Biology subjects

Thomas, U.

Publications and source records attributed to Thomas, U..

5 recordsLinked to original sources

bioRxiv2026

Presynaptic PMCA interacts with Dlg, regulating in vivo synaptic calcium in Drosophila larval NMJ

Precise regulation of presynaptic Ca2+is essential for neurotransmitter release and synaptic function. At the Drosophila larval neuromuscular junction (NMJ), the plasma membrane Ca2+-ATPase (PMCA) mediates presynaptic Ca2+; extrusion and is enriched in peri-active-zone membrane domains. Here, we investigated how PMCA distribution is regulated and contributes to synaptic function. Co-immunoprecipitation and super-resolution STED microscopy revealed that PMCA interacts and co-localizes with the MAGUK scaffold protein Discs large (Dlg) in discrete presynaptic membrane clusters. Neuronal depletion of the DlgS97 isoform disrupted PMCA clustering without substantially altering its abundance, redistributing the pump into smaller membrane puncta. Reporter assays further showed that PDZ-binding motifs in the PMCA C-terminus mediate its interaction with Dlg and are required for normal cluster formation. Electrophysiological analyses revealed that reducing either PMCA or Dlg increased spontaneous neurotransmitter release and enhanced asynchronous release following high-frequency stimulation. PMCA depletion also prolonged postsynaptic current decay during repetitive stimulation, implicating PMCA in the rapid regulation of presynaptic Ca2+; signals. Together, these findings identify Dlg as a key organizer of presynaptic PMCA and establish a link between synaptic scaffold organization and Ca2+; homeostasis in vivo. Our results demonstrate that PMCA contributes to maintaining resting presynaptic Ca2+; levels, restoring Ca2+; homeostasis after repetitive stimulation, and regulating Ca2+; dynamics following a single stimulus. These findings highlight the importance of the nanoscale organization of Ca2+; extrusion machinery in controlling neurotransmitter release at the Drosophila NMJ.

neuroscience↗

bioRxiv2025

Glycaemic variability underlies myocyte dysfunction and myocardial injury risk in diabetes

Heart disease is the leading cause of morbidity and mortality in individuals with diabetes, due largely to risks associated with ischaemic injuries such as myocardial infarction (MI). We use human population genetic data to demonstrate that current biomarkers of hyperglycaemia do not account for risk of post-MI mortality in diabetes patients. This study therefore systematically evaluates glycaemic stress underpinning cardiovascular risk in diabetes. Using in vivo and in vitro models, we demonstrate that glycaemic variability rather than hyperglycaemia alone is a dominant risk factor for heart muscle dysfunction and myocardial injury sensitivity in diabetes. These findings provide new preclinical models for mechanistic and drug discovery studies and inform strategies for managing cardiovascular outcomes in patients with diabetes.

cell biology↗

bioRxiv2025

Monoamine-induced diacylglycerol signaling rapidly accumulates Unc13 in nanoclusters for fast presynaptic potentiation

Neuromodulators control mood, arousal, and behavior by inducing synaptic plasticity via G-protein coupled receptors. Long-term potentiation of presynaptic neurotransmitter release requires structural changes, but how fast potentiation is achieved within minutes remains enigmatic. Using the Drosophila melanogaster neuromuscular junction, we show that on the timescale of one minute, octopamine, the invertebrate analog of nor-epinephrine, rapidly potentiates evoked neurotransmitter release by a G protein coupled pathway involving presynaptic OAMB receptors and phospholipase C. No changes of presynaptic calcium influx were seen, but confocal signals of the release factor Unc13A and the scaffolding protein Bruchpilot increased within one minute of octopamine treatment. On the same timescale, live, single-molecule imaging of endogenously tagged Unc13 revealed its instantly reduced motility and its increased concentration in synaptic nanoclusters with potentiation. Presynaptic knockdown of Unc13A fully blocked fast potentiation and removal of its N-terminal localization sequence delocalized the protein fragment to the cytosol, but it was rapidly recruited to the plasma membrane by DAG analog phorbol esters and octopamine, pointing to a role in C-terminal domains. Point mutation of endogenous Unc13 disrupting diacylglycerol-binding to its C1 domain blocked plasticity-induced nanoscopic enrichment and synaptic potentiation. The mutation increased basal neurotransmission but reduced Unc13 levels, revealing a gain of function and potential homeostatic compensation. The mutation also blocked phorbol ester-induced potentiation, decreased the calcium-sensitivity of neurotransmission and caused short-term synaptic depression. At the organismal level, the mutation reduced locomotion and survival while enhancing reproduction. Thus, the Unc13 C1 domain mediates acute subsynaptic compaction of Unc13 under monoamine-induced potentiation and influences short-term plasticity, locomotion, reproduction, and survival.

neuroscience↗

bioRxiv2022

Drosophila Atlastin regulates synaptic vesicle mobilization independent of Bone Morphogenetic Protein signaling

Motor neurons are highly dependent on membrane trafficking, in which the endoplasmic reticulum (ER) and its contact sites with endosomes, confer the ER the role of a long-distance communicator. Atlastin (Atl), a large GTPase located on the ER membrane is required for its function and its tubular structural dynamics. Atl also downregulates, by a yet unknown mechanism, the BMP (Bone Morphogenic Protein) pathway. In humans, Atl mutations are the second more common cause of Hereditary Spastic Paraplegia (HSP), a genetic disease characterized by spasticity of the lower extremities. Here, we explore the molecular basis of Atl-dependent defects on synaptic vesicle (SV) traffic in Drosophila under the hypothesis that those defects are the direct consequence of the atl-knock-down and not of the Atl-dependent BMP signaling upregulation. Motor neuronal knockdown of atl (Atl-KD) leads to an increase in synaptic and satellite bouton number similar to the increase in BMP signaling activity (TKV-CA). Neuronal Atl-KD also associates to a reduction in the boutons of the abundance of the SV markers CSP (Cysteine string protein) and VGLUT (vesicular glutamate transporter) as well as in TKV-CA larvae, both phenotypes are suppressed by decreasing the function of BMP receptor wishful thinking expressing one copy of the mutant receptor (wit /+). Surprisingly, we determined in Atl-KD larvae an increase in the CSP peripheral density and distribution, dependent on synaptic stimulation, that was not replicated in Tkv-CA larvae, suggesting that there could be differences in the mechanisms that underlie the reduction in CSP abundance. Additionally, we determined that Atl-KD associates to an increase in FM 1-43 unload but not in TKV-CA larvae. Moreover, one copy of wit was not able to suppress the FM-143 in Atl-KD larvae (Atl-KD, wit), supporting that BMP signaling does not participate in this phenotype. Together with the stimuli-dependent changes in the SV distribution and dynamics determined in Atl-KD larvae, we measured an increase in Rab11/CSP colocalization, suggesting changes in SV traffic through late recycling endosomes. Together our results suggest a mechanism by which the loss of an ER structuring protein in the motor neuron could, through its role in regulating SV and endosomal trafficking, explain defects in SV accumulation and synaptic dysfunction.

cell biology↗

bioRxiv2021

A quick and versatile protocol for the 3D visualization of transgene expression across the whole body of larval Drosophila

Larval Drosophila are used as a genetically accessible study case in many areas of biological research. Here we report a fast, robust and user-friendly procedure for the whole-body multifluorescence imaging of Drosophila larvae; the protocol has been optimized specifically for larvae by systematically tackling the pitfalls associated with clearing this small but cuticularized organism. Tests on various fluorescent proteins reveal that the recently introduced monomeric infrared fluorescent protein (mIFP) is particularly suitable for our approach. This approach comprises an effective, low-cost clearing protocol with minimal handling time and reduced toxicity in the reagents employed. It combines a success rate high enough to allow for small-scale screening approaches and a resolution sufficient for cellular-resolution analyses with light sheet and confocal microscopy. Given that publications and database documentations typically specify expression patterns of transgenic driver lines only within a given organ system of interest, the present procedure should be versatile enough to extend such documentation systematically to the whole body. As examples, the expression patterns of transgenic driver lines covering the majority of neurons, or subsets of chemosensory, central brain or motor neurons, are documented in the context of whole larval body volumes (using nsyb-Gal4, IR76b-Gal4, APL-Gal4 and mushroom body Kenyon cells, or OK371-Gal4, respectively). Notably, the presented protocol allows for triple-color fluorescence imaging with near-infrared, red and yellow fluorescent proteins.

neuroscience↗