SynLight: a dicistronic strategy for simultaneous active zone and cell labeling in the Drosophila nervous system.
| العنوان: | SynLight: a dicistronic strategy for simultaneous active zone and cell labeling in the Drosophila nervous system. |
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| المؤلفون: | Aimino MA; Department of Neuroscience, Vickie and Jack Farber Institute of Neuroscience, Thomas Jefferson University, Bluemle Life Sciences Building, Philadelphia, PA 19107., Humenik J; Department of Neuroscience, Vickie and Jack Farber Institute of Neuroscience, Thomas Jefferson University, Bluemle Life Sciences Building, Philadelphia, PA 19107., Parisi MJ; Department of Neuroscience, Vickie and Jack Farber Institute of Neuroscience, Thomas Jefferson University, Bluemle Life Sciences Building, Philadelphia, PA 19107., Duhart JC; Department of Neuroscience, Vickie and Jack Farber Institute of Neuroscience, Thomas Jefferson University, Bluemle Life Sciences Building, Philadelphia, PA 19107., Mosca TJ; Department of Neuroscience, Vickie and Jack Farber Institute of Neuroscience, Thomas Jefferson University, Bluemle Life Sciences Building, Philadelphia, PA 19107. |
| المصدر: | BioRxiv : the preprint server for biology [bioRxiv] 2023 Jul 17. Date of Electronic Publication: 2023 Jul 17. |
| نوع المنشور: | Preprint |
| اللغة: | English |
| بيانات الدورية: | Country of Publication: United States NLM ID: 101680187 Publication Model: Electronic Cited Medium: Internet NLM ISO Abbreviation: bioRxiv Subsets: PubMed not MEDLINE |
| مستخلص: | At synapses, chemical neurotransmission mediates the exchange of information between neurons, leading to complex movement behaviors and stimulus processing. The immense number and variety of neurons within the nervous system makes discerning individual neuron populations difficult, necessitating the development of advanced neuronal labeling techniques. In Drosophila , Bruchpilot-Short and mCD8-GFP, which label presynaptic active zones and neuronal membranes, respectively, have been widely used to study synapse development and organization. This labeling is often achieved via expression of two independent constructs by a single binary expression system, but expression can weaken when multiple transgenes are expressed by a single driver. Ensuring adequate expression of each transgene is essential to enable more complex experiments; as such, work has sought to circumvent these drawbacks by developing methods that encode multiple proteins from a single transcript. Self-cleaving peptides, specifically 2A peptides, have emerged as effective sequences for accomplishing this task. We leveraged 2A ribosomal skipping peptides to engineer a construct that produces both Bruchpilot-Short and mCD8-GFP from the same mRNA, which we named SynLight. Using SynLight, we visualized the putative synaptic active zones and membranes of multiple classes of olfactory, visual, and motor neurons and observed correct separation of signal, confirming that both proteins are being generated separately. Furthermore, we demonstrate proof-of-principle by quantifying synaptic puncta number and neurite volume in olfactory neurons and finding no difference between the synapse densities of neurons expressing SynLight or neurons expressing both transgenes separately. At the neuromuscular junction, we determined that synaptic puncta number labeled by SynLight was comparable to endogenous puncta labeled by antibody staining. Overall, SynLight is a versatile tool for examining synapse density in any nervous system region of interest and allows new questions to be answered about synaptic development and organization. |
| التعليقات: | Update in: G3 (Bethesda). 2023 Sep 27;:. (PMID: 37757863) |
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| معلومات مُعتمدة: | P40 OD018537 United States OD NIH HHS; R00 DC013059 United States DC NIDCD NIH HHS; R01 NS110907 United States NS NINDS NIH HHS |
| تواريخ الأحداث: | Date Created: 20230728 Latest Revision: 20231104 |
| رمز التحديث: | 20231104 |
| مُعرف محوري في PubMed: | PMC10370149 |
| DOI: | 10.1101/2023.07.17.549367 |
| PMID: | 37502901 |
| قاعدة البيانات: | MEDLINE |
| DOI: | 10.1101/2023.07.17.549367 |
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