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Zinn, K.

Publications and source records attributed to Zinn, K..

12 recordsLinked to original sources

Next-generation hybridization chain reaction tools with enhanced sensitivities to detect challenging targets

Compared to traditional enzyme-based in situ amplification methods, Hybridization Chain Reaction v3.0 (HCR v3.0) offers high specificity for spatial RNA visualization but lacks the sensitivity required to robustly detect short or low-abundance targets, particularly in thick tissue with high autofluorescence. Here, we describe three HCR variants that combine the specificity of HCR v3.0 with additional signal amplification through catalytic reporter deposition (HCR-Cat), immunostaining (HCR-Immuno), or iterative HCR and immunodetection (HCR-Multi). These methods substantially enhance detection sensitivity, enabling robust spatial visualization of low-abundance transcripts, improved performance in challenging tissue environments, and compatibility with fluorescence- and alkaline phosphatase-based chromogenic detection. These methods enable transcript-resolution imaging, even when using a limited number of probes, thereby expanding the range of biological applications accessible to HCR.

neuroscience↗

A multiplex extracellular interactome screening method employing high-avidity nanoparticles

Metazoan cells signal to each other via direct contact between cell surface proteins (CSPs) and by interactions of CSP receptors with secreted ligands. CSP extracellular domain (ECD) interactions control organ development and physiology and are perturbed in disease states. However, because they cannot be accurately assessed using standard high-throughput screening techniques, they are underrepresented in protein interaction databases. Many ECD interactions are of low affinity, and their detection in vitro requires taking advantage of avidity effects, typically by multimerization of fusion proteins. Assays that test only one or a few interactions in each binding reaction are inadequate for global interactome screening. Here we describe a new multiplex method that uses purified dimeric ECD fusion proteins coupled to 60-mer nanoparticles as soluble prey, and the same dimers coupled to spectrally distinguishable fluorescent microspheres (beads) as bait. We add one prey to a mixture of up to 500 baits in a single well, then use a Luminex FLEXMAP 3D (FM3D) instrument to read out bait identity and prey binding. The FM3D measures the fluorescent dye ratio for each bead and simultaneously determines the amount of epitope-tagged prey bound to that bead. We use the method, denoted as the Multiplex Interactome Assay (MPIA), to analyze a proof-of-concept (PoC) set of 41 CSPs and secreted protens that is derived from larger collections examined in two interactome screens that used ELISA-based binding assays. By analyzing interactions among PoC proteins, we compared the MPIA with earlier screening methods. The MPIA has a dynamic range that is at least 30-fold greater than ELISA-based assays and appears to be more sensitive. By coupling the MPIA to an automated protein production and purification platform, we hope to be able to conduct a screen for interactions among thousands of human CSPs and secreted ligands.

molecular biology↗

An Automated End-to-End Workflow for Production of Secreted Proteins in Transfected Mammalian Cells

The advancement of automation technologies has helped to enable a surge in large-scale screening efforts across fields such as molecular biology, protein biochemistry, cell biology, and structural biology. In the context of this "omics"-driven research, there is a need to generate automation platforms that are more flexible and less expensive, so that they can be utilized for basic research conducted by small groups. A key challenge in automation lies in developing methods that can replicate fine motor techniques that are normally performed manually by researchers at the bench. We are engaged in a large-scale project to map interactions among human cell-surface and secreted proteins and assess their effects on cells. This project involves production of a library of more than 2000 recombinant His-tagged fusion proteins secreted from transfected Expi293 cells. To execute such a project with a small group at an academic institution required construction of an affordable automated system that could also be used by other investigators. This led us to develop a high-throughput, 96-well format automation platform for end-to-end protein production. The workflow includes transformation of E. coli, plasmid DNA preparation, transient transfection, protein purification, desalting and buffer exchange, protein quantification, and normalization of protein concentrations, resulting in assay-ready proteins. The system is built around an in-house engineered modular robotic platform that integrates liquid handling with a suite of interchangeable plug-and-play mobile enclosed device modules. Housed within a BSL-2 sterile environment, the platform enables flexible, fully automated workflows and can be readily customized for diverse user-defined protocols.

biochemistry↗

FETCH enables fluorescent labeling of membrane proteins in vivo with spatiotemporal control in Drosophila

Fluorescent labeling approaches are crucial for elucidating protein function and dynamics. While enhancer trapping in Drosophila has been useful for the characterization of gene transcription, protein-specific visualization in vivo has been more elusive. To overcome these limitations, we developed Fluorescent Endogenous Tagging with a Covalent Hook (FETCH) to label cell surface proteins (CSPs) in vivo through a stable covalent bond mediated by the DogTag-DogCatcher peptide partner system1. FETCH leverages a spontaneous covalent isopeptide bond that forms between the 23-amino acid DogTag and the 15-kDa DogCatcher. Unlike most tags that work best at protein termini, DogTag is optimized for function in protein loops, expanding the range of sites that can be targeted in proteins. In FETCH, DogTag is introduced into extracellular loops of CSPs through genome engineering, enabling covalent bond formation with a genetically encoded DogCatcher-GFP fusion protein that can be secreted from a variety of cell types. We describe a flow cytometry-based platform for the identification of efficient DogTag insertion sites in vitro and demonstrate the ability to visualize both tagged DIP- and Dpr10 in vivo, two immunoglobulin superfamily proteins that facilitate neuronal target recognition at Drosophila neuromuscular junctions and brain synapses. The versatility of FETCH enables fluorescent labeling with precise temporal and spatial control in vivo, enabling applications previously unfeasible.

neuroscience↗

An extracellular vesicle targeting ligand that binds to Arc proteins and facilitates Arc transport in vivo

Communication between distant cells can be mediated by extracellular vesicles (EVs) that deliver proteins and RNAs to recipient cells. Little is known about how EVs are targeted to specific cell types. Here we identify the Drosophila cell-surface protein Stranded at second (Sas) as a targeting ligand for EVs. Full-length Sas is present in EV preparations from transfected Drosophila Schneider 2 (S2 cells). Sas is a binding partner for the Ptp10D receptor tyrosine phosphatase, and Sas-bearing EVs preferentially target to cells expressing Ptp10D. We used co-immunoprecipitation and peptide binding to show that the cytoplasmic domain (ICD) of Sas binds to dArc1. dArc1 and mammalian Arc are related to retrotransposon Gag proteins. They form virus-like capsids which encapsulate Arc and other mRNAs and are transported between cells via EVs. The Sas ICD contains a motif required for dArc1 binding that is shared by the mammalian and Drosophila amyloid precursor protein (APP) orthologs, and the Sas and APP ICDs also bind to mammalian Arc. Sas facilitates delivery of dArc1 capsids bearing dArc1 mRNA into distant Ptp10D-expressing recipient cells in vivo.

cell biology↗

Identification of orphan ligand-receptor relationships using a cell-based CRISPRa enrichment screening platform.

Secreted proteins, which include cytokines, hormones and growth factors, are extracellular ligands that control key signaling pathways mediating cell-cell communication within and between tissues and organs. Many drugs target secreted ligands and their cell-surface receptors. Still, there are hundreds of secreted human proteins that either have no identified receptors ("orphans") and are likely to act through cell surface receptors that have not yet been characterized. Discovery of secreted ligand-receptor interactions by high-throughput screening has been problematic, because the most commonly used high-throughput methods for protein-protein interaction (PPI) screening do not work well for extracellular interactions. Cell-based screening is a promising technology for definition of new ligand-receptor interactions, because multimerized ligands can enrich for cells expressing low affinity cell-surface receptors, and such methods do not require purification of receptor extracellular domains. Here, we present a proteo-genomic cell-based CRISPR activation (CRISPRa) enrichment screening platform employing customized pooled cell surface receptor sgRNA libraries in combination with a magnetic bead selection-based enrichment workflow for rapid, parallel ligand-receptor deorphanization. We curated 80 potentially high value orphan secreted proteins and ultimately screened 20 secreted ligands against two cell sgRNA libraries with targeted expression of all single-pass (TM1) or multi-pass (TM2+) receptors by CRISPRa. We identified previously unknown interactions in 12 of these screens, and validated several of them using surface plasmon resonance and/or cell binding. The newly deorphanized ligands include three receptor tyrosine phosphatase (RPTP) ligands and a chemokine like protein that binds to killer cell inhibitory receptors (KIRs). These new interactions provide a resource for future investigations of interactions between the human secreted and membrane proteomes.

cell biology↗

Affinity Requirements For Control Of Synaptic Targeting And Neuronal Cell Survival By Heterophilic IgSF Cell Adhesion Molecules

Neurons in the developing brain express many different cell adhesion molecules (CAMs) on their surfaces, and CAM interactions are essential for the determination of synaptic connectivity patterns. CAM binding affinities can vary by more than 200-fold, but the significance of affinity differences among CAMs is unknown. Here we provide a systematic characterization of the in vivo consequences of altering CAM affinity. Interactions between DIP- and its binding partners Dpr6 and Dpr10 control synaptic targeting and cell survival for Drosophila optic lobe neurons. We generated mutations that change DIP-::Dpr10 binding affinity and introduced these into the endogenous loci. We show that cell survival and synaptic targeting have different affinity requirements, and that there is a threshold affinity required for targeting. Reducing affinity causes graded loss-of-function phenotypes, while increasing affinity rescues cells that would normally die. Affinity reduction can be compensated for by increasing gene copy number.

neuroscience↗

Sticks and Stones, a conserved cell surface ligand for the Type IIa RPTP Lar, regulates neural circuit wiring in Drosophila

Control of tyrosine phosphorylation is an essential element of many cellular processes, including proliferation, differentiation neurite outgrowth, and synaptogenesis. Receptor-like protein-tyrosine phosphatases (RPTPs) have cytoplasmic phosphatase domains and cell adhesion molecule (CAM)-like extracellular domains that interact with cell-surface ligands and/or co-receptors. We identified a new ligand for the Drosophila Lar RPTP, the immunoglobulin superfamily CAM Sticks and Stones (Sns). Lar is orthologous to the three Type IIa mammalian RPTPs, PTPRF (LAR), PTPRD (PTP{delta}), and PTPRS (PTP{sigma}). Lar and Sns bind to each other in embryos and in vitro. The human Sns ortholog, Nephrin, binds to PTPRD and PTPRF. Genetic interaction studies show that Sns is essential to Lars functions in several developmental contexts in the larval and adult nervous systems. In the larval neuromuscular system, Lar and sns transheterozygotes (Lar/sns transhets) have synaptic defects like those seen in Lar mutants and Sns knockdown animals. Lar and Sns reporters are both expressed in motor neurons and not in muscles, so Lar and Sns likely act in cis (in the same neurons). Lar mutants and Lar/sns transhets have identical axon guidance defects in the larval mushroom body in which Kenyon cell axons fail to stop at the midline and do not branch. Pupal Kenyon cell axon guidance is similarly affected, resulting in adult mushroom body defects. Lar is expressed in larval and pupal Kenyon cells, but Sns is not, so Lar-Sns interactions in this system must be in trans (between neurons). Lastly, R7 photoreceptor axons in Lar mutants and Lar/sns transhets fail to innervate the correct M6 layer of the medulla in the optic lobe. Lar acts cell-autonomously in R7s, while Sns is only in lamina and medulla neurons that arborize near the R7 target layer. Therefore, the Lar-Sns interactions that control R7 targeting also occur in trans.

neuroscience↗

Neurogenetic and genomic approaches reveal roles for Dpr/DIP cell adhesion molecules in Drosophila reproductive behavior

Drosophila reproductive behaviors are directed by fruitless neurons (fru P1 isoforms). A reanalysis of genomic studies shows that genes encoding dpr and DIP Immunoglobulin superfamily (IgSF) members are expressed in fru P1 neurons. Each fru P1and dpr/DIP (fru P1 {cap} dpr/DIP) overlapping expression pattern is similar in both sexes, with dimorphism in neuronal morphology and cell number. Behavioral studies of fru P1 {cap} dpr/DIP perturbation genotypes point to the mushroom body functioning together with the lateral protocerebral complex. Functionally, we find that perturbations of sex hierarchy genes and DIP-{varepsilon} changes sex-specific morphology of fru P1 {cap} DIP- neurons. A single-cell RNA-seq analysis shows that the DIPs have high expression in a restricted set of fru P1 neurons, whereas the dprs are expressed in larger set of neurons at intermediate levels, with a myriad of combinations.

genomics↗

Interactions with presynaptic photoreceptors mediated by the Dpr11 and DIP-γ cell surface proteins control selection and survival of Drosophila amacrine neurons

Drosophila R7 UV photoreceptors (PRs) are divided into yellow (y) and pale (p) subtypes with different wavelength sensitivities. yR7 PRs express the Dpr11 cell surface protein and are presynaptic to Dm8 amacrine neurons (yDm8) that express Dpr11s binding partner DIP-{gamma}, while pR7 PRs synapse onto DIP-{gamma}-negative pDm8 neurons. Dpr11 and DIP-{gamma} expression patterns define yellow and pale medulla color vision circuits that project to higher-order areas. DIP- {gamma} and dpr11 mutations affect the morphology of yDm8 arbors in the yellow circuit. yDm8 neurons are generated in excess during development and compete for presynaptic yR7 partners. Transsynaptic interactions between Dpr11 and DIP-{gamma} are required for generation of neurotrophic signals that allow yDm8 neurons to survive. yDm8 and pDm8 neurons do not normally compete for neurotrophic support, but can be forced to do so by manipulating R7 subtype fates. DIP-{gamma}-Dpr11 interactions allow yDm8 neurons to select yR7 PRs as their home column partners.

neuroscience↗

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↗

Transsynaptic interactions between IgSF proteins DIP-α and Dpr10 are required for motor neuron targeting specificity in Drosophila

The Drosophila larval neuromuscular system provides an ideal context in which to study synaptic partner choice, because it contains a small number of pre- and postsynaptic cells connected in an invariant pattern. The discovery of interactions between two subfamilies of IgSF cell surface proteins, the Dprs and the DIPs, provided new candidates for cellular labels controlling synaptic specificity. Here we show that DIP- is expressed by two identified motor neurons, while its binding partner Dpr10 is expressed by postsynaptic muscle targets. Removal of either DIP- or Dpr10 results in loss of specific axonal branches and NMJs formed by one motor neuron, MNISN-1s, while other branches of the MNISN-1s axon develop normally. The temporal and spatial expression pattern of dpr10 correlates with muscle innervation by MNISN-1s during embryonic development. We propose a model whereby DIP- and Dpr10 on opposing synaptic partners interact with each other to generate proper motor neuron connectivity.

neuroscience↗