gamma protocadherins (Pcdh-γs) resemble classical cadherins and have the potential to engage in cell-cell interactions with homophilic properties. Emerging evidence suggests non-conventional roles for some protocadherins in neural development. We sought to determine whether Pcdh-γ trafficking in neurons is consistent with an intracellular role for these molecules. Here we show that, in contrast to the largely surface localization of classical cadherins, endogenous Pcdh-γs are primarily intracellular in rat neurons in vivo and are equally distributed within organelles of subsynaptic dendritic and axonal compartments. A strikingly higher proportion of Pcdh-γ-containing organelles in synaptic compartments was observed at postnatal day 16. To determine the origin of Pcdh-γ-trafficking organelles, we isolated organelles with Pcdh-γ antibody-coupled magnetic beads from brain organelle suspensions. Vesicles with high levels of COPII and endoplasmic reticulum-Golgi intermediate compartment (ERGIC) components were isolated with the Pcdh-γ antibody but not with the classical cadherin antibody. In cultured hippocampal neurons, Pcdh-γ immunolabeling partially overlapped with calnexin- and COPII-positive puncta in dendrites. Mobile Pcdh-γ-GFP profiles dynamically codistributed with a DsRed construct coupled to ER retention signals by live imaging. Pcdh-γ expression correlated with accumulations of tubulovesicular and ER-like organelles in dendrites. Our results are consistent with the possibility that Pcdh-γs could have a unique function within the secretory pathway in addition to their documented surface roles.

LC3-dependent intracellular membrane tubules induced by gamma-protocadherins A3 and B2: a role for intraluminal interactions.

Clustered protocadherins (Pcdhs) are a family of cadherin-like molecules arranged in gene clusters (alpha, beta, and gamma). gamma-Protocadherins (Pcdh-gammas) are involved in cell-cell interactions, but their prominent intracellular distribution in vivo and different knock-out phenotypes suggest that these molecules participate in still unidentified processes. We found using correlative light and electron microscopy that Pcdh-gammaA3 and -gammaB2, but not -gammaC4, -alpha1, or N-cadherin, generate intracellular juxtanuclear membrane tubules when expressed in cells. These tubules recruit the autophagy marker MAP1A/1B LC3 (LC3) but are not associated with autophagic vesicles. Lipidation of LC3 is required for its coclustering with Pcdh-gamma tubules, suggesting the involvement of an autophagic-like molecular cascade. Expression of wild-type LC3 with Pcdh-gammaA3 increased tubule length whereas expression of lipidation-defective LC3 decreased tubule length relative to Pcdh-gammaA3 expressed alone. The tubules were found to emanate from lysosomes. Deletion of the luminal/extracellular domain of Pcdh-gammaA3 preserved lysosomal targeting but eliminated tubule formation whereas cytoplasmic deletion eliminated both lysosomal targeting and tubule formation. Deletion of the membrane-proximal three cadherin repeats resulted in tubes that were narrower than those produced by full-length molecules. These results suggest that Pcdh-gammaA and -gammaB families can influence the shape of intracellular membranes by mediating intraluminal interactions within organelles.