Biology subjects

Duhart, J. C.

Publications and source records attributed to Duhart, J. C..

3 recordsLinked to original sources

bioRxiv2023

SynLight: a dicistronic strategy for simultaneous active zone and cell labeling in the Drosophila nervous system

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.

neuroscience↗

bioRxiv2023

Localized modulation of DNA supercoiling, triggered by the Shigella anti-silencer VirB, is sufficient to relieve H-NS-mediated silencing

In Bacteria, nucleoid structuring proteins govern nucleoid dynamics and regulate transcription. In Shigella spp., at [≤] 30 {degrees}C, the histone-like nucleoid structuring protein (H-NS) transcriptionally silences many genes on the large virulence plasmid. Upon a switch to 37 {degrees}C, VirB, a DNA binding protein and key transcriptional regulator of Shigella virulence, is produced. VirB functions to counter H-NS-mediated silencing in a process called transcriptional anti-silencing. Here, we show that VirB mediates a loss of negative DNA supercoils from our plasmid-borne, VirB-regulated PicsP-lacZ reporter, in vivo. The changes are not caused by a VirB-dependent increase in transcription, nor do they require the presence of H-NS. Instead, the VirB-dependent change in DNA supercoiling requires the interaction of VirB with its DNA binding site, a critical first step in VirB-dependent gene regulation. Using two complementary approaches, we show that VirB:DNA interactions in vitro introduce positive supercoils in plasmid DNA. Subsequently, by exploiting transcription-coupled DNA supercoiling, we reveal that a localized loss of negative supercoils is sufficient to alleviate H-NS-mediated transcriptional silencing, independently of VirB. Together, our findings provide novel insight into VirB, a central regulator of Shigella virulence and more broadly, a molecular mechanism that offsets H-NS-dependent silencing of transcription in bacteria.

molecular biology↗

bioRxiv2020

γ-secretase promotes postsynaptic maturation through the cleavage of a Wnt receptor

An emerging feature of neurodegenerative disease is synaptic dysfunction and loss, leading to the suggestion that mechanisms required for synaptic maturation may be linked to disease. Synaptic maturation requires the transmission of signals between nascent synaptic sites and the nucleus, but how these signals are generated is not well understood. We posit that proteolytic cleavage of receptors, which enables their translocation to the nucleus, may be a shared molecular mechanism between the events that promote synaptic maturation and those linked to later-onset disorders of the nervous system, including neurodegenerative disease. Here we show during synaptic development, that cleavage of synaptic maturation molecules requires {gamma}-secretase, a protein complex linked to Alzheimers Disease, a devastating neurodegenerative condition, is required for postsynaptic maturation. In the absence of {gamma}-secretase, Drosophila neuromuscular synapses fail to appropriately recruit postsynaptic scaffolding and cytoskeletal proteins, and mutant larvae display behavioral deficits. At the NMJ, {gamma}-secretase promotes synaptic maturation through the cleavage of the Wnt receptor Fz2, and the subsequent entry of its C-terminus into the nucleus. A developmental synaptic role for {gamma}-secretase is also conserved in both the Drosophila central nervous system and mammalian cortical neuron dendrites. Finally, we found that similar maturation defects are evident in fly models for ALS, Alzheimers, Huntingtons, and Parkinsons Diseases. The previously unknown, but conserved, role for {gamma}-secretase coupled with its well-known role in neurodegenerative disease suggest that neurodevelopmental defects may be common to diverse neurodegenerative disease models.

neuroscience↗