Structural origins of clustered protocadherin-mediated neuronal barcoding

Rubinstein, Rotem; Goodman, K.M.; Maniatis, Tom; Shapiro, Lawrence; Honig, Barry

doi:10.1016/j.semcdb.2017.07.023

Cited by 42 publications

(46 citation statements)

References 72 publications

(151 reference statements)

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“…Outside of these well-characterized proteins, cadherin superfamily members containing more than five EC repeats have been loosely termed protocadherins (pcdhs). The largest groups within the protocadherin branch are the clustered protocadherins (pcdha, pcdhb, and pcdhg), encoded by a contiguous gene cluster subject to alternative splicing (Mountoufaris et al, 2018;Rubinstein et al, 2017), and the non-clustered d-protocadherins -subjects of the current study-whose genes are dispersed in the genome (Kim et al, 2007;Light and Jontes, 2017;Morishita and Yagi, 2007;Redies et al, 2005). Both families encode singlepass transmembrane proteins containing six or seven EC domains and are closely related in their extracellular regions ($30%-40% identity).…”

Section: Introductionmentioning

confidence: 99%

Family-wide Structural and Biophysical Analysis of Binding Interactions among Non-clustered δ-Protocadherins

et al. 2020

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Graphical Abstract Highlights d d-pcdh adhesive interactions are preferentially homophilic with distinct affinities d d1-and d2-pcdhs form canonical adhesive dimers with divergent specificity regions d Ectodomain-mediated cis interactions are not observed, in contrast to clustered pcdhs SUMMARYNon-clustered d1-and d2-protocadherins, close relatives of clustered protocadherins, function in cell adhesion and motility and play essential roles in neural patterning. To understand the molecular interactions underlying these functions, we used solution biophysics to characterize binding of d1-and d2-protocadherins, determined crystal structures of ectodomain complexes from each family, and assessed ectodomain assembly in reconstituted intermembrane junctions by cryoelectron tomography (cryo-ET). Homophilic trans (cell-cell) interactions were preferred for all d-protocadherins, with additional weaker heterophilic interactions observed exclusively within each subfamily. As expected, d1-and d2-protocadherin trans dimers formed through antiparallel EC1-EC4 interfaces, like clustered protocadherins. However, no ectodomain-mediated cis (same-cell) interactions were detectable in solution; consistent with this, cryo-ET of reconstituted junctions revealed dense assemblies lacking the characteristic order observed for clustered protocadherins.Our results define non-clustered protocadherin binding properties and their structural basis, providing a foundation for interpreting their functional roles in neural patterning.

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Section: Introductionmentioning

confidence: 99%

Family-wide Structural and Biophysical Analysis of Binding Interactions among Non-clustered δ-Protocadherins

et al. 2020

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“…Clustered Pcdh isoforms interact strictly homophilically in trans, engaging in anti-parallel interactions involving EC1-EC4, while EC5 and EC6 mediate promiscuous cis dimer formation between γ isoforms, as well as with α-and β-Pcdhs [15][16][17][18][19]. These two types of interactions result in a multimeric lattice of dimers between cell membranes sharing the same isoform composition [20,21], and indeed, homophilic specificity is observed at the multimer level [18,19]. In this way, the 58 cPcdh isoforms generate thousands of distinct recognition signals.…”

Section: Introductionmentioning

confidence: 99%

“…While α, β and γ isoforms can all contribute to multimer formation [21], the γ-Pcdhs are particularly critical for neural development [11]. Mice lacking either the entire Pcdhg cluster or the γC3-C5 V exons exhibited neonatal lethality, with excessive apoptosis of neuronal subtypes in the spinal cord and hypothalamus [31][32][33][34].…”

Section: Introductionmentioning

confidence: 99%

CRISPR/Cas9 interrogation of the mouse Pcdhg gene cluster reveals a crucial isoform-specific role for Pcdhgc4

et al. 2019

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The mammalian Pcdhg gene cluster encodes a family of 22 cell adhesion molecules, the gamma-Protocadherins (γ-Pcdhs), critical for neuronal survival and neural circuit formation. The extent to which isoform diversity-a γ-Pcdh hallmark-is required for their functions remains unclear. We used a CRISPR/Cas9 approach to reduce isoform diversity, targeting each Pcdhg variable exon with pooled sgRNAs to generate an allelic series of 26 mouse lines with 1 to 21 isoforms disrupted via discrete indels at guide sites and/or larger deletions/ rearrangements. Analysis of 5 mutant lines indicates that postnatal viability and neuronal survival do not require isoform diversity. Surprisingly, given reports that it might not independently engage in trans-interactions, we find that γC4, encoded by Pcdhgc4, is the only critical isoform. Because the human orthologue is the only PCDHG gene constrained in humans, our results indicate a conserved γC4 function that likely involves distinct molecular mechanisms.

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“…The Pcdh gene cluster is capable of generating, through epigenetic regulation of ~60 different isoforms (Guo et al, 2012;Hirayama et al, 2012;Monahan et al, 2012;Yin et al, 2017), what has been considered an adhesive "barcode" (Rubinstein et al, 2017;Mountoufaris et al, 2018) on the surfaces of individual neurons. What this code does during neural development is still being elaborated.…”

Section: Discussionmentioning

confidence: 99%

Gamma-protocadherin localization at the synapse corresponds to parameters of synaptic maturation

LaMassa

Sverdlov²,

Mambetalieva

et al. 2019

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Clustered protocadherins (Pcdhs) are a large family of ~60 cadherin-like proteins (divided into the subclasses α, β, and γ) that compose a surface "barcode" in individual neurons. The code is generated through combinatorial expression via epigenetic regulation at a large gene cluster that encodes the molecules. During early neural development, Pcdhs were shown to mediate dendrite self-recognition and avoidance in some neuronal types through a still uncharacterized anti-adhesive mechanism. Pcdhs have also been postulated to participate in synaptogenesis and the specificity of connectivity. Some synaptic defects were noted in knockout animals including synaptic number and physiology but the role of these molecules in synaptic development is not understood. We have shown previously that Pcdh-γs are highly enriched in intracellular compartments located in dendrites and spines with less frequent localization at the synaptic membrane. We compared synapses that expressed endogenous Pcdh-γs versus those that did not for parameters of synaptic maturation including pre-and postsynaptic punctum size, postsynaptic perforations and spine morphology in mature cultured hippocampal neurons. We also performed serial section immuno-electron microscopy in intact hippocampal CA1 and measured synaptic size and perforation status of Pcdh-γ positive and negative synapses. At both the light and ultrastructural levels, synapses immunopositive for Pcdh-γs were found to be larger in diameter with more frequent perforations. Analysis of spines in cultured neurons revealed that mushroom spines were more frequently immunopositive for Pcdh-γs at their tips than thin spines. Taken together, these results suggest that Pcdh-γ function at the synapse may be related to synaptic maturation and stabilization.

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Structural origins of clustered protocadherin-mediated neuronal barcoding

Cited by 42 publications

References 72 publications

Family-wide Structural and Biophysical Analysis of Binding Interactions among Non-clustered δ-Protocadherins

Family-wide Structural and Biophysical Analysis of Binding Interactions among Non-clustered δ-Protocadherins

CRISPR/Cas9 interrogation of the mouse Pcdhg gene cluster reveals a crucial isoform-specific role for Pcdhgc4

Gamma-protocadherin localization at the synapse corresponds to parameters of synaptic maturation

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