Biology subjects

Ruchti, E.

Publications and source records attributed to Ruchti, E..

4 recordsLinked to original sources

bioRxiv2026

Spontaneous neurotransmitter release is regulated by Unc-5

The spontaneous quantal release of single vesicles of neurotransmitter in the absence of an action potential is a universal feature of all neuronal chemical synapses. These miniature events have long been thought to be stochastic and unregulated, leading to measurement of their frequency as a widely employed estimate of the number of synaptic connections within neuronal networks. Here we show using high resolution live imaging of Drosophila adult glutamatergic synapses that the spontaneous release of neurotransmitter occurs from only a subset of synaptic release sites. We discover that the proportion of release sites participating in spontaneous neurotransmitter release is regulated by a novel synaptic function of the transmembrane signalling receptor Unc-5 which is heparan sulfate proteoglycan dependent, but Netrin independent. We show that synaptic Unc-5 forms a complex with the SNARE protein Syntaxin to regulate neurotransmitter release. The depletion of Unc-5 in adult synapses diminishes miniature events, inducing terminal degeneration and behavioural decline. Our results reveal that spontaneous neurotransmitter release is a singular, essential and independently regulated property of synapses that is critical for the structural integrity and behavioural contribution of synaptic connections.

neuroscience↗

bioRxiv2026

Tools for synchronous temporal control of independent transgenes in discrete Drosophila tissues

Exogenous binary gene regulatory expression systems are a linchpin technology critical for many model organism genetic manipulations including of Drosophila melanogaster. Subsequent to the initial establishment of the Gal4/UAS binary expression system, LexA/LexOp and QF/QUAS binary gene expression systems have been adopted to supplement and expand the tissue specific control of genes expression in Drosophila. Here, we have developed a compendium of modular vectors that enable robust, reproducible, and high throughput parallel construction of transgenes that produce either Gal4, LexA or QF2 transcription factors under specific gene enhancers and compatible partner vectors that allow transgenes to be regulated by these factors under UAS, LexOp, or QUAS control. Expanding upon this foundation, we have generated a novel hybrid binary gene regulatory system derived from QF and Gal4 - QFG4, that enables simultaneous coordinate regulation of UAS and QUAS transgenes by Gal80, allowing synchronous temporal control of independent transgene expression in distinct cells or tissues. We envision these new tools will facilitate novel and increasingly sophisticated interrogation of cell and tissue interactions particularly in contexts where temporal control is paramount.

genetics↗

bioRxiv2025

Highly frequent undesired insertional mutagenesis during Drosophila genome editing

CRISPR/Cas9 based genome editing employing Homology Directed Repair (HDR) from template vector sequences is a widely used technique to enable precise insertions, deletions or modifications to genes. Here, we describe an undesired and highly frequent editing event when using conventional CRISPR/Cas9 plus HDR methods for Drosophila melanogaster germline genome editing. We find that the template vector employed for HDR repair unwantedly and commonly inserts into the genome. We observe this deviation from the desired edit at multiple genomic locations, with different HDR vectors and with multiple genome editing designs. To avoid these events, we have generated a novel HDR template vector that enables animals with these undesired insertions to be identified and excluded. Our results suggest that HDR based genome edited animals must be carefully screened for unwanted vector template genomic integration in order to avoid misleading interpretations of genome editing outcomes.

molecular biology↗

bioRxiv2021

Intact Drosophila Whole Brain Cellular Quantitation reveals Sexual Dimorphism

Establishing with precision the quantity and identity of the cell types of the brain is a prerequisite for a detailed compendium of gene and protein expression in the central nervous system. Currently however, strict quantitation of cell numbers has been achieved only for the nervous system of C.elegans. Here we describe the development of a synergistic pipeline of molecular genetic, imaging, and computational technologies designed to allow high-throughput, precise quantitation with cellular resolution of reporters of gene expression in intact whole tissues with complex cellular constitutions such as the brain. We have deployed the approach to determine with exactitude the number of functional neurons and glia in the entire intact larval Drosophila central nervous system (CNS), revealing fewer neurons and more glial cells than previously predicted. We also discover an unexpected divergence between the sexes at this juvenile developmental stage, with the female CNS having significantly more neurons than that of males. Topological analysis of our data establishes that this sexual dimorphism extends to deeper features of CNS organisation. We additionally extended our analysis to quantitate the expression of voltage-gated potassium channel family genes throughout the CNS and uncover substantial differences in abundance. Our methodology enables robust and accurate quantification of the number and positioning of cells within intact organs, facilitating sophisticated analysis of cellular identity, diversity, and gene expression characteristics.

neuroscience↗