Gamma-protocadherins (Pcdh-γs) are good candidates to mediate specificity in synaptogenesis but their role in cell-cell interactions is a matter of debate. We proposed that Pcdh-γs modify preformed synapses via trafficking of Pcdh-γs-containing organelles, insertion into synaptic membranes and homophilic transcellular interaction. Here we provide evidence in support of this model. We show for the first time that Pcdh-γs have homophilic properties and that they accumulate at dendrodendritic and axo-dendritic interfaces during neuronal development. Pcdh-γs are maintained in a substantial mobile intracellular pool in dendrites and cytoplasmic deletion shifts the molecule to the surface and reduces the number and velocity of the mobile packets. We monitored Pcdh-γ temporal and spatial dynamics in transport organelles. Pcdh-γ organelles bud and fuse with stationary clusters near synapses. These results suggest that Pcdh-γ-mediated cell-cell interactions in synapse development or maintenance are tightly regulated by control of intracellular trafficking via the cytoplasmic domain.

Clustered protocadherins (Pcdhs) are a family of cadherinlike molecules arranged in gene clusters (␣, ␤, and ␥). ␥-Protocadherins (Pcdh-␥s) 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-␥A3 and -␥B2, but not -␥C4, -␣1, 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-␥ tubules, suggesting the involvement of an autophagic-like molecular cascade. Expression of wild-type LC3 with Pcdh-␥A3 increased tubule length whereas expression of lipidation-defective LC3 decreased tubule length relative to Pcdh-␥A3 expressed alone. The tubules were found to emanate from lysosomes. Deletion of the luminal/extracellular domain of Pcdh-␥A3 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-␥A and -␥B families can influence the shape of intracellular membranes by mediating intraluminal interactions within organelles. Clustered protocadherins (Pcdhs)3 comprise ϳ50 different cadherin-like transmembrane proteins arranged into three genomic clusters, termed ␣, ␤, and ␥ (1, 2). The Pcdh genes within the ␣ and ␥ clusters, numbering 14 and 22, respectively, share constant cytoplasmic domains within their respective clusters by alternative splicing (3). The Pcdh-␥s participate in cell-cell interactions (4, 5) with homophilic properties (6) and have been localized at synapses (6 -8), but also have a prominent intracellular distribution in cultured cells and in vivo (6,8). For Pcdh-␣s, conflicting evidence has been presented as to whether they mediate cell-cell interactions (9 -11), but a synaptic localization has been reported (1).Complete genetic deletion of Pcdh-␥s reduced the number of synaptic specializations in spinal cord but also caused substantial apoptosis of spinal cord interneurons (7, 12). Conditional knock out in retina also increased apoptosis of some retinal cell types but, unlike in spinal cord, did not affect synaptic connectivity (13). Conditional knock out in astrocytes did not cause apoptosis but delayed synaptic development (14). The multiple effects of Pcdh-␥ deletion may indicate still unidentified cellular roles for these molecules.We show here that Pcdh-␥A3 and -␥B2 generate intracellular membrane tubules that recruit the autophagic vesicle protein LC3, the mammalian homologue of yeast Atg8. LC3 is lipidated via a reaction similar to the ubiquitin ligase cascade and targets to nascent autophagosomes where it is thought...

The variable portion of the γ-protocadherin (Pcdh-γ) cytoplasmic domain (VCD) controls Pcdh-γ trafficking and organelle tubulation in the endolysosome system. Active VCD segments are conserved in Pcdh-γA and Pcdh-γB subfamilies.

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