Sigulinsky, C. | Search bioRxiv

bioRxiv2024

Uncovering the electrical synapse proteome in retinal neurons via in vivo proximity labeling

AbstractElectrical synapses containing Connexin 36 (Cx36) represent the main means for direct electrical communication among neurons in the mammalian nervous system. However, little is known about the protein complexes that constitute these synapses. In the present study, we applied different BioID strategies to screen the interactomes of Connexin 36 and its zebrafish orthologue Cx35b in retinal neurons. For in vivo proximity labeling in mice, we took advantage of the Cx36-EGFP strain and expressed a GFP-nanobody-TurboID fusion construct selectively in AII amacrine cells. For in vivo BioID in zebrafish, we generated a transgenic line expressing a Cx35b-TurboID fusion under control of the Cx35b promoter. Both strategies allowed us to capture a plethora of molecules that were associated with electrical synapses and showed a high degree of evolutionary conservation in the proteomes of both species. Besides known interactors of Cx36 such as ZO-1 and ZO-2 we have identified more than 50 new proteins, such as scaffold proteins, adhesion molecules and regulators of the cytoskeleton. Moreover, we determined the subcellular localization of these proteins in mouse retina and tested potential binding interactions with Cx36. Amongst these new interactors, we identified signal induced proliferation associated 1 like 3 (Sipa1l3), a protein that has been implicated in cell junction formation and cell polarity, as a new scaffold of electrical synapses. Interestingly, Sipa1l3 was able to interact with ZO-1, ZO-2 and Cx36, suggesting a pivotal role in electrical synapse function. In summary, our study provides the first detailed view of the electrical synapse proteome in retinal neurons, which is likely to apply to electrical synapses elsewhere.

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bioRxiv2018

Heterocellular coupling between amacrine cell and ganglion cells

All superclasses of retinal neurons display some form of electrical coupling including the key neurons of the inner plexiform layer: bipolar cells (BCs), amacrine or axonal cells (ACs) and ganglion cells (GCs). However, coupling varies extensively by class. For example, mammalian rod bipolar cells form no gap junctions at all, while all cone bipolar cells form class-specific coupling arrays, many of them homocellular in-superclass arrays. Ganglion cells are unique in that classes with coupling predominantly form heterocellular cross-class arrays of ganglion cell::amacrine cell (GC::AC) coupling in the mammalian retina. Ganglion cells are the least frequent superclass in the inner plexiform layer and GC::AC gap junctions are sparsely arrayed amidst massive cohorts of AC::AC, bipolar cell BC::BC, and AC::BC gap junctions. Many of these gap junctions and most ganglion cell gap junctions are suboptical, complicating analysis of specific ganglion cells. High resolution 2 nm TEM analysis of rabbit retinal connectome RC1 allows quantitative GC::AC coupling maps of identified ganglion cells. Ganglion cells classes apparently avoid direct cross-class homocellular coupling altogether even though they have opportunities via direct membrane touches, while transient OFF alpha ganglion cells and transient ON directionally selective (DS) ganglion cells are strongly coupled to distinct amacrine / axonal cell cohorts.\n\nA key feature of coupled ganglion cells is intercellular metabolite flux. Most GC::AC coupling involves GABAergic cells ({gamma}+ amacrine cells), which results in significant GABA flux into ganglion cells. Surveying GABA coupling signatures in the ganglion cell layer across species suggests that the majority of vertebrate retinas engage in GC::AC coupling.\n\nMulti-hop synaptic queries of the entire RC1 connectome clearly profiles the coupled amacrine and axonal cells. Photic drive polarities and source bipolar cell class selec-tivities are tightly matched across coupled cells. OFF alpha ganglion cells are coupled to OFF {gamma}+ amacrine cells and transient ON DS ganglion cells are coupled to ON {gamma}+ amacrine cells including a large interstitial axonal cell (IAC). Synaptic tabulations show close matches between the classes of bipolar cells sampled by the coupled amacrine and ganglion cells. Further, both ON and OFF coupling ganglion networks show a common theme: synaptic asymmetry whereby the coupled {gamma}+ neurons are also presynaptic to ganglion cell dendrites from different classes of ganglion cells outside the coupled set. In effect, these heterocellular coupling patterns enable an excited ganglion cell to directly inhibit nearby ganglion cells of different classes. Similarly, coupled {gamma}+ amacrine cells engaged in feedback networks can leverage the additional gain of bipolar cell synapses in shaping the signaling of a spectrum of downstream targets based on their own selective coupling with ganglion cells.

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