Biology subjects

Özkan, E.

Publications and source records attributed to Özkan, E..

10 recordsLinked to original sources

bioRxiv2026

Evolution of NELL binding by dual-ligand-responsive axon guidance receptor Robo

Robo receptors are conserved across bilaterians and best known for their ability to mediate axonal repulsion in response to Slit family ligands. In mammals, this applies to Robo1 and Robo2, but mammalian Robo3 binds NELL proteins instead of Slits. The evolutionary origin of NELL-Robo interactions and the possible existence of dual-ligand responsiveness across species remain unknown. Here, we systematically analyzed Robo and NELL homologs across bilaterians and found that NELL-Robo binding is conserved among chordate Robos, but not in protostomes. We show that cephalochordate Robo and NELL can mediate axon repulsion in vitro, suggesting conserved functionality. We observed that conformational masking of the NELL-binding site is prevalent among chordate Robos, modulating NELL-Robo affinity. We also demonstrate that NELL-Robo complexes undergo liquid-liquid phase separation in vitro, a property preserved from cephalochordates to mammals. Our findings support a model in which an ancestral chordate Robo receptor was dual-responsive to Slit and NELL, still the case for some extant Robos, and vertebrate paralogs subfunctionalized, with full ligand specialization emerging in mammals.

biochemistry↗

bioRxiv2026

Reciprocal repulsions enforce heterotypic dendrite segregation in an olfactory circuit

How dendrites of different neurons segregate into discrete spatial domains during neural circuit assembly is poorly understood. Here, using the Drosophila olfactory system, we found that heterophilic interactions between two cell-surface proteins Teneurin-m (Ten-m) and Capricious (Caps) drive dendrite segregation. Ten-m and Caps are expressed in largely inverse patterns across projection neuron (PN) types when PNs are establishing their dendritic territories. Loss of Ten-m in Ten-m+ PNs causes their dendrites to invade Caps+ territories, whereas loss of Caps in Caps+ PNs causes dendrite invasion into Ten-m+ territories. Structure-guided mutations that abolish Ten- m-Caps binding disrupt dendrite segregation, whereas the same mutation on Ten-m preserves its homophilic attraction in a synaptic partner matching assay. These results support a model in which mutual repulsions between two inversely expressed cell-surface proteins drive dendrite segregation into discrete glomerular territories.

neuroscience↗

bioRxiv2025

Structural insights into wiring specificity in the neuromuscular system through the Beat-Side complex

Nervous system assembly is guided by the actions of cell surface receptors. In Drosophila, members of the Beaten Path (Beat) and Sidestep (Side) protein families have been described as axon guidance receptor-cue pairs, in addition to roles in specifying synaptic connectivity in the optic lobe. To understand the molecular basis and specificity of Beat-Side interactions, we report here the first Beat-Side structure, Beat-Vc bound to Side-VI. The structure showed a binding topology similar to other neuronal immunoglobulin superfamily receptors, especially Nectins, SynCAMs, Dprs and DIPs, despite lack of established evolutionary relationships. Using a structure-based rational approach, we engineered and validated point mutations to break the binding between Beats and Sides. Using these mutant variants, we demonstrated in developing Drosophila larvae that the interaction between Beat-Ia and Side is required for establishing proper connectivity of motor neurons with muscles.

neuroscience↗

bioRxiv2025

Repulsive interactions instruct synaptic partner matching in an olfactory circuit

Neurons exhibit extraordinary precision in selecting synaptic partners. Although cell-surface proteins (CSPs) mediating attractive interactions between developing axons and dendrites have been shown to instruct synaptic partner matching1,2, the degree to which repulsive interactions play a role is less clear. Here, using a genetic screen guided by single-cell transcriptomes3,4, we identified three CSP pairs--Toll2-Ptp10D, Fili-Kek1, and Hbs/Sns- Kirre--in mediating repulsive interactions between non-partner olfactory receptor neuron (ORN) axons and projection neuron (PN) dendrites in the developing Drosophila olfactory circuit. Each CSP pair exhibits inverse expression patterns in the select ORN-PN partners. Loss of each CSP in ORNs led to similar synaptic partner matching deficits as the loss of its partner CSP in PNs, and mistargeting phenotypes caused by overexpressing one CSP could be suppressed by loss of its partner CSP. All CSP pairs are also differentially expressed in other brain regions. Together, our data reveal that multiple repulsive CSP pairs work together to ensure precise synaptic partner matching during development by preventing neurons from forming connections with non-cognate partners.

neuroscience↗

bioRxiv2024

Nematode Extracellular Protein Interactome Expands Connections between Signaling Pathways

Multicellularity was accompanied by the emergence of new classes of cell surface and secreted proteins. The nematode C. elegans is a favorable model to study cell surface interactomes, given its well-defined and stereotyped cell types and intercellular contacts. Here we report our C. elegans extracellular interactome dataset, the largest yet for an invertebrate. Most of these interactions were unknown, despite recent datasets for flies and humans, as our collection contains a larger selection of protein families. We uncover new interactions for all four major axon guidance pathways, including ectodomain interactions between three of the pathways. We demonstrate that a protein family known to maintain axon locations are secreted receptors for insulins. We reveal novel interactions of cystine-knot proteins with putative signaling receptors, which may extend the study of neurotrophins and growth-factor-mediated functions to nematodes. Finally, our dataset provides insights into human disease mechanisms and how extracellular interactions may help establish connectomes.

biochemistry↗

bioRxiv2023

WFIKKN2 is a bifunctional axon guidance cue that signals through divergent DCC family receptors

Axon pathfinding is controlled by attractive and repulsive molecular cues that activate receptors on the axonal growth cone, but the full repertoire of axon guidance molecules remains unknown. The vertebrate DCC receptor family contains the two closely related members DCC and Neogenin with prominent roles in axon guidance and three additional, divergent members - Punc, Nope, and Protogenin - for which functions in neural circuit formation have remained elusive. We identified a secreted Punc/Nope/Protogenin ligand, WFIKKN2, which guides mouse peripheral sensory axons through Nope-mediated repulsion. In contrast, WFIKKN2 attracts motor axons, but not via Nope. These findings identify WFIKKN2 as a bifunctional axon guidance cue that acts through divergent DCC family members, revealing a remarkable diversity of ligand interactions for this receptor family in nervous system wiring. One-Sentence SummaryWFIKKN2 is a ligand for the DCC family receptors Punc, Nope, and Prtg that repels sensory axons and attracts motor axons.

developmental biology↗

bioRxiv2023

Neuronal wiring receptors Dprs and DIPs are GPI anchored and this modification contributes to their cell surface organization

The Drosophila Dpr and DIP proteins belong to the immunoglobulin superfamily of cell surface proteins (CSPs). Their hetero- and homophilic interactions have been implicated in a variety of neuronal functions, including synaptic connectivity, cell survival, and axon fasciculation. However, the signaling pathways underlying these diverse functions are unknown. To gain insight into Dpr-DIP signaling, we sought to examine how these CSPs are associated with the membrane. Specifically, we asked whether Dprs and DIPs are integral membrane proteins or membrane anchored through the addition of glycosylphosphatidylinositol (GPI) linkage. We demonstrate that Dprs and DIPs are GPI anchored to the membrane of insect cells and validate these findings for some family members in vivo using Drosophila larvae, where GPI anchor cleavage results in loss of surface labeling. Additionally, we show that GPI cleavage abrogates aggregation of insect cells expressing cognate Dpr-DIP partners. To test if the GPI anchor affects Dpr-DIP localization, we replaced it with a transmembrane domain and observed perturbation of sub-cellular localization on motor neurons and muscles. These data suggest that membrane anchoring of Dprs and DIPs through GPI linkage is required for localization and that Dpr-DIP intracellular signaling likely requires transmembrane co-receptors.

neuroscience↗

bioRxiv2022

Structural insights into the formation of repulsive Netrin guidance complexes

Netrins can dictate attractive and repulsive responses during axon growth and cell migration, where presence of the receptor UNC-5 on target cells results in Netrin-mediated repulsion. Molecular details of Netrin-UNC-5 interactions and how they signal remain elusive. Here, we show that nematode UNC-5 is a heparin-binding protein, and the UNC-5-heparin affinity can be modulated using directed evolution or via rational design using our novel structure of UNC-5 with a heparin fragment. Furthermore, UNC-5 and nematode UNC-6/Netrin form a large, stable and rigid oligomeric complex in the presence of heparin, which can incorporate the attractive UNC-40/DCC receptor, demonstrating binary and ternary ectodomain complexes at preparative scale. C. elegans with a heparin-binding deficient UNC-5 fail to establish proper gonad morphology due to abrogated distal tip cell migration, which relies on repulsive UNC-5 signaling in response to UNC-6. Our findings establish Netrin responses to be mediated through glycosaminoglycan-regulated large macromolecular complexes.

biochemistry↗

bioRxiv2021

Molecular and Structural Basis of Olfactory Sensory Neuron Coalescence by Kirrel Receptors

Projections from sensory neurons of olfactory systems coalesce into glomeruli in the brain. The Kirrel receptors are believed to homodimerize via their ectodomains and help separate sensory neuron axons into Kirrel2- or Kirrel3-expressing glomeruli. Here we present the crystal structures of homodimeric Kirrel receptors and show that the closely related Kirrel2 and Kirrel3 have evolved specific sets of polar and hydrophobic interactions, respectively, disallowing heterodimerization while preserving homodimerization, likely resulting in proper segregation and coalescence of Kirrel-expressing axons into glomeruli. We show that the dimerization interface at the N-terminal IG domains is necessary and sufficient to create homodimers, and fail to find evidence for a secondary interaction site in Kirrel ectodomains. Furthermore, we show that abolishing dimerization of Kirrel3 in vivo leads to improper formation of glomeruli in the mouse accessory olfactory bulb as observed in Kirrel3-/- animals. Our results provide strong evidence for Kirrel3 homodimerization controlling axonal coalescence.

neuroscience↗

bioRxiv2018

A new family of neural wiring receptors across bilaterians defined by phylogenetic, biochemical and structural evidence

The evolution of complex nervous systems was accompanied by the expansion of groups of protein families, most notably cell adhesion molecules, surface receptors and their ligands. These proteins mediate axonal guidance, synapse targeting, and other neuronal wiring-related functions. Recently, members of a set of thirty interacting cell surface proteins belonging to two newly defined families of the immunoglobulin superfamily (IgSF) in fruit flies were discovered to label different subsets of neurons in the brain and ventral nerve cord. They have been shown to be involved in synaptic targeting and morphogenesis, retrograde signaling, and neuronal survival. Here we show that these proteins, denoted as Dprs and DIPs, belong to a family of two and three-Ig domain molecules in bilaterians generally known for neuronal wiring functions. In protostomes, the ancestral Dpr/DIP gene has duplicated to form heterophilic partners, such as Dprs and DIPs, while in deuterostomes, they have evolved to create the IgLON family of neuronal receptors. In support of this phylogeny, we show that IgLONs interact with each other, and that their complexes can be broken by mutations designed using homology models based on Dpr and DIP structures. Similarly, the nematode orthologs ZIG-8 and RIG-5 can form heterophilic and homophilic complexes structurally matching Dpr-DIP and DIP-DIP complexes. The evolutionary, biochemical and structural relationships we demonstrate here provides insights into neural development and the rise of complexity in metazoans.\n\nSignificance StatementCell surface receptors assign and display unique identities to neurons, and direct proper and robust wiring of neurons to create functional neural circuits. Recent work has identified two new classes of receptors in fruit flies, called the Dpr and DIP families with 30 members, which interact in 38 pairwise combinations. These proteins are implicated in neural identity, wiring and survival in many parts of the fly nervous system. Here, using evolutionary, biochemical and structural evidence, we show that Dprs and DIPs are members of an ancient bilaterian family of receptors. Members of this family share functional roles relevant to wiring across species, and are likely crucial in the emergence of the bilaterian nervous systems common to vertebrate and invertebrate animals.

biochemistry↗